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Communication

Production of Atosiban’s Key Intermediate Pentapeptide: Synthetic Approaches to the Development of a Peptide Synthesis with Less Racemization and Simplifier Purification Process

Department of New Drug Research and Development, Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, China
*
Author to whom correspondence should be addressed.
Molecules 2022, 27(6), 1920; https://doi.org/10.3390/molecules27061920
Submission received: 16 December 2021 / Revised: 10 March 2022 / Accepted: 14 March 2022 / Published: 16 March 2022

Abstract

:
The key intermediate NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH of Atosiban was prepared from N-Boc-S-Bzl-cysteine by the stepwise lengthening of the chain according to the repetitive N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) strategy. This synthetic route required no chromatography purification and can be readily performed, yielding a highly pure pentapeptide compound.

1. Introduction

Atosiban (Figure 1) is a competitive antagonist of oxytocin receptors (OTR) that has been approved in Europe for the short-term treatment of preterm labor. It can inhibit oxytocin from binding to its receptors, which are expressed by the myoepithelial cells of the mammary gland, and in both the myometrium and endometrium of the uterus [1,2,3,4]. The solid-phase method has been widely adopted to synthesize Atosiban [5,6,7,8]; however, it is inappropriate to start large-scale productions due to its high cost. A solution synthesis route, which was accomplished by the addition of three polypeptide fragments end to end, was suggested for the preparation of Atosiban, and the pentapeptide NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH (Figure 1) was a key intermediate in the liquid synthesis [9].
However, most repetitive solution-phase methodologies used superstoichiometric amounts of a coupling reagent and activated amino acids to ensure high coupling efficiency, and later, the excess of the reagent had to be neutralized by additional reactions, followed by a purification procedure using several acidic and basic aqueous extractions [10,11,12,13]. These post-synthetic treatments caused the synthetic procedure to become complicated and time-consuming, and the severe conditions they used might have destroyed the peptide products and introduced undesirable impurities. In our previous study, a method using BSA/NHS (Figure 1) as coupling agents for the dipeptide synthesis was proven to be simple and efficient [14]. In order to further understand the BSA/NHS strategy in the synthesis of peptide, the key intermediate, NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH, was synthesized using the BSA/NHS strategy. In comparison, the synthesis of the key pentapeptide utilized five different coupling methods [15].

2. Results and Discussion

Our previous experiments confirmed that unprotected amino acid could react with Boc-protected NHS ester with the assistance of BSA at room temperature in a high coupling efficiency for dipeptide synthesis [14]. In our current study, Boc-Cys(Bzl)-Pro-COOH was obtained using this approach (Figure 2) in a 92.4% yield.
In addition, in the synthesis of Atosiban’s key intermediate pentapeptide, all the excessive reagents and increased byproducts could be removed just using water or saturated sodium chloride solution rather than certain amounts of acidic and basic aqueous extractions [16]. All the excessive reactants, byproducts and the racemization products were undetected, according to the results of the NMR analysis (Figure 3).
The N-Boc protecting group was subsequently cleaved using trifluoroacetic acid/dichloromethane (TFA/CH2Cl2) (1:1), and the pure deprotected dipeptide NH2-Cys(Bzl)-Pro-COOH was obtained after the additional recrystallization from diethyl ether (94.0% yield). More impurities would be observed when just trifluoroacetic acid was utilized as a deprotection reagent, and therefore the yield would be low as well as the purification process would be more complicated. When peptide was produced in the form of hydrochloride salt, it would be more hygroscopic. According to the results of the HPLC analysis (Figure 4), no epimerization happened during the deprotection process either. Above all, we could synthesis dipeptide products in good yield and high purity in significantly shorter reaction times and with a simple purification process.
Then, the peptide sequence could be extended by repeating the same coupling and deprotection cycle after the removal of the Boc-protecting group. The synthetic routine for the NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH was demonstrated in Scheme 1.
Before the synthesis of the pentapeptide intermediate, the N-Boc-protected amino acid fragments, including N-Boc-S-benzyl-cysteine (Cys), N-Boc-asparagine (Asn), N-Boc-O-benzyl threonine (Thr) and N-Boc-isoleucine (Ile), were derivatized with NHS ester at the C-terminus in advance [17].
Based on the above study, the deprotected dipeptide TM2 was obtained in good yield as colorless bulk crystals. Then, the reaction between TM2 and Boc-Asn-ONHS afforded the Boc-protected tripeptide Boc-TM3 in 92.1% yield, which was recrystallized from ethyl acetate. The Boc group was then cleaved and TM3 precipitated as a white solid after the addition of anhydrous diethyl ether (93.7% yield). The resulting peptide TM3 reacted with Boc-Thr(Bzl)-ONHS to produce Boc-TM4 in 91.2% yield. After the same deprotection steps as above, the deprotected tetrapeptide TM4 was reacted with Boc-Ile-ONHS to give Boc-TM5 after recrystallization from ethyl acetate/diethyl ether in the yield of 88.6%. After the same deprotection steps, the final product, TM5, was obtained in 90.9% yield. Both the deprotected tetrapeptide TM4 and deprotected pentapeptide TM5 were recrystallized from anhydrous diethyl ether. The HPLC analysis of the deblocked pentapeptide, TM5, demonstrated that even after four coupling cycles, no further purification was required (Figure 5).

