An approach to photolabile, fluorescent protecting groups

Article abstract of DOI:10.1021/jo9702608

The photolablie and fluorescent system 1 was prepared via a convergent route which involved thymidine 3'-functionalized with a proline residue. Photodecomposition of 1 was examined using 360 nm irradiation. Without any additives, 5'-silylated thymidine was not formed, but the N(α)-deprotected cyclization precursor II accumulated instead. However, in the presence of ethanolamine, 5'-silylated thymidine formed at a rate which increases with the concentration of ethanolamine.

Full text of DOI:10.1021/jo9702608

J . Org. Chem. 1997, 62, 5165-5168
5165
An Ap p r oa ch to P h otola bile, F lu or escen t P r otectin g Gr ou p s
Kevin Burgess,* Swanee E. J acutin, Dongyeol Lim, and Aroonsiri Shitangkoon
Department of Chemistry, Texas A & M University, College Station, Texas 77843-3255
Received February 12, 1997X
The photolabile and fluorescent system 1 was prepared via a convergent route which involved
thymidine 3′-functionalized with a proline residue. Photodecomposition of 1 was examined using
360 nm irradiation. Without any additives, 5′-silylated thymidine was not formed, but the NR-
deprotected cyclization precursor II accumulated instead. However, in the presence of ethanolamine,
5′-silylated thymidine formed at a rate which increases with the concentration of ethanolamine.
For several years we1 and others2-6 have been working
Sch em e 1. P h otoch em ica l Activa tion of a
F lu or escen tly-Ta gged Sa fety Ca tch Mech a n ism
on closely related chemical methods for sequencing DNA.
Central to our approach are nucleoside triphosphates I.
These are identical to the natural deoxynucleosides,
except that they bear chemically labile 3′-protecting
groups that also fluoresce at distinctive wavelengths to
spectroscopically encode for the aromatic heterocyclic
component of the nucleoside. Ultimately, we hope to
establish that solid-supported DNA can be sequenced by
a series of addition, detection, and deprotection steps.
Specifically, the primer-template complex would be sub-
jected to (i) biocatalytic addition of the appropriate
nucleoside triphosphate analog; (ii) fluorescence detection
to determine which base added to the primer (hence the
complement in the template strand); and (iii) 3′-depro-
tection. Each cycle of these three steps would extend the
primer by one base. Increased throughput of sequence
information would follow if 100-300 bases could be
sequenced because many DNA fragments could be pro-
cessed in parallel on a single chip. The time and labor
intensive aspects of gel electrophoresis in sequencing
thereby would be circumvented.
activation are shown in Scheme 1. Irradiation at ap-
proximately 360 nm was anticipated to remove the NVOC
(3,4-dimethoxy-6-nitrobenzyloxycarbonyl) protecting group
giving intermediate II which would tend to ring close to
give the diketopiperazine 2 and the free nucleoside 3. In
this way the 3′-terminus of the nucleoside would be
unmasked and the fluorescent groups would be cleaved
in a mild photochemical process that would not signifi-
cantly perturb DNA strands.
Many hurdles must be surmounted to realize the
sequencing method outlined above. First amongst these
are construction of nucleoside triphosphates I with labile
and fluorescent 3′-protecting groups. This paper de-
scribes a synthesis of compound 1 which has a fluorescent
tag supported on a safety catch linker that can be
rendered labile via a photochemical trigger. The antici-
pated sequence of events that would follow photochemical
Our synthesis of the target molecule 1 consisted of two
branches that converged in the final step. The first
branch began with a series of routine manipulations of
lysine protected at the R- and ω-termini by tert-butyloxy-
carbonyl and benzyl groups, respectively {i.e. BOC-Lys-
(Bn)}. Hydrogenolysis to remove the benzyl group and
dansylation gave BOC-Lys(dansyl) in 91% overall yield.
Removal of the BOC protecting group using TFA and
X Abstract published in Advance ACS Abstracts, J uly 1, 1997.
(1) Metzker, M. L.; Raghavachari, R.; Richards, S.; J acutin, S. E.;
Civitello, A.; Burgess, K.; Gibbs, R. A. Nucl. Acids Res. 1994, 22, 4259-
67.
