Chlorin e6-cholesterol conjugate and its copper complex. Simple synthesis and entrapping in phospholipid vesicles

Article abstract of DOI:10.1016/j.bmcl.2010.03.041

Synthesis of 13′[(cholest-5-en)-3β-yloxyethoxycarbamoyl]-chlorin e6 starting from methylpheophorbide and 3β(2-hydroxy)-ethoxycholest-5-ene is presented, as well as the preparation of related copper complex. Both conjugates obtained may be simply incorporated in phosphatidyl choline vesicles.

Full text of DOI:10.1016/j.bmcl.2010.03.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2872–2875
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chlorin e6–cholesterol conjugate and its copper complex. Simple synthesis
and entrapping in phospholipid vesicles
Irina A. Nikolaeva a, Alexander Yu. Misharin a, Gelii V. Ponomarev a, Vladimir P. Timofeev b,
,
*
Yaroslav V. Tkachev b
a V.N. Orekhovich Institute of Biomedical Chemistry RAMS, Moscow, Russia
b V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32, Vavilov Street, Moscow 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Synthesis of 130[(cholest-5-en)-3b-yloxyethoxycarbamoyl]-chlorin e6 starting from methylpheophorbide
and 3b(2-hydroxy)-ethoxycholest-5-ene is presented, as well as the preparation of related copper com-
plex. Both conjugates obtained may be simply incorporated in phosphatidyl choline vesicles.
Ó 2010 Elsevier Ltd. All rights reserved.
Article history:
Received 17 February 2010
Revised 5 March 2010
Accepted 6 March 2010
Available online 11 March 2010
Keywords:
Chlorin e6
Cholesterol
Bioconjugates
Tetrapyrrolic macrocyles, that is, porphyrins and chlorins, ow-
ing to their unique spectral, photochemical, photophysical, and
metal chelating properties, have wide range of biomedical appli-
cations, such as optical imaging, fluorescent labeling, photody-
namic inactivation of microbial infections, and photodynamic
therapy of solid tumors.1–6 A key challenge to the implementa-
tion of tetrapyrrolic macrocyles for biomedicine entails tailoring
the molecules either with hydrophilic substituents to achieve its
water solubility,7,8 or with lipophilic substituents for the incor-
poration into liposomes and lipid micelles.9,10 Coupling of phtha-
locyanine and pyropheophorbide macrocycles with estradiol and
3b-oleoyloxyanrost-5-en-17-amine was shown to be an efficient
approach for the receptor-dependent targeting of macrocycles to
cells.11,12
Modification of tetrapyrrolic macrocycle with lipophilic cho-
lesterol moiety may be of interest, since cholesterol is essential
component of mammalian membranes. The resulting conjugates
are supposed to have affinity to membranes, and may be used as
photosensitizers, being entrapped in liposomes. Insertion of
paramagnetic, for example, copper ion, into the coordination
sphere of macrocycle may convert them to spin probes suitable
for membrane studies. Herein, the simple synthesis of chlorin
e6–cholesterol conjugate from easily available methylpheophor-
bide,13 preparation of related copper complex, and entrapping
both these conjugates into phosphatidyl choline vesicles, are
presented.
The synthetic pathway is shown in Scheme 1. 3b(2-Hydroxy)-
ethoxycholest-5-ene 2 was prepared from cholesterol 1 according
to published method;14 substitution of hydroxyl group for amino
group was carried out by three step procedure, the resulting
3b(2-amino)-ethoxycholest-5-ene15 3 was obtained in 62% overall
yield (based on cholesterol 1). For the coupling of sterol and mac-
rocycle fragments the known reaction of nucleophilic opening of
exocycle E in methylpheophorbide by amines was used:16 incuba-
tion of methylpheophorbide with 1.5 equiv of aminosterol 3 in THF
at 40 °C for 48 h led to target amide conjugate17 4 isolated in 90%
yield. The copper complex of conjugate 5 was prepared in quanti-
tative yield by heating of conjugate 4 with Cu(CH3COO)2 excess in
CH2Cl2–MeOH mixture (1:3) at 45 °C for 1 h with subsequent iso-
lation of product18 5 by silica gel flash chromatography. The forma-
tion of copper complex was confirmed by HRMS peak
corresponding to molecular ion, hypsochromic shift of long wave
maximum in absorption spectrum, and characteristic EPR
spectrum.
