Synthesis of bifunctional cationic compound for gene delivery

Article abstract of DOI:10.1016/S0040-4039(00)02221-8

Bifunctional cationic compound carrying trivalent galactosides as the cell targeting ligand and DAB-dendr-(NH₂)₈ (generation 2.0) as the DNA binding domain was synthesized for gene delivery to hepatocytes. DAB-dendr-(NH₂)₄ (generation 1.0) conjugated with a hydrocarbon chain was used as a scaffold for the attachment of three galactosides, while the other hydrocarbon end was linked with DAB-dendr-(NH₂)₈ (generation 2.0) through a carbamate bond. This design provided an effective entry for the synthesis of a polyamine compound having the cell targeting galactosyl ligand. Preliminary in vitro transfection results demonstrated that the bifunctional cationic compound could effectively deliver the gene into HepG2 cells.

Full text of DOI:10.1016/S0040-4039(00)02221-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 1007–1010
Synthesis of bifunctional cationic compound for gene delivery
Tan Ren, Guisheng Zhang and Dexi Liu*
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA
Received 8 November 2000; accepted 28 November 2000
Abstract—Bifunctional cationic compound carrying trivalent galactosides as the cell targeting ligand and DAB-dendr-(NH2)8
(generation 2.0) as the DNA binding domain was synthesized for gene delivery to hepatocytes. DAB-dendr-(NH2)4 (generation
1.0) conjugated with a hydrocarbon chain was used as a scaffold for the attachment of three galactosides, while the other
hydrocarbon end was linked with DAB-dendr-(NH2)8 (generation 2.0) through a carbamate bond. This design provided an
effective entry for the synthesis of a polyamine compound having the cell targeting galactosyl ligand. Preliminary in vitro
transfection results demonstrated that the bifunctional cationic compound could effectively deliver the gene into HepG2 cells.
© 2001 Elsevier Science Ltd. All rights reserved.
cytes, the hydrophobic chain in the middle provides a
sheath around the DNA that protects it against degra-
dation by the biological fluids.
The biological process where hepatic asialoglycoprotein
receptor can recognize and uptake b- -galactoside ter-
D
minated glycoproteins is well known.1 Previous efforts
in understanding this recognition event through the
probes of artificial glycopeptides having galactoside
residues revealed that an enormous affinity enhance-
ment was achieved by multivalent binding.2 Recently,
the use of asialoglycoprotein receptor as a target
formed a basis for targeted gene delivery into hepato-
cytes.3 As part of our program dealing with cationic
compound-mediated gene delivery and cell targeting,4
here we combined the clustered galactosyl ligand and
poly(propylene imine) dendrimer onto a hydrocarbon
chain. We expected that this bifunctional cationic com-
pound could serve as a targetable DNA-vector which
could efficiently deliver the gene into hepatocytes
through an asialoglycoprotein receptor. The rationale
for our design is that the DAB-dendr-(NH2)8 should
spontaneously compact the DNA via an electrostatic
interaction, the galactose residues on the other end of
the molecule serve as the targeting ligand for hepato-
b-D-galactose was converted to its hexyl spacer-armed
derivative containing an activated imidazole carboxylic
ester as the reactive end group (Scheme 1). The synthe-
sis was started by chemoselective removal of anomeric
acetyl group in pentaacetate b-D-galactose by hydrazine
acetate.5 Subsequent treatment of the resulting hemiac-
etal with large excess amounts of trichloroacetonitrile6
in the presence of a catalytic amount of DBU7 for 1.5
h at 0°C, gave the thermodynamically more stable
a-trichloroacetimidate in an excellent yield. AgOTf pro-
moted O-glycosylation gave the corresponding b-gly-
coside derivative
1
in 97% isolated yield.8 The
stereochemistry of compound 1 was confirmed by its 1H
NMR spectrum with the anomeric proton exhibiting a
doublet with a coupling constant of 7.9 Hz centered at
4.44 ppm. Desilylation of 1 with tetrabutylammonium
fluoride by a standard method released the primary
Scheme 1. Reagents and conditions: (a) NH2NH2–AcOH (1.1 equiv.), DMF, 60°C, 30 min, 100%; (b) CCl3CN–CH2Cl2 (1:4=v/v),
,
cat. DBU, 90%; (c) 4 A MS, HO(CH2)6OTBDMS (0.5 equiv.), then AgOTf (0.3 equiv.), 0°C, 2 h, 97%; (d) Bu4NF (1.5 equiv.),
THF, rt, 12 h, 90%; (e) CDI (1.25 equiv.), DMAP (0.2 equiv.), CH2Cl2, rt, 1 h, 95%. DBU=1,8-diazabicyclo[5,4,0]undec-7-ene,
CDI=1,1%-carbonyldiimidazole, DMAP=4-(dimethylamino)pyridine.