(2) Cheeseman, P. C. U.S. Patent US 5,302,509, 1994.
(3) Wilhelm, A. German Patent DE 41 41 178 A1, 1993.
(4) Rosenthal, A.; Close, K.; Brenner, S. WO 93/21340, 1993.
(5) Tsien, R. Y.; Ross, P.; Fahnestock, M.; J ohnston, A. J . WO 91/
06678, 1991.
(6) Canard, B.; Sarfati, R. S. Gene 1994, 148, 1-6.
S0022-3263(97)00260-0 CCC: $14.00 © 1997 American Chemical Society

5166 J . Org. Chem., Vol. 62, No. 15, 1997
Burgess et al.
replacement of this with an NVOC group7,8 (Na2CO3,
dioxane) gave NVOC-Lys(dansyl) 4 in 54% yield. The
second branch of the synthesis involved coupling 5′-(tert-
butyldiphenylsilyl)thymidine (5′-TBDPS-T) with N-(R,R-
dimethyl-3,5-dimethoxybenzyloxycarbonyl)-L-proline (DDZ-
Pro) and then deprotection (5% TFA in CH2Cl2) to give
compound 5 in 50% yield. It was necessary to use the
relatively acid labile DDZ group9 so that the proline
nitrogen could be deprotected cleanly; BOC protection
and TFA cleavage gave some desilylation of the nucleo-
side fragment. The synthesis culminated with the cou-
pling of fragments 4 and 5 (EDCI, HOBt, NMM, CH2Cl2,
89%) to give the target molecule 1.
Photolysis of compound 1 was performed using a
relatively inexpensive strip light that generates irradia-
tion in the 360 nm region; these very useful lamps are
available from Southern New England Ultraviolet Com-
pany. Progress of the reaction in 7:3 acetonitrile:water
was monitored by analytical HPLC. Using our weak
light source, the half life for decomposition of compound
1 was determined to be approximately 30 min. Analysis
of the products revealed that neither 5′-TBDPS-T or the
diketopiperazine 2 was formed even after 5 h irradiation.
In fact, the only new peak on the HPLC trace was an
“intermediate” with a retention time and UV spectrum
very similar to 5′-TBDPS-T. Formation of 5′-TBDPS-T3
and the diketopiperazine 2 were observed (as proven by
HPLC analyses of authentic samples), however, when the
irradiation was performed with ethanolamine as an
additive. Figure 1 shows HPLC traces reflecting the
composition of the mixture immediately after 30 min
irradiation times in the presence of 0 to 100 equiv of
ethanolamine. As the amount of ethanolamine in the
solution increases, so does the amount of 5′-TBDPS-T and
diketopiperazine 2. When 100 equiv of ethanolamine
were used, no significant amount of the intermediate was
observed. In other experiments we observed that decay
of the intermediate and concomitant increase of 5′-
TBDPS-T and diketopiperazine 2 occurs in the absence
of light, i.e. the transformation is a thermal one. Conver-
sion of the intermediate to 5′-TBDPS-T was complete
after 4 h in the presence of 10 equiv of ethanolamine or
more.
F igu r e 1. HPLC traces showing the photodecomposition of 1
in the presence of different amounts of ethanolamine. All
samples were taken at an irradiation time corresponding
approximately t1/2 for the photodecomposition (30 min).
Ta ble 1. P h otod ecom p osition of 1 in to 5′-TBDP S-T a s a
F u n ction of Ad d ed Eth a n ola m in e
ethanolamine
(equiv relative to 1)
photodecomposition conversion of 1 into
of 1 (%)a
5′-TBDPS-T (%)a,b
0
4
10
25
50
100
47
54
52
48
48
52
c
d
55
58
60
57
a
After 30 min irradiation and 4 h equilibration time (to allow
the intermediate to decompose into 5′-TBDPS-T); throughout the
table, the data quoted is for the average of two experiments
measured with 5-nitroindole as an internal standard (shown to
b
be inert under these conditions). Conversion is based upon
percentage of photodecomposed 1 that is transformed into TBDPS-
d
T. c 5′-TBDPS-T was not formed in this experiment. Conversion
of the intermediate formed after photolysis into 5′-TBDPS-T was
incomplete after 4 h.
formation increases with the concentration of that base
(Figure 1). In other work, we have observed that hydra-
zine also promotes conversion of the same intermediate
into 5′-TBDPS-T, though not so effectively.
