Synthesis and characterization of photolabeling probes of miltefosine

Article abstract of DOI:10.1016/j.jfluchem.2005.02.019

Miltefosine is an ether-phospholipid analogue showing remarkable anti-cancer and anti-leishmanial activity thanks to its cell membrane-targeting properties. In order to study the mechanisms responsible for the biological effects of miltefosine using a photolabeling approach, we designed, synthesized and characterized photolabeling probes for studying the effects of miltefosine. In these probes, the photoactivatable tetrafluorophenylazido group is incorporated either at the polar head or in the alkyl chain of miltefosine. The probes showed fast, clear-cut photochemical reactions, which suggests that they are promising tools for use in photolabeling studies.

Full text of DOI:10.1016/j.jfluchem.2005.02.019

Journal of Fluorine Chemistry 126 (2005) 739–743
www.elsevier.com/locate/fluor
Synthesis and characterization of photolabeling
probes of miltefosine
Fei Huang a, Fanqi Qu a, Qing Peng a, Yi Xia a, Ling Peng a,b,
*
a College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China
b
´ ´
AFMB CNRS UMR 6098, Departement de Chimie, Universite Aix-Marseille II,
163 Avenue de Luminy, 13288 Marseille Cedex, France
Received 23 September 2004; received in revised form 26 January 2005; accepted 11 February 2005
Available online 7 April 2005
Abstract
Miltefosine is an ether-phospholipid analogue showing remarkable anti-cancer and anti-leishmanial activity thanks to its cell membrane-
targeting properties. In order to study the mechanisms responsible for the biological effects of miltefosine using a photolabeling approach, we
designed, synthesized and characterized photolabeling probes for studying the effects of miltefosine. In these probes, the photoactivatable
tetrafluorophenylazido group is incorporated either at the polar head or in the alkyl chain of miltefosine. The probes showed fast, clear-cut
photochemical reactions, which suggests that they are promising tools for use in photolabeling studies.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Miltefosine; Photolabeling; Photolabeling probes; Tetrafluorophenylazides; Alkylphosphocholine
1. Introduction
required for ether-phospholipids to be capable of anti-
neoplastic activity led to the elimination of the glycerol
backbone, leaving the alkylphosphocholines as the most
likely candidates [6]. Among the various alkylphosphocho-
lines, miltefosine was the first to be approved in 1992 as an
anti-cancer drug for the topical treatment of certain forms of
cutaneous cancer. Recently, miltefosine has attracted
increasing attention because of its potential as an anti-
leishmanial drug [7], and it has by now been registered for
the oral treatment of visceral leishmaniasis in India, where it
has achieved a success rate of up to 97% [8]. It has also
proved to be an efficient means of treating immunodeficient
patients with visceral leishmaniasis [9] and patients with
cutanous leishmaniasis [10].
Milltefosine (hexadecylphosphocholine, Scheme 1) is an
alkyl phosphocholine showing remarkable anit-neoplastic,
anti-tumor and anti-leishmanial activities [1,2]. It is one of
the ether-phospholipid analogues and is generally thought to
target the cell membranes. Miltefosine was originally
developed as an anti-cancer drug [3], based on the seminal
studies by Munder et al. [4], describing the immunomodu-
latory activities of lysophosphatidylcholines (Scheme 1).
Lysophosphatidylcholines are a special kind of phospholi-
pids, which are generated by the hydrolysis of a fatty acid
present in the sn-2 position of various phospholipids. Since
lysophosphatidylcholines can be easily hydrolyzed by
several phospholipases and esterases in biological systems,
more stable lysophosphatidylcholine analogues such as
ether-phospholipids have been synthesized and character-
ized with a view to developing potential anti-tumor
candidates [5]. Studies on the minimum structural features
Although miltefosine has achieved considerable clinical
success, little is known about its mechanism of action on
either tumor cell lines or parasites. Due to its amphiphilic
properties, miltefosine interacts mainly with the cell
membranes of cancer cells as well as those of protozoa
[1,2]. Many hypotheses have been proposed to explain the
anti-cancer activity of miltefosine. The possible modes of
action proposed so far include modulating membrane
* Corresponding author. Tel.: +33 4 91 82 94 90; fax: +33 4 91 82 94 91.
E-mail address: ling@afmb.cnrs-mrs.fr (L. Peng).
