Solid-phase synthesis of acridine-based threading intercalator peptides

Article abstract of DOI:10.1016/S0960-894X(00)00388-7

The preparation of a novel acridine-based amino acid is reported. This N-Alloc-protected monomer can be coupled and deprotected under solid-phase peptide synthesis procedures to create acridine-peptide conjugates as potential threading intercalators. A peptide containing this novel amino acid undergoes spectral changes in the presence of duplex DNA and RNA consistent with intercalative binding. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00388-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1979±1982
Solid-Phase Synthesis of Acridine-Based Threading
Intercalator Peptides
Coby B. Carlson and Peter A. Beal*
Department of Chemistry, University of Utah, Salt Lake City, UT 81112, USA
Received 11 May 2000; accepted 29 June 2000
AbstractÐThe preparation of a novel acridine-based amino acid is reported. This N-Alloc-protected monomer can be coupled and
deprotected under solid-phase peptide synthesis procedures to create acridine±peptide conjugates as potential threading inter-
calators. A peptide containing this novel amino acid undergoes spectral changes in the presence of duplex DNA and RNA con-
sistent with intercalative binding. # 2000 Elsevier Science Ltd. All rights reserved.
Solid-phase synthesis of combinatorial libraries, anity
chromatography and mass spectrometry are experimental
techniques that have converged to create a productive
approach to the discovery of small molecules that bind to
biological receptors.1,2 Our laboratory is using this
approach in the search for compounds that bind with
high anity and speci®city to nucleic acids.3 Such com-
pounds are of interest as potential therapeutic agents
and new tools in nucleic acid biochemistry.4 7
acids makes them good candidates for selective ligands.
These grooves provide unique molecular recognition
surfaces that can be exploited to distinguish di€erent
nucleic acid structures.
Many derivatives of acridine substituted at both the 4-
and 9-positions have been synthesized.13 15 We recently
reported the preparation of combinatorial libraries of
acridine±peptide conjugates by modifying a 9-anilinoacri-
dine with a variable peptide appendage at the 4-position.3
We are currently screening these libraries, whose major
groove appendage is randomized, for structure±speci®c
nucleic acid ligands via anity chromatography selection
with mass spectrometric detection. To advance these
studies further, we devised a synthetic route to attach a
variable peptide appendage at the 9-position of an acri-
dine intercalator. In this letter, we report the synthesis
of 9-(40-methylamino-allyloxycarbamate)-anilinoacridine-
4-carboxylic acid 1, a non-natural acridine-based amino
acid, and its incorporation into a ®xed peptide
sequence. This peptide demonstrated spectral changes
upon titration with either duplex DNA or RNA con-
sistent with intercalative binding. Thus, using this new
amino acid and solid-phase peptide synthesis (SPPS),
acridine derivatives can be rapidly prepared that place
peptide appendages at the 4- and 9-positions, which are
directed into the grooves of nucleic acid duplexes in
intercalation complexes.
The primary modes of noncovalent binding of small
molecules to DNA and RNA are intercalation, groove
binding, coulombic attraction or a combination of these.7,8
Threading intercalation is a binding mode wherein the
intercalating group directs substituents into both
grooves of a duplex simultaneously.9 This typically
requires a bulky side chain to pass between base pairs, or
thread, during the binding event. A number of compounds
have been shown to bind DNA by the threading mechan-
ism, including 9-anilinoacridine-4-carboxamides.10 In the
intercalation complex, acridine substituents at the 4- and
9-positions are located in the major and minor grooves
with the acridine chromophore sandwiched between base
pairs (Fig. 1). This binding mode allows for base-speci®c
groove binding by the substituents to take place. Indeed,
the 4-substituent of a 9-aminoacridine-4-carboxamide
derivative was recently shown by X-ray crystallography
to make speci®c contacts in the major groove of a DNA
double helix.11,12
The ability of threading intercalators to direct function-
ality into both the major and minor grooves of nucleic
Synthesis
The synthesis of this novel amino acid (Scheme 1) ®rst
required selective protection of the primary amino
group of 4-aminobenzylamine. We chose the Alloc
*Corresponding author. Tel.: +1-801-585-9719; fax: +1-801-581-8433;
e-mail: beal@chemistry.utah.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00388-7

1980
C. B. Carlson, P. A Beal / Bioorg. Med. Chem. Lett. 10 (2000) 1979±1982
Scheme 1. Preparation of Alloc-protected amino acid (1).19 (i) allyl 1-
benzotriazolyl carbamate/TEA/THF/rt/6 h/92%; (ii) TEA/CH3CN/
re¯ux/24 h/75%; (iii) LiOH/H2O/THF/rt/12 h/72%.
