Establishment of One-Pot Disulfide-Driven Cyclic Peptide Synthesis with a 3-Nitro-2-pyridinesulfenate

Abstract

We have developed a new one-pot disulfide-driven cyclic peptide synthesis. The entire process is carried out in the solid phase, thus eliminating complicated work up procedures to remove by-products and unreacted reagents and enabling production of high-purity cyclic disulfide peptides by simple cleavage of a peptidyl resin. The one-pot synthesis of oxytocin was accomplished in this way with an isolated yield of 28% over 13 steps. These include peptide chain elongation from an initial resin, sulfenylation of the protected side chain of a cysteine (Cys) residue, disulfide ligation between thiols in an additional peptide fragment and a 3-nitro-2-pyridinesulfenyl-protected cysteine (Cys(Npys))-containing peptide resin, subsequent intramolecular amide bond formation of the disulfide-connected fragments by an Ag⁺-promoted thioester method, followed by deprotection and HPLC purification.

Introduction

Disulfide bonds in peptides and proteins contribute to the immobilization and rigidity of their structures, leading to the expression of functional activity and resistance to metabolic enzymes.¹⁾ In recent research into drug development, disulfide cyclic peptides are useful because this immobilization can result in benefits such as improved pharmacological activity and pharmacokinetics. Many cyclic peptides with disulfide bonds have been approved by U.S. Food and Drug Administration (FDA). For instance, Plecanatide, containing two disulfides is an agonist of guanylate cyclase-C and is used for the treatment of chronic idiopathic constipation in adults,²⁾ and Setmelanotide acts as a selective melanocortin-4 receptor agonist used for the treatment of rare genetic diseases associated with obesity.³⁾ As a peptide-chelator-radionuclide conjugates used in positron emission tomography imaging, [⁶⁸Ga]-DOTATOC and [⁶⁴Cu]-DOTATATE are cyclic disulfide 7-mer peptides used to target somatostatin receptor and were recently approved by FDA in 2019 and 2020, respectively.^4,5)

Efficient and convenient synthetic methodology of disulfide peptides could promote the development of peptide drugs and radioactive diagnostic agents. Disulfide peptides are conventionally synthesized in solution by oxidation with air or iodine of free or protected thiol groups of linear peptides which in turn are prepared by cleavage of peptidyl resins constructed by 9-fluorenylmethyloxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) (Fig. 1A). However, these oxidations in solution have some practical limitations: i) the reactions are carried out with dilute solutions to avoid formation of undesired oligomers by intermolecular reactions; ii) due to the low solubility of peptides, the available solvents for the reaction can be limited; iii) a cumbersome purification step is necessary after each reaction to isolate the desired peptides.

Fig. 1. Synthetic Routes to Disulfide Peptides: A) Conventional in-Solution Method; B) Disulfide-Driven Cyclic Peptide Synthesis (DdCPS) Method; C) One-Pot DdCPS Method

To establish a synthetic methodology to produce disulfide compounds efficiently, we focused on reactions of the 3-nitro-2-pyridinesulfenyl (Npys) group.^6–8) Npys-protected cysteine, Cys(Npys) is prepared from the reaction of Npys-Cl with the thiol group of cysteine. Because it functions as an active disulfide, Cys(Npys) readily reacts with unprotected thiol groups, forming the corresponding disulfide products. Previously, we developed one-pot solid-phase disulfide ligation (SPDSL) which uses an Npys-Cl resin. A component A, containing a t-Bu-protected thiol is loaded onto Npys-resin via an active disulfide. After washing the resin, a disulfide exchange reaction between an unprotected thiol-containing component B and an active disulfide on the resin results in efficient production in solution of a compound with a disulfide connection between the two components with high purity, whereas unreacted active disulfide and thiopyridone by-product are remaining on the resin^9–11) (Chart S1 in Supplementgary Materials).

