CC BY
21Calixarenes
Каликсарены
Макрогэтэроцмклы
Review
http://macroheterocycles.isuct.ru
Обзор
DOI: 10.6060/mhc171150k
Calixarene Derivatives for (Nano)Biotechnologies
Roman Rodik, Mykola Poberezhnyk, and Vitaly Kalchenko@
The article is devoted to 75th anniversary of Academician A. Yu. Tsivadze
Institute of Organic Chemistry, National Academy of Sciences of Ukraine, 02660 Kyiv, Ukraine @Corresponding author E-mail: vik@ioch.kiev.ua
This review summarizes the author's research in the field of bio-medical application of modified calixarenes as well as their nano-associates. The paper includes results of design and investigation of the calixarenes as intracellular transport modulators of Ca andNa cations, as well as effective and selective phosphatases inhibitors. The antibacterial and anticoagulant properties of series water-soluble positively or negatively charged calixarenes are described. Also, utilization of calixarenes for hierarchical formation of nanoparticles for nanobiotechnological application (DNA transfection and bio-imaging) is discussed.
Keywords: Calixarenes, nanobiotechonogies, intracellular transport, phosphatases inhibitors, DNA transfection, bio-imaging.
Производные каликсаренов для (нано)биотехнологий
Р. Родик, Н. Побережник, В. Кальченко
Статья посвящена 75-летию со дня рождения академика А. Ю. Цивадзе
Институт органической химии, Национальная академия наук Украины, 02660 Киев, Украина @Е-таИ: vik@ioch.kiev.ua
В работе представлены результаты дизайна и исследования каликсаренов как внутриклеточных модуляторов транспорта катионов Са и Ыа, а также эффективных и селективных ингибиторов фосфатаз. Описаны антибактериальные и антикоагулянтные свойства серии водорастворимых положительно или отрицательно заряженных каликсаренов. Обсуждается также использование каликсареновых наночастиц для трансфекции ДНК и создания флуоресцентных маркеров для медицинской диагностики.
Ключевые слова: Каликсарены, нанобиотехнологии, внутриклеточный транспорт, ингибиторы фосфатаз, трансфекция ДНК, флуоресцентные маркеры.
Introduction
Calixarenes are synthetically readily available nano-sized macrocyclic compounds111 which possess an exceptional three-dimensional cone-shaped topology. The upper and the lower rim of calixarenes can be grafted with a large variety of bio affine groups, which may carry many bio-inspired functions. Due to unique receptor properties of the calixarene family concerning bio-cation, bio-anion, bio-molecules and biopolymers a wide possibilities of its bio-medical application are discussed.[2] Bio-medical potential of the calixarenes: the antiviral, bactericidal, antithrom-bothic, antituberculosis, anticancer activities, transfection ability, specific protein complexation, enzyme mimic, mem-branotropic properties etc., is well documented.[3]
This review summarizes the author's research of the bio-medical application of modified calixarenes as well as their nano-associates. The paper includes results of design and investigation of the calixarenes as intracel-lular transport modulators of Ca and Na cations, as well as effective and selective phosphatases inhibitors. The antibacterial and anticoagulant properties a series of water-soluble positively or negatively charged calixarenes are described. Also, utilization of calixarenes for hierarchical formation of nanoparticles for nanobiotechnolical application (DNA transfection and bio-imaging) is discussed.
Calixarenes Modulators of Contractile Function and Ca2+ Ions Exchange in Smooth Muscle Cells
Inhibition of Calcium Pumps
The effect of sulfonylamidine calixarenes on the activity of calcium pumps of smooth muscle cells was investigated. [4-6] It was shown that calixarene-tetrasulfonylamidine 1 reduces the transport activity of the ruthenium red insensitive, oxalate-stimulated, Mg2+, ATP-dependent calcium pump of sarcoplasmic reticulum by 75 %. A similar result was found for the Mg2+, ATP-dependent calcium pump of the plasma membrane (PM, I05=34.6 ^M). On the other hand, the ruthenium red sensitive Ca2+-transport in mitochondria was not affected by calixarene 1 as well as other membrane pumps (Figure 1).[5,6]
Further investigation of macrocycles 2, 3 showed that di-substituted calixarenes as well as model compounds 4 and 5 possess less activity (Table 1). So, inhibition efficiency ofthe Ca2+, Mg2+-ATPase ofPMby the sulfonylamidine calix[4] arenes correlates with quantity of phenylsulfonylamidine groups on the calixarene upper rim. It should be noted that the inhibitory effects of the studied calix[4]arenes also depended on the relative location of phenylsulfonylamidine groups on the upper rim of the calix[4]arene "bowl".[7]
Calixarene-bisamide 6 in concentration 100 ^M acts multidirectional^ on the functionally different Mg2+-dependent ATP-hydrolase enzymatic systems. Calixarene 6 activates effectively the actomyosin ATPase (Ka=52±11 ^M). Also, 6 effectively reduces basal Mg2+-ATPase activity, insignificantly activating Na+, K+-ATPase, but has no influence on transporting Ca2+, Mg2+-ATPase activity of plasmatic membranes.[6] The results of the comparative investigation of 6 are shown in the Figure 2.