previously in our laboratory,3 gave compound 4 in high
yield.17 Subsequent saponi®cation of the ester a€orded
N-Alloc-protected amino acid 1.18
Figure 1. Generalized structure of 4,9-disubstituted acridines and
schematic of their binding to duplex DNA by threading intercalation.
To demonstrate that 1 could be used in SPPS, the ®xed
peptide sequence N-Ser-Val-Acr-Lys-C was prepared,
where Acr refers to the 4,9-disubstituted acridine
(Scheme 2). Amino acid 1 was activated as the N-hydroxy-
succinimide (NHS) ester and coupled to the solid-
supported primary amino group of N-e-Boc-lysine. To
remove the Alloc group and reveal the benzyl amine for
subsequent elaboration by peptide synthesis, we chose a
set of conditions similar to those previously reported to
be compatible with resin-bound peptides (Pd(PPh3)4 in
CHCl3:AcOH:NEM, rt, 2 h).20 Monitoring the eciency
group for two reasons. First, we needed a base stable
protecting group for our conditions of 9-substitution of
the acridine ring. Secondly, we desired a protecting
group that could be removed under conditions that
would leave acid-labile side chain protecting groups
intact, allowing this amino acid to be incorporated at any
point during SPPS using N-a-Fmoc amino acids. The
protection was accomplished using allyl-1-benzotriazolyl
carbonate to yield the Alloc-protected amine 2 in 92%
yield.16 Reaction of 2 with acridine ester 3, synthesized
Scheme 2. Incorporation of acridine-based amino acid (1) into the ®xed peptide sequence N-Ser-Val-Acr-Lys-C (5). (i) NHS/DCC/THF/rt/12 h; (ii)
Rink Amide AM resin-supported N-e-Boc-lysine/TEA/rt/12 h; (iii) Pd(PPh3)4/CHCl3:AcOH:NEM (37:2:1)/rt/2 h; (iv) SPPS incorporation of Fmoc-
Val-OH; (v) SPPS incorporation of Fmoc-Ser(Trt)-OH; (vi) TFA:TIS:H2O (95:2.5:2.5)/rt/18 h.

C. B. Carlson, P. A Beal / Bioorg. Med. Chem. Lett. 10 (2000) 1979±1982
1981
of Alloc removal in our case was not possible by the
usual colorimetric assays, since the beads took on an
orange color themselves upon coupling of the acridine
amino acid to the resin. Instead, essentially quantitative
Alloc removal under the above conditions was con-
®rmed by spectroscopic evaluation of Fmoc release after
the subsequent amino acid coupling. Upon completion
of the solid-phase synthesis, the tetrapeptide was released
and the side chains were deprotected by treatment of the
resin with TFA:TIS:H2O (95:2.5:2.5) to give 5. The
structure of 5 was con®rmed by electrospray mass spec-
trometry (Fig. 2). Tandem mass spectrometric analysis
(ES-MS/MS) of m/z peak 657.3 gave daughter ion peaks
consistent with the expected fragmentation pattern of
this peptide.21
Nucleic Acid Binding
The binding of intercalators to nucleic acids has been
studied by a number of methods.22 These experiments
usually evaluate the structural changes in the helix or the
spectroscopic changes of the chromophore. Acridines
have been shown to undergo bathochromic and hypo-
chromic shifts in their visible absorption spectra upon
binding DNA.23 To determine if such changes could be
observed with 5, calf thymus DNA was titrated into a
bu€ered aqueous solution of 5. A bathochromic shift
(12 nm) and decrease in extinction coecient was observed
for the visible absorption maximum (Fig. 3).
