Synthesis, DNA binding and anti-leukemic activity of an aminoacyl nucleolipid

Article abstract of DOI:10.1016/j.bmcl.2013.07.030

The synthesis and characterization of a new class of DNA binding molecule exhibiting potent and selective anti-leukemic activity is described. The synthesis of an aminoacyl nucleolipid was developed from an efficient EEDQ coupling strategy, in which a series of seven bioconjugates were synthesized in yields of 53-78%. Guanosine bioconjugate 7, was used as building block for the synthesis of a target aminoacyl nucleolipid 14. Its GRP78 DNA binding affinity was confirmed by gel shift assay, CD spectroscopy, T_m measurements and dynamic light scattering experiments. Moreover, in a single dose (10 μM) screen against a panel of 60 cancer cell lines, aminoacyl nucleolipid 14 was found to selectively trigger greater than 90% cell death in a SR human leukemia cancer cell line. The reported aminoacyl nucleolipid represents a useful model for a new class of DNA binding molecules for the development of potent and selective anti-cancer agents.

Full text of DOI:10.1016/j.bmcl.2013.07.030

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5086–5090
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, DNA binding and anti-leukemic activity of an aminoacyl
nucleolipid
Pradeepkumar Patel a, Emi Hanawa a, Reeta Yadav b, Uri Samuni b, Cecilia Marzabadi a, David Sabatino a,
⇑
a Department of Chemistry and Biochemistry, Seton Hall University, 400 South Orange Avenue, South Orange, NJ 07079, United States
b Department of Chemistry and Biochemistry, Queens College, City University of New York, 65-30 Kissena Blvd., Flushing, NY 11367, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and characterization of a new class of DNA binding molecule exhibiting potent and selec-
tive anti-leukemic activity is described. The synthesis of an aminoacyl nucleolipid was developed from an
efficient EEDQ coupling strategy, in which a series of seven bioconjugates were synthesized in yields of
53–78%. Guanosine bioconjugate 7, was used as building block for the synthesis of a target aminoacyl
nucleolipid 14. Its GRP78 DNA binding affinity was confirmed by gel shift assay, CD spectroscopy, Tm
Received 26 May 2013
Revised 6 July 2013
Accepted 16 July 2013
Available online 23 July 2013
measurements and dynamic light scattering experiments. Moreover, in a single dose (10 lM) screen
Keywords:
Aminoacyl nucleolipid
GRP78 DNA
Bioconjugate
Anti-leukemic activity
against a panel of 60 cancer cell lines, aminoacyl nucleolipid 14 was found to selectively trigger greater
than 90% cell death in a SR human leukemia cancer cell line. The reported aminoacyl nucleolipid repre-
sents a useful model for a new class of DNA binding molecules for the development of potent and selec-
tive anti-cancer agents.
Published by Elsevier Ltd.
Aminoacyl nucleolipids represent an interesting class of biocon-
jugates owning the ability to form stable, higher-ordered struc-
tures for potential applications in medicinal chemistry.1a For
example, they form complexes with short-interfering RNA (siRNA),
applicable in siRNA transfection leading to the down-regulation of
mRNA and protein expression in cancer cells.1b–d
These bioconjugates may also have the ability to bind and sta-
bilize double-stranded DNA by a series of non-covalent interac-
tions. These include, H-bond base-pairing and
p-stacking of the
nucleoside base, as seen in the canonical Watson–Crick2a and Hoo-
gsteen base-pairing interactions of ligands with duplex DNA.2b,c
Favorable ionic interactions can occur between the positively
charged aminoacyl and the anionic phosphodiester DNA backbone
as illustrated by the condensation of DNA with poly-Arg and poly-
Lys sequences.3 Lastly, the van der Waals/hydrophobic interactions
of the lipids create an amphiphilic microenvironment for DNA
complex formation and cell permeability4 (Fig. 1). In the fight
against cancer, stable DNA-bioconjugate interactions may form
the basis for an effective gene therapy strategy.5
The glucose-regulated protein 78 (GRP78) is a chaperone pro-
tein that functions to regulate protein folding events in the lumen
of the endoplasmic reticulum (ER) of all cell types.6 In cancer,
GRP78 is overexpressed and exclusively localized on the cell sur-
face where it exhibits a variety of signal transduction pathways
associated with tumorogenesis and resistance towards apoptosis.7
Figure 1. Chemical structure and DNA-binding interactions of aminoacyl nucleo-
lipid, 14.
