Cholesterol-based α-phenyl-N-tert-butyl nitrone derivatives as antioxidants against light-induced retinal degeneration

Article abstract of DOI:10.1016/j.bmcl.2010.10.037

Two cholesterol-based α-phenyl-N-tert-butyl nitrone derivatives were synthesized as antioxidants against light-induced retinal degeneration. Whereas nitrone 10 significantly protected retina against bright fluorescent light exposure when injected into the

Full text of DOI:10.1016/j.bmcl.2010.10.037

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7405–7409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cholesterol-based
a-phenyl-N-tert-butyl nitrone derivatives as
antioxidants against light-induced retinal degeneration
Fanny Choteau a, Grégory Durand a, , Isabelle Ranchon-Cole b, Christine Cercy b, Bernard Pucci a,
⇑
⇑
a Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels, Université d’Avignon et des Pays de Vaucluse, Faculté des Sciences, 33 Rue Louis Pasteur,
F-84000 Avignon, France
b Laboratoire de Biophysique des Handicaps Sensoriels (UE 2667), Université d’Auvergne, 28 Place Henri Dunant, F-63000 Clermont-Ferrand, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Two cholesterol-based
a-phenyl-N-tert-butyl nitrone derivatives were synthesized as antioxidants
Received 2 September 2010
Revised 6 October 2010
Accepted 7 October 2010
Available online 20 October 2010
against light-induced retinal degeneration. Whereas nitrone 10 significantly protected retina against
bright fluorescent light exposure when injected into the vitreous at 1 mM, no protection was observed
with nitrone 6. The parent compound
at 9 mM but not at 1 mM. This suggests that nitrone 10 may be a candidate for the treatment of retinal
a-phenyl-N-tert-butyl nitrone also exhibited protective activity
diseases.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
a-Phenyl-N-tert-butyl nitrone
Amphiphiles
Antioxidants
Reactive oxygen species
Retinal degeneration
The retina is a light sensitive tissue lining the inner surface of
the eye and is constituted of several cellular types. Among them
are the photoreceptor cells whose function is to transform the light
signal into an electrophysiological signal, which is then transmit-
ted to the brain. Retinal degeneration is characterised by a progres-
sive loss of photoreceptor cells leading to visual loss. In inherited
and aged-related retinal degeneration, the photoreceptor cells un-
dergo apoptosis as the final common death pathway converging
the primary defects.1 Because apoptotic cell death underlies
light-induced retinal damage,2 the excessive light-induced photo-
receptor cells death represents a suitable model system to study
retinal degeneration.3 For instance, synthetic antioxidants and free
radical scavengers such as dimethylthiourea,4,5 cyclic nitroxides,6
expression of multiple apoptosis-associated genes.11,12 On the basis
of these data, the selective targeting of nitrones as therapeutics
has been an active field of research for the past decade. For exam-
ple, with the aim to target cellular membranes, lipophilic nitrones
bearing one or two long alkyl chains have been developed13–15 as
well as a lipophilic b-cyclodextrin cyclic nitrone conjugate16 or a
cholesteryl ester of DEPMPO17 (Fig. 1).
edaravone7 and -phenyl-N-tert-butyl nitrone (PBN)8 have already
a
shown potency against light-induced retinal degeneration.
Indeed, since the seminal work of Novelli et al.,9 nitrone spin-
traps have been widely used as protective agents in several biolog-
ical models with particular attention to PBN (Fig. 1).10 Originally
designed as a probe for the scavenging of oxygen and carbon-
centred free radicals, PBN has also been shown to have several
pharmacologic effects, such as preventing the induction of inducible
nitric oxide synthase (iNOS), inhibiting the expression of multiple
cytokine genes, activating transcription factors, or inhibiting the
⇑
Corresponding authors. Tel.: +33 (0)4 9014 4445 (G.D.); tel.: +33 (0)4 9014 4442
(B.P.).
E-mail addresses: gregory.durand@univ-avignon.fr (G. Durand), bernard.pucci@
univ-avignon.fr (B. Pucci).
