Model Studies for New o-Nitrobenzyl Photolabile Linkers: Substituent Effects on the Rates of Photochemical Cleavage

Article abstract of DOI:10.1021/jo961602x

Both a model phenacyl and o-nitrobenzyl photolabile linker from the literature along with four new o-nitrobenzyl linkers were prepared and the kinetics of their photolytic cleavage examined in solution. The linkers were prepared by amidation of the carboxylic acid anchoring tether with benzylamine, and the cleavable benzylic substituent was chosen to be either acetic acid or acetamide. Irradiation of the linkers in four solvents (methanol, p-dioxane, and aqueous buffer ± dithiothreitol) at 365 nm and analysis via HPLC afforded kinetic rates of cleavage suitable for comparative purposes. The phenacyl linker was found to cleave slowly under aqueous conditions with no detectable cleavage being observed in the organic solvents. Known o-nitrobenzyl linker 4 showed modest rates of cleavage in aqueous and organic solvents. Incorporation of two alkoxy groups in the benzene ring to generate the veratryl-based linker 13a increased the rate of cleavage dramatically, and introduction of an additional benzylic methyl group (13b) increased the rate of cleavage by an additional 5 fold. Increasing the length of the anchoring carboxylic acid tether from acetic to butyric acid (19) improved the cleavage kinetics modestly in organic media and slightly diminished the rates in water. The amide model linker 21 cleaved from 3 to 7 times faster than the corresponding ester linkage 19. An amide-generating linker 26 was prepared, and its performance to generate photolabile solid supports was briefly examined. The stability of the linker and subsequent cleavage upon photolysis from the support of an isotopically enriched 4-thiazolidinone was demonstrated by gel phase ¹³C NMR.

Full text of DOI:10.1021/jo961602x

2370
J . Org. Chem. 1997, 62, 2370-2380
Mod el Stu d ies for New o-Nitr oben zyl P h otola bile Lin k er s:
Su bstitu en t Effects on th e Ra tes of P h otoch em ica l Clea va ge
Christopher P. Holmes
Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, California 94304
Received August 19, 1996 (Revised Manuscript Received February 3, 1997X)
Both a model phenacyl and o-nitrobenzyl photolabile linker from the literature along with four
new o-nitrobenzyl linkers were prepared and the kinetics of their photolytic cleavage examined in
solution. The linkers were prepared by amidation of the carboxylic acid anchoring tether with
benzylamine, and the cleavable benzylic substituent was chosen to be either acetic acid or acetamide.
Irradiation of the linkers in four solvents (methanol, p-dioxane, and aqueous buffer ( dithiothreitol)
at 365 nm and analysis via HPLC afforded kinetic rates of cleavage suitable for comparative
purposes. The phenacyl linker was found to cleave slowly under aqueous conditions with no
detectable cleavage being observed in the organic solvents. Known o-nitrobenzyl linker 4 showed
modest rates of cleavage in aqueous and organic solvents. Incorporation of two alkoxy groups in
the benzene ring to generate the veratryl-based linker 13a increased the rate of cleavage
dramatically, and introduction of an additional benzylic methyl group (13b) increased the rate of
cleavage by an additional 5 fold. Increasing the length of the anchoring carboxylic acid tether
from acetic to butyric acid (19) improved the cleavage kinetics modestly in organic media and slightly
diminished the rates in water. The amide model linker 21 cleaved from 3 to 7 times faster than
the corresponding ester linkage 19. An amide-generating linker 26 was prepared, and its
performance to generate photolabile solid supports was briefly examined. The stability of the linker
and subsequent cleavage upon photolysis from the support of an isotopically enriched 4-thiazoli-
dinone was demonstrated by gel phase 13C NMR.
In tr od u ction
4-(bromomethyl)-3-nitrobenzoic acid was first described
by Rich.4 Support 1 has been widely employed by many
researchers for the generation of both peptide acids and
amides5 and recently small molecules6 and tagging moi-
eties.7 Several phenacyl linkers first described by Wang8
and later evolved to the p-alkoxy derivative 29 have also
been attached to supports to afford peptide acids, al-
though less extensively than the o-nitrobenzyl supports.
The rapidly growing field of combinatorial chemistry
involving libraries of molecules has renewed interest in
the use of solid phase organic synthesis techniques as a
convenient means of assembling molecules.1,2 As the
need for diversity of chemistry amenable to a solid phase
approach has greatly expanded, so too has the need for
improved and novel linkers anchoring the molecules to
the support. While many groups have focused their
efforts on chemically-cleavable linkers (i.e. acid-, base-,
transition metal-cleavable linkers), we have been pursu-
ing linkers which can be cleaved with UV light. Photo-
lytic cleavage offers a mild method of cleavage from the
support which is particularly attractive in combinatorial
library screening, as the release of the molecules can
occur after removal of any protecting groups and exten-
sive washing of the resin, thereby affording the liberated
molecules suitable for biological assay without contami-
nation by cleavage reagents. Photolabile linkers also
afford additional flexibility in compound synthesis as
many linkers are stable to both acidic and basic condi-
tions suitable for small molecule synthesis. The use of
a photolabile molecule3 as an orthogonal linker for the
cleavage of molecules from solid supports dates back two
decades since the o-nitro benzyl support 1 derived from
(4) Rich, D. H.; Gurwara, S. K. J . Chem. Soc., Chem. Commun. 1973,
610-611.
(5) (a) Rich, D. H.; Gurwara, S. K. J . Am. Chem. Soc. 1975, 97,
1575-1579. (b) Rich, D. H.; Gurwara, S. K. Tetrahedron Lett. 1975,
301-304. (c) Hammer, R. P.; Albericio, F.; Gera, L.; Barany, G. Int. J .
Pept. Protein Res. 1990, 36, 31-45. (d) LLoyd-Williams, P.; Albericio,
F.; Giralt, E. Int. J . Pept. Protein Res. 1991, 37, 58-60. (e) Lloyd-
Williams, P.; Gair´ı, M; Albericio, F.; Giralt, E Tetrahedron 1993, 49,
10069-10078. (f) Giralt, E.; Albericio, F.; Dalcol, I.; Gair´ı, M.; Lloyd-
Williams, P.; Rabanal, F. In Innovation and Perspectives in Solid Phase
Synthesis; Epton, R., Ed.; Mayflower Worldwide Ltd.: Birmingham,
UK, 1994; pp 51-60.
X Abstract published in Advance ACS Abstracts, March 15, 1997.
(1) Combinatorial chemistry reviews: (a) Terrett, N. K., Gardner,
M., Gordon, D. W., Kobylecki, R. J ., Steele, J . Tetrahedron 1995, 51,
8135-8173. (b) Gallop, M. A.; Barrett, R. W.; Dower, W. J .; Fodor, S.
P. A.; Gordon, E. M. J . Med. Chem. 1994, 37, 1233-1251. (c) Gordon,
E, M.; Barrett, R. W.; Dower, W. J .; Fodor, S. P. A.; Gallop, M. A. J .
Med. Chem. 1994, 37, 1385-1401.
(2) Solid phase organic synthesis reviews: (a) Thompson, L. A.;
Ellman, J . A. Chem. Rev. 1996, 96, 555-600. (b) Fru¨chtel, J . S.; J ung,
G. Angew. Chem. 1996, 35, 17-41. (c) Patel, D. V.; Gordon, E. M. Drug
Disc. Today 1996, 4, 134-144.
(6) (a) Burbaum, J . J .; Ohlmeyer, M. H. J .; Reader, J . C.; Henderson,
I.; Dillard, L. W.; Li, G.; Randle, T. L.; Sigal, N. H.; Chelsjy, D.;
Baldwin, J . J . Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6027-6031.
(7) Ohlmeyer, M. H. J .; Swanson, R. N.; Dillard, L. W.; Reader, J .
C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 10922-10926.
(3) For a general review of the use of photolabile supports see:
Lloyd- Williams, P.; Albericio, F.; Giralt, E. Tetrahedron 1993, 49,
11065-11133. For the general use of photoprotecting groups see:
Pallai, V. N. R. Synthesis 1980, 1-26.
(8) Wang, S. W. J . Org. Chem. 1976, 41, 3258-3261.
S0022-3263(96)01602-7 CCC: $14.00 © 1997 American Chemical Society

New o-Nitrobenzyl Photolabile Linkers
J . Org. Chem., Vol. 62, No. 8, 1997 2371
Although the original photolabile supports and their
close analogs have been widely employed in the peptide
arena, their usefulness in small molecule synthesis
remains to be validated. As the realm of solid phase
chemistries is expanded beyond biopolymers, the inter-
play between rate of cleavage from the support and any
inherent instability the molecules may have to UV light
becomes increasingly important. Peptides are relatively
stable to >350 nm UV light, whereas complex hetero-
cycles may not be stable, and hence minimizing overex-
posure to UV light is crucial to the success of such
photolinkers. Both the o-nitrobenzyl and phenacyl link-
ers suffer from unduly slow cleavage kinetics, with typical
photolysis times reported in the literature ranging from
12 to 24 h.3,5 These long photolysis times may in fact
serve to exacerbate the undesired photooxidation of
sensitive functionalities.10 Methionine for example, has
been observed to be particularly sensitive to photooxi-
dation during cleavage.5 Moreover, the o-nitrobenzyl
linkers generate a reactive nitroso-aldehyde on the
support upon cleavage which can potentially trap any
liberated compound or may act as an internal light filter
to slow the rate of cleavage.11 The principal chemical
liability of the phenacyl linker 2 appears to be the
reactive carbonyl group of the linker participating in
undesired cyclizations and small amounts of diketopi-
perazine formation while at the dipeptide stage.9b,12 The
sensitivity of linker 2 toward mildly nucleophilic agents9
additionally restricts the range of chemistries possible
with supports bearing linkers of this type.
R-methyl-6-nitroveratryl alcohol.19 The use of veratryl-
based nitrobenzyl groups which incorporate two ad-
ditional alkoxy groups onto the benzene ring greatly
facilitates the photolytic cleavage with >350 nm UV light
by shifting the principle chromophore into this region
(vide infra). The inclusion of an R-methyl group onto the
benzylic carbon atom enhances the relative cleavage
kinetics and additionally renders the photolysis byprod-
ucts much less reactive toward the liberated com-
pounds.20 The rapid cleavage rates and minimally-
reactive byproducts have allowed us to obtain rapid and
high yields of peptides containing sensitive amino acids
including methionine,19 as well as affording high yields
of small molecules such as thiazolidinones21a and â-lac-
tams.21b In developing these new linkers we had occasion
to examine their photokinetic properties in solution as
well as the cleavage properties of several related analogs
and compared them to those of known linkers 1 and 2.
We now wish to report on these findings.