Thus, GRP78 functions as an ideal molecular marker for the selec-
tive detection and treatment of cancer cells.8
In an effort to develop potent GRP78 DNA binders for anti-
cancer applications, we describe the development of an aminoacyl
nucleolipid, 14. This aminoacyl nucleolipid, as a valuable represen-
tative of a new class of DNA binding molecules has been validated
by its ability to bind to the promoter region of GRP78 DNA, while
soliciting potent and selective anti-leukemic activity.
The synthetic strategy begins with the generation of N-isobu-
tyrroyl 5’-carboxy 20,30-bis-O-(carbobenzyloxy) guanosine, 4,
(Scheme 1) as the building block component for furnishing biocon-
jugates (Table 1). In the preparation of 4, N-isobutyrroyl 50-dime-
thoxytrityl guanosine, 1, was used as commercially available
starting material to initiate the synthesis strategy. The Cbz
⇑
Corresponding author. Tel.: +1 973 313 6539.
E-mail address: david.sabatino@shu.edu (D. Sabatino).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.07.030

P. Patel et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5086–5090
5087
Scheme 1. Synthesis of N-isobutyrroyl 50-carboxy 20,30-bis-O-(carbobenzyloxy) guanosine, 4. Reagents and conditions: (a) DMAP, benzyl chloroformate, DCM, 0 °C then rt,
72 h, 77%; (b) 3% TCA:DCM, rt, 15 min, 98%; (c) TEMPO, BAIB, MeCN:H2O (50:50 v/v), rt, 15 h, 82%.
Table 1
Bio-conjugation of aminoacyl and lipid nucleosides
Entry
R1-OH or R2-NH2
X
Yield (%)
5
6
73
55
7
8
9
53
78
67
10
11
70
70
protecting groups were installed using an excess of benzylchloro-
formate and DMAP in DCM.9 Following carbomylation, 2, was det-
ritylated in 98% yield using 3% trichloroacetic acid in DCM.10 N-
isobutyrryol-20,30-bis-O-(carbobenzyloxy)guanosine, 3, was oxi-
dized in 82% yield using a TEMPO oxidation to the corresponding
carboxylic acid, 4.11
With building block 4 in hand, a small library of aminoacyl and
lipid conjugates were prepared. Coupling conditions were opti-
mized based on literature precedence,12 by using N-ethoxycar-
bonyl 1,2-ethoxy-1,2-dihydroquinoline, EEDQ, (2 equiv) as
coupling reagent and N,N-diisopropylethylamine, DIEA (2 equiv)
as the base in DMF. Briefly, alkyl amines or alcohols (Table 1, en-
tries 5, 6, 11) were coupled with 4 to their corresponding amides
or esters, respectively, in yields of 55–73%. In similar fashion, ami-
no esters (Table 1, entries 7–10) were coupled to their correspond-
ing amides with building block 4 in yields ranging from 53% to 78%.
The pure bio-conjugates were characterized by NMR, IR, MS and
polarimetry (see Supplementary data).
Bioconjugate 7, was selected for the preparation of aminoacyl
nucleolipid, 14, following
a
simple three-step procedure

5088
P. Patel et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5086–5090
Scheme 2. Synthesis of aminoacyl nucleolipid, 14. Reagents and conditions: (a) H2, 10% Pd/C, EtOH, rt, 1.5 h, >99%; (b) palmitoyl chloride, DMAP, py, 2 h, >99%; (c) 50%
TFA:DCM, 2 h, rt, then 1 M HCl, 3 h, >99%.
(Scheme 2). Firstly, the Cbz protecting groups were removed fol-
lowing traditional hydrogenation conditions,9 generating diol 12
in quantitative yields. The product was dissolved in pyridine and
acylated with an excess of palmitoyl chloride13 to afford the pro-
tected aminoacyl nucleolipid 13 in >99% yield. Following purifica-
tion, the Boc-protecting group of 13 was removed using 50%
TFA:DCM, followed by salt exchange with aqueous HCl. The desired
product, 14, obtained in quantitative yield was lyophilized to dry-
ness and characterized (see Supplementary data). Attempts to
Figure 2. Gel shift assay for GRP78 DNA with 14: from lane 3 to 7, increasing
amount of ligand 14 from 0 to 100 equiv. Compound 14/P (0–3.9 mM). Lane 1 and 2
are single stranded GRP78 DNA.
selectively deprotect the guanine NH-i-Bu using mildly basic con-
ditions14 resulted in concomitant ester cleavage of the palmitoyls
and methyl ester groups. Thus, we rationalized that partial protec-
tion of aminoacyl nucleolipid, 14 may confer greater resistance
between duplex DNA with and without 14 demonstrated 50% du-
plex binding affinity (KD) at 0.7 mM.
