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Synlett
DOI: 10.1055/a-2536-8969
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Small Molecules in Medicinal Chemistry

Design and Synthesis of a C 2-Symmetric Cyclic Decapeptide and Its Peptidomimetic as Potential Inhibitors against Amyloid-β Aggregation

Takuya Kobayakawa‡
,
Kenichi Yamamoto‡
,
Aiichiro Fukutome
,
Marisa Arioka
,
Kohei Tsuji
,
Hirokazu Tamamura  ∗

This work was supported in part by grants from the Japan Society for the Promotion of Science (JSPS), KAKENHI [24K02144 (to H.T.) and 23K14318 (to T.K.)], the Japan Agency for Medical Research and Development (AMED) [JP24ama121043 (Research Support Project for Life Science and Drug Discovery, BINDS) (to H.T.)], and the Japan Science and Technology Agency (JST), SPRING [JPMJSP2180 (to K.Y.)].
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Abstract

A C 2-symmetric cyclic decapeptide and its peptidomimetic based on the KLVFF fragment of amyloid-β (Aβ) are designed and synthesized as potential inhibitors against Aβ aggregation in Alzheimer’s disease. These compounds are efficiently synthesized using solution- and solid-phase peptide synthesis. Thioflavin-T assays reveal that both the cyclic decapeptide and its chloroalkene dipeptide isostere (CADI)-containing peptidomimetic significantly inhibit Aβ aggregation. These results demonstrate the potential of C 2-symmetric cyclic peptides and peptidomimetics as effective Aβ aggregation inhibitors for Alzheimer’s disease therapies.


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Key words

cyclic decapeptide - peptidomimetic - C 2-symmetric - peptide synthesis - Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and memory loss.[1] The number of AD patients in the world is estimated to reach 100 million by 2050, therefore AD has the potential to become a major global health concern.[2] While the exact cause of AD remains unclear, the amyloid cascade hypothesis has gained widespread acceptance.[3] This hypothesis posits that the aggregation of amyloid-β (Aβ) peptides, particularly Aβ1-42, into oligomers and fibrils plays a central role in AD pathogenesis.[4] [5]

The Aβ16-20 fragment (KLVFF) has been identified as a critical region for Aβ self-assembly and aggregation.[6] [7] Consequently, numerous studies have focused on the development of KLVFF-based peptides as potential Aβ aggregation inhibitors.[7c,8,9] However, linear peptides frequently suffer from poor metabolic stability and blood-brain barrier permeability.[10] To compensate for these shortcomings, cyclic peptides have emerged as promising candidates due to their enhanced stability and pharmacokinetic properties.[11] [12]

The development of cyclic peptides and peptidomimetics as Aβ aggregation inhibitors has been extensively explored. For instance, Soto et al. demonstrated that beta-sheet breaker peptides, including cyclic structures, effectively inhibit Aβ fibrillogenesis in a rat brain model of amyloidosis, highlighting their potential for Alzheimer’s disease therapy.[7a] In addition, Arai et al. developed a cyclic KLVFF-derived peptide that showed potent inhibition of Aβ1-42 aggregation and toxicity, further supporting the utility of cyclic peptides in targeting Aβ aggregation.[7b] Furthermore, Etienne et al. reported that peptides containing alternative α,α-disubstituted amino acids can stoichiometrically inhibit Aβ aggregation, providing a basis for the design of peptidomimetics with enhanced rigidity and binding affinity.[7c] These studies collectively underscore the potential of cyclic peptides and peptidomimetics as effective inhibitors of Aβ aggregation.

In this study, we aimed to develop novel compounds that effectively inhibit Aβ aggregation by incorporating two crucial structural elements into our molecular design concept: the ‘Aβ16-20 fragment (KLVFF)’ and ‘C 2-symmetric molecules’. The KLVFF sequence has been recognized as playing a pivotal role in Aβ self-assembly and aggregation processes, as described above.[6] [7] Concurrently, C 2-symmetric structures have been identified as important structural features in various bioactive molecules.[13–15] Accordingly, by combining these insights, we designed the C 2-symmetric cyclic decapeptide 1 (Figure [1]).

