Design and studies of novel polyoxysterol-based porphyrin conjugates

Article abstract of DOI:10.1016/j.steroids.2012.07.005

New types of steroid-porphyrin conjugates derived from 20-hydroxyecdysone (20E) and 24-epibrassinolide (EBl) were synthesized. An exceptional regioselectivity in the reaction of both steroids with porphyrin boronic acids was found to give side-chain-conjugated boronic esters as sole products. UV-Vis-, fluorescence and NMR spectroscopy yielded similar data for all the studied compounds confirming the solvent driven supramolecular assembly with formation of J-aggregates. CD measurements of water diluted solutions showed a clear difference between 20E and EBl conjugates. The latter showed a strong supramolecular chirality, whereas 20E J-aggregates did not.

Full text of DOI:10.1016/j.steroids.2012.07.005

Steroids 77 (2012) 1169–1175
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Design and studies of novel polyoxysterol-based porphyrin conjugates
a,b
a
a
b
Halina A. Zhylitskaya , Vladimir N. Zhabinskii , Raisa P. Litvinovskaya , Raffaella Lettieri ,
Donato Monti , Mariano Venanzi , Vladimir A. Khripach , Pavel Drašar c
b
b
a,
⇑
a
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich str., 5/2, 220141 Minsk, Belarus
Dipartimento di Scienze e Tecnologie Chimiche, Univ. Roma ‘‘Tor Vergata’’, I-00133 Rome, Italy
Institute of Chemical Technology, Technická 5, CZ-166 28 Praha 6, Czech Republic
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 May 2012
Received in revised form 28 June 2012
Accepted 3 July 2012
Available online 21 July 2012
New types of steroid-porphyrin conjugates derived from 20-hydroxyecdysone (20E) and 24-epibrassin-
olide (EBl) were synthesized. An exceptional regioselectivity in the reaction of both steroids with porphy-
rin boronic acids was found to give side-chain-conjugated boronic esters as sole products. UV–Vis-,
fluorescence and NMR spectroscopy yielded similar data for all the studied compounds confirming the
solvent driven supramolecular assembly with formation of J-aggregates. CD measurements of water
diluted solutions showed a clear difference between 20E and EBl conjugates. The latter showed a strong
supramolecular chirality, whereas 20E J-aggregates did not.
Keywords:
Brassinosteroids
Ecdysteroids
Porphyrins
Ó 2012 Elsevier Inc. All rights reserved.
Conjugates
J-aggregates
1
. Introduction
Steroid frame has been quite widely utilized as a building block
agrochemicals. BS and ES share similar structural elements involv-
ing a diol function in the cycle A and a polyoxygenated side chain.
Specific molecular structure of these compounds possessing a fixed
rigid stereochemistry along with a number of chiral centres and
functional groups makes them possible to be used as chiral ligands
and potential sensor components for selective recognition of com-
plex natural compounds and their synthetic analogs in instrumen-
tal analysis.
For a number of years, we have been interested in various as-
pects of pyrrole–steroid conjugates [5,13,23–30] having in mind
their expected value as new fluorescent probes for biochemical
studies and immunoanalysis [31], and as possible sensing elements
in special analytical devices [32] for recognizing the corresponding
hormones perception. The main purposes of the present work were
(1) to synthesize new steroid–porphyrin conjugates based on oxy-
genated sterols belonging to BS and ES and (2) to investigate phys-
icochemical properties of the newly prepared compounds.
for supramolecular materials [1]. It is large, rigid, chiral, and thus
suitable for creating of extended architectures with well-defined
conformations, capable in principle to enantiodiscrimination [2].
Among numerous supramolecular systems based on the use of ste-
roids, those derived from conjugation with porphyrins should be
mentioned in the first place. They are used as photosensitizers
for photodynamic therapy [3], in saccharide sensing [4–9], and an-
ion binding [10]. Nearly all studies have been limited so far to bile-
acid based structures [11–14], with a few exceptions of porphyrin
conjugates of estrogens [15–17], androgens [18,19] and cholesterol
[
20].
Oxygenated sterols form a large group of naturally occurring
steroids widely spread in plants and animals and playing an impor-
tant role as physiologically active substances. Brassinosteroids (BS,
hormones of plants) [21] and ecdysteroids (ES, hormones of arthro-
pods) [22] belong to the most remarkable members of the series.
Representatives of these hormones are promising potential
medications and active ingredients for new ecologically safe
2
. Experimental
2.1. General
Melting points were determined on a Boetius hot stage. Mass
Abbreviations: 20E, 20-hydroxyecdysone; BS, brassinosteroids; DDQ, 2,3-
dichloro-5,6-dicyanobenzoquinone; DMA, dimethylacetamide; EBl, 24-epibrassin-
olide; ES, ecdysteroids.
spectra were recorded on a LCQ™ ion-trap MS instrument (Thermo
Electron, D-Dreich) (EI), N served as sheath and auxiliary gas, and
2
⇑
Corresponding author. Tel./fax: +375 172 678 647.
E-mail address: khripach@iboch.bas-net.by (V.A. Khripach).
He (purity >99.9990%, Messer–Griesheim, D-Krefeld) as collision
gas. Mass spectra of conjugates were obtained on Bruker-Daltonics
0
039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.07.005

1
170
H.A. Zhylitskaya et al. / Steroids 77 (2012) 1169–1175
Biflex IV MALDI-TOF mass spectrometer equipped with standard
nitrogen laser (337 nm) in a reflector mode. For evaluation of MAL-
DI-TOF MS spectra mMass software was used [33]. High resolution
ESIMS were obtained on Thermo Fischer Scientific LTQ Orbitrap
Velos. Analysis was carried by positive and negative ionization.
2.2.3. General procedure for porphyrin deprotection
Porphyrin (0.1 mmol) was suspended in 4:1 mixture of THF/
2
H O (7.5 mL) and concentrated HCl (2.5 mL) was added. Resulting
mixture was stirred for 12 h at room temperature and red suspen-
sion became green. Reaction mixture was diluted with water and
precipitate was filtered through fine glass filter and washed several
times with water until neutral pH. The resulting green powder was
further used without additional purification.