0022-1139/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2005.02.019

740
F. Huang et al. / Journal of Fluorine Chemistry 126 (2005) 739–743
function relationships occurring upon the modification of
the polar head. In probe 2, the photoactivatable group is
located in the alkyl chain, which was designed to probe the
interactions in which miltesoine is involved in the
hydrophobic membrane core. The molecular design on
which these probes were based was inspired by previous
studies on photolabling probes designed for investigating the
effects of phospholipids in biomemebranes and membrane
proteins, where photoactivatable groups were introduced
either into the polar head or into the fatty acid chain of the
phospholipids [15,16]. Here, we report on the synthesis and
characterization of the two photolabeling probes designed
for studying the biological effects of miltefosine.
2. Results and discussion
Scheme 1. Miltefosine, lysophosphocholine and the designed photolabel-
We first attemped to synthesize 1 by directly coupling
miltefosine to N-succinimidyl-4-azido-tetrafluorobenzoate
3 (Scheme 2) [13]. However, product 1 could not be obtained
in pure form in this way because it was contaminated with
the by-product 4-azido-tetrafluorobenzoic acid and could
not be separated from this impurity using either a silica gel
column or TLC seperation methods. We then performed the
synthesis of 1 de novo by coupling POCl3 to hexadecanol
and then to N-(4-azido-tetrafluorobenzoyl)-ethanoamine 4
(Scheme 2), where 4 was obtained by treating ethanolamine
with N-succinimidyl-4-azido-tetrafluorobenzoate 3. After
being extracted from the acidic aqueous solution (pH 1.0), 1
was obtained in pure form by performing column
chromatography. Although TLC monitoring showed that a
significant amount of 1 was formed during the reaction, the
yield was rather low. One possible reason for the low yield
here might be the poor solubility of 1, which resulted in the
loss of product during the work-up procedures.
ing probes 1 and 2.
permeability and membrane lipid composition, regulating
phospholipid metabolism, alternating proliferation signal
transduction pathways and inducing apoptosis [1]. Few
studies have been carried out so far on the anti-leishmanial
activity of miltefosine [2,11]. Since miltefosine heads for
more than one cellular target, its action may involve complex
mechanisms possibly affecting its overall biological proper-
ties. In order to study the molecular mechanisms involved, it
was proposed first of all to identify the biological
macromolecules interacting with miltefosine. For this
purpose, we propose to use a photoaffinity labeling approach.
Photoaffinity labeling [12] is an efficient method for
studying the interactions between drugs and their biolo-
gical targets. This method involves the use of photo-
activatable probes, which, upon being exposed to
photoirradiation, generate highly reactive species, such
as nitrene or carbene, which cross-link covalently with the
targets at the binding sites. This process can be used to
identify the biological targets that interact with the drugs
and to map the binding sites on the targets, and thus to study
the drug–target interactions. Aryl azides are frequently
used as photoaffinity probes because they can be easily
synthesized, as well as being chemically stable in the dark
and highly reactive upon exposure to irradiation. Among
the arylazides, fluorinated aryl azides [13] are exception-
ally promising because they result in much more efficient
photolabeling than non-fluorinated arylazides thanks to
the retard of phenyl nitrene cyclization [14]. With a view
to studying the mechanisms underlying the biological
activity of miltefosine using photolabeling procedures, we
designed two complementary photolabeling probes having
fluorinated aryl azides (Scheme 1).
The synthesis of 2 required the use of tetrafluorophenyl
azide containing alcohol 6, which was prepared by coupling
the 1,8-octanediol to pentafluorobenzoic acid and subse-
In probe 1, the photoactivatable fluorinated arylazido
group is introduced into the polar head. It is therefore
proposed to probe the interactions between the probe and the
lipid–water interface as well as to study the structure–
Scheme 2. Synthesis of probe 1.

F. Huang et al. / Journal of Fluorine Chemistry 126 (2005) 739–743
741
Scheme 3. Synthesis of probe 2.
quently introducing an azido group into the aromatic ring of
5 via nucleophilic substitution using NaN3 (Scheme 3). This
two-step procedure readily provided 6 with a good yield. By
coupling POCl3 successively to 6 and to choline tosylate
(Scheme 3), 2 was obtained with a 35% yield without
requiring any further optimization.
adding SDS (Fig. 1). This result indicates that probe 1 still
retains the amphiphilic nature of miltefosine. Similar
findings has been reported on the modification of
phospholipid probes designed to investigate the lipid–water
interfaces of biomembranes [16].
