INEOS OPEN, 2023, X (X), XX–XX
Journal of Nesmeyanov Institute of Organoelement Compounds
Mono(Amino Acid) Derivatives of Fullerenes, Hybrid Structures
on Their Basis and Their Biological Activity
Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, str. 1, Moscow, 119334 Russia
Corresponding author: N. Yu. Shepeta, e-mail: email@example.com
Received 7 March 2023; accepted 10 May 2023
The synthesis and biological properties of mono(amino acid) derivatives of fullerenes (MAADFs) as well as hybrid nanostructures (HNSs) on their basis are reviewed. It is shown that these derivatives are highly effective non-toxic compounds in various fields of medicine and can be further used as potential drugs.
Key words: amino acid derivatives of fullerenes, hybrid nanostructures, biological activity.
A major challenge of modern nanotechnologies is the creation of new materials for medicine based on the production of hybrid nanostructures. Among modern nanomaterials, of particular interest are the nanocarbon structures, including fullerenes. Research in this filed is focused on the creation of water-soluble derivatives of fullerenes, which have high biocompatibility and exhibit a broad spectrum of biological activity. The number of amino acid or peptide moieties and the methods for their binding with a fullerene core can be different. Therefore, the physicochemical and biological properties of the resulting compounds will also differ significantly from each other.
It was found that the water-soluble derivatives of fullerene C60 display antioxidant [1, 2], membranotropic , anti-ischemic [4, 5], neuroprotective , antiviral , antibacterial , and antitumor  properties and can be used as effective low-toxic agents  for target-specific drug delivery in various diseases [11, 12]. The polyaddition of amino acids to a fullerene gives rise to new biologically active compounds. Thus, the penta(amino acid) derivatives of fullerene C60 show great promise as a basis for creating highly effective potential drugs for the treatment of pathological processes associated with the development of type 2 diabetes .
Mono(amino acid) derivatives of fullerenes
The mono(amino acid) and peptide derivatives of fullerene C60 (AADFs) stand somewhat apart. This is explained by the fact that the addition of one amino acid or peptide molecule leads to the appearance of a proton on the fullerene core, which readily enters the substitution reactions (Scheme 1).
Scheme 1. Synthesis of amino acid derivatives of fullerenes.
These compounds were isolated and characterized for the first time in 1994 at the Nesmeyanov Institute of Organoelement Compounds of the Russian Academy of Sciences (INEOS RAS) [12–15]. These derivatives exhibit high solubility in water, low toxicity, and different types of biological activity [16–21].
In experiments on mice and rabbits, it was demonstrated that the fullerene derivatives of l-alanine, l-serine, l-alanyl-l-alanine, which do not have their own immunogenicity, exhibit pronounced adjuvant activity [18, 20]. The presence of a free carboxy group in the amino acid and peptide moieties of AADFs allows one to conjugate the resulting derivatives with other biologically active compounds. The addition of the well-known adjuvant, namely, a peptide derivative of muramic acid widely used in biology and medicine, to N-(monohydro)fullerenyl-ε-aminocaproic acid led to the formation of a new adjuvant (compound III, Fig. 1) which displayed more than an order of magnitude higher activity than the initial adjuvant .
Figure 1. Adjuvant based on a fullerene and muramic acid.
The conjugation of N-(monohydro)fullerenyl-ε-aminocaproic acid with a 24-mer peptide—a fragment of the foot-and-mouth disease (FMD) virus protein that exhibits the anti-FMD activity—afforded antigen H-C60-Z-(136–159) (Z = -NH(CH2)5C(O)-) which immunogenicity was several orders of magnitude higher than that of the original 24-mer peptide—(136–159) = Y-S-A-G-G-M-G-R-R-G-D-L-E-P-L-A-A-R-V-A-A-Q-L-P .
It is known that human cytomegalovirus (CMV) can cause severe illness in adults, especially in immunodeficient conditions, affect young children, and lead to death of the fetus and newborns. The CMV infection is one of the most common opportunistic infections in AIDS. The drugs used in the treatment of the CMV infection are ganciclovir and foscarnet, which are highly toxic. Furthermore, their application is often associated with the development of the drug-resistant CMV infection. To expand the range of substances with the anti-CMV activity, the fullerene derivatives of aminobutyric and aminocaproic acids were explored. The antiviral activity was studied using a primary culture of human embryonic diploid lung fibroblasts (HELFs). The sodium salts of the mentioned acids were studied. These compounds do not have acute cytotoxicity both in vitro and in vivo up to high concentrations (the experiments were carried out on cells, mice, and rabbits), while the chronic cytotoxicity is observed at doses 2–3 orders of magnitude higher than the effective antiviral dose. It was shown that the introduction of the aminobutyric acid derivative into infected HELFs results in a decrease in the concentration of the viral proteins in cells to the values approaching the protein concentration in an uninfected cell culture . In HIV infection, the best results were demonstrated by N-(monohydro)fullerenyl-ε-aminocaproic acid, which, in ultra-low doses, effectively inhibited intracellular reproduction of HIV under conditions of acute and chronic infection and suppressed the infectivity of the mature extracellular virus .
Tritium-labeled fullerene derivative of aminocaproic acid T-C60-NT(CT2)5COOK was shown to be excreted from the body in 72 h via the kidneys .
However, the presence of a proton on the fullerene core promotes significant expansion of the application scope of these substances in biology and medicine by creating biocompatible hybrid nanostructures through binding of the second addend to the fullerene core instead of a proton.