1
13
H NMR and C NMR spectra were recorded as solutions in CD
N or CDCl on a Bruker AVANCE-500 spectrometer or on Var-
ian 300 spectrometer. Chemical shift values are given in d (ppm)
3
OD,
5
C D
5
3
relative to the residual solvent peaks: d
and d 135.91 for C N; d 7.26 and d
H
3.31 for CD
77.00 for CDCl
3
OD; d
; d
H
7.58
2.50
C
5
D
5
H
C
3
H
2.2.4. 5,10,15,20-Tetrakis(4-dihydroxylborylphenyl)porphyrin (9)
Product 9 (26 mg, 92%) was prepared from 5,10,15,20-tetrakis[4-
for DMSO-d
in Hz.
6
as references, and coupling constants are reported
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]porphyrin
(40 mg, 0.036 mmol). m.p.: >350 °C. H NMR (300 MHz, CD OD) d:
3
6
1
Column chromatography and TLC were carried out using Merck
silica gel 60: 70–230 mesh and precoated silica gel 60 F254 plates,
respectively. Spots on TLC were visualized under UV light and by
7.93 (d, J = 7 Hz, 8 H, Ph-H), 8.14 (d, J = 7 Hz, 8 H, Ph-H), 8.76 (s, 8
+
H, b-pyrrole-H); ESIMS m/z (rel. int.%): 805 [M+H+14] (7), 791
+
+
+
spraying with anisaldehyde–H
2
SO
4
or ammonium cerium (IV) sul-
[M+H] (82), 763 [M+H-28] (100), 735 [M+H-56] (4).
fate–H SO reagents followed by heating. Solvents were dried and
2
4
freshly distilled according to common practice.
2.2.5. 5,15-Bis(4-dioxylborylphenyl)porphyrin (10)
UV spectra were measured on UV–Visible Spectrophotometer
Varian Cary 1E in DMSO and DMA solutions at room temperature.
Fluorescence spectra were collected on Jasco 7850, and FluoroMax-
Product 10 (16 mg, 74%) was synthesized from 5,15-bis[4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]porphyrin
7
(prepared according to [35], 30 mg, 0.042 mmol). m.p.: >350 °C.
H NMR (300 MHz, CD OD) d: 8.05 (d, J = 8 Hz, 4H, Ph-H), 8.27 (d,
3
1
2
Spectrofluorophotometers in 30 nM solutions in DMSO and DMA.
CD spectra were measured on JASCO J-600, equipped with a
thermostated cell holder and purged with ultra-pure nitrogen
J = 8 Hz, 4H, Ph-H), 9.07 (d, J = 5 Hz, 4H, b-pyrrole-H), 9.41 (d,
J = 5 Hz, 4H, b-pyrrole-H), 10.35 (s, 2H, meso-H). ESIMS m/z (rel.
int.%): 607 [M+H+56] (5), 579 [M+H+28] (14), 565 [M+H+14]+
+
+
gas, in DMA and DMSO solution (1 mkM) and in DMA–H
2
O (20–
+
+
6
0% v/v water content), DMSO–H O (20–60% v/v water content)
2
(74), 551 [M+H] (100), 523 [M+H-28] (40). HR ESIMS m/z
+
mixtures in 5 h and in 1 day after water addition.
24 2 4 4
551.2063 [M+H] (calcd for C32H B N O +H, 551.2062).
Solvent-induced aggregation was studied by examining UV and
fluorescence spectra changes against water content and time.
2.2.6. 5,15-Bis(4-dioxylborylphenyl)-10,20-
1
2
0–80% v/v H O–DMSO and H
2
O–DMA solutions were measured
bispentafluorophenylporphyrin (11)
directly after water addition and in 1 h, 2 h, 5 h, 1 day and
Product 11 (20 mg, 80%) was prepared from porphyrin 8 (30 mg,
0.029 mmol). m.p.: >350 °C. H NMR (300 MHz, CDCl ) d: 7.29 (d,
3
J = 8 Hz, 4H, Ph-H), 8.06 (d, J = 8 Hz, 4H, Ph-H), 8.79 (d, J = 5 Hz,
1
6
1
days. Conjugate concentration for aggregation studies was
mkM and 30 nM for UV and fluorescence spectroscopy
correspondingly.
4H, b-pyrrole-H), 9.00 (d, J = 5 Hz, 4H, b-pyrrole-H); ESIMS m/z
+
+
+
(
(
rel. int.%): 896 [M+H+14] (4), 883 [M+H] (100), 855 [M+H-28]
36), 827 [M+H+56] (10). HR ESIMS m/z 883.1744 [M+H]+ (calcd
+
2.2. Synthesis of compounds
for C44
H
22
2
B F
10
N
4
O
4
+H, 883.1746).
2
2
.2.1. 5,10,15,20-Tetrakis[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
-yl)phenyl]porphyrin (6)
5
2.2.7. General procedure for porphyrin-steroid conjugate preparation
Corresponding porphyrin (0.025 mmol) and steroid (1.1 equiv.
per each boronic acid group) were dissolved in methanol
(12.5 mM solution). The resulting mixture was stirred at room
temperature overnight. Formed purple crystals were filtered and
washed with methanol (about 40% of product was isolated). The
filtrate was evaporated under reduced pressure and residue was
purified on silica gel (eluent EtOAc-MeOH – 10/1–5/1 for 20E and
EtOAc – EtOAc-MeOH (10/1) for EBl) to give purple solid (about
50% of product).