A photochemical study was carried out with 1 in
CH2Cl2/MeOH and 2 in phosphate buffer at pH 7.4. Fig. 2
Both probes 1 and 2 were characterized by 1H NMR, 13
C
NMR, 19F NMR, IR, UVand MS, giving satisfactory results.
The maximum UV absorption wavelengths of these probes
are 260 nm, and the molar absorption coefficients are around
5000 molÀ1 L cmÀ1, which are the values characteristic of
aryl azides.
The probes 1 and 2 are stable in the dark. 1 is only
marginally soluble in MeOH/CH2Cl2, while 2 is soluble in
various solvents, such as MeOH, MeOH/CH2Cl2 and
phosphate buffer. The poor solubility of probe 1 might be
due to the introduction of the photoactivatable fluorinated
arylazido group at the polar head. We tried to solubilize
probe 1 in the phosphate buffer in the presence of the
detergent sodium dodecylsulfate (SDS). The solubility of
probe 1 in the buffer can in fact be significantly increased by
Fig. 1. UV spectral recording of the increase in the solubility of 1 in buffer
solution (100 mM phosphate, pH 7.4 at 20 8C) observed upon adding the
detergent, sodium dodecylsulfate (SDS).
Fig. 2. Evolution of the UV absorption spectra when probes 1 and 2 were
irradiated at a wavelength of 300 nm at 20 8C. The spectra were taken every
30 s. (a) 1 in MeOH/CH2Cl2 and (b) 2 in 100 mM phosphate, pH 7.4.

742
F. Huang et al. / Journal of Fluorine Chemistry 126 (2005) 739–743
shows the photodecomposition of 1 and 2, which
occurred in response to irradiation at 300 nm. Both probes
underwent a fast, clear-cut process of photodecomposition
upon being irradiated at wavelength ꢀ 300 nm. Irradiation
of both 1 and 2 quickly led to the disappearance of the
absorption band. The isobestic points observed indicated
that the photochemical reaction was a single photo
decomposition process. The fact that all these probes can
be activated at wavelength ꢀ300 nm is an important point,
since it means that the biological macromolecules will not
be damaged by UV irradiation at this wavelength. These
results indicate that both 1 and 2 retain the photochemical
properties of fluorinated phenylazide and are therefore
promising photolabeling probes for studying the mole-
cular mechanisms underlying the biological activity of
miltefosine.
4: Ethanolamine (0.50 mL, 8.25 mmol) was added to a
solution of 3 (280 mg, 0.84 mmol) in dry CH2Cl2 (10 mL).
The reaction mixture was stirred at 20 8C for 2 h. The organic
solvent was then removed. After extracting the product with
CH2Cl2, the extract was dried over MgSO4 and evaporated
under reduced pressure. The residue obtained was purified by
performing chromatography on silica gel with ethyl acetate/
petroleum ether 1/1, yielding 4 (70%) as white solid. Mp:
1
139–140.5 8C. H NMR (300 MHz, CD3OD): d = 3.69 (t,
J = 5.7 Hz, 2H), 3.49 (t, J = 5.7 Hz, 2H). 13C NMR
(150 MHz, CDCl3 + CD3OD): d = 163.4, 142.8 (1JCF
=
255 Hz), 140.2 (1JCF = 240 Hz, JCF = 17.4 Hz), 118.1,
114.9, 59.4, 41.4. 19F NMR (564.6 MHz, CDCl3 + CD3OD):
d = À139.2 (m, 2F), À151.3 (m, 2F). IR (KBr, cmÀ1): 3279
(NH), 2925, 2849 (C–H), 2123 (N N N), 1661 (C O). ESI-
MS: m/z 278.4 [M]+.
2
1: A solution of hexadecanol (145 mg, 0.60 mmol) in dry
THF (10 mL) was added dropwise to a mixture of freshly
distilled phosphorus oxychloride (0.60 mL, 0.64 mmol) and
triethylamine (1.0 mL, 7.2 mmol) in THF (5.0 mL), under
nitrogen at 0 8C for 30 min. After 30 min at 0 8C, the
mixture was left to warm up to 25 8C and then stirred for a
further 2 h. A solution of 4 (165 mg, 0.59 mmol) in THF
(10 mL) was then added dropwise to this mixture at 0 8C.
