Photoamplification and multiple tag release in a linear peptide-based array of dithiane adducts

Article abstract of DOI:10.1002/anie.200701512

(Chemical Equation Presented) Binary encoding and amplification can be achieved in linear polypeptide-based scaffolds of photoactive dithiane adducts. QA masked sensitizer is tethered to lysine and incorporated in a peptide chain terminated with a sensiti

Full text of DOI:10.1002/anie.200701512

Angewandte
Chemie
DOI: 10.1002/anie.200701512
Photoamplification
Photoamplification and Multiple Tag Release in a Linear Peptide-
Based Array of Dithiane Adducts**
Kavitha Majjigapu, Janaki R. R. Majjigapu, and Andrei G. Kutateladze*
Externally sensitized photoinduced fragmentation in dithiane
adducts of carbonyl compounds[1] requires the presence of an
electron-transfer sensitizer, and therefore can be made
contingent on a molecular recognition event. This event
“arms” the binary photoactive system by bringing the adduct
and the sensitizer into the immediate proximity of each
other.[2] Such conditional photorelease of dithianes, which are
readily detectable at subpicomolar levels, presents new
opportunities for useful bioanalytical applications.
We have recently developed this concept into a funda-
mentally novel methodology for direct screening of solution-
phase combinatorial libraries, in which various 2-alkyl-
substituted dithianes are used as encoding digits.[3] By
immobilizing dithiane-masked benzophenone units on poly-
meric beads or dendrimers, we have further shown that one
molecular recognition event can trigger the release of multi-
ple copies of the encoding dithiane tags, which amounts to
amplification on the surface.[4] Such photoamplification is
possible because each externally sensitized fragmentation of
the benzophenone–dithiane adducts unmasks more sensitizer
which, in turn, unmasks its neighbors carrying the amplifica-
tion chain and therefore boosting the sensitivity.
The next logical question is whether this photoamplifica-
tion methodology can be implemented in a linear array of
masked sensitizers, and—if such amplification is possible—
whether the propagation of the effect and the release of
dithiane tags occurs sequentially (that is, not unlike the one-
dimensional propagation in the Bickford fuse), randomly, or
Scheme 1. Synthesis of photoactive lysine conjugates. EDC=1-ethyl-3-
[3-dimethylaminopropyl]carbodiimide; Fmoc=9-fluorenylmethoxycar-
bonyl.
in some other unusual order. Herein, we report on photo-
amplification in linear polypeptide scaffolds.
esters 5a–d, were utilized in solid-state peptide synthesis on
TentaGel beads (0.48 mmolgÀ1). The peptides were capped
with lysine-tethered benzophenone as the photoinitiator. As
linear amplification in a single strand was the focus of our
study, we chose the low-loading beads as a scaffold mimicking
infinite dilution in solvent (that is, preventing interstrand
sensitization; see Supporting Information).
The synthesis of masked benzophenone sensitizers teth-
ered to Fmoc-protected lysine is outlined in Scheme 1. We
chose four 2-substituted dithianes, ethyl (a), propyl (b), pentyl
(c), and octyl (d), to positionally encode the lysine residues in
the polypeptide chain. The lithiated dithianes were reacted
with 3-benzoylbenzoic acid sodium salt (1), thus furnishing
adducts 2a–d, which were converted into N-hydroxysuccini-
mide (NHS) esters 3a–d and tethered to Fmoc-protected
lysine. The modified lysines 4a–d, in the form of their NHS
The following tetra-, hepta-, and decapeptides, which
contain photoactive lysine residues and spacers, were synthe-
sized:
LysBP-LysDT1-LysDT2-LysDT3-BEAD (6a)
LysBP-Gly-LysDT1-Gly-LysDT2-Gly-LysDT3-BEAD (6b)
LysBP-Met-LysDT1-Met-LysDT2-Met-LysDT3-BEAD (6c)
[*] K. Majjigapu, Dr. J. R. R. Majjigapu, Prof. Dr. A. G. Kutateladze
Department of Chemistry and Biochemistry
University of Denver
LysBP-Gly-Gly-LysDT1-Gly-Gly-LysDT2-Gly-Gly-LysDT3
-
BEAD (6d)
2190 E. Iliff Ave., Denver, CO 80208-2436 (USA)
Fax: (+1)303-871-2254
E-mail: akutatel@du.edu
where LysDTX represents a lysine residue outfitted with
benzophenone masked with 2-alkyldithiane, and LysBP is
lysine that carries “free” tethered sensitizer. The standard
synthetic Fmoc–piperidine protocol was used, except that to
assure completion at each step of the peptide synthesis, the
time for coupling of NHS esters 5a–d was increased to 3 days
Homepage: http://www.biochem.du.edu/kgroup
[**] This work is supported by NSF and NIH.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2007, 46, 6137 –6140
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6137