-[4-(4,4,5,5)-Tetramethyl-1,3,2-dioxaborolan-2-yl)-phenyl]-
dipyrromethane 4 (prepared according to [34], 174 mg, 0.5 mmol)
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzalde-
hyde 1 (prepared according to [34], 116 mg, 0.5 mmol) were dis-
solved in dry dichloromethane (50 mL), the resulting solution
was degassed under reduced pressure and flushed with argon. Bor-
on trifluoride diethyl etherate complex (23 mkL, 0.186 mmol) was
added dropwise and the reaction mixture was stirred at room tem-
perature for 1 h, then DDQ (170 mg, 0.75 mmol) was added in one
portion and the mixture was stirred for additional 1 h. Triethyl-
amine (26 mkL, 0.186 mmol) was added and the resulting dark
solution was filtered through a pad of silica and eluted with dichlo-
2.2.8. Conjugate of 5,10,15,20-tetrakis(4-dihydroxylborylphenyl)por-
phyrin with 20-hydroxyecdysone (12)
Product 12 (31 mg, 80%) was prepared as a purple solid from
porphyrin 9 (12 mg, 0.015 mmol) and 20E (30 mg, 0.062 mmol).
romethane to give porphyrin 6 as purple solid (105 mg, 38%). m.p.:
1
>270 °C, H NMR (300 MHz, CDCl
3 3
) d: 1.50 (s, 48H, CH ), 8.22 (m,
m.p. (MeOH): >350 °C. UV kmax, nm (
e
, L/(cmÁmol)): [DMA] 419
(438000), [DMSO] 420 (488000). H NMR (500 MHz, C N) d:
À2.29 (s, 2H, NH), 0.72 (s, 12H, C18–H), 0.90 (s, 12H, C19–H),
.10 (s, 24H, C26/C27–H), 1.19 (s, 12H, C21–H), 2.99 (m, 4H,
1
1
6H, Ph-H), 8.82 (s, 8H, b-pyrrole-H).
5 5
D
1
2
.2.2. 5,15-Bis[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
C17–H), 3.09 (d, J = 11 Hz, 4H, C5–H), 3.65 (m, 4H, C9–H), 4.24
(br s, 4H, C2–H), 4.31 (br s, 4H, C3–H), 4.64 (d, J = 9 Hz, 4H, C22–
H), 5.85 (s, 4H, OH), 6.10 (s, 4H, OH), 6.19 (s, 4H, OH), 6.34 (s, 4H,
C7–H), 6.58 (s, 4H, OH), 8.57 (s, 16H, Ph-H), 9.21 (s, 8H, pyrrole-
yl)phenyl]-10,20-bispentafluorophenylporphyrin (8)
Product 8 (90 mg, 35%) was synthesized from 5-[4-(4,4,5,5)-
tetramethyl-1,3,2-dioxaborolan-2-yl)-phenyl]dipyrromethane 4
1
3
(
prepared according to [34], 174 mg, 0.5 mmol) and pentafluoro-
5 5
H). C NMR (125 MHz, C D N) d: 18.31, 22.04, 23.16, 24.00,
benzaldehyde 3 (98 mg, 0.5 mmol) according to the procedure
25.45, 28.10, 31.06, 31.26, 32.59, 33.46, 35.38, 38.96, 39.73,
43.02, 48.77, 52.41, 53.75, 69.08, 69.15, 70.43, 85.23, 87.26,
88.43, 121.94, 122.95, 134.89, 135.66, 146.27, 166.46, 204.48. MS
1
described for compound 6. m.p.: >270 °C. H NMR (300 MHz, CDCl
d: 1.51 (s, 24H, CH ), 8.23 (s, 8H, Ph-H), 8.79 (d, J = 5 Hz, 4H, b-pyr-
role-H), 8.94 (d, J = 5 Hz, 4H, b-pyrrole-H).
3
)
3
+
+
MALDI-TOF m/z: 2591 [M+Na] (32), 2568 [M+H] (49), 2096

H.A. Zhylitskaya et al. / Steroids 77 (2012) 1169–1175
1171
(
(
49), 2118 (24), 1802 (12), 1680 (33), 1624 (52), 1652 (100), 1207
42), 1222 (14), 1180 (32).
2.2.9. Conjugate of 5,10,15,20-tetrakis(4-dihydroxylborylphenyl)por-
phyrin with 24-epibrassinolide (13)
Product 13 (38 mg, 78%) was obtained as a purple solid from
porphyrin 9 (15 mg, 0.019 mmol) and EBl (prepared according to
[
36], 40 mg, 0.083 mmol). m.p. (MeOH): >350 °C. UV kmax, nm (e,
1
Fig. 1. Chemical structures of 20-hydroxyecdysone (20E) and 24-epibrassinolide
L/(cmÁmol)): [DMA] 420 (312000), [DMSO] 420 (465000).
H
(
EBl).
NMR (500 MHz, C
5
D
5
N) d: À2.32 (s, 2H, NH), 0.74 (s, 12H,
C18–H), 0.91 (d, J = 6 Hz, 12H, C–21H), 1.10 (m, 36H, C19–H, C26/
C27–H), 1.20 (d, J = 5 Hz, 12H, C24–H), 3.67 (dd, J = 11, 4 Hz, 4H,
C5–H), 4.01–4.24 (m, 12H, C2–H, C7–H), 4.33 (m, 4H, C23–H),
pyrrole-H), 9.62 (d, J = 5 Hz, 4H, pyrrole-H), 10.61 (s, 2H, meso-H).