Our method involves the synthesis of a single linear pentapeptide comprising five amino acids (KLVFF) and its subsequent dimerization to obtain the desired decapeptide. This approach is expected to simplify the synthetic process and improve the overall yield. Furthermore, we incorporated a chloroalkene dipeptide isostere (CADI), which was developed as a peptidomimetic by us, to enhance the rigidity of the cyclic structure and potentially improve the biological activity (Figure [2]).[16] [17] [18] [19] [20] [21]

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Figure 1 Design of a C 2-symmetric decapeptide based on the KLVFF fragment
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Figure 2 Design of a C 2-symmetric peptidomimetic containing a CADI

The introduction of a peptidomimetic aims to increase the structural constraint of the peptide backbone and optimize interactions with the target molecule. The retrosynthetic analyses of the C 2-symmetric decapeptide 1 and its peptidomimetic 2 are shown in Scheme [1].

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Scheme 1 Retrosynthetic analyses of the C 2-symmetric decapeptide 1 (a) and its peptidomimetic 2 (b)

As shown in Scheme [1a], we planned that the C 2-symmetric decapeptide 1 would be constructed through an intermolecular annulation of the linear pentapeptide 3 between the Lys and Phe residues. Peptide 3 was envisioned to be synthesized from the Phe-bound resin 4 and commercially available protected amino acids 5–8. In a similar manner, we intended to construct peptidomimetic 2 via an intermolecular annulation of the linear peptidomimetic 9 between the Val and Leu residues (Scheme [1b]). The linear peptidomimetic 9 is designed to be synthesized from a Leu-bound resin 10, the Fmoc-protected CADI Fmoc-Phe-ψ[(Z)-CCl=CH]-Phe-OH (12), and commercially available protected amino acids 6 and 11. This strategic approach allows a modular and efficient synthesis of both the C 2-symmetric decapeptide and its peptidomimetic analogue based on solid-phase peptide synthesis techniques and key cyclization reactions.

In accordance with the retrosynthetic analysis, the C 2-symmetric decapeptide 1 was synthesized utilizing an established solid-phase peptide synthesis (SPPS) protocol (Scheme [2]).[22] The protected resin-bound peptide 14 was constructed by Fmoc-based SPPS on the Phe-tethered 2-chlorotrityl (Clt) resin 4. The resin 4 was sequentially coupled with Fmoc-Phe-OH (5), Fmoc-Val-OH (6), Fmoc-Leu-OH (7) and Boc-Lys(Fmoc)-OH (8) according to the Fmoc-SPPS protocol, which was performed using N,N′-diisopropylcarbodiimide (DIPCI) and 1H-benzo[d][1,2,3]triazol-1-ol monohydrate (HOBt·H2O) as coupling reagents. Each coupling was followed by removal of the N α-Fmoc group with piperidine. Treatment of the obtained resin-bound peptide 14 with trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (95:2.5:2.5) provided the N ε-Fmoc-protected peptide 3, which was then treated with 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol (HOAt), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) to afford the cyclic decapeptide 15 through intermolecular annulation. Finally, deprotection of the N ε-Fmoc groups on the side chains of the two Lys residues of 15 with piperidine and subsequent HPLC purification provided the desired C 2-symmetric decapeptide 1 in 0.05% yield from the resin 4.

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Scheme 2 Synthesis of the C 2-symmetric decapeptide 1

Next, Fmoc-Phe-ψ[(Z)-CCl=CH]-Phe-OH (12) was produced by published synthetic methods.[8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] As shown in Scheme [3], the (Z)-γ,γ-dichloro-α,β-unsaturated ester 18 was prepared from the aldehyde 16, corresponding to the side chain of the N-terminal amino acid, and the tert-butylsulfinamide 17 [23]as a chiral auxiliary. Diastereoselective allylic alkylation utilizing an organocopper reagent, prepared from CuCN and benzylzinc chloride, afforded the desired chloroalkene product 19 in a high yield and excellent diastereoselectivity. Deprotection of the N-tert-butylsulfonyl (Bus) group with AlCl3 and anisole[24] followed by Fmoc protection led to the ester 20. The ester group of 20 was reduced to the corresponding aldehyde with DIBAL-H at –78 °C, and this was followed by Pinnick oxidation to provide the desired Fmoc-protected CADI 12 in 48% yield.

