A three-component photoreversible tag for thiols

Article abstract of DOI:10.1021/jo0481396

(Chemical Equation Presented) A one-pot coupling of a 1,3-diketone, an aldehyde, and an alkanethiol has been developed to produce a protected sulfide. Through use of an o-nitrophenylbenzaldehyde, this method provides a one-step route to a photochemically reversible thiol-protecting group. The kinetics of photolysis were established using ¹H NMR analysis, which allows for the rate to be based on the entire reaction scheme.

Full text of DOI:10.1021/jo0481396

SCHEME 1. Synthesis and Processing of
o-Nitrobenzyl Derivatives (1) as a Photoremovable
Thiol Modificationa
A Three-Component Photoreversible Tag
for Thiols
Kristine M. Clarke, James J. La Clair, and
Michael D. Burkart*
Department of Chemistry and Biochemistry,
University of California San Diego, 9500 Gilman Drive,
La Jolla, California 92093-0358
mburkart@ucsd.edu
Received October 20, 2004
a Key: (a) CH2Cl2, dried SiO2; (b) addition of 5, CH2Cl2; (c)
CDCl3, λ ) 365 nm; (d) Zn, TFA, CH2Cl2/MeOH or 1,4-dithiothrei-
tol.
on coupling of a thiol to an alkyl halide or an R,â-
unsaturated diketone.5,6 While effective, these reactions
are often complicated by the synthetic complexity of
forming appropriate thiol-reactive labels.
A one-pot coupling of a 1,3-diketone, an aldehyde, and an
alkanethiol has been developed to produce a protected
sulfide. Through use of an o-nitrophenylbenzaldehyde, this
method provides a one-step route to a photochemically
reversible thiol-protecting group. The kinetics of photolysis
To achieve a photocleavable Michael acceptor, we
propose the in situ generation of a 2-ene-1,3-dione via a
Knoevenagel condensation.7 Surprisingly, this approach
has not yet been advanced as a general tool for thiol
modifications. Using this methodology, we developed a
one-pot, three-component method to generate a photo-
cleavable-protected thiol (Scheme 1). Thioether 1 repre-
sents the general components of this system: a “tuner”,
an “activator”, and a target thiol. The tuner is an
o-nitrobenzaldehyde, the cleavage wavelength of which
has been demonstrated to be attenuated by ether func-
tionalities at positions 4 and 5.8 The activator is a reactive
nucleophilic 1,3-diketone that forms an enedione upon
Knoevenagel condensation with the tuner aldehyde. To
investigate the utility of this o-nitrophenyl protecting
group, a simple alkylthiol, ethanethiol, was protected by
combining 5,5-dimethylcyclohexane-1,3-dione (2b) and
6-nitroveratraldehyde (3c) to yield the protected alkyl-
thiol 7 in 63% yield. As shown in Scheme 1, diketone
activator 2 and aldehyde tuner 3 couple via Knoevenagel
condensation to form intermediate 4 in situ. Enone 4 then
serves as a Michael acceptor for thiol 5. The protected
thiol is deprotected by light irradiation, yielding disulfide
6 and the photolysis byproduct 4. Enone 4 is further
hydrolyzed to o-nitrobenzaldehyde 3 according to 1H-
NMR.
1
were established using H NMR analysis, which allows for
the rate to be based on the entire reaction scheme.
The modification of functional groups with photocleav-
able moieties is a versatile tool for organic synthesis,1
the development of small molecule libraries,2 and the
decoding of proteomic and genomic queries.3 Modifica-
tions by appropriate photocleavable moieties allows for
orthogonal protection in complex synthetic pathways and
in situ deprotection where exposure to a photon flux is
allowed. While the development of photocleavable pro-
tecting groups for alcohols and amides has been estab-
lished for solution, bead, and surface arrays,3b,4 only a
few methods exist for thiols. Established methods focus
(1) (a) Pillai, V. N. R. Synthesis 1980, 1-26. (b) Bochet, C. G. J.