13
5 5
C NMR (125 MHz, C D N) d: 9.59, 11.59, 11.97, 15.78, 16.87
4
.45 (br.s, 4H, C3–H), 4.68 (d, J = 3 Hz, 4H, C22–H), 8.55 (d,
2
4
6
1
1.06, 22.44, 24.86, 27.72, 28.12, 29.83, 33.00, 38.45, 39.63,
1.62, 41.68, 42.56, 42.67, 44.86, 51.22, 52.76, 58.30, 68.31,
8.60, 70.14, 83.17, 83.83, 106.06, 119.30, 131.27, 132.46, 133.98,
J = 6 Hz, 8H, Ph-H), 8.61 (d, J = 6 Hz, 8H, Ph-H), 9.14 (s, 8H, pyr-
role-H). C NMR (125 MHz, C
1
4
7
1
3
5
D
5
N) d: 9.48, 11.54, 11.91, 15.78,
6.69, 21.04, 22.39, 24.81, 27.59, 28.07, 32.99, 38.42, 39.57,
34.81, 144.54, 145.71, 147.27, 158.42, 176.49. MS MALDI-TOF
1.59, 42.49, 42.63, 44.84, 51.17, 52.68, 58.25, 68.31, 68.60,
0.10, 83.07, 83.89, 120.71, 133.80, 134.63, 145.28, 176.51. MS
+
m/z (rel. int.%): 1440 [M+H] (41), 1001 (30), 967 (100). HR ESIMS
m/z: 1439.8561 [M+H] (calcd for C88H B N O12+H, 1439.8541).
112 2 4
+
+
+
MALDI-TOF m/z (rel. int.%): 2591 [M+Na] (3), 2569 [M+H] (53),
097 (100), 1624 (35).
2
2.2.12. Conjugate of 5,15-bis[4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]-10,20-bispentafluorophenylporphyrin with
2
.2.10. Conjugate of 5,15-bis(4-dioxylborylphenyl)porphyrin with 20-
20-hydroxyecdysone (16)
hydroxyecdysone (14)
Product 14 (35 mg, 93%) was obtained as a purple solid from
porphyrin 10 (14 mg, 0.025 mmol) and 20E (27 mg, 0.055 mmol).
Conjugate 16 (30 mg, 85%) was prepared as purple solid from
porphyrin 11 (17 mg, 0.020 mmol) and 20E (21 mg, 0.044 mmol).
m.p. (MeOH): >350 °C. UV kmax, nm ( , L/(cmÁmol)): 417 (255000)
DMA], 418 (263000) [DMSO]. H NMR (500 MHz, C N) d:
À2.54 (s, 2H, NH), 1.16 (s, 6H, C18–H), 1.27 (s, 6H, C19–H), 1.55
d, J = 4 Hz, 12H, C26/C27–H), 1.71 (s, 6H, C21–H), 3.01 (t,
J = 8 Hz, 2H, C17–H), 3.09 (dd, J = 13, 3 Hz, 2H, C5–H), 3.66 (m,
e
1
m.p. (MeOH): >350 °C. UV kmax, nm (
e
, (L/cmÁmol)): [DMA] 407
335000), [DMSO] 409 (323000). H NMR (500 MHz, C N) d:
À2.65 (s, 2H, NH), 1.17 (s, 6H, C18–H), 1.31 (s, 6H, C19–H), 1.57
d, J = 4 Hz, 12H, C26/C27–H), 1.73 (s, 6H, C–21H), 3.03 (t,
J = 8 Hz, 2H, C17–H), 3.11 (dd, J = 13, 2 Hz, 2H, C5–H), 3.68 (m,
[
5 5
D
1
(
5 5
D
(
(
2
4
H, C9–H), 4.25 (br d, J = 12 Hz, 2H, C2–H), 4.31 (br s, 2H, C3–H),
.65 (d, J = 11 Hz, 2H, C22–H), 6.34 (s, 2H, C7–H), 6.59 (s, 2H,
2
4
H, C9–H), 4.26 (br d, J = 11 Hz, 2H, C2–H), 4.32 (br s, 2H, C3–H),
.67 (d, J = 11 Hz, 2H, C22–H), 6.37 (s, 2H, C7–H), 6.62 (s, 2H,
OH), 8.54 (d J = 8 Hz, 4H, Ph-H), 8.59 (d, J = 8 Hz, 4H, Ph-H), 9.31
d, J = 4 Hz, 4H, pyrrole-H), 9.49 (d, J = 4 Hz, 4H, pyrrole-H). 13
N) d: 18.69, 22.42, 23.56, 24.37, 25.82,
C
OH), 8.47 (d, J = 8 Hz, 4H, pyrrole-H), 8.60 (d, J = 8 Hz, 4H, pyr-
role-H), 9.25 (d, J = 4 Hz, 4H, Ph-H), 9.63 (d, J = 4 Hz, 4H, Ph-H),
(
5 5
NMR (125 MHz, C D
1
2
3
8
1
0.62 (s, 2H, meso-H). 13C NMR (125 MHz, C
5 5
D N) d: 16.91, 20.62,
2
4
1
8.48, 31.44, 31.62, 32.98, 33.83, 35.75, 39.35, 40.11, 43.39,
9.15, 52.78, 54.13, 69.44, 69.51, 70.77, 85.59, 87.66, 88.83,
04.25, 123.33, 123.39, 135.31, 136.01, 145.70, 166.80, 204.83.
1.77, 22.62, 24.03, 26.74, 29.67, 29.84, 31.20, 32.06, 33.96,
7.55, 38.33, 41.62, 47.37, 51.01, 52.36, 67.65, 67.73, 68.98,
3.80, 85.80, 86.95, 105.79, 119.12, 121.54, 131.00, 132.20,
MS MALDI-TOF m/z (rel. int.%): 1772 [M+H] (100). HR ESIMS m/z:
+
33.58, 134.47, 143.95, 145.39, 147.00, 165.08, 203.08. MS MAL-
1
771.7524 [M+H] (calcd for C98
102 2 10 4
H B F N O14+H, 1771.7497).
+
DI-TOF m/z (rel. int.%): 1440 [M+H] (100), 996 (60). HR ESIMS
m/z: 1439.7839 [M+H] (calcd for C86H B N O14+H, 1439.7813).