Figure 1. Inhibition of myometrium smooth muscle cells transporting ATP-ases by calixarene 1 (0.1 mM): 1 - Mg2+, ATP-depen-dent Ca2+ accumulation in the miometrial sarcoplasmic reticulum;
2 - Mg2+, ATP-dependent calcium pump of the plasma membrane;
3 - ouabain-suppressed Na+, K+-ATPase; 4 - Mg2+, ATP-dependent Ca2+ accumulation in the mitochondria; 5 - Ca2+-independent Mg2+-ATPase ("basal" Mg2+-ATPase).
Table 1. Inhibitory effect of the sulfonylamidine calix[4]arenes 1-3, bisamide calix[4]arene 4 and the model compound 5 on specific enzymatic activity of Ca2+, Mg2+-ATPase of plasma membrane of myometrium cells.
Calix[4]arene Inhibition coefficient, I0.5' The remaining enzymatic activity at concentration 100 ^M, %
1 20.2±0.5 25.0±0.3
2 53.4±3.6 38.5±1.9
3 165±20 54.1±0.6
4 - 79.3±0.9
5 - 90.7±2.4
1
Figure 2. Influence of transporting ATP-ases of smooth muscle cells of miometrium by calixarene 6: 1 - Mg2+, ATP-dependent Ca2+ accumulation in the mitochondria; 2 - Mg2+, ATP-dependent Ca2+ accumulation in the miometrial sarcoplasmic reticulum; 3 - ouabain-suppressed Na+,K+-ATPase; 4 - Mg2+, ATP-dependent calcium pump of the plasma membrane; 5 - Ca2+-independent Mg2+-ATPase ("basal" Mg2+-ATPase).
inhibited enzymatic activity of Na+, K+-ATPase by 86-98 % and had almost no influence on activity of Mg2+-ATPase. These calixarenes are more efficient than ouabain in reduction of enzymatic activity of the sodium pump (Table 2). In the presence of calixerenes 7, 9, 10 the value of the apparent constant of inhibition I05 was < 0.1 ^M.[8] The model compound 8 (a fragment of the calixarene 7) in concentrations 10 nM - 0.4 mM practically didn't change the enzymatic activities as Na+, K+-ATPase and basal Mg2+-ATPase.
Hence the influence of calixarene 7 on the Na+, K+-ATPase activity is caused by cooperative action of two fragments 8 on the calixarene scaffold. Calixarenes 7 and 9 increase affinity of the Na+, K+-ATPase to the cardiac glycoside ouabain.[9]
Table 2. Inhibition of activity of Na+, K+-ATPase of myometrium cell plasma membranes by the calixarenes 7, 9, 10 and ouabain.
„„.... ■ J. Pseudo constant TT.„ .
T , ., Full inhibition ■,■ T Hill coefficient,
Inhibitors ^ ,, of inhibition, i„ „
concentration, цМ ^m nH
7
9
10
ouabain
~ 10 ~ 10 1000 1000
54+6 33+4 98+8 21000+5000
0.40+0.08 0.38+0.06 0.12+0.03 0.54+0.07
6
Calixarene Effectors of Sodium-Potassium Pump
Effect of calix[4]arenes 7, 9, 10 functionalized by aminophosphonic acid, hydroxyphosphonic acid, and methylene-bisphosphonic acid groups on enzymatic activity of oubain-sensitive Na+, K+-ATPase and oubain resistant basal Mg2+-ATPase was studied.[89]
This pump indirectly regulates intracellular concentration of Ca2+ due to conjugate action with Calcium-Sodium exchanger of PM. It was found that calixarene phosphonic acids 7, 9, 10 at concentration of 100 ^M
Further investigation showed, that alkylation of calixarene lower rim (derivatives 11 and 12) lead to loss of their activity. Probably, strong intramolecular hydrogen bonds O-HOPr at the lower rim stabilize the molecule 10 in the cone conformation and favor to high inhibition of Na+, K+-ATPase of plasma membrane myometrium cell.[9]
OH
Огр-LoH v^/OH
11
12
Calixarenes Influence on Ca2+ Accumulation in Subcellular Structures
Effects of calixarene chalconamides on Mg2+, ATP-dependent Ca2+ transport in smooth muscle subcellular structures were also studied.[10] The results are presented in Figures 3 and 4.
Calixarenes 13a and 13b stimulate Ca2+ accumulation in the mitochondria and don't have modulating effect on Ca2+ accumulation in the sarcoplasmic reticulum. It was also shown that model compound 15 does not modulate Ca2+ accumulation in the mitochondria and sarcoplasmic reticulum and 4'-acetamidochalcone 16 inhibits accumulation in the mitochondria by 65 % and in the sarcoplasmic reticulum by 35 %. Acid 14 completely suppresses energy dependent Ca2+ accumulation both in the mitochondria and in sarcoplasmic reticulum.
a Chalc=
r 0
^yV00 V N -sL \
NHChalc 0 b Chalc :
ChalcHN
13 a,b
Л^у0 ч0^ ysy0 0
3H J Д_ 0H PhHN J Л 0^0H /=° 0^NHPh }=<
=0
0H
14 H
HsC^N
15
16 0
200 180 160 140 120 ^ 100 80 60 40 20 0
I
III.
control 14
13a
13b
15
16
Figure 3. Calixarene 13-16 effects on Mg2+, ATP-dependent Ca2+ accumulation in rat uterus smooth muscle mitochondria.