Figure 1. Chemical structures of PBN, 5-ChEPMPO, LPBNH15 and LPBNAH.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.037

7406
F. Choteau et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7405–7409
In our group, we have demonstrated that amphiphilic nitrones,
to compound 3 in 96% yield. Then, the nitro group of 3 was selec-
tively reduced using Zn/NH4Cl in a 3:1 (v/v) THF/H2O mixture,
according to our general procedure,23 to give hydroxylamine 4 in
40% yield. The nitrone functional group was obtained by reaction
of the hydrophobic cholesterol-based N-tert-butyl hydroxylamine
comprised of a hydrophilic polar head and a lipophilic group,
exhibited high potency in preventing oxidative stress-mediated
damages in in vitro,18,19 ex vivo20 and in vivo models.21,22 The
amphiphilic nature is very likely responsible for improved mem-
brane crossing ability and therefore enhanced bioavailability and
bioactivity. Very recently, two amphiphilic amide nitrones called
LPBNH15 and LPBNAH (Fig. 1) were found to rescue cell cultures
and aquatic organisms exposed to lethal doses of various oxidotox-
ins.23 They were also found to decrease electron and proton leak-
age in mitochondria at nanomolar concentrations suggesting that
amphiphilic amide nitrones are not only free radical scavengers
but may act as bioenergetic agents.23 However, because of their
relatively short C7H15 alkyl chain, these amphiphilic amide nitro-
nes, exhibiting very good water solubility, may have a poor mem-
brane crossing ability and so would exhibit limited efficiency as
neuroprotective agents.
Therefore, with the expectation that the presence of a bulky
lipophilic group would impart better membrane affinity and higher
lipophilicity, we have designed two new amide nitrones 6 and 10,
in which the alkyl chain is replaced by a cholesterol moiety.
Whereas in nitrone 6 the cholesterol is grafted onto the N-tert bu-
tyl group, in nitrone 10 the cholesterol is grafted onto the aromatic
ring. In order to evaluate the neuroprotective effect of these new
compounds, we used the light-induced retinal degeneration mod-
el.5 Albino rats were treated by intravitreal injection of nitrone
agents 18 h before being exposed for 24 h to a bright fluorescent
light, and the retinal status was evaluated by histology.
The convergent synthetic pathway of the cholesteryl nitrone 6,
bearing cholesterol on the N-tert-butyl group, is based on three key
steps:24 (i) synthesis of a polar lactobionamide benzaldehyde from
4-cyanobenzaldehyde according to the previously reported proce-
dure;25 (ii) synthesis of a lipophilic cholesterol-based N-tert-butyl-
hydroxylamine from 2-methyl-2-nitropropanol (Scheme 1); (iii)
formation of the nitronyl function by condensation of the hydrox-
ylamine to the benzaldehyde under inert atmosphere (Scheme 1).
Cholesterol was first grafted onto tert-butyl acrylate in the pres-
ence of NaH to give, after purification by flash chromatography,
compound 1 in 72% yield. Removal of the tert-butyl protective
group was carried out under acidic condition in a 1:1 (v/v) TFA/
CH2Cl2 mixture to give compound 2 in quantitative yield. The cou-
pling reaction of 2-methyl-2-nitro-propylamine18 to compound 2
was next achieved using DCC/HOBt as coupling reagents to lead
4
with N-(4-formylbenzyl)octa-O-acetyl-lactobionamide25 in
a
3:2 (v/v) THF/AcOH mixture at 60 °C. In order to avoid the oxida-
tion of the hydroxylamine, the reaction was carried out in the dark
and under argon atmosphere and small amounts of hydroxylamine
were regularly added to the mixture. It is important to note that
despite the bulky cholesterol and lactobionamide groups, the ki-
netic of the coupling reaction was dramatically increased by addi-
tion of acetic acid to the milieu as previously observed.23 Extensive
purification by flash chromatography on silica gel (eluent: EtOAc)
and gravity size exclusion chromatography on Sephadex LH-20 re-
sin (eluent: CH2Cl2/MeOH 1:1 (v/v)), led to the pure nitrone 5 in
58% yield. Finally, Zemplén de-O-acetylation of the lactobionamide
group followed by purification by size exclusion chromatography
led to the lipophilic nitrone 6 in 84% yield.