Communications
per residue. Mono- and bis(glycyl) spacers were introduced to
probe the distance dependence, whereas methionine was
tested as a potential co-sensitizer or quencher. The beads
were suspended in acetonitrile, degassed, and irradiated using
a 330-nm long-pass filter to selectively excite the sensitizer.
Subsequent release of dithianes into solution was monitored
by GC–MS. After 10 min of irradiation all three dithianes
were found in the solution, thus indicating that linear
amplification and binary encoding in a linear array of dithiane
tags are possible.
This finding is critical to the development of our method-
ology for encoding and screening of solution-phase combina-
torial libraries. A single library member can now be readily
encoded with a complete stringed set of tags, which are
necessary for its subsequent identification. Beyond this, the
unusual ordering of the initial relative efficiencies presented
an interesting opportunity to probe the mechanism of the
release. The bar graph in Figure 1 shows the relative dithiane
Figure 1. Release of dithianes after irradiation for 10 min as a function
of their position in polypeptides 6a–d (sensitizer is at position 0).
peak intensities after irradiation for 10 min as a function of
their position in the polypeptide chain. The results illustrate
that the overall efficiency of release from the position most
distal to the sensitizer is generally higher.
A more detailed analysis of the release is shown in
Figure 2. As a result of the secondary photooxidation of the
released dithiane, its concentration over the course of
photolysis reaches a maximum and then decreases. We
approximate this release by a trinomial function A = at3 +
bt2 + ct, where term c gives us @A/@t at time zero, which
allows an accurate comparison of the initial rates of release
(values of c are bold in Figure 2). Again, as a rule, the most
distal dithianes were released with higher initial efficiency.
We suggest that the outcome of photoamplification in
peptides is governed by the migration of the formed radical
cation from one dithianyl moiety to the next in the chain. Such
charge transfer between photoactive residues should be
unbiased and random, because the oxidation potentials of
alkyl dithianes are nearly degenerate and the system does not
have thermodynamic sinks, other than the wasteful back-
electron-transfer (BET) trap. The probability of BET from
the benzophenone radical anion is the lowest for the most
distal dithiane, and accounts for the experimental observation
Figure 2. Release of dithianes from peptides 6a–d; A=area. Position
~
&
*
from the sensitizer: proximal, middle, distal.
6138
www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 6137 –6140

