Poly-histidine grafting leading to fishbone-like architectures

Abstract

A small series of Morita-Baylis-Hillman adduct (MBHA) derivatives was synthesized and made to react with imidazole, N-acetylhistidine, and N-acetylhexahistidine as models of poly-histidine derivatives. Intriguingly, the reaction of MBHA derivatives 1a and b with imidazole in acetonitrile-phosphate buffered saline (PBS) gave the imidazolium salt biadducts 3a and b as the main reaction products. These results were confirmed by experiments performed with N-acetylhistidine and 1b and suggested the possible occurrence of these structures in the products of poly-histidine labeling with MBHA derivatives 1a and b. These compounds were then transformed into the corresponding water-soluble derivatives 1c-e by introducing oligo(ethylene glycol) chains and their reactivity was evaluated in preliminary experiments with imidazole and then with N-acetylhexahistidine in PBS. The structure of polymeric materials Ac-His-6-MBHA-1d and Ac-His-6-MBHA-1e obtained using ten-fold excesses of compounds 1d and e was investigated using mass spectrometry, NMR spectroscopy, and photophysical studies, which suggested the presence of biadduct residues in both polymeric materials. These results provide the basis for the preparation of fishbone-like polymer brushes, the characterization of their properties, and the exploration of their potential applications in different fields of science such as in vivo fluorogenic labeling, fluorescence microscopy, protein PEGylation, up to the production of smart materials and biosensors.

Fig. 1. Grafting N-acetylhexahistidine (Ac-His-6) with Morita–Baylis–Hillman adduct (MBHA) derivatives.

Scheme 1. Synthesis of MBHA derivative 1a. Reagents: (i) propargyl bromide, K₂CO₃, acetonitrile; (ii) methyl acrylate, DABCO, CH₃OH, THF; (iii) CH₃COCl, TEA, CH₂Cl₂.

Scheme 2. Synthesis of MBHA derivative 1b. Reagents: (i) ethynyltrimethylsilane, Pd(PPh₃)₂Cl₂, CuI, TEA, THF; (ii) K₂CO₃, MeOH; (iii) DABCO, methyl acrylate, MeOH, THF; (iv) CH₃COCl, TEA, CH₂Cl₂.

Scheme 3. Reaction of MBHA derivatives 1a,b with imidazole. Reagents: (i) imidazole, CH₃CN, PBS. Substituents: R = –OCH₂CCH (1a, 2a, 3a); R = –CCH (1b, 2b, 3b).

Fig. 2. Structure of imidazolium salt 3a obtained by crystallography. The ellipsoids enclose 50% probability.

Scheme 4. Reaction of MBHA derivative 1b with N-acetylhistidine. Reagents: (i) N-acetylhistidine, CH₃CN, PBS.

Scheme 5. Synthesis of water-soluble MBHA derivatives 1c,d and their reactivity towards imidazole. Reagents: (i) CH₃O(CH₂CH₂O)₁₁CH₂CH₂COCl, TEA, CH₂Cl₂; (ii) imidazole, PBS. Substituents: R = OCH₂CCH (1c, 3a, 6a); CCH (1d, 3b, 6b).

Scheme 6. Coupling of water-soluble MBHA derivative 1d with Ac-His-6. Reagents: (i) PBS, D₂O.

Fig. 3. (a) MALDI-TOF mass spectrum (positive-ion mode) and (b) ESI mass spectrum (positive-ion mode) of the starting Ac-His-6 sample.

Fig. 4. MALDI-TOF mass spectrum (positive-ion mode) of Ac-His-6-MBHA-1d obtained by reaction of Ac-His-6 with a ten-fold excess of 1d. In each assignment, the value in parentheses is the number of naphthalene substituents for each Ac-His molecule. M9 denotes the peaks assigned to the highly substituted species bearing compound 9 as a counterion of the imidazolium salt moieties.

Fig. 5. ¹H NMR spectrum (500 MHz, CDCl₃) of Ac-His-6-MBHA-1d obtained by reaction of Ac-His-6 with a ten-fold excess of 1d compared with that of biadduct 3c. The peaks marked by 9 were attributed to compound 9, which was produced in the concerted addition–elimination process and remained as a counterion of the imidazolium salt moieties.

Fig. 6. ¹³C NMR spectrum (125, CDCl₃) of Ac-His-6-MBHA-1d obtained by reaction of Ac-His-6 with a ten-fold excess of 1d compared with that of biadduct 3c. The peaks marked by 9 were attributed to compound 9, which was produced in the concerted addition–elimination process and remained as a counterion of the imidazolium salt moieties.

Scheme 7. Synthesis of water-soluble MBHA derivative 1e. Reagents: (i) CH₃(OCH₂CH₂)₉N₃, CuSO₄, sodium ascorbate, tert-butanol, PBS.

Scheme 8. Coupling of water-soluble MBHA derivative 1e with Ac-His-6. Reagents: (i) PBS, D₂O.

Fig. 7. ¹H NMR spectra (400 MHz, PBS-D₂O) of the mixtures obtained by reaction of Ac-His-6 with a ten-fold excess of compound 1e. The peaks marked with A (attributed to the acetyl moiety of 1e) and B (attributed to acetate 10 produced by the concerted addition–elimination process) were used to follow the reaction progress.

Fig. 8. MALDI-TOF mass spectrum (positive-ion mode) of the polymeric material Ac-His-6-MBHA-1e obtained by reaction of Ac-His-6 with a ten-fold excess of compound 1e. The polymerization degree of the corresponding Ac-His molecule is reported for each monoisotopic mass peak along with the grafting degree (i.e. the number of naphthalene substituents for each Ac-His molecule) noted in parentheses.

Fig. 9. ¹³C NMR spectrum (CDCl₃) of Ac-His-6-MBHA-1e obtained by reaction of Ac-His-6 with a ten-fold excess of 1e compared with that of Ac-His-6-MBHA-1d obtained by reaction of Ac-His-6 with a ten-fold excess of 1d. The peaks marked by 9 were attributed to compound 9, which was produced in the concerted addition–elimination process and remained as a counterion of the imidazolium salt moieties.

Fig. 10. Fishbone-like architecture assumed for some organized sequences of poly-histidine derivatives grafted with naphthalene derivatives.

Fig. 11. Optical properties of imidazole derivative 2a (left) and 2b (right). Normalized absorption and emission spectra obtained in methanol (black solid line) and emission spectrum of the powder (dashed blue line).

Fig. 12. Optical properties of imidazole derivative 2d. Left panel: normalized absorption and emission spectra obtained in methanol (black solid line) and emission spectrum of the powder (dashed blue line). Right panel: fluorescence microscopy image (290 μm width) obtained with solid 2d.

Fig. 13. Optical features of imidazolium salts 3a and 3b. Normalized absorption and emission spectra obtained in methanol (black solid line) and emission spectrum of the powder (dashed blue line).

Fig. 14. Optical features of histidine derivatives 2c and 3c. Normalized absorption and emission spectra obtained in methanol (black solid line) and emission spectrum of the powder (dashed blue line).

Fig. 15. Optical features of polymeric materials Ac-His-6-MBHA-1d and Ac-His-6-MBHA-1e. Normalized absorption and emission spectra obtained in methanol (black solid line) and emission spectrum of the powder (dashed blue line).

Fig. 16. ¹H NMR spectrum (400 MHz, D₂O – PBS) of Ac-His-6.

Fig. 17. ¹H NMR spectrum (CDCl₃) of Ac-His-6-MBHA-1e obtained by reaction of Ac-His-6 with a ten-fold excess of 1e.