Chem. Soc., Perkin Trans. 1 2002, 125-142. (c) Greene, T. W.; Wuts,
P. G. M. Protective Group in Organic Synthesis, 3rd ed.; Wiley: New
York, 1999; pp 545-547.
(2) (a) Smith, A. B., III; Savinov, S. N., Manjappara, U. V.; Chaiken,
I. M. Org. Lett. 2002, 4, 4041-4044. (b) Tan, D. S.; Foley, M. A.;
Stockwell, B. R.; Shair, M. D.; Schreiber, S. L. J. Am. Chem. Soc. 1999,
121, 9073-9087. (c) Baldwin, J. J.; Burbaum, J. J.; Henderson, I.;
Ohlmeyer, M. H. J. J. Am. Chem. Soc. 1995, 117, 5588-5589.
(3) (a) Pirrung, M. C.; Wang, L.; Montague-Smith, M. P. Org. Lett.
2001, 3, 1105-1108. (b) Seo, T. S.; Bai, X.; Ruparel, H.; Li, Z.; Turro,
N. J.; Ju, J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5488-5493. (c)
Bai, X.; Li, Z.; Jockusch, S.; Turro, N.; Ju, J. Proc. Natl. Acad. Sci.
U.S.A. 2004, 100, 409-413. (d) Curley, K.; Lawrence, D. S. J. Am.
Chem. Soc. 1998, 120, 8573-8547. (e) Chang, C.; Fernandez, T.;
Panchai, R.; Bayley, H. J. Am. Chem. Soc. 1998, 120, 7661-7662. (f)
Ghosh, M.; Ichetovkin, I.; Song, X.; Condeelis, J. S.; Lawrence, D. S.
J. Am. Chem. Soc. 2002, 124, 2440-2441.
Traditionally, the extent of photolytic cleavage is
quantified by UV-vis spectroscopy,9 time-resolved FTIR
(5) (a) Hazum, E.; Gottlieb, P.; Amit, B.; Patchornik, A.; Fridkin,
M. In Peptides: Proc. Eur. Pept. Symp., 16th; Brunfeldt, K., Ed.;
Scriptor Publ.: Copenhagen, 1981; p 105. (b) Marriot, G.; Heidecker,
M. Biochemistry 1996, 35, 3170.
(6) Golan, R.; Zehavi, U.; Naim, M.; Patchornik, A.; Smirnoff, P.
Biochim. Biophys. Acta 1996, 1293, 238-242.
(7) Fuchs, K.; Paquette, L. A. J. Org. Chem. 1994, 59, 528-532.
(8) (a) Bochet, C. G. Tetrahedron Lett. 2000, 41, 6341-6346. (b)
Blanc, A.; Bochet, C. G. J. Org. Chem. 2002, 67, 5567-5577.
(4) (a) Pirrung, M. C.; Bradley, J. C. J. Org. Chem. 1995, 60, 1116-
1117. (b) Furuta, T.; Hirayama, Y.; Iwamura, M. Org. Lett. 2001, 3,
1809-1812. (c) Hamada, T.; Nishida, A.; Yonemitsu, O. Tetrahedron
Lett. 1989, 30, 4241-4244.
10.1021/jo0481396 CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/26/2005
J. Org. Chem. 2005, 70, 3709-3711
3709

increases at 295 nm and 340 nm. The absence of a clear
isosbestic point at 275 nm indicates the formation of
multiple products (8 and 3c), which is in agreement with
1
the HNMR.
While the rates of o-nitrophenyl photocleavage reac-
tions are often linked to decomposition of aci-nitro
intermediates,12 photolytic deprotection has been shown
to be orders of magnitude slower than the breakdown of
the aci-nitro group.9a By comparison of the disappearance
of starting material and the formation of the product with
1H NMR, we were able to monitor photolytic kinetics of
the complete reaction. This obviated the need to deter-
mine whether the rate-determining step consists of the
decomposition of aci-nitro group or the decay of secondary
intermediates.
