Solid-Phase Synthesis of β-Peptoids with Chiral Backbone

Letter pubs.acs.org/OrgLett

Cite This: Org. Lett. 2017, 19, 5912-5915

Solid-Phase Synthesis of β‑Peptoids with Chiral Backbone Substituents Using Reductive Amination Jumpei Morimoto,* Yasuhiro Fukuda, and Shinsuke Sando* Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *

ABSTRACT: A new submonomeric synthetic method of β-peptoids that allows introduction of chiral backbone substituents is established. The synthesis of β-peptoids with various backbone substituents on β-carbons and spectroscopic studies of synthesized oligomers are described.

S

reported by Hamper and co-workers.14 They devised a facile two-step synthetic method in which acryloyl chloride and primary amines are used as submonomers. Due to their good pharmacokinetic properties and the facile synthetic method, βpeptoids are potentially useful molecules for biomedical applications.17,22−27 However, β-peptoids are conformationally flexible and do not stably form well-defined conformations because β-peptoids lack some structural features that direct folding of peptides and proteins. More specifically, first, βpeptoids are not able to form intramolecular hydrogen bond networks on their backbones. Second, β-peptoids do not have chiral centers on their backbone; thus, there is no intrinsic handedness. Third, tertiary amides have low energy barriers between the cis and trans isomers compared to secondary amides of regular peptides.28 To overcome, or compensate for, the conformational flexibility, chiral N-substituents have been introduced to β-peptoids (Figure 1b).29−32 This strategy has been shown to improve folding propensities of β-peptoids; however, the requirement of bulky and chiral amines as submonomers limits the utility of the strategy for producing structurally diverse β-peptoids with well-defined folded structures. Introduction of backbone chiral substituents is expected to offer a complementary strategy for increasing the folding propensity of β-peptoids (Figure 1c). This strategy would decrease the degree of freedom of backbone dihedral angles, endow β-peptoids with backbone handedness, and potentially bias the ratio of cis and trans isomers. However, the original submonomeric protocol using acryloyl chloride and primary amines does not allow the synthesis of β-peptoids with chiral backbone substituents. Synthesis and structural study of βpeptoids with chiral backbone substituents therefore have not

ynthetic peptidomimetic oligomers that fold into particular conformations and display functionalities to well-defined three-dimensional space have advantages of better proteolytic resistance and larger structural diversity than natural peptides and proteins. Thus, there is considerable interest in designing such synthetic oligomers for biomedical applications. To investigate the folding principles and pharmacokinetic properties of such synthetic oligomers, it is important to devise facile methods for synthesizing sequence-defined oligomers using readily available monomers. Over the past few decades, solidphase synthetic schemes of various synthetic oligomers such as β-peptides,1,2 peptoids,3−5 oligocarbamates,6 oligoureas,7 oligotriazoles,8 γ-AApeptides,9 oligopyrrolidines,10 azapeptides,11 aromatic oligoamides,12 and COPAs13 have been established. These synthetic methods have greatly facilitated investigations and biological applications of the oligomers. β-Peptoids, i.e., oligo-N-substituted β-alanines14−16 (Figure 1a), are a class of such synthetic oligomers. Because of their N-

Figure 1. General structures of (a) β-peptoids, (b) β-peptoids with chiral N-substituents, and (c) β-peptoids with chiral backbone substituents.

substituted amide bonds, β-peptoids are highly resistant to proteolytic degradation17 and are expected to be more membrane-permeable than natural peptides like other classes of N-substituted peptides, such as N-methyl peptides18 and oligo-N-substituted glycines (α-peptoids).19−21 In 1998, a solidphase synthetic scheme of sequence-defined β-peptoids was © 2017 American Chemical Society

Received: September 17, 2017 Published: October 17, 2017 5912

DOI: 10.1021/acs.orglett.7b02909 Org. Lett. 2017, 19, 5912−5915

Letter

Organic Letters

S1). Products were obtained in yields of 64, 55, and 71%, respectively. Product purity was checked by analytical HPLC (Figure S2), and the identity of the desired product was confirmed by HRMS (Table 1) and 1H and 13C NMR analyses (Figures S3−S8). These results demonstrated that β-peptoid structures with various chiral backbone substituents and Nsubstitutions can be efficiently synthesized using Fmoc-β-amino acids and aldehydes as submonomers. We next synthesized homo-oligomers of β-peptoids using the established submonomeric synthetic method. The dimer (4), trimer (5), tetramer (6), and pentamer (7) of N-benzyl-β3homoalanine were synthesized (Table 2) by repeating the

