Synthesis and photolysis properties of a photolabile linker based on 3′-methoxybenzoin

Full text of DOI:10.1021/jo950822s

1526
J . Org. Chem. 1996, 61, 1526-1529
Sch em e 1
Syn th esis a n d P h otolysis P r op er ties of a
P h otola bile Lin k er Ba sed on
3′-Meth oxyben zoin †
Ronald S. Rock and Sunney I. Chan*
Arthur Amos Noyes Laboratory of Chemical Physics,
127-72 California Institute of Technology,
Pasadena, California 91125
Carboxylic and phosphate esters of appropriately sub-
stituted benzoins cleanly and rapidly photolyze in high
yields when exposed to light between 308 and 366 nm.
A suitable linker may be derived from this compound by
incorporation of a carboxylic acid moiety. Thus, in
compound 4, a carboxy group was added to the 3′-
methoxy substituent, to provide the second functional
group for linkage. The benzoyl ring was left unsubsti-
tuted, as the results of Sheehan et al. showed that such
substitution lowered the overall photolysis yield.12
The synthesis of the carboxylic acid 4 proceeded as
outlined in Scheme 2. The central step was the addition
of 2-phenyl-1,3-dithiane to 1, via the lithium anion, to
form the dithiane 2. The hydroxyl of 3-hydroxybenzal-
dehyde was protected as the TBDMS ether, to circumvent
dianion solubility problems. The phenolic hydroxyl was
conveniently and selectively alkylated by treatment with
TBAF in the presence of methyl bromoacetate, to yield
the methyl ester 3. The carboxylate was then depro-
tected by lithium iodide in refluxing pyridine, providing
the dithiane-protected benzoin linker 4 in an overall yield
of 65%.
Received May 2, 1995 (Revised Manuscript Received
December 6, 1995 )
In recent years the applications of photolabile com-
pounds, or compounds that unmask a functional group
upon absorption of light, have become more diverse.
Since such compounds can provide an effective means of
orthogonal protection, they have been used to facilitate
the synthesis of complex, polyfunctional organic mole-
cules.1-3 These materials also have been used to “cage”
compounds by protection of an essential functional group,
so that a chemical reaction may be initiated by a pulse
of light. In this manner, mixing difficulties can be
circumvented in kinetic measurements.4,5 Applications
of this method include the photolysis of caged ATP for
studies of muscle fiber contraction, where diffusion of
ATP into the muscle fiber is slow;6 of caged fluorescent
probes that only emit light after photolysis;7 and even of
caged enzymes by incorporating photolabile groups on
essential side chains.8
Several photolabile moieties are currently available.
The photolysis properties of compounds such as nitroben-
zyl esters,5 phenacyl esters,9 and benzoin esters10,11 of
carboxylates and phosphates have been well studied. On
the other hand, researchers searching for a photolabile
linker, or a moiety that holds two compounds together
until released by exposing the sample to light (Scheme
1), have fewer options. The photolabile compounds
described above have only a single functional group for
attachment, rendering them unsuitable for linkage. To
provide more options in this area, a photolabile linker
based on 3′-methoxybenzoin has been synthesized, and
its photolysis properties were studied. Applications for
this compound include linkers in solid phase synthesis,
methods of caging by encapsulation, and protein folding
studies.
One difficulty in the synthesis of caged compounds is
premature photolysis. In the described synthesis, this
problem is circumvented by dithiane protection of the
benzoyl carbonyl. Photolysis of the linker is therefore
prevented, until the dithiane “safety-catch” is removed
(data not shown). Since dithianes may be removed under
a variety of mild conditions, many substrates may be
caged and manipulated with no special precautions
against photolysis, until the dithiane is removed just
prior to photolysis. This safety-catch offers a significant
advantage over other currently available cage compounds
and photolabile protecting groups.
