Supporting Information
Silica Nanoparticles Surface-Modified with Thiacalixarenes
Selectively Adsorb Oligonucleotides and Proteins
Elena A. Yuskova, Patricia Ignacio-de Leon, Ivan I. Stoikov,* and Ilya Zharov*
1.
Synthesis of p-tert-butyl thiacalix[4]arene containing guanidinium fragment
The monosubstituted compound 2 in the cone conformation was obtained in 67% yield by alkylation of p-tert-butyl
thiacalix[4]arene 1 with N-(2-bromoethyl)phthalimide in the presence of cesium carbonate (Scheme 1). Compound
2 is a suitable precursor for the synthesis of various p-tert-butyl thiacalix[4]arens substituted at the lower rim. The
presence of phthalimide fragment that is synthetically inert in alkylation as well as application of alkali metal
carbonates as bases made it possible to trialkylate the thiacalix[4]arene 2. The phthalimide fragment can also be
converted into the amino group by the Gabriel reaction. The reaction of macrocycle 2 with ethyl bromoacetate in
the presence of sodium carbonate in acetone resulted in the formation of the product of complete alkylation 3 with
three ester groups in the cone conformation in 51% yield (Scheme 1). Hydrolysis of thiacalix[4]arene 3 in the
presence of the excess lithium hydroxide in aqueous tetrahydrofuran gave the compound 4 in 94% yield.
Scheme 1.
Next, we studied the transformation of amino groups of the macrocycle 4 to guanidinium groups (Scheme
1). The introduction of guanidinium fragments into the structure of macrocycles is a complex problem. Different
approaches have been proposed to the conversion of amino groups to guanidinium fragment. The cyanamide,
N1,N2-(Di-Boc)-N3-guanidinium trifluoroacetate, thiourea or 3,5-dimethyl-1H-pyrazole-1-carboxamidine nitrate
were used for this purpose. Selective introduction of a single guanidinium fragment into the thiacalix[4]arene
structure with preservation of other binding sites (carboxyl groups) was achieved by one of the most selective
reagent, i.e., 3,5-dimethyl-1H-pyrazole-1-carboxamidine nitrate, which is sufficiently reactive in amidination of
primary and secondary amines. Macrocycle 5 with guanidinium fragment was obtained by the reaction of 3,5dimethyl-1H-pyrazole-1-carboxamidine nitrate with compound 4 in the presence of triethylamine in acetonitrile with
47% yield.
The structure and composition of the compounds 4 and 5 were confirmed by 1D 1H,
13
C and 2D 1H-1H
NOESY NMR, IR spectroscopy, mass spectrometry and elemental analysis. The 1H NMR spectrum of p-tert-butyl
thiacalix[4]arene 5 includes the signals of the protons of the tert-butyl groups as two broaden singlets with integral
intensities equal to 1:1. The aromatic protons of the macrocycle give four broaden singlets, the oxymethylene
protons of OCH2CO groups two singlets with the integral intensities equal to 1:2, methylene protons of OCH2CH2N
group and the proton of COOH groups broadened singlets. The broadening of the signals in the 1H NMR spectra of
compound 5 is due to the fast exchange processes in the system. The shift of the protons of the CH2N fragment
toward weaker field (Δδ = 0.08) in the 1H NMR spectrum of thiacalix[4]arene 5 compared to that of compound 4, is
an indicator of the replacement of amino group of compound 4 with guanidinium fragment.
The 3D structure of p-tert-butyl thiacalix[4]arene 5 substituted at the lower rim was also confirmed by twodimensional 1H-1H NOESY spectroscopy.
Apart from the cross-peaks attributed to dipole-dipole interaction
between protons of the aromatic rings of the cone stereoisomer of the macrocycle with protons of tert-butyl groups,
the cross-peaks were referred to the dipole-dipole interaction between the neighboring oxymethylene protons and
protons of the aromatic rings with protons of carboxylic groups. This indicates the proximity of the carboxyl groups
with aromatic rings of the macrocycle.
2.
Synthesis of p-tert-butyl thiacalix[4]arene with hydrazide and amine fragments
The preparation of disubstituted p-tert-butyl thiacalix[4]arene 6 was studied for the synthesis of other p-tert-butyl
thiacalix[4]arenes substituted at the lower rim with pyridine, amine, and hydrazide fragments. The reaction of
macrocycle 1 with ethyl glycolate performed under Mitsunobu conditions resulted in the formation of compound 6
in 73% yield (Scheme 2). Its hydrolysis by excess of lithium hydroxide in aqueous tetrahydrofuran gave compound
7 in 62% yield. Next, p-tert-butyl thiacalix[4]arene 7 was converted into the corresponding acyl chloride by
refluxing in SOCl2, followed by acylation with 4-aminimethylpyridine in tetrahydrofuran to give the
monosubstituted compound 8 in 75% yield. It was previously suggested that only one acyl chloride group reacts
with 4-aminimethylpyridine in a stepwise acylation while the second one is hydrolyzed to the carboxylic group
(Scheme 2). The latter forms an intramolecular hydrogen bond with the nitrogen atom of the pyridine ring and
hence prevents further acylation. In addition, free phenolic hydroxyl groups present in thiacalix[4]arene may affect
subsequent hydrolysis of oxymethylene fragment to form the monosubstituted compound 8 (Scheme 2).
