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Solid-phase synthesis of oligopeptides containing sterically hindered amino acids on non-swellable resin using 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1yl)phosphonium hexafluorophosphate (PyNTP) as the condensing reagent Rintaro Iwata Hara, Yuta Mitsuhashi, Keita Saito, Yuske Maeda, and Takeshi Wada ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00184 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018
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ACS Combinatorial Science
Solid-phase synthesis of oligopeptides containing sterically hindered amino acids on non-swellable resin using 3-nitro-1,2,4-triazol-1-yltris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as the condensing reagent Rintaro Iwata Hara,§ Yuta Mitsuhashi,§ Keita Saito,‡ Yusuke Maeda,¶ and Takeshi Wada§,* §
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan. ¶ Course of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. Supporting Information Placeholder ‡
ABSTRACT: Peptides are still difficult to synthesize when they
contain sterically hindered amino acids such as α,α-di-substituted amino acids and N-substituted amino acids. In this study, solidphase syntheses of oligopeptides containing multiple α-aminoisobutyric acid (Aib) residues were performed in high yields by using a non-swellable resin as the solid-support and 3-nitro-1,2,4-triazol1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as the condensing reagent.
Highly reactive condensing reagents for solid-phase peptide synthesis, such as HATU and COMU (Figure. 1), have enabled the synthesis of many difficult peptide sequences.1 By contrast, some peptides are still difficult to be synthesized even when using these conventional condensing reagents.2 One typically difficult peptide sequence is a peptide containing many sterically hindered amino acids, such as α,α-di-substituted amino acids and N-substituted amino acids.2a Furthermore, the synthesis of these peptides seems to be important because some of them are useful and/or show interesting properties. For example, oligopeptides containing α-aminoisobutyric acid (Aib) residues are known to have helicity,3 and some antibiotics based on Aib-containing peptides have been reported to work as ion channels because of their helicity.4 N-Methyl amino acids are seen in microbially derived peptides, including cyclic oligopeptides. Moreover, N-methylation of hydrophilic peptides is known to be a potential strategy for endow them with membrane permeability.5 In our previous study, we first reported 3-nitro-1,2,4-triazol-1-yltris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) (Figure 1) as a phosphonium azolide type condensing reagent, which had never been used for peptide synthesis. PyNTP enabled to synthesize peptides with both quick condensation and little epimerization compared with HATU.6 PyNTP was also applicable to Aib-containing peptide synthesis, although the yields of oligopeptides containing multiple Aib residues were moderate. In the current paper, we studied the potential of PyNTP for the synthesis of various Aib-containing peptides. We also compared non-swellable and swellable resins as solid supports in the solid-phase synthesis. Swellable resins are commonly used in peptide syntheses,7 whereas
non-swellable resins are commonly used in the solid-phase syntheses of nucleic acids8 and are rarely used for peptides.9 The use of nonswellable resins is advantageous because a variety of solvents are applicable for such resins and the resins can be dried in vacuo between respective steps. In particular, the latter feature is important in the case of highly reactive condensing reagents because the activated intermediates are susceptible to hydrolysis.