The synthesis of the reverse analogue 10, bearing the choles-
terol on the aromatic ring, used a connected synthetic pathway
based on three key steps:24 (i) synthesis of a polar lactobionamide
N-tert-butylhydroxylamine
from
2-methyl-2-nitropropanol
according to the procedure recently described;23 (ii) synthesis of
a lipophilic cholesterol-based N-4-formyl benzylamide from 4-cya-
nobenzaldehyde (Scheme 2); (iii) condensation of the hydroxyl-
amine to the benzaldehyde to lead to the nitrone derivative
(Scheme 2). First, the cholesterol derivative 2 was grafted onto 4-
[1,3]dioxolan-2-ylbenzylamine25 in the presence of DCC/HOBt to
give compound 7 in 72% yield. Then, dioxolane group removal
was carried out in a 7:3 (v/v) AcOH/H2O mixture to give compound
8 in quantitative yield. Nitrone 9 was prepared following the same
procedure as described above. The lactobionamide-based N-tert-
butyl hydroxylamine previously published23 was grafted onto the
cholesterolyl benzaldehyde 8 in a 3:2 (v/v) THF/AcOH mixture.
The reaction was carried out in the dark under argon atmosphere
and small amounts of hydroxylamine were regularly added to
the mixture. After purification by flash chromatography on silica
gel (eluent: EtOAc) and gravity size exclusion chromatography on
Sephadex LH-20 resin (eluent: CH2Cl2/MeOH 1:1 (v/v)), acetylated
nitrone 9 was isolated in 82% yield. Finally, Zemplén de-O-acetyla-
tion gave the lipophilic nitrone 10 in 96% yield. The lipophilic nit-
rones 6 and 10 and their acetylated derivatives 5 and 9 were
Scheme 1. Synthesis of 6. Reagents and conditions: (a) cholesterol, NaH, THF, rt, 16 h, 72%; (b) TFA/CH2Cl2 2:8 (v/v), rt, 2 h, 81%; (c) 2-methyl-2-nitro-propylamine, DCC,
HOBt, DIEA, CH2Cl2, 1 h, rt, 96%; (d) Zn dust, NH4Cl, THF/H2O 3:1 (v/v), 0 °C, 1 h, 40%; (e) N-(4-formylbenzyl)octa-O-acetyllactobionamide, THF/AcOH 3:2 (v/v), dark, 60 °C,
24 h, 58%; (f) MeONa, MeOH, rt, 3 h, 84%.

F. Choteau et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7405–7409
7407
Scheme 2. Synthesis of 10. Reagents and conditions: (a) 4-[1,3]dioxolan-2-ylbenzylamine, DCC, HOBt, DIEA, CH2Cl2, rt, 16 h, 72%; (b) AcOH/H2O 7:3 (v/v), rt, 12 h, 92%; (c) N-
(octa-O-acetyl-lactobionyl)-2-methyl-2-hydroxylaminopropanamide, THF/AcOH 3:2 (v/v), dark, 60 °C, 8 h, 82%; (d) MeONa, MeOH, rt, 3 h, 96%.
characterised by 1H and 13C NMR as well as mass spectrometry.
The purity of 6 and 10 was confirmed by C18 reverse phase HPLC
(UV detection at 298 nm) and was higher than 95%.