Figure 4. Calixarenes 13-16 effects on Mg2+, ATP-dependent Ca2+ accumulation in rat uterus smooth muscle sarcoplasmic reticulum.
Myometrium Actomyosin and Myosin Inhibition
It was shown that the calix[4]arene 9 bearing one meth-ylene-bisphosphonic acid fragment inhibits the ATPase
activity of myometrium actomyosin (inhibition coefficient I05=84±2 дМ) and myometrium myosin subfragment-1 (I05=83±7 дМ).[11] Investigation of calixarene 9 effects on myometrium myosin subfragment-1 ATPase showed that in a concentration of 10 дМ it almost didn't influence ATPase activity. The dose-dependent inhibition of myosin head ATPase was observed at growing calixarene 9 concentrations, and at 100 дМ the inhibiting effect vs. control was 60±5 %. According to the linearized graph in Hill coordinates (Figure 5), inhibition coefficient I0 5=83±7 дМ.
Figure 5. Catalytic titration of myosin subfragment-1 ATPase activity with calixarene 9.
To understand the molecular mechanism of calixarene 9 inhibition of myosin subfragment-1 the molecular docking for the interaction of calixarene 9 (as a separate ligand or coupled with ATP) with myometrium myosin subfragment-1 in the presence of Mg2+ as a cofactor (Figure 6) was performed (Mg cation takes part in the binding of АТР to the active site, and in the process of its hydrolysis).
Figure 6. Docking of calixarene 9 as a separate ligand into the active site of myosin subfragment-1 (А) and docking of the paired ligand "9 + АТР" (B) in the presence of Mg2+ as a cofactor.
Leu and Gln
676 678
It was suggested that Asp320, Asn321 may be involved in the coordination of the calixarene phosphonate groups. These data are obtained based on analysis of the docking of calixarene 9 into the ligand binding site of myosin subfragment-1.
Ph
0
Ph
0
NHPh
Ph
Highly Effective Calixarene-based Phosphatase Inhibitors
Inhibitors of Alkaline Phosphatases
Inhibition of nonspecific alkaline phosphatases is of significant current medical interest, since these enzymes catalyze the hydrolysis of phosphate monoesters, which are involved in a number of important biochemical pathways.
Calix[4]arenes 9 and 17 bearing one or two methylene-bisphosphonic acid fragments displayed stronger inhibition of calf intestine alkaline phosphatase than model methylene-bisphosphonic acid 18 or 4-hydroxyphenyl methylene-bisphosphonic acids 19.[12]
17
18
OH
19
The inhibitory activities of the compounds 9, 17-19 were investigated in the calf intestine alkaline phosphatase catalyzed hydrolysis of /-nitrophenylphosphate. The influence of compounds 9, 17-19 on the enzyme activity is in agreement with mixed-type or partial mixed-type inhibition. The mechanism of catalysis by alkaline phosphatase includes formation of the enzyme-substrate complex, phos-phoryl transfer from this complex to a nucleophilic serine group, the hydrolysis of the covalent phosphorylated enzyme intermediate, the formation of a non-covalent enzyme-phosphate complex and the release of inorganic phosphate (Figure 7).
Figure 7. Schematic mechanism of alkaline phosphatase action and inhibition.
The dissociation constants of the enzyme inhibitor K and the enzyme-substrate inhibitor Kcomplexes with 9, 1719 are presented in the Table 3.
The inhibitory activity of calixarene 17 is the consequence of the preorganization of two methylene-bisphos-phonic acid moieties. The efficient coordination of 17 with the metal cations in the enzyme's active site and the additional van der Waals interactions brought about by the scaffold itself are probably responsible for its high activity.
Further investigations of inhibitory effect of chiral calixarene-aminophosphonic acids 20, 21 to alkaline phosphatase of different domestic animals were carried out.[13]
Table 3. Inhibition constants K. and K.' for the inhibitors 9, 17-19 of
i i y
calf intestine alkaline phosphatase.
Inhibitor
K, ^M
K.\ ^M
9 2.5 46
17 0.38 2.8
18 67±5 750±100
19 22±4 290±110
J \
20 a R-isomer, b S-isomer
21a RR- diastereomer b SS - diastereomer
22 S-isomer
Compounds 20, 21 display almost identical affinities to bovine intestinal alkaline phosphatase (BIAP). But the inhibition of porcine kidney alkaline phosphatase (PKAP) is sensitive to the absolute configuration of the calixarene a-aminophosphonic acids. The K. value for S-isomer 20b is about two times smaller than for its ^-counterpart 20a. The enantioselectivity of the inhibition is drastically increased in bisaminophosphonic acids 21a,b. Namely, the ,R,,R-isomer 21a binds to PKAP about 50 times stronger than the S,S-isomer 21b, which corresponds to a difference in free energy of 2.3 kcal/mol (Table 4).