104 2 4
+
2.2.13. Conjugate of 5,15-bis[4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]-10,20-bispentafluorophenylporphyrin with
24-epibrassinolide (17)
2.2.11. Conjugate of 5,15-bis(4-dioxylborylphenyl)porphyrin with 24-
epibrassinolide (15)
Product 15 (20 mg, 85%) was obtained as a purple solid from
porphyrin 10 (8 mg, 0.015 mmol) and EBl (prepared according to
Conjugate 17 (19 mg, 80%) was prepared as purple solid from
porphyrin 11 (12 mg, 0.014 mmol) and EBl (prepared according
to [36], 15 mg, 0.031 mmol). m.p. (MeOH): >350 °C. H NMR
1
1
[
(
(
(
36], 15 mg, 0.032 mmol). m.p. (MeOH): >350 °C.
H
NMR
(500 MHz, C
5
D
5
N) d: À2.68 (s, 2H, NH), 0.73 (s, 6H, C18–H), 0.90
500 MHz, C
5
D
5
N) d: À2.68 (s, 2H, NH), 0.75 (s, 6H, C18–H), 0.94
(d, J = 7 Hz, 6H, C21–H), 1.08 (m, 18H, C19–H, C26/C27–H), 1.19
(d, J = 6 Hz, 6H, C24–H), 3.67 (dd, J = 12, 4 Hz, 2H, C5–H), 3.99–
4.25 (m, 6H, C2–H, C7–H), 4.33 (dd, J = 9, 5 Hz, 2H, C23–H), 4.45
(br.s, 2H, C3–H), 4.68 (d, J = 4.5 Hz, 2H, C22–H), 8.52 (d, J = 8 Hz,
4H, Ph-H), 8.61 (d, J = 8 Hz, 4H, Ph-H), 9.27 (d, J = 5 Hz, 4H, pyr-
role-H), 9.46 (d, J = 5 Hz, 4H, pyrrole-H). MS MALDI-TOF m/z (rel.
d, J = 7 Hz, 6H, C21–H), 1.12 (m, 18H, C19–H, C26/C27–H), 1.26
d, J = 6 Hz, 6H, C24–H), 3.68 (dd, J = 12, 4 Hz, 2H, C5–H), 4.04–
4
(
4
.23 (m, 6H, C2–H, C7–H), 4.38 (dd, J = 9, 5 Hz, 2H, C23–H), 4.46
br.s, 2H, C3–H), 4.71 (d, J = 5 Hz, 2H, C22–H), 8.46 (d, J = 8 Hz,
H, Ph-H), 8.64 (d, J = 8 Hz, 4H, Ph-H), 9.22 (d, J = 5 Hz, 4H,
Scheme 1. Reagents and conditions: (a) i: BF
3
ÁEt
2 2 2 2
O, CH Cl , 45 min, r.t., ii: DDQ, 1 h, r.t.; (b) HClconc, THF–H O (4:1).

1
172
H.A. Zhylitskaya et al. / Steroids 77 (2012) 1169–1175
Scheme 2. Reagents and conditions: (a) MeOH, r.t., 12 h.
compounds are the most available in the appropriate group of ste-
roids. 20E is the most abundant among natural ecdysteroids [22],
content of which in plants can reach 4% percent per dry weight.
EBl is the first brassinosteroid which is produced for agricultural
application [37]. The presence of two diol functions in the mole-
cules of both 20E and EBl is a structural feature which is of interest
for conjugation purposes. This allowed developing a general strat-
egy for the conjunction process useful both for EBl and 20E.
After having evaluated several possibilities for the conjugation
of steroid and porphyrin fragments, we selected those based on
the use of boronate esters [38]. Porphyrin–boronic acid derivatives
are well known compounds [6,39–42] that can form covalent
bonds with 1,2-diols [43–45]. There was an evident concern to
manage two diol functions present in the molecules of 20E and
EBl. However, preliminary experience with 24-epibrassinolide
methylboronates [46] suggested a possibility to achieve a selective
formation of the side-chain functionalized conjugates.
The porphyrins 6–8 were synthesized using Lindsey’s method
[
47] by the reaction of aldehydes 1–3 and dipyrromethanes 4,5
in the presence of a catalytic amount of boron trifluoride–etherate
Fig. 2. UV–Vis absorption spectra of conjugate 14 in DMA in aggregation condi-
tions: (a): (0 h) 1 – 30% H O, 2 – 40% H O, 3 – 50% H O, 4 – 60% H O, 5 –70% H O, 6 –
0% H O; (b) (80% H O): 1 – in 0 h, 2 – in 1 h, 3 – in 5 h.
2
2
2
2
2
8
2
2
int.%): 1772 [M+H] (100), 1299 (65). HR ESIMS m/z: 1771.8252
+
[
M+H] (calcd for C100
. Results and discussion
Out of the numerous oxygenated sterols belonging to ES and BS
110 2 10 4
H B F N O12+H, 1771.8225).
3
Fig. 3. Fluorescence emission (kexc 420 nm) spectra in aggregation conditions in
series, we have chosen 20-hydroxyecdysone (20E) and 24-epibras-
sinolide (EBl) as ligands for different porphyrin cores (Fig. 1). Both
DMSO for compound 12 (measurements directly after water addition): 1 – 0% H
2
O,
2 – 20% H O, 3 – 40% H O, 4 – 50% H O, 5 – 60% H O.
2
2
2
2

H.A. Zhylitskaya et al. / Steroids 77 (2012) 1169–1175
1173
Fig. 4. 1H NMR spectra (aromatic region) of compound 14 in: (a) DMSO-d
; (b) DMSO-d –D O (1:1); (c) DMSO-d –D O (3:7).
6 6 2 6 2
complex with subsequent DDQ oxidation (Scheme 1). Compound 7
was identical in measured parameters to that described in the lit-
erature [35].
were the same, although 20E conjugates being more polar required
higher water content to initiate aggregation (20–30% for EBl
against 40–50% for 20E). Porphyrin part had no significant influ-
ence on the spectral characteristics and aggregation properties.
