Hydrogen Bonding Drives Helical Chirality via 10-Membered Rings in Dipeptide Conjugates of Ferrocene-1,1'-Diamine

Abstract

Considering the enormous importance of protein turns as participants in various biological events, such as protein-protein interactions, great efforts have been made to develop their conformationally and proteolytically stable mimetics. Ferrocene-1,1'-diamine was previously shown to nucleate the stable turn structures in peptides prepared by conjugation with Ala (III) and Ala-Pro (VI). Here, we prepared the homochiral conjugates of ferrocene-1,1'-diamine with l-/d-Phe (32/35), l-/d-Val (33/36), and l-/d-Leu (34/37) to investigate (1) whether the organometallic template induces the turn structure upon conjugation with amino acids, and (2) whether the bulky or branched side chains of Phe, Val, and Leu affect hydrogen bonding. Detailed spectroscopic (IR, NMR, CD), X-ray, and DFT studies revealed the presence of two simultaneous 10-membered interstrand hydrogen bonds, i.e., two simultaneous β-turns in goal compounds. A preliminary biological evaluation of d-Leu conjugate 37 showed its modest potential to induce cell cycle arrest in the G0/G1 phase in the HeLa cell line but these results need further investigation.

Keywords: Density Functional Theory (DFT); X-ray; biological activity; chirality; conformational analysis; ferrocene; hydrogen bonds; peptidomimetic.

Figure 1

The hydrogen-bonding patterns in peptidomimetics containing turn-inducing ferrocene templates –CO–Fn–CO– (I and IV), –NH–Fn–CO– (II and V), and –NH–Fn–NH– (III and VI).

Scheme 1

Synthesis of homochiral conjugates of ferrocene-1,1′-diamine with l- and d-Phe (32/35), l- and d-Val (33/36) and l- and d-Leu (34/37). (i) 1. HClgaseous, 2. Et₃N, 3. Boc–AA–OH, HOBT/EDC; (ii) 1. HClgaseous, 2. NEt₃, 3. AcCl; (iii) NaOH/H₂O, MeOH; (iv) 1. Et₃N, 2. ClCOOEt, 3. NaN₃; (v) t-BuOH/Δ.

Figure 2

(a) The most stable conformers of 32 (l-Phe) and enantiomeric 35 (d-Phe). Stereochemical descriptors for P- and M-isomers depending on the value of pseudo-torsion angle ω. (b) The most stable conformers of 33 (l-Val) and 34 (l-Leu). Nonpolar hydrogen atoms are omitted for clarity. (c) Intramolecular hydrogen bonds (IHBs) pattern with two 10-membered rings (β-turns). Numbering scheme of each 10-membered ring is shown by the green and red color.

Figure 3

The (a) NH and (b) CO stretching vibrations in IR spectra of ferrocene-1,1′-diamine conjugates with l-Phe (32), l-Val (33), and l-Leu (34) in CH₂Cl₂ (c = 5 × 10⁻² M) and ratios of free and associated NH bands; (c) the NH stretching vibrations of compounds 32–34 (2 mg) in KBr (200 mg).

Figure 4

Changes in chemical shifts (Δδ) observed for NH_Fn and NH_Ac in peptides 32–34 at high (50 mM) vs. low concentrations (6.25 mM).

Figure 5

Changes in chemical shifts (Δδ) of NH_Fn and NH_Ac in peptides 32–34 in CDCl₃ (c = 1 × 10⁻³ M) from 258–328 K.

Figure 6

Changes in chemical shifts (Δδ) of NH_Fn and NH_Ac in peptides 32–34 in the presence of 55% d₆-DMSO in CDCl₃ (c = 25 mM, 298 K).

Figure 7

The interstrand NOE contacts in the spectra of conjugates 33 and 34 are depicted with arrows. The NOE contacts attributed to interstrand HBs are not observed in spectrum of conjugate 32.

Figure 8

The Cotton effects in chirality-organized ferrocene peptides 32–37 (a–c) in solution (CH₂Cl₂, c = 1 × 10⁻³ M, (—) and CH₂Cl₂, c = 1 × 10⁻³ M containing 20% of DMSO (– – –)) and (d) in solid state (2 mg in 200 mg KBr).

Figure 9

Conformations of l-Val peptide 33 (left) and d-Val peptide 36 (right) with intramolecular hydrogen bonds shown as cyan dashed lines.

Figure 10

A hydrogen bonded chain parallel to [100] in crystal packing of l-Val peptide 33. Intramolecular hydrogen bonds are shown as cyan and intermolecular as dark blue dashed lines.

Figure 11

Dose response curves for tested compounds 32–37 in Hela (a), Hep G2 (b), and MCF-7 (c) cells. Cells were treated with 5 different concentrations of compounds (5, 10, 50, 100, and 350 uM) and their viability was assessed by MTT test after 72h of incubation. Average ± s.d. from 3 biological replicates is shown.

Figure 12

The effect of peptidomimetic 37 on apoptosis of HeLa cells. Cells were treated with four different concentrations of the tested compound (26 µM, 44 µM, 61 µM, and 105 µM) for 24 h. Bar graphs present the percentage of different cell subpopulations: live cells, early apoptotic cells, late apoptotic, and necrotic cells. Total apoptotic cells are the sum of the percentages of early apoptotic and late apoptotic/necrotic cells. Values are presented as mean ± SEM from two independent experiments.

Figure 13

Cell cycle profiles for control HeLa cells and cells treated with four different concentrations of compound 37 (26 µM, 44 µM, 61 µM, and 105 µM) for 24 h. Cell cycle distribution was calculated as the percentage of cells in the G0/G1, S, and G2/M phase. Data are representative of two independent experiments and were calculated as mean ± SEM. Statistical significance was considered if * p < 0.05.