To demonstrate the utility of this methodology, an 11-
membered set of protected thiols was prepared from
Fmoc-cystamine, four diketone activators (2a-d), and
three nitrobenzadehydes (3a-c) (Figure 3). The yields
ranged from 41% to 96%. Initially, the derivatives
containing 1,3-indandione (11a and 11b) were isolated
and purified. However, upon standing the 1,3-indandione
Knoevenagel products proved to be poor Michael accep-
tors. As a result, the product readily undergoes â-elim-
ination of the thiol, reverting to the enone intermediate.
Each of the purified compounds of the library was
subjected to photolysis at 365 nm. The percent of al-
kanethiol cleaved ranged from 55% to 86%.14 The rate of
each deprotection was monitored by 1H NMR. The rates
of photocleavage are on the same order as current
literature.9a As expected, the 6-veratraldehyde deriva-
tives cleaved twice as fast as the 6-nitro and 6-nitro-
piperonal derivatives. This increased rate of thiol cleav-
age and amount of thiol cleaved, as compared to the nitro
and piperonal derivatives, may be attributed to the 3,4-
dimethoxy substituents on the nitro benzaldehyde in 1c,
9c, and 10c.8
We also sought to demonstrate the utility of this
protecting group by assessing its stability under condi-
tions used in organic synthesis.1c The protected thiol 7
was found to be stable under basic conditions (NaH, Pyr,
TEA, piperidine, and NaOMe); Lewis acid (TsOH); and
strong acid (TFA). The protecting group was stable to
heat (<100 °C). The photolytic cleavage may also be
performed in the presence of other protecting groups such
as N-(9-fluorenylmethoxycarbonyl) (Fmoc).
We have demonstrated rapid entry into a three-
component protection strategy that provides flexibility
for additional functionality or specific photochemical
reactivity. The three component thiol protecting group
is also stable to many conditions commonly used in
organic synthesis. This methodology should find useful
applications in organic synthesis, the development of
small molecule libraries,and the decoding proteomic and
genomic queries.
FIGURE 1. 1H NMR analysis of photolytic cleavage of 7 (red)
with periodic irradiation at λ ) 365 nm in CDCl3 to 8 (blue)
and 9 (magenta). The CDCl3 peak has been removed (*) to
simplify the spectra.
FIGURE 2. UV-vis spectral monitor of the photolysis of 7
at λ ) 365 nm in CDCl3.
spectroscopy,9a,10 or HPLC.11 This thiol protecting group,
however, provides a convenient handle to monitor pho-
tolytic cleavage by 1H NMR. Figure 1 displays an 1H
NMR time course of the photolytic deprotection of
ethanethiol at 365 nm over 60 min. The change in
hybridization of the â-carbon of protected ethanethiol 7
to photolytic product 8, from sp3 f sp2, provided a means
to monitor the photolytic reaction. Upon photolytic cleav-
age, the proton signal at δ 6.33 ppm (7) disappears and
a new signal at δ 8.28 ppm appears. In addition to the
formation of disulfide and enone 8, a third product
6-nitroveratraldehyde 3c arises from the hydrolysis of
1
enone 8. Both photolytic byproducts had H NMR and
13C NMR identical to authentic samples. Photolytic
deprotection could also be followed by UV-vis. Figure 2
depicts the photolysis of protected thiol 7 between 220
and 600 nm over 60 min. Photolytic cleavage provides
the formation of photolytic byproduct 8 whose absorption
(12) (a) Walker, J. W.; Reid, G. P.; McCray, J. A.; Trentham, D. R.
J. Am. Chem. Soc. 1988, 110, 7170-7177. (b) Barth, A.; Corrie, J. E.
T.; Gradwell, M. J.; Maeda, Y.; Mantele, W.; Meier, T.; Trentham, D.
R. J. Am. Chem. Soc. 1997, 119, 4149-4159.
(13) Peng, L.; Goeldner, M. J. Org. Chem. 1996, 61, 185-191.
(14) The percent thiol cleaved was calculated based monitoring the
disappearance of the benzylic proton at δ 6.33 ppm and standardizing
it to doublet of the Fmoc protecting group.