Figure 1. Structure of condensing reagents, HATU, COMU, PyAOP, PyOxim, and PyNTP. First, we compared PyNTP with other conventional condensing reagents, namely, HATU, COMU, PyAOP,10 and PyOxim (Fig. 1),11 for the synthesis of hexapeptide Ac-(Aib)4-Lys-Tyr-NH2. Peptide synthesis was conducted according to Scheme 1 on a swellable or non-swellable resin as the solid support. In this study, Fmoc-NHSAL-PEG Resin XV®, based on aminoethyl polyethylene glycol resin, was used as a swellable resin (260 μmol/g), and Custom Primer Support 200 amino®, based on highly-crosslinked polystyrene, functionalized with Fmoc-protected Rink amide12 was used as a non-swellable resin (125 μmol/g). After the syntheses, the crude products were analyzed by RP-HPLC. The results using the swellable resin are presented in Table 1. When using HATU or PyAOP, the hexapeptide was scarcely observed and short peptides were detected regardless of the solvent used (Entries 1, 2, 5, and 6). Among the other three condensing reagents, modest results were obtained using COMU and PyOxim. The condensation with COMU afforded a better yield in acetonitrile (27%) than in DMF. The condensation with PyOxim afforded a better yield in DMF (45%) than in acetonitrile as shown in Entries 3, 4, 7, and 8. The peptide synthesis using PyNTP in acetonitrile afforded an even better yield of the hexapeptide (66%, Figure 2a) than in DMF (29%), as shown in
Scheme 1 Solid-phase synthesis of oligopeptides
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coupling reactions in other solvents. By comparing these results that in the Entry 6, it is clear that acetonitrile is the best solvent for this peptide synthesis. In THF or 1,4-dioxane, coupling did not sufficiently proceed, and many short oligomers were detected (Entries 10 and 11). DMF was suggested to be an applicable solvent on the non-swellable resin (Entry 9), and this result was contrast to the coupling on the swellable resin (Entry 9 in Table 1). It should be noted that these differences in the results were not entirely derived from difference in the property of swellable or non-swellable resin and loading capacity of each resin might affect the coupling efficiency. In this study, we used commercially available swellable resin whose linker units were loaded to almost all resin-derived amino groups: i.e. the ratio of actual-loading/loading-capacity was ca. 1. On the other hand, the non-swellable resin was prepared from a commercial source (loading capacity: ca. 200 μmol/g) and the ratio of actual-loading/loading-capacity was ca. 0.6 in the nonswellable resin. This selection of less-sterically hindered amino groups might have partly had advantage for coupling reactions.
(b)
(a)
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Table 2 Reaction conditions and HPLC yield in the synthesis of Ac-(Aib)4-Lys-Tyr-NH2 on non-swellable resin.
Figure 2. RP-HPLC profiles of crude peptides synthesized under the conditions of Table 1, Entry 10 (a) and Table 2, Entry 5 (b) (detection at 280 nm). The peak at 22 min was identified as Ac-(Aib)4-Lys-Tyr-NH2 by mass spectrometry.
Entry
Condensing
DIPEA
reagent/equiv
(equiv)
Solvent
Coupling time (min)
Table 1 Reaction conditions and HPLC yield in the synthesis of Ac-(Aib)4-Lys-Tyr-NH2 on swellable resin. Entry
1 2
Condensing
DIPEA
reagent/equiv
(equiv)
HATU HATU
20 20
20 20
Solvent
DMF MeCN
Coupling time
HPLC
HPLC Yield (%)
1
HATU
20
20
MeCN
15
4
2
COMU
20
20
MeCN
15
31
3
PyAOP
20
20
MeCN
15
3
4
PyOxim
20
20
MeCN
15
33
5
PyNTP
20
20
MeCN
15
90 88
(min)
Yield (%)
15
1
6
PyNTP
20
20
MeCN
5
3
7
PyNTP
40
40
MeCN
15
81
8
PyNTP
80
80
MeCN
15
11*
15
3
COMU
20
20
DMF
15
11
4
COMU
20
20
MeCN
15
27
9
PyNTP
20
20
DMF
5
76
3
10
PyNTP
20
20
THF
5
36
2
11
PyNTP
20
20
1,4-
5
22
5 6
PyAOP PyAOP
20 20
20 20
DMF MeCN
15 15
7
PyOxim
20
20
DMF
15
45
8
PyOxim
20
20
MeCN
15
32
9
PyNTP
20
20
DMF
15
29
10
PyNTP
20
20
MeCN
15
66
dioxane *Non-swellable resin (113 μmol/g) was used.
.