Chlorin e6–cholesterol conjugates 4 and 5 may be simply
incorporated in phospholipids bilayers. Mixed vesicles consisted
of egg yolk phosphatidyl choline (PC) and compounds 4 and 5
were prepared according to known procedure19 developed earlier
for the preparation of unilamellar vesicles from pure PC, and PC–
cholesterol mixtures. Incorporation of conjugates 4 and 5 in PC
vesicles20 provides their solubilization in aqueous medium, and
leads to notable changes in absorption spectra when compared
* Corresponding author. Tel.: +7 499 135 9859; fax: +7 499 135 1405.
E-mail address: tim@eimb.ru (V.P. Timofeev).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.041

I. A. Nikolaeva et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2872–2875
2873
1) TsCl / Py
2) AcONa / MeOH
3) HOCH2CH2OH /dioxane, H+
HO
2
1
O
HO
1) TsCl / Py
2) NaN3 / DMF
3) LiAlH4 / Et2O
N
NH
N
+
HN
H2N
3
O
O
COOMe
COOMe
N
NH
N
HN
COOMe
NH
O
O
COOMe
4
Cu(CH3COO)2
N
N
N
N
Cu
COOMe
NH
O
O
COOMe
5
Scheme 1.
with these of CH2Cl2 solutions. Bathochromic shifts, apparently
caused by increasing of the medium polarity, thus confirming
the dye exposure on the bilayer surface, were observed both
for Soret bands and for long wave maxima (Fig. 1).
Hamiltonian anisotropy) it is equal to parallel component of 63Cu
nucleus hyperfine tensor (typically about 200G), while motions
comparable to hyperfine tensor anisotropy leads to decreasing
of A value.
EPR spectra21 of conjugate 5 powder (1, Fig. 2A) and PC ves-
icles containing conjugate 5 in aqueous solution (2, Fig. 2A) look
similar to well-known ones for porphin-like copper complexes.22
High-field perpendicular manifold splits into two major compo-
nents due to second-order effects (‘angular anomalies’),23 and
this is typical for systems with highly anisotropic spin Hamilto-
nian. Angles about 70° (with respect to magnetic field) contrib-
ute mainly to the most high-field component,23 thus providing
some spatial resolution in perpendicular region. Splitting be-
tween low-field peaks (referenced as A parameter, Fig. 2A) pro-
vides a measure of motional spectrum narrowing. In rigid-limit
Compared to EPR spectrum of conjugate 5 in CH3Cl solution
(Fig. 2B), the spectrum of conjugate
5 in PC vesicles (2,
Fig. 2A) displays much higher value of A (209.7G vs 110.6G). This
clearly confirms an entrapping of conjugate 5 into PC vesicles,
because of s(free conjugate) ꢀ
s(vesicle). Moreover, the value of A at
293 K for PC-entrapped complex is even larger than in powder
spectrum of free one (209.7G vs 201.5G), approaching rigid-limit
(211.9G, as measured at 77 K) indicating intramolecular fast
oscillations being hindered by the bilayer. This allows relating
membrane director tilt angles with hyperfine splitting.
Additionally, in spectrum of compound 5 powder (1, Fig. 2A),
the super-hyperfine splitting in the region of perpendicular mani-
state (no motion, with correlation time
s much larger than spin

2874
I. A. Nikolaeva et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2872–2875
Acknowledgements
Authors are acknowledged to Mr. Oleg Kharybin and Mrs. Maria
Zavialova (from Orekhovich Institute of Biomedical Chemistry
RAMS, Moscow) for HRMS measurements. This study was sup-
ported by Russian Foundation for Basic Research (grant 09-04-
00454-a).
References and notes
1. Licha, K. Top. Curr. Chem. 2002, 222, 1.
2. Perfetto, S. P.; Chattopadhyay, P. K.; Roederer, M. Nat. Rev. Immunol. 2004, 4,
648.
3. Hamblin, M. R.; Hasan, T. Photochem. Photobiol. Sci. 2004, 3, 436.
4. Pandey, R. K.; Zheng, G.. In The Porphyrin Handbook; Kadish, K. M., Smith, K. M.,
Guilard, R., Eds.; Academic: San Diego, CA, 2000; Vol. 6, pp 157–230.
5. Bonnett, R. Chemical Aspects of Photodynamic Therapy; Gordon and Breach
Science: Amsterdam, 2000.
6. Detty, M. R.; Gibson, S. L.; Wagner, S. J. J. Med. Chem. 2004, 47, 3897.
7. Fan, D.; Taniguchi, M.; Yao, Z.; Dhanalekshmi, S.; Lindsey, J. S. Tetrahedron 2005,
61, 10291.