Resu lts a n d Discu ssion
The most common method of preparing photolabile
supports is to anchor preformed linkers22 bearing a
carboxylic acid tether onto the support as amides, thereby
affording maximum control over the chemistry and level
of substitution of the support. The alternative approach
of synthesizing the linker in situ on the support has at
least in one case led to difficulties due to competing
chemistries of the linker and the polymer support.13
Amide-generating linkers are thus incorporated as pro-
tected (e.g. Boc or Fmoc) amino acids whereas acid-
generating linkers are typically attached as hydroxy-
methyl acids, which are subsequently esterified by a
number of methods.2 We chose to initially survey the
photolysis kinetics of several model linkers in solution
to access their relative cleavage rates in order to deter-
mine what effects differing substituents about the ring
would have. The best of these linkers would then be
transferred to the support and their usefulness confirmed
for solid phase organic synthesis. The model linkers were
prepared as either their O- or N-acetates to represent a
generic acid or amide release, respectively, and were
amidated with benzylamine on their carboxy terminus
to mimic the local environment which would be present
on a typical solid support. It is difficult to extrapolate
relative cleavage rates for linkers 1 and 2 from the data
reported in the literature, as it is not yet common to
report UV light intensity when describing photolysis
conditions.23 We therefore prepared and investigated the
solution photokinetics of benzylamide 4 as an analog of
1 and prepared benzylamide 7 as an analog of 2 in order
to directly compare the new linkers under investigation
with those previously employed.
Other researchers have addressed these shortcomings
and several additional photocleavable linkers have ap-
peared recently. Pillai introduced an R methyl group
onto the benzylic carbon of an o-nitrobenzyl linker, but
observed poor release of peptides longer than five resi-
dues, presumably due to poor swelling of the resin.13 The
use of a 2′-nitrobenzhydryl support has also been reported
to give high yields of peptide acids14 and of peptide
amides15 after 20-24 h of photolysis. Brown has intro-
duced a new linker with particular application for the
mass spectral characterization of compounds released
from it.16 Greenberg has described several o-nitrobenzyl
linkers and documented their use in DNA synthesis.17
Chan has reported a new linker based on 3′-methoxy-
benzoin.18 We recently described two new o-nitrobenzyl
linkers based on R-methyl-6-nitroveratrylamine and
(9) (a) Tjoeng, F. S.; Heavner, G. A. J . Org. Chem. 1983, 48, 355-
359. (b) Bellof, D.; Mutter, M. Chimia 1985, 39, 317-320.
(10) The undesired oxidation most likely stems from the prolonged
exposure to UV light and not from interaction of the products with
the resin-bound photolysis byproducts. Workers have noted that
photolysis of methionine in solution for 16 h in the absence of an
o-nitrobenzyl linker resulted in a 1:1 mixture of methionine:methionine
sulfoxide; see ref 5c.
(11) (a) Patchnornik, A.; Amit, B.; Woodward, R. B. J . Am. Chem.
Soc. 1970, 92, 6333-6335. (b) Reid, W.; Wilk, M. J ustus Liebigs Ann.
Chem. 1954, 590, 91.
(12) Tjoeng, F. S.; Tam, J . P.; Merrifield, R. B. Int. J . Pept. Protein
Res. 1979, 14, 262-274.
(13) Ajayaghosh, A; Pillai, R. Tetrahedron 1988, 44, 6661-6666.
(14) Ajayaghosh, A.; Pillai, V. N. R. J . Org. Chem. 1987, 52, 5714-
5717.
(15) Ajayaghosh, A.; Pillai, V. N. R. Tetrahedron Lett. 1995, 36, 777-
780.
(16) Brown, B. B.; Wagner, D. S.; Geysen, H. M. Mol. Diversity 1995,
1, 4-12.
(19) (a) Holmes, C. P.; J ones, D. G. J . Org. Chem. 1995, 60, 2318-
2319. (b) Holmes, C. P.; J ones, D. G.; Frederick, B. T.; Dong, L.-C. In
Peptides: Chemistry, Structure and Biology (Proceedings of the 14th
American Peptide Symposium); Kaumaya, P. T. P., Hodges, R. S., Eds.;
Mayflower Scientific Ltd: Birmingham, 1995; pp 44-45.
(20) The molecules lead to the nitrosoacetophenone; the character-
ization and subsequent reactivity of the nitrosoacetophenone photo-
product will be reported in due course.
(21) (a) Holmes, C. P.; Chinn, J . P.; Look, G. C.; Gordon, E. M.;
Gallop, M. A. J . Org. Chem. 1995, 60, 7328-7333. (b) Ruhland, B.;
Bhandari, A.; Gordon, E. M.; Gallop, M. A. J . Am. Chem. Soc. 1996,
118, 253-254.
(17) (a) McMinn, D. L.; Greenberg, M. M. Tetrahedron 1996, 52,
3827-3840. (b) Venkatesan, H.; Greenberg, M. M. J . Org. Chem. 1996,
61, 525-529. (c) Yoo, D. J .; Greenberg, M. M. J . Org. Chem. 1995, 60,
3358-3364. (d) Greenberg, M. M.; Gilmore, J . L. J . Org. Chem. 1994,
59, 746-753.
(22) Alternatively referred to as “handles”.
(23) Since photolytic cleavage is first order with respect to UV light
intensity, it is the combination of lamp intensity and photolysis time
(in addition to wavelength) which should be reported to quantify the
amount of light delivered.
(18) Rock, R. S.; Chan, S. I. J . Org. Chem. 1996, 61, 1526-1529.

2372 J . Org. Chem., Vol. 62, No. 8, 1997
Holmes
Sch em e 1a
Sch em e 2a
a
Reagents: (a) benzylamine, DCC, HOBt, DMF; (b) 2-bro-
mopropionyl chloride, AlCl3, CS2; (c) HOAc, DIEA, EtOAc.
Model linker 4 was synthesized via amidation of
benzoic acid 324 with benzylamine and DCC (Scheme 1).
The phenacyl derivative 7 was prepared starting with
amide 525 via acylation with 2-bromopropionyl chloride
to generate intermediate bromoacyl amide 6 in 85% yield.
Partial halogen exchange was observed during the
Friedel-Crafts reaction to afford 6 contaminated with
approximately 5% of the corresponding chloro com-
pound.26 This proved to be of no concern as subsequent
displacement of the halogen(s) with acetic acid gave the
desired phenacyl model linker 7 in 82% yield.
Synthesis of two veratryl-based linkers started with
either vanillin or acetovanillone (Scheme 2). Alkylation
of vanillin 8a with tert-butyl bromoacetate gave the
aldehyde-ester 9a in quantitative yield. Subsequent
nitration with 70% nitric acid effected both the ring
nitration and cleavage of the tert-butyl ester to give the
acid 10a in 91% yield. Amidation with benzylamine gave
the amide 11a in 72% yield, which was reduced with
NaBH4 to give the benzyl alcohol 12a in low yield.
Alcohol 12a proved to be surprisingly insoluble in most
organic solvents and was difficult to prepare in modest
scale. It was found that a two-step, one-pot conversion
of the aldehyde group to the acetoxymethyl group af-
forded the best yields of model linker 13a , which was
obtained in 70% overall yield from 11a . Conversion of
acetovanillone 8b to model linker 13b proceeded without
incident in analogy to the above procedure. Thus,
alkylation of 8b with tert-butyl bromoacetate, followed
by nitration, amidation with benzylamine, and conversion
of the ketone to the acetoxyethyl moiety afforded ample
quantities of model linker 13b for photokinetic evalua-
tion.
a
Reagents: (a) tert-butyl bromoacetate, K2CO3, DMF; (b) HNO3,
Ac2O; (c) benzylamine, EDC, HOBt, DMF; (d) NaBH4, THF; (e)
Ac2O, pyridine, CH2Cl2.
have on the photolytic cleavage. We therefore prepared
linker 19 which possesses a butyric acid anchoring tail
in contrast to the acetic tail present in linkers 7, 13a ,
and 13b. Greenberg has recently described extending
the tether to butyric acid after observing â-elimination
with propionic acid as the tether.17a Linker 19 was
assembled via a five-step sequence beginning with ace-
tovanillone 8b (Scheme 3). Alkylation of 8b with methyl
4-bromobutyrate afforded the keto-ester 14 in quantita-
tive yield and subsequent nitration afforded the nitrated
compound 15 in 66% yield. Saponification of the ester
and conversion to the corresponding benzyl amide gener-
ated amide 17 in excellent overall yield. Reduction of
the ketone with NaBH4 followed by acetylation afforded
the desired linker 19 in near quantitative yield for the
two steps.
Model amide linker 21, which would represent a typical
acylation of a support-bound amine, was additionally
prepared to compare the relative rates of an amide bond
cleavage. A one-pot, two-step conversion of the Fmoc-
protected linker 26 (described later in this paper) to the
acetate-acid 21 proceeded in 88% yield. Amidation with
benzylamine gave the desired model linker 21 in good
yield.
The nature of the tether anchoring the linker to the
support has played an important role in the usefulness
of various linkers in solid phase synthesis.27 We were
curious to explore what effect increasing the relative
electron-donating ability of the para substituent may
The UV spectra of the model linkers exhibited the
anticipated bathochromic shift of the principle absor-
bance bands upon substitution of the phenyl ring by
alkoxy groups (Figure 1). Thus, the veratryl-based
linkers 13a and 13b showed a dramatic increase in
absorbance in the targeted deprotection wavelength
region centered near 365 nm in comparison to linkers 4
and 7. Our previous work and work from others had
(24) Barany, G.; Albericio, F. J . Am. Chem. Soc. 1985, 107, 4936-
4942.
(25) Dermer, O. C.; King, J . J . Org. Chem. 1943, 8, 168-173.
(26) Drefahl, G.; Fisher, F. J ustus Liebigs Ann. Chem. 1956, 598,
159.
(27) The heightened susceptibility toward acidic cleavage as one
increases the chain length of the anchoring tail is well known; see for
example ref 2 and Albericio, F.; Barany, G. Int. J . Pept. Protein Res.
1990, 30, 206-216.

New o-Nitrobenzyl Photolabile Linkers
J . Org. Chem., Vol. 62, No. 8, 1997 2373
Ta ble 1. Mea su r ed P h otolysisa Ha lf Lives for th e
P h otola bile Lin k er s (m in )
Sch em e 3a
solventb
linker
PBS
348
14.1
12.9
1.74
2.87
0.69
DTT/PBS
MeOH
p-dioxane
7
4
13a
13b
19
21
259
c
362
16.2
5.81
3.85
0.51
c
47.2
7.32
1.98
2.86
0.66
36.8
4.00
0.64
0.50
0.17
a
Photolysis in indicated solvent containing 1% DMSO with
Hg(Xe) Arc lamp, 350-450 nm wavelength, and a power of 10 mW/
b
cm2 at 365 nm. PBS is phosphate-buffered saline, pH 7.4; DTT/
c
PBS is 10 mM DTT in pH 7.4 PBS. No photolysis was observed.