In an effort to examine the photolysis properties of the
described benzoin linker, the acetate 6 was synthesized
by aminolysis of the methyl ester 3 to form the amide 5,
followed by acylation with acetic anhydride and a cata-
lytic quantity of DMAP. The dithiane was then hydro-
lyzed by treatment with mercuric perchlorate in aqueous
acetonitrile to yield 7.
Resu lts a n d Discu ssion
The linker described below is based on the 3′-meth-
oxybenzoin protecting group described by Sheehan et al.12
Irradiation of 7 resulted in a clean conversion to the
phenylbenzofurans 8 and 9. Steady-state photolysis spec-
tra of 7 show two isosbestic points throughout the course
of the photolysis (Figure 1). The two isomeric photo-
products 8 and 9 were produced in a 98% yield at a ratio
of 3:1 as determined by GCMS and NMR of the isolated
phenylbenzofurans, along with 1 equiv of acetic acid.
Due to the large absorption change at 310 nm (∆ꢀ )
35000 M-1 cm-1), transient absorption studies of the
formation of 8 and 9 were performed. Quenching studies
by Sheehan et al. showed that photolysis of 3′,5′-
dimethoxybenzoin acetate is extremely rapid, with a rate
* To whom correspondence should be addressed.
† Contribution no. 9088.
(1) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 2nd ed.; J ohn Wiley & Sons: New York, 1991; p 473.
(2) Cameron, J . F.; Fre´chet, J . M. J . J . Am. Chem. Soc. 1991, 113,
4303.
(3) Gravel, D.; Herbert, J .; Thoraval, D. Can. J . Chem. 1983, 61,
400.
(4) Gurney, A. M.; Lester, H. A. Physiol. Rev. 1987, 67, 583.
(5) McCray, J . A.; Trentham, D. R. Annu. Rev. Biophys. Biophys.
Chem. 1989, 18, 239.
(6) Goldman, Y. E.; Hibberd, M. G.; McCray, J . A.; Trentham, D. R.
Nature 1982, 300, 701.
(7) Cummings, R. T.; Krafft, G. A. Tetrahedron Lett. 1988, 29, 65.
(8) Mendel, D.; Ellman, J . A.; Schultz, P. G. J . Am. Chem. Soc. 1991,
113, 2758.
(9) Baldwin, J . E.; McConnaughie, A. W.; Moloney, M. G.; Pratt, A.
J .; Shim, S. B. Tetrahedron 1990, 46, 6879.
(10) Corrie, J . E. T.; Trentham, D. R. J . Chem. Soc. Perkin Trans.
1 1992, 2409.
on the order of 1010 s-1 12
Rapid photolysis seems to have
.
been preserved in the benzoin acetate 7. Photolysis of 7
with a frequency tripled Nd:YAG laser at 355 nm
(12) Sheehan, J . C.; Wilson, R. M.; Oxford, A. W. J . Am. Chem. Soc.
1971, 93, 7222.
(11) Pirrung, M. C.; Shuey, S. W. J . Org. Chem. 1994, 59, 3890.
0022-3263/96/1961-1526$12.00/0 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 4, 1996 1527
Sch em e 2
F igu r e 1. Steady-state photolysis of benzoin acetate 7. A 47.7
µM solution of 7 in 1:1 methanol/Tris‚HCl (0.05 M, pH 7.40)
was irradiated with an Oriel 66011 Hg vapor lamp; irradiation
time in seconds: A, 0; B, 2; C, 5; D, 10; E, 15; F, 20; G, 30; H,
40; I, 90.
Sch em e 3
avoid acidolysis of the linker. This peptide was then
photolyzed to yield three products: the N-terminal pep-
tide, and two C-terminal peptides corresponding to the
two isomeric forms of the phenylbenzofuran photoproduct
(Figure 2).
resulted in a rapid absorption increase at 310 nm. The
course of this increase could not be measured within the
instrument response time of approximately 30 ns, which
places a lower limit on the photolysis rate of 3 × 107 s-1
(data not shown).
