Solid-phase synthesis of acridine-peptide conjugates and their analysis by tandem mass spectrometry

Article abstract of DOI:10.1021/ol005809v

(Matrix presented) A novel and high-yielding synthesis of 9-anilinoacridine-4-carboxylic acid is reported. This acid has been used in the solid-phase synthesis of a small combinatorial library of acridine-peptide conjugates. Tandem mass spectrometry (ES-M

Full text of DOI:10.1021/ol005809v

ORGANIC
LETTERS
2000
Vol. 2, No. 10
1465-1468
Solid-Phase Synthesis of
Acridine−Peptide Conjugates and Their
Analysis by Tandem Mass Spectrometry
Coby B. Carlson and Peter A. Beal*
Department of Chemistry, UniVersity of Utah, 315 South 1400 East,
Salt Lake City, Utah 84112-0850
beal@chemistry.chem.utah.edu
Received March 14, 2000
ABSTRACT
A novel and high-yielding synthesis of 9-anilinoacridine-4-carboxylic acid is reported. This acid has been used in the solid-phase synthesis
of a small combinatorial library of acridine−peptide conjugates. Tandem mass spectrometry (ES-MS/MS) can be used for structure determination
of these compounds at a sensitivity of ∼10 pmol. This work makes possible the generation of acridine−peptide libraries for the discovery of
structure-specific nucleic acid ligands via affinity chromatography selection with mass spectrometric detection.
Low molecular weight compounds that bind selectively to
nucleic acids and inhibit the formation of protein‚nucleic acid
complexes have considerable potential as new cancer che-
motherapeutics, antiviral or antibacterial agents, and new
tools in nucleic acid research.1-4 Molecules with high affinity
and specificity for nucleic acid targets have been discovered
via the synthesis and screening of combinatorial libraries
biased for DNA or RNA binding.5-7 For this purpose, we
are preparing libraries of nucleic acid binding molecules by
elaborating a known intercalating group with a variable
peptide appendage. These molecules are designed such that
the intercalation domain will direct the peptide into a groove
of double helical secondary structure where sequence-specific
contacts are possible.
For our initial libraries, we have chosen to prepare
acridines with the substitution pattern of SN 16713, a
derivative of the antitumor drug amsacrine (Figure 1).8 SN
(1) Nielsen, P. E. Bioconj. Chem. 1991, 2, 1-12.
(2) Li, K.; Fernandez-Saiz, M.; Rigl, C. T.; Kumar, A.; Ragunathan, K.
G.; McConnaughie, A. W.; Boykin, D. W.; Schneider, H.-J.; Wilson, W.
D. Bioorg. Med. Chem. 1997, 5, 1157-1172.
(3) Michael, K.; Tor, Y. Chem. Eur. J. 1998, 4, 2091-2098.
(4) Pearson, N. D.; Prescott, C. D. Chem. Biol. 1997, 4, 409-414.
(5) Wong, C.-H.; Hendrix, M.; Manning, D. D.; Rosenbohm, C.;
Greenberg, W. A. J. Am. Chem. Soc. 1998, 120, 8319-8327.
(6) Guelev, V. M.; Harting, M. T.; Lokey, R. S.; Iverson, B. L. Chem.
Biol. 2000, 7, 1-8.
Figure 1. SN 16713, a 9-anilinoacridine-4-carboxamide that binds
DNA at G‚C-rich sequences and HIV’s TAR RNA.
16713 has been shown to bind duplex DNA at G‚C-rich
(7) Shipps, G. W.; Prior, K. E.; Xian, J.; Skyler, D. A.; Davidson, E.
H.; Rebek, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11833-11838.
sequences and HIV’s TAR RNA via intercalation.9,10 Aniline
10.1021/ol005809v CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/21/2000