The photolysis rates of the model linkers were mea-
sured by photolyzing aliquots of the linkers in solution
for various times and determining the rate of disappear-
ance of the starting linker via HPLC analysis. While this
technique did not allow us to quantify the amount of
product produced, it did provide an expedient method to
achieve our goal, namely the determination of the relative
cleavage kinetics for the compounds. The lamp used was
a 1000 W Hg(Xe) Arc lamp equipped with a 350-450
dichroic reflector thus affording primarily 365 nm il-
lumination. The power level was adjusted to 10 mW/
cm2.29 Times were chosen to cleave from 1 to 10% of the
starting material in order to derive kinetic parameters,
although the cleavage showed excellent linearity over the
course of complete cleavage. No temperature control of
the sample was attempted as the sample did not warm
appreciably over the course of photolysis. Four solvents
were chosen for the photolysis study: MeOH and p-
dioxane to represent protic and aprotic organic solvents,
respectively, whereas pH 7.4 phosphate buffer (PBS) with
and without 10 mM dithiothreitol (DTT) were chosen to
represent typical biological assay buffers containing an
optional scavenger.30 Table 1 summarizes the observed
kinetics for the six linkers examined with the rates
expressed as photolysis half-lives.
The phenacyl linker 7 was found to photolyze much
more slowly in comparison to the other linkers examined
(Table 1). The cleavage of 7 is strikingly solvent depend-
ent, and we could not observe any cleavage in organic
solvents under the conditions examined, whereas modest
rates of cleavage was noted for the aqueous solvents.
Given that previous researchers have successfully em-
ployed phenacyl linkers such as 7, we conclude that
substantially higher light intensities must have been
utilized in order to achieve the reported yields. The
parent o-nitrobenzyl linker 4 exhibited a moderate
increase in photolysis kinetics and displays an interesting
dichotomy: its cleavage appears abnormally fast in the
aqueous environment lacking DTT relative to the organic
solvents. This is in contrast to the other model o-
nitrobenzyl linkers described in this paper and related
compounds examined in our laboratory and is similar to
the observed solvent dependence of phenacyl linker 7.
The presence of the additional carbonyl in conjugation
a
Reagents: (a) methyl 4-bromobutryate, K2CO3, DMF; (b)
HNO3, Ac2O; (c) NaOH, MeOH; (d) benzylamine, 1-(3-(dimethyl-
amino)propyl)-3-ethylcarbodiimide hydrochloride, HOBt, DMF; (e)
NaBH4, THF; (f) Ac2O, pyridine, CH2Cl2; (g) (i) NaOH, MeOH, (ii)
Ac2O, dioxane/H2O.
F igu r e 1. UV trace for photolabile linkers in p-dioxane (0.1
mM): (- - -) 4; (- - -) 7; (- - -) 13a ; (s) 13b.
(28) (a) Holmes, C. P.; Adams, C, L.; Kochersperger, L. M.; Mortens-
en, R. B.; Aldwin, L. A. Biopolymers 1995, 37, 199-211. (b) Pease, A.
C.; Solas, D.; Sullivan, E. J .; Cronan, M. T.; Holmes, C. P.; Fodor, S.
P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (c) Pirrung,
M. C.; Bradley, J .-C. J . Org. Chem. 1995, 60, 116-117.
(29) For comparison, a typical hand-held UV light source emits 2-3
mW/cm2.
demonstrated that both peptides28a and oligonucleotides28b,c
are relatively stable to photolysis with 365 nm UV light
which can be easily achieved from a Hg ARC lamp or
transilluminator. Linkers 19 and 21 exhibited essen-
tially the same UV spectra as 13a and 13b and are not
shown for clarity.
(30) Walker, J . W.; Reid, G. P.; McCray, J . A.; Trentham, D. R. J .
Am. Chem. Soc. 1988, 110, 7170-7177.

2374 J . Org. Chem., Vol. 62, No. 8, 1997
Holmes
Sch em e 4a
with the principle nitrobenzyl chromophore may play a
role in distinguishing 4 from the other o-nitrobenzyl
linkers.
Gratifyingly, the veratryl-based linkers 13a , 13b, 19,
and 21 exhibited the anticipated increase in photolysis
rates upon 365 nm irradiation relative to the parent
o-nitrobenzyl linker 4, presumably due to the increase
in absorbance in this region. The kinetics of cleavage of
13a are representative of this class and similar to what
we have observed for related o-nitrobenzyl protecting
groups, namely the decrease in rate under protic condi-
tions relative to aprotic environments. Thus, the pho-
tolysis rates of 13a displays a three-fold rate increase
when changing from PBS and to dioxane as solvent. The
effect of the R-methyl group is apparent when comparing
13b to 13a , which shows roughly a five-fold increase in
relative cleavage rates when the R-methyl substituent
of 13b is present. Hess and co-workers have also
observed a five-fold increase in the rate of decay of the
key aci-nitro intermediate when comparing an R-proton
versus R-methyl as benzylic substituents in a related
series of o-nitrobenzyl protecting groups.31
Model linker 19 with the longer anchoring tail exhib-
ited faster kinetics in the organic solvents compared to
the shorter tail analog 13b, yet curiously this trend
reversed itself under the aqueous conditions. The dra-
matic increase in photolysis rates observed in organic
solvents for linkers 13b and 19, which is 60-90 times
faster than the previously known linker 4 under identical
conditions, serves to illustrate the kinetic advantage of
these new linkers. The rapid photolysis (2-3 min half
lives) for 13b and 19 in an aqueous environment also
bodes well for the use of linkers such as these for the
photolytic release of compounds under biologically-
relevant conditions. The linkers containing an R-methyl
group do not display any dramatic kinetic dependence
on whether DTT is present, whereas linkers 4 and 13a
lacking a benzylic substituent show a kinetic effect.
While the mode of action of DTT is thought to intercept
a key intermediate with o-nitrobenzyl molecules,30 it is
less clear how DTT could account for the modest increase
in photolysis rate of the phenacyl linker 7.
The amide-releasing linker 21 displayed the fastest
kinetics of all the model linkers examined. Its kinetic
profile is in agreement with the other R-methyl veratryl-
based linkers, namely faster photolysis times being
observed in organic environments and being relatively
insensitive to the presence of DTT. The 10 second half
life for cleavage of 21 observed in dioxane as compared
to 36 minutes for 4 underscores the advantages that the
veratryl-based linkers impart.
a
Reagents: (a) H2NOH‚HCl, pyridine/H2O; (b) (i) H2, Pd/C,
HOAc, (ii) TFAA, pyridine; (c) HNO3; (d) NaOH, MeOH; (e) Fmoc-
Cl, H2O/dioxane; (f) NaBH4, THF.
The somewhat longer approach involving a two-step
transformation of the ketone to an amino group was
therefore optimized to afford sufficient quantities of 26
for further study. Treatment of 14 with hydroxylamine
occurred in quantitative yield to afford crystalline oxime
22, which could be used without any purification. A two-
step procedure of hydrogenation to give an intermediate
amine, followed by acylation with TFAA in pyridine gave
the trifluoroacetate 23 in 80% overall yield from aceto-
vanillone 8b. We found pyridine to be crucial to obtain
high yields during the acylation with TFAA, as the
liberated TFA if not neutralized decomposed the inter-
mediate amine, which itself bears similarity to conven-
tional acid-cleavable linkers. Trifluoroacetate was chosen
to render 23 stable to the strongly acidic conditions of
nitration (70% HNO3), which was found to give 24 in 81%
yield after recrystallization. Once the nitro group is
installed in the benzene ring the molecule was quite
stable to acidic conditions (vide infra). Removal of both
the trifluoroacetyl and methyl ester protecting groups
was accomplished upon treatment with NaOH in reflux-
ing MeOH/H2O to generate the free amino acid 25, which
could be protected in the same flask by simply concen-
trating the reaction mixture and treating with 9-fluor-
enylmethyl chloroformate. In this manner we could
obtained a yield of 81% for the desired photolabile linker
26. Of particular note is that this seven-step procedure
from commercially available acetovanillone does not
Having identified the benefit of the butyric acid tether
and the R-methyl group in the model series, the next step
was to assemble linkers suitable for attaching to solid
supports and to explore their use. We chose to initially
explore the use of an amide-generating linker and
embarked on the synthesis of the fluorenylmethoxycar-
bonyl (Fmoc) protected amine linker 26 (Scheme 4). A
route to 26 was initially explored involving the reductive
amination of ketones 15 or 16, but this proved to be
intractable as no satisfactory conditions could be found.32
(31) Ramesh, D.; Wieboldt, R.; Billington, A. P.; Carpenter, B. K.;
Hess, G. P. J . Org. Chem. 1993, 58, 4599-5605.
(32) Other workers have reported poor yields for the reductive
amination of 2-nitro-4,5-dimethoxyacetophenone: Wilcox, M.; Viola,
R. W.; J ohnson, K. W.; Billington, A. P.; Carpenter, B. K.; McCray, J .
A.; Guzikowski, A. P.; Hess, G. P. J . Org Chem. 1990, 55, 1585-1589.

New o-Nitrobenzyl Photolabile Linkers
J . Org. Chem., Vol. 62, No. 8, 1997 2375
F igu r e 2. Gel phase 13C NMR trace for resin 28. Trace A: natural abundance polyethylene glycol (PEG) is at 71 ppm, 13C-C2
glycine appears at 44 ppm, and 13C-C2 of the thiazolidinone appears at 65 ppm. Trace B: resin 28 after exposure to TFA/H2O) for
1 h. Trace C: resin 28 after irradiation at 365 nm for 1 h. Trace D: resin 28 after irradiation at 365 nm for 3 h.
require any chromatography, as all the intermediates are
solids which were purified by recrystallization. The
alcohol-based linker 27 was prepared for solid phase
studies through a two-step, one-pot conversion of keto
ester 15 to the intermediate hydroxy ester, followed by
saponification to 27 (Scheme 4). The synthetic routes to
both 26 and 27 are easily scaleable, and we have been
able to prepare both on a 0.5 kg scale without incident.33
The chemistry of linker 26 was first explored as an
amide-generating linker for both peptides19a and small
molecules.21 Coupling of the acid anchoring tail as an
amide to a variety of commercial amine-supports with
standard carbodiimide coupling agents affords quantita-
tive loadings to generate the corresponding photolabile
supports. Despite the support’s sensitivity toward pho-
tolytic cleavage, the use of subdued laboratory lights or
foil-wrapped vessels has generally allowed us to employ
these supports without altering routine operations.
analyzing the supernatant from irradiated supports via
HPLC to assess the quality of the liberated material, we
sought to employ direct analysis of the support via fast
13C NMR34 to establish complete cleavage of the bound
compound(s). We therefore assembled support 28 con-
taining linker 26 coupled to C-R 13C labeled glycine as
internal reference and subsequently formed a thiazoli-
dinone derived from a second molecule of glycine, 13C
labeled benzaldehyde. and mercaptoacetic acid using
conditions previously described.21a The use of isotopically
enriched reagents meant that analysis of the resin could
be performed rapidly in a conventional NMR probe,
thereby affording a simple tool to provide qualitative
information on the performance of the linker(s). Analysis
of support 28 via gel phase 13C NMR showed revealed
two resonances for the diastereomeric C-2 of the thiazo-
lidinone and a single resonance for the glycine carbon
(Figure 2a). The polyethylene glycol tether of the sup-
port35 appears as a sharp singlet at ≈71 ppm. The
Linker 26 was used to illustrate the synthesis and
cleavage of a typical support-bound small molecule,
namely 4-thiazolidinone 29 (Figure 2). In addition to
(34) Look, G. C.; Holmes, C. P.; Chinn, J . P.; Gallop, M. A. J . Org.