As shown in Fig. 1B, we recently have developed the disulfide-driven cyclic peptide synthesis (DdCPS) which proceeds in two steps: i) formation of an asymmetric disulfide peptide by SPDSL from two different sulfur-containing peptide fragments; and ii) subsequent cyclization of this disulfide peptide using thioester ligation or conventional coupling reagents. After the prior formation of the disulfide cross-linking between two peptide fragments by SPDSL, the intramolecular cyclization is assisted by the proximity effect of each reaction center, and more efficient and higher regioselectivity can be expected than in the intermolecular amide bond formation.^12,13) We have achieved practical syntheses of oxytocin and human endothelin-2 by DdCPS, which indicates its utility as a synthetic method to produce cyclic disulfide peptides.^12,14) However, since these syntheses involve in-solution reactions of intramolecular cyclization and deprotection of N-terminal Fmoc-group and other protecting groups, extensive HPLC purification is necessary to remove the reagents and by-products after these reactions.

If a “one-pot” DdCPS methodology in which all the reactions are carried out on the resin could be established, simple filtration after each reaction can avoid purification steps to remove the reagents and by-products. This can also be expected to produce the pure desired disulfide peptides by simple cleavage from the resin and removal of protecting groups (Fig. 1C). Consequently, in this study, we investigated the one-pot DdCPS of oxytocin, a cyclic peptide containing a disulfide. This one-pot reaction includes peptide chain elongation from initial resin-bound amino acid and on-resin reactions including Npy-sulfenylation, disulfide ligation, intramolecular cyclization, and cleavage/deprotection of peptide from the resin. For the on-resin Npy sulfenylation reaction, we used 3-nitro-2-pyridinesulfenates (Npy sulfenates, Npys-ORs) that are disulfide bond formation reagents in the new Npys category.^15–17) As a representative compound, 4-fluorophenyl 3-nitro-2-pyridinesulfenate (Npys-OPh(pF)) has a high reactivity to side chain t-Bu, acetamidomethyl and 4-methoxybenzyl protected-Cys as well as a conventional reagent, Npys-Cl and efficiently yields the corresponding active disulfide (Cys(Npys)).¹¹⁾ A major drawback of Npys-Cl is its instability in the presence of light or moisture, but the physicochemical stability of Npys-OPh(pF) is superior that of Npys-Cl.¹⁶⁾

Experimental

General Information

Solvents and reagents were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan), Kokusan Chemical Co., Ltd. (Tokyo, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and Watanabe Chemical Industries, Ltd. (Hiroshima, Japan). Mass spectra (TOF MS ES+) were obtained on a Waters MICRO MASS LCT-premier mass spectrometer. Preparative HPLC was performed using a C18 reversed-phase column (19 × 150 mm; SunFire™ Prep C18 OBDTM 5 µm) with a binary solvent system. Analytical HPLC was performed using a C18 reversed-phase column (4.6ID × 150 mm; LaChrom II C18 (5 µm) P/N 889-0911) with a binary solvent system.