substitution at the 9-position of the acridine elevates the ring
nitrogen pKa, such that a significant fraction of the acridine
is cationic at physiological pH.11,12 Carboxamide substitution
at the 4-position of acridine has been shown to control DNA
binding specificity by allowing for base-specific major
groove contacts by the substituent.13,14 Several 9-aminoacri-
dine-4-carboxamides have been previously prepared.11,15,16
Typically, the 4-carboxamide bond is formed first by reaction
of an amine with 9-chloroacridine-4-carbonyl chloride fol-
lowed by amine substitution at the 9-position under acidic
conditions. This approach is not directly applicable to the
solid-phase synthesis of derivatives using standard peptide
synthesis protocols. In this Letter, we describe the first
reported preparation of 9-anilinoacridine-4-carboxylic acid,
its application to the synthesis of 9-anilinoacridine-peptide
libraries, and the analysis of these compounds by tandem
mass spectrometry (ES-MS/MS).
zoles.18 This reaction has been used successfully in the
synthesis of nucleoside analogues from uridine and thymidine
derivatives.19 The triazole intermediates formed react selec-
tively with a number of different nitrogen and oxygen
nucleophiles under mildly basic conditions to give substitu-
tion products in good yield. We investigated the utility of
this reaction for substitution of the acridine ring because we
believed these conditions would be compatible with the
acridone-4-carboxylic acid protected as a hindered ester.
Upon treatment of the acridone isopropyl ester with an
acetonitrile solution of the triazolating reagent formed from
1,2,4-triazole, POCl3, and TEA, the starting material was
slowly consumed and a new product, presumably the
9-triazoleacridine derivative (2), was formed. This compound
was not isolated but was allowed to react with aniline in
acetonitrile at reflux in the presence of TEA to give ester 3
in high yield. Ester deprotection with LiOH in THF/H2O
gave acid 4 in 88% overall yield from 1.
To determine if acid 4 could be used directly in solid-
phase synthesis of acridine-peptide conjugates, it was
applied to the preparation of a 9-anilinoacridine-4-carboxa-
mide with a short peptide fused to the acridine through a
4-carboxamide linkage to the N-terminus. The tripeptide
H2N-Gly-Arg-Ser-COOH was synthesized using standard
Fmoc peptide synthesis procedures on Rink amide resin. Acid
4 was activated as the NHS ester 5, and this compound was
allowed to react with the free amino terminus of the side
chain-protected, solid support-bound peptide to give 6
(Scheme 2). Removal of unreacted 5 by filtration was
The known 9(10H)-acridone-4-carboxylic acid (1), avail-
able in two steps from 2-chlorobenzoic and anthranilic acids,
was protected as the isopropyl ester via reaction of carbon-
yldiimidazole (CDI) and 2-propanol in THF (Scheme 1).17
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) CDI, THF, iso-PrOH, rt, 6 h, 95%;
(b) 1,2,4-triazole, POCl3, TEA, CH3CN, reflux, 96 h; (c) aniline,
TEA, CH3CN, reflux, 24 h, 94% (two steps); (d) LiOH, THF, H2O,
rt, 12 h, 99%.
The ester was then subjected to reaction conditions known
to convert oxygen-substituted heterocycles to 1,2,4-tria-
(8) Wakelin, L. P. G.; Chetcuti, P.; Denny, W. A. J. Med. Chem. 1990,
33, 2039-2044.
(9) Bailly, C.; Denny, W. A.; Mellor, L. E.; Wakelin, L. P. G.; Waring,
M. J. Biochemistry 1992, 31, 3514-3524.
(10) Bailly, C.; Colson, P.; Houssier, C.; Hamy, F. Nucl. Acids Res. 1996,
24, 1460-1464.
(11) Denny, W. A.; Cain, B. F.; Atwell, G. J.; Hansch, C.; Panthananickal,
A.; Leo, A. J. Med. Chem. 1982, 25, 276-315.
(12) Crenshaw, J. M.; Graves, D. E.; Denny, W. A. Biochemistry 1995,
34, 13682-13687.
(13) Todd, A. K.; Adams, A.; Thorpe, J. H.; Denny, W. A.; Wakelin, L.
P. G. J. Med. Chem. 1999, 42, 536-540.
(14) Adams, A.; Guss, J. M.; Collyer, C. A.; Denny, W. A.; Wakelin,
L. P. G. Biochemistry 1999, 38, 9221-9233.
a Reagents and conditions: (a) resin-bound peptide + 5 (3 equiv),
THF, rt, 12 h; (b) TFA:TIS:H2O (95:2.5:2.5), rt, 18 h.
(15) Bourdouxhe-Housiaux, C.; Colson, P.; Houssier, C.; Waring, M.
J.; Bailly, C. Biochemistry 1996, 35, 4251-4264.
(16) Bourdouxhe-Housiaux, C.; Colson, P.; Houssier, C.; Bailly, C.
Anticancer Drug Des. 1996, 11, 509-525.
followed by deprotection of the side chains and cleavage
from the support with TFA:TIS:H2O, giving acridine-
1466
Org. Lett., Vol. 2, No. 10, 2000

