Phosphorylated 1,6-diphenyl-1,3,5-hexatriene

Article abstract of DOI:10.1016/j.bmcl.2004.01.005

A lipophilic dye consisting of a (1E,3E,5E)-1,6-diphenyl-1,3,5-hexatriene (DPH) fluorophore attached to a phosphate diester was prepared, and its fluorescence behavior in different solvent systems and in a liposomal membrane bilayer was examined. The key step in the synthesis of the functionalized end of the dye is a Sonogashira coupling of protected iodophenol with propargyl alcohol; the remaining phenyl ring and double bonds of the all-trans polyene core arise from a Wittig reaction with trans-cinnamaldehyde. Like DPH itself, the emission intensity of its phosphorylated derivative is quenched in polar media.

Full text of DOI:10.1016/j.bmcl.2004.01.005

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1075–1078
Phosphorylated 1,6-diphenyl-1,3,5-hexatriene
Ling Ma, Jessica C. Morgan, Wendy E. Stancill and William E. Allen*
Department of Chemistry, East Carolina University, Greenville, NC 27858-4353, USA
Received 28 September 2003; revised 16 December 2003; accepted 7 January 2004
Abstract—A lipophilic dye consisting of a (1E,3E,5E)-1,6-diphenyl-1,3,5-hexatriene (DPH) fluorophore attached to a phosphate
diester was prepared, and its fluorescence behavior in different solvent systems and in a liposomal membrane bilayer was examined.
The key step in the synthesis of the functionalized end of the dye is a Sonogashira coupling of protected iodophenol with propargyl
alcohol; the remaining phenyl ring and double bonds of the all-trans polyene core arise from a Wittig reaction with trans-cinna-
maldehyde. Like DPH itself, the emission intensity of its phosphorylated derivative is quenched in polar media.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
for these first two steps were also achieved using tert-
butyldimethylsilyl as the -OH protecting group.16
Treatment of alkyne 4 with LiAlH4 in THF at reflux
slowly effected reduction to allylic alcohol 5, the E ste-
reochemistry of which was confirmed by 1H NMR
spectrometry (JCH=CH=15.9 Hz). To convert alcohol 5
to phosphonium salt 6, the syntheses of precursor cin-
namyl halides were attempted first. Both iodination (I2,
The phosphorylation of tyrosine residues of membrane-
spanning kinase enzymes, to give cytosolic phosphotyro-
sine (pTyr) units, is an early step in the transmission
of biochemical signals from the outside to the inside of
cells.1,2 Artificial mimics of pTyr can serve to probe
and/or inhibit the protein–protein recognition events
that are required for cell signaling, and therefore such
mimics show promise in the treatment of diseases
that arise when signaling goes awry.3ꢀ6 We reasoned that
direct attachment of a phosphate moiety to one of the
phenyl rings of fluorescent (1E,3E,5E)-1,6-diphenyl-
1,3,5-hexatriene (DPH) would yield a pTyr model with
the ability to report on its binding state, as the emission
behavior of DPH is known to be sensitive to the polar-
izability of its local environment.7,8 Furthermore, a
DPH-based system would be expected to localize in
lipid bilayers,9ꢀ14 thereby modeling a key aspect of the
native tyrosine kinase enzymes themselves. This paper
addresses the feasibility of using DPH as a pTyr scaf-
fold, both from a synthetic standpoint, and by quanti-
fiying the effects of phosphate substitution on the
photophysical behavior of the fluorophore.