Many of the properties of the substituted benzoin
described above combine to make it an ideal photolabile
linker for a variety of applications. Photolysis is ex-
tremely rapid, producing a high yield of isolated photo-
products. The linker may be photolyzed by 308-366 nm
light, which allows photolysis to proceed without compet-
ing absorption by organic moieties that absorb further
to the blue. On the other hand, the linker itself is
sufficiently transparent from 308-366 nm to prevent
inner filter effects from interfering with the course of the
photolysis. The phenylbenzofuran photoproducts are
expected to be inert,11 and their production may be easily
monitored at 310 nm. Finally, the linker 4 is photo-
chemically inert until removal of the dithiane. Com-
bined, these attributes should make 4 a valuable photo-
labile linker.
As a demonstration of a potential application for this
compound, the linker was incorporated within the back-
bone of a decapeptide (Scheme 3). Such a system would
be useful for studies of protein conformation and folding,
in that the amino acid composition of a target peptide
could be drastically altered with a photolysis pulse. In
order to facilitate the peptide synthesis, the benzylic
hydroxyl of the linker 4 was protected as the Fmoc-ester
10. This protected linker may be included in any
synthetic sequence by standard Fmoc solid-phase syn-
thesis protocols.13 After preparation of the decapeptide,
the dithiane was removed with a solution of bis(trifluo-
roacetoxy)iodobenzene prior to TFA cleavage, in order to
(13) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis,
2nd. ed.; Springer-Verlag: New York, 1994.

1528 J . Org. Chem., Vol. 61, No. 4, 1996
Notes
Hz, 1 H), 2.936 (d, J ) 3.76 Hz, 1 H), 2.739-2.610 (m, 4 H),
1.942-1.879 (m, 2 H), 0.935 (s, 9 H), 0.111 (s, 6 H). 13C NMR
(CDCl3, TMS) δ 154.43, 138.89, 137.47, 130.42, 128.00, 127.69,
127.36, 121.23, 119.89, 119.54, 80.74, 66.36, 27.22, 26.93, 25.65,
24.69, 18.03, -4.40. Anal. Calcd C23H32O2S2Si: C, 63.84; H,
7.45. Found: C, 63.83; H, 7.26.
Syn th esis of (()-1-Hyd r oxy-1-[3-((ca r bom eth oxy)m eth -
oxy)p h en yl]-2-p h en yl-2-(1,3-d ith ia n -2-yl)eth a n e (3). A so-
lution of 2 (28.12 g, 65 mmol) and methyl bromoacetate (12.43
g, 81.25 mmol) in 150 mL of dry THF was prepared under a
nitrogen atmosphere. The solution was treated with 1 M TBAF
in THF (68.25 mL, 68.25 mmol) dropwise. The solution was
allowed to react overnight and then was poured into ethyl
acetate (200 mL) and washed with water (5 × 50 mL). The
organic phase was dried with Mg2SO4 and evaporated. The
residue was dissolved in 200 mL of diethyl ether, filtered through
a small quantity of neutral alumina and activated charcoal, and
dried in vacuo. The product was crystallized from ethyl acetate/
hexanes, to afford a white powder. Yield: 23.61 g (93%). Mp
122-122.5 °C. IR: 3471 (br), 1760, 1595, 1441, 1211, 714 cm-1
.
1H NMR (CDCl3, TMS) δ 7.679 (dd, J ) 8.16, 1.55 Hz, 2 H),
7.265-7.325 (m, 3 H), 7.045 (t, J ) 7.92 Hz, 1 H), 6.804-6.782
(m, 1 H), 6.553 (d, J ) 7.61 Hz, 1 H), 6.302 (s, 1 H), 4.960 (d, J
) 3.51 Hz, 1 H), 4.367 (s, 2 H), 3.778 (s, 3 H), 3.023 (d, J ) 3.52
Hz), 2.757-2.620 (m, 4 H), 1.951-1.891 (m, 2 H). 13C NMR
(CDCl3, TMS) δ 169.17, 156.61, 138.88, 137.40, 130.46, 128.08,
127.98, 127.49, 121.76, 115.35, 113.68, 80.73, 66.32, 52.11, 27.30,
26.99, 24.74. Anal. Calcd for C20H22O4S2: C, 61.51; H, 5.68.