Keywords: galactoside; polyamine dendrimer; targeting ligand; carbamate; 1,1%-carbonyldiimidazole; gene delivery.
* Corresponding author.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)02221-8

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T. Ren et al. / Tetrahedron Letters 42 (2001) 1007–1010
(Scheme 2). Treatment of 1,12-diol with 0.5 equiv. of
TBDMSCl in the presence of imidazole afforded the
corresponding mono-TBDMS derivative. The primary
alcohol in the mono-TBDMS derivative was activated
with CDI in the presence of 4-(dimethylamino)pyridine
(DMAP) to give the imidazolide 3 in an excellent yield.
Compound 3 was treated with excess amounts of DAB-
dendr-(NH2)4 to give carbamate 4, the excess amounts
of unreacted DAB-dendr-(NH2)4 were removed by
alcohol, which was activated with 1,1%-carbonyldiimida-
zole (CDI)9 under DMAP10 to afford the imidazole
carboxylic ester 2.11
Commercially available 1,12-diol was chosen as a
hydrophobic linker, since it has two hydroxyl function-
alities available for generating two carbamate bonds,
which could bridge the cell surface targeting ligand—
galactoside and the DNA binding domain—polyamine
Scheme 2. Reagents and conditions: (a) TBDMSCl (0.5 equiv.), imidazole (1 equiv.), DMF, 78%; (b) CDI (1.25 equiv.), DMAP
(0.2 equiv.), CH2Cl2, rt, 1 h. 90%; (c) DAB-dendr-(NH2)4 (4 equiv.), dry MeCN, DMAP (0.2 equiv.), rt, 1 h. 85%; (d) Compound
2 (3.5 equiv.), THF, DMAP, reflux, 6 h, 50%; (e) Bu4NF (1.5 equiv.), THF, rt, 18 h, 85%; (f) CDI (1.5 equiv.), DMAP (0.2
equiv.), CH2Cl2, rt, 2 h, 88%; (g) DAB-dendr-(NH2)8 (4 equiv.), THF, reflux, 1.5 h; (h) 0.04 M NaOMe–MeOH, rt, 2 h; then
Dowex 50WX4-100. ca. 70% for two steps.

T. Ren et al. / Tetrahedron Letters 42 (2001) 1007–1010
1009
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activated galactoside 2 in an SN2 manner. Indeed,
when compound 4 was reacted with an excess amount
of imidazolide 2 in refluxing tetrahydrofuran (THF)
for 6 h, the clustered trisaccharide derivative 5 was
obtained in 50% separated yield.12 Exposure of clus-
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tered trisaccharide silyl ether
5
to tetra-
butylammonium floride in THF slowly liberated the
primary alcohol, which was further activated with
CDI to give the imidazole carboxylic ester 6.13 Treat-
ment of 6 with excess amounts of DAB-dendr-(NH2)8
(generation 2.0) furnished the construction of the final
carbamate bond. Removal of excess amounts of unre-
acted DAB-dendr-(NH2)8 by careful workup with 5%
potassium hydroxide aqueous solution14 led to the
compound 7.15 Deprotection of peracetate in 7 was
achieved under Zemple´n conditions. After neutraliza-
tion with acidic resin and concentration, the reaction
residues were dialyzed against water in a membrane
with molecular weight cut-off of 1000. Finally,
lyophilization of the dialyzed compound afforded the
desired product 8 in ca. 70% yield for the last two
steps.16
Transfection efficiency of 8 was initially tested in
HepG2 liver cells. Five nmol of compound 8 mixed
with 1 mg of pCMV-Luc plasmid DNA was used for
the transfection of HepG2 liver cells (5×104 cells per
well in a 48-well plate). Using a standard transfection
assay with luciferase as the reporter,17 we harvested
64 ng of luciferase protein per mg of extracted
proteins. These results demonstrated that the bifunc-
tional cationic compound 8 could effectively deliver a
gene into HepG2 cells.