The synthesis of phosphorylated DPH 1 is shown in
Scheme 1. Commercially available 4-iodophenol 2 was
protected as the p-methoxybenzyl (PMB) ether, then the
resultant iodide 3 was coupled to propargyl alcohol
under Sonogashira conditions.15 Excellent yields (>99%)
* Corresponding author. Tel.: +1-252-328-9779; fax: +1-252-328-
6210; e-mail: allenwi@mail.ecu.edu
Scheme 1.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.005

1076
L. Ma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1075–1078
Table 1. Photophysical data for 1
PPh3, imidazole, CH3CN) and bromination (CBr4, PPh3,
CH2Cl2) gave products that were too unstable to be
isolated. As an alternative, direct synthesis of 6 from 5
using N-bromosuccinimide (NBS) and excess PPh3 did
not require isolation of the presumed cinnamyl bromide
intermediate.17,18
Solvent
lmax (abs.),
nm
lmax (em.),
nm
ÈF
Benzene
Dichloromethane
Methanol
DMPC/aq. phosphatea
347, 362, 382
343, 359, 379
337, 353, 372
343, 360, 379
428
431
431
429
0.20
0.13
0.032
Not
determined
The central C¼C bond of the DPH moiety was installed
via a Wittig reaction19,20 between the ylide formed from
6 and trans-cinnamaldehyde, giving dye 7 as a yellow
solid with an intense blue fluorescence on ordinary silica
gel TLC plates (lexcit=365 nm). Although yields for this
step were never greater than 40%, the product was iso-
lated in analytically pure form simply by filtering it from
the reaction medium and washing to remove inorganic
salts and Ph3P=O. Subsequent removal of the PMB
group of 7 proved to be problematic. Oxidative clea-
vage using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ)21 in CH2Cl2–H2O (20:1, v:v) at ambient temp-
erature was incomplete after two days, while catalytic
hydrogenolysis (5% Pd/C, H2, EtOH)22 gave only
non-fluorescent products, presumably arising from hydro-
genation of the triene unit. Disappearance of fluores-
cence was observed with other deprotection methods, as
well.23,24 Ultimately, phenol 8 was formedꢁin 30% yield
by treating 7 with excess acetic acid at 90 C.25 Attach-
ment of a dibenzylphosphate ester26 to 8 provided
desired product 1 in 41% yield after column chromato-
graphy. Consistent with the structure of 1, all but one
of its 13C NMR signals appear between 124 and 142
ppm, in the region expected for sp2-hybridized carbon
atoms.
a Liposomes prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine,
containing 1 mol% 1.
cence intensity of 1 is sensitive to solvent polarizability/
polarity. When excited at 360nm, the fluorescence
quantum yield of 1 is about six times greater in C6H6
than in CH3OH.27 As expected, quenching is also
observed when adding water to ethanol solutions of 1.
In 4:1 (v:v) CH3CH2OH–H2O, the integrated emission
intensity of 1 from 370–600 nm is approximately 15%
of that observed in neat ethanol. For DPH under the
same conditions, this value is 22%. When incorporated
into liposomes of 1,2-dimyristoyl-sn-glycero-3-phos-
phocholine (DMPC) in aqueous phosphate buffer solu-
tion,28 the electronic absorption and fluorescence
emission spectra of 1 resemble those acquired in CH2Cl2
(Fig. 1). This suggests that the fluorophore localizes
within the hydrophobic portion of the bilayer. Analo-
gues of 1 bearing deprotected, anionic phosphate would
presumably have a greater likelihood of residing near
the polar head groups of the membrane.
2. Experimental
The presence of the phosphate group in 1 has a modest
influence on the photophysical properties of the diphe-
nylhexatriene chromophore. As shown in Table 1, dye 1
displays three absorption peaks in benzene, dichloro-
methane, and methanol, with lmax appearing at 362,
359, and 353 nm, respectively. Like DPH, the lumines-
2.1. 4-Iodo-1-[(4-methoxyphenyl)methoxy]benzene (3)
4-Iodophenol 2 (10.84 g, 49.27 mmol), K2CO3 (7.49 g,
54.2 mmol), and a catalytic amount of tetra-
butylammonium iodide were combined in 120mL of
Figure 1. Absorption and emission spectra of 1 in aerated CH2Cl2 at 23 ꢁC. lexcit=360nm.

