Thiazole Orange-Peptide Conjugates: Sensitivity of DNA Binding to Chemical Structure

Article abstract of DOI:10.1021/ol0362818

(Equation presented) Derivatives of the highly fluorescent and DNA-binding dye thiazole orange (TO) are described that feature appended peptides. Functionalization of TO can be achieved at either of the endocyclic nitrogens, and the photophysical properties and DNA-binding modes are sensitive to the position of the tethered peptide. A series of TO-peptide conjugates are described, demonstrating the utility of a solid-phase synthesis approach to their preparation and illustrating how the photophysical and DNA-binding properties of the compounds are influenced by chemical structure.

Full text of DOI:10.1021/ol0362818

ORGANIC
LETTERS
2004
Vol. 6, No. 4
517-519
Thiazole Orange−Peptide Conjugates:
Sensitivity of DNA Binding to Chemical
Structure
Jay R. Carreon,† Kerry P. Mahon, Jr.,† and Shana O. Kelley*
Boston College, Eugene F. Merkert Chemistry Center,
Chestnut Hill, Massachusetts 02467
shana.kelley@bc.edu
Received November 21, 2003
ABSTRACT
Derivatives of the highly fluorescent and DNA-binding dye thiazole orange (TO) are described that feature appended peptides. Functionalization
of TO can be achieved at either of the endocyclic nitrogens, and the photophysical properties and DNA-binding modes are sensitive to the
position of the tethered peptide. A series of TO−peptide conjugates are described, demonstrating the utility of a solid-phase synthesis approach
to their preparation and illustrating how the photophysical and DNA-binding properties of the compounds are influenced by chemical structure.
The development of biomimetic molecules has provided
useful probes of the structure and function of nucleic acids.1
Peptide-intercalator conjugates, with an intercalating moiety
serving as an anchor for nucleic-acid binding and an array
of functional groups displayed on amino acids imparting
increased sequence specificity or chemical reactivity, are a
promising but relatively unexplored class of nucleic acid
probes.2,3 Only a few examples of peptidointercalator con-
jugates have been reported, but the successful isolation of
compounds exhibiting RNA cleavage,3a DNA hydrolysis
activity,3b,c or sequence specificity2a,c-e highlights the po-
tential of this type of architecture.
Our efforts toward developing peptidointercalator conju-
gates focus on the use of thiazole orange (TO) as an
intercalating scaffold that will deliver an appended peptide
to the DNA helix. TO is an ideal photophysical probe for
DNA binding, as it displays a high quantum yield when DNA
is bound and is essentially nonfluorescent when uncomplexed
in aqueous solution.4,5 Derivatives of TO appended to
peptide-nucleic acids have been prepared that provide
fluorescence-based detection of triple helix formation,6 but
peptide appendages have not been explored systematically.
Our initial studies of these compounds revealed that TO-
peptide conjugates containing reactive aromatic residues
exhibit photonuclease activity and provide model systems
for amino acid promoted DNA damage.7 To explore the
DNA-binding properties of TO-peptide conjugates system-
† These authors contributed equally to this work.
(1) (a) Helene, C. Curr. Opin. Biotechnol. 1993, 4, 29. (b) Erkkila, K.
E.; Odom, D. T.; Barton, J. K. Chem. ReV. 1999, 99, 2777. (c) Dervan, P.
B. Bioorg. Med. Chem. 2001, 9, 2215.
(2) (a) Guelev, V. M.; Harting, M. T.; Lokey, R. S.; Iverson, B. L. Chem.
Biol. 2000, 7, 1. (b) Carlson, C. B.; Beal, P. A. Bioorg. Med. Chem. Lett.
2000, 10, 1979. (c) Bailly, C.; Sun, J. S.; Colson, P.; Houssier, C.; Helene,
C.; Waring, M. J.; Henichart, J. P. Bioconjugate Chem. 1992, 3, 100. (d)
Sardesai, N. Y.; Lin, S. C.; Zimmerman, K.; Barton, J. K. Bioconjugate
Chem. 1995, 6, 302. (e) Thompson, M.; Woodbury, N. W. Biochemistry
2000, 39, 4327. (f) Bailly, F.; Bailly, C.; Helbecque, N.; Pommery, N.;
Colson, P.; Houssier, C.; Heinchart, J. P. Anticancer Drug Des. 1992, 7,
83.
(4) Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers 1998, 46, 39.
(5) Netzel, T. L.; Nafisi, K.; Zhao, M.; Lenhard, J. R.; Johnson, I. J.
Phys. Chem. 1995, 99, 17936.
(3) (a) Tung, C. H.; Wei, Z.; Leibowitz, M. J.; Stein, S. Proc. Natl. Acad.
Sci. U.S.A. 1992, 89, 7114 (b) Copeland, K. D.; Fitzsimons, M. P.; Houser,
R. P.; Barton, J. K. Biochemistry 2002, 41, 343. (c) Fitzsimons, M. P.;
Barton, J. K. J. Am. Chem. Soc. 1997, 119, 3379.
(6) Svanvik, N.; Westman, G.; Wang, D.; Kubista, M. Anal. Biochem.
2000, 281, 26.
(7) Mahon, K. P., Jr.; Ortiz-Meoz, R. F.; Prestwich, E. G.; Kelley, S.
O. Chem. Commun. 2003, 1956.
10.1021/ol0362818 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/23/2004