Table 1 relates the number of equivalents of ethanol-
amine used to the percentage of the starting material 1
that was consumed after 30 min irradiation and 4 h
equilibration and the percentage conversion of that
material into 5′-TBDPS-T. Several conclusions can be
drawn from these data. First, the extent of photodecom-
position of 1 was 50 ( 4% throughout and does not
depend on the amount of ethanolamine used. Similarly,
the percent conversion of 1 into 5′-TBDPS-T after ther-
mal equilibration is 55-60%, irrespective of the ethanol-
amine concentration. However, 5′-TBDPS-T is not formed
in the absence of ethanolamine, and the rate of its
Photolysis of compound 1 gave the free amine inter-
mediate II as demonstrated by HPLC and MALDI-MS
analyses. Intermediate II was shown to be stable for at
least three days in MeCN(aq) at ambient temperature.
Addition of ethanolamine to this intermediate, however,
rapidly generated the diketopiperazine 2 and 5′-TBDPS-T
3. These experiments, coupled with those features in
Figure 1 and Table 1, indicate that addition of ethanol-
amine is necessary for the formation of the products 2
and 3 and that it does not matter if the amine is added
before or after photolysis.
(7) Patchornik, A.; Amit, B.; Woodward, R. B. J . Am. Chem. Soc.
1970, 92, 6333-5.
(8) Amit, B.; Zehavi, U.; Patchornik, A. J . Org. Chem. 1974, 39, 192-
At this stage we hypothesized that ethanolamine
promotes the cyclization of the intermediate II and
simultaneously reacts with the nitrosoaldehyde byprod-
6.
(9) Birr, C.; Lochinger, W.; Stahnke, G.; Lang, P. Liebigs Ann. Chem.
1972, 763, 162-72.

Photolabile, Fluorescent Protecting Groups
J . Org. Chem., Vol. 62, No. 15, 1997 5167
200 MHz, 13C at 50 MHz) spectrometer. 1H and 13C chemical
shifts are reported in δ (ppm). Multiplicities in 1H NMR are
reported as (br) broad, (s) singlet, (d) doublet, (t) triplet, and
(m) multiplet. Thin layer chromatography was performed
using silica gel 60 F254 plates from Whatman. Flash chroma-
tography was performed using SP Silica Gel 60 (230-600 mesh
ASTM). CH2Cl2 was distilled from LiAlH4. Other chemicals
were purchased from commercial suppliers and used as
received unless otherwise specified.
Nr-(ter t-Bu toxycar bon yl)-NE-(5-(dim eth ylam in o)-1-n aph -
th a len esu lfon yl)-L-lysin e. Boc-Lys-OH (4 g, 14 mmol) was
dissolved in 50 mL of CH2Cl2. Et3N (6 mL, 41.1 mmol, 3 equiv)
was added at 25 °C, followed by dansyl chloride (4.4 g, 16
mmol, 1.2 equiv). After 12 h stirring under N2, the reaction
mixture was concentrated under vacuum, and the residue was
purified by flash column chromatography (eluant 1:10 MeOH/
uct to form the oxazolidine 6. To probe for the final
byproduct from the photodecomposition of 1 (which we
presume is the oxazolidine 6), N-(3,4-dimethoxy-6-ni-
trobenzyloxycarbonyl)ethanolamine 7 was synthesized
and then photolyzed in the presence of ethanolamine.
This solution was analyzed by HPLC and was also
coinjected with a sample from the photolysis of compound
1. In this way it was shown that the diketopiperazine 2
and the putative oxazolidine 6 coeluted under the HPLC
conditions that were used throughout this study.