Peptide Synthesis

Peptide fragments were synthesized using the Fmoc/t-Bu-SPPS protocol. The Fmoc-amino acids used include Fmoc-Cys(Trt)-OH, Fmoc-Cys(t-Bu)-OH, Fmoc-Cys(St-Bu)-OH, Fmoc-glycine (Gly)-OH, Fmoc-isoleucine (Ile)-OH, Fmoc-leucine (Leu)-OH, Fmoc-asparagine (Asn)(Trt)-OH, Fmoc-D-Asn(Trt)-OH, Fmoc-proline (Pro)-OH, Fmoc-glutamine (Gln)(Trt)-OH, Fmoc-tyrosine (Tyr)(Ot-Bu)-OH and Fmoc-arginine (Arg)(Pbf)-OH. To the H-Rink amide ChemMatrix resin or H-Asn(Trt)-Trt(2-Cl) chloride resin in a reaction vessel, N,N-dimethylformamide (DMF) was added and removed after 20 min to swell the resin. The coupling steps were then carried out using the Fmoc-amino acid (3.0 equivalent (equiv.)) and 1-hydroxybenzotriazole monohydrate (HOBt·H₂O, 3.0 equiv.) in DMF, and N,N′-diisopropylcarbodiimide (DIPCI, 3.0 equiv.). The resin and solutions were mixed (30 min). By repeating the Fmoc-deprotection and coupling reaction steps, the peptidyl resins with protecting groups were obtained. After washing the resin with DMF and MeOH, the cleavage and deprotection steps of resin was carried out by treatment with trifluoroacetic acid (TFA) cocktail (5 mL) for 3 h. Then the TFA solutions were removed in a N₂ stream. The crude peptide was precipitated with cold Et₂O and centrifuged twice. Finally, the crude peptide was dissolved in H₂O with 0.1% TFA/CH₃CN and purified by preparative reverse-phase HPLC.

Synthesis of Peptide Thioester, Fmoc-Cys-Tyr-Ile-Gln-Asn-SCH₂CH₂SO₃Na (2)

The peptide thioester (2) was synthesized by the method of Hojo et al.^18,19) Fmoc-Rink amide resin (1.00 g, 0.45 mmol) was treated with 20% (v/v) piperidine/DMF solution for 25 min (2×). Then, Fmoc-Arg(Pbf)-OH (931 mg, 1.35 mmol), HOBt·H₂O (207 mg, 1.35 mmol), and DIPCI (209 µL, 1.35 mmol) were added to the resin. With the same procedure, another Arg residue was introduced to give the Fmoc-[Arg(Pbf)]₂-NH-resin. After removal of the Fmoc group, a solution of Fmoc-Asn(Trt)-(Et)Cys(Trt)-OH^18,19) (873 mg, 0.90 mmol), HOBt·H₂O (207 mg, 1.35 mmol) and DIPCI (209 µL, 1.35 mmol) in DMF was added to the resin, and the mixture was vortex-stirred overnight at room temperature (r.t.). The peptide chain, Fmoc-Asn(Trt)-(Et)Cys(Trt)-[Arg(Pbf)]₂-NH-resin was elongated by the Fmoc-SPPS method, yielding the Fmoc-Cys(Trt)-Tyr(t-Bu)-Ile-Gln(Trt)-Asn(Trt)-(Et)Cys(Trt)-[Arg(Pbf)]₂-NH-resin. The resin was treated with a TFA cocktail (TFA:H₂O:triisopropylsilane (TIS):1,2-ethanedithiol = 94 : 2.5 : 1.0 : 2.5, v/v/v/v, 20 mL), and the mixture was stirred for 3 h at r.t. After filtration, the TFA was removed in an N₂ stream. The residue was precipitated with ice-chilled Et₂O, washed twice with ice-chilled Et₂O, and dried in vacuo. The crude (679 mg) was dissolved in 40 mL of a solution of 50% MeCN aq. containing 6 M urea, 5% (v/v) acetic acid and sodium 2-mercaptoethane sulfonate (2.10 g). The mixture was stirred for 115 h at r.t. Subsequent purification using reversed-phase HPLC gave the peptide thioester (2) (36.24 mg, 36.75 µmol, 7% yield, HPLC chart is shown in Supplementary Fig. S2) as a white solid. High resolution (HR)MS (electrospray ionization (ESI)) m/z Calcd for C₄₄H₅₆N₇O₁₃S₃ [M + H]⁺ 986.3098. Found 986.3098.