the DMSO-exposed group the area under the ONL thickness curve
of the inferior side is significantly reduced by 23% (p = 0.047) com-
pared to the control, while that of the superior side is reduced by
51% (p = 0.004). Injection of PBN at 9 mM into the vitreous offered
a significant protection (p <0.009) to the retina from the damaging
effect of light compared to the DMSO-exposed group, with the ONL
thickness in both inferior and superior sides similar to that of the
control (Fig. 2A). Obviously, no significant reduction of the area un-
der the inferior or the superior ONL thickness in the PBN group was
also observed (Fig. 2C). This is in full agreement with our previous
observations where PBN was found to exhibit neuroprotective ef-
fects against light-induced retinal degeneration when injected
intraperitoneally.8 However, when PBN was injected intravitreally
at 1 mM, no protection was observed showing that this concentra-
tion is too low to provide protection. With regard to the choles-
terol-based PBN derivatives administered into the vitreous at
1 mM, the outer nuclear layer was thicker in the 10-treated group
than in the DMSO-exposed one (Fig. 2B). The area under the supe-
rior side of the ONL thickness curve of the 10-treated group was
also significantly higher (p <0.003) than that of the DMSO-exposed
group. Therefore, nitrone 10 at 1 mM can efficiently protect retina
from light-induced damage, this protection being similar to that of
PBN at 9 mM. On the contrary, the outer nuclear layer thicknesses
in the 6-treated group and in the DMSO-exposed group were sim-
ilar, and the area under the curve was not significantly different
between these two groups. This clearly demonstrates that nitrone
10 but not nitrone 6 protects the photoreceptor cells from light-in-
duced degeneration. Since both compounds possess a very close
lipophilic character, as demonstrated by their log k0W values, one
can expect them to have similar membrane crossing ability. There-
fore, the difference of protection is very likely due to other param-
eters. As reported with LPBNAH and LPBNH15, a simple reversal of
the position of the polar head and the hydrophobic chain on the
nitrone group can alter the scavenging properties but also the anti-
oxidant potency of the nitrones.23 Although LPBNAH and LPBNH15
are structural isomers as they are differentiated only from the po-
sition of the polar head and the hydrophobic chain, LPBNH15
exhibited higher potency than LPBNAH.23 The data herein supports
the conclusion that amphiphilic nitrones possessing the polar head
group on the tert-butyl group and the lipophilic group on the aro-
matic ring are more potent than their reverse analogues in pre-
venting oxidative stress-mediated damages. Moreover, as the
injection of nitrones was done 18 h before inducing the light stress
and considering the short half-live of PBN in the retina (ꢀ2 h),8 it is
likely that the concentration of molecules still present into the
Despite the presence of a lactobionamide group, the two nitro-
nes were found to be insoluble in water at concentration ꢀ0.5 g/L
demonstrating that the cholesterol group provides a high lipophilic
character to the molecule. The relative lipophilicities of these two
analogues were further measured by a chromatographic method.24
Whereas, PBN exhibits a log kW0 value of ꢀ1.7,23 the two cholesteryl
nitrones 6 and 10 were found to be much more lipophilic with val-
ues of 8.83 and 8.98, respectively. When comparing to the log kW0
value of amphiphilic amide nitrones LPBNAH (2.76) and LPBNH15
(2.86),23 this demonstrates the effect of the very hydrophobic cho-
lesterol group on the overall lipophilicity of the nitrone derivatives.
One can also note that the grafting of the cholesterol onto the aro-
matic ring or the N-tert-butyl group does not affect the lipophilicity
of the nitrone. This confirms our recent data on amphiphilic amide
nitrones showing that reverse analogues exhibit only slightly high-
er log k0W values.23
We next focused our attention on the effects of this novel series
of cholesterol-based nitrones against retinal damage induced by
light exposure. Although, the primary function of photoreceptors
is to absorb light, they are very sensitive to light26 and it has been
demonstrated that excessive light exposure induces reactive oxy-
gen species overproduction,3 which can lead to retinal dysfunction
and cell death. To evaluate the protective effect of nitrones on pho-
toreceptor cells, we measured the thickness of the outer nuclear
layer (ONL), which contains the photoreceptor nuclei, after expo-
sure to damaging light.24 An unexposed-untreated-group was done
in parallel for control as well as a DMSO 2%-treated group. Indeed,
both cholesterol-based PBN derivatives 6 and 10 were insoluble in
PBS 1ꢁ at 1 mM and so DMSO was used instead, whereas PBN was
normally dissolved in PBS 1ꢁ. However, it has to be noted that the
effects of intravitreal injection of PBS 1ꢁ and DMSO 2% against the
damaging effect of light were previously assayed, and as a result no
protection of the ONL thickness was observed.2 Therefore, in the
present study DMSO 2% group is used as equivalent to an un-
treated-group. The ONL thickness measurements give an estimate
of the number of photoreceptor cells in the retina and therefore are
a good estimate of the potency of the nitrone, the thicker the layer,
the stronger the protection by the nitrone. In the control group, the
ONL was 35–40 lm thick all along the retina. As already observed
by us2,8 and others,27,28 a 24-h exposure to bright fluorescent light
induced a significant reduction of the ONL thickness of the retina,
with a stronger effect in the superior side. As shown in Figure 2C, in

7408
F. Choteau et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7405–7409
In conclusion, two novel amide nitrones bearing both a choles-
60
50
40
30
20
10
0
A
terol and lactobionamide group were synthesized through a con-
vergent synthetic route. We investigated the protective effects of
these two nitrone derivatives against light-induced retinal damage
as well as those of the parent compound PBN. Treatment with PBN
at 9 mM resulted in a significant protection of the retina while no
protection was observed when used at 1 mM. The nitrone 10 bear-
ing the cholesterol group on the aromatic ring was found to signif-
icantly protect the retina against fluorescent light exposure at only
1 mM but no protection was observed with nitrone 6, its reverse
analogue, which bears the cholesterol group on the tert-butyl
group. These findings confirm that oxidative stress plays a crucial
role in light-induced retinal damage and that administration of
nitrone-type antioxidants 10 and PBN prevents light-induced
photoreceptor degeneration. This suggests that the cholesterol-
based nitrone 10 may be a candidate for the treatment of retinal
diseases.