been reported for a long time. Recently, Lim and co-workers synthesized β-peptoids bearing methyl substituents on backbone α-carbons by using N-substituted β2-homoalanines as monomers (Figure 1c, Rα = CH3, Rβ = H).33 The oligomers, so-called α-ABpeptoids, exhibited unique CD signals indicating that the introduction of backbone side chains induced folding of β-peptoids. However, their synthetic protocol requires the synthesis of individual N-substituted β-amino acids in solution; therefore, synthesis of diverse β-peptoids with chiral backbone substituents is labor-intensive. Here we report a new submonomeric synthetic method for the preparation of β-peptoids using reductive amination that allows the facile synthesis of β-peptoids with chiral backbone substituents. As a demonstration of the utility of this synthetic scheme, the synthesis of β-peptoids with various chiral substituents on backbone β-carbons (Figure 1c, Rα = H, Rβ ≠ H) is shown. We assumed that β-peptoids could be synthesized using Fmoc-β-amino acids and aldehydes as submonomers by utilizing reductive amination (Scheme 1). Reductive amination

Table 2. Structures and Analytical Data of β-Peptoid Oligomers 4−7

mass [M + H]+

Scheme 1. Submonomeric Synthetic Scheme for Preparation of β-Peptoids Using Reductive Amination

compd

n

yield (%)

calcd

found

4 5 6 7

2 3 4 5

48 51 30 47

368.2333 543.3330 718.4327 893.5324

368.2321 543.3343 718.4316 893.5340

coupling and reductive amination procedures. For all of the oligomers, the desired products were observed as the major peak on the HPLC chromatograms (Figure S9), and yields did not significantly decrease even in the case of the pentamer (Table 2), demonstrating the efficiency of the synthetic procedure for oligomer synthesis. The purity of the product was checked by HPLC (Figure S10), and formation of the desired products was confirmed by HRMS (Table 2). To determine whether any racemization proceeds during the oligomer synthesis, we synthesized two stereoisomers, RR (4) and SR (8), of N-benzyl-β-homoalanine dimer and then analyzed them by HPLC using a C18 column (Figure 2 and Figure S11). The chromatograms showed that the two stereoisomers 4 and 8 were eluted at different times from the column, demonstrating that homochiral dimers, RR or SS, and heterochiral dimers, SR or RS, are separable on the reversedphase column. Therefore, the formation of undesired

has been employed to synthesize N-acylated oligoamines,34 γAApeptides,9 and α-peptoids with chiral substituents on the αcarbons35 on solid phase, suggesting that reductive amination is a reliable method for introducing N-substitution on solid phase. To demonstrate the feasibility of the submonomeric procedure for constructing β-peptoid structures with backbone substituents on solid phase, we first synthesized β-peptoid monomers 1−3 using three Fmoc-β3-amino acids bearing methyl, benzyl, or isobutyl substituents on β-carbon and three aldehydes of different functionalities (Table 1). Fmoc-β3-amino acids were coupled on resin using HATU and HOAt as coupling reagents, and N-substituents were introduced by reductive amination using aldehyde and sodium borohydride. Synthesized compounds were cleaved from resin and purified by HPLC (Figure Table 1. Structures and Analytical Data of N-Substituted βPeptoid Monomers 1−3

mass [M + H]+ compd

yield (%)

calcd

found

1 2 3

64 55 71

193.1335 235.1805 225.1598

193.1341 235.1776 225.1616

Figure 2. HPLC chromatograms of N-benzyl-β3-homoalanine dimer 4 (RR) (top), 8 (SR) (middle), and coinjection of the two diastereomers (bottom). 5913