Found: C, 61.34; H, 5.75.
F igu r e 2. Steady-state photolysis of peptide 11, as monitored
by reversed-phase (C18) HPLC. The two C-terminal phenyl-
benzofuran-containing peptides elute at 28.5 and 31.0 min,
while the N-terminal peptide elutes in the void volume. Some
photodegradation of the benzofuran is also apparent at reten-
tion times below 30 min. Gradient: 0-35% acetonitrile in
water, 0.1% TFA, detection: 214 nm. Irradiation time in
minutes: A, 0; B, 5; C, 10.
Syn th esis of (()-1-Hyd r oxy-1-[3-(ca r boxym eth oxy)p h en -
yl]-2-p h en yl-2-(1,3-d ith ia n -2-yl)eth a n e (4). A solution of
anhydrous lithium iodide (2.68 g, 20 mmol, Aldrich) in 25 mL
of dry pyridine was brought to reflux under a nitrogen atmo-
sphere and treated with 3 (1.95 g, 5 mmol). The reaction was
refluxed for 6 h and then allowed to cool to room temperature
under a stream of nitrogen. The solution was poured into 1 N
HCl (300 mL) and extracted with ethyl acetate (3 × 50 mL).
The combined ethyl acetate layers were extracted with 5%
sodium bicarbonate (4 × 50 mL). The aqueous phase was
acidified to pH 2 and extracted with ethyl acetate (3 × 50 mL).
The organic phase was dried with Mg2SO4, filtered through
activated charcoal, evaporated, and triturated with hexanes to
yield a white solid. Yield: 1.71 g (91%). Mp 99-101 °C. IR:
Exp er im en ta l Section
Gen er a l. THF was refluxed over sodium and benzophenone
and was distilled prior to use. 3-Hydroxybenzaldehyde (Fluka)
was dissolved in diethyl ether, filtered through a plug of neutral
alumina, and evaporated. The 1.0 M tetrabutylammonium
fluoride (TBAF) solution in THF was dried over 3A molecular
seives. All other starting materials were from Aldrich and used
without further purification. IR spectra were acquired from a
thin film of the sample on a polyethylene substrate. 1H and 13C
NMR spectra were collected at 500 and 125 MHz, respectively.
Syn t h esis of 3-(ter t-Bu t yld im et h ylsilyloxy)b en za ld e-
h yd e (1). To a solution of 3-hydroxybenzaldehyde (12.21 g, 100
mmol) in 600 mL THF was added tert-butyldimethylsilyl chloride
(TBDMSCl, 18.84 g, 125 mmol). The solution was cooled to 0
°C and triethylamine (12.65 g, 17.4 mL, 125 mmol) was added
dropwise. The reaction mixture was brought to room temper-
ature and stirred 5 h. The mixture was filtered and the THF
removed under reduced pressure. The oil was repeatedly
dissolved in 200 mL portions of THF and evaporated, until no
more triethylamine hydrochloride precipitated. The oil was then
dissolved in 150 mL diethyl ether, filtered through a plug of
neutral alumina and activated charcoal to remove the salt and
the yellow color, and evaporated. The colorless, mobile oil was
dried in vacuo overnight. Yield: 21.43 g (91%). IR: 1703, 1583,
3448 (br), 1735, 1595, 1462, 1232, 719 cm-1 1H NMR (CDCl3,
.