Table 4. Inhibition constants K. and K.' (uM) for the inhibition of PKAP by compounds 20, 21 and (S)-4-hydroxybenzyl-2-aminomethylphosphonic acid 22. Free energies AG (kcal/mol) for the formation of enzyme-inhibitor complexes of PKAP
Inhibitor K. 1 K ' 1 AG1
20a (R) 73±13 540±90 -5.6
20b (S) 32±5 780±100 -6.1
21a (RR) 1.7±3 130±30 -7.8
21b (SS) 86±11 610±210 -5.5
22 (S) 580±110 6800±840 -4.4
0.1 M tris-HCI buffer (pH 9), 23 oC, JG=-RTln(Ki"1).
So, the strength of the enzyme-inhibitor interactions is determined both by cooperative effects and the goodness of the fit of the chiral inhibitor to the chiral space around the binding site. To the best of our knowledge compound 21a is the first alkaline phosphatase inhibitor of R-aminophosphonic acid type that has an affinity for the enzyme in the micromolar range.
Inhibitors of Protein Tyrosine Phosphatases (PTPs)
The next stage of phosphatase survey was study a series of (thia)calix[4]arene phosphonous acids in inhibition of protein tyrosine phosphatases (PTP). Named enzymes regulate a number of biochemical processes which proceed
through dephosphorylation of phosphotyrosine residues in proteins, including cell growth, differentiation, metabolism, and movement. Because of importance of the protein phosphorylation-dephosphorylation cycle in living cells, the activities of these enzymes are associated with diabetes, obesity, cancer and other human diseases.
Preliminary, inhibition of Yersinia protein tyrosine phosphatase by calix[4]arene and thiacalix[4]arene tetrakis(methylphosphonic) acids has been investigated.[14] The kinetic studies revealed that 23 and 24 are potent competitive inhibitors of Yersinia PTP with inhibition constants 0.92 and 0.22 ^M. It was found the phosphonate groups as well as macrocyclic structure is essential for the activities of these compounds. Model docking studies revealed similar binding mode for calixarene inhibitors covering the active site, with location of the phosphonate residues around the entry of the phosphotyrosine binding cavity.
23
OH
24
Protein tyrosine phosphatase 1B (PTP1B) belongs to the superfamily of PTPs and involves in insulin and leptin signal transduction by dephosphorylation of activated insulin receptor or leptin receptor-associated kinase, JAK2. As a negative regulator of insulin and leptin signalling, PTP1B is one of the most promising therapeutic targets for treatment of type 2 diabetes.[15] Calix[4]arenes bearing methylene-bisphosphonic 9 and 17 or hydroxymethylene-bisphosphonic 25 and 26 acid fragments at the wide rim of the macrocycle were studied as inhibitors of PTP1B.[16]
Next part of the study operated with calix[4]arene mono- and bis-a-hydroxymethylphosphonic acids 27 and 10.[17] The calixarene a-ketophosphonous acids 28 and 29 were also investigated and it was found that these macrocycles are able to inhibit PTPIB with IC50 values in the micromolar range displaying lesser efficiency but selectivity over TC-PTP, MEG1, MEG2, SHP2, LAR and PTP-p.[18] Some of the inhibitors showed IC50 values in the micromolar range and good selectivity in comparison with other protein tyrosine phosphatases such as TC-PTP, PTPb, LAR, and CD45 (Table 5). Kinetic studies indicated that the calix[4]arene derivatives influence PTP1B activity as slow-binding inhibitors. The a-ketophosphonate moiety together with macrocyclic platform was supposed to bind to the active site of PTP1B by reversible slow-binding inhibition mechanism. Molecular docking performed on example of PTP1B indicated that hydroxymethylphospho-nate fragment of the inhibitor 27 is able to mimic the binding of the substrate in the enzyme active site region. To clarify the mechanism of inhibition, the possible enzymeinhibitor complexes have been considered using several crystal structures of PTP1B and all stereoisomeric forms of the inhibitors.
In contrast to inhibition of alkaline phosphatases, better activity showed mono-substituted calixarene derivatives compared to disubstituted.
28
29
Last part of this systematic investigation involved tetrasubstituted thiacalix[4]arenes. It was demonstrated that the thiacalix[4]arene derivatives with covalently attached phosphonic acid monoester groups 30a,b exhibit strong submicromolar inhibition of PTP1B and selectivity over some other PTPs.[19] Compound 30a is an approximately 11-fold more potent inhibitor of PTP1B as compared to TC-PTP, Meg1, SHP2, and 8-fold more active towards Meg2. Molecular interactions between the phosphonate monoesters and HSA were detected to demonstrate a possible mechanism of their bioavailability. The presented data provide new insight into properties of the phosphonate monoesters creating the basis for further development of potent macrocyclic inhibitors of PTP1B.
ho oh
30 aR = n-C2Hs b R = n-C^jHg
Anticoagulant and Antithrombotic Properties of Calixarenes
Inappropriate coagulation of blood (thrombosis) may occur within the circulatory system in pathological states such as atherosclerosis or in response to a variety of insults, including surgery and implantation of medical devices. Prevention of thrombosis is considered a crucial part of the treatment regimen for patients at risk for the thrombi development.