LiOH
THF/H2O
O
O
S
OH
S
S S
HO HO
OH
OH
TPP/DEAD
THF
S
S
O
O
HO
OH
O
1
S
S S
O
O
S
O
O
OH
O
NH2
S
S
O
10
71%
NH2
S
O O
N
S
S
S
O O
O
S
NH2
NH2
9
32%
H2N
N
O
O
N2H4* H2O
C2H5OH
62%
O
HO
O
O
O
HO
1) SOCl2
2) THF, NEt3,
HN
S S
O
7
73%
6
HO
OH
Сs2CO3, (CH3)2CO
O
N
N
O
N
O
O
Br
O
S
S
S
OH
HO
OH
8
S
S
O
O
HN
75%
O
O
N
Scheme 2.
The uncharacteristic position of proton signals was observed in the 1H NMR spectrum of macrocycle 8 (Figure 1).
The chemical shifts of tert-butyl and aromatic protons of one of the aromatic ring were observed at 0.32 and 6.32
ppm respectively, indicating strong shielding of one of the aromatic fragment. The chemical shifts observed suggest
that thiacalix[4]arene 8 exists in a pinched cone conformation, in which one of the aromatic rings of
thiacalix[4]arene partially occupies the macrocyclic cavity.
(CH3)3C
S
O
NH
H1
H2
S
OH
S S
OH O
OH
O
8 HN
H4
N
N
H3
Ar-H
H1,H4
H2,H3
-O-CH2-C(O)-NH-CH2-
-NHppm
Figure 1. 1H NMR spectrum of compound 8 ((CD3)2SO, 25ºC, 300 MHz).
CH3
CH3
H
H
S
S
S
OH HO
HO
S
O
O
8
HN
N
Figure 2. Two-dimensional 1H-1H NOESY NMR spectrum of compound 8 ((CD3)2SO, 25°C, 500 MHz).
To confirm this structure, compound 8 was studied using 2D 1H-1H NOESY NMR spectroscopy. The cross-peaks
caused by dipole-dipole interaction between the aromatic protons at 6.32 ppm with neighboring aromatic protons
and protons of the tert-butyl group of macrocycle at 1.27 ppm were observed for compound 8. This confirms the
position of the tert-butyl group in the cavity of the macrocycle (Figure 2). The shift of tert-butyl and aromatic
proton signals to the stronger fields also indicate the distorted cone conformation.
The reaction of monosubstituted derivative 8 with N-(3-bromopropyl)phthalimide was performed in the presence of
cesium carbonate in acetone. Monosubstituted derivative 9 was obtained in 32% yield (Scheme 2). IR, 1H NMR
spectroscopy and mass spectrometry indicated simultaneous alkylation and hydrolysis of the amide fragment.
Analysis of the 1H-1H NOESY NMR spectra of 9 confirmed the partial cone conformation of the thiacalix[4]arene.
The cross-peaks in the 1H-1H NOESY NMR spectra were related to dipole-dipole interaction between the protons of
OCH2C(O) group and aromatic protons of the macrocycle, as well as between the protons of OCH2СН2CH2N and
phtalimide groups with each other. Such interactions indicate the partial cone conformation of thiacalix[4]arene 9.
Hydrazinolysis was studied for the conversion of phthalimide fragments into the amino groups. The reaction of ptert-butyl thiacalix[4]arene 9 in the partial cone conformation with hydrazine hydrate in ethanol resulted in the
formation of the macrocycle 10, with three amino and one hydrazide group, in 71% yield (Scheme 2). The reaction
of compound 9 with hydrazine hydrate in ethanol involved not only the phthalimide fragment but also the carboxyl
group, which was converted into the ester while refluxing in ethanol. The product reacted with hydrazine with the
formation of the hydrazide group.
The structure and composition of the synthesized p-tert-butylthiacalix[4]arenes 5, 8-10 tetrasubstituted at
the lower rim were determined by 1H and
13
C NMR, IR spectroscopy, mass spectrometry and elemental analysis.
The configurations of the macrocycles were established by 1D 1H and 2D 1H-1H NOESY NMR spectroscopy. Thus,
macrocycles 5 and 10 with different functional groups, potentially able to coordinate both proteins (via carboxyl and
amino groups) and DNA through the guanidinium fragments, were synthesized.
3.