TMS) δ 7.656 (dd, J ) 8.00, 1.71 Hz, 2 H), 7.299-7.254 (m, 3
H), 7.036 (t, J ) 7.98 Hz, 1 H), 6.794-6.773 (m, 1 H), 6.573 (d,
J ) 7.55 Hz, 1 H), 6.251 (s, 1 H), 4.955 (s, 1 H), 4.357 (s, 2 H),
2.728-2.620 (m, 4 H), 1.914-1.868 (m, 2 H). 13C NMR (CDCl3,
TMS) δ 173.47, 156.32, 139.01, 137.40, 130.50, 128.12, 128.07,
127.56, 122.02, 115.43, 113.66, 80.56, 66.13, 64.73, 27.26, 26.96,
24.67. Anal. Calcd for C19H20O4S2: C, 60.62; H, 5.35. Found:
C, 60.36; H, 5.21.
Syn t h esis of (()-1-H yd r oxy-1-[3-(ca r b a m ylm et h oxy)-
p h en yl]-2-p h en yl-2-(1,3-d ith ia n -2-yl)eth a n e (5). A solution
of 4 (391 mg, 1 mmol) was prepared in 50 mL of methanol with
gentle warming. The solution was cooled to 0 °C, and gaseous
ammonia was bubbled through for 30 min. The flask was
wrapped in a towel, securely stoppered, and allowed to come to
room temperature. After 2 h, the solvent was removed under
reduced pressure to yield a white solid. Yield: 348 mg (93%).
Mp 140-141°C. IR: 3460, 3346 (br), 1680, 1586, 1442, 1252,
1482, 1278, 1145, 840 cm-1
.
1H NMR (CDCl3, TMS) δ 9.927 (s,
1 H), 7.447 (d, J ) 7.50 Hz, 1 H), 7.379-7.335 (m, 2 H), 7.096-
7.074 (m, 1 H), 0.994 (s, 9 H), 0.215 (s, 6 H). 13C NMR (CDCl3,
TMS) δ 191.60, 156.34, 138.03, 130.03, 126.34, 123.46, 119.70,
25.59, 18.12, -4.52. Anal. Calcd for C13H20O2Si: C, 66.05; H,
8.53. Found: C, 66.13; H, 8.53.
1058, 714 cm-1 1H NMR (CDCl3, TMS) δ 7.686 (dd, J ) 7.82,
.
1.70 Hz, 2 H), 7.336-7.291 (m, 3 H), 7.088 (t, J ) 7.93 Hz, 1 H),
6.774 (dd, J ) 8.09, 2.55 Hz, 1 H), 6.626 (d, J ) 7.67 Hz, 1 H),
6.463 (s, br, 1 H), 6.335 (s, 1 H), 5.582 (s, br, 1 H), 4.971 (d, J )
3.16 Hz, 1 H), 4.245 (s, 2 H), 3.092 (d J ) 3.24 Hz, 1 H), 2.777-
2.635 (m, 4 H), 1.962-1.905 (m, 2 H). 13C NMR (CDCl3, TMS)
δ 170.699, 156.059, 139.293, 137.529, 130.437, 128.245, 127.696,
122.261, 114.772, 114.270, 80.706, 67.077, 66.462, 27.342,
27.002, 24.729. Anal. Calcd for C19H21NO3S2: C, 60.77; H, 5.64;
N, 3.73. Found: C, 60.93; H, 5.79; N, 3.76.
Syn t h esis of (()-1-Acet oxy-1-[3-(ca r b a m ylm et h oxy)-
p h en yl]-2-p h en yl-2-(1,3-d ith ia n -2-yl)eth a n e (6). To a solu-
tion of 5 (192 mg, 0.5 mmol) in 10 mL of THF were added DMAP
(2 mg), triethylamine (70 µL, 0.5 mmol), and acetic anhydride
(94 µL, 1.0 mmol). The solution was stirred at room temperature
for 4 h and then partitioned between ethyl acetate (50 mL) and
5% sodium bicarbonate (50 mL). The organic phase was washed
Syn th esis of (()-1-Hyd r oxy-1-[3-(ter t-bu tyld im eth ylsi-
lyloxy)p h en yl]-2-p h en yl-2-(1,3-d ith ia n -2-yl)eth a n e (2).