In general, agents affecting blood hemostasis are divided into three categories: agents who interfere with portions
Table 5. Experimental inhibitory activity (IC50, ^M) of calix[4]arene phosphonous acids 9, 10, 17, 25-29, 30a,b as inhibitors of PTP1B and other protein tyrosine phosphatases.
Inhibitor PTP1B TC-PTP PTPP CD45 SHP2 LAR
9 1.2±0.2 9.3±1.9 13±1 5.7±0.9 1.8±0.5 >100
17 2.5±0.2 4.9±1.5 32±7 22±6 2.6±0.6 >100
25 1.9±0.2 27±6 18±1 8.4±2.1 3.4±0.3 >100
26 8.3±1.1 22±2 12±2 59±10 9.2±0.4 >100
27 1.6±0.4 11±3 3.5±0.7 4.5±0.9 0.64±0.15 >100
10 8.4±2.1 59±14 17±4 45±11 7.6±1.9 >100
28 3.2±0.4 27±3 32±4 11±3 40±6 72±5
29 6.7±0.2 51±10 30±10 16±3 53±5 56±3
30a 0.34±0.07 5.2±1.4 - - 3.60±0.85 -
30b 0.32±0.03 2.8±0.5 - 3.8±0.9 -
of the coagulation cascade (anticoagulants), agents, which interfere with platelet activation and aggregation (antiplatelet drugs) and agents, which promote disintegration of blood clots (thrombolytics). Currently available anticoagulant drugs are limited to the heparin-like compounds, which are active only when given intravenously, and to the oral cou-marin drugs. Heparin is an endogenous polysulfonated gly-cosaminoglycan which serves as catalyst for the reaction between antithrombin and various activated factors in the coagulation cascade. But, both heparin and coumarin drugs are limited in their usefulness by their hemorrhagic potential, because effective antithrombotic doses of these drugs also concomitantly produce excessive systemic anticoagulation.
The effect on fibrin polymerization of calixarene tetrakis-methylene-bisphosphonic acid 31 was studied in two kinds of assay.[20]
owOHHO OH
V \ /
OH
31
Turbidity analysis showed that 31 decreased the maximum rate of fibrin polymerization in the thrombin-fibrinogen reaction by 50 % at a molar ratio of compound to starting fibrinogen of 1.7:1 (Figure 8). The lag-time was also increased strongly in the presence of 31, indicating either a decrease of the rate of protofibril formation or, conceivably, an increase of protofibril critical length (Figure 8B). Similar
Figure 8. Turbidity analysis of the influence of 31 on fibrin polymerization in the fibrinogen + thrombin reaction. The dependence on calixarene 31 concentration is shown for: A - the maximum rate of fibrin polymerization Vmax; B - the lag-time t; C - the final turbidity of fibrin clots Ah.
ABC
[31]-10-6M [31]-10-6M [311-10-6 M
Figure 9. Turbidity analysis of the influence of 31 on fibrin desAB polymerization. The dependence on calixarene 31 concentration is shown for: A - the maximum rate of fibrin polymerization Vmax; B - the lag-time t, and C - the final turbidity of fibrin clots Ah.
results were obtained when calixarene 31 inhibited the reassociation of dispersed desAB fibrin (Figure 9), in this case, IC=1.26-10-6 M. Electron microscopy confirmed that 31 inhibits the first stage of fibrin polymerization (i.e. the formation of protofibrils).
It is of interest that the inhibitory activity of 17, which has the two methylene bisphosphonic acid substituents, is much less (Table 6) (IC50=1.3110-4 M, indicating that the calixarene scaffold and the number of phosphoiyl groups in the molecule play a crucial role in the inhibitory effect.
Table 8. Clotting potential (CP), haemostasis potential (HP), fibrinolytic potential (FP) and ratios of Clotting and Fibrinolytic potentials (CP/FP) in rabbit plasma before (0), and 2 and 4 h after an IV injection of 31.
Time after injection, h CP HP FP CP/FP Clot half-life time, h
0 91.8 64.9 26.9 0.29 740
2 53.4 41.4 12 0.22 740
4 8.8 5.7 3.1 0.35 550
Table 6. Concentration values of compounds that cause 50 % inhibition of the maximum rate of the polymerization of fibrin produced in the fibrinogen + thrombin reaction.
Compound IC50
31 1.26 10-6 M
17 1.3110-4 M
18 0.72-10"4 M
19 >1.0010- 4 M
Probably, this inhibition is a result of the blocking of site 'A' (Aa17-19, GlyProArg) by the calixarene in a 'knob-hole' manner. HPLC study[21] of the complex formation between 31 and the synthetic peptide Gly-Pro-Arg-Pro, a synthetic analogue of the A knob was carried out. The association constants of calixarene 31 complexes with amino acids Gly, Pro, Arg and tetrapeptide Gly-Pro-Arg-Pro in a methanol-water mobile phase (50:50, vV) presented in Table 7. The highest association constant 3395 M-1, is observed for tetrapeptide Gly-Pro-Arg-Pro. Analysis of the dependence of peptide retention time on the calixarene concentration in the mobile phase indicates 1:1 ratio of the host and guest in the complex.