Synthesis of Peptidyl Resin (3), H-Cys(t-Bu)-Pro-Leu-Gly-NH-resin

H-Rink amide ChemMatrix resin with a loading rate of 0.45 mmol/g (210 mg, 0.09 mmol), was elongated by the Fmoc-SPPS method, yielding the H-Cys(t-Bu)-Pro-Leu-Gly-NH-resin (3) (237 mg, 0.09 mmol). To analyze the reaction, the peptidyl resin (3) (1.1 mg) treated with a TFA cocktail (TFA:H₂O:TIS = 95 : 2.5 : 2.5, v/v/v, 1 mL), and the mixture was stirred for 1 h at r.t. and the TFA was removed in an N₂ stream. H₂O was added to the residue and the mixture was lyophilized. The crude was product was dissolved in 50% MeCN aq. (205 µL) for analysis by HPLC and HR-MS. The desired t-butyl protected form (3′) was obtained with 90% purity (linear gradient from 15 to 65% MeCN over 50 min, t_R:13.01 min, the HPLC chart is shown in Fig. 2A in main text). HR-MS (ESI) m/z Calcd for C₂₀H₃₈N₅O₄S [M + H]⁺ 444.2645. Found 444.2644.

Fig. 2. HPLC of Crude Peptides Cleaved from: A) Peptidyl Resin 3; B) 4; C) 5; D) 6 and E) Crude Oxytocin (1)

A value in the parentheses in the charts shows the purity of the peptide formed on the resin. *Non-peptide peak.

On-resin Sulfenylation

The peptidyl resin (3) (9.1–11.2 mg, 3.5–4.2 µmol) was treated with 20 mM Npys-OPh(pF) (1.0 equiv.) or Npys-OMe (1.0 equiv.) solution in DMF, 10% (v/v) AcOH/DMF or 50% (v/v) AcOH/DMF, AcOH or 0.4 M LiCl/AcOH), with shaking for 1 h at r.t. The resin was then washed with DMF and MeOH, and dried in vacuo. This led to the H-Cys(Npys)-Pro-Leu-Gly-NH-resin (4) (8.6–11.3 mg) was obtained. To analyze the reaction, the cleavage of peptidyl resin (4) (1.0–1.8 mg) was treated with a TFA cocktail (TFA:H₂O:TIS = 95 : 2.5 : 2.5, v/v/v, 1 mL), and the mixture was stirred for 1 h at r.t. The TFA was removed in an N₂ stream. H₂O was added to the residue and the mixture was lyophilized. The crude product was dissolved in 50% MeCN aq. (200–330 µL) for analysis by HPLC and HR-MS. The desired Npys protected form (4′) produced under reaction condition of Entry 5 in Table 1 was obtained with >99% purity (linear gradient from 15 to 65% MeCN over 50 min, t_R:14.39 min, HPLC chart is shown in Fig. 2B in the main text). HR-MS (ESI) m/z Calcd for C₂₁H₃₂N₇O₆S₂ [M + H]⁺ 542.1855. Found 542.1856.

Table 1. Conversion of Cys(t-Bu) to Cys(Npys) on Peptidyl Resin^a)

Entry	Solvent	Npys derivative	Conversion rate (%) to resin 4b)
1	DMF	Npys-OPh(pF)	0
2	10% (v/v) AcOH/DMF	Npys-OPh(pF)	0
3	50% (v/v) AcOH/DMF	Npys-OPh(pF)	0
4	AcOH	Npys-OPh(pF)	26
5	0.4 M LiCl/AcOH	Npys-OPh(pF)	100
6	0.4 M LiCl/AcOH	Npys-OMe	21

a) Reaction conditions: Npys derivative (1.0 equiv.) in each solvent was 20 mM in a reaction for 1 h at r.t. b) Purity of the peptide was determined by RP-HPLC analysis of crude products after the treatment of peptidyl resins with TFA:TIS:H₂O (95 : 2.5 : 2.5, v/v/v) at r.t. for 1 h. Conversion rate (%) to resin (4) was calculated by the following formula; [peak area of peptide 4′/(peak area of peptide 3′ + peak area of peptide 4′)] × 100.