Entries 9 and 10. From these results, the use of PyNTP in acetonitrile was the best reaction condition for the synthesis of the hexapeptide. In all cases, the by-products were identified to mainly be short oligopeptides. Therefore, the differences between the results reflect the difference in coupling efficiency rather than the appearance of competitive side reactions. The results of the same reactions on the non-swellable resin are presented in Table 2. The yields of the hexapeptide only significantly could be improved in the case of PyNTP (90%, Figure 2b), as shown in Entries 1–5. From these results, the combination of PyNTP with a non-swellable resin was demonstrated to be effective for synthesizing the Aib-containing hexapeptide. Entry 6 shows that a coupling time of 5 min, shorter than those in Entries 1–5, was enough to synthesize the hexapeptide (88%). Entries 7—8 studied the dose dependency of PyNTP. As shown in Entry 8, the product yield was dramatically lowered using 80 equivalents of PyNTP and DIPEA compared to Entries 5 and 7. From the HPLC profiles of the crude products in these experiments, side reactions occurred in the presence of excessive reagents (see the Supporting Information). However, the by-products were not identified because there were numerous kinds of by-products. Entries 9–11 show the results of
Table 3 Reaction conditions and HPLC yield in the synthesis of Ac-(Aib)10-Lys-Tyr-NH2 on non-swellable resin. Entry
Condensing
DIPEA
reagent/equiv
(equiv)
Solvent
Coupling time
HPLC
(min)
Yield (%)
1
PyNTP
20
20
MeCN
5
72*
2
PyNTP
20
20
MeCN
15
73
*Non-swellable resin (113 μmol/g) was used.
On the basis of these results, we conducted the synthesis of long oligopeptides containing Aib residues by using the PyNTP/acetonitrile/non-swellable-resin system. First, a dodecapeptide containing 10 successive Aib residues, Ac-(Aib)10-Lys-Tyr-NH2, was synthesized. As shown in Table 3, the dodecapeptide was obtained in a good yield in each of the coupling time used (both 5 min and 15 min with 72% and 73% yields, respectively, Figure 3). These results strongly demonstrated that this system can construct successive Aib sequences. Finally, we conducted the synthesis of the Aib67, Aib68-modified ACP (65—74) decapeptide model [Ac-ValGln-Aib-Aib-Ile-Asp-Tyr-Ile-Asn-Gly-NH2], which is conventionally used to evaluate the efficiency of novel synthetic methods.13 In
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ACS Combinatorial Science the synthe sis, each condensation with the Aib monomer was conducted for a coupling time of 10 or 20 min and that of the other amino acids was conducted for 5 min. Table 4 clearly shows that the Aib-ACP decapeptide could be synthesized in a good yield, even with 10 min Aib coupling times (75%, Entry 1). Furthermore, the long coupling time of 20 min afforded the decapeptide in a high yield (92%, Figure 4) as shown in Entry 2. These results showed that the Aib-ACP decapeptide can be efficiently synthesized using short coupling times compared to other previous studies.13
As described above, we demonstrated that the application of a nonswellable resin, which is rarely used for peptide synthesis, is useful for the solid-phase synthesis of oligopeptides containing multiple Aib residues by using PyNTP as the condensing reagent. High coupling efficiency was achieved without additional activation, such as microwave treatment or heating.
Supporting Information The Supporting Information is available free of charge on the ACS Publications website. r_ACSComb_PyNTP_ver1.pdf General Information Standard procedure of oligopeptide synthesis on a non-swellable resin HPLC profiles
AUTHOR INFORMATION Corresponding Author *
[email protected] Notes The authors declare no competing financial interests. Figure 3. RP-HPLC profiles of crude peptides synthesized under the conditions of Table 3, Entry 2 (detection at 280 nm). The peak at 32 min was identified as Ac-(Aib)10-LysTyr-NH2 by mass spectrometry.
ACKNOWLEDGMENT We thank Dr. Louis A. Watanabe (Watanabe Chemical Industries Inc.) for the helpful suggestions and the provision of the reagents.
REFERENCES Table 4 Reaction conditions and HPLC yield in the synthesis of Ac-Val-Gln-Aib-Aib-Ile-Asp-Tyr-Ile-AsnGly-NH2 on non-swellable resin. Entry
Condensing
DIPEA
reagent/equiv
(equiv)
Solvent
Coupling time (min)
HPLC
Aib/others
Yield (%)
1
PyNTP
20
20
MeCN
10/5
75
2
PyNTP
20
20
MeCN
20/5
92
(a)
(b)
Figure 4. RP-HPLC profiles (detection at 215 nm (a) and 280 nm (b)) of crude peptides synthesized under the conditions of Table 4, Entry 2. The peak at 34 min was identified as Ac-Val-Gln-Aib-Aib-Ile-Asp-Tyr-Ile-Asn-GlyNH2 by mass spectrometry.