L. Ma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1075–1078
1077
acetone. With stirring, 4-methoxybenzyl chloride (8.00
mL, 59.0mmol) was then added dropwise at rt. After
the addition was completed, the solution was heated to
reflux for 48 h, during which time a white precipitate
formed. The mixture was cooled to rt. The solid was
collected by suction filtration, washed with water, and
dried under vacuum to afford 15.89 g (95%) of 3. Mp
heated to reflux in the dark for 24 h. Upon cooling to rt,
the product precipitated. It was collected by filtration
and washed with ice-cold THF to afford 8.00 g (68%) of
6. Mp 168 ꢁC (dec); H NMR (CDCl3): d 7.90–7.13 (m,
1
23H), 7.08 (d, 2H), 6.69 (d, 2H), 6.50 (dd, 1H), 5.85 (dd,
1H), 5.00(d, 2H), 4.94 (s, 2H), 4.30(s, 2H), 3.80(s, 3H);
13C NMR (CDCl3): d 159.5, 159.1, 139.8, 135.0, 134.1,
130.4, 129.2, 128.8, 128.7, 127.9, 118.6, 118.0, 115.0,
138–139 ꢁC; H NMR (CDCl3): d 7.54 (d, 2H), 7.33 (d,
1
.
2H), 6.91 (d, 2H), 6.72 (d, 2H), 4.94 (s, 2H), 3.81 (s,
3H); 13C NMR (CDCl3): d 159.8, 158.9, 138.4, 129.4,
117.6, 114.3, 83.1, 70.1, 55.6. Anal. calcd for C14H13IO2:
C 49.43, H 3.85; found: C 49.35, H 3.87.
114.0, 111.0, 70.0, 55.3. Anal. calcd for C35H32BrO2P 1/
3H2O: C 69.89, H 5.47; found: C 69.86, H 5.63.
2.5. (1E,3E,5E)-1-{4-[(4-Methoxyphenyl)methoxy]phenyl}-
6-phenylhexa-1,3,5-triene (7)
2.2. 3-{4-[(4-Methoxyphenyl)methoxy]phenyl}prop-2-yn-
1-ol (4)
A suspension of phosphonium salt 6 (7.76 g, 13.0mmol)
in 200 mL of THF at ꢀ78 ꢁC was treated dropwise with
n-BuLi (9.77 mL of a 1.6 M solution, 15.6 mmol). After
30min, the flask was wrapped in aluminum foil to
exclude light, and trans-cinnamaldehyde (1.64 mL, 13.0
mmol) was added dropwise. The mixture was allowed to
warm to rt over 24 h. The crude product was collected
by filtration and washed with water then diethyl ether.
Drying under vacuum afforded 1.68 g (35%) of func-
tionalized DPH 7 as a yellow solid. Mp 205 ꢁC (dec); 1H
NMR (CDCl3): d 7.50–6.40 (m, 13H; m, 6H), 5.00 (s,
2H), 3.82 (s, 3H); 13C NMR (CDCl3): d 135.2, 134.4,
134.3, 134.1, 132.7, 132.6, 132.2, 130.6, 130.5, 129.5,
129.4, 128.9, 127.8, 127.6, 127.4, 126.5, 115.3, 114.2,
70.1, 55.5; UV–vis (CH2Cl2) lmax (e Mꢀ1 cmꢀ1) 352 (sh,
ꢂ52,000), 366 (63,000), 384 (45,000). Anal. calcd for
C26H24O2: C 84.75, H 6.57; found: C 84.84, H 6.46.
Protected iodide
3 (6.44 g, 18.9 mmol), tetra-
kis(triphenylphosphine)palladium(0) (0.67 g, 0.58
mmol), and copper(I) iodide (0.72 g, 3.8 mmol) were
combined in 120mL of toluene, and the mixture was
stirred for 2 h at rt. The reaction flask was cooled in an
ice bath, then a solution of diethylamine (13.9 mL, 134
mmol) and propargyl alcohol (5.00 mL, 85.9 mmol) was
added dropwise. The reaction was stirred for 2 h and
was then allowed to warm to rt for a further 10h.
Saturated aqueous NH4Cl (100 mL) was added, the
layers were separated, and the aqueous phase was
extracted with EtOAc. The combined organic layers
were washed with brine, dried over MgSO4, and filtered.