8. Muresan, A. Z.; Lindsey, J. S. Tetrahedron 2008, 64, 11440.
9. Leroux, J.; Roux, E.; Le Garrec, D.; Hong, K.; Drummond, D. C. J. Controlled
Release 2001, 72, 71.
10. Polo, L.; Bianco, G.; Reddi, E.; Jori, G. Int. J. Biochem. Cell Biol. 1995, 27, 1249.
11. Zheng, G.; Li, H.; Zhang, M.; Lund-Katz, S.; Chance, B.; Glickson, J. D.
Bioconjugate Chem. 2002, 13, 392.
Figure 1. Absorption spectra of compounds 4 and 5 in CH2Cl2 (curves 1 and 2,
respectively) and mixed vesicles of compounds 4 and 5 with PC in aqueous solution,
pH 7.4 (curves 3 and 4, respectively).
12. Khan, E. H.; Ali, H.; Tian, H.; Rousseau, J.; Tessier, G.; Shafiullaha; van Lier, J. E.
Bioorg. Med. Chem. Lett. 2003, 13, 1287.
fold (arising from four nitrogen nuclei of chlorin ring) is almost
unresolved, while in presence of PC it is clearly emphasized. There-
fore, entrapping of cholesterol moiety of conjugate 5 into PC vesi-
cle, leads to exposure of its chlorin ring on the surface of bilayer,
hindering its reorientational motion, and preventing copper cen-
ters from spin–spin interaction due to spatial separation, which
otherwise leads to line broadening.
In conclusion, chlorin e6–cholesterol conjugates were synthe-
sized and characterized, as well as the related mixed vesicles with
PC. The affinity of cholesterol moiety of conjugates to phospholip-
ids ensures efficient incorporation in lipid aggregates. Being used
as spin probes, chlorin e6–cholesterol conjugates containing para-
magnetics, may provide structural (distinguishing between paral-
lel, perpendicular, and 70° orientations) and dynamical
13. Methyl pheophorbide was isolated from Spirulina platencis according to the
method described in: Ponomarev, G. V.; Tavrovskij, L. D.; Zaretskij, A. M.;
Ashmarov, V. V.; Baum, R.F. Patent RU 22276976 C2, 2006. Egg yolk
phosphatidyl choline and phosphate buffered saline were purchased from
‘Sigma’; cholesterol and other reagents were purchased from ‘Acros Organics’.
14. Misharin, A. Y.; Malugin, A. V.; Steinschneider, A. Y.; Kosykh, V. A.; Novikov, D.
K. Med. Chem. Res. 1993, 3, 451. Analytical data for 3b(2-hydroxy)-ethoxycholest-
5-ene 1: white needles, mp 104 °C (from CH3CN); 1H NMR (here and below
were obtained on a Bruker AMX-III instrument, 400 MHz, in CDCl3): 0.67 (s, 3H,
H-18); 0.85 and 0.86 (each d, J = 6.6 Hz, 3H, H-26 and H-27); 0.91 (d, J = 6.6 Hz,
H-21); 1.00 (s, 3H, H-19); 3.19 (m, 1H, H-3); 3.58 (m, 2H, COCH2); 3.70 (m, 2H,
HOCH2); 5.34 (m, 1H, H-6); 13C NMR: 12.78; 19.61; 20.22; 21.95; 23.38; 23.60;
24.70; 25.12; 29.02; 28.78; 29.23; 32.70; 32.71; 36.53; 36.97; 37.60; 37.96;
39.86; 40.25; 40.55; 43.05; 50.89; 56.85; 57.40; 62.59; 69.55; 79.89; 121.71;
140.71.
15. 3b(2-Amino)-ethoxycholest-5-ene 2 was isolated as white solid homogenous by
TLC and indicated positive reaction with ninhydrin. 1H NMR: 0.67 (s, 3H, H-
18); 0.85 and 0.86 (each d, J = 6.6 Hz, 3H, H-26 and H-27); 0.91 (d, J = 6.6 Hz,
3H, H-21); 1.00 (s, 3H, H-19); 3.20 (m, 1H, H-3); 3.34 (m, 2H, NCH2); 3.65 (m,
2H, COCH2); 5.34 (m, 1H, H-6); 13C NMR: 12.02; 18.89; 19.53; 21.25; 22.70;
22.95; 24.01; 24.45; 28.16; 28.38; 28.65; 32.08; 35.95; 36.38; 37.06; 37.42;
39.36; 39.69; 39.98; 42.41; 42.50; 50.40; 56.37; 56.96; 70.24; 79.43; 121.72;
141.09. Analytical data for intermediates: 3b(2-p-tolyolsulfonyloxy)-
information (with maximum sensitivity at
s
ꢁ 1 ns and less). Being
incorporated in PC vesicles, chlorin e6–cholesterol conjugates are
efficiently taken up by the cultured cells, enabling them to be con-
sidered as potential sensitizers for photodynamic therapy.