Synthesis and Characterization
5,11,17,23-Tetra-tert-butyl-25,26,27-trihydroxy-28-[(4pyridylmethylamidocarbonyl)-methoxy]-2,8,14,20-
2
CH3
1
H3C
tetrathiacalix[4]arene (cone-8). White powder, yield: 1.54 g (75%),
3
CH3
4
H
H
mp 298 °C.
2 g, (2.40 mmol) of acid 7 were put into a round-bottom flask and SOCl2
(10 mL, 84.0 mmol) was added. The mixture was refluxed for 1.5 hrs,
S
S
S
OH OH OH O
S
O
excess of SOCl2 was removed; reminder was dried under reduced
pressure for 2 hrs. A solution of 4-aminomethylpyridine (49.69 mmol)
5
HN
and triethylamine (1.54 ml, 7.86 mmol) in 30 mL of tetrahydrofuran was
added.
The mixture was stirred at rt overnight, the remainder was
N
separated, organic layer was evaporated in vacuo. The remainder was
crystallized from the acetonitrile.
1
H NMR (300 MHz, 298 K, (CD3)2SO)  0.32 (s, 9H, (CH3)3C), 1.10 (s, 9H, (CH3)3C), 1.27 (br.s, 18H, (CH3)3C),
4.48 (d, 2H, 3JHH = 6.2 Hz, СH2NH), 5.54 (s, 2H, OCH2CO), 6.32 (s, 2H, Ar-H), 7.33 (s, 2H, Ar-H), 7.38 (br.s, 2Н,
O-CH2-CH2-NH), 7.38 (d, 2H, 3JHH = 5.6 Hz, о-Ar-H), 7.50 (d, 2H, 4JHH = 2.6 Hz, Ar-H), 7.54 (d, 2H, 4JHH = 2.6 Hz,
Ar-H), 8.49 (d, 2H, 3JHH = 5.6 Hz, m-Ar-H), 8.81 (t, 1H, 3JHH = 6.7 Hz, NH).
13
C NMR (125 MHz, (CD3)2SO)  29.9, 31.5, 32.5, 33.5, 121.8, 124.2, 124.4, 127.3, 128.5, 131.9, 134.1, 134.2,
135.3, 135.4, 136.6, 140.2, 149.2, 150.6, 158.7, 170.1.
1
H – 1H NOESY NMR (NОE) (the most important cross-peaks): H1 / H5, H3 / H5, H4 / H5.
IR (KBr)max 1244 (COC); 1673 (С=О); 2962 (Ar-H); 3210 (NH).
ESI mass spectra: calcd for [M+H]+ m/z = 869.3, [M+Na]+ m/z = 891.3, [M+K]+ m/z = 907.3, found m/z = 869.3,
891.3, 907.3.
El. Anal. Calcd for C48H56N2O5S4: C, 66.32; H, 6.49; N, 3.22; S, 14.76. Found: C, 66.25; H, 6.49; N, 3.06; S,
14.69.
5,11,17,23-Tetra-tert-butyl-25,26,27-tris[3'-(Nphthalimido)propoxy]-28-[(hydroxycarbonyl)methoxy]2,8,14,20-tetrathiacalix[4]arene (partial cone-9).
Light
O
HO
H
2H C
2
O
brown powder, yield: 1.48 g (32%), mp 246 °C.
A mixture of 3.00 g (3.45 mmol) of monosubstituted
compound
8,
6.20
g
(23.2
mmol)
of
1
S
S
S
O
O O
N-(3-
4 CH2
bromopropyl)phthalimide and 7.50 g (23.0 mmol) of cesium
O
N
carbonate were refluxed in 150 mL of dry acetone for 72 h.
The solvent was evaporated in vacuo. The reminder was
S
CH2 3
N
O
N
O
O
O
dissolved in 70 mL of CHCl3 and mixed with 2 M HCl
12
9H
aqueous solution (70 ml), the organic phase was dried over
10 H
Na2SO4, and the solvent was evaporated. A pure product
H
O
5
H
H8
H
H11
6
H7
was obtained by recrystallization from acetonitrile/methanol.
1
H NMR (300 MHz, 298 K, (CDCl3)  1.17 (s, 18H, (CH3)3C), 1.20 (s, 9H, (CH3)3C), 1.24 (s, 9H, (CH3)3C), 1.61-
169 (m, 4H, O-CH2-CH2-CH2-N), 1.83-1.90 (m, 2H, O-CH2-CH2-CH2-N), 3.59-3.67 (m, 4H, O-CH2-CH2-CH2-N),
3.73 (t, 2H, 3JHH = 7.3 Hz, O-CH2-CH2-CH2-N), 3.89-3.95 (m, 2Н, O-CH2-CH2-NH), 4.05-4.11 (m, 2Н, O-CH2CH2-NH), 4.30 (s, 2H, OCH2CO), 7.25 (s, 2H, Ar-H), 7.28 (d, 2H, 4JHH = 2.4 Hz, Ar-H), 7.32 (d, 2H, 4JHH = 2.6 Hz,
Ar-H), 7.48 (s, 2H, Ar-H), 7.69-7.72 (m, 4Н, ArPht-H), 7.72-7.74 (m, 2Н, ArPht-H), 7.81-7.83 (m, 4Н, ArPht-H), 7.867.89 (m, 2Н, ArPht-H).