Table 7. The association constants of calixarene 31 with amino acids and tetrapeptide.
Guest KA, M-1 (RSD, %)
Gly 280 (10)
Pro 814 (26)
Arg 2576 (21)
Gly-Pro-Arg-Pro 3395 (19)
Furthermore, calixarene 31 doubles both the PT and APTT in normal human blood plasma at concentrations of 7.1310-5 M and 1.1010-5 M, respectively.
In vivo studies demonstrated1221 that 31, being injected intravenously into rabbit's bloodstream, acts as effective anticoagulant agent and its effects correspond to those shown in vitro (Table 8). According to the results calix[4]arene 31 can be considered as a potential anti-thrombotic agent with narrow effect range.
Direct study of 31 action on endothelial cells was carried-out using PAE cell culture that was synchronized in G0/G1 phase of cell cycle.[23] However, cell culture studies demonstrated antiapoptotic and proliferation-stimulating effects of 31 on PAE (Table 9).
Fast decrease of platelets count by 31 could provide additional anticoagulant effect but on the other hand it
Table 9. Density of apoptotic cells and distribution of endothelial cells in the presence of 31 (300 ^M).
Apoptotic Cell cycle stages
cells, % G0/G1 G2/M S
Control 24.0±2.1 85.71±2.30 4.85±0.50 9.71±0.70
31 19±1 34.13±1.40 33.19±1.80 32.68±1.30
made obligatory the instant control of platelet viability during treatment with 31 and, possibly, concomitant therapy targeted to platelet restoration.
Intravenous administration of 31 did not affect endothelial cells that was confirmed by determining the level of tPA which is the marker of endothelial activation. However, the study of 31 effects on endothelial cells culture allowed to conclude anti-apoptotic and proliferation-stimulating effects of 31 that could be promising during revascularization of tissues after ischemia.
Antibacterial Properties of Calixarenes
The antibacterial and antibiofilm activities of several amphiphillic tetracationic calixarenes functionalized with methylimidazolium 32a,b, choline 33a,b and N,N-dimethyl-N-(2-aminoethyl)ammonium 34a,b groups were tested and revealed that their MIC values are close to those of antibiotics and better or equal to those of QAS (Table 10). Obtained results demonstrated the clear dependence of antibacterial activity from the alkyl chain length on the lower rim of calixarene. Tetrapropoxycalixarenes have 10-50 times higher antibacterial activity than tetraoctylcalixarenes with the same cationic residues on the upper rim. The nature of the cationic groups at their macrocycle upper rim, defines their mode of interaction with bacterial membrane structures. Calixarene 32a grafted with more lipophillic and planar methylimidazolium groups showed the best activity among all investigated calixarenes. At the same time, tetrapropoxycalixarene 34a shows high inhibitory activity against Staphylococcus aureus biofilms.[24] Another deep investigation of calixarenes 33a,b also confirmed previously described results.[25]
Calixarene-based Nanoparticles
for Gene Transfection and Bio-Imaging
A series of amphiphilic calixarenes 32b, 33a-d, 34b bearing cationic groups at the upper rim and the alkyl chains at the lower rim were synthesized.[26-28] The molecular design
32 aR = n-C3H7,
bR = n-CgHi7
OH
HO
33
34 aR-n-QB,,
bR = n-C8H17
my
0 p 1 0
RÎ R2 1 R1
R2
aRl = R2 = n-C3H7,
b R1 = R2 =n-CgH17
cRl = R2 = n-C6H13
d R1 = : = D.-O22H25
e R1 = 11-C3H7, R2 =
7 cells. It was found that longer alkyl chains favor formation of remarkably small sizes ~40 nm particles with plasmid DNA. These nano-aggregates are characterized by higher transfection efficiency and lower cytotoxicity.
o
/'10 \ Alk Alk \ Alk Alk
35, n = 1, cone Alk = C3H7
36, n = 1,1,3-alt, Alk = C3H7
37, n = 1, cone Alk = C5H13
38,n = 3,Alk=CH3
39,n = 5,Alk=CH3
Table 10. MIC values of calixarenes 32a-34a, ^gmL-1 (^M).
„ S. aureus Calixarene ATCC 6538 P. aeruginosa ATCC 9027 K. pneumoniae ATCC 10031 E. coli ATCC 25922
32a <10 (<9) 50 (45) <10 (<9) <10 (<9)
33a <10 (<9) 1000 (870) 100(90) 100 (90)
34a <10 (<5) 50 (26) 500 (260) 500 (260)
of these calixarenes was directed to increase their affinity for DNA (compared to 35-39[29]) due to formation ofthe additional hydrogen bonds between the hydroxyl group of choline residues and the carbohydrate and phosphate groups of DNA in the case of 33 or additional electrostatic interaction between protonated aminogroups of 34b and phosphate groups of DNA or to improve n-stacking interaction with the nucleic bases of DNA in the case of 32b.