By an example of compound 14, Fig. 4 shows the H NMR spec-
tral changes of these conjugates during the aggregation process. It
The removal of the pinacol protecting groups was achieved by
treatment of the porphyrins 6–8 with concentrated HCl in THF/
water to give the desired unprotected products 9–11 in 74–92%
yields. Their structures were confirmed by disappearance of the
1
can be seen that by adding 50% of deuterated water to DMSO-d
6
1
signals corresponding to pinacol methyls in the H NMR spectra.
solution of 14, which corresponds to an intermediate state (partial
aggregation), the proton signals of porphyrin part show complex
picture: additional broadened upfield shifted signals are appeared
(Fig. 4b) – monomers, dimers, trimers, etc. might be present in the
solution. When 70% water content was reached (complete aggrega-
tion conditions), higher aggregated species were formed. Thus the
The reaction of the porphyrins 9–11 with slight excess (1.1 eq.)
of 20E and EBl in methanol gave the corresponding products 12–17
(Scheme 2) resulting from interaction of 20,22-diol group of 20E or
2
2,23-diol group of EBl with boronic acid moieties of the porphy-
rins. All products were isolated as the purple solids in good to
excellent yields (78–93%). These compounds were found to be sta-
ble in the solid state as well as in the solution. It is worth mention-
ing that no conjugates attached to another diol in the cycle A was
observed. Evidently that the formation side-chain functionalized
conjugates as sole products of this reaction is favored by thermo-
dynamic factors, because 2,3-diol functions in 20E and EBl are less
hindered than those in the side chain.
1
H NMR spectrum (aromatic region) of J-aggregates was not ob-
served because of inhomogeneous broadening of the resonances
(Fig. 4c) [52]. It should be pointed out that there were no
1
H NMR spectra of the newly synthesized compounds showed
remarkable low-field shifts in the signals of the methyne protons
at C-22 in 20E conjugates 12, 14 and 16 (from 3.87 to 4.64–
4
4
.67 ppm) and at C-22/C-23 in EBl conjugates 13, 15 and 17 (from
.04 to 4.68–4.71 and 3.71 to 4.33–4.38 ppm).
It’s well known that porphyrins and their conjugates could eas-
ily form aggregates upon different conditions [48–50]. At room
temperature 20E and EBl conjugates are well soluble in polar or-
ganic solvents such as dimethylsylfoxide (DMSO) and dimethyl-
acetamide (DMA), and they have
a sharp Soret band at
kmax = 420, 407 or 416 nm depending on the structure of the por-
phyrin part. Addition of water to the DMA or DMSO solutions of
conjugates resulted in gradual decreasing of the absorption inten-
sity signals which indicates random aggregates’ formation. The red
shift in UV absorption spectra was observed with time (up to sev-
eral hours) for all conjugates studied confirming the formation of
organized and orientated J-aggregates [51]. A typical change of
UV–Vis spectra as a function of water content and time is shown
in Fig. 2 for the conjugate 14.
The water-driven aggregation process for steroid–porphyrin
1
conjugates was also confirmed by fluorescence, CD- and H NMR
spectra. Thus, fluorescence emission observed in DMSO and DMA
for all conjugates decreased with water addition (Fig. 3). In general,
absorption and fluorescence properties of 20E and EBl conjugates
Fig. 5. CD spectra changes with water content increasing for compound 13 in: (a)
DMA, 1 – 0% H
40% H O.
2 2 2 2 2
O, 2 – 30% H O, 3 – 40% H O; (b) DMSO, 1 – 0% H O, 2 – 20% H O, 3 –
2

1
174
H.A. Zhylitskaya et al. / Steroids 77 (2012) 1169–1175
significant changes of steroidal signals. As in previous cases, the
same tendency was observed for all conjugates.
[9] Bonar-Law RP, Sanders JKM. Polyol recognition by a s teroid-capped porphyrin.
enhancement and modulation of misfit guest binding by added water or
methanolW. J Am Chem Soc 1995;117:259–71.
10] Kolehmainen E, Koivukorpi J, Sievänen E, Král V. Novel porphyrin-cholic acid
conjugates as receptors for biologically important anions. Supramol Chem
Taken as a whole, very similar data were obtained using UV–
Vis-, fluorescence and NMR spectroscopy suggesting the water dri-
ven formation of J-aggregates for all the studied compounds. Their
CD spectra in pure DMSO and DMA yielded no signals. However,
CD measurements of water diluted solutions showed a clear differ-
ence between 20E and EBl conjugates. An intense bisignate band
with a negative peak at 416 nm, and a positive peak at 430 nm ap-
peared when water was added to the EBl conjugate solutions both
in DMA and DMSO (Fig. 5). At the same time, no changes were ob-
served in CD spectra of 20E conjugates on water addition. Thereby
EBl J-aggregates showed a strong supramolecular chirality whereas
[
[
2005;17:437–41.
11] Virtanen E, Kolehmainen E. Use of bile acids in pharmacological and
supramolecular applications. Eur J Org Chem 2004;2004:3385–99.
[12] Tamminen J, Kolehmainen E. Bile acids as building blocks of supramolecular
hosts. Molecules 2001;6:21–46.
13] Huong NTT, Klímková P, Sorrenti A, Mancini G, Drašar P. Synthesis of
spiroannulated oligopyrrole macrocycles derived from lithocholic acid.
Steroids 2009;74:715–20.
14] Weiss J. Zinc(II)-porphyrin receptors in multi-point molecular recognition:
recent progress. J Inclusion Phenom Macrocyclic Chem 2001;40:1–22.
15] Swamy N, James DA, Mohr SC, Hanson RN, Ray R. An estradiol-porphyrin
conjugate selectively localizes into estrogen receptor-positive breast cancer
cells. Bioorg Med Chem 2002;10:3237–43.
16] Bats JW, Haake G, Meier A, Montforts F-P, Scheurich G. Synthesis of oxochlorins
from the red blood pigment heme and their transformation into potentially
biologically active chlorin derivatives. Liebigs Ann 1995;1995:1617–31.