Chem. 1994, 59, 7588-7590.
(35) A polyethyleneglycol-polystyrene based support (TentaGel) was
used.
(33) Baer, T. A.; Raillard, S. P. Unpublished results.

2376 J . Org. Chem., Vol. 62, No. 8, 1997
Holmes
Sch em e 5
and 27 dramatically enhances the cleavage rates upon
photolysis at 365 nm relative to the previously known
linker 4. The new linkers are not so sensitive as to
exclude their use in normal laboratory settings, however,
and we are continuing to explore their use in combina-
torial organic synthesis. The rapid cleavage kinetics in
organic solvents and the ability to release compounds into
biologically-relevant solvents now opens the way for high
throughput screening techniques to employ linkers of this
type.
Exp er im en ta l Section
Gen er a l. All melting points are uncorrected. Unless
otherwise noted, materials were of the highest grade available
from commercial sources and used without further purification.
Commercial resin (TentaGel) was obtained from Rapp Poly-
mere, Tu¨bingen, Germany and was used without further
characterization other than determination of the loading.
Benzaldehyde (carbonyl-13C) and Fmoc-glycine (2-13C) were
obtained from Cambridge Isotope Labs, Andover, MA. 1H and
13C NMR data were measured in the indicated solvent with
tetramethylsilane as internal reference. Mass spectra were
obtained with either APCI or ESI as ionization method.
Combustion analyses were performed by the Microanalytical
Laboratory, University of California at Berkeley.
stability toward typical TFA deprotection conditions
widely used in peptide synthesis schemes was examined
by incubating 28 with 95% TFA/5% H2O for 1 h at room
temperature. We were gratified to find that both the
linker and thiazolidinone were stable as evidenced by
complete retention of the benzylic resonance at 65 ppm
(Figure 2b). Support 28 was also stable toward exposure
to a standard TFA-scavenger cocktail containing phenol,
thioanisole, water, and ethanedithiol for 2 h at room
temperature (data not shown). Cleavage in PBS buffer
containing 5% DMSO to simulate a biological assay
medium was performed by irradiating a slurry of support
28 in a glass vial for several different times. The 13C
NMR spectra from 1.5 and 3 h of photolysis are shown
in Figure 2, parts c and d, respectively, and represent
roughly 60 and 95% cleavage. The liberated thiazolidi-
none was recovered in high yield (≈90%) and in high
purity (95%) and was identical with authentic material
as judged by HPLC and NMR analysis.
The support-bound photolysis is considerably slower
than the corresponding solution photolysis due to several
factors which include light scattering, shielding, or
shadowing effects of the resin, and the swelling and
solvation properties of the support. Estimates based on
analysis via HPLC of the liberated compounds and NMR
examination of the irradiated resin for the half life are
20-30 min in PBS, consistent with Figure 2D showing
approximately 95% cleavage. Photolysis of resin 28 is
significantly faster in organic solvents which parallels
the observed solution kinetics, with 1-2 h of irradiation
being sufficient to achieve high yields of liberated com-
pound. To date we have employed linker 26 for the
production of numerous small molecule amide libraries
and have observed that basic scavengers such as hydra-
zine or ethanolamine (2-5 equiv) dramatically improves
the rates of cleavage from the support. The use of less
mass of resin also shortens the time required for complete
cleavage, presumably due to less light scattering and
shadowing effects. The hydroxyethyl linker 27 for the
generation of small molecule acids also performs admi-
rably, and its use and application will be reported in due
course.
N-Ben zyl-4-(a cetoxym eth yl)-3-n itr oben za m id e (4). To
a solution of 4-acetoxy-3-nitrobenzoic acid (1.02 g, 4.26 mmol),
benzylamine (930 mg, 8.68 mmol), and 1-hydroxybenzotriazole
hydrate (650 mg, 4.81 mmol) in 25 mL of DMF was added 1,3-
dicyclohexylcarbodiimide (1.00g, 4.85 mmol), and the solution
was stirred for 23 h. The reaction mixture was partitioned
between EtOAc and saturated NaCl and was washed (satu-
rated NaHCO3, 1 N HCl), dried (MgSO4), and evaporated to
give a brown oil. Chromatography on silica gel (CH2Cl2 to 5%
acetone/CH2Cl2) afforded acetate 4 (0.81 g, 58% yield) as a light
1
yellow oil which slowly solidified: mp 132-134 °C; H NMR
(300 MHz, CDCl3) δ 2.16 (s, 3 H), 4.63 (d, J ) 6.3 Hz, 2 H),
5.51 (s, 2 H), 6.85 (br t, J ) 6.3 Hz, 1 H), 7.24-7.37 (m, 5 H),
7.65 (d, J ) 8.4 Hz, 1 H), 8.10 (dd, J ) 8.4, 2.0 Hz, 1 H), 8.48
(d, J ) 2.0 Hz, 1 H); 13C NMR (75.5 MHz, CDCl3) δ 20.7, 44.3,
62.6, 123.4, 127.7, 127.9, 128.7, 129.1, 132.2, 135.0, 135.3,
137.5, 147.1, 164.6, 170.2; MS (APCI) m/ z 329 (MH)+. Anal.
Calcd for C17H16N2O5‚0.25H2O: C, 61.35; H, 5.00; N, 8.42.
Found: C, 61.02; H, 5.02; N, 8.28.
N-Ben zyl-(4-(2-br om opr opion yl)ph en oxy)acetam ide (6).
To a solution of amide 5 (3.40 g, 14.1 mmol) and 2-bromopro-
pionyl chloride (1.65 mL, 16.3 mmol) in 35 mL of carbon
disulfide warmed to ≈40 °C was added aluminum trichloride
(2.35 g, 17.6 mmol). The resultant black slurry was heated to
reflux for 10 h and was then cooled to 0 °C and was quenched
with water. The reaction mixture was partitioned between
EtOAc and 1 N HCl, and the combined organic phase was
washed (saturated NaCl), dried (MgSO4), and evaporated to
give the crude product as a white solid. Chromatography on
silical gel (50% EtOAc/hexanes) afforded the product bro-
mophenacyl amide 6 (4.52 g, 85% yield) as a white solid: mp
102-105 °C; 1H NMR (300 MHz, CDCl3) δ 1.89 (d, J ) 7.5
Hz, 3 H), 4.55 (d, J ) 6.3 Hz, 2 H), 4.63 (s, 2 H), 5.24 (q, J )
7.5 Hz, 1 H), 6.80 (br t, 1 H), 6.96-7.02 (m, 2 H), 7.25-7.39
(m, 5 H), 7.99-8.07 (m, 2 H), a resonance for a minor isomer
was observed: 1.73 (d, J ) 7.5 Hz); 13C NMR (75.5 MHz,
CDCl3) δ 20.1, 41.3, 43.1, 67.3, 114.6, 127.7, 128.2, 128.8, 131.4,
131.5, 137.5, 161.0, 167.1, 191.8; MS (APCI) m/ z 378 (MH)+,
GCMS indicated the presence of ≈5% of the corresponding
chloro derivative: 331 (MH)+. Anal. Calcd for C20H18NO3Br:
C, 57.46; H, 4.82; N, 3.72. Found: C, 58.00; H, 5.09; N, 3.69.
Repeated attempts to purify 6 to homogeneity were unsuc-
cessful.
In conclusion we have described model studies leading
to new o-nitrobenzyl cleavable linkers. Relying primarily
on kinetic rates of cleavage to guide our selection process,
we have developed the R-methyl-6-nitroveratryl-based
chromaphore into linkers for solid phase organic synthe-
sis. The addition of both a benzylic methyl group and
incorporation of two alkoxy substituents to produce 26
N -B e n z y l-(4-(2-a c e t o x y p r o p i o n y l)p h e n o x y )a c e -
ta m id e (7). A solution of bromophenacyl amide 6 (500 mg,
1.33 mmol), HOAc (0.76 mL, 13.3 mmol), and diisopropylethyl-
amine (2.31 mL, 13.3 mmol) was stirred at room temperature
for 57 h. The reaction mixture was partitioned between EtOAc

New o-Nitrobenzyl Photolabile Linkers
J . Org. Chem., Vol. 62, No. 8, 1997 2377
199.6; MS (APCI) m/ z 270 (MH)+. Anal. Calcd for C11H11
NO7: C, 49.08; H, 4.12; N, 5.20. Found: C, 48.84; H, 4.45; N,
5.10.
and saturated NaCl, and the combined organic phase was
dried over MgSO4. Removal of the solvent under reduced
pressure and chromatography of the residue on silical gel (50%
EtOAc/hexanes) afforded pure acetate 7 (389 mg, 82% yield)
as a white solid: mp 124-125 °C; 1H NMR (300 MHz, CDCl3)
δ 1.52 (d, J ) 7.6 Hz, 3 H), 2.14 (s, 3 H), 4.55, (d, J ) 6.3 Hz,
2 H), 4.62 (s, 2 H), 5.92 (q, J ) 7.6 Hz, 1 H), 6.81 (br t, J ) 6.3
Hz, 1 H), 6.95-7.05 (m, 2 H), 7.25-7.38 (m, 5 H), 7.91-7.98
(m, 2 H); 13C NMR (75.5 MHz, CDCl3) δ 17.2, 20.7, 43.0, 67.2,
71.1, 114.6, 127.7, 128.5, 128.7, 130.9, 130.9, 137.5, 161.0,
167.1, 170.3, 195.1; MS (APCI) m/ z 356 (MH)+. Anal. Calcd
for C20H21NO5‚0.05H2O: C, 67.42; H, 5.97; N, 3.43. Found:
C, 67.11; H, 6.26; N, 3.87.
ter t-Bu tyl (4-for m yl-2-m eth oxyp h en oxy)a ceta te (9a ).
A slurry of vanillin (25.00 g, 164.3 mmol), tert-butyl bromo-
acetate (34.35 g, 176.1 mmol), and potassium carbonate (34.4
g, 249 mmol) was stirred at room temperature for 48 h. Water
was added to the reaction mixture until all the salts had
dissolved, and the mixture was partitioned between EtOAc and
saturated NaCl acidified to pH 2 with 1 N HCl. The combined
organic phase was dried (MgSO4) and evaporated to give the
aldehyde-ester 9a (43.55 g, quantitative) as a white solid: mp
95-96 °C; 1H NMR (300 MHz, CDCl3) δ 1.48 (s, 9 H), 3.95 (s,
3 H), 4.68 (s, 2 H), 6.86 (d, J ) 8.4 Hz, 1 H), 7.40-7.46 (m, 2
H); 13C NMR (75.5 MHz, CDCl3) δ 27.8, 55.9, 65.9, 82.6, 109.6,
111.8, 125.9, 130.7, 149.7, 152.5, 166.8, 190.6; MS (GCMS) m/ z
266 (M)+. Anal. Calcd for C14H18O5: C, 63.15; H, 6.81.
Found: C, 63.05; H, 7.08.