3. Materials and Methods

All raw materials, reagents and solvents were purchased from commercial suppliers and used without further purification. NMR spectra were acquired in chloroform (CDCl3), methanol-d4 and dimethylsulfoxide-d6 (DMSO-d6) using a Varian Inova 400 (400 MHz) instrument. The tetramethylsilane, as a reference, was used.
Chromatographic conditions: Instrument: Waters 2695; chromatographic column: Diamonsil C18 (5 uM, 250 * 4.6 mm); flow rate: 1 mL/min; column temperature: 30 °C; mobile phase: phase A: CH3CN, 0.1% TFA; phase B: water, 0.1% TFA; gradient conditions: 0.01 min → 25.0 min: 5% phase A → 70% phase A; 25.0 min → 30 min: 70% phase A → 90% phase A.
General Procedure for the formation of amide bond using BSA/NHS strategy. Under argon protection, BSA (2.2 equiv.) was added to amino precursor (1.1 equiv.) in anhydrous CH2Cl2. After the mixture was stirred for 1–24 h at 25 °C, a solution of N-Boc protected NHS ester (1 equiv.) in dichloromethane was added. The reaction mixture was stirred at 25 °C under argon until all active ester was consumed, as judged by the TLC analysis. The reaction mixture was washed with brine, dried over Na2SO4 and concentrated in vacuo to provide a white solid. The isolated product was recrystallized from diethyl ether/n-hexane to yield the targeted compound.
General Procedure for the Boc-deprotected reaction. The material was dissolved in CH2Cl2 and a solution of TFA/CH2Cl2 (1:2) (10 equiv.) was added. After the mixture was stirred for 4 h at 25 °C, the reaction mixture was concentrated in vacuo to yield a yellow oil. Afterwards, the pure product was recrystallization from diethyl ether as a white solid.

4. Conclusions

In summary, we have successfully developed a rapid, large-scale solution-phase synthesis of the key pentapeptide intermediate of Atosiban in a repetitive BSA/NHS strategy. Less racemization happened, shorter numbers of unit operation were necessary and the purification process was more simplified than other repetitive solution-phase methodologies. Above all, the repetitive BSA/NHS strategy has the potential to be applied in the further commercial-scale manufacturing of more peptide drugs.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules27061920/s1. Figure S1: The 1H NMR of Boc-Cys(Bzl)-ONHS; Figure S2: The 13C NMR of Boc-Cys(Bzl)-ONHS; Figure S3: The 1H NMR of Boc-Asn-ONHS; Figure S4: The 13C NMR of Boc-Asn-ONHS; Figure S5: The 1H NMR of Boc-Thr(Bzl)-ONHS; Figure S6: The 13C NMR of Boc-Thr(Bzl)-ONHS; Figure S7: The 1H NMR of Boc-Ile-ONHS; Figure S8: The 13C NMR of Boc-Ile-ONHS; Figure S9: The 1H NMR of Boc-TM2; Figure S10: The 13C NMR of Boc-TM2; Figure S11: The 1H NMR of TM2; Figure S12: The 13C NMR of TM2; Figure S13: The 1H NMR of Boc-TM3; Figure S14: The 13C NMR of Boc-TM3; Figure S15: The 1H NMR of TM3; Figure S16: The 13C NMR of TM3; Figure S17 The MS spectrum of TM3; Figure S18: The 1H NMR of Boc-TM4; Figure S19: The 13C NMR of Boc-TM4; Figure S20: The 1H NMR of TM4; Figure S21: The 13C NMR of TM4; Figure S22: The MS spectrum of TM4; Figure S23: The 1H NMR of Boc-TM5; Figure S24: The 13C NMR of Boc-TM5; Figure S25: The 1H NMR of TM5. Figure S26: The 13C NMR of TM5; Figure S27: The MS spectrum of TM5.