The filtrate was evaporated, and the crude product was
recrystallized from diethyl ether–hexanes (1:1, v:v) to
afford 4.55 g (90%) of 4 as a light yellow solid. Mp 120–
121 ꢁC; 1H NMR (CDCl3): d 7.38 (m, 4H), 6.92 (m, 4H),
4.99 (s, 2H), 4.48 (s, 2H), 3.82 (s, 3H); 13C NMR
(CDCl3): d 159.8, 159.5, 133.1, 129.6, 128.7, 115.2,
115.1, 114.2, 86.0, 85.9, 70.1, 55.6, 51.9. Anal. calcd for
C17H16O3: C 76.10, H 6.01; found: C 75.88, H 5.90.
2.6. (1E,3E,5E)-4-(6-Phenylhexa-1,3,5-trienyl)phenol (8)
Compound 7 (1.96 g, 5.32 mmol) and acetic acid (350
mL) were heated to 90 ꢁC for 72 h in the dark. The
acetic acid was evaporated under reduced pressure, and
the residue was partitioned between CH2Cl2 and water.
The organic layer was washed with saturated aqueous
NaHCO3 then brine, and was dried over Na2SO4. Eva-
poration of the filtrate gave 0.43 g (30%) of 8 as a light
yellow solid. Mp 190 ꢁC (dec); 1H NMR (CDCl3): d
7.42–6.48 (m, 9H; m, 6H); 13C NMR (CDCl3): d 158.0,
137.9, 135.1, 133.5, 132.6, 132.1, 130.2, 129.4, 128.9,
2.3. (2E)-3-{4-[(4-Methoxyphenyl)methoxy]phenyl}prop-
2-en-1-ol (5)
With stirring, a solution of alkyne 4 (3.11 g, 11.6 mmol)
in THF (30mL) was added dropwise to a suspension of
lithium aluminum hydride (0.88 g, 23 mmol) in 30 mL
of THF at 0 ꢁC. After 2 h, the temperature was raised to
reflux for 48 h. Ice water was cautiously added, and the
mixture was filtered through a pad of Celite1. The fil-
trate was dried over MgSO4 and evaporated under
reduced pressure. Recrystallization from diethyl ether-
hexanes (1:1, v:v) gave 2.65 g (84%) of light yellow 5.
Mp 141–142 ꢁC; 1H NMR (CDCl3): d 7.33 (m, 4H), 6.92
(d, 4H), 6.55 (d, 1H), 6.25 (m, 1H), 4.99 (s, 2H), 4.30(d,
2H), 3.82 (s, 3H), 1.59 (br s, 1H); 13C NMR (CDCl3): d
159.7, 158.8, 131.2, 129.8, 129.4, 129.1, 127.9, 126.5,
115.2, 114.2, 70.1, 64.2, 55.5. Anal. calcd for C17H18O3:
C 75.53, H 6.71; found: C 75.63, H 6.56.
.
128.5, 128.0, 126.9, 116.3. Anal. calcd for C18H16O 1/
2H2O: C 84.01, H 6.66; found: C 83.76, H 6.34.
2.7. Phosphoric acid dibenzyl ester (1E,3E,5E)-4-(6-phenyl-
hexa-1,3,5-trienyl)phenyl ester (1)
A solution of phenol 8 (0.20 g, 0.80 mmol) in 30 mL of
CH3CN was cooled to ꢀ10 ꢁC in an ice/salt bath. Car-
bon tetrachloride (0.58 mL, 6.1 mmol) was then added.
After 10min, the stirred mixture was treated dropwise
with N,N-diisopropylethylamine (0.46 mL, 2.7 mmol)
via syringe, followed by 4-(dimethylamino)pyridine
(0.0157 g, 0.13 mmol) and dibenzyl phosphite (0.42 mL,
1.9 mmol). The reaction was stirred at ꢀ10 ꢁC for 20h,
then 0.5 M aqueous Na2HPO4 (10mL) was added. The
mixture was warmed to rt and extracted with EtOAc.