A
B
Figure 2. X-band EPR spectra of conjugate 5. (A) Solid-state powder spectrum (1); spectrum of conjugate 5—PC mixed vesicles in aqueous solution, pH 7.4 (2). (B) Spectrum in
CHCl3 solution.

I. A. Nikolaeva et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2872–2875
2875
ethoxycholest-5-ene (white needles from hexane, mp 115 °C); 1H NMR: 0.67 (s,
3H, H-18); 0.85 and 0.86 (each d, J = 6.6 Hz, 3H, H-26 and H-27); 0.91 (d,
J = 6.6 Hz, H-21); 0.96 (s, 3H, H-19); 2.43 (s, 3H, CH3, tosyl); 3.09 (m, 1H, H-3);
3.64 (m, 2H, COCH2); 4.14 (m, 2H, SOCH2); 5.31 (m, 1H, H-6); 7.33 (d, J = 8.0 Hz,
tosyl); 7.80 (d, J = 8.0 Hz, tosyl); 13C NMR: 12.02; 18.90; 19.49; 21.24; 21.78;
22.71; 22.96; 24.01; 24.45; 28.17; 32.08; 32.11; 28.17; 28.38; 32.11; 35.95;
36.38; 36.98; 37.27; 39.05; 39.70; 39.97; 42.51; 50.35; 56.37; 56.96; 66.56;
69.81; 79.79; 121.95; 128.17; 129.92; 133.50; 140.77; 144.82. 3b(2-Azido)-
ethoxycholest-5-ene (white cubes from EtOH, mp 60 °C); 1H NMR: 0.67 (s, 3H,
H-18); 0.85 and 0.86 (each d, J = 6.6 Hz, 3H, H-26 and H-27); 0.91 (d, J = 6.6 Hz,
H-21); 1.00 (s, 3H, H-19); 3.20 (m, 1H, H-3); 3.34 (m, 2H, NCH2); 3.65 (m, 2H,
COCH2); 5.34 (m, 1H, H-6); 13C NMR: 12.02; 18.90; 19.53; 21.26; 22.71; 22.96;
24.01; 24.46; 28.17; 28.39; 28.49; 32.09; 32.12; 35.95; 36.39; 37.02; 37.36;
39.16; 39.70; 39.99; 42.52; 50.40; 51.22; 56.38; 56.98; 66.91; 79.88; 121.93;
140.90.
31.97; 31.99; 35.86; 36.30; 36.93; 37.25; 37.99; 39.18; 39.63; 39.86; 40.90;
42.41; 49.36; 50.26; 51.65; 52.25; 53.24; 56.28; 56.84; 66.49; 70.67; 71.72;
79.57; 93.82; 98.89; 101.46; 102.58; 121.58; 121.72; 121.87; 128.51; 129.60;
130.12; 130.27; 134.72; 134.92; 135.01; 135.30; 136.07; 139.05; 140.72;
144.73; 148.84; 153.92; 166.94; 169.02; 169.44; 173.56; 173.81; HRMS (here
and below were obtained on a Bruker ‘Apex Ultra’ FT ICR MS instrument, at ion
positive electro spray ionization mode) calculated for [C67H98N5O6]+:
1068.7517; found: 1068.7444; kmax(nm) (here and below: absorption
spectrum
spectrophotometer) in CH2Cl2: 398; 498; 660.
18. 13’[(Cholest-5-en)-3b-yloxyethoxycarbamoyl]-chlorin e6–Cu complex
were
obtained
on
a
‘Thermospectronic
Helios
a’
5
was
isolated by silica gel flash chromatography in CH2Cl2 containing MeOH (1%
v/v). HRMS calculated for [C67H97CuN5O6]+: 1129.6657; found: 1129.6620;
kmax,(nm) in CH2Cl2: 398 (sh), 410, 634.