13
C NMR (125 MHz, CDCl3)  28.7, 29.5, 29.9, 31.2, 31.3, 31.4, 34.4, 35.6, 36.1, 64.8, 67.6, 69.5, 123.4, 123.7,
126.1, 127.9, 128.4, 128.6, 130.4, 132.1, 132.3, 134.1, 134.3, 146.8, 147.3, 147.8, 152.8, 156.9, 157.5, 168.3, 168.4,
170.2.
1
H – 1H NOESY NMR (NОE) (the most important cross-peaks): H1 / H2, H4 / H3, H5 / H9, H5 / H10, H5 / H11, H5 /
H12, H6 / H9, H6 / H10, H6 / H11, H6 / H12, H7 / H9, H7 / H10, H7 / H11, H7 / H12, H8 / H9, H8 / H10, H8 / H11, H8 / H12.
IR (KBr)max 1244 (COC); 1673 (С=О); 2962 (Ar-H); 3342 (OH).
ESI mass spectra: calcd for [M+Na]+ m/z = 1362.4, [M+K]+ m/z = 1378.4, found m/z = 1361.4, 1377.4.
El. Anal. Calcd for C75H77N3O12S4: C, 67.19; H, 5.79; N, 3.13; S, 9.57. Found: C, 66.88; H, 6.58; N, 3.74; S, 8.24.
5,11,17,23-Tetra-tert-butyl-25,26,27-tris[3'-aminopropoxy]-28-[(hydrazidocarbonyl)methoxy]-2,8,14,20tetrathiacalix[4]arene (partial cone-10). Yellow powder, yield: 1.00 g (71%), mp 272 °C.
A mixture of 2.00 g (1.49 mmol) of compound 9, 20 mL (0.4 mol) of
NH2
hydrazine hydrate was refluxed in 30 mL of ethanol for 30 h. The
solvent was evaporated in vacuo. The reminder was dissolved in 20
HN
O
1
CH3
mL of CHCl3 and mixed with 20 % NH4OH aqueous solution (20 ml),
the organic phase was dried over Na2SO4, and the solvent was
evaporated. The remainder was washed of 50 mL of distilled water
O
S
S
O
H
S
O O
S
and pure product was obtained.
1
3
CH2
H NMR (300 MHz, 298 K, (CDCl3)  0.69-0.88 (m, 6H, O-CH2-CH2-
CH2-N), 1.25 (s, 18H, (CH3)3C), 1.29 (s, 9H, (CH3)3C), 1.30 (s, 9H,
(CH3)3C), 2.46 (t, 4H, 3JHH = 6.9 Hz, O-CH2-CH2-CH2-NH2), 2.61 (t,
2
NH2
NH2
NH2
4H, 3JHH = 6.7 Hz, O-CH2-CH2-CH2-NH2), 3.88-3.93 (m, 2Н, O-CH2-CH2-NH2), 3.96-4.02 (m, 2Н, O-CH2-CH2NH2), 4.16 (t, 2H, 3JHH = 7.3 Hz, O-CH2-CH2-NH2), 4.45 (s, 2H, OCH2CO), 7.27 (d, 2H, 4JHH = 2.3 Hz, Ar-H), 7.31
(s, 2H, Ar-H), 7.35 (m, 1H, NH), 7.39 (d, 2H, 4JHH = 2.3 Hz, Ar-H), 7.40 (s, 2H, Ar-H).
13
C NMR (125 MHz, CDCl3)  29.9, 31.4, 31.5, 31.6, 33.4, 33.8, 34.5, 39.6, 39.8, 65.9, 67.5, 70.0, 127.1, 127.8,
128.6, 128.8, 145.8, 146.8, 147.4, 157.2, 167.7.
1
H – 1H NOESY NMR (NОE) (the most important cross-peaks): H1 / H2, H2 / H3.
IR (KBr)max 1244 (COC); 1673 (С=О); 2962 (Ar-H); 3210 (NH).
ESI mass spectra: calcd for [M+H]+ m/z = 964.4, [M+Na]+ m/z = 986.4, [M+K]+ m/z = 1002.4, found m/z = 964.4,
986.5, 1002.4.
El. Anal. Calcd for C51H73N5O5S4: C, 63.51; H, 7.63; N, 7.26; S, 13.30. Found: C, 63.19; H, 7.64; N, 6.26; S,
11.99.