In summary, we have developed an efficient route
toward the synthesis of bifunctional cationic com-
pound 8. DAB-dendr-(NH2)4 (generation 1.0) conju-
gated with a hydrocarbon chain was used as a
scaffold for the attachment of three galactosides,
while the other hydrocarbon end was linked with
DAB-dendr-(NH2)8 (generation 2.0) through a carba-
mate bond. This design provided an effective entry
for the synthesis of a polyamine compound having
the cell targeting galactosyl ligand. Preliminary in
vitro transfection results demonstrated that the
bifunctional cationic compound could effectively
deliver a gene into HepG2 cells. Application of this
compound for delivering a gene into the targeted cells
in vivo is now underway.
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T.; Zhang, G. S.; Song, Y. K.; Liu, D. J. Drug Target
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Acknowledgements
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10. For DMAP accelerated the formation of thioimidazolide,
see: Nicolaou, K. C.; Vassilikogiannakis, G.; Kranich, R.;
This work was supported by the grants from the
National Institute of Health (CA72925) and Target
Genetics Corporation.

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T. Ren et al. / Tetrahedron Letters 42 (2001) 1007–1010
Baran, P. S.; Zhong, Y. L.; Natarajan, S. Org. Lett. 2000,
2, 1895.
H-5 and alkyl chain H-1%), 3.47 (m, 3H, alkyl chain H-1%),
3.20 (brm, 8H, 4×CH2NHCO), 2.50–2.35(m, 12H, 2×
N(CH2)3), 2.15 (s, 9H, 3×Ac), 2.07 (s, 18H, 6×Ac), 1.98
(s, 9H, 3×Ac), 1.70–1.50 (m, 24H, 12×CH2), 1.50–1.20
(m, 32H, 16×CH2) ppm.
11. Compound 2: 1H NMR (300 MHz, CDCl3) l 8.14 (s, 1H,
imidazole ring), 7.44 (s, 1H, imidazole ring), 7.08 (s, 1H,
imidazole ring), 5.39 (d, J=3.2 Hz, 1H, H-4), 5.20 (dd,
J=10.4, 8 Hz, 1H, H-2), 5.01 (dd, J=10.4, 3.4 Hz, 2H,
H-3), 4.45 (d, J=8 Hz, 1H, H-1), 4.41 (t, J=6.6 Hz, 2H,
CH2OCO), 4.20 (dd, J=11.2, 4.7 Hz, 1H, H-6a), 4.12
(dd, J=11.6, 6.9 Hz, 1H, H-6b), 3.90 (m, 2H, H-5 and
alkyl chain H-1%), 3.48 (m, 1H, alkyl chain H-1%), 2.15 (s,
3H, Ac), 2.05 (s, 6H, 2×Ac), 1.99 (s, 3H, Ac), 1.78 ( m,
2H, CH2), 1.60 (m, 2H, CH2), 1.42 (m, 4H, 2×CH2) ppm.