Next, the GRP78 DNA binding stability with and without 14 was
evaluated by thermal denaturation (Fig. 3). Changes in duplex DNA
UV-absorption at 260 nm was monitored with increasing temper-
towards degradation, while inhibiting formation of higher-order
self-assemblies (i.e. guanine-tetraplexes) that may influence DNA
binding and biological activity.15 CD spectroscopy of aminoacyl
nucleolipid 14 (0.2–3.9 mM) did not exhibit a guanine-tetraplex
structure (see Supplementary data, Fig. S2). They are usually char-
acterized by an intense positive band near 264 nm and a negative
one near 245 nm (for parallel tetraplex structures), and a positive
band near 290 nm and negative ones near 260 and 245 nm (for
anti-parallel tetraplex structures).16
The promoter region of GRP78 DNA encompassing nucleotides
252–26917 was synthesized by conventional automated solid-
phase oligonucleotide synthesis.18 The oligonucleotides were puri-
fied by reverse-phase ion-pairing HPLC (RP-IP HPLC)19 and charac-
terized by mass spectrometry (see Supplementary data, Fig. S1).
atures (5-90 °C). DNA samples (1.1 lM) were prepared by anneal-
ing complementary single strands in physiological phosphate
binding buffer (140 mM KCl, 1 mM MgCl2, 5 mM Na2PHO4 adjusted
to pH 7.2). The melting temperature (Tm) of native GRP78 DNA was
evaluated at 66 °C, whereas the addition of ligand (0.2–3.9 mM)
produced drastic changes in the DNA melting curves. For example,
at 0.2 mM, 14 exhibited a stabilizing effect on GRP78 DNA duplex
(DTm = +4 °C), whereas at higher concentrations (0.9–3.9 mM)
changes in duplex DNA hyperchromicities with increasing temper-
atures were indicative of higher-order structure transitions.21
In order to gain greater insight into the structural changes of na-
tive GRP78 DNA with the addition of ligand 14, the circular dichro-
ism (CD) spectrum was collected (Fig. 4). The samples were
prepared as previously described for the thermal denaturation
studies. The native GRP78 DNA duplex exhibited a CD structure
consistent with a B-type helix, with characteristic broad maximum
band between 260 and 280 nm and a minimum at 248 nm.22 The
amplitudes of the molar ellipticities at these characteristic wave-
lengths were found to diminish upon titration with 14 (0.2–
3.9 mM) suggesting that the ligand-bound structure compromises
DNA duplex helicity until the formation of a higher-order complex
Complementary sequences (0.57 lM) were annealed in 15 lL of
30% sucrose in 5ꢀ TAE buffer (89 mM Tris/acetate, 2.5 mM EDTA,
pH 4.2) and incubated at 37 °C for 36 h with and without ligand
14, at 0, 5, 25, 50, and 100 equiv, based on the ratio of 14/DNA
phosphate groups, which corresponds to the following concentra-
tions: 0, 0.2, 0.9, 1.9, 3.9 mM, respectively. A 24% native PAGE gel
shift binding assay was performed to evaluate ligand binding to
GRP78 DNA (Fig. 2). Following electrophoresis, gradual disappear-
ance of the DNA bands was visualized by UV-shadowing at 260 nm.
The observed trend is typical of higher-order complex structures,
which results in the diminished intensity of the UV-bands corre-
sponding to duplex DNA.20 Quantitative analysis of the ratio

P. Patel et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5086–5090
5089
Figure 5. Cell death of human leukemia cell lines following treatment with 14
(10 M) for 48 h.
l
Figure 3. Thermal denaturation curves. Changes in % hyperchromicity at 260 nm
and cell viability was determined following 48 h incubation with
cancer cells at 37 °C. A sulforhodamine B (SRB cell viability assay)24
was performed to determine cell death in the presence of 14. Ami-
noacyl nucleolipid 14 was found to exhibit potent and selective
anti-cancer activity (see Supplementary data, Fig. S3), with greater
than 90% cell death against a SR human lymphoblastic leukemia
cancer cell line (Fig. 5). Interestingly, GRP78 expression levels are
elevated in leukemic blasts of adult patients and early relapse in
versus temperature of GRP78 DNA (1.1
(0.2–3.9 mM).
lM) in the presence of ligand 14
childhood leukemia25 suggesting
a correlation between the
GRP78 DNA binding affinity and anti-leukemic activity of amino-
acyl nucleolipid 14. We are currently investigating the bio-molec-
ular basis for the potent and selective activity of 14 in leukemia
cells.