Figure 3. UV±visible spectrum of 5 (30 mM in 0.01 M PIPES bu€er pH
7, 0.01 M NaCl, 1 mM EDTA) upon titration with calf thymus DNA.
(a) no DNA (unbound, lmax=442 nm); (b) 10 mM base pairs (bp); (c)
20 mM bp; (d) 30 mM bp; (e) 40 mM bp; (f) 50 mM bp; (g) 60 mM bp
(bound, lmax=454 nm).
derivatives that have been shown to bind via intercala-
tion by other experimental techniques.12,27,28
Conclusion
The magnitudes of these changes are similar to those
observed when other 9-anilinoacridine-4-carboxamides
bind calf thymus DNA.24 Upon titration with poly-
Iꢀ polyC, a similar shift and change in extinction coecient
was observed, indicating that 5 can bind both B-form and
A-form helices (data not shown).25 These experiments
illustrate that the chemical modi®cations made to the
acridine ring system did not prevent its interaction with
DNA or RNA. Although these results are not proof of
intercalation by 5, they are consistent with intercalative
binding.26 Furthermore, they indicate that this acridine±
peptide conjugate binds in a manner similar to acridine
We have reported the preparation of an acridine-based
amino acid derivative and its incorporation into a peptide
using solid-phase peptide synthesis. The structure of this
compound is reminiscent of known threading inter-
calators, such as other 9-anilinoacridine-4-carbox-
amides.10 A peptide containing this nonnatural amino
acid undergoes spectral changes in the presence of DNA
and RNA consistent with intercalative binding. The syn-
thetic approach reported here may be a useful drug-design
strategy, since other 9-anilinoacridines have been shown
to be potent inhibitors of the cancer chemotherapy target
topoisomerase II.29,30 Furthermore, threading inter-
calators have the potential for regiospeci®c delivery of
variable functional groups to the two di€erent grooves of
DNA or RNA duplexes. Both of these characteristics make
these intercalator peptides good candidates for future
study.
Acknowledgements
P.A.B. would like to acknowledge support from the
University of Utah Research Foundation. C.B.C was
supported by a National Institutes of Health training
grant (GM-08573).
References and Notes
1. Kelly, M. A.; Liang, H.; Sytwu, I.-I.; Vlattas, I.; Lyons, N. L.;
Bowen, B. R.; Wennogle, L. P. Biochemistry 1996, 35, 11747.
Figure 2. Electrospray mass spectrum of intercalator peptide N-Ser-
Val-Acr-Lys-C after reverse-phase HPLC puri®cation.

1982
C. B. Carlson, P. A Beal / Bioorg. Med. Chem. Lett. 10 (2000) 1979±1982
2. Huang, P. Y.; Carbonell, R. G. Biotech. Bioeng. 1999, 63,
633.
2H), 6.82 (d, 2H), 5.96 (m, 1H), 5.33 (dd, 1H), 5.30 (m, 1H iso-
propyl methine), 5.23 (dd, 1H), 5.11 (bs, 1H), 4.63 (d, 2H),
4.39 (d, 2H), 1.44 (d, 6H); FABHRMS calcd mass for
C28H28N3O4: 470.206, found 470.208.
3. Carlson, C. B.; Beal, P. A. Org. Lett. 2000, 2, 1465.
4. Li, K.; Fernandez-Saiz, M.; Rigl, C. T.; Kumar, A.; Ragu-
nathan, K. G.; McConnaughie, A. W.; Boykin, D. W.;
Schneider, H.-J.; Wilson, W. D. Bioorg. Med. Chem. 1997, 5,
1157.