UV spectra of the diketopiperazine 2 and the putative
oxazolidine 6 give additional information. The two
compounds have different spectra; that of the diketopip-
erazine 2 shows a λmax at 220 nm, while oxazolidine 6
has an absorbance minimum at 220 nm and a λmax at 264
nm. The HPLC peak detected in the photolysis of
compound 1 has a UV spectrum similar to that of pure
diketopiperazine 2. However, we suspect that this peak
is really derived from coelution of 2 and 6, but oxazolidine
6 has a much lower molar absorptivity (ꢀ). Unfortu-
nately, an attempt to isolate and characterize the oxazo-
lidine was unsuccessful, so we were unable to measure
the relative molar absorptivities of 2 and 6.
Safety-catch deprotection via formation of diketopip-
erazines is not novel to this study since Bray and
co-workers have used a similar scheme to generate acid
labile linkers for syntheses of peptides on pins.10,11 This
work is the first, however, to demonstrate proof of concept
for photochemical triggering of a safety catch bearing a
fluorescent flag. With respect to our proposed sequencing
procedure, the dansylated system is a prototype with
convenient physical properties for the chemical synthesis.
However, the fluorescence of the dansyl group is rela-
tively weak, so this is a suboptimal choice with respect
to detection sensitivity. Moreover, compound 1 does not
afford 5′-TBDPS-T with high conversion (55-65%).
Though it is possible that diminished yields in the
conversion of the protected nucleoside into the 3′-unpro-
tectected was due to inefficient conversion to the inter-
mediate II, we regard photoreactions of the dansyl group
prior to the desired sequence of events to be a more likely
explanation. The reason for this conclusion is that dansyl
groups are known to decompose to highly reactive species
under these conditions,12 and because we were unable
to observe any stable byproducts other than those already
discussed. Our thoughts are now focused upon refine-
ments of 1 wherein the dansyl functionality is replaced
by other fluorescent groups with higher extinction coef-
ficients and for which more efficient photodeprotection
can be achieved.
CHCl3, Rf ) 0.28) to give 6.6 g (91%) of the product Boc-Lys-
(dansyl)-OH. 1H NMR (300 MHz, CD3OD) δ 8.53 (d, J ) 8.7
Hz, 1H), 8.34 (d, J ) 8.7 Hz, 1H), 8.17 (d, J ) 7.5 Hz, 1H),
7.65-7.5 (m, 2H), 7.25 (d, J ) 7.5 Hz, 1H), 4.01 (br, 1H), 3.95-
3.75 (m, 1H), 3.25-3.08 (m, 2H), 2.86 (s, 6H), 2.9-2.78 (m,
2H), 1.78 (br, 1H), 1.69-1.15 (m, 5H), 1.41 (s, 9H). 13C NMR
(75 MHz, CD3OD) δ 176.1, 157.9, 152.9, 136.9, 131, 130.8,
130.1, 129, 124.3, 120.5, 116.4, 79.4, 54.6, 45.8, 43.5, 32.1, 30,
28.7, 23.8. IR (KBr) 3324, 2977, 2867, 1698, 1575, 1506, 1367,
1317, 1162, 1143 cm-1. FAB-MS m/ z 479 [M]+.
Nr-(3,4-Dim et h oxy-6-n it r ob en zyloxyca r b on yl)-NE-(5-
(dim eth ylam in o)-1-n aph th alen esu lfon yl)-L-lysin e (4). Boc-
Lys(dansyl)-OH (0.1 g, 0.19 mmol) was dissolved in 0.5 mL of
CH2Cl2, and 2 mL of 50% TFA/CH2Cl2 was added at 25 °C.
After 2 h of stirring, the reaction mixture was concentrated
under vacuum. Repeated evaporation with MeOH/CH2Cl2
gave 0.14 g of the crude NR-deprotected amino acid. This was
used in the following step without further purification.