In conclusion, with the synthesis of derivative 14, we have dem-
onstrated the utility of a new class of aminoacyl nucleolipid, which
can have great relevance due to their tight binding with GRP78
DNA and marked ability to trigger potent and selective anti-leuke-
mic activity. This work paves the way for additional studies into
the mechanism of aminoacyl nucleolipid anti-cancer activity.
Moreover, 14 may form the basis for novel ligand design and devel-
opment for anti-cancer applications.
Figure 4. Circular dichroism (CD) spectrum of GRP78 DNA (1.1
plexing ligand 14 (0–3.9 mM) in phosphate buffer.
lM) with com-
Acknowledgments
persists at higher ligand concentrations (i.e. 100 equiv 14/P,
3.9 mM).
To further characterize the size of the complex formed between
GRP78 DNA and 14, dynamic light scattering (DLS) studies were
performed relative to native DNA and aminoacyl nucleolipid 14.
The authors thank Seton Hall University, the Queens College
Research Enhancement and PSC-CUNY Research Award for
financial support. The testing of 14 was performed by the
Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer Institute and the URL
to the Program’s website is http://dtp.cancer.gov. The authors also
thank Mark Hail at Novatia Inc. for DNA mass spectrometry
analyses.
DNA (1.1 lM) was prepared with and without aminoacyl nucleoli-
pid 14 (3.9 mM) and incubated in phosphate buffer for 36 h prior
to DLS measurements (Table 2). The observed effective diameters
for the particle sizes shows fixed values for DNA (ꢁ1.8 nm) and
aminoacyl nucleolipid 14 (ꢁ460 nm). Alternatively, the complex
formed between DNA and 14 illustrates a greater than two fold in-
crease in particle sizes from 0 to 36 h, suggesting the formation of a
higher-order complex structure.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.0
7.030.
In order to assess the anti-cancer activity of aminoacyl nucleo-
lipid 14, a cancer cell line screening assay was conducted against a
panel of 60 cell lines selected at the National Cancer Institute
(NCI).23 A single dose (10
lM) screen was performed with 14,
References and notes
1. (a) Simeone, L.; Irace, C.; Di Pascale, A.; Ciccarelli, D.; D’Errico, G.;
Montesarchio, D. Eur. J. Med. Chem. 2012, 57, 429; (b) Patil, S. P.; Yi, J. W.;
Bang, E. K.; Jeon, E. M.; Kim, B. H. Med. Chem. Commun. 2011, 2, 505; (c) Yang, H.
W.; Yi, J. W.; Bang, E. K.; Jeon, E. M.; Kim, B. H. Org. Biomol. Chem. 2011, 9, 291;
(d) Ceballos, C.; Prata, C. A. H.; Giorgio, S.; Garzino, F.; Payet, D.; Barthélémy, P.;
Grinstaff, M.; Camplo, M. Bioconjugate Chem. 2009, 20, 193.
2. (a) Watson, J.; Crick, H. Nature 1953, 171, 737; (b) Hoogsteen, K. Acta
Crystallogr. 1963, 16, 907; (c) Hopton, S. R.; Thompson, A. S. Biochemistry
2011, 50, 4143.
Table 2
Effective diameters (nm) of aminoacyl nucleolipid, 14, and DNA+14 at 25 °C
Compound
14
Time (h)
Effective diameters (nm)
0
36
0
468 50
400 50
980 50
DNA+14
3. Mann, A.; Thakur, G.; Shukla, V.; Singh, A. K.; Khanduri, R.; Naik, R.; Jiang, Y.;
Kalra, N.; Dwarakanath, B. S.; Langel, U.; Ganguli, M. Mol. Pharm. 2011, 8, 1729.
4. Safinya, C. R. Curr. Opin. Struct. Biol. 2001, 11, 440.
4500 100
9300 100
36

5090
P. Patel et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5086–5090
5. (a) Chien, P. Y.; Wang, J.; Carbonaro, D.; Lei, S.; Miller, B.; Sheikh, S.; Ali, S. M.;
Ahmad, M. U.; Ahmad, I. Cancer Gene Ther. 2005, 12, 321; (b) Ward, C. M. Curr.