Author Contributions

C.M. and W.F. conceived and designed the experiments. C.M., J.L. and Y.Z. performed the experiments. C.M. wrote the manuscript. Y.Z. and X.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (No. 81903432), the Fundamental Research Funds for the Central Universities and the 2019 Key R&D Plan Program of Shandong (Major Scientific and Technological Innovation Project) (No. 2019JZZY020601).

Institutional Review Board Statement

Not applicable for studies not involving humans or animals.

Informed Consent Statement

Not applicable for studies not involving humans.

Data Availability Statement

Data are contained within the article or the Supplementary Material.

Acknowledgments

We thank the National Natural Science Foundation of China (No. 81903432) and the Fundamental Research Funds for the Central Universities, 2019 Key R&D Plan Program of Shandong (Major Scientific and Technological Innovation Project) (No. 2019JZZY020601) for the financial support of this project.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

References

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Figure 1. Structure of Atosiban, pentapeptide intermediate, BSA and NHS ester.
Figure 1. Structure of Atosiban, pentapeptide intermediate, BSA and NHS ester.
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Figure 2. Synthesis of Boc-Cys(Bzl)-Pro-COOH using BSA/NHS as coupling agents.
Figure 2. Synthesis of Boc-Cys(Bzl)-Pro-COOH using BSA/NHS as coupling agents.
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Figure 3. NMR analysis of Boc-Cys(Bzl)-Pro-COOH synthesized on BSA/NHS strategy.
Figure 3. NMR analysis of Boc-Cys(Bzl)-Pro-COOH synthesized on BSA/NHS strategy.
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Figure 4. HPLC analysis of NH2-Cys(Bzl)-Pro-COOH.
Figure 4. HPLC analysis of NH2-Cys(Bzl)-Pro-COOH.
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Scheme 1. Solution-phase synthesis of NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH using repetitive BSA/NHS strategy.
Scheme 1. Solution-phase synthesis of NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH using repetitive BSA/NHS strategy.
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Figure 5. HPLC analysis of NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH synthesized on BSA/NHS strategy.
Figure 5. HPLC analysis of NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH synthesized on BSA/NHS strategy.
Molecules 27 01920 g005
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Ma, C.; Zheng, Y.; Liu, J.; Li, X.; Feng, W. Production of Atosiban’s Key Intermediate Pentapeptide: Synthetic Approaches to the Development of a Peptide Synthesis with Less Racemization and Simplifier Purification Process. Molecules 2022, 27, 1920. https://doi.org/10.3390/molecules27061920

AMA Style

Ma C, Zheng Y, Liu J, Li X, Feng W. Production of Atosiban’s Key Intermediate Pentapeptide: Synthetic Approaches to the Development of a Peptide Synthesis with Less Racemization and Simplifier Purification Process. Molecules. 2022; 27(6):1920. https://doi.org/10.3390/molecules27061920

Chicago/Turabian Style

Ma, Chunying, Yixuan Zheng, Jinwei Liu, Xiaobao Li, and Wenhua Feng. 2022. "Production of Atosiban’s Key Intermediate Pentapeptide: Synthetic Approaches to the Development of a Peptide Synthesis with Less Racemization and Simplifier Purification Process" Molecules 27, no. 6: 1920. https://doi.org/10.3390/molecules27061920

APA Style

Ma, C., Zheng, Y., Liu, J., Li, X., & Feng, W. (2022). Production of Atosiban’s Key Intermediate Pentapeptide: Synthetic Approaches to the Development of a Peptide Synthesis with Less Racemization and Simplifier Purification Process. Molecules, 27(6), 1920. https://doi.org/10.3390/molecules27061920

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