A
solution of 2-phenyl-1,3-dithiane (15.71 g, 80 mmol) in 125 mL
of THF was prepared. The solution was treated at 0 °C under
a nitrogen atmosphere with 40 mL of n-butyllithium (2.0 M in
cyclohexane, 80 mmol). After 30 min, 1 (18.91 g, 80 mmol) was
added. The solution was stirred for 1 h at 0 °C and then poured
into 100 mL of 1 N HCl and extracted with methylene chloride
(4 × 50 mL). The organic phase was washed with brine, dried
with Mg2SO4, filtered through a plug of activated charcoal and
silica gel, and evaporated under reduced pressure. The resulting
oil was crystallized from ethanol/water to form a white powder.
Yield: 28.98 g (84%). Mp 75-76 °C. IR: 3449 (br), 1601, 1484,
1275, 1152, 834 cm-1
7.50 Hz, 2 H), 7.308-7.235 (m, 3 H), 6.937 (t, J ) 7.79 Hz, 1 H),
6.682-6.660 (m, 1 H), 6.427-6.404 (m, 2 H), 4.926 (d, J ) 3.73
.
1H NMR (CDCl3, TMS) δ 7.70 (d, J )

Notes
J . Org. Chem., Vol. 61, No. 4, 1996 1529
with water (3 × 50 mL), dried with Mg2SO4, and evaporated to
Samples were acquired at a 10 Hz photolysis pulse repetition
rate, and scans represented the average of 20 pulses.
yield a colorless oil. Yield 205 mg (98%). IR: 3479, 3331 (br),
1747, 1694, 1589, 1443, 1224, 1033, 910, 718 cm-1
.
1H NMR
Syn th esis of (()-1-(F lu or en ylm eth oxyca r bon yloxy)-1-[3-
(ca r b oxym et h oxy)p h en yl]-2-p h en yl-2-(1,3-d it h ia n -2-yl)-
eth a n e (10). A solution of 4 (1.57 g, 4.17 mmol) in 50 mL of
THF was prepared under a nitrogen atmosphere. The solution
was cooled to -78 °C, and n-butyllithium (2.0 M in cyclohexane,
Aldrich) was added until the dianion precipitated and a persis-
tent yellow color remained (approximately 4 mL, 8 mmol). The
suspension was treated with 9-fluorenylmethyl succinimidyl
carbonate (Fmoc-OSu, 3.18 g, 9.43 mmol, Bachem), and the cold
bath was removed. After 1 h, the suspension was poured into 1
N HCl and extracted with ethyl acetate. The organic phase was
dried and evaporated under reduced pressure. The resulting
oil was purified by reversed-phase HPLC (C18, 70% to 90%
acetonitrile in water, 0.1% TFA, 40 min). Yield: 1.06 g (42%).
(CDCl3, TMS) δ 7.740 (dd, J ) 8.15, 1.55 Hz, 2 H), 7.348-7.281
(m, 3 H), 7.111 (t, J )7.96 Hz, 1 H), 6.801 (dd, J ) 8.18, 2.53
Hz, 1 H), 6.686 (d, J ) 7.59 Hz, 1 H), 6.521 (s, 2 H), 6.312 (s, 1
H), 6.136 (s, 1 H), 4.238 (s, 2 H), 2.751-2.597 (m, 4 H), 2.104 (s,
3 H), 1.933-1.857 (s, 1 H). 13C NMR (CDCl3, TMS) δ 171.080,
169.318, 156.012, 136.974, 130.771, 128.402, 128.045, 127.738,
122.586, 115.072, 114.635, 79.890, 66.982, 63.133, 27.270,
27.109, 24.568, 20.848. Anal. Calcd for C21H23NO4S2: C, 60.41;
H, 5.55; N, 3.35. Found: C, 59.92; H, 5.76; N, 3.14.