12. Compound 5: 1H NMR (300 MHz, CDCl3) l 5.51 (br,
4H, 4×NHCO2), 5.36 (d, J=3 Hz, 3H, H-4), 5.19 (dd,
J=10.4, 7.9 Hz, 3H, H-2), 4.99 (dd, J=10.5, 3.3 Hz, 3H,
H-3), 4.42 (d, J=7.8 Hz, 3H, H-1), 4.10 (dd, J=11.5, 7
Hz, 3H, H-6a), 4.07 (dd, J=11.2, 6.9 Hz, 3H, H-6b), 3.99
(t, J=6.5 Hz, 8H, 4×CH2OCO), 3.88 (m, 6H, H-5 and
alkyl chain H-1%), 3.56 (t, J=6.6 Hz, 2H, CH2OTBDMS),
3.44 (m, 3H, alkyl chain H-1%), 3.17 (brm, 8H, 4×
CH2NHCO2), 2.50–2.40 (m, 12H, 2×N(CH2)3), 2.12 (s,
9H, 3×Ac), 2.02 (s, 18H, 6×Ac), 1.95 (s, 6H, 2×Ac),
1.70–1.50 (m, 24H, 12×CH2), 1.40–1.20 (32H, 16×CH2),
0.86 (s, 9H, t-Bu), 0.02 (s, 6H, 2×CH3) ppm.
14. When the reaction mixture diluted with 50 mL of chloro-
form and washed with saturated NaCl aqueous solution,
a formation of emulsions occurred. However, when the
organic layer was treated with 5% KOH aqueous solution
combined with 10 mL of methanol, a better phase separa-
tion was obtained.
15. Compound 7: 1H NMR (300 MHz, CDCl3) l 5.55 (br,
5H, 5×NHCO2), 5.33 (d, J=3 Hz, 3H, H-4), 5.14 (dd,
J=10.4, 8 Hz, 3H, H-2), 4.96 (dd, J=10.5, 3.3 Hz, 3H,
H-3), 4.40 (d, J=7.9 Hz, 3H, H-1), 4.10 (dd, J=11.1, 6.7
Hz, 3H, H-6a), 4.08 (dd, J=11.1, 6.6 Hz, 3H, H-6b), 3.96
(t, J=6.6 Hz, 10H, 5×CH2OCO), 3.85 (m, 6H, H-5 and
alkyl chain H-1%), 3.40 (m, 3H, alkyl chain H-1%), 3.20 (m,
10H, 5×CH2NHCO), 2.66 (t, J=7 Hz, 14H, 7×CH2NH2),
2.45–2.22 (m, 48H, 8×N(CH2)3), 2.09 (s, 9H, 3×Ac), 2.00
(s, 18H, 6×Ac), 1.93 (s, 9H, 3×Ac), 1.70–1.20 (m, 98H,
7×NH2 and 42×CH2) ppm.
1
16. Compound 8: H NMR (300 MHz, CD3OD) l 4.17 (d,
13. Compound 6: 1H NMR (300 MHz, CDCl3) l 8.13 (s, 1H,
imidazole ring), 7.43 (s, 1H, imidazole ring), 7.07 (s, 1H,
imidazole ring), 5.48 (br, 4H, 4×NHCO2), 5.39 (d, J=3
Hz, 3H, H-4), 5.19 (dd, J=10.4, 8 Hz, 3H, H-2), 4.98
(dd, J=10.4, 3.4 Hz, 3H, H-3), 4.45 (d, J=8 Hz, 3H,
H-1), 4.41 (t, J=6.6 Hz, 2H, CH2OCO), 4.18 (dd, J=
11.3, 6.7 Hz, 3H, H-6a), 4.12 (dd, J=11.3, 7 Hz, 3H,
H-6b), 4.02 (t, J=6.6 Hz, 8H, 4×CH2OCO), 3.92 (m, 6H,
J=6.9 Hz, 3H, H-1), 3.98 (m, 10H, 5×CH2OCO), 3.90–
3.80 (m, 6H, alkyl chain H-1% and H-4), 3.70 (m, 6H,
H-6a and H-6b), 3.54–3.45 (m, 12H, H-2, H-3, H-5 and
alkyl chain H-1%), 3.15 (t, J=6.6 Hz, 10H, 5×
CH2NHCO2), 2.75 (m. 14H, 7×CH2NH2), 2.45 (m, 48H,
8×N(CH2)3), 170–1.20 (m, 84H, 42×CH2) ppm.
17. For detailed experimental procedure of in vitro transfec-
tion see: reference 4b.
.
.