atically, we generated a series of compounds that feature
peptides of different lengths and compositions, and we mon-
itored the effect of appending peptides to the two endocyclic
nitrogens within the heterocyclic TO structure. The photo-
physical properties of the DNA-bound conjugates reveal that
the binding modes are altered by subtle structural changes.
Two derivatives of TO suitable for peptide coupling were
prepared (Scheme 1) using an adaptation of previously
minor groove, while the benzothiazole nitrogen is oriented
toward the major groove. Therefore, the isolation of the TOQ
and TOZ derivatives may permit the delivery of appended
functional groups to different regions of DNA’s structure.
To study the effects of peptide length, peptide sequence,
and linkage position on the DNA-binding characteristics of
the peptidointercalators, a series of compounds was synthe-
sized (Scheme 1) using TO derivatives functionalized with
a carboxylate-terminated tether and a solid-phase synthetic
approach. Peptides were prepared on solid support function-
alized with a Wang linker, capped with the TO derivative
using standard Fmoc synthetic methods, and cleaved from
the resin. The individual sequences selected for this study
were designed to include a diverse array of functional groups.
Scheme 1. Synthesis of TOZ and TOQ Derivatives and
TO-Peptide Conjugatesa
The synthetic procedure produced highly pure materials,
with purities of ∼95% as judged by HPLC. The conjugates
were then characterized using MALDI-TOF mass spectrom-
etry, UV-vis spectroscopy, and fluorescence spectroscopy,
and DNA binding affinities were measured by fluorescence.
The characterization methods and spectral data are described
in the Supporting Information.
All of the TO-peptide conjugates displayed the same
DNA-dependent fluorescence as the parent compound (Table
1). Fluorescence quantum yields in the absence of DNA were
very low (<0.0001), with 80- to 1300-fold increases in
intensity observed upon the addition of calf thymus (CT)
DNA (Table 1). TO is only emissive when the monomethine
bridge connecting the two heterocyclic structures is rigidified
through intercalation and exhibits a quantum yield of 0.11
when DNA bound.5 Therefore, the observation of comparable
quantum yields for most of the TO-peptide conjugates
indicates that an intercalative binding mode is maintained.
The conjugates with the lowest quantum yields are the
decapeptide derivatives featuring the GAQVWGFSAK se-
quence. Compounds with this sequence appeared to display
very weak DNA binding, as evidenced also by low hypo-
chromicity values (Table 1). A second-generation set of
decapeptide conjugates were modified to incorporate ad-
ditional cationic and polar residues that might facilitate DNA
binding. These sequence changes resulted in significantly
increased quantum yields, as reflected in the values measured
for conjugates containing a KAQSWGKSAK sequence
(bolded positions represent altered residues). Thus, the
appended peptide appears to significantly alter the DNA-
binding properties of a conjugate. A comparison of the
properties of TO-K and TO-WK (Table 1) indicates that
even more subtle changes in peptide sequence affect the
binding mode and photophysical properties of the conjugates.
The TO-WK conjugates containing a bulky aromatic residue
maintain high DNA affinity but exhibit significantly lower
quantum yields.
a See the Supporting Information for synthetic protocols and
compound characterization.
developed methods.6,8 One derivative featured a carboxylate-
terminated tether attached to the quinoline nitrogen (TOQ,
3), and the other featured the same linker attached to the
benzothiazole nitrogen (TOZ, 6). Derivatives of quinoline and
benzothiazole were modified with 11-bromoundecanoic acid
(selected to minimize cyclization reactions that occurred with
shorter linkers for TOZ) and subsequently coupled with the
appropriate heterocyclic quaternary salt to yield the cyanine
dye containing a carboxylate functionality. The motivation
for preparing these two isomers comes from the analysis of
the NMR structure of a dimeric TO derivative, which shows
the two nitrogens pointing toward different faces of the DNA
helix.9 The quinoline nitrogen points directly into the DNA
Interestingly, most of the DNA-bound conjugates based
on the TOQ derivative displayed higher quantum yields than
those with the TOZ scaffold. This trend may signify that the
(8) (a) Benson, S. C.; Singh, P.; Glazer, A. N. Nucl. Acids Res. 1993,
21, 5727. (b) Privat, E.; Asseline, U. Bioconj. Chem. 2001, 12, 757.
(9) Staerk, D.; Hamed, A. A.; Pedersen, E. B.; Jacobsen, J. P. Biocon-
jugate Chem. 1997, 8, 869.
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Org. Lett., Vol. 6, No. 4, 2004