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Scheme 3 Synthesis of Fmoc-protected CADI 12

In accordance with the retrosynthetic analysis, the C 2-symmetric peptidomimetic 2 was synthesized utilizing an established SPPS protocol (Scheme [4]).[22] The protected resin-bound peptide 21 was constructed by Fmoc-based SPPS on a Leu-tethered Clt resin 10. The resin 10 was coupled with Fmoc-Lys(Boc)-OH (11) using DIPCI and HOBt·H2O as coupling reagents, followed by removal of the N α-Fmoc group with piperidine to afford the resin-bound peptide 21. The Fmoc-protected CADI 12 was successfully condensed with the resin 21 using DIPCI and Oxyma. This was followed by removal of the N α-Fmoc group with piperidine to give the resin-bound peptidomimetic 22. The resin 22 was subsequently coupled with Fmoc-Val-OH (6) using DIPCI and HOBt·H2O, followed by removal of the N α-Fmoc group with piperidine to afford the desired resin-bound peptidomimetic 23. Treatment of 23 with AcOH/2,2,2-trifluoroethanol (TFE)/CH2Cl2 (1:1:3) provided the protected peptidomimetic 24, which was then cyclized utilizing HOAt, HATU and DIPEA to yield the protected cyclic decapeptidomimetic 25. Finally, deprotection of the N ε-Boc groups on the side chains of the two Lys residues of 25 with TFA/TIS/H2O (95:2.5:2.5) and subsequent HPLC purification provided the desired CADI-containing cyclic peptidomimetic 2 in 0.75% yield from the resin 10.

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Scheme 4 Synthesis of the C 2-symmetric peptidomimetic 2; ψCl = ψ[(Z)-CCl=CH].

The aggregation inhibitory activities of the synthesized C 2-symmetric decapeptide 1 and its CADI-containing peptidomimetic 2 were evaluated using a thioflavin-T (ThT) dye assay (Table [1]).[25]

Table 1 The Inhibitory Effects of Aggregation of Aβ by a Thioflavin-T (ThT) Dye Assay

Entry

Compound

ThT fluorescence intensity (%)a,b,c

% Inhibitiond

(ψCl = ψ[(Z)-CCl=CH])

1

cyclo[-Lys-Leu-Val-Phe-Phe-]2 1

3.7 ± 2.2a

96

2

cyclo[-Lys-Leu-Val-Phe-ψCl -Phe-]2 2

3.2 ± 0.60a

97

3

H-Lys-Leu-Val-Phe-Phe-OH 26

90 ± 8.3b

10

4

H-Lys-Leu-Val-ψCl -Phe-Phe-OH 27

80 ± 16b

20

5

cyclo[-Lys-Leu-Val-Phe-Phe-] 28

20 ± 1.9b (78 ± 2.5)c

80 (22)

6

cyclo[-Lys-Leu-Val-ψCl -Phe-Phe-] 29

25 ± 0.90b (30 ± 3.3)c

75 (70)

7

morin (30)

11 ± 0.40a (36 ± 8.9)b

89 (64)

a Relative fluorescence intensity of Aβ 1-42 (10 μM) + test compound (20 μM) vs A β1-42 (10 μM) alone after incubation for 6 h. All data shown are mean values of at least three independent experiments.

b Reported values; relative fluorescence intensity of Aβ 1-42 (20 μM) + test compound (20 μM) vs Aβ 1-42 (20 μM) alone after incubation for 6 h.[20]

c Relative fluorescence intensity of Aβ 1-42 (20 μM) + test compound (10 μM) vs Aβ 1-42 (20 μM) alone after incubation for 6 h.[20]

d % Inhibition = 100 – ThT fluorescence intensity.