The organic extracts were washed with water and brine,
then were dried over MgSO4. Filtration and evapora-
tion of the filtrate gave the crude product, which was
2.4. (2E)-3-{4-[(4-Methoxyphenyl)methoxy]phenyl}prop-
2-en-1-triphenylphosphoniumbromide ( 6)
A solution of allylic alcohol 5 (5.41 g, 20.0 mmol), tri-
phenylphosphine (13.08 g, 49.9 mmol), and N-bromo-
succinimide (4.26 g, 23.9 mmol) in 300 mL of THF was

1078
L. Ma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1075–1078
purified by flash column chromatography on silica gel
using CH2Cl2 as the eluent. A total of 0.17 g (41%) of 1
was obtained as a yellow solid. Mp 99–102 ꢁC; 1H NMR
(CDCl3): d 7.44–6.54 (m, 25H; m, 6H), 7.33 (d, 2H),
5.14 (s, 4H); 13C NMR (CDCl3): d 141.6, 139.3, 138.0,
137.4, 136.8, 135.1, 133.6, 133.0, 132.8, 132.6, 132.5,
132.1, 132.0, 131.5, 131.4, 130.2, 124.2, 124.1, 74.4; UV–
vis (CH2Cl2) lmax (e Mꢀ1 cmꢀ1) 343 (41,000), 359
(55,000), 379 (40,000). Anal. calcd for C32H29O4P: C
75.58, H 5.75; found: C 75.48, H 5.98.
6. Boerner, R. J.; Kassel, D. B.; Barker, S. C.; Ellis, B.;
Delacy, P.; Knight, W. B. Biochemistry 1996, 35, 9519.
7. Cehelnik, E. D.; Cundall, R. B.; Lockwood, J. R.; Palmer,
T. F. J. Phys. Chem. 1975, 79, 1369.
8. For a review of diphenylpolyene photophysics and pho-
tochemistry, see: Saltiel, J.; Sun, Y.-P. in Photochromism,
Molecules and Systems; Durr, H., Bouas-Laurent, H.,
Eds.; Elsevier: Amsterdam, 1990; p 64.
9. Magzoub, M.; Eriksson, L. E. G.; Graslund, A. Biophys.
Chem. 2003, 103, 271.
10. Hofmann, M.; Schumann, C.; Zimmer, G.; Henzel, K.;
Locher, U.; Leuschner, U. Chem. Phys. Lipids 2000, 110,
165.
11. Jones, J. D.; Gierasch, L. M. Biophys. J. 1994, 67, 1534.
12. Lentz, B. R. Chem. Phys. Lipids 1993, 64, 99.
13. Parasassi, T.; De Stasio, G.; Rusch, R. M.; Gratton, E.
Biophys. J. 1991, 59, 466.
14. Mulders, F.; van Langen, H.; van Ginkel, G.; Levine,
Y. K. Biochim. Biophys. Acta 1986, 859, 209.
2.8. Liposome preparation
A stock solution of 1,2-dimyristoyl-sn-glycero-3-phos-
phocholine (DMPC) was prepared in absolute ethanol
such that the concentration was 0.040 M. Dye 1 was
also dissolved in ethanol, to a concentration of
0.0035 M. Aliquots of the lipid solution (50 mL) and dye
solution (5.0 mL) were combined and slowly injected
into 15 mL of stirring phosphate buffer (comprised of
0.026 M KH2PO4 and 0.041 M Na2HPO4 in water). The
resulting mixture was sonicated at 40 ꢁC for 5 min, and
was used immediately. For samples used in fluorescence
measurements, an electronic absorption spectrum was
acquired to ensure that the OD was less than 0.1.
15. Sato, K.; Yoshimura, T.; Shindo, M.; Shishido, K. J. Org.
Chem. 2001, 66, 309.
16. Data for the tert-butyldimethysilyl analogue of 4: 1H
NMR (CDCl3) d 7.33 (d, 2H), 6.79 (d, 2H), 4.48 (s, 2H),
1.79 (br s, 1H), 0.99 (s, 9H), 0.20 (s, 6H). Reduction of the
triple bond in this compound using LiAlH4 in THF was
extremely sluggish at rt. Increasing to reflux temperature
resulted in loss of the TBDMS protecting group.