-4
-2
0
2
4
Distance from the optic nerve (mm)
60
50
40
30
20
10
0
B
Acknowledgements
F.C. is the recipient of a fellowship from the ‘Région Provence
Alpes Côte d’Azur’ and Targeting System Pharma company.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.037.
References and notes
-4
-3
-2
-1
0
1
2
3
4
Distance from the optic nerve (mm)
1. Remé, C. E.; Grimm, C.; Hafezi, F.; Wenzel, A.; Williams, T. P. News Physiol. Sci.
2000, 15, 120.
2. Perche, O.; Doly, M.; Ranchon-Cole, I. Invest. Ophthalmol. Vis. Sci. 2007, 48,
2753.
3. Wenzel, A.; Grimm, C.; Samardzija, M.; Remé, C. E. Prog. Retin. Eye Res. 2005, 24,
275.
4. Organisciak, D. T.; Darrow, R. M.; Jiang, Y. L.; Marak, G. E.; Blanks, J. C. Invest.
Ophthalmol. Vis. Sci. 1992, 33, 1599.
5. Ranchon, I.; Gorrand, J. M.; Cluzel, J.; Droy-Lefaix, M. T.; Doly, M. Invest.
Ophthalmol. Vis. Sci. 1999, 40, 1191.
6. Tanito, M.; Li, F.; Elliott, M. H.; Dittmar, M.; Anderson, R. E. Invest. Ophthalmol.
Vis. Sci. 2007, 48, 1900.
7. Imai, S.; Inokuchi, Y.; Nakamura, S.; Tsuruma, K.; Shimazawa, M.; Hara, H. Eur. J.
Pharmacol. 2010, 642, 77.
8. Ranchon, I.; Chen, S.; Alvarez, K.; Anderson, R. E. Invest. Ophthalmol. Vis. Sci.
2001, 42, 1375.
9. Novelli, G. P.; Angiolini, P.; Tani, R.; Consales, G.; Bordi, L. Free Radic. Res. 1986,
1, 321.
C160
+
+
+
+
+
+
+
+
120
80
40
0
*
* *
10. Floyd, R. A.; Kopke, R. D.; Choi, C.-H.; Foster, S. B.; Doblas, S.; Towner, R. A. Free
Radic. Biol. Med. 2008, 45, 1361.
11. Hensley, K.; Robinson, K. A.; Gabbita, S. P.; Salsman, S.; Floyd, R. A. Free Radic.
Biol. Med. 2000, 28, 1456.
12. Floyd, R. A.; Hensley, K.; Kelleher-Andersson, J. A.; Wood, P. L. Mech. Ageing Dev.
2002, 123, 1021.
13. Morandat, S.; Durand, G.; Polidori, A.; Desigaux, L.; Bortolato, M.; Roux, B.;
Pucci, B. Langmuir 2003, 19, 9699.
14. Stolze, K.; Udilova, N.; Rosenau, T.; Hofinger, A.; Nohl, H. Biochem. Pharmacol.
2003, 66, 1717.