-
N-Ben zyl-(4-for m yl-2-m et h oxy-5-n it r op h en oxy)a cet -
a m id e (11a ). To a solution of aldehyde-acid 10a (1.004 g,
3.934 mmol), benzylamine (0.850 mL, 7.78 mmol), and 1-hy-
droxybenzotriazole hydrate (1.06 g, 7.84 mmol) in 16 mL of
DMF was added 1-(3-(dimethylamino)propyl)-3-ethylcarbodi-
imide methiodide (1.137 g, 5.931 mmol). The reaction mixture
was stirred for 15 h and was then partitioned between EtOAc
and saturated NaCl. The combined organic phase was washed
(saturated NaHCO3, 1 N HCl, saturated NaCl), dried (MgSO4),
and evaporated to give an off-white solid. Chromatography
on silical gel (CH2Cl2 to 5% acetone/CH2Cl2 to 15% acetone/
CH2Cl2) to afford the product aldehyde-amide 11a (0.97 g, 72%
1
yield) as a light yellow solid: mp 176-178 °C; H NMR (300
MHz, CDCl3) δ 3.93 (s, 3 H), 4.57 (d, J ) 6.3 Hz, 2 H), 4.71 (s,
2 H), 7.02 (br t, 1 H), 7.27-7.40 (m, 5 H), 7.41 (s, 1 H), 7.65 (s,
1 H), 10.46 (s, 1 H); 13C NMR (75.5 MHz, CDCl3) δ 43.2, 56.7,
68.9, 110.3, 110.6, 127.7, 127.8, 128.8, 137.5, 143.4, 149.8,
153.7, 166.4, 187.4; MS (APCI) m/ z 345 (MH)+. Anal. Calcd
for C17H16N2O6: C, 59.30; H, 4.68; N, 8.14. Found: C, 59.48;
H, 5.08; N, 7.85.
N-Ben zyl-(4-a cet yl-2-m et h oxy-5-n it r op h en oxy)a cet -
a m id e (11b). To a solution of nitro-acid 10b (1.015 g, 3.770
mmol), benzylamine (0.825 mL, 7.55 mmol), and 1-hydroxy-
benzotriazole hydrate (562 mg, 4.16 mmol) in 25 mL of DMF
was added 1,3-dicyclohexylcarbodiimide (880 mg, 4.26 mmol).
The reaction mixture was stirred at room temperature for 27
h and was then partitioned between EtOAc and saturated
NaCl. The organic phase was washed (saturated NaHCO3, 1
N HCl), dried (MgSO4), and evaporated to give a light yellow
oil. Chromatography on silica gel (CH2Cl2 to 5% acetone/CH2-
Cl2) gave pure keto-amide 11b (0.80 g, 59% yield) as a light
ter t-Bu tyl (4-Acetyl-2-m eth oxyp h en oxy)a ceta te (9b).
A slurry of acetovanillone 8b (10.50 g, 63.2 mmol), tert-butyl
bromoacetate (13.50 g, 69.2 mmol), and K2CO3 (14.4 g, 104
mmol) in 75 mL of DMF was stirred at room temperature for
48 h. Water was added until all the salts were dissolved, and
the reaction mixture was partitioned between EtOAc and
saturated NaCl. The combined organic phase was washed
(saturated NaCl), dried (MgSO4), and evaporated to give the
keto-ester 9b (17.80 g, quantitative) as a white solid: mp 48-
1
yellow solid: mp 138-140 °C; H NMR (300 MHz, CDCl3) δ
2.50 (s, 3 H), 3.86 (s, 3 H), 4.56 (d, J ) 6.3 Hz, 2 H), 4.67 (s, 2
H), 6.75 (s, 1 H), 7.04 (br s, 1 H), 7.27-7.40 (m, 5 H), 7.68 (s,
1 H); 13C NMR (75.5 MHz, CDCl3) δ 30.4, 43.1, 56.5, 69.2,
109.2, 110.6, 127.7, 127.8, 128.8, 134.6, 137.5, 138.2, 147.2,
154.5, 166.7, 199.6; MS (APCI) m/ z 359 (MH)+. Anal. Calcd
for C18H18N2O6: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.14;
H, 5.17; N, 7.83.
1
49 °C; H NMR (300 MHz, CDCl3) δ 1.48 (s, 9 H), 2.56 (s, 3
H), 3.94 (s, 3 H), 4.66 (s, 2 H), 6.78 (d, J ) 8.3 Hz, 1 H), 7.50-
7.56 (m, 2 H); 13C NMR (75.5 MHz, CDCl3) δ 26.1, 27.9, 55.9,
66.0, 82.6, 110.7, 111.6, 122.7, 131.3, 149.2, 151.4, 167.1, 196.6;
MS (ESI) m/ z 281 (MH)+. Anal. Calcd for C15H20O5: C, 64.27;
H, 7.19. Found: C, 64.22; H, 7.28.
N-Ben zyl-(4-(h yd r oxym et h yl)-2-m et h oxy-5-n it r op h e-
n oxy)a ceta m id e (12a ). To a solution of aldehyde-amide 11a
(0.28 g, 0.813 mmol) in 25 mL of THF was added NaBH4 (80
mg, 2.11 mmol) at room temperature. The reaction mixture
was stirred for 16 h and was then partitioned between EtOAc
and saturated NH4Cl. The combined organic phase was dried
(MgSO4) and evaporated to give a yellow residue. Chroma-
tography on silica gel (1% MeOH/CHCl3 to 5% MeOH/CHCl3)
afforded the alcohol-amide 12a (63.6 mg, 23% yield) as a yellow
solid: mp 177-188 °C dec; 1H NMR (300 MHz, CDCl3 plus
several drops of DMSO-d6) δ 3.90 (s, 3 H), 4.51 (d, J ) 6.3 Hz,
2 H), 4.64 (s, 2 H), 4.99 (d, J ) 6.3 Hz, 2 H), 5.10 (t, J ) 6.3
Hz, 1 H), 7.23-7.38 (m, 5 H), 7.47 (s, 1 H),, 7.65 br t, 1 H),
7.77 (s, 1H); 13C NMR (75.5 MHz, DMSO-d6) δ 41.9, 56.1, 60.1,
68.1, 110.0, 110.3, 126.8, 127.2, 128.2, 135.8, 138.1, 139.1,
145.4, 153.9, 167.2; MS (APCI) 347 (MH)+. Anal. Calcd for
C17H16N2O6: C, 58.96; H, 5.24; N, 8.09. Found: C, 58.84; H,
5.22; N, 8.12.
(4-For m yl-2-m eth oxy-5-n itr oph en oxy)acetic Acid (10a).
A solution of aldehyde-ester 9a (5.00 g, 18.8 mmol) in 15 mL
of acetic anhydride was added to a solution of 100 mL of 70%
HNO3 and 15 mL of acetic anhydride at 0 °C. The resultant
orange solution was stirred for 2 h and then allowed to warm
to room temperature, and stirring was continued for an
additional 4 h. The reaction mixture was poured into water,
the pH adjusted with solid NaOH to 3-4, and the solution
saturated with solid NaCl. The aqueous phase was extracted
with EtOAc and the combined organic phase dried (MgSO4)
and evaporated to give a yellow solid. Chromatography on
silica gel (1% HOAc/5% MeOH/94% CHCl3) gave pure alde-
hyde-acid 10a (4.35 g, 91% yield) as a pale yellow solid: mp
1
163-164 °C; H NMR (300 MHz, CDCl3) δ 4.04 (s, 3 H), 4.82
(s, 2 H), 7.43 (s, 1 H), 7.57 (s, 1 H), 10.43 (s, 1 H); 13C NMR
(75.5 MHz, DMSO-d6) δ 56.5, 65.4, 108.7, 110.5, 125.2, 143.2,
150.3, 152.6, 169.2, 188.5; MS (EI) m/ z 255 (M)+. Anal. Calcd
for C20H22N2O7‚0.2H2O: C, 46.41; H, 3.66; N, 5.41. Found: C,
46.16; H, 3.84; N, 5.72.
N-Ben zyl-(4-(1-h yd r oxyet h yl)-2-m et h oxy-5-n it r op h e-
n oxy)a ceta m id e (12b). To a solution of keto-amide 11b (207
mg, 0.578 mmol) in 20 mL of THF was added NaBH4 (78 mg,
2.06 mmol) at room temperature. The reaction mixture was
stirred for 31 h and was then partitioned between EtOAc and
saturated NH4Cl. The combined organic phase was dried over
MgSO4 and evaporated to give a yellow solid. Chromatography
on silica gel (5% MeOH/CHCl3) afforded pure alcohol 12b (210
(4-Acetyl-2-m eth oxy-5-n itr op h en oxy)a cetic Acid (10b).
A solution of keto-ester 9b (5.06 g, 18.0 mmol) in 15 mL of
acetic anhydride was added to a solution 15 mL of 70% HNO3
and 10 mL of acetic anhydride cooled to 0 °C. The solution
was stirred for 2 h and then allowed to warm to room
temperature. After stirring an additional 4 h, the reaction
mixture was poured into water and chilled to 4 °C overnight.
The product was isolated by filtration, and the precipitate was
washed extensively with water. Recrystalization from MeOH/
H2O afforded the product nitro-acid 10b as light yellow
1
mg, 97% yield) as a yellow solid: mp 152-153 °C; H NMR
(300 MHz, CDCl3) δ 1.55 (d, J ) 7.1 Hz, 3 H), 2.27 (br d, J )
2.5 Hz, 1 H), 3.85 (s, 3 H), 4.55 (d, J ) 6.3 Hz, 2 H), 4.63 (s,
2H), 5.59 (dq, J ) 7.1, 2.5 Hz, 1 H), 7.14 (br t, 1 H), 7.26 (s, 1
H), 7.27-7.39 (m, 5 H), 7.63 (s, 1 H); 13C NMR (75.5 MHz,
CDCl3 + several drops of DMSO-d6) δ 24.7, 42.6, 55.8, 64.5,
69.1, 109.2, 111.3, 127.1, 127.2, 128.3, 137.5, 138.7, 140.1,
144.9, 153.8, 167.1; MS (APCI) m/ z 361 (MH)+. Anal. Calcd
1
crystals: mp 201-203 °C; H NMR (300 MHz, CDCl3) δ 2.50
(s, 3 H), 3.99 (s, 3 H), 4.75 (s, 2H), 6.69 (s, 1 H), 7.57 (s, 1H);
13C NMR (75.5 MHz, CDCl3 + several drops of DMSO-d6) δ
30.2, 56.5, 65.7, 108.5, 108.8, 133.4, 137.8, 147.6, 154.1, 169.4,

2378 J . Org. Chem., Vol. 62, No. 8, 1997
Holmes
for C18H20N2O6: C, 59.99; H, 5.59; N, 7.77. Found: C, 59.97;
H, 5.68; N, 7.52.
mL (15 mmol) of 1 N NaOH in 100 mL of MeOH were stirred
at room temperature for 21 h before being partitioned between
EtOAc and sat NaCl acidified to pH 2 with 1 N HCl. The
organic phase was washed (saturated NaCl), dried (MgSO4),
and evaporated to dryness. The crude product was chromato-
graphed on silica gel (1% HOAc/1% MeOH/98% CHCl3 to 1%
HOAc/5% MeOH/94% CHCl3) to afford recovered 15 (0.78 g)
and pure acid 16 as a yellow solid (1.55 g, 97% yield based on
recovered 15): mp 159-160 °C; 1H NMR (300 MHz, CDCl3 +
2 drops of DMSO-d6) δ 2.17 (pentet, J ) 6.7 Hz, 2 H), 2.50 (s,
3 H), 2.53 (t, J ) 6.7 Hz, 2 H), 3.97, (s, 3H), 4.18 (t, J ) 6.7
Hz, 2 H), 6.78 (s, 1 H), 7.63 (s, 1 H); 13C NMR (75.5 MHz,
DMSO-d6) δ 23.9, 29.9, 30.0, 56.6, 68.2, 108.0, 109.8, 131.1,
138.3, 148.5, 153.3, 173.9, 199.2; MS (EI) m/ z 297 (M+), 196.