17. Imperiali, B.; Roy, R. S. J. Org. Chem. 1995, 60, 1891.
18. Nicolaou, K. C.; Veale, C. A.; Webber, S. E.; Kater-
inopoulos, H. J. Am. Chem. Soc. 1985, 107, 7515.
19. Saltiel, J.; Krishnamoorthy, G.; Huang, Z.; Ko, D.-H.;
Wang, S. J. Phys. Chem. A 2003, 107, 3178.
Acknowledgements
This work was supported by the Research Corporation
(Grant #CC5510to W.E.A.) and by the Research and
Creative Activity Committee of ECU. J.C.M. was a
GlaxoSmithKline Undergraduate Research Fellow for
Summer 2002. The authors thank Eric C. Holloway,
Kim V. McCumber, and Kerry L. Partis for synthetic
assistance, and Dr. Nathan L. Brandstater for consult-
ing on the photophysics and membrane behavior of
DPH.
20. Allen, M. T.; Miola, L.; Whitten, D. G. Tetrahedron 1987,
43, 1477.
21. Yadav, J. S.; Chandrasekhar, S.; Sumithra, G.; Kache, R.
Tetrahedron Lett. 1996, 37, 6603.
22. Sajik, H.; Kuno, H.; Hirota, K. Tetrahedron Lett. 1997,
38, 399.
23. Yu, W.; Su, M.; Gao, X.; Yang, Z.; Jin, Z. Tetrahedron
Lett. 2000, 41, 4015.
24. White, J. D.; Amedio, J. C., Jr. J. Org. Chem. 1989, 54, 736.
25. Hodgetts, K. J.; Wallace, T. W. Synth. Commun. 1994, 24,
1151.
26. Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron
Lett. 1996, 37, 771.
References and notes
27. Fluorescence quantum yield determinations were carried
out according to: Fery-Forgues, S.; Lavabre, D. J. Chem.
Educ. 1999, 76, 1260using a single-photon counting
instrument. Samples were prepared in N2-purged spectro-
photometric grade solvents. In all cases, the excitation
wavelength was 360nm, and the emission was integrated
from 370–600 nm. The absorbance at 360 nm was
between 0.04 and 0.06 for each sample. 7-Amino-4-
methylcoumarin (ÈF=0.33, CH3OH) was used as fluo-
rescence standard. Dioxygen has a pronounced effect on
the fluorescence efficiency of 1; emission intensities of
benzene solutions saturated with O2 are 40–45% of those
saturated with N2.
1. Burke, T. R., Jr.; Lee, K. Acc. Chem. Res. 2003, 36, 426.
2. Cole, P. A.; Courtney, A. D.; Shen, K.; Zhang, Z.; Qiao, Y.;
Lu, W.; Williams, D. M. Acc. Chem. Res. 2003, 36, 444.
3. Yan, Z.; Kahn, M.; Qabar, M.; Urban, J.; Kim, H.-O.;
Blaskovich, M. A. Bioorg. Med. Chem. Lett. 2003, 13, 20 83.
4. Shakespeare, W.; Yang, M.; Bohacek, R.; Cerasoli, F.;
Stebbins, K.; Sundaramoorthi, R.; Azimioara, M.; Vu,
C.; Pradeepan, S.; Metcalf, C.; Haraldson, C.; Merry, T.;
Dalgarno, D.; Narula, S.; Hatada, M.; Lu, X.; van
Schravendijk, M. R.; Adams, S.; Violette, S.; Smith, J.;
Guan, W.; Bartlett, C.; Herson, J.; Iuliucci, J.; Weigele,
M.; Sawyer, T. Proc. Natl. Acad. Sci. 2000, 97, 9373.
5. Lynch, B. A.; Loiacono, K. A.; Tiong, C. L.; Adams,
S. E.; MacNeil, I. A. Anal. Biochem. 1997, 247, 77.
28. Gramlich, G.; Zhang, J.; Winterholter, M.; Nau, W. M.
Chem. Phys. Lipids 2001, 113, 1.