(1) (a) Chantell, C. A.; Onaiyekan, M. A.; Menakuru, M. Fast conventional Fmoc solid-phase peptide synthesis: a comparative study of different activators. J. Peptide Sci. 2012, 18, 88. (b) El-Faham, A.; Albericio, F. COMU: A third generation of uronium-type coupling reagents. J. Peptide Sci. 2010, 16, 6. (c) Subiros-Funosas R.; Acosta, G. A.; El-Faham, A. A.; Albericio, F. Microwave irradiation and COMU: a potent combination for solid-phase peptide synthesis. Tetrahedron Lett. 2009, 50, 6200. (d) El-Faham, A.; Subiros-Dunosas, R.; Prohens R.; Albericio, F. COMU: A Safer and More Effective Replacement for Benzotriazole-Based Uronium Coupling Reagents. Chem. Eur. J. 2009, 15, 9404. (2) (a) Coin, I.; Beyermann, M.; Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat. Protocol 2007, 12, 3247. (b) Paradis-Bas, M.; Tulla-Puche, J.; Albericio, F. The road to the synthesis of “difficult peptides” Chem. Soc. Rev. 2016, 45, 631. (3) Jones, J. E.; Diemer, V.; Adam, C.; Raftery, J.; Ruscoe, R. E.; Sengel, J. T.; Wallace, M. I.; Bader, A.; Cockroft, S. L.; Clayden, J.; Webb, S. J. Length-Dependent Formation of Transmembrane Pores by 310-Helical α‑Aminoisobutyric Acid Foldamers. J. Am. Chem. Soc. 2016, 138, 888. (4) a) Sansom, M. S. P. Alamethicin and related peptaibols - model ion channels. Eur. Biophys. J. 1993, 22, 105. (b) Duclohier, H. Peptaibiotics and Peptaibols: An Alternative to Classical Antibiotics? Chem. Biodivers. 2007, 4, 1023. (5) (a) Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D. The future of peptidebased drugs. Chem. Biol. Drug Des. 2013, 81, 136. (b) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. N-Methylation of Peptides: A New Perspective in Medicinal Chemistry. Acc. Chem. Res. 2008, 41, 1331. (6) Saito, K.; Wada, T. 3-Nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as a condensing reagent for solidphase peptide synthesisTetrahedron Lett. 2014, 55, 1991. (7) (a) Amblard, M.; Fehrentz, J.-A.; Martinez, J.; Subra, G. Methods and protocols of modern solid phase peptide synthesis. Mol. Biotech. 2006, 33, 239. (b) Fields, G. B.; Noble, R. L. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Peptide Protein Res. 1990, 35, 161. (8) (a) Beaucage, S. L.; Iyer, R. P. Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. Tetrahedron, 1992, 48, 2223. (b) Roy, S.; Caruthers, M. Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries. Molecules 2013, 18, 14268. (9) (a) Schuster, M.; Wang, P.; Paulson, J. C.; Wong, C.-H. Solid-Phase Chemical-Enzymic Synthesis of Glycopeptides and Oligosaccharides. J. Am. Chem. Soc. 1994, 116, 1135. (b) Kofoed, T.; Hansen; H. F.; Ørum; H.;
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Koch, T. PNA synthesis using a novel Boc/acyl protecting group strategy. J. Peptide Sci. 2001, 7, 402. (10) Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.; Kates, S. A. On the use of PyAOP, a phosphonium salt derived from HOAt, in solid-phase peptide synthesis. Tetrahedron Lett. 1997, 38, 4853. (11) Subirós-Funosas, R. R.; El-Faham, A.; Albericio, F. PyOxP and PyOxB: the Oxyma-based novel family of phosphonium salts. Org. Biomol. Chem. 2010, 8, 3665. (12) Rink H. Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin. Tetrahedron Lett. 1987, 28, 3787. (13) (a) Jad, Y. E.; Acosta, G. A.; Khattab, S. N.; Torre, B. G. de la; Govender, T.; Kruger, H. G.; El-Faham, A.; Albericio, F. Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives. Org. Biomol. Chem. 2015, 13, 2393. b) Carpino, L. A.; Ionescu, D.; El-Faham, A.; Bayermann, M.; Henklein, P.; Hanay, C.; Wenschuh, H.; Bienert, M. Com-
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