The amino acid formed as described above (0.14 g, 0.4 mmol)
was dissolved in 7 mL of H2O and NaHCO3 (0.09 g, 1.1 mmol,
3 equiv) was added. NVOCCl (3,4-dimethoxy-6-nitrobenzyl-
oxycarbonyl chloride)8 (0.2 g, 0.7 mmol, 2 equiv) in dioxane
(10 mL) was added at 25 °C under N2, and the solution was
stirred for 12 h. The solution was concentrated under high
vacuum. The residue was dissolved in EtOAc and then
acidified with 10 mL of 0.5 N HCl, and the organic layer was
washed with water then brine and dried over Na2SO4. After
evaporation of solvent, the residue was purified via flash
chromatography (eluant 1:10 MeOH/CHCl3, Rf ) 0.14) to give
a 0.12 g of the product 4 (52% yield). 1H NMR (300 MHz, CD3-
OD) δ 8.53 (d, J ) 8.7 Ηz, 1H), 8.33 (d, J ) 8.7 Ηz, 1H), 8.17
(d, J ) 7.2 Hz, 1H), 7.73 (s, 1H), 7.6-7.5 (m, 2H), 7.25 (d, J )
8.1 Hz, 1H), 7.17 (s, 1H), 5.47 (d, J ) 15.6 Hz, 1H), 5.40 (d, J
) 15.6 Hz, 1H), 3.98-3.83 (m, 2H), 3.92 (s, 3H), 3.89 (s, 3H),
Exp er im en ta l Section
Gen er a l P r oced u r es. High field NMR spectra were
recorded on a Varian XL-400 (1H at 400 MHz), UnityPlus 300
(1H at 300 MHz, 13C at 75.4 MHz), or Varian XLAA 200 (1H at
(10) Bray, A. M.; Maeji, N. J .; Valerio, R. M.; Campbell, R. A.;
Geysen, H. M. J . Org. Chem. 1991, 56, 6659-66.
(11) Bray, A. M.; Lagniton, L. M.; Valerio, R. M.; Maeji, N. J .
Tetrahedron Lett. 1994, 35, 9079-82.
(12) Pillai, V. N. R. In Organic Photochemistry; Padwa, A., Ed.;
Marcel Dekker, Inc.: New York, 1987; Vol. 9, pp 225-323.

5168 J . Org. Chem., Vol. 62, No. 15, 1997
Burgess et al.
3.69-3.52 (m, 1H), 2.86 (s, 6H), 2.9-2.75 (m, 2H), 1.75-1.57
(br, 1H), 1.58-1.15 (m, 5H). 13C NMR (50 MHz, CDCl3 ) δ
175.3, 155.9, 153.6, 151.4, 147.8, 139.1, 134.6, 130.2, 129.5,
129.4, 129.2, 128.1, 123.2, 123.1, 118.8, 115.2, 109.7, 107.9,
77.2, 63.8, 56.4, 56.2, 53.9, 45.3, 42.6, 28.7, 22. IR (KBr) 3330,
2941, 1711, 1582, 1519, 1463, 1439, 1328, 1278, 1220, 1161,
1142, 1069 cm-1. MS m/ z 619 [M + H]+. HRMS (FAB, NaI):
calcd for [M + Na]+ 641.1893; found 641.1924.
3′-O-{N-(r,r-Dim eth yl-3,5-d im eth oxyben zyloxyca r bon -
yl)-L-p r olin e}-5′-O-(ter t-b u t yld ip h en ylsilyl)t h ym id in e.
DDZ-Pro-OH (0.5 g, 1.5 mmol) and 5′-TBDPS protected thy-
midine (0.78 g, 1.6 mmol, 1.1 equiv) were dissolved in 10 mL
of CH2Cl2, and then N-hydroxybenzotriazole (0.4 g, 3 mmol,
2.0 equiv) and N-methylmorpholine (0.81 mL, 5 equiv) were
added. The solution was stirred for 5 min under N2 at 0 °C,
and then dicyclohexylcarbodiimide (0.61 g, 2.9 mmol, 2.0 equiv)
was added and reaction mixture was stirred 12 h at 25 °C. A
colorless precipitate formed (presumably a urea derivative) and
was removed by filtration. The solvent was evaporated, the