The effect of the length of aliphatic chains and the head group structure on their self-assembly, interaction with DNA, properties of their DNA complexes, the transfection efficiency and cytotoxicity were studied. Calixarene amphiphiles 32-34 form micelles of 6-9 nanometers in water solutions. Such micelles bind to negatively charged DNA surfaces and compensate their charges. Gene transfection experiments were performed in HeLa and COS-
The most promising results are observed for 33d-DOPE complex at N/P 2 ratio, which shows much higher rate of transfected cells (~80%) compared to 33b (~36%) and even to jetPEI (~70%), which is one of the most efficient transfection agents. The rate of transfected cells grows in the following order: 33a<34b<33c<32b<33e~33b<33d. In the presence of serum only 33d molecule showed detectable transfection among the studied calixarenes. Moreover, 33d was the only calixarene that showed detectable transfection without DOPE. Thus, long alkyl chains and the largest hydrophobic domain of 33d improve significantly the transfection efficiency. On the other hand, presence of four additional amino groups, which could be protonated at pH<7 affected only stoichiometry of the complexes with DNA without affecting their transfection efficiency or cytotoxicity. The obtained results provide new insights for designing new artificial viruses based on macrocyclic molecules.
Stable nanoparticles with a diameter of 7 nm, coated with chemically bound fragments of fluorescent cyanine dyes (Figure 11) were synthesized on the basis of cationic amphiphilic calixarene 40 with reactive acetylene groups.[30] The nanoparticles are characterized by a maximum of fluorescence in the region of 550 nm. They are twice as bright as the commercial cadmium-telluric nanoparticles QD-585, which are used as biomarkers. An example of a HeLa
Figure 10. A schematic representation of the binding of polycationic calixarene (33b) to a DNA molecule. Макрогетер0циmbl /Macroheterocycles 2017 10(4-5) 421-431
biological cell shows that nanoparticles penetrate biological membranes, selectively accumulate only in certain areas of the cell and stain them. These nanoparticles can be used as markers in cytological and histological studies, as well as in molecular biochemistry.
Conclusion
Nano-sized calixarene-based macrocycles, function-alized with various bio-affine groups were synthesized and investigated. Systematic experimental study of these calixarenes showed their broad spectrum of bio-activity. Sulfonylamidine calixarenes selectively inhibit active transport of Ca2+ in smooth muscle cells due to binding to Ca2+, Mg2+-ATPase of plasmatic membrane. Modification of the calixarene upper rim by the hydroxyphosphonous and aminophosphonous acid groups leads to specific highly effective inhibitors of Na+, K+-ATPase (Sodium-Potassium exchanger) plasmatic membrane of the same cells. On the other hand, calixarenes modified by amide groups, especially amidochalcone moieties effectively stimulate accumulation of Ca2+ in mitochondria of smooth muscle cells. Hydroxy-, amino-, methylene-bis- and hydroxym-ethylene-bisphosponic acid derivatives of (thia)calixarenes efectively and enantio selectively bind to various alkaline phosphatases, glutathione transferase, and protein tyrosine phosphatase 1B and block their catalytic action. Calix-arene modified with four methylene-bisphosphonic acid groups due to formation of strong complex with Gly-Pro-Arg-Pro tetrapeptide effectively inhibits fibrin polymerization and act as anthitrombotic agent in vivo. Polycationic water-soluble calixarenes showed good antibacterial properties often better than quaternary ammonium compounds used in medicine as antiseptics. Amphiphilic polycationic calixarenes form the micelle-like particles in water with diameter of 6 nm. These particles can bind DNA, compact and transport it into living cells promoting transfection. On the other hand, chemical stabilization of these nano-particles with fluorescent cyanine dyes leads to formation useful instrument for bio-imaging (diagnostic) and target drug delivery.
Acknowledgements. The authors thank Professors S.O. Kosterin, T.V. Lugovskoy, S.V Komisarenko, A.I. Vovk, Y. Mely and HDR A.S. Klymchenko for their great contribution to the Bio-Calix project and for helpful discussions.
References
1. Calixarenes in the Nanoworld(Vicens J., Harrowfield J., Eds.), Dordrecht: Springer, 2007.
2. Calixarenes and Beyond (Neri P., Sessler J.L., Wang M.-X., Eds.), Switzerland: Springer, 2016.
3. Rodik R.V., Boyko V.I., Kalchenko V.I. Front. Med. Chem. 2016, 8, 206-301.
4. Rodik R.V., Boyko V.I., Danylyuk O.B., Suwinska K., Tsym-bal I.F., Slinchenko N.V., Babich L.G., Shlykov S.O., Kosterin S.O., Lipkowski J., Kalchenko V.I. Tetrahedron Lett. 2005, 46, 7459.
5. Labyntseva R.D., SlinchenKO N.M., Veklich T.O., Rodik R.V., Cherenok S.O., Boyko V.I., Kalchenko V.I., Kosterin S.O. Ukr. Biochem. J. 2007, 79, 44-54.
6. Veklich T.A., Shkrabak A.A., Slinchenko N.N., Mazur I.I., Rodik R.V., Boyko V.I., Kalchenko V.I., Kosterin S.A. Biochemistry 2014, 79, 417-424.