Hybrid nanostructures of fullerenes
Kotel'nikov et al. [23, 24] suggested a new approach to further modification of the amino acid derivatives of fullerenes. A significant expansion of the range of AADFs was found to be possible by creating hybrid structures based on fullerenes (for example, V) obtained by the addition of the second addend (ADDEND-2) to fullerene derivative IV by replacing the hydrogen atom introduced into the fullerene structure in the process of the amino acid addition (Scheme 2). These hybrid fullerene derivatives (HFDs) or hybrid fullerene compounds (HFCs) opened the way to a wide range of biocompatible compounds using combinations of two different addends: one of them, the amino acid component, imparts water solubility and membranotropic properties to the fullerene core, while the second one imparts additional biological activity, in particular, antioxidant activity, which gives the ability to release nitric oxide, to act as a photosensitizer, or to inhibit key enzymes.
Scheme 2. Synthesis of hybrid fullerene nanostructures.
The replacement of a hydrogen atom for the second substituent usually proceeds in pyridine under the action of the corresponding halogen-containing compound.
The quantum-chemical calculations showed that the addition of amino acids or peptides to fullerene C60 occurs at the double bond of six-membered rings of the polyene system with the formation of 1,2-isomers . However, a hydrogen atom of the fullerene core in the resulting adduct is labile ; therefore, the introduction of the second substituent instead of it can lead to steric hindrances in vicinal positions 1 and 2, facilitating the appearance of 1,4-isomers. The structure of the second substituent affects not only the chemical properties of the ensuing compounds, but also the contact area of the fullerene core with water and, as a consequence, the solvation mechanism .
To improve the biological efficacy of fullerene derivatives, recent advances in the field of the physiological activity of nitric oxide were used. It is known that nitric oxide controls vascular tone, serves as a modulator of oxidative reactions, apoptosis process, and immune responses .
The nitro derivative of fullerene C60 HO-CH2CH2-С60-Pro-C(O)CH2CH2ONO2 (VI) (Scheme 3) was tested as an antihypertensive agent. The effect of this compound on blood pressure and heart rate in Wistar rats was studied. A lower effect on the NO-dependent indicators of the cardiovascular system of rats than in the case of nitroglycerin was detected. These results indicate the possibility of creating original vasodilating compounds for antihypertensive therapy based on this class of compounds [29, 30].
Scheme 3. Synthesis of the nitro derivatives of N-(monohydro)fullerenyl-l-proline.
The synthesis of compounds VII–X (Fig. 2) was carried out analogously.
Figure 2. HNSs based on N-(monohydro)fullerenyl-l-proline methyl ester.
To evaluate the antitumor potential of fullerene derivatives, the hybrid structures based on a fullerenyl-substituted proline (VII–X) with the biologically active NO donors and antioxidant carnosine (XI) were used.
These nanostructures exhibited significant antitumor effects. Hybrid nanostructures VII–XI acted as effective chemosensitizers, which caused the recovery of up to 67% of animals with P388 leukemia when the compounds were administered in combination with the clinically used cytostatic cyclophosphamide. Similar hybrid molecules act as pronounced inhibitors of metastasis when administered in combination with a cytostatic. Therewith, the therapeutic dose of the cytostatic agent is decreased tenfold, which reduces its toxicity and prevents the development of resistance . The fullerene derivative of proline IIi showed no antitumor activity under these conditions.
The effect of nitroxy HFDs O2NOCH2CH2-С60-l-Pro-OMe (VIII) and O2NOCH2CH(ONO2)CH2-С60-l-Pro-OMe (X) on the enzymatic activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase was studied. These HFDs were shown to be the inhibitors of the SR Ca2+-ATPase . Thus, mononitrate VIII decelerates the ATP hydrolysis with the inhibition constant Ki = 1.92∙10–6 M and the transmembrane transfer of Ca2+ ions with the inhibition constant Ki = 3.79∙10–6 M. Dinitrate X, although it is a close analog of mononitrate VIII and noncompetitively inhibits both functions of the enzyme, differs from mononitrate by the lower values (by two orders of magnitude) of the inhibition constants (Ki): 2.38∙10–8 and 3.08∙10–8 M, respectively. These experimental facts suggest an increased sensitivity of the enzyme hydrolytic function to the action of dinitrate X and may indicate a partial uncoupling of the SR Ca2+-ATPase function. It was noted that the results on the induced modulation of the activity of the SR Ca2+-ATPase, associated with a change in the ratio of extra- and intracellular contents of Ca2+ ions, suggest the existence of the antimetastatic properties of the nitroxy HFD explored.
Photodynamic therapy (PDT) is an actively developing area of medicine, which is based on the selective effect of low-toxic dye molecules, photosensitizers (PS), on tumors or microorganisms, when they are excited by light of a certain wavelength—in the tissue transparency window of 700–950 nm. The photoexcitation results in the generation of highly toxic free radicals: singlet oxygen, superoxide anion radical, and other reactive oxygen species (ROS), which inhibit the growth of tumors or microorganisms. The main advantages of PDT over the conventional methods of treating malignant neoplasms (surgery, chemotherapy, and radiotherapy) are non-invasiveness, high efficiency, and selectivity of the effect on a tumor in the absence of a noticeable effect on healthy body tissues, the absence of toxicity of the drugs in use, and the possibility of multiple application [33–44].
Some HNSs based on poly(amino acid) derivatives of fullerenes were reported, for which the possibility of a significant increase in the photodynamic activity of the dye owing to the interaction with the fullerene core was demonstrated [45–49]. But not all of the compounds obtained appeared to be water-soluble. To expand the panel of HNSs, the conjugates of chlorin (XII) (Scheme 4) and pyropheophorbide (XIII) with the fullerene mono(amino acid) derivatives were synthesized that showed high efficiency in PDT.
Scheme 4. Synthesis of the HNSs based on chlorin and fullerene mono(amino acid) derivatives.
The fullerene derivatives of pyropheophorbide XIIIa and XIIIb were obtained analogously (Fig. 3).