residue was dissolved in 10 mL of EtOAc and acidified with
0.5 N HCl, and the organic layer was washed with water then
brine and dried over Na2SO4. After evaporation of the solvent,
L-pr olin e}-5′-O-(ter t-bu tyldiph en ylsilyl)th ym idin e (1). The
acid 4 (0.16 g, 0.27 mmol, 1.05 equiv), amine 5 (0.15 g, 0.25
mmol), and N-hydroxybenzotriazole (0.07 g, 0.5 mmol, 2 equiv)
were dissolved in 2 mL of CH2Cl2, and N-methylmorpholine
(0.08 mL, 0.75 mmol, 3 equiv) was added. The solution was
stirred at 0 °C under N2 for 5 min, and then 1-(3-(dimethyl-
amino)propyl)-3-ethylcarbodiimide hydrochloride (0.01 g, 0.5
mmol, 2 equiv) was added. Reaction mixture was stirred for
12 h at 25 °C. The solvent was evaporated, and the residue
was dissolved in 10 mL of EtOAc, washed with water then
brine, and dried over Na2SO4. The solvent was removed, and
the residue was purified via flash chromatography (1:10
MeOH/CHCl3, Rf ) 0.61) to give a 0.26 g (89%) of the product
1. 1H NMR (400 MHz, CDCl3) δ 8.63 (br, 1H), 8.51 (d, J ) 8.4
Hz, 1H), 8.33 (d, J ) 8.8 Hz, 1H), 8.18 (d, J ) 7.2 Hz, 1H),
7.68 (s, 1H), 7.67-7.58 (m, 4H), 7.54-7.32 (m, 9H), 7.21 (d, J
) 6.4 Hz, 1H), 6.99(s, 1H), 6.38-6.32 (m, 1H), 5.86-5.77 (m,
2H), 5.57-5.4 (m, 3H), 4.5-4.4 (m, 1H), 4.08-4.0 (br, 1H), 4.0-
3.95 (br, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.95-3.82 (m, 1H),
3.8-3.64 (m, 2H), 2.92 (s, 6H), 2.95-2.77 (m, 2H), 2.35-1.9
(m, 6H), 1.82 (br, 1H), 1.7-1.32 (m, 5H), 1.46 (s, 3H), 1.06 (s,
9H). IR (KBr) 3072, 2933, 2854,1737, 1673, 1519, 1438, 1203,
1178, 1137 cm-1. MS m/ z 1178 [M + H]+. HRMS (MALDI/
TOF): calcd for [M + Na]+ 1200.4395; found 1200.4403.
Gen er a l P r oced u r e for th e P h otolysis Exp er im en ts.
Throughout the study, the solvent used was a 7:3 acetonitrile:
water mixture. 5-Nitroindole was used as an internal stan-
dard prior to photolysis; other experiments showed 5-nitroin-
dole was inert under the reaction conditions. The sample was
placed in an Eppendorf tube (Brinkmann Instruments, Inc.,
Westbury, NY) and irradiated at ca. 1 cm above the UV-
fluorescent tube. After photolysis, each sample was analyzed
by HPLC immediately and analyzed again after 4 h equilibra-
tion time.
Gen er al P r ocedu r e for th e HP LC An alyses. All samples
were analyzed on an analytical HPLC (SSI, State College, PA)
equipped with a Reliasil C18 column (25 cm × 4.6 mm, 5 µm
particle size) (Column Engineering, Inc., Ontario, CA); a diode-
array UV detector was used, although most of the measure-
ments were made at 220 nm. The separations were performed
using a linear gradient (mobile phase A: 5% B in 0.1% TFA/
water; mobile phase B: 0.1% TFA/acetonitrile; 50 to 100% B
in 10 min, then at 100% B for 6 min) with a flow rate of 1
mL/min.