15. Hay, A.; Burkitt, M. J.; Jones, C. M.; Hartley, R. C. Arch. Biochem. Biophys. 2005,
435, 336.
16. Han, Y.; Liu, Y.; Rockenbauer, A.; Zweier, J. L.; Durand, G.; Villamena, F. A. J. Org.
Chem. 2009, 74, 5369.
17. Hardy, M.; Ouari, O.; Charles, L.; Finet, J.-P.; Iacazio, G.; Monnier, V.;
Rockenbauer, A.; Tordo, P. J. Org. Chem. 2005, 70, 10426.
18. Durand, G.; Polidori, A.; Salles, J.-P.; Pucci, B. Bioorg. Med. Chem. Lett. 2003, 13,
859.
Figure 2. Effects of nitrones on retinal damage induced by exposure to light. The
outer nuclear layer was measured from the optic nerve (0) to the superior and
inferior ora serata. Control rats were untreated and unexposed to the damaging
light. Two-microliter intravitreal injections of compounds were done 18 h before
exposure to a 24-h bright fluorescent light. (A) Control rats (d), PBN at 9 mM (4),
PBN at 1 mM (ꢁ), and DMSO 2% (j). (B) Nitrone 10 (}) and nitrone 6 (s) at 1 mM,
PBN at 9 mM (4), and DMSO 2% (j). (C) Area under the ONL thickness curves in the
superior and the inferior retina calculated using the programme Origin 6.0
(Microcal). * compared to unexposed group; + compared to DMSO 2% group. One
symbol: p <0.05; two symbols: p <0.01.
19. Durand, G.; Prosak, R. A.; Han, Y.; Ortial, S.; Rockenbauer, A.; Pucci, B.;
Villamena, F. A. Chem. Res. Toxicol. 2009, 22, 1570.
20. Tanguy, S.; Durand, G.; Reboul, C.; Polidori, A.; Pucci, B.; Dauzat, M.; Obert, P.
Cardiovasc. Drugs Ther. 2006, 20, 147.
retina at the time of light exposure is extremely low. This suggests,
in agreement with the literature,10–12 that the protective effects
provided by the treatment with nitrone agents is not dependent
on their free radical scavenging capacities. Therefore, further exper-
iments are needed to elucidate the molecular mechanism explain-
ing the neuroprotective effect of PBN and nitrone 10 compared to
nitrone 6.
21. Poeggeler, B.; Durand, G.; Polidori, A.; Pappolla, M. A.; Vega-Naredo, I.; Coto-
Montes, A.; Boeker, J.; Hardeland, R.; Pucci, B. J. Neurochem. 2005, 95, 962.
22. Asanuma, T.; Yasui, H.; Inanami, O.; Waki, K.; Takahashi, M.; Iizuka, D.;
Uemura, T.; Durand, G.; Polidori, A.; Kon, Y.; Pucci, B.; Kuwabara, M. Chem.
Biodivers. 2007, 4, 2253.
23. Durand, G.; Poeggeler, B.; Ortial, S.; Polidori, A.; Villamena, F. A.; Böker, J.;
Hardeland, R.; Pappolla, M. A.; Pucci, B. J. Med. Chem. 2010, 53, 4849.

F. Choteau et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7405–7409
7409
24. Details for the synthesis and characterization of compounds 1–10 as well as for
in vivo experiments are given in Supplementary data.
25. Ouari, O.; Chalier, F.; Bonaly, F.; Pucci, B.; Tordo, P. J. Chem. Soc., Perkin Trans. 2
1998, 2299.
26. Noell, W. K.; Walker, V. S.; Kang, B. S.; Berman, S. Invest. Ophthalmol. 1966, 5,
450.
27. Rapp, L. M.; Naash, M. I.; Wiegand, R. D.; Joel, C. D.; Nielsen, J. C.; Anderson, R. E.
In Retinal Degeneration: Experimental an Clinical Studies; LaVail, M. M.,
Hollyfield, J. G.; Anderson, R. E., Eds.; Alan R. Liss: New York, 1985; pp 421–437.
28. Káldi, I.; Martin, R. E.; Huang, H.; Brush, R. S.; Morrison, K. A.; Anderson, R. E.
Mol. Vis. 2003, 9, 337.