The reaction mixture was stirred at 0 8C for another 30 min
and then at 25 8C for 24 h before adding H2O (2.0 mL), and
stirred again at 25 8C for 1 h. The organic solvent was then
removed. After extracting the solution (pH 1.0) with
CH2Cl2, the extract was dried over MgSO4 and evaporated
under reduced pressure. The residue obtained was purified
by chromatography on silica gel with MeOH/CH2Cl2 1/7,
yielding 1 (20%) in the form of a white solid. Mp: 90 8C
(decomposition) 1H NMR (300 MHz, CDCl3 + CD3OD):
d = 3.98 (m, 2H), 3.82 (m, 2H), 3.63 (m, 2H), 1.59 (m, 2H),
1.26 (m, 26H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz,
CDCl3 + CD3OD + CF3COOH): d = 158.4, 141.1, 137.7,
114.1 (1JCF = 284 Hz), 113.5 (1JCF = 282 Hz), 66.5, 64.0,
52.5, 31.0, 29.3, 28.7, 28.4, 28.3, 24.6, 21.7, 13.0. 19F NMR
(564.6 MHz, CDCl3 + CD3OD): d = À138.3 (dd, J = 20.9,
9.60 Hz, 2F), À147.9 (dd, J = 20.9, 9.60 Hz, 2F). IR (KBr,
cmÀ1): 3347 (NH), 2919, 2851 (C–H), 2122 (N N N),
1666 (C O). ESI-MS: m/z 581.0 [M À H]À. UV: (MeOH/
CH2Cl2) lmax 257.8 nm (e 4910 molÀ1 L cmÀ1).
5: Pentafluorobenzoic acid (424 mg, 2.0 mmol) was
added to SOCl2 (10 mL), and then refluxed for 20 h. After
removing the SOCl2, the residue was dissolved in THF
(10 mL). The solution was added dropwise to a solution of
1,8-octanediol (584 mg, 4.0 mmol) in THF (10 mL) at 0 8C.
After 30 min at 0 8C, the mixturewas left towarm up to 25 8C
and then stirred for another 8 h. The organic solvent was
removed, and the residue obtained was purified by
chromatography on silica gel with ethyl acetate/petroleum
ether 1/5, yielding 5 (62%) in the form of a white solid. Mp:
34.5–35.5 8C. 1H NMR (300 MHz, CDCl3): d = 4.36 (t,
J = 6.6 Hz, 2H), 3.63 (t, J = 6.6 Hz, 2H), 1.70 (m, 2H), 1.55
(m, 2H), 1.34 (m, 8H). 13C NMR (150 MHz, CDCl3):
3. Conclusion
Two photolabeling probes based on miltefosine, with the
photoactivatable fluorinated arylazido group incorporated at
the polar head (1) and in the alkyl chain (2), were
synthesized with a view to studying the mechanisms
responsible for the biological effects of miltefosine using
a photolabeling approach. The process of photodecomposi-
tion of 1 and 2 was studied and found to involve a fast, clear-
cut photochemical reaction. Probes 1 and 2 therefore both
constitute promising tools for studying the mechanisms
underlying the biological effects of miltefosine using a
photolabeling approach. Further studies are now under way
to investigate the interactions of these probes with biological
membranes.
4. Experimental part
4.1. General
All reagents and solvents used for the synthesis of the
ether-phospholipids were freshly purified or distilled before
use. All the compounds were purified by flash chromato-
graphy on silica gel (200–300 mesh), which was purchased
from Qingdao Ocean Chemical Plant. The H NMR, 19F
1
NMR and 13C NMR spectra were recorded at 300, 564.6 and
150 MHz, respectively, on Varian Mercury-VX300 and
Varian Inova-600 spectrometers. Chemical shifts were
recorded in parts per million (ppm) with TMS as the
internal references and CF3COOH as the external reference.
ESI mass spectra were determined using a Finnigan LCQ
Advantage mass spectrometer. IR spectra were recorded
using an Avatar 360 FT-IR spectrophotometer. UV absorp-
tion spectra were recorded using a Perkin-Elmer Lambda
35 UV–vis spectrophotometer. The N-succinimidyl-4-
azido-tetrafluorobenzoate 3 was synthesized using a pre-
viously described procedure [13].