the residue was purified by flash chromatography (1:10 MeOH/
CHCl3, Rf ) 0.51) to give a 0.6 g (50%) of the product, 3′-O-
(DDZ-Pro)-5′-O-TBDPS-T as a mixture of two rotamers {1H
and 13C NMR spectra show two sets of peaks in CDCl3 (1:1)
and in CD3OD (3:1)}. 1H NMR (300 MHz, CD3OD) δ 7.82 (s,
1H), 7.72-7.62 (m, 4H), 7.52 (s, 1H), 7.48-7.33 (m, 6H), 6.49
(s, 1H), 6.48 (s, 1H), 6.36-6.22 (m, 2H), 5.59-5.42 (m, 1H),
4.5-4.42 (m, 1H), 4.12-4.04 (m, 1H), 4.02-3.88 (m, 2H), 3.71
(s, 3H), 3.69 (s, 3H), 3.8-3.32 (m, 2H), 2.46-2.16 (m, 3H),
2.14-1.8 (m, 3H), 1.75 (s, 3H), 1.67 (d, J ) 14.1 Hz, 3H), 1.59
(d, J ) 55.8 Hz, 3H), 1.07 (s, 9H). 13C NMR (75 MHz, CD3-
OD) δ 173.6 166.1, 162.2, 162.1, 154.8, 152.1, 150.1, 136.8,
136.6, 136,4, 134.3, 133.5, 131.3, 131.2, 129.1, 129, 111.9,
103.9, 99.6, 99.3, 85.9, 85.6, 83.5, 79.5, 76.8, 65.3, 60.4, 60.3,
55.8, 55.7, 47.8, 38.6, 30.2, 27.5, 24.5, 20.2, 12.3. IR (KBr)
3072, 2958, 2935, 2856, 1747, 1668, 1596, 1486, 1398, 1157,
1114 cm-1
.
FAB-MS m/ z 822 [M + Na]+. HRMS (FAB,
NaI): calcd for [M + Na]+ 822.3398; found 822.3409.
3′-O-L-P r olin yl-5′-(O-t er t -b u t yld ip h e n ylsilyl)t h ym i-
d in e (5). 3′-O-(DDZ-Pro)-5′-O-TBDPS-T (0.22 g, 0.3 mmol) in
1 mL of CH2Cl2 and 5% TFA/CH2Cl2 (4.4 mL) were mixed at
25 °C. After 1 h stirring, the reaction was concentrated under
Ack n ow led gm en t. We thank Mr Alex J . Zhang for
performing the MALDI-MS measurements. Financial
support was provided by The Texas Advanced Technol-
ogy Program and The Robert A. Welch Foundation, and
K.B. thanks the NIH for a Research Career Develop-
ment Award and the Alfred P. Sloan Foundation for a
fellowship. NMR facilities were provided by the NSF
via the chemical instrumentation program.
vacuum. The residue was purified via flash chromatography
(1:10 MeOH/CHCl3, Rf ) 0.31) to give 0.15 g (97%) of the
product 5. 1H NMR (300 MHz, CD3OD) δ 7.89 (s, 1H), 7.76-
7.6 (m, 4H), 7.53 (s, 1H), 7.49-7.32 (m, 6H), 6.32(dd, J ) 5.7,
8.7 Hz, 1H), 5.61 (brd, J ) 6.3 Hz, 1H), 4.49 (t, J )7.8 Hz,
1H), 4.19 (brd, J ) 1.8 Hz, 1H), 4.08-3.95 (m, 2H), 3.46-3.33
(m, 2H), 2.61-2.34 (m, 3H), 2.22-1.96 (m, 3H), 1.5 (s, 3H),
1.13 (s, 9H). 13C NMR (75 MHz, CD3OD) δ 169.9, 166.1, 152.2,
136.9, 136.7, 136.4, 134.3, 133.5, 131.3, 131.2, 129.1, 112.1,
86.0, 85.9, 78.5, 65.4, 60.6, 47.3, 38.4, 29.3, 27.5, 24.6, 20.2,
12.2. IR (KBr) 3382, 3052, 2958, 2931, 2857, 1686, 1477, 1203,
1134, 1113 cm-1. FAB-MS m/ z 578 [M + H]+.
Su p p or tin g In for m a tion Ava ila ble: Synthesis scheme
of 1, 4, and 5; copies of 1H NMR spectra for all the compounds;
data for photodecomposition of 1 giving t1/2; HPLC traces
showing thermal decomposition of the intermediate; HPLC
traces showing the coelution of compounds 2 and 6; UV spectra
of 2, 6, and the HPLC peak derived from coelution of 2 and 6
upon photolysis of 1 (10 pages). This material is contained in
libraries on microfiche, immediately follows this article in the
microfilm version of the journal, and can be ordered from the
ACS; see any current masthead page for ordering information.
3′-O-{Nr-(3,4-Dim et h oxy-6-n it r ob en zyloxyca r b on yl)-
NE-(5-(d im eth yla m in o)-1-n a p h th a len esu lfon yl)-L-lysin e-
J O9702608