Table 1. Photophysical Properties and DNA-Binding Affinities for TO-Peptide Conjugatesa
compd
Φrel (DNA)b
Kd (µM)
hypochromicityc (%)
kq (× 1012 M-1 s-1
)
displacement (%)
TOQ-K
TOZ-K
TOQ-WK
0.13 ( 0.01
1.7 ( 0.2
0.9 ( 0.1
1.2 ( 0.3
3.1 ( 0.4
not measured
not measured
0.4 ( 0.1
29 ( 2
31 ( 2
26 ( 1
37 ( 2
9 ( 3
12 ( 1
21 ( 1
31 ( 2
1.9 ( 0.2
4.7 ( 0.1
4.4 ( 0.5
12 ( 2
30 ( 9
38 ( 4
33 ( 2
30 ( 5
40 ( 6
26 ( 5
34 ( 4
36 ( 2
33 ( 2
21 ( 1
0.043 ( 0.001
0.078 ( 0.003
0.021 ( 0.002
0.008 ( 0.002
0.012 ( 0.001
0.13 ( 0.01
TOZ-WK
TOQ-GAQVWGFSAK
TOZ-GAQVWGFSAK
TOQ-KAQSWGKSAK
TOZ-KAQSWGKSAK
1.4 ( 0.1
3.4 ( 0.1
0.042 ( 0.002
0.5 ( 0.1
a See the Supporting Information for methods used to obtain values tabulated. Bolded residues in decapeptide conjugates represent positions varied to
improve DNA binding. b Measured in samples containing 1.5 µM TO-peptide, 45 µM bp CT DNA and reported relative to a fluorescein standard. c Measured
at λmax (∼500 nm).
attachment of the peptide chain to the benzothiazole nitrogen
impedes intercalation. Many intercalators bind from the
minor groove of DNA;10 therefore, the addition of substit-
uents to a TO heteroatom that faces into the major groove
might interfere with binding or alter the binding mode by
requiring threading of the appendage through the DNA helix.
While the peptide conjugates featuring the TOZ or TOQ
derivatives exhibited comparable DNA-binding affinities,
there were significant changes in their photophysical proper-
ties (Table 1). To further test the idea that the binding modes
of the isomeric derivatives differed, quenching of *TO by
Ru(NH3)63+ was monitored (Figure 1). The reactivity of this
is consistent with the quantum yield values that suggested
that the binding mode of the TOZ derivatives involves a
conformation with less stacking and protection than observed
for the TOQ derivatives.
The binding modes of DNA-bound TO-peptide deriva-
tives were also monitored using a distamycin displacement
assay (Table 1).12 Distamycin binds to the minor groove of
DNA and can block the binding of ligands that bind to DNA
intercalatively, particularly if large functional groups reside
in the minor groove. Interestingly, the binding of many of
the TOQ derivatives was more sensitive to the presence of
distamycin than the TOZ counterparts. This trend may suggest
that the TOZ derivatives thread the attached peptide into the
major groove, which could permit simultaneous binding of
distamycin in the minor groove and the TOZ-peptide
conjugates.
TO-peptide conjugates represent a new class of DNA-
binding compounds that may have utility as probes of nucleic
acids structure and function, as evidenced by recent studies
of DNA cleavage reactions facilitated by these agents.7 The
systematic analysis of a family of TO-peptides, prepared
by an efficient and facile solid-phase synthetic approach,
revealed that the DNA-binding properties of the conjugates
were affected by the position of the appended peptide within
the heterocyclic dye structure and the composition of the
peptide sequence. The generation of two isomeric TO deriv-
atives as the core for peptide conjugates presents the potential
to deliver functional groups to the minor and major grooves
of DNA, providing a versatile framework for the develop-
ment of model systems mimicking protein-DNA complexes.
Figure 1. Stern-Volmer analysis of quenching of *TOQ-WK and
3+
*TOZ-WK by Ru(NH3)6
.
Acknowledgment. We acknowledge the following sources
for support of this work: the Dreyfus Foundation for a New
Faculty Award to S.O.K., the Research Corporation for a
Research Innovation Award, and Boston College for a
Research Incentive Award.
diffusible quencher with the different conjugates provides
an estimate of the solution accessibility of TO.11 For the sets
of mono-, di-, and decapeptide conjugates, the TOZ isomers
exhibited higher Stern-Volmer kq values (Table 1), indicat-
ing that the fluorophore is more accessible when the peptide
is tethered to the benzothiazole nitrogen. This observation
Supporting Information Available: Experimental pro-
cedures and full characterization of compounds 1-6, 3a-
d, and 6a-d. This material is available free of charge via
the Internet at http://pubs.acs.org.
(10) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I. Nucleic Acids:
Structures, Properties, and Functions; University Science Books: Sausalito,
CA, 2000.
(11) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic/Plenum: New York, 1999.
OL0362818
(12) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick, M.
P. J. Am. Chem. Soc. 2001, 123, 5878.
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