Anal. Calcd for C13H15NO7: C, 52.53; H, 5.09; N, 4.71.
Found: C, 52.49; H, 5.09; N, 4.61.
N-Ben zyl-(4-(a cet oxym et h yl)-2-m et h oxy-5-n it r op h e-
n oxy)a ceta m id e (13a ). To a solution of aldehyde-amide 11a
(74.5 mg, 0.216 mmol) in 10 mL of THF was added NaBH4
(75 mg, 1.98 mmol) at room temperature. After stirring for 1
h, the reaction was quenched with excess glacial acetic acid
until all gas evolution had ceased. Pyridine (1 mL) and acetic
anhydride (0.5 mL, 5.3 mmol) were added, and the solution
was stirred an additional 22 h. The reaction mixture was
partitioned between EtOAc and saturated NaCl acidified to
pH 2 with 1 N HCl. The combined organic phase was
evaporated to dryness to give crude acetate-amide 13a as a
light yellow solid. Chromatography on silica gel (CH2Cl2 to
5% acetone/CH2Cl2) afforded pure acetate-amide 13a (58.3 mg,
1
70% overall yield for both steps): mp 128-131 °C; H NMR
(300 MHz, CDCl3 plus several drops of DMSO-d6) δ 2.17 (s, 3
H), 3.85 (s, 3 H), 4.55 (d, J ) 6.3 Hz, 2 H), 4.64 (s, 2 H), 5.50
(s, 2 H), 7.01 (s, 1 H), 7.11 (br t, 1 H), 7.27-7.40 (m, 5 H), 7.76
(s, 1 H); 13C NMR (75.5 MHz, CDCl3) δ 20.8, 43.0, 56.1, 63.0,
69.4, 111.0, 112.3, 127.6, 127.7, 128.7, 128.9, 137.6, 139.8,
145.9, 154.0, 167.1, 170.1; MS (APCI) m/ z 389 (MH)+. Anal.
Calcd for C19H20N2O7: C, 58.76; H, 5.19; N, 7.21. Found: C,
58.78; H, 5.04; N, 7.19.
N-Ben zyl-(4-(1-a cet oxyet h yl)-2-m et h oxy-5-n it r op h e-
n oxy)a ceta m id e (13b). A mixture of alcohol 12b (112 mg,
0.311 mmol), pyridine (1 mL), acetic anhydride (0.5 mL), and
DMAP (5 mg) in 10 mL of CH2Cl2 was stirred for 48 h. The
reaction mixture was partitioned between EtOAc and satu-
rated NaCl, and the organic phase was dried (MgSO4) and
evaporated. The crude product was chromatographed on silica
gel (CH2Cl2 to 5% acetone/CH2Cl2) to afford pure acetate 13b
(105.8 mg, 85% yield) as a pale yellow solid: mp 115-116 °C;
1H NMR (300 MHz, CDCl3) δ 1.60 (d, J ) 6.7 Hz, 3 H), 2.08 (s,
3 H), 3.84 (s, 3 H), 4.55 (d, J ) 6.3 Hz, 2 H), 4.62 (s, 2 H), 6.46
(q, J ) 6.7 Hz, 1 H), 7.03 (s, 1 H), 7.14 (br t, 1 H), 7.25-7.39
(m, 5 H), 7.63 (s, 1 H); 13C NMR (75.5 MHz, CDCl3) δ 21.1,
21.9, 43.0, 56.1, 68.1, 69.4, 108.6, 111.8, 127.6, 127.7, 128.7,
135.2, 137.6, 139.7, 145.7, 154.1, 167.2, 169.5; MS (APCI) m/ z
403 (MH)+. Anal. Calcd for C20H22N2O7: C, 59.70; H, 5.51;
N, 6.96. Found: C, 59.58; H, 5.56; N, 7.03.
N-Ben zyl-4-(4-a cet yl-2-m et h oxy-5-n it r op h en oxy)b u -
tyr a m id e (17). To a solution of acid 16 (503 mg, 1.69 mmol),
benzylamine (0.37 mL, 3.39 mmol), and 1-hydroxybenzotri-
azole hydrate (459 mg, 3.39 mmol) in 8 mL of DMF was added
1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide methiodide
(542 mg, 2.83 mmol). The reaction mixture was stirred for 15
h and was then partitioned between EtOAc and saturated
NaCl. The combined organic phase was washed (1 N HCl,
saturated NaHCO3), dried (MgSO4), and evaporated to give a
white solid. Chromatography on silical gel (CH2Cl2 to 5%
acetone/CH2Cl2 to 15% acetone/CH2Cl2) afforded the product
amide 17 (0.58 g, 89% yield) as a white solid: mp 142-143
1
°C; H NMR (300 MHz, CDCl3) δ 2.25 (pentet, J ) 7.1 Hz, 2
H), 2.46 (t, J ) 7.1 Hz, 2 H), 2.48 (s, 3 H), 3.89 (s, 3 H), 4.15
(t, J ) 7.1 Hz, 2 H), 4.44 (d, J ) 6.3 Hz, 2 H), 5.93 (br t, 1 H),
6.72 (s, 1 H), 7.21-7.35 (m, 5 H), 7.59 (s, 1 H); 13C NMR (75.5
MHz, CDCl3) δ 24.7, 30.3, 32.5, 43.6, 56.5, 68.5, 108.0, 108.7,
127.5, 127.7, 128.7, 132.8, 138.1, 138.4, 148.7, 154.1, 171.6,
200.0; MS (ESI) m/ z 387 (MH+), 409 (M + Na)+. Anal. Calcd
for C20H22N2O6: C, 62.17; H, 5.74; N, 7.25. Found: C, 62.16;
H, 5.73; N, 7.22.
N-Ben zyl-4-(4-(1-h yd r oxyeth yl)-2-m eth oxy-5-n itr op h e-
n oxy)bu tyr a m id e (18). To a solution of 17 (397 mg, 1.03
mmol) in 20 mL of THF was added NaBH4 (83 mg, 2.19 mmol)
at room temperature. The reaction mixture was stirred for
26 h and was then partitioned between EtOAc and saturated
NH4Cl. The combined organic phases were dried (MgSO4) and
evaporated to give crude alcohol. Chromatography on silica
gel (1% MeOH/CHCl3 to 5% MeOH/CHCl3) gave alcohol 18 (398
Meth yl 4-(4-Acetyl-2-m eth oxyp h en oxy)bu ta n oa te (14).
A slurry of acetovanillone (41.00 g, 246.7 mmol), methyl
4-bromobutyrate (49.63 g, 274.1 mmol), and K2CO3 (51.1 g,
370 mmol) in 200 mL of DMF was stirred at room temperature
for 16 h. Water was added to the reaction mixture until all
the salts were dissolved, and the solution was partitioned
between EtOAc and saturated NaCl. The organic phase was
dried (MgSO4), filtered, and evaporated to dryness to afford
67.90 g (100% crude yield) of product keto-ester 14 as a
1
mg, 100% yield) as a light yellow solid: mp 124-125 °C; H
NMR (300 MHz, CDCl3) δ 1.54 (d, J ) 6.3 Hz, 3 H), 2.21
(pentet, J ) 6.7 Hz, 2 H), 2.46 (t, J ) 6.7 Hz, 2 H), 2.63 (br s,
1 H), 3.87 (s, 3 H), 4.09 (t, J ) 6.7 Hz, 2 H), 4.42 (d, J ) 6.3
Hz, 2 H), 5.55 (dq, J ) 6.3, 2.9 Hz, 1 H), 6.08 (br t, J ) 6.3 Hz,
1 H), 7.20-7.33 (m, 6 H), 7.53 (s, 1 H); 13C NMR (75.5 MHz,
DMSO-d6) δ 24.7, 25.2, 31.5, 42.0, 56.0, 63.9, 68.2, 108.3, 109.1,
126.7, 127.1, 128.2, 138.0, 138.9, 139.6, 146.2, 153.4, 171.4;
MS (ESI) m/ z 389 (MH+), 411 (M + Na)+. Anal. Calcd for
C20H24N2O6: C, 61.85; H, 6.23; N, 7.21. Found: C, 62.01; H,
6.24; N, 7.21.
1
colorless oil which slowly solidified: mp 48-49 °C; H NMR
(300 MHz, CDCl3) δ 2.19 (pentet, J ) 7.3 Hz, 2 H), 2.56 (t, J
) 7.3 Hz, 2 H), 2.56 (s, 3 H), 3.70 (s, 3 H), 3.91 (s, 3 H), 4.15
(t, J ) 7.3 Hz, 2 H), 6.89 (d, J ) 8.4 Hz, 1 H), 7.51-7.78 (m,
2 H); 13C NMR (75.5 MHz, CDCl3) δ 24.2, 26.1, 30.2, 51.5, 55.9,
67.6, 110.4, 111.2, 123.1, 130.4, 149.2, 152.5, 173.3, 196.7; MS
(EI) m/ z 266 (M+). Anal. Calcd for C14H18O5: C, 63.15; H,
6.81. Found: C, 62.81; H, 6.83.
N-Ben zyl-4-(4-(1-a cetoxyeth yl)-2-m eth oxy-5-n itr op h e-
n oxy)bu tyr a m id e (19). A solution of alcohol 18 (145 mg,
0.373 mmol) and 1 mL of pyridine in 10 mL of CH2Cl2 was
treated with acetic anhydride (0.5 mL, 5.3 mmol) and catalytic
DMAP for 4 h. The reaction mixture was partitioned between
EtOAc and saturated NaCl, washed (1 N HCl), dried (MgSO4),
and evaporated to give crude acetate product as a pale yellow
solid. Chromatography on silica gel (CH2Cl2 to 5% acetone/
CH2Cl2) gave pure acetate 19 (170.0 mg, quantitative) as a
pale yellow solid: mp 114-115 °C; 1H NMR (300 MHz, CDCl3)
δ 1.61 (d, J ) 6.7 Hz, 3 H), 2.07 (s, 3 H), 2.22 (pentet, J ) 7.0
Hz, 2 H), 2.45 (t, J ) 7.0 Hz, 2 H), 3.88 (s, 3 H), 4.10 (t, J )
7.0 Hz, 2 H), 4.43 (d, J ) 6.3 Hz, 2 H), 6.04 (br t, 1 H), 6.46 (q,
J ) 6.7 Hz, 1 H), 6.98 (s, 1 H), 7.20-7.33 (m, 5 H), 7.55 (s, 1
H); 13C NMR (75.5 MHz, CDCl3) δ 21.1, 22.0. 24.8, 32.8, 43.6,
56.2, 68.3, 68.3, 108.1, 109.0, 127.5, 127.7, 128.6, 133.2, 138.2,
139.8, 147.1, 153.8, 169.6, 171.8; MS (ESI) m/ z 431 (MH)+.