Syn th esis of (()-O-Acetyl-3′-(ca r ba m ylm eth oxy)ben zoin
(7). To a solution of 6 (110 mg, 0.26 mmol) in 5 mL 9:1 (v/v)
acetonitrile/water was added mercuric perchlorate (148 mg, 0.33
mmol). The solution was stirred for 15 min, filtered through a
0.45 µm PTFE syringe filter (Gelman) into a 5% sodium
bicarbonate solution (10 mL), and extracted with 50 mL of
methylene chloride. The organic phase was dried and evapo-
rated under reduced pressure to yield a colorless oil. Samples
for analysis were evaporated from methanol, dissolved in warm
water, and lyophilized. Yield: 65 mg (76%). IR: 3445, 1743,
IR: 1747, 1597, 1462, 1256, 730 cm-1
.
1H NMR (CDCl3, TMS)
δ 7.778 (d, J ) 6.85 Hz, 2 H), 7.739 (dd, J ) 7.40, 2.47 Hz, 2 H),
7.590 (dd, J ) 12.18, 7.47 Hz, 2 H), 7.404-7.237 (m, 7 H), 7.115
(t, J ) 7.94 Hz, 1 H), 6.842 (dd, J ) 8.24, 2.38 Hz, 1 H), 6.710
(d, J ) 7.64 Hz, 1 H), 6.346 (s, 1 H), 6.024 (s, 1 H), 4.430-4.393
(m, 3 H), 4.298-4.256 (m, 2 H), 2.778-2.603 (m, 4 H), 1.984-
1.856 (m, 2 H). 13C NMR (CDCl3, TMS) δ 173.41, 156.42, 154.02,
143.32, 143.25, 141.25, 141.24, 136.79, 136.17, 130.78, 128.44,
128.13, 127.13, 125.26, 122.51, 119.98, 115.92, 114.17, 83.94,
70.19, 64.77, 62.93, 46.66, 27.26, 27.18, 24.48. Anal. Calcd for
1694, 1462, 1236, 1075, 720 cm-1
.
1H NMR (CDCl3, TMS) δ
7.936 (d, J ) 7.82 Hz, 2 H), 7.529 (t, J ) 7.56 Hz, 1 H), 7.412 (t,
J ) 7.57 Hz, 2 H), 7.319 (t, J ) 7.86 Hz, 1 H), 7.139 (d, J ) 7.51
Hz, 1 H), 7.051 (s, 1 H), 6.884 (dd, J ) 8.21, 2.75 Hz, 1 H) 6.835
(s, 1 H), 6.558 (s, 1 H), 6.148 (s, 1 H), 4.462 (s, 2 H), 2.207 (s, 3
H). 13C NMR (CDCl3, TMS) δ 193.577, 170.825, 170.348,
157.593, 135.473, 134.494, 133.637, 130.516, 128.768, 128.711,
122.371, 115.205, 115.140, 77.144, 67.115, 20.715. Anal. Calcd
for C18H17NO5‚H2O: C, 62.59; H, 5.54; N, 4.05. Found: C, 62.53;
H, 5.12; N, 3.90.
C
34H30O6S2‚H2O: C, 66.21; H, 5.23. Found C, 66.44; H, 5.44.
Syn th esis of P h otola bile P ep tid e 11. The sequence Ac-
Val-Gly-Glu-Arg-Gly-linker-Gly-Arg-Nle-Lys-Glu-NH2 was pre-
pared on an Fmoc-amide resin (Applied Biosystems, Inc.) on a
0.1 mmol scale. Couplings were performed with 1 mmol each
of DCC, HOBt, and Fmoc-AA or 10 in NMP for 1 h, with the
exception of Gly-5 which was coupled to the benzylic hydroxyl
of the linker with 1 mmol DCC and 0.01 mmol DMAP for 24 h.