F. Huang et al. / Journal of Fluorine Chemistry 126 (2005) 739–743
743
2
d = 159.6, 145.9 (1JCF = 255 Hz, JCF = 13.5 Hz), 141.2
20 8C. The absorption spectra of the irradiated samples were
recorded using a CARY UV spectrophotometer.
(1JCF = 240 Hz, JCF = 16.5 Hz), 123.3, 108.3 (2JCF
=
2
15 Hz), 66.9, 63.2, 32.9, 29.9, 29.3, 28.6, 25.9, 25.8. 19F
NMR (564.6 MHz, CDCl3): d = À138.8 (m, 2F), À149.3 (m,
1F), À160.8 (m, 2F). ESI-MS: m/z 341.0 [M + H]+.
Acknowledgements
6: NaN3 (80.7 mg, 1.24 mmol) was added to a solution of
5 (422 mg, 1.24 mmol) in H2O (5.0 mL) and acetone
(5.0 mL). The reaction mixture was stirred at 70 8C for 12 h.
The organic solvent was then removed. After extracting the
residual mixture with CH2Cl2, the extract was dried over
MgSO4 and evaporated under reduced pressure. The residue
obtained was purified by chromatography on silica gel with
ethyl acetate/petroleum ether 1/5, yielding 6 (92%) in the
form of a white solid. Mp: 44.5–45.5 8C. 1H NMR
(300 MHz, CDCl3): d = 4.36 (t, J = 6.3 Hz, 2H), 3.62 (t,
J = 6.6 Hz, 2H), 1.74 (m, 2H), 1.56 (m, 2H), 1.34 (m, 8H).
13C NMR (150 MHz, CDCl3): d = 159.5, 145.3
We are grateful to Professor Andre Samat for giving us
access to the irradiation equipment used here. We thank Dr.
Jessica Blanc for revising the English manuscript. Financial
support from the Ministry of Science and Technology in
China (N8 2003CB114400, 2003AA2Z3506), Cheung Kong
Scholar Foundation, Wuhan University and CNRS is
gratefully acknowledged.
References
2
(1JCF = 255 Hz), 140.4 (1JCF = 255 Hz, JCF = 15 Hz),
[1] V. Jendrossek, R. Handrick, Curr. Med. Chem. Anti-Cancer Agents 3
123.2, 108.0 (2JCF = 15.3 Hz), 67.0, 63.0, 32.7, 29.7,
29.3, 28.4, 25.7. 19F NMR (564.6 MHz, CDCl3):
d = À139.2 (m, 2F), À151.4 (m, 2F). IR (KBr, cmÀ1):
3369 (OH), 2933, 2859 (C–H), 2130 (N N N), 1737
(C O). ESI-MS: m/z 362.3 [M À H]+.
(2003) 343–353.
ˆ
[2] S.L. Croft, K. Seifert, M. Duchene, Mol. Biochem. Parasitol. 126
(2003) 165–172.
[3] H. Eibl, C. Unger, Cancer Treat Rev. 17 (1990) 233–242.
[4] P.G. Munder, M. Modolell, E. Ferber, H. Fischer, Biochem. Z. 344
(1966) 310–313.
2: A solution of 6 in dry CH2Cl2 (10 mL) was added
dropwise to a mixture of freshly distilled phosphorus
oxychloride (1.2 mL, 1.3 mmol) and triethylamine (1.0 mL,
7.2 mmol) in THF (5.0 mL) at 0 8C under nitrogen. After
30 min at 0 8C, the mixture was left to warm up to 25 8C and
then stirred for 1 h. A solution of choline tosylate (875 mg,
3.0 mmol) in freshly distilled pyridine (10 mL) was added to
this mixture. The resulting mixture was stirred under
nitrogen for 3 days at 25 8C. Water (1.0 mL) was added, and
the solution obtained was stirred for 1 h. After being
evaporated under reduced pressure, the residue was purified
by chromatography on silica gel with H2O/MeOH/CH2Cl2
4/35/65, yielding 2 (35%) in the form of a yellowish solid.
[5] D. Berkovic, Gen. Pharmacol. 31 (1998) 511–517.
[6] P. Hilgard, T. Klenner, J. Stekar, C. Unger, Cancer Chemother.
Pharmacol. 32 (1993) 90–95.
[7] B.L. Herwaldt, N. Engl. J. Med. 341 (1999) 1840–1842.