Anal. Calcd for C22H26N2O7: C, 61.39; H, 6.09; N, 6.51.
Found: C, 61.47; H, 6.25; N, 6.59.
Met h yl 4-(4-Acet yl-2-m et h oxy-5-n it r op h en oxy)b u -
ta n oa te (15). A solution of keto-ester 14 (5.00 g, 18.8 mmol)
in 15 mL of acetic anhydride was slowly added to a solution
of 100 mL of 70% HNO3 and 20 mL of acetic anhydride at 0
°C. After stirring for 2.5 h the reaction mixture was poured
into water and chilled to 4 °C overnight. The precipitate was
collected by filtration and washed extensively with water.
Recrystalization from MeOH/H2O afforded the nitro-ester 15
as yellow crystals (3.87 g, 66% yield): mp 112-113 °C; 1H
NMR (300 MHz, CDCl3) δ 2.20 (pentet, J ) 7.1 Hz, 2 H), 2.49
(s, 3 H), 2.56 (t, J ) 7.1 Hz, 1 H), 3.70 (s, 3 H), 3.95 (s, 3 H),
4.15 (t, J ) 7.1 Hz, 2 H), 6.75 (s, 1 H), 7.67 (s, 1 H); 13C NMR
(75.5 MHz, CDCl3) δ 24.1, 30.2, 30.4, 51.7, 56.6, 68.4, 108.0,
108.7, 132.8, 138.2, 148.8, 154.3, 173.2, 200.0; MS (EI) m/ z
311 (M+), 280. Anal. Calcd for C14H17NO7: C, 54.02; H, 5.50;
N, 4.50. Found: C, 53.98; H, 5.60; N, 4.49.
4-(4-Acetyl-2-m eth oxy-5-n itr op h en oxy)bu ta n oic Acid
(16). A solution of nitro-ester 15 (2.44 g, 7.84 mmol) and 15

New o-Nitrobenzyl Photolabile Linkers
J . Org. Chem., Vol. 62, No. 8, 1997 2379
4-(4-(1-Acet a m id oet h yl)-2-m et h oxy-5-n it r op h en oxy)-
bu ta n oic Acid (20). To a solution of Fmoc-linker 26 (217
mg, 0.417 mmol) in 35 mL of MeOH and 10 mL of water was
added 1 N NaOH (6.0 mL, 0.60 mmol). The solution was
stirred for 16 h before being concentrated under reduced
pressure to 5-10 mL. p-Dioxane (35 mL) and water (10 mL)
were added to the resultant slurry, and the pH was adjusted
to 7-8 with 6 N HCl. Acetic anhydride (2 mL) was added,
and the solution was stirred at room temperature for 18 h.
The reaction mixture was partitioned between Et2O (washings
discarded) and saturated NaHCO3. The aqueous phase was
acidified to pH 1 with 1 N HCl and extracted with EtOAc. The
combined organic phase was dried (MgSO4) and evaporated
to afford the crude acetate product as a tan oil. Chromatog-
raphy on silical gel (1% HOAc/15% MeOH/CHCl3) gave pure
acetate-acid 20 (125.0 mg, 88% yield) as a yellow solid: mp
188-192 °C; 1H NMR (300 MHz, CDCl3 plus several drops of
DMSO-d6) δ 1.48 (d, J ) 7.1 Hz, 3 H), 1.95 (s, 3 H), 2.13 (pentet,
J ) 7.1 Hz, 2 H), 2.49 (t, J ) 7.1 Hz, 2 H), 3.93 (s, 3 H), 4.10
(t, J ) 7.1 Hz, 2 H), 5.59 (dq, J ) 7.1, 7.1 Hz, 1 H), 7.09 (s, 1
H), 7.53 (s, 1 H), 7.70 (d, J ) 7.1 Hz, 1 H); 13C NMR (75.5
MHz, CDCl3 plus several drops of DMSO-d6) δ 21.3, 22.6, 23.9,
30.0, 45.5, 56.0, 67.9, 109.0, 109.5, 134.8, 139.9, 146.3, 153.4,
169.2, 174.9; MS (APCI) m/ z 341 (MH)+. Anal. Calcd for
C15H20N2O7‚0.1H2O: C, 52.66; H, 5.95; N, 8.19. Found: C,
52.35; H, 5.95; N, 7.89.
filtered after 5 days, and the solvent was removed under
vacuum. The oily residue was taken up in 600 mL of water
and acidified to pH 1 with 6 N HCl. The aqueous phase was
extracted with Et2O (washings discarded), and the aqueous
phase was basified with solid NaOH to pH 11 and extracted
with EtOAc. The EtOAc phase was dried (MgSO4), filtered,
and evaporated to dryness to afford the intermediate amine
as a colorless oil.
The crude amine was taken up in 300 mL of pyridine, cooled
to 0 °C with an ice bath, and treated with trifluoroacetic
anhydride (31 mL, 219 mmol) for 1 h. The reaction mixture
was worked up by partitioning between EtOAc and saturated
NaCl. The organic phase was dried (MgSO4), filtered, and
evaporated to dryness to give the crude trifluoroacetate as a
light yellow solid. The solid was recrystallized from CH2Cl2/
hexanes to afford trifluoroacetate 23 (71.62 g, 80% overall yield
from acetovanillone) as a white solid: mp 96-97 °C; 1H NMR
(300 MHz, CDCl3) δ 1.58 (d, J ) 7.5 Hz, 3 H), 2.15 (pentet, J
) 7.1 Hz, 2 H), 2.54 (t, J ) 7.1 Hz, 2 H), 3.68 (s, 3 H), 3.86 (s,
3 H), 4.05 (t, J ) 7.1 Hz, 2 H), 5.09, (dq, J ) 7.5, 7.5 Hz, 1 H),
6.38 (br d, J ) 7.5 Hz, 1 H), 6.80-6.90 (m, 3 H); 13C NMR
(75.5 MHz, CDCl3) δ 20.8, 24.4, 30.4, 49.5, 51.6, 56.0, 67.9,
110.4, 113.4, 118.2, 133.7, 148.2, 149.7, 173.6, (the resonances
for NCOCF3 and NCOCF3 were not observed); MS (APCI) m/ z
364 (MH+), 251 (MH-H2NCOCF3)+. Anal. Calcd for
C16H20F3NO5: C, 52.89; H, 5.55; N, 3.86. Found: C, 52.76; H,
5.45; N, 3.59.
N-Ben zyl-4-(4-(1-a cet a m id oet h yl)-2-m et h oxy-5-n it r o-
p h en oxy)bu tyr a m id e (21). To a solution of acetate-acid 20
(83.2 mg, 0.244 mmol), benzylamine (80 µL, 0.732 mmol), and
1-hydroxybenzotriazole hydrate (66 mg, 0.488 mmol) in 3 mL
of DMF was added 1,3-diisopropylcarbodiimide (40 µL, 0.255
mmol). The reaction mixture was stirred at room temperature
for 22 h and was then partitioned between EtOAc and
saturated NaCl. The organic phase was washed (1 N HCl,
saturated NaHCO3, sat NaCl), dried (MgSO4), and evaporated
to give a light yellow oil. Chromatography on silica gel (50%
acetonitrile/CH2Cl2 to 2% HOAc/49% acetonitrile/49% CH2Cl2)
afforded amide 21 (75.7 mg, 72% yield) as a light yellow solid:
mp 202-204 °C; 1H NMR (300 MHz, CDCl3) δ 1.53 (d, J ) 7.0
Hz, 3 H), 1.98 (s, 3 H), 2,21 (pentet, J ) 7.0 Hz, 2 H), 2.45 (t,
J ) 7.0 Hz, 2 H), 3.86 (s, 3 H), 4.10 (t, J ) 7.0 Hz, 2 H), 4.44
(d, J ) 6.3 Hz, 2 H), 5.47 (dq, J ) 7.0, 7.0 Hz, 1 H), 5.98 (br t,
1 H), 6.39 (br d, J ) 7.0 Hz, 1 H), 6.87 (s, 1 H), 7.21-7.34 (m,
5 H), 7.53 (s, 1H); 13C NMR (75.5 MHz, CDCl3 plus several
drops of DMSO-d6) δ 21.3, 22.6, 24.6, 32.2, 43.0, 45.6, 55.9,
68.3, 109.2, 109.6, 126.8, 127.3, 128.1, 134.8, 138.4, 139.8,
146.3, 153.7, 169.1, 171.8; MS (ESI) m/ z 430 (MH)+. Anal.
Calcd for C22H27N3O6‚0.3H2O: C, 60.76; H, 6.40; N, 9.66.
Found: C, 60.40; H, 6.42; N, 9.26.
Meth yl 4-(4-(1-(Hyd r oxyim in o)eth yl)-2-m eth oxyp h e-
n oxy)bu ta n oa te (22). To a solution of the keto-ester 14 (68.4
g) in 225 mL of 2:1 pyridine:H2O was added hydroxylamine
hydrochloride (21.46 g, 309 mmol). After stirring at room
temperature for 14 h the reaction mixture was partitioned
between EtOAc and saturated NaCl. The organic phase was
dried (MgSO4), filtered, and evaporated to dryness to afford
oxime 22 (69.94 g, 100% crude yield) as a white solid: mp 82-
83 °C; 1H NMR (300 MHz, CDCl3) δ 2.16 (pentet, J ) 8.0 Hz,
2 H), 2.26 (s, 3 H), 2.55 (t, J ) 8.0 Hz, 2 H), 3.68 (s, 3 H), 3.88
(s, 3 H), 4.09 (t, J ) 8.0 Hz, 2 H), 6.87 (d, J ) 8.8 Hz, 1 H),
7.12 (dd, J ) 2.1, 8.8 Hz, 1 H), 7.25 (d, J ) 2.1 Hz, 1 H); 13C
NMR (75.5 MHz, CDCl3) δ 11.9, 24.4, 30.4, 51.6, 55.9, 67.8,
109.0, 112.4, 119.1, 129.5, 149.3, 149.4, 155.6, 173.6; MS
(APCI) m/ z 282 (MH+). Anal. Calcd for C14H19NO5: C, 59.78;
H, 6.81; N, 4.98. Found: C, 59.62; H, 6.75; N, 4.81.