[17] Shuhua C, Yan C, Hengguang L. Synthesis, configuration and properties of
estrogenic steroid-porphyrin enzyme model compounds. Acta Chim Sinica
[
[
[
2
0E J-aggregates did not. The observed difference may be due to
[
various stacking of the porphyrin macrocyclic rings [53]. Supramo-
lecular chirality in EBl derivatives could originate from the helical
edge-to-edge stacked aggregates, and the lack of chirality for 20E
conjugates resulted from linearly or randomly stacking.
1997;55:716–22.
[
[
18] Girgenti E, Ricoux R, Mahy J-P. Design and synthesis of a Mn(III)-porphyrin
steroid conjugate used as a new cleavable affinity label: on the road to semi-
synthetic catalytic antibodies. Tetrahedron 2004;60:10049–58.
19] Vollmer MS, Effenberger F, Stumpfig T, Hartschuh A, Port H, Wolf HC. Steroid-
bridged anthryloligothienylporphyrins: synthesis and study on the
intramolecular energy transfer. J Org Chem 1998;63:5080–7.
4
. Conclusion
A number of new conjugates of 20-hydroxyecdysone and 24-
epibrassinolide with three different porphyrins modified by boro-
nic acid residue as a linker were synthesized. Exceptionally high
regioselectivity in derivatization of a steroid molecule by porphy-
rin boronic acids was shown, which resulted in the formation of
the side-chain-conjugated boronic esters as sole products. Studies
of supramolecular assembly in water-organic solvent systems of
the obtained conjugates were carried out by spectral methods.
UV–Vis-, fluorescence and NMR spectroscopy confirmed the water
driven formation of J-aggregates for all the studied compounds. CD
measurements delivered an evidence for the presence of
supramolecular chirality in EBl J-aggregates and for its absence in
[20] Hombrecher HK, Schell C, Thiem J. Synthesis and investigation of a
galactopyranosyl-cholesteryloxy substituted porphyrin. Bioorg Med Chem
Lett 1996;6:1199–202.
[
21] Khipach VA, Zhabinskii VN, de Groot A. Brassinosteroids: a new class of plant
hormones. San Diego: Academic Press; 1999.
[22] Smagghe G. Ecdysone: Structures and functions. Springer science + Business,
Media BV; 2009.
[
23] Zelenka K, Trnka T, Tišlerová I, Monti D, Cinti S, Naitana ML, et al.
Spectroscopic, morphological, and mechanistic investigation of the solvent-
promoted aggregation of porphyrins modified in meso-positions by
glucosylated steroids. Chem Eur J 2011;17:13743–53.
[
24] Dukh M, Drašar P, Cˇ ern y´ I, Pouzar V, Shriver JA, Král V et al. Novel deep cavity
calix[4]pyrroles derived from steroidal ketones. Supramol Chem 2002; 14.
[25] Monti D, Venanzi M, Gatto E, Mancini G, Sorrenti A, Št eˇ pánek P, et al. Study of
the supramolecular chiral assembly of meso-‘‘C-glucoside’’-porphyrin
derivatives in aqueous media. New J Chem 2008;32:2127–33.
2
0E J-aggregates.
[
[
[
[
[
[
[
[
26] Št eˇ pánek P, Dukh M, Šaman D, Moravcová J, Knie zˇ o L, Monti D, et al. Synthesis
and solvent driven self-aggregation studies of meso-‘‘C-glycoside’’-porphyrin
derivatives. Org Biomol Chem 2007;5:960–70.
27] Zelenka K, Trnka T, Tišlerová I, Král V, Dukh M, Drašar P. Synthesis of porphyrin
receptors modified by glycosylated steroids. Collect Czech Chem Commun
Acknowledgments
This work was supported by by the Ministry of Education,
Youth and Sports of CR by Projects No. P304/10/1951, P503/11/
2004;69:1149–60.
28] Raichyonok TF, Khripach VA, Zhabinskii VN, Konstantinova OV, Drasar PB,
Monti D. Synthesis and spectral-luminescence properties of the conjugate of
0
616, MSM6046137305, and NATO Grant CBP.EAP.CLG.982972.
24-epibrassinolide with porphyrin. J Appl Spectr 2009;76:542–6.
We thank to Mgr. Martin Svoboda and Mgr. Martina Vermachová,
Dept. of Biochemistry and Microbiology, ICT Prague for MALDI
spectra measurements.
29] Lettieri R, Monti D, Zelenka K, Trnka T, Drašar P, Venanzi M. Glucosylated
steroid-porphyrins as new tools for nanotechnology applications. New J Chem
2012;36:1246–54.
30] Št eˇ pánek P, Šimák O, Nováková Z, Wimmer Z, Drašar P. Asymmetrically
substituted calix[4]pyrrole with chiral substituents. Org Biomol Chem
2011;9:682–3.
References
31] Khripach VA, Zhabinskii VN, Litvinovskaya RP. Immunoassays of
brassinosteroids. In: Hayat S, Ahmad A, editors. Brassinosteroids: a class of
plant hormone. Dordrecht: Springer; 2011. p. 375–92.
32] Rumlerová-Lipšová A, Barek J, Drašar P, Zelenka K, Pecková K. Polarographic and
voltammetric detrmination of meso-tetrakis(4-sulphonatophenyl)porphyrin
tetrasodium salt at mercury electrodes. Int J Electrochem Sci 2007;2:235–47.
33] Strohalm M, Kavan D, Novák P, Voln y´ M, Havli cˇ ek V. MMass 3: a cross-
platform software environment for precise analysis of mass spectrometric
data. Anal Chem 2010;82:4648–51.
[
[
1] Ungaro R, Dalcanale E. Supramolecular science. Where it is and Where it is
Going. Springer; 1999.
2] Luliano A, Ruffini A. Effect of the presence of a free hydroxyl group on the
enantiodiscrimination properties of cholic acid based CSPs bearing 2-
naphthylcarbamate and 3,5-dinitrophenylcarbamate moieties in the HPLC
resolution of racemic compounds. Tetrahedron Assymmetry 2005;16:3820–
3
828.