On-resin Disulfide Exchange Reaction

A solution of Fmoc-Cys-Tyr-Ile-Gln-Asn-SCH₂CH₂SO₃Na (2) (14.4 mg, 14.6 µmol, 10 mM) in 80% DMF/0.1 M sodium acetate buffer (pH 4.5) was added to the sulfenylated peptidyl resin (4) (28.7 mg, 14.6 µmol), and shaken for 1 h at r.t. The resin was washed with DMF and MeOH, and dried in vacuo, producing the disulfide peptidyl resin 5 (33.7 mg). To analyze the reaction, the cleavage products of the peptidyl resin (5) (1.2 mg) were treated with a TFA cocktail (TFA:H₂O = 95 : 5, v/v, 1 mL), and the mixture was stirred for 1 h at r.t. The TFA was removed in an N₂ stream. The residue was washed and precipitated twice with Et₂O (1 mL), then dried in vacuo. The crude was dissolved in 50% MeCN aq. (102 µL) for analysis by HPLC and HR-MS. The desired disulfide peptide (5′) was obtained in 76% purity (linear gradient from 15% to 65% MeCN over 50 min, t_R:23.97 min, HPLC chart is shown in Fig. 2C in main text). HR-MS (ESI) m/z Calcd for C₆₀H₈₃N₁₂O₁₇S₄ [M + H]⁺ 1371.4882. Found 1371.4878.

On-Resin Cyclization by Ag⁺-Promoted Thioester Method

A solution of AgNO₃ (1.4 mg, 8.3 µmol), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) (4.1 mg, 24.9 µmol) and N,N-diisopropylethylamine (DIPEA) (2.1 µL, 12.5 µmol) in DMF (277 µL) was added to the disulfide peptidyl resin (5) (5.2 mg, 1.66 µmol), and shaken for 6 h at r.t. in the dark. The resin was washed with DMF and MeOH, and dried in vacuo. Then, Fmoc-Cys*-Tyr-Ile-Gln-Asn-Cys*-Pro-Leu-Gly-NH-resin (6) (* = disulfide bond) (5.0 mg) was obtained. To analyze the reaction, the cleavage of peptidyl resin (6) was carried out with a TFA cocktail (TFA:H₂O = 95 : 5, v/v, 1 mL), and the mixture was stirred for 1 h at r.t. The TFA was removed in an N₂ stream and the residue was washed and precipitated twice with Et₂O (1 mL), and dried in vacuo. The crude product was dissolved in 50% MeCN aq. (332 µL) for analysis by HPLC and HR-MS. The desired cyclic peptide 6′ was obtained with 61% purity (linear gradient from 15 to 65% MeCN over 50 min, t_R:30.97 min, HPLC chart is shown in Fig. 2D in main text). HR-MS (ESI) m/z Calcd for C₅₈H₇₆N₁₂O₁₄S₂ [M + H]⁺ 1229.5124. Found 1229.5129.

Fmoc-Deprotection and Cleavage from the Resin

The peptidyl resin (6) (25.3 mg, 8.0 µmol) was treated with 20% (v/v) piperidine/DMF (1.0 mL), and shaken for 20 min at r.t. The resin was washed with DMF and MeOH, and dried in vacuo. The resin was treated with a TFA cocktail (TFA:H₂O = 95 : 5, v/v, 4 mL). After stirring the mixture for 3 h at r.t., the TFA was removed in an N₂ stream. The residue was washed and precipitated twice with Et₂O (10 mL), and dried in vacuo. The desired product, oxytocin (1) was obtained with 51% purity (linear gradient from 10 to 40% MeCN over 30 min, t_R:16.86 min, HPLC chart is shown in Fig. 2E in main text). Subsequent purification using reversed-phase HPLC gave oxytocin (1) (2.52 mg, 2.25 µmol, 28% yield, the HPLC chart is shown in Supplementary Fig. S3) as a white solid. HR-MS (ESI) m/z Calcd for C₄₃H₆₇N₁₂O₁₄S₂ [M + H]⁺ 1007.4443. Found 1007.4424.