(9) (a) Il’ichev, Y.; Schwo¨rer, M.; Wirz, J. J. Am. Chem. Soc. 2004,
126, 4581-4595. (b) Abbruzzetti, S.; Carcelli, M.; Rogolino, D.;
Viappiani, C. Photochem. Photobiol. Sci. 2003, 2, 796-800.
(10) Corrie, J. E. T.; Barth, A.; Munasinghe, V. R. N.; Trentham,
D. R.; Hutter, M. C. J. Am. Chem. Soc. 2003, 125, 8546-8554.
(11) Specht, A.; Goeldner, M. Angwe. Chem., Int. Ed. 2004, 43,
2008-2012.
3710 J. Org. Chem., Vol. 70, No. 9, 2005

FIGURE 3. Set of protected alkanethiols. Key: (a) yield of purified protected thiol from Knoevenagel condensation and Michael
addition; (b) percent thiol cleaved was determined by 1HNMR analysis after 50 min of irradiation; (c) quantum yield (Φ) determined
by measuring the number of reactions/photon. This can be compared to 8.71 × 10-14 for 1-(2-nitrophenyl)ethylcholine (Φr ) 0.27).13
Experimental Section
Acknowledgment. We thank Dr. Yongxuan Su and
the Mass Spectrometry Facility at the University of
California, San Diego, for running the mass spectrom-
etry on all compounds. We also thank Dr. Tammy
Dwyer at the University of San Diego for assistance
with NMR experiments and helpful discussions. We
gratefully acknowledge the University of California, San
Diego Department of Chemistry and Biochemistry, for
funding. K.M.C. was partially supported by a US
Department of Education GAANN fellowship. This
manuscript is warmly dedicated to Chris Walsh on the
occasion of his 60th birthday.
General Procedure for Knoevenagel Condensation:
Michael Addition. 2-[(4,5-Dimethoxy-2-nitrophenyl)ethyl-
sulfanylmethyl]-3-hydroxy-5,5-dimethylcyclohex-2-
enone (7) (Representative Example). 1,3-Cyclohexadione 2b
(500 mg, 3.6 mmol) was added to 6-nitrobenzadehyde 3c (1.29
g, 6.1 mmol) in CH2Cl2 (20 mL) containing dried silica gel (50
mg). The reaction vessel was purged with Ar, and ethanethiol
(0.29 mL, 4.0 mmol) was added in CH2Cl2 (5 mL). After 4 h, the
reaction was concentrated to 10% volume and purified via flash
chromatography (hexanes, EtOAc) to yield a yellow foam 7: 63%
yield; Rf (1:2 hexanes/ethyl acetate) 0.44; 1H NMR (CDCl3) δ
(ppm) 0.95 (3H, s), 1.00 (3H, s), 1.24 (3H, t, 7.2 Hz), 2.17 (H, d,
J ) 16.3 Hz), 2.25 (H, d, 16.9), 2.40 (H, d, 17.6 Hz), 2.52 (H, d,
17.6 Hz), 2.59-2.72 (2H, m), 3.80 (3H, s), 3.81 (3H, s), 6.18 (H,
s), 6.90 (H, s), 7.44 (H, s), 10.70 (H, s); 13C NMR (CDCl3) δ (ppm)
14.0, 27.7, 27.6, 29.1, 31.9, 39.7, 44.1, 50.6, 56.4, 56.6, 108.9,
110.8, 110.9, 129.6, 140.8, 148.1, 153.0, 174.5, 197.1;1 IR (thin
film) (max/cm-1) 2961, 1614, 1578, 1521, 1274, 1058; MS [M -
H]- 393.98; HRMS (FAB) calcd for C19H26O6NS 396.1475 [M +
H]+, found 396.1479 m/z.
Supporting Information Available: Experimental pro-
cedures, characterization data, and 1H NMR and 13C NMR for
compounds 1a-c, 6-8, 9a-c, and 10a-c. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO0481396
J. Org. Chem, Vol. 70, No. 9, 2005 3711