7. Veklich T.O., Kosterin S.O., Rodik R.V., Cherenok S.O., Boyko V.I., Kalchenko V.I. Ukr. Biochem. J. 2006, 78, 70-86.
8. Veklich T.O., Shkrabak O.A., Kosterin S.O., Rodik R.V., Cherenok S.O., Boyko V.I., Kalchenko V.I. Ukr. Biochem. J. 2006, 78, 53-63.
9. Veklich T.O., Shkrabak A.A., Cherenok S.O., Kalchenko V.I., Kosterin S.O. Ukr. Biochem. J. 2012, 84, 49-57.
10. Klyachina M.A., Boyko V.I., Yakovenko A.V., Babich L.G., Shlykov S.G., Kosterin S.O., Khilya V.P., Kalchenko V.I. J. Inclusion Phenom. 2008, 60, 131-137.
11. Bevza A.A., Labintseva R.D., Rodik R.V., Cherenok S.O., Kosterin S.O., Kalchenko V.I. Ukr. Biochem. J. 2009, 81, 49-58.
12. Vovk A.I., Kalchenko V.I., Cherenok S.A., Kukhar V.P., Muzychka O.V., Lozynsky M.O. Org. Biomol. Chem. 2004, 2, 3162-3166.
13. Cherenok S., Vovk A., Muravyova I., Shivanyuk A., Kukhar V., Lipkowski J., Kalchenko V. Org. Lett. 2006, 8, 549-552.
14. Vovk A.I., Kononets L.A., Tanchuk V.Yu., Cherenok S.O., Drapailo A.B., Kalchenko V.I., Kukhar V.P. Bioorg. Med. Chem. Lett. 2010, 20, 483-487.
15. Koren S., Fantus I.G. BestPract. Res. Clin. Endocrinol. Metab. 2007, 21, 621-640.
16. Trush V.V., Cherenok S.O., Tanchuk V.Yu., Kukhar V.P., Kalchenko V.I., Vovk A.I. Bioorg. Med. Chem. Lett. 2013, 23, 5619-5623.
17. Trush V.V., Tanchuk V.Y., Cherenok S.O., Kalchenko V.I., Vovk A.I. Journal of Organic and Pharmaceutical Chemistry 2014, 12, 39-42.
18. Trush V., Cherenok S., Tanchuk V., Kalchenko V., Vovk A. Chem. Biol. Lett. 2015, 2, 1-5.
19. Vovk A., Trush V., Kharchenko S., Tanchuk V., Kalchenko V. Org. Biomol. Chem. 2015, 13, 8803-8806.
20. Lugovskoy E.V., Gritsenko P.G., Koshel T.A., Cherenok S.O., Kalchenko VI., Komisarenko S.V. FEBS J. 2011, 278, 1244-1251.
21. Kalchenko O.I., Lipkowski J., Kalchenko V.I., Vysotsky M.A., Markovsky L.N. J. Chrom. Sci. 1998, 36, 269-273.
22. Chernyshenko V.O., Korolova D.S., Dosenko V.E., Pashe-vin D.O., Kalchenko V.I., Pirogova L.V., Chernyshenko T.M., Lugovska O.E., Kravchenko N.A., Makogonenko Y.M., Lugovskoy E.V, Komisarenko S.V. Pharm. Anal. Acta 2015, 6:406.
23. Chernyshenko V.O., Korolova D.S., Nikolaienko T.V., Dosenko V.E., Pashevin D.O., Kalchenko V.I., Cherenok S.O.,
Khranovska N.N., Garmanchuk L.V., Lugovskoy E.V., Komisarenko S.V. Biotechnologia Acta 2016, issue 3, 37-43.
24. Melezhyk I.O., Rodik R.V., Iavorska N.V., Klymchenko A.S., Mely Y., Shepelevych V.V., Skivka L.M., Kalchenko V.I. Anti-Infective Agents 2015, 13, 87-94.
25. Ukhatskaya E., Kurkov S.V., Hjâlmarsdôttir M.A., Karginov V.A., Matthews S.E., Rodik R.V., Kalchenko V.I., Loftsson T. Int. J. Pharm. 2013, 458, 25-30.
26. Mchedlov-Petrossyan N.O., Vilkova L.N., Vodolazkaya N.A., Yakubovskaya A.G., Rodik R.V., Boyko V.I., Kalchenko V.I. Sensors 2006, 6, 962-977.
27. Rodik R.V., Klymchenko A.S., Jain N., Miroshnichenko S.I., Richert L., Kalchenko V.I., Mély Y. Chem. Eur. J. 2011, 17, 5526-5538.
28. Rodik R.V., Anthony A.-S., Kalchenko V.I., Mély Y., Klymchenko A.S. New J. Chem. 2015, 39, 1654-1664.
29. Shi Y., Schneider H.J. J. Chem. Soc, Perkin Trans. 2 1999, 1797-1803.
30. Shulov I., Rodik R.V., Arntz Y., ReischA., Kalchenko V.I., Klymchenko A.S. Angew. Chem. Int. Ed. 2016, 55, 15884-15888.
Received 27.11.2017 Accepted 03.12.2017