Angewandte
Chemie
in which the most efficient release is from the distal position
in 6b–d (Scheme 2). As a result of BET, the proximal radical
cation does not live long enough to fragment efficiently. This
stochastic approach provides rationale for most of the
Scheme 3. Charge-density migration through dimeric radical cations.
even though Met+C is known to form 2c3e bonds with various
heteroatoms.[7]
To assess the effect of the polymeric matrix on the
amplified release, the photoactive dithiane adducts were
immobilized in the reverse order of the glycine-separated
heptapeptide 6b:
Scheme 2. Radical-cation hopping in the polypeptide chain.
LysDT3-Gly-LysDT2-Gly-LysDT1-Gly-LysBP-BEAD (7b)
that is, the lysine carrying the sensitizer was immobilized
first. Irradiation of 7b produced the same preferential release
of the distal dithiane (from the terminal LysDT3), thus attesting
to the generality of the described phenomenon.
The second control was to utilize the dithiane adducts of
4-formylbenzoic acid, which are not capable of amplification.
Upon irradiation of 8b (Figure 3) we see the most distal
experimental observations, if one recalls that as soon as the
fragmentation occurs a fresh copy of the sensitizer is
unmasked. The unmasked sensitizer is in turn excited,
which initiates similar radical-cation hopping away from the
radical anion, with the most distal position this time being the
one closest to the original terminal benzophenone.
One peculiar experimental observation is the release from
tetrapeptide 6a (Figure 2), which has no spacers between the
photoactive lysine residues. In this peptide, the release of the
two distal dithianes seems to occur simultaneously. This result
may point to a specific mode of radical-cation propagation in
a system where dithiane adducts are spatially close to each
other and, therefore, are capable of forming interdithiane
À
two-center, three-electron (2c3e) S S bonds. Such 2c3e bonds
are well-precedented in the literature.[5]
Previously, we have also shown that additional stabiliza-
tion in bis(dithianyl) radical cations considerably increases
their quantum yields of fragmentation.[6] We therefore suggest
that in tight systems, such as 6a, propagation of the radical
cation may occur through the direct formation of 2c3e bonds,
so that the charge density migrates in the form of a dimeric
dithiane radical cation (Scheme 3). Release of either of the
dithianes from these pairs has near-equal probability, which
explains the comparable initial efficiencies of release from the
second (1203) and third (997) adducts. The dimer formed by
the first and second dithianes is again at a disadvantage of
BET. The introduction of methionine (Met) as a spacer (cf. 6b
and 6c) produced an overall effect of a competitive quencher,
Figure 3. Peptide 8b based on benzaldehyde adducts, which is not
capable of amplification.
dithiane eventually released into solution, whereas the
combined relative quantum efficiency of the release from
the aldehyde-based linear array is an order of magnitude
smaller. Most likely this is caused by the premature reduction
of the only sensitizer in the strand during the extended
photolysis needed to fragment all three dithiane adducts. In
the amplified release from 6a–d and 7b every fragmentation
Angew. Chem. Int. Ed. 2007, 46, 6137 –6140
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
6139

Communications
event replenishes the sensitizer, which carries the photo-
amplification chain.
[1] W. McHale, A. G. Kutateladze, J. Org. Chem. 1998, 63, 9924 –
9931.
[2] S. Lakkakula, O. D. Mitkin, R. A. Valiulin, A. G. Kutateladze,
Org. Lett. 2007, 9, 1077 – 1079.
[3] R. Kottani, R. A. Valiulin, A. G. Kutateladze, Proc. Natl. Acad.
Sci. USA 2006, 103, 13917 – 13921.
[4] R. Kottani, J. R. R. Majjigapu, A. N. Kurchan, K. Majjigapu, T. P.
Gustafson, A. G. Kutateladze, J. Am. Chem. Soc. 2006, 128,
14794 – 14795.
[5] a) K. D. Asmus, Acc. Chem. Res. 1979, 12, 436 – 442; b) M.
Bonifacic, K. D. Asmus, J. Org. Chem. 1986, 51, 1216 – 1222;
c) W. K. Musker, P. B. Roush, J. Am. Chem. Soc. 1976, 98, 6745 –
6746; d) D. K. Maity, J. Am. Chem. Soc. 2002, 124, 8321 – 8328.
[6] Z.-G. Li, A. G. Kutateladze, J. Org. Chem. 2003, 68, 8236 – 8239.
[7] P. Brunelle, A. Rauk, J. Phys. Chem. A 2004, 108, 11032 – 11041.
In conclusion, we have demonstrated that binary dithiane
encoding and amplification can be implemented in linear
polypeptide-based arrays of photoactive, externally sensitized
dithiane adducts. The variable efficiency of dithiane release
also opens up an exciting opportunity for developing a
positionally encoded molecular barcode system beyond
simple binary encoding.
Received: April 6, 2007
Revised: May 19, 2007
Published online: July 12, 2007
Keywords: combinatorial chemistry · electron transfer ·
.
peptides · photoamplification · sensitizers
6140 www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 6137 –6140