The Fmoc groups were removed by 3 × 3 min treatments with
30% piperidine in NMP. Couplings were monitored by the
absorbtion of the dibenzofulvene-piperidine adduct or the quali-
tative Kaiser test.14 The N-terminal valine was capped with 1
mmol of acetic anhydride and 0.01 mmol of DMAP, and the
dithiane was removed by treatment with 0.5 mmol of bis-
(trifluoroacetoxy)iodobenzene in 9:1 acetonitrile:water for 10
min. The resin was dried and then cleaved with 1.8 mL of TFA,
0.1 mL of water, and 100 mg of phenol for 90 min. The crude
peptide was isolated by precipitation in methyl butyl ether and
purified by reversed-phase HPLC (C18, water/acetonitrile gradi-
ent containing 0.1% TFA).
Stea d y-Sta te P h otolysis of 7. A 47.7 µM solution of 7 in
1:1 methanol/Tris‚HCl (0.05 M, pH 7.40) was prepared in a 1
cm pathlength quartz cuvette. The sample was irradiated by
an Oriel 66011 Hg vapor lamp operating at 450 W and filtered
with a water cooled Schott glass UG11 filter. At intervals, the
sample was removed, and the UV absorption spectrum from
210-400 nm was taken by a HP 8452 spectrophotometer.
Complete photolysis occurred within a 90 s exposure.
For isolation of the photoproduct, a 25.6 mg sample of 7 in
50 mL of methanol was irradiated in 3 mL batches as above,
until no further change was observed in the absorption spectrum
of the sample. The methanol was removed under reduced
pressure to yield 20.6 mg (99%) of the photoproduct. The
composition of this material was 74% 2-phenyl-5-(carbamyl-
methoxy)benzofuran (8), 24% 2-phenyl-7-(carbamylmethoxy)-
benzofuran (9), and 2% other material, as determined by GCMS.
Standard samples were obtained by preparative-TLC (silica gel/
diethyl ether) of the crude photolyzed sample and identified by
1H NMR and IR.
Stea d y-Sta te P h otolysis of P ep tid e 11. The purified
peptide HPLC fractions were diluted 10:1 with 25 mM sodium
acetate buffer, pH 5.5, and irradiated as for the photolysis of 7.
Aliquots were periodically analyzed by reversed-phase HPLC.
Maximum photolysis occurred after 10 min.
Tr a n sien t P h otolysis of 7. A 9.54 µM solution of 7 in 1:1
methanol/Tris‚HCl (0.05 M, pH 7.40) was prepared in a 1 cm
pathlength quartz cuvette. The sample was photolyzed using
the third harmonic at 355 nm from a Q-switched Spectra Physics
DCR-12 Nd:YAG laser. Typical pulses were 10-20 ns (FWHM)
in duration at an energy of 1.5 mJ /pulse. The sample was
monitored with a 75 W xenon arc lamp filtered with a Schott
glass UG11 filter placed between the arc lamp and the cuvette.
The probe light exiting the cuvette was then wavelength-selected
by a SA 1690B double monochromator set at 310 nm and was
detected with a photomultiplier. The signal was amplified with
a Keithly 427 current amplifier and digitized by a Tektronix
R710 200 MHz transient digitizer interfaced to a microcomputer.
Ack n ow led gm en t. The authors would like to thank
Michael H. B. Stowell for synthetic advice, and Theodore
J . DiMagno for assistance with the transient absorption
studies. This work was supported in part by NIH grant
GM 22432 from the National Institute of General
Medical Sciences, U. S. Public Health Service.
J O950822S
(14) Fields, G. B.; Noble, R. L. Int. J . Pept. Protein Res. 1990, 35,
161.