5. Michael, K.; Tor, Y. Chem. Eur. J. 1998, 4, 2091.
6. Pearson, N. D.; Prescott, C. D. Chem. Biol. 1997, 4, 409.
7. Nielsen, P. E. Bioconj. Chem. 1991, 2, 1.
8. Bogdan, F. M.; Chow, C. S. Chem. Rev. 1997, 97, 1489.
9. Liaw, Y.-C.; Gao, Y.-G.; Robinson, H.; van der Marel, G.
A.; van Boom, J. H.; Wang, A. H.-J. Biochemistry 1989, 28,
9913.
10. Wakelin, L. P. G.; Chetcuti, P.; Denny, W. A. J. Med.
Chem. 1990, 33, 2039.
11. Todd, A. K.; Adams, A.; Thorpe, J. H.; Denny, W. A.;
Wakelin, L. P. G. J. Med. Chem. 1999, 42, 536.
12. Adams, A.; Guss, J. M.; Collyer, C. A.; Denny, W. A.;
Wakelin, L. P. G. Biochemistry 1999, 38, 9221.
13. Denny, W. A.; Cain, B. F.; Atwell, G. J.; Hansch, C.;
Panthananickal, A.; Leo, A. J. Med. Chem. 1982, 25, 276.
14. Bourdouxhe-Housiaux, C.; Colson, P.; Houssier, C.;
Waring, M. J.; Bailly, C. Biochemistry 1996, 35, 4251.
15. Bourdouxhe-Housiaux, C.; Colson, P.; Houssier, C.;
Bailly, C. Anti-Cancer Drug Des. 1996, 11, 509.
18. Preparation of 9-(40-methylamino-allyloxycarbamate)-
anilinoacridine-4-carboxylic acid (1). To a solution of ester 3
(0.250 g, 0.533 mmol) in THF (5 mL), was added lithium
hydroxide monohydrate (0.046 g, 1.1 mmol, 2 equiv) in water
(0.6 M). The resulting solution was stirred at rt for 12 h. The
reaction solution was neutralized with 2N HCl, and the solvent
was removed under reduced pressure. The brick red residual oil
was puri®ed by ¯ash chromatography (EtOAc:hexanes 9:1 w/
late addition of methanol) on a column of silica gel to give
0.163 g (72%) of acid 1 as a reddish-orange solid. 1H NMR
(500 MHz, CDCl3) d 8.40 (bs, 1H), 8.25 (bs, 1H), 7.94 (bs,
1H), 7.77 (t, 1H), 7.69 (bs, 2H), 7.22 (m, 3H), 6.93 (bs, 2H),
5.92 (m, 1H), 5.29 (dd, 1H), 5.18 (dd, 1H), 4.51 (d, 2H), 4.20
(d, 2H); FABHRMS calcd mass for C25H22N3O4: 428.161,
found 428.161.
19. Abbreviations used in the synthetic schemes are: AcOH,
acetic acid; DCC, 1,3-dicyclohexylcarbodiimide; NEM, N-
ethylmorpholine; NHS, N-hydroxysuccinimide; TEA, triethy-
lamine; TFA, tri¯uoroacetic acid; THF, tetrahydrofuran; TIS,
triisopropylsilane.
20. Kates, S. A.; Daniels, S. B.; Sole, N. A.; Barany, G. In
Peptides, Chemistry, Structure & Biology, Proc. 13th American
Peptide Symposium; Hodges, R. S., Smith, J. A., Eds.;
ESCOM: Leiden, 1994; Vol. 13, pp 113±115.