DOI: 10.1021/acs.orglett.7b02909 Org. Lett. 2017, 19, 5912−5915

Letter

Organic Letters

negative signal at around 195 nm. With extension of the chain length, the signal at around 195 nm intensified, and the longest oligomer, pentamer 7, showed a strong local minimum at 193 nm. This result indicates that trimer or longer oligomers of Nbenzyl-β3-homoalanine form ordered structures that are repeated over the chain and the structure is gradually stabilized by chain extension. The spectral shape differs from that of any previously reported β-peptoids, indicating that β-peptoids with chiral substituents on β-carbons form unique folded structures. Next, we investigated the concentration dependence of CD spectra of the β-peptoid pentamer 7 (Figure S12a). The spectrum remained almost unchanged for 10−100 μM, suggesting that the spectral shape is not originated from aggregation of the peptoid. We then recorded the CD spectrum of the pentamer at temperatures in the range 5−65 °C (Figure 3b). The gradual decrease in signal intensity upon heating suggested temperature-mediated denaturation of the folded structure. The thermal denaturation behavior is similarly observed for previously reported β-peptoids such as β-peptoids bearing 1-(1-naphtyl)ethyl N-substituents.31 In particular, the peak at 193 nm significantly decreased upon increasing temperature, indicating that the peak is indicative of a folded structure. By decreasing the temperature to 25 °C after heating to 65 °C, the spectral shape returned to the original one, demonstrating that the observed temperature-dependent spectral change was not due to decomposition of oligomers but rather due to denaturation by heating (Figure S12b). To understand how structures of N-substituents affect folding propensities of β-peptoids, we also synthesized N-isobutyl-β3homoalanine oligomers 11−14 (Figure S10) and measured CD spectra of the oligomers (Figure S13). The oligomers did not show any chain length or temperature dependence on CD, indicating that the oligomers do not form stable folded structures. These results indicate that combinations of N- and backbone substituents determine the folding propensities of βpeptoids. Next, we assessed how introduction of chiral substituents on β-carbons affect the cis/trans ratio of N-substituted amides by measuring 1H NMR spectrum of the β-peptoid dimer 4 (Figure S14). The methine proton on backbone β-carbon of C-terminal residue was observed as two separate multiplets. From the integration of these proton peaks, the ratio of cis and trans isomers of this compound was estimated to be Kcis/trans = 1.7 (Figure S15). This result indicates that introduction of chiral backbone subsituents bias cis/trans ratio of β-peptoids, although the degree of induction is not as significant as the 1-(1-naphtyl)ethyl N-substituent.32 β-peptoids with bulkier backbone substituents than methyl are interesting future research subjects that may exhibit larger bias on cis/trans Nsubstituted amides and afford more thermally stable folded structures. In summary, we report a new synthetic method for βpeptoids using reductive amination and demonstrated the utility for synthesizing β-peptoids bearing chiral backbone substituents. Yields of the oligomers are comparable to βpeptoids synthesized by other methods.14,31 CD spectroscopic studies suggest that some β-peptoids bearing chiral backbone substituents exhibit folding propensities. However, CD does not provide concrete evidence of folded structures; therefore, further structural studies by X-ray crystallography and NMR need to be performed to elucidate detailed conformations of the β-peptoids. The established method utilizes readily available Fmoc-β-amino acids and aldehydes as submonomers; thus, β-

diastereomers is observable on HPLC chromatograms. The chromatograms showed no racemization. We also synthesized two tetramers 9 and 10 (Table 3), each of which possesses multiple different chiral backbone Table 3. Structures and Analytical Data of β-Peptoid Tetramers 9 and 10

mass [M + H]+ compd

yield (%)

calcd

found

9 10

32 22

716.4745 800.4957

716.4717 800.4949

substituents and N-substituents, to demonstrate the utility of the established method for synthesizing sequence-defined βpeptoid hetero-oligomers. During the oligomer synthesis, reductive amination using piperonal was found to proceed less efficiently than with other aldehydes. Therefore, the reaction time for imine formation with piperonal was elongated to 2 h. Also, reductive amination was conducted twice for this aldehyde. In future studies, other electron-rich aldehydes may also require modified procedures as used here for piperonal. After synthesis and HPLC purification, these hetero-oligomers 9 and 10 were obtained in yields of 32% and 22%, respectively (Table 3). HRMS analysis confirmed the formation of the target sequence of β-peptoid tetramers. These results demonstrated that the established procedure enables the synthesis of sequence-defined homo- and hetero-oligomers with various chiral backbone substituents and N-substituents. β-Peptoids bearing chiral substituents on backbone βcarbons have never been reported in the literature to the best of our knowledge. Therefore, we conducted spectroscopic studies to investigate the folding propensity of the synthesized β-peptoid oligomers. We first measured CD spectra of Nbenzyl-β3-homoalanine oligomers 4−7 at 25 °C (Figure 3a). The spectrum of the dimer had a broad positive signal at 195− 220 nm, whereas the spectrum of the trimer had a weak

Figure 3. CD spectra of N-benzyl-β3-homoalanine oligomers. All spectra were obtained using 100 μM solution of the respective compound in acetonitrile. Y-Axis was normalized to molar ellipticity per N-substituted amide bond: (a) CD spectra of 4−7 at 25 °C; (b) CD spectra of 7 at 5−65 °C. 5914

DOI: 10.1021/acs.orglett.7b02909 Org. Lett. 2017, 19, 5912−5915

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peptoids bearing various backbone substituents and Nsubstituents can be easily synthesized. This scheme will facilitate investigations into folding propensities and biological applications of β-peptoids with chiral backbone substituents.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b02909. Experimental details and analytical data of compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jumpei Morimoto: 0000-0002-8393-9616 Shinsuke Sando: 0000-0003-0275-7237 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Y. Goto and H. Suga at the University of Tokyo for the use of a CD spectrometer. This work was supported by JSPS KAKENHI Grant No. JP17K13265 and CREST (JPMJCR13L4), Japan Science and Technology Agency. Y.F. was supported by the Graduate Program for Leaders in Life Innovation from MEXT, Japan.

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REFERENCES

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DOI: 10.1021/acs.orglett.7b02909 Org. Lett. 2017, 19, 5912−5915