peptide conjugate 7. Tandem mass spectrometry (ES-MS/
MS) was used to analyze this compound (Figure 2). The
Figure 3. Structure of a small combinatorial library of 9-anilino-
acridine-peptides. The tripeptide appendage has one fixed residue
(G) and two randomized positions, Y and Z, each with four different
amino acids (A, G, K, and S).
addition to the singly charged ions shown, the lysine-
containing conjugates were also detected as the [M + 2H]2+
ions (data not shown). The ion at m/z ) 545 was selected
for daughter ion analysis and the result confirmed its identity
as the 9-anilinoacridine-GSS conjugate (Figure 4, inset).
In summary, we have developed a high-yielding synthesis
to 9-anilinoacridine-4-carboxylic (4), and this acid has been
applied to the solid-phase synthesis of 9-anilinoacridine-4-
carboxamides. Peptides linked to acridine via a 4-carboxa-
mide linkage can be readily prepared using standard Fmoc
peptide synthesis techniques and the NHS ester of 4. We
have shown that structures of these acridine-peptide con-
jugates can be determined with less than 10 pmol of
compound by tandem mass spectrometry. We are currently
screening 9-anilinoacridine-peptide libraries prepared with
these methods by affinity chromatography selection with
Figure 2. Electrospray mass spectrum of the 9-anilinoacridine-
peptide conjugate ACR-GRS (7). Inset: Daughter ion analysis of
m/z ) 614 peak.
electrospray mass spectrum showed a peak at m/z ) 614,
corresponding to [M + H]+. Fragmentation of this ion
generated daughter ions at 597, 510, 354, and 297, corre-
sponding to cleavage at the four amide bonds in the molecule
(Figure 2, inset).
Importantly, these results indicate that the presence of the
9-anilinoacridine does not interfere with mass spectrometry
sequencing of these peptide conjugates. Furthermore, the
sensitivity of the ES-MS/MS technique (∼10 pmol per
peptide) allows for the determination of their structures with
very little material. For comparison, a typical peptide
synthesis with 0.2 g of Rink amide resin corresponds to 80-
160 µmol of peptide.
Using a split and pool strategy with Fmoc-protected amino
acids,20 Rink amide resin, and NHS ester 5, a small acridine-
peptide library was prepared. This library had one fixed and
two randomized positions. At each randomized position, four
different amino acids (Ala, Gly, Ser, and Lys) were
incorporated to give 16 sequences. After deprotection of the
N-terminal glycine, 5 was allowed to react with the library.
Removal of unreacted ester by filtration, side chain depro-
tection, and cleavage from the support yielded the acridine-
peptide library 8 (Figure 3). ES-MS/MS was used to analyze
components of approximately 120 pmol of this library
(Figure 4). Nine of the 10 different masses expected for the
16 components of this library were detected as the [M +
H]+ ions in the electrospray mass spectrum. The GKK
component was detected as the [M + Na]+ adduct. In
(17) Rewcastle, G. W.; Denny, W. A. Synthesis 1985, 217-220.
(18) Webb, T. R.; Matteucci, M. D. Nucl. Acids Res. 1986, 14, 7661-
7674.
(19) Sung, W. L. Nucl. Acids Res. 1981, 9, 6139-6151.
(20) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein
Res. 1991, 37, 487-493.
Figure 4. ES-MS/MS fragmentation pattern of the 9-anilinoacri-
dine-peptide library, indicating that masses corresponding to the
16 components are detected. The peak at m/z ) 582 represents an
unknown impurity. Inset: daughter ion analysis of m/z ) 545 peak.
Org. Lett., Vol. 2, No. 10, 2000
1467

mass spectrometric detection to identify members that bind
with high affinity and selectivity to predefined nucleic acid
structures.21
assistance and Professor James McCloskey for helpful
discussions.
Supporting Information Available: Experimental pro-
cedures and NMR and mass spectral data for compounds
listed in Scheme 1. Additionally, the general procedure for
the synthesis and ES-MS/MS analysis of a representative
9-anilinoacridine-peptide conjugate is included. This infor-
mation is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. P.A.B acknowledges support from the
University of Utah Research Foundation. C.B.C was sup-
ported by a National Institutes of Health training grant (GM-
08573). The authors also thank Dr. Vajira Nanayakkara of
the University of Utah mass spectrometry facility for his
(21) Kelly, M. A.; Liang, H.; Sytwu, I.-I.; Vlattas, I.; Lyons, N. L.;
Bowen, B. R.; Wennogle, L. P. Biochemistry 1996, 35, 11747-11755.
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