[
[
3] Sternberg ED, Dolphin D. Porphyrin-based photosensitizers for use in
photodynamic therapy. Tetrahedron 1998;54:4151–202.
[
[
34] Yu L, Lindsey JS. Investigation of two rational routes for preparing p-
phenylene-linked porphyrin trimers. Tetrahedron 2001;57:9285–98.
35] Senge MO, Shaker YM, Pintea M, Ryppa C, Hatscher SS, Ryan A, et al. Synthesis
of meso-substituted ABCD-type porphyrins by functionalization reactions. Eur
J Org Chem 2010;2010:237–58.
4] Koivukorpi J, Sievänen E, Kolehmainen E, Král V. Synthesis, characterization,
and saccharide binding studies of bile acid À porphyrin conjugates. Molecules
2
007;12:13–24.
[
[
[
[
5] Dukh M, Šaman D, Lang K, Pouzar V, Cˇ ern y´ I, Drašar P, et al. Steroid-porphyrin
conjugate for saccharide sensing in protic media. Org Biomol Chem
[
[
36] Khripach VA, Zhabinskii VN, Olkhovik VK, Ivanova GI, Zhernosek EV,
Kotyatkina AI. Improved synthesis of epibrassinolide. Russ
994;30:1735–40.
J Org Chem
2
003;1:3458–63.
1
6] Hirata O, Kubo Y, Takeuchi M, Shinkai S. Mono- and oligosaccharide sensing by
phenylboronic acid-appended 5,15-bis(diarylethynyl)porphyrin complexes.
Tetrahedron 2004;60:11211–8.
37] Khripach V, Zhabinskii V, de Groot A. Twenty years of brassinosteroids:
steroidal plant hormones warrant better crops for the XXI century. Ann Bot
2000;86:441–7.
7] Hirata O, Yamamoto M, Sugiyasu K, Kubo Y, Ikeda M, Takeuchi M, et al.
[
[
38] Hall DG, editor. Boronic Acids: Preparation and Applications in Organic
Synthesis and Medicine. Weinheim: WILEY-VCH Verlag; 2005.
39] Imada T, Kijima H, Takeuchi M, Shinkai S. Selective binding of glucose-6-
Allosteric saccharide sensing by
a phenylboronic-acids-appended 5,15-
bis(triarylethynyl) porphyrin. J Supramol Chem 2003;2:133–42.
8] Kalenius E, Koivukorpi J, Kolehmainen E, Vainiotalo P. Noncovalent saccharide
recognition by means of a tetrakis(bile acid)-porphyrin conjugate: selectivity,
cooperation, and stability. Eur J Org Chem 2010;6:1052–8.
phosphate, 3,4-dihydroxyphenylalanine (DOPA) and their analogs with
boronic-acid-appended metalloporphyrin. Tetrahedron 1996;52:2817–26.
a

H.A. Zhylitskaya et al. / Steroids 77 (2012) 1169–1175
1175
[
40] Sugasaki A, Ikeda M, Takeuchi M, Shinkai S. Novel oligosaccharide binding to
[47] Ravikanth M, Strachan J-P, Li F, Lindsey JS. Trans-substituted porphyrin
building blocks bearing iodo and ethynyl groups for applications in bioorganic
and materials chemistry. Tetrahedron 1998;54:7721–34.
iv
Ce bis(porphyrinate) double decker: effective amplification of a binding
signal through positive homotropic allosterism. Angew Chem, Int Ed
2
000;39:3839–42.
[48] Zhang Y, Wu Y. Different J-type aggregates of meso-tetrakis (4-
[
41] Sugasaki A, Sugiyasu K, Ikeda M, Takeuchi M, Shinkai S. First successful
molecular design of an artificial lewis oligosaccharide binding system utilizing
positive homotropic allosterism. J Am Chem Soc 2001;123:10239–44.
42] Zhang C, Suslick KS. Syntheses of boronic-acid-appended metalloporphyrins as
potential colorimetric sensors for sugars and carbohydrates. J Porphyrins
Phthalocyanines 2005;9:659–66.
hydroxyphenyl) porphyrin (H
Chem Lett 2005;16:534.
[49] White WI. In: Dolphin D, editor. The Porphyrins. New York: Academic Press;
1978.
[50] Würthner F, Kaiser TE, Saha-Möller CR. J-Aggregates: from serendipitous
discovery to supramolecular engineering of functional dye materials. Angew
Chem Int Ed 2011;50:3376–410.
[51] Mishra A, Behera RK, Behera PK, Mishra BK, Behera GB. Cyanines during the
1990s: a review. Chem Rev 2000;100:1973–2011.
[52] Ma HL, Wu JJ, Liang WJ, Chao JB. Study on the association phenomenon of
cyclodextrin to porphyrin J-aggregates by NMR spectroscopy. J Inclusion
Phenom Macrocyclic Chem 2007;58:221–6.
[53] Liu L, Li Y, Liu M. Acidichromism and supramolecular chirality of tetrakis(4-
sulfonatophenyl)porphyrin in organized molecular films. J Phys Chem (C)
2008;112:4861–6.
2
THPP) formed in different solvents. Chin
[
[
[
[
[
43] Mader HS, Wolfbeis OS. Boronic acid based probes for microdetermination of
saccharides and glycosylated biomolecules. Microchim Acta 2008;162:1–34.
44] Jin S, Cheng Y, Reid S, Li M, Wang B. Carbohydrate recognition by
boronolectins, smallmolecules, and lectins. Med Res Rev 2010;30:171–257.
45] Hargrove AE, Reyes RN, Riddington I, Anslyn EV, Sessler JL. Boronic acid
porphyrin receptor for ginsenoside sensing. Org Lett 2010;12:4804–7.
46] Khripach VA, Zhabinskii VN, Zhiburtovich YY, Ivanova GV, Konstantinova OV,
Tsavlovskii DV, et al. Preparation and synthetic application of partially
protected brassinosteroids. Steroids 2010;75:27–33.