[8] T.K. Jha, S. Sundar, C.P. Thakur, P. Bachmann, J. Karbwang, C.
Fischer, K. Junge, A. Voss, J. Berman, N. Engl. J. Med. 341 (1999)
1795–1800;
S. Sundar, T.K. Jha, C.P. Thakur, J. Engel, H. Sindermann, C. Fischer,
K. Junge, A. Bryceson, J. Berman, N. Engl. J. Med. 347 (2002) 1739–
1746.
[9] H.W. Murray, Antimicrob. Agents Chemother. 44 (2000) 3235–3236.
[10] J. Soto, J. Toledo, P. Gutierrez, R.S. Nicholls, J. Padilla, J. Engel, C.
Fischer, A. Voss, J. Berman, Clin. Infect. Dis. 33 (2001) E57–E61.
[11] N.K. Verma, C.S. Dey, Antimicrob. Agents Chemother. 48 (2004)
3010–3015.
´
[12] G. Dorman, G.D. Prestwich, Trends Biotechnol. 18 (2000) 64–77;
´
G. Dorman, Top. Curr. Chem. 211 (2000) 169–225;
1
Mp: > 70 8C (decomposition). H NMR (300 MHz, D2O):
d = 4.12 (m, 4H), 3.72 (m, 2H), 3.55 (m, 2H), 3.12 (s, 9H),
1.49 (m, 4H), 1.16 (m, 8H). 13C NMR (150 MHz, D2O):
d = 159.4, 145.3 (1JCF = 255 Hz), 141.1 (1JCF = 225 Hz,
2JCF = 15 Hz), 123.6, 107.1, 66.9, 66.2, 59.4, 54.2, 30.5,
29.1, 28.9, 28.2, 25.5. 19F NMR (564.6 MHz, D2O):
d = À141.3 (s, br, 2F), À153.4 (s, br, 2F). IR (KBr,
cmÀ1): 3390 (OH), 2933, 2858 (C–H), 2130 (N N N),
1733 (C O). ESI-MS: m/z 529.2 [M + H]+. UV: (H2O) lmax
267.1 nm (e 6973 molÀ1 L cmÀ1).
F. Kotzyba-Hibert, I. Kapfer, M. Goeldner, Angew. Chem. Int. Ed.
Engl. 34 (1995) 1296–1312.
[13] J.F.W. Keana, S.X. Cai, J. Fluorine Chem. 43 (1989) 151–154;
J.F.W. Keana, S.X. Cai, J. Org. Chem. 55 (1990) 3640–3647.
[14] K.A. Schnapp, R. Poe, E. Leyva, N. Soundararajan, M.S. Platz,
Bioconjugate Chem. 4 (1993) 172–177;
´
N.P. Gritsan, A.D. Gudmundsdottir, D. Tigelaar, Z. Zhu, W.L. Karney,
C.M. Hadad, M.S. Platz, J. Am. Chem. Soc. 123 (2001) 1951–1962.
[15] L. Peng, M.-L. Alcaraz, P. Klotz, F. Kotzyba-Hibert, M. Goeldner,
FEBS Lett. 346 (1994) 127–131.
[16] Y. Ogawa, W. Hahn, P. Garnier, N. Higashi, D. Massotte, M.-H. Metz-
Boutigue, B. Rousseau, M. Kodaka, J. Sunamoto, G. Ourisson, Y.
Nakatani, Chem. Eur. J. 8 (2002) 1843–1849;
4.2. General photolytic procedure
Y. Ogawa, W. Hahn, P. Garnier, N. Higashi, D. Massotte, M.-H. Metz-
Boutigue, B. Rousseau, J. Sunamoto, G. Ourisson, Y. Nakatani,
Angew. Chem. Int. Ed. 40 (2001) 944–946;
Compounds 1 and 2 were dissolved in MeOH/CH2Cl2
and in 100 mM phosphate buffer at pH 7.4, respectively, to a
concentration of approximately 1.5 Â 10À4 mol LÀ1. The
solutions (1.5 mL) were photolyzed under stirring using a
150 W USHIO Xenon Short Arc Lamp for 0–10 min at
M.-L. Alcaraz, L. Peng, P. Klotz, M. Goeldner, J. Org. Chem. 61
(1996) 192–201;
J.M. Delfino, S.L. Schreiber, F.M. Richards, J. Am. Chem. Soc. 115
(1993) 3458–3474.