Synthesis of Fmoc-(5-D-Asn)-oxytocin (D-6′)

Fmoc-(5-D-Asn)-oxytocin (D-6′) was prepared by a synthetic procedure.¹⁵⁾ Fmoc-Rink amide resin (108 mg, 40 µmol) was subjected to the Fmoc-SPPS method using the automatic protocol of the peptide synthesizer Prelude^® to obtain H-Tyr(t-Bu)-Ile-Gln(Trt)-D-Asn(Trt)-Cys(St-Bu)-Pro-Leu-Gly-NH-resin. After swelling the resin, Fmoc-Cys(St-Bu)-OH (51.9 mg, 120 mmol), HOBt·H₂O (18.4 mg, 120 mmol) and DIPCI (18.6 µL, 120 mmol) were added to the resin, which was shaken for 1.5 h at r.t. The resin was washed with DMF and MeOH, and dried in vacuo. Fmoc-Cys(St-Bu)-Tyr(t-Bu)-Ile-Gln(Trt)-D-Asn(Trt)-Cys(St-Bu)-Pro-Leu-Gly-NH-resin (168 mg) was obtained. Part of the resin (19.6 mg, 4.7 µmol) was treated with a solution of tributylphosphine (PBu₃, 115.5 µL, 468 µmol) in trifluoroethanol (TFE) cocktail (TFE:H₂O:PBu₃ = 90 : 5 : 5, v/v/v, 2.3 mL), and shaken for 1 h at r.t. The resin was washed with DMF, MeOH and Et₂O, and dried in vacuo. Then, thiol free containing peptidyl resin, Fmoc-Cys-Tyr(t-Bu)-Ile-Gln(Trt)-D-Asn(Trt)-Cys-Pro-Leu-Gly-NH-resin (15.3 mg) was obtained. This resin (14.1 mg, 4.3 µmol) was treated with a solution of 5 mM Npys-OMe (2 equiv.) in 0.4 M LiCl/DMF, and the mixture was stirred for 3 h at r.t. The resin was washed with DMF, MeOH and Et₂O, and dried in vacuo. The resin was treated with a TFA cocktail (TFA:H₂O:TIS = 95 : 2.5 : 2.5, v/v/v, 3 mL). After stirring the mixture for 3 h at r.t., reversed-phase HPLC gave Fmoc-(5-D-Asn)-oxytocin (D-6′) (0.7 mg, 0.57 µmol, 13% yield, HPLC chart is shown in Supplementary Fig. S4) as a white solid. HR-MS (ESI) m/z Calcd for C₅₈H₇₆N₁₂O₁₄S₂ [M + H]⁺ 1229.5124. Found 1229.5123.

Results and Discussion

In one-pot DdCPS, as shown in Chart 1, the sequence of oxytocin (1, H-C*YIQNC*PLG-NH₂; * disulfide bond) was divided into two fragments at the ligation site between Asn5 and Cys6. Two fragments, an N-terminal pentapeptide alkyl thioester, Fmoc-CYIQN-SCH₂CH₂SO₃Na (2) and a C-terminal Cys(t-Bu) containing tetrapeptidyl resin, H-C(t-Bu)PLG-resin (3), were prepared. It was assumed that in on-resin sulfenylation and subsequent disulfide exchange reactions, a Cys residue located at the N-terminus may be sterically less hindered and more accessible to reactants with an Npy sulfenate and to an SH group of a peptide thioester 2. In this study, we chose as a suitable solid-support, a ChemMatrix resin which can swell in both organic and aqueous buffer solvents.²⁰⁾ A part of the resin collected after each reaction step was treated with a TFA cocktail (TFA:TIS:H₂O (95 : 2.5 : 2.5, v/v/v) or TFA:H₂O (95 : 5, v/v)) and the resulting crude products (indicated by primed compound numbers) were analyzed by RP-HPLC to determine their purity for evaluating each reaction.