Meth yl 4-(2-Meth oxy-5-n itr o-4-(1-(tr iflu or oa ceta m id o)-
eth yl)p h en oxy)bu ta n oa te (24). Trifluoroacetate 23 (9.40 g,
25.9 mmol) was slowly added to 200 mL of 70% HNO3 cooled
to 0 °C. The solution turned orange in color and was quenched
after 2 h by pouring into water and adjusting the total volume
to 2 L. The resultant slurry was chilled to 4 °C overnight and
filtered to give a light yellow solid. The solid was washed with
water and recrystallized from MeOH/H2O to afford the nitrated
acetate 24 (9.07 g, 86% yield) as a light yellow solid: mp 156-
1
157 °C; H NMR (300 MHz, CDCl3) δ 1.62 (d, J ) 7.9 Hz, 3
H), 2.18 (pentet, J ) 7.5 Hz, 2 H), 3.70 (s, 3 H), 3.94 (s, 3 H),
4.12 (t, J ) 7.5 Hz, 2 H), 5.50 (dq, J ) 7.9, 7.9 Hz, 1 H), 6.87
(s, 1 H), 7.37 (br d, J ) 7.9 Hz, 1 H), 7.61 (s, 1 H); 13C NMR
(75.5 MHz, CDCl3) δ 20.1, 24.2, 30.3, 48.4, 51.7, 56.4, 68.2,
110.2, 111.0, 130.9, 140.5, 147.6, 154.0, 156.6, 173.3, the
resonance for NCOCF3 was not observed; MS (APCI) m/ z 409
(MH+). Anal. Calcd for C16H19F3N2O7‚0.05H2O: C, 46.96; H,
4.70; N, 6.85. Found: C, 46.59; H, 4.78; N, 6.89.
4-(4-(1-(9-F lu or en ylm et h oxyca r b on yla m in o)et h yl)-2-
m eth oxy-5-n itr op h en oxy)bu ta n oic Acid (26). To a solu-
tion of the nitrated compound 24 (12.36 g, 30.27 mmol) in 250
mL of warm MeOH was added 1 N NaOH (100 mL, 100 mmol),
and the resultant solution was heated to reflux for 5 h. The
solution was cooled to room temperature and concentrated to
approximately 100 mL with a rotary evaporator. p-Dioxane
(150 mL) and H2O (100 mL) were added, and the pH of the
solution was adjusted to pH 9 with 6 N HCl. A solution of
Fmoc-Cl (9.83 g, 38.0 mmol) in 100 mL of dioxane was added,
and an additional 25 mL of dioxane was added to create a
homogeneous solution. The pH of the solution was adjusted
with 1 N NaOH to pH 8 over the next 30 min, and a light
yellow precipitate formed during this time. The reaction was
quenched after 18 h by adding 100 mL of 1 N HCl and
adjusting the total volume to 1 L with H2O. The precipitate
was collected, taken up in 1 L of hot EtOAc, dried over MgSO4,
and filtered while hot. The solvent was removed under
reduced pressure, affording a light yellow solid which was
triturated with 1 L of hot Et2O. The solid was collected and
was recrystallized from MeOH to afford the Fmoc linker 26
(12.78 g, 81% yield for two steps) as a light yellow solid: mp
200-201 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.43 (d, J ) 7.4
Hz, 3 H), 1.95 (pentet, J ) 7.3 Hz, 2 H), 2.39 (t, J ) 7.3 Hz, 2
H), 3.87 (s, 3 H), 4.05 (t, J ) 7.3 Hz, 2 H), 4.16 (t, J ) 6.9 Hz,
1 H), 4.27 (m, 2 H), 5.19 (dq, J ) 7.4 Hz, 1 H), 7.25 (s, 1 H),
7.29 (t, J ) 7.3 Hz, 2 H), 7.39 (t, J ) 7.3 Hz, 2 H), 7.48 (s, 1
H), 7.64 (d, J ) 8.3 Hz, 2 H), 7.87 (d, J ) 8.3 Hz, 2 H), 8.06 (d,
J ) 8.3 Hz, 1 H); 13C NMR (75.5 MHz, DMSO-d6) δ 21.9, 24.0,
29.9, 46.0, 46.7, 56.2, 65.2, 67.9, 108.2, 109.4, 120.1, 125.0,
126.9, 127.6, 135.5, 139.9, 140.7, 143.6, 143.9, 146.3, 153.4,
Meth yl 4-(2-Meth oxy-4-(1-(tr iflu or oa ceta m id o)eth yl)-
p h en oxy)bu ta n oa te (23). A slurry of the oxime 22 from
above (146.7 mmol) and 10% palladium on charcoal (2.5 g) in
400 mL of glacial acetic acid was degassed twice by placing
the flask under reduced pressure with an aspirator and
subsequently filling the flask with H2. The reaction mixture
was placed under 1.1 atm of hydrogen gas via a balloon and
vigorously stirred at room temperature. An additional 2 g of
catalyst was added after 18 h, and the balloon was refilled
with hydrogen as the gas was consumed. An additional 2 g of
catalyst was added after 2 days. The reaction mixture was

2380 J . Org. Chem., Vol. 62, No. 8, 1997
Holmes
155.3, 174.0; MS (APCI) m/ z 551 (MH+), 282, 179. Anal.
Calcd for C28H28N2O8‚0.3H2O: C, 63.94; H, 5.48; N, 5.33.
Found: C, 63.57; H, 5.44; N, 5.54.
dride (prepared from 182 mg of Fmoc-Gly-OH and 50 µL of
DIC in 0.6 mL of DMF) was coupled to the resin for 1 h
followed by washing as before. Deprotection of the Fmoc group
with piperidine, washing, and drying as above gave roughly
150 mg of dry resin. A portion of the resin (40 mg) was
transferred into a 4-mL vial, acetonitrile (2 mL), 3 Å molecular
sieves (20-30 pellets), benzaldehyde (carbonyl-13C, 99%) (152
µL), and mercaptoacetic acid (300 µL) were added, and the vial
was heated to 70 °C for 2 h. The resin was transferred to a
disposable filter tube and washed extensively (3 × 5 mL of
CH2Cl2, 3 × 5 mL of DMF, 3 × 5 mL of CH2Cl2, 3 × 5 mL of
MeOH, 3 × 5 mL of CH2Cl2, 3 × 5 mL of Et2O).
4-(4-(1-Hyd r oxyeth yl)-2-m eth oxy-5-n itr op h en oxy)bu -
ta n oic Acid (27). To a solution of ester 15 (24.19 g, 77.72
mmol) in 400 mL of THF and 200 mL of MeOH was added
NaBH4 (3.61 g, 95.4 mmol) at room temperature. An ad-
ditional 1 g (26.4 mol) of NaBH4 was added after stirring for
1 h. The reaction mixture was stirred for 15 h at room
temperature by which time TLC indicated that complete
reduction had taken place. 1 N NaOH (200 mL, 0.200 mmol)
and H2O (100 mL) were added and the reaction stirred 7 h.
The reaction mixture was concentrated under reduced pres-
sure to approximately 400 mL, carefully acidified with 6 N
HCl, and then extracted with EtOAc. The combined organic
phase was dried (MgSO4) and evaporated to give a yellow
residue. Recrystallization from EtOAc/hexane afforded pho-
tolinker 27 (16.61 g, 71% yield) as a pale yellow solid: mp 164-
Gen er a l P h otolysis Con d ition s. Solution photolyses
were conducted with a 1000 W Hg(Xe) ARC lamp fitted with
a water filter and a 350-450 nm dichroic reflector on a
horizontal axis. The output was passed through a collimating
lens (Oriel Corp.) and the sample (0.10 mM) irradiated by
placing a quartz 0.2 cm path length cuvette in the beam for
various times. The power level at the cuvette was adjusted
to 10 mW/cm2 as determined with a Optical Associates Inc.
UV powermeter with a 365 nm wavelength probe. The percent
remaining starting material was determined via reverse-phase
C18 HPLC analysis of the photolyzed solutions with 220 nm
detection.
1
168 °C dec; H NMR (300 MHz, CDCl3 plus several drops of
DMSO-d6) δ 1.49 (d, J ) 7.0 Hz, 2 H), 2.15 (pentet, J ) 7.0
Hz, 2 H), 2.52 (t, J ) 7.0 Hz, 2 H), 3.98 (s, 3 H), 4.13 (t, J )
7.0 Hz, 2 H), 4.60 (bs, 1 H), 5.53 (q, J ) 7.0 Hz, 1 H), 7.40 (s,
1 H), 7.57 (s, 1 H); 13C NMR (75.5 MHz, CDCl3 plus several
drops of DMSO-d6) δ 23.9, 24.7, 30.0, 56.0, 64.6, 68.0, 108.5,
108.7, 138.3, 138.8, 146.2, 153.8, 174.6; MS (APCI) 300 (MH)+.
Anal. Calcd for C13H17NO7: C, 52.17; H,, 5.73; N, 4.68.
Found: C, 52.50; H, 5.63; N, 4.72.
Resin photolyses were conducted with 50 mg of resin
suspended in 1-2 mL of pH 7.4 PBS buffer containing 5% of
DMSO. Photolyses were conducted by irradiating the samples
with a 500 W Hg ARC lamp fitted with a 350-450 nm dichroic
mirror on a vertical axis at a 10 mW/cm2 power level measured
at 365 nm. The samples were irradiated from above with
gentle mixing from an orbital shaker table. After photolysis
the supernatant was analyzed by reverse-phase HPLC and the
resin analyzed via gel phase 13C NMR for the disappearance
of the thiazolidinone resonance.
4-Th iazolidin on e-P h otolin ker -Glycin e-Ten taGel (28).
TentaGel-S-NH2 (0.50 g, 0.30 mmol/g loading) was washed
with DMF, and a 0.25 M solution of Fmoc-glycine (2-13C, 99%)
symmetrical anhydride (prepared from 182 mg of Fmoc-Gly-
OH and 50 µL of DIC in 1.2 mL of DMF) was coupled to the
resin for 1 h, by which time ninhydrin had revealed that a
complete reaction had taken place. The resin was washed with
DMF, capped with 20% Ac2O, 30% pyridine, and 50% CH2Cl2
for 30 min, and washed with CH2Cl2, DMF, and MeOH. The
Fmoc group was removed by incubating the resin in 30%
piperidine/DMF for 30 min, followed by washing with DMF.
The resin was treated with a 0.20 M solution of OBt-activated
Fmoc-photolinker 26 (prepared from 310 mg of 26, 92 mg of
HOBt, 95 µL of DIC in 3 mL of DMF) for 16 h. Ninhydrin
test indicated a complete reaction had taken place. The resin
was washed with DMF and CH2Cl2 and was then capped with
20% Ac2O, 30% pyridine, and 50% CH2Cl2 for 30 min, followed
by washing with DMF and CH2Cl2. The resin was deprotected
with 30% piperidine/DMF for 30 min and then washed with
DMF. A 0.5 M solution of Fmoc-glycine symmetrical anhy-
Sta bility of 28 Tow a r d TF A Tr ea tm en t. A portion of
resin 28 was treated with 95% TFA/5% H2O for 1 h, followed
by washing with CH2Cl2, MeOH, and Et2O. In a separate
experiment, an additional 20 mg of resin was treated for 2 h
at room temperature with a solution of phenol (75 mg),
thioanisole (50 µL), water (50 µL), ethanedithiol (25 µL), and
TFA (1 mL) followed by washing as above. Gel 13C NMR
analysis of the resin indicated no loss of thiazolidinone or
photolabile linker, as evidenced by the relative integration of
the two labeled carbons.
J O961602X