Opin. Mol. Ther. 2000, 2, 182; (c) Yin, Z.; Liu, N.; Ma, M.; Wang, L.; Hao, Y.;
Zhang, X. Int. J. Nanomedicine 2012, 7, 4625; (d) Wang, H. Y.; Yi, W. J.; Qin, S. Y.;
Li, C.; Zhuo, R. X.; Zhang, X. Z. Biomaterials 2012, 33, 8685; (e) Yang, F.; Nickols,
N. G.; Li, B. C.; Marinov, G. K.; Said, J. W.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A.
2013, 110, 1863.
13. Kaczmarek, O.; Brodersen, N.; Bunge, A.; Löser, L.; Huster, D.; Herrmann, A.;
Arbuzova, A.; Liebscher, J. Eur. J. Org. Chem. 2008, 11, 1917.
14. Fan, Y.; Gaffney, B. L.; Jones, R. A. Org. Lett. 2004, 6, 2555.
15. Simeone, L.; Milano, D.; De Napoli, L.; Irace, C.; Di Pascale, A.; Boccalon, M.;
Tecilla, P.; Montesarchio, D. Chemistry 2011, 17, 13854.
16. Karsisiotis, A. I.; Hessari, N. M.; Novellino, E.; Spada, G. P.; Randazzo, A.; Webba
da Silva, M. Angew. Chem., Int. Ed. 2011, 50, 10645.
6. (a) Ni, M.; Zhang, Y.; Lee, A. S. Biochem. J. 2011, 434, 181; (b) Pfaffenbach, K. T.;
Lee, A. S. Curr. Opin. Cell Biol. 2011, 23, 150; (c) Quinones, Q. J.; de Ridder, G. G.;
Pizzo, S. V. Histol. Histopathol. 2008, 23, 1409; (d) Ni, M.; Lee, A. S. FEBS Lett.
2007, 581, 3641.
17. Ting, J.; Lee, A. S. DNA 1988, 7, 275.
18. Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.; Fisher, E. F.;
McBride, L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J. Y. Methods Enzymol. 1987,
154, 287.
7. (a) Fu, Y.; Li, J.; Lee, A. S. Cancer Res. 2007, 67, 3734; (b) Lee, A. S. Cancer Res.
2007, 67, 3496; (c) Li, J.; Lee, A. S. Curr. Mol. Med. 2006, 6, 45; (d) Arap, M. A.;
Lahdenranta, J.; Mintz, P. J.; Hajitou, A.; Sarkis, A. S.; Arap, W.; Pasqualini, R.
Cancer Cell 2004, 6, 275.
19. Gilar, M.; Fountain, K. J.; Budman, Y.; Neue, U. D.; Yardley, K. R.; Rainville, P. D.;
Russell, R. J.; Gebler, J. C. J. Chromatogr. A 2002, 958, 167.
20. Kim, S. W.; Kim, N. Y.; Choi, Y. B.; Park, S. H.; Yang, M.; Shin, S. J. Controlled
Release 2010, 143, 335.
8. (a) Kim, Y.; Lillo, A. M.; Steiniger, S. C.; Liu, Y.; Ballatore, C.; Anichini, A.;
Mortarini, R.; Kaufmann, G. F.; Zhou, B.; Felding-Habermann, B.; Janda, K. D.
Biochemistry 2006, 45, 9434; (b) Liu, Y.; Steiniger, S. C. J.; Kim, Y.; Kaufmann, G.
F.; Felding-Habermann, B.; Janda, K. D. Mol. Pharm. 2007, 4, 435.
9. Johnson, D. C.; Widlanski, T. S. Org. Lett. 2004, 6, 4643.
21. Arigon, J.; Prata, C. A. H.; Grinstaff, M. W.; Barthelemy, P. Bioconjugate Chem.
2005, 16, 864.
22. Kypr, J.; Kejnovska, I.; Renciuk, D.; Vorlickova, M. Nucleic Acids Res. 2009, 37,
1713.
23. Showmaker, R. H. Nat. Rev. Cancer 2006, 6, 813.
10. Mourani, R.; Damha, M. J. Nucleosides, Nucleotides Nucleic Acids 2006, 25, 203.
11. Foitzik, R. C.; Devine, S. M.; Hausler, N. E.; Scammells, P. J. Tetrahedron 2009, 65,
8851.
24. Voigt, W. Methods Mol. Med. 2005, 110, 39.
25. Wey, S.; Luo, B.; Tseng, C. C.; Ni, M.; Zhou, H.; Fu, Y.; Bhojwani, D.; Carroll, W.
L.; Lee, A. S. Blood 2012, 119, 817.
12. Matulic-Adamic, J.; Beigelman, L.; Dudycz, W. L.; Gonzalez, C.; Usman, N.
Bioorg. Med. Chem. Lett. 1995, 5, 2721.