Chart 1. One-Pot DdCPS of Oxytocin

Reagent and conditions: a) Fmoc/t-Bu-SPPS; b) Npys-OPh(pF) (20 mM) in 0.4 M LiCl/AcOH, room temperature (r.t.), 1 h; c) Fmoc-CYIQN-SCH₂CH₂SO₃Na (2), 80% (v/v) DMF/0.1 M NaOAc buffer (pH 4.5), r.t., 1 h; d) AgNO₃, HOOBt, DIPEA, DMF, r.t., 6 h; e) i) 20% (v/v) piperidine/DMF, r.t., 20 min, ii) TFA: H₂O = 95 : 5 (v/v), r.t., 1 h, HPLC purification, 28% (13 steps).

The one-pot DdCPS of oxytocin began with preparation of the peptidyl resin (3) in 8 steps from H-Rink amide ChemMatrix resin using a standard Fmoc/t-Bu solid phase peptide synthesis (SPPS) protocol, obtaining the desired resin with 90% purity (Fig. 2A). To investigate the conversion rate of Cys(t-Bu) to Cys(Npys) on resin, a 20 mM Npys-OPh(pF) solution in each of the solvents was added to H-C(t-Bu)PLG-resin (3, 1 equiv.), and shaken for 1 h at r.t. As shown in Entries 1–3 in Table 1, under low-acidic reaction conditions in DMF with 10 or 50% (v/v) AcOH/DMF, production of the Cys(Npys)-containing peptide, H-C(Npys)PLG-NH₂ (4′) was not observed. However, the desired peptide was produced in conversion rate with 26% when glacial AcOH was used (Entry 4). To improve the on-resin Npy-sulfenylation, LiCl was used as an additive because lithium salt additives can also increase resin swelling and improve the efficiency of the sulfenylation.¹⁵⁾ With 0.4 M LiCl/AcOH, the production of Npys-OPh(pF) bound to resin (4) was dramatically increased (Entry 5, Fig. 2B, purity >99%), but Npys-OMe¹⁵⁾ a mild oxidizing reagent was less reactive (21%, Entry 6). This result indicates that the phenoxide Npys-OPh(pF) with pK_a = 10²¹⁾ is more reactive than the methoxide (pK_a 16²¹⁾). After washing the peptidyl resin, the disulfide exchange reaction between the compound (4) and peptide thioester, Fmoc-CYIQN-SCH₂CH₂SO₃Na (2), proceeded smoothly in 80% (v/v) DMF/0.1 M NaOAc buffer (pH 4.5) at rt for 1 h, resulting producing the peptidyl resin (5) with satisfactory purity (76%) as shown in Fig. 2C.

With the peptidyl resin (5) in hand, we investigated the on-resin intramolecular cyclization between an alkyl thioester at Asn and an amino group at Cys using an Ag⁺-promoted thioester method. As shown in Table 2, according to synthetic procedure in-solution, when using HOOBt and an Ag⁺ source, AgCl as a thioester activator, in the presence of DIPEA in dimethyl sulfoxide (DMSO) for 24 h at r.t., the conversion rate to resin 6 was 78% (Entry 1). In order to further improve the cyclization reaction, the Ag⁺ source was changed to AgNO₃ which has been reported by Teruya et al. to be an activator of thioester²²⁾ and the conversion rate in the presence of AgNO₃ in DMSO or DMF, was increased to 100 or 96%, respectively (Entries 2 and 3). In DMSO, reducing the reaction time to 6 h gave a 100% (Entry 4, Fig. 2D, purity: 61%), but reduction of the reaction time to 3 h decreased the production rate of resin 6 to 81% (Entry 5). The level of racemization at Asn position of crude Fmoc-oxytocin (6′) in the reaction conditions of Entry 4 in Table 2, was checked because it was reported that the thioester method risks racemization of the C-terminal amino acid at the ligation site.²²⁾ In HPLC analysis and mass measurement of the crude product, it was found that Fmoc-(5-D-Asn)-oxytocin (D-6′) could not be detected (see Supplementary Fig. S1 and Mass spectra in Supplementary Materials), indicating that on-resin cyclization via the thioester method could be proceed efficiently without racemization of Asn at the ligation site. In addition, aspartimide formation²³⁾ at Asn position did not occur (see Supplementary Chart S2 and Mass spectra).