In our previous study, a linear KLVFF peptide 26 and its CADI-containing peptidomimetic 27 showed slight inhibitory activity against Aβ aggregation (Table [1], entries 3 and 4).[20] In addition, a cyclic KLVFF peptide 28, which was previously reported by Arai et al.,[7b] and its CADI-containing peptidomimetic 29,[20] showed higher inhibitory activities than the corresponding linear peptide and peptidomimetic 26 and 27, respectively, and the potencies of 28 and 29 were comparable with that of the control compound morin (30)[2] (entries 5–7). Notably, the CADI-containing cyclic peptidomimetic 29 highly inhibits aggregation at a lower concentration (10 μM) than the cyclic peptide 28.[20] The C 2-symmetric peptide 1 and its peptidomimetic 2, which were synthesized in this study, exhibited remarkably higher inhibitory activities than the corresponding KLVFF-related compounds, and the potencies of 1 and 2 were superior to that of morin (30) (entries 1 and 2). The order of Aβ aggregation inhibitory activity was found to be as follows: C 2-symmetric CADI-containing cyclic decapeptide mimetic 2 > C 2-symmetric cyclic decapeptide 1 > CADI-containing cyclic pentapeptide mimetic 29 > cyclic pentapeptide 28 > CADI-containing linear pentapeptide mimetic 27 > linear pentapeptide 26. According to these results, it is suggested that the increased rigidity of the overall peptide structure enhances the interaction with Aβ, which leads to improved aggregation inhibitory activity.[19] [20] Furthermore, the C 2-symmetric peptide 1 and its peptidomimetic 2 possess two KLVFF sequence recognition sites, which can effectively interact with two Aβ strands and tend to be inserted between Aβ strands and to contribute to their high aggregation inhibitory activity. In addition, the CADI moiety may contribute to enhanced hydrophobic interactions with Aβ.[19] [20] [21]

In conclusion, we have successfully designed, synthesized and evaluated the C 2-symmetric cyclic decapeptide 1 and its peptidomimetic 2 containing a CADI moiety as potential inhibitors against Aβ aggregation. Our results show the potential of C 2-symmetric cyclic peptides and peptidomimetics as being promising for the development of effective inhibitors against Aβ aggregation. In addition, the incorporation of the CADI element provides an additional tool for fine-tuning the structural and pharmacophore properties of these compounds. This research contributes to the ongoing efforts to develop novel therapeutic approaches for Alzheimer’s disease and demonstrates the potential of the rational design of effective inhibitors against Aβ aggregation.


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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

This research is based on the Cooperative Research Project of the Research Center for Biomedical Engineering.

Supporting Information

    Supporting information for this article is available online at https://doi-org.accesdistant.sorbonne-universite.fr/10.1055/a-2536-8969.
  • Supporting Information

Corresponding Author

Hirokazu Tamamura
Laboratory for Biomaterials and Bioengineering, Institute of Integrated Research, Institute of Science Tokyo
2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062
Japan   

Publication History

Received: 31 December 2024

Accepted after revision: 10 February 2025

Accepted Manuscript online:
10 February 2025

Article published online:
04 April 2025

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   Permissions and Reprints All articles of this category
Zoom Image
Figure 1 Design of a C 2-symmetric decapeptide based on the KLVFF fragment
Zoom Image
Figure 2 Design of a C 2-symmetric peptidomimetic containing a CADI
Zoom Image
Scheme 1 Retrosynthetic analyses of the C 2-symmetric decapeptide 1 (a) and its peptidomimetic 2 (b)
Zoom Image
Scheme 2 Synthesis of the C 2-symmetric decapeptide 1
Zoom Image
Scheme 3 Synthesis of Fmoc-protected CADI 12
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Scheme 4 Synthesis of the C 2-symmetric peptidomimetic 2; ψCl = ψ[(Z)-CCl=CH].