16. Preparation of allyl 4-aminobenzylamino carbamate (2).
To a mixture of allyl 1-benzotriazolyl carbamate (0.580 g,
2.65 mmol, 1.2 equiv) in THF (2.5 mL), was added dropwise 4-
aminobenzylamine (0.250 mL, 2.21 mmol) and TEA (0.260 mL,
1.2 equiv). The resulting yellow solution was stirred at rt for
6 h. When the reaction was complete by thin layer chromato-
graphy (TLC) (acetone:TEA 98:2), the solvent was evaporated
under reduced pressure. The residual liquid was puri®ed by
¯ash chromatography on a column of silica gel, eluting with
EtOAc:hexanes (3:2) to give 0.390 g (92%) of protected amine
21. ES-MS/MS data for 5: m/z 657.3 (N-Ser-Val-Acr-Lys-C);
570.4 (N-Val-Acr-Lys-C); 512.2 (N-Ser-Val-Acr-C); 425.2 (N-
Val-Acr-C); 326.1 (N-Acr-C).
22. Long, E. C.; Barton, J. K. Acc. Chem. Res. 1990, 23, 273.
23. Denny, W. A.; Wakelin, L. P. G. Cancer Res. 1986, 46, 1717.
24. Searcey, M.; Martin, P. N.; Howarth, N. M.; Madden, B.;
Wakelin, L. P. G. Bioorg. Med. Chem. Lett. 1996, 6, 1831.
25. The RNA-binding experiment was conducting in the same
fashion as for DNA. Titration of compound 5 (30 mM in
0.01 M PIPES bu€er pH 7, 0.01 M NaCl, 1 mM EDTA) with
polyIꢀ polyC RNA (10 mM bp increments). A bathochromic
shift of 10 nm was observed and saturation was reached at
approximately the same concentration as with DNA (60 mM).
26. As are many acridine derivatives, compound 5 is ¯uor-
escent, with excitation and emission maxima at 400 and
450 nm, respectively. The emission maximum was blue-shifted
and the intensity decreased in the presence of excess calf thy-
mus DNA, again consistent with intercalation.
27. Westhof, E.; Sundaralingam, M. Proc. Natl. Acad. Sci.
U.S.A. 1980, 77, 1852.
28. Sakore, T. D.; Reddy, B. S.; Sobell, H. M. J. Mol. Biol.
1979, 114, 763.
29. Figgitt, D. P.; Denny, W. A.; Gamage, S. A.; Ralph, R. K.
Anti-Cancer Drug Des. 1994, 9, 199.
30. Froelich-Ammon, S. J.; Oshero€, N. J. Biol. Chem. 1995,
270, 21429.
1
1 as a viscous liquid. H NMR (500 MHz, CDCl3) d 7.01 (d,
2H, J=8.4 Hz), 6.56 (d, 2H, J=8.4 Hz), 5.89 (m, 1H), 5.51 (bs,
1H amide), 5.26 (dd, 1H, J=17.1, 1.5 Hz), 5.18 (dd, 1H,
J=10.2, 1.5 Hz), 4.54 (d, 2H, J=6 Hz), 4.18 (d, 2H, J=5.7 Hz),
3.75 (s, 2H aniline); FABHRMS calcd mass for C11H15N2O2:
207.124, found 207.123.
17. Preparation of iso-propyl-9-(40-methylaminoallyl-oxycar-
bamate)anilinoacridine-4-carboxylate (4). To a solution of
ester 3 (0.75 mmol) in anhydrous acetonitrile (8 mL), was
added amine 1 (3 mmol, 4 equiv) and TEA (1 mL, 7.5 mmol,
10 equiv). The resulting mixture was heated under re¯ux for
24 h. When the reaction was complete by TLC (EtOAc:hex-
anes:MeOH 90:9:1), the solution was allowed to cool to rt, and
solvent was evaporated under reduced pressure. The residue
was puri®ed by ¯ash chromatography on a column of alumina
oxide, eluting with hexanes:EtOAc (4:1) to give 0.262 g (75%)
1
of ester 4 as an orange solid. H NMR (500 MHz, CDCl3) d
11.32 (bs, 1H), 8.24 (bs, 1H), 7.35 (d, 1H), 7.25 (d, 2H), 7.18 (d,