Table 2. On-Resin Intramolecular Cyclization Using Ag⁺-Promoted Thioester Method^a)

Entry	Ag+ Source	Solvent	Time (h)	Conversion rate (%) to resinb)
1	AgCl	DMSO	24	78
2	AgNO3	DMSO	24	100
3	AgNO3	DMF	24	96
4	AgNO3	DMF	6	100
5	AgNO3	DMF	3	81

a) Reaction conditions: Ag⁺ source (5.0 equiv.), DIPEA (7.5 equiv.) and HOOBt (15 equiv.) at r.t. b) Conversion rate (%) to resin 6 was calculated by the following formula; [peak area of peptide 6′/(peak area of peptide 5′ + peak area of peptide 6′)] × 100.

Finally, the N-terminal Fmoc group of peptidyl resin 6 was deprotected by treatment with 20% (v/v) piperidine in DMF. After washing and drying the resin, the final deprotection and cleavage of the peptidyl resin with TFA cocktail (TFA:H₂O = 95 : 5, v/v) at r.t. for 1 h gave crude oxytocin (purity: 51%, Fig. 2E). Subsequent RP-HPLC purification afforded oxytocin with an isolated yield of 28% prepared in 13 steps from the initial H-Rink amide ChemMatrix resin. In an alternative route, oxytocin (1) could be prepared by using the unprotected side chain of the Cys-containing pentapeptide, Boc-CY(t-Bu)IQ(Trt)N(Trt)-OH (7) instead of the peptide thioester (2). The on-resin intramolecular cyclization between a carboxylic acid of Asn and the amine of Cys smoothly proceeded with conventional coupling reagents, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 1-hydroxy-7-azabenzotriazole in the presence of DIPEA. Consequently, oxytocin (1) was produced in 13% yield over 12 steps (see Supplementary Materials 7–11). These results indicated that an on-resin intramolecular cyclization from peptide fragment and peptidyl resin is compatible with both thioester and conventional condensation methods.

Conclusion

We have successfully developed a one-pot disulfide-driven cyclic peptide synthesis, in which all the reaction steps were carried out on a resin. This method could avoid laborious purification steps instead simply requiring filtration of the reaction mixture. To demonstrate this concept, a one-pot synthesis of oxytocin (1) was achieved. This involved peptide elongation by Fmoc/t-Bu-SPPS from the initial resin, on-resin sulfenylation at the protected side chain of a Cys residue, disulfide ligation between Cys(Npys) containing a peptidyl resin and a peptide thioester, intramolecular cyclization by Ag⁺-promoted thioester method and cleavage from cyclized peptidyl resin. In the intramolecular cyclization, the racemization of C-terminal amino acid at ligation site was not observed. HPLC purification of crude oxytocin easily afforded the product with high purity. This method can therefore provide efficient synthesis of cyclic disulfide peptides and can be used in the preparation of more complex disulfide peptides and artificial peptides.

Acknowledgments

The authors acknowledge Mr. H. Fukaya of Tokyo University of Pharmacy and Life Sciences for mass spectral analysis. This work was supported by the Japan Society for the Promotion of Science (JSPS), KAKENHI, a Grant-in-Aid for Young Scientists (B) 16K18914, Early-Career Scientists 19K16324 and Scientific Research (B) 19H03356 and JST SPRING, Grant Number JPMJSP2134.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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