19. Batzri, S.; Korn, D. E. Biochim. Biophys. Acta 1973, 298, 1015. Mixed vesicles
16. Ellsworth, P. A.; Storm, C. B. J. Org. Chem. 1978, 43, 281.
were prepared by injection of iso-propanol solution of lipids (30 lL of 100 mM
17. 13’[(Cholest-5-en)-3b-yloxyethoxycarbamoyl]-chlorin e6 4 was isolated by silica
gel flash chromatography in CH2Cl2 containing MeOH (1% v/v). 1H NMR
(assignment of signals was obtained from COSY spectra): ꢂ1.88 (broad s, 1H,
NH in chlorin e6 moiety); ꢂ1.40–1.90 (broad, 1H, NH in chlorin e6 moiety);
0.64 (s, 3H, H-18 in cholesterol moiety); 0.84 and 0.85 (each d, J = 6.6 Hz, 3H,
H-26 and H-27 in cholesterol moiety); 0.90 (d, J = 6.6 Hz, H-21 in cholesterol
moiety); 0.93 (s, 3H, H-19 in cholesterol moiety); 1.71 (d, J = 7.2 Hz, 3H, CH3CH
in chlorin e6 moiety); 1.72 (t, J = 7.2 Hz, 3H, CH3CH2C@ in chlorin e6 moiety);
3.21 (m, 1H, H-3 in cholesterol moiety); 3.31, 3.48, 3.58, 3.61, and 3.71 (each s,
3H, CH3CC@ and CH3OO in chlorin e6 moiety); 3.80 (q, 2H, J = 7.5 Hz,
CH3CH2C@ in chlorin e6 moiety); 3.82 (m, 2H, NCH2CH2O); 4.03 (m, 2H,
NCH2CH2O); 4.40 (dd, J = 9.7 Hz and J = 1.9 Hz, 1H, CH3CHCH in chlorin e6
moiety); 4.47 (q, J = 7.2 Hz, CH3CH2C@ in chlorin e6 moiety); 5.29 (d,
J = 18.9 Hz, 1H, CCH2COO in chlorin e6 moiety); 5.32 (m, 1H, H-6 in
cholesterol moiety); 5.56 (d, J = 18.9 Hz, 1H, CCH2COO in chlorin e6 moiety);
6.13 (dd, J = 11.5 Hz and J = 1.4 Hz, 1H, H2C@CH (trans) in chlorin e6 moiety);
6.35 (dd, J = 17.7 Hz and J = 1.4 Hz, 1H, H2C@CH (cis) in chlorin e6 moiety);
6.72 (broad m, 1H, HNCO); 8.07 (dd, J = 11.5 Hz and J = 17.7 Hz, 1H, H2C@CH in
chlorin e6 moiety); 8.80, 9.64, and 9.71 (each s, 1H, b-H, CHC@ in chlorin e6
moiety); 13C NMR: 11.42; 11.92; 12.04; 12.22; 17.75; 18.81; 19.42; 19.79;
21.14; 22.64; 22.89; 23.15; 23.94; 24.35; 28.10; 28.30; 28.54; 29.82; 31.22;
PC solution containing 2 mM of conjugates 4 and 5) into 3 mL of phosphate
buffered saline.
20. Stoichiometric composition of PC vesicles containing conjugates 4 and 5 was
determined as follows: 1 mL of obtained vesicles was extracted with CHCl3/
MeOH (2:1 by vol.) mixture (3 ꢃ 3 ml), followed by quantitative
determination of PC concentration according to known method: Vaskovsky,
V. E.; Kostetsky, E. V.; Vasendin, I. M. J. Chromatogr. 1975, 114, 129–141; and
chlorin e6–cholesterol conjugates concentration from absorption spectra
(suggesting e660 value for compound 4, and e630 value for compound 5 to be
equal to 36,000). The molar ratio of conjugate/PC was found to be 1:50 in both
cases. Compound 4—PC mixed vesicles: kmax (nm) in H2O: 406, 505, 668.
Compound 5—PC mixed vesicles: kmax (nm) in H2O: 401(sh), 413, 503,
592(sh), 639.
21. EPR spectra of 13’[(cholest-5-en)-3b-yloxyethoxycarbamoyl]-chlorin e6–Cu
complex 5 (as a powder, mixed with solid silica gel, as solution in CHCl3, and as
mixed vesicles with PC in phosphate buffered saline, pH 7.4) were obtained on
a Varian E-104A X-band (9.15 GHz) spectrometer at room temperature (293 K),
with microwave irradiation level of 20 mW, modulation amplitude of 4G and
sweep width of 1000G.
22. Bohandy, J.; Kim, B. F. J. Magn. Reson. 1977, 26, 341.
23. Rollman, L. D.; Chan, S. I. J. Chem. Phys. 1969, 50, 3416.