Peptoid dendrimers-microwave-assisted solid-phase synthesis and transfection agent evaluation

Article abstract of DOI:10.1016/j.tetlet.2007.11.122

Three generations of peptoid-based dendrimers were synthesised by solid-phase methods, using N-Fmoc-N-(6-N′-Fmoc-aminohexyl)-glycine as both the initiator core and the monomer unit, which offer an unusual dendrimeric periphery composed of both secondary and primary amines. The third generation compound proved to be an efficient mediator of transfection while displaying minimal cytotoxicity.

Full text of DOI:10.1016/j.tetlet.2007.11.122

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 923–926
Peptoid dendrimers—microwave-assisted solid-phase synthesis
and transfection agent evaluation
Juan J. Diaz-Mochon, Mario A. Fara, Rosario M. Sanchez-Martin, Mark Bradley *
School of Chemistry, Joseph Black Building, West Mains Road, University of Edinburgh, EH9 3JJ Edinburgh, United Kingdom
Received 3 October 2007; revised 31 October 2007; accepted 9 November 2007
Available online 21 December 2007
Abstract
0
Three generations of peptoid-based dendrimers were synthesised by solid-phase methods, using N-Fmoc–N-(6-N -Fmoc-aminohexyl)-
glycine as both the initiator core and the monomer unit, which offer an unusual dendrimeric periphery composed of both secondary and
primary amines. The third generation compound proved to be an efficient mediator of transfection while displaying minimal cytotoxicity.
Ó 2007 Elsevier Ltd. All rights reserved.
Dendrimers are fascinating compounds, offering many
of the properties of more conventional polymers but with
a highly defined shape, molecular mass and with a structure
which displays a large number of functional groups on the
periphery, while ‘hiding’ much internal functionality. Two
dominant synthetic methodologies have been used to gen-
erate dendrimers: (i) a linear approach as initially described
PAMAM dendrimers have found extensive practical
application in the area of gene delivery and a number of
3
other biomedical applications such as magnetic resonance
4
,5
imaging (MRI) and delivery of RNAi into mammalian
6
cells. Another family of dendrimers, which have seen prac-
tical application, are those based on repeating and amplify-
ing amino acid residues, most notably the lysine dendrimers
1
7
by V o¨ gtle, where surface functionalities grow at an
introduced by Tam which offer a chemical route to the
8
exponential rate while the numbers of synthetic steps
increases linearly and (ii) a convergent synthesis strategy
development of high-density antigenic peptides.
A new step forward, as reported in this Letter, is a
hybrid combination of PAMAM and peptide dendrimers
in which peptoids (N-alkylglycines) are used both as an ini-
tiator and a monomer unit. In this Letter, we describe the
2
as introduced by Fr e´ chet, where dendrimeric fragments
are synthesised before condensation to give the full
dendrimeric structure.
9
–11
Polyamidoamines (PAMAMs) can be regarded as the
classical dendrimeric material and are typically constructed
by the reaction of methyl acrylate with an initiation unit
such as ammonia, with subsequent treatment with dia-
mines followed by repetitive treatment with methyl acrylate
and diamine. Such dendrimers display multiple primary
amines on their external surface which can vary from 4
solid-phase synthesis of peptoid dendrimers
using N-
0
Fmoc–N-(6-N -Fmoc-aminohexyl)-glycine 5 (rather than
Fmoc-Lys(Fmoc) or methyl acrylate and diamine) and
their evaluation as transfection agents. The monomer unit
1
0,11
5 was prepared
by mono protection of 1,6-diamino-
hexane 1 with Boc O before alkylation with ethyl bromo-
2
acetate to give ester 3. Saponification of the ester
followed by N-Fmoc protection with Fmoc-succinimide
gave acid 4. Boc deprotection with TFA followed by N-
Fmoc protection with Fmoc-succinimide gave rapid access
(
generation 0) to 4096 (generation 10) as well as having a
host of primary amides and tertiary amines within the
global structure.
1
2
to 5 (Scheme 1).
*
The dendrimers were assembled with high efficiency
using monomer 5 on PS-Rink amide resin 6 using HOBt/
Corresponding author. Tel.: +44 131 650 4820; fax: +44 131 650 6453.
E-mail address: mark.bradley@ed.ac.uk (M. Bradley).
0
040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.11.122

924
J. J. Diaz-Mochon et al. / Tetrahedron Letters 49 (2008) 923–926
O
O
NH
O
O
O
O
O
O
NH
2
O
6 O
NH
NH
(
i)
(ii)
NH
(iii)
(iv)
(
v)
N
O
OH
6
6 O
6
(vi)
6
O
O
NH
2
N
NH
2
HN
OH
OEt
O
2
3
4
5
1
0
10,12
Scheme 1. Synthesis of N-Fmoc–N-(6-N -Fmoc-aminohexyl)-glycine 5.
Reagents and conditions: (i) Boc
2
O (0.3 equiv), dioxane, 24 h, 91%; (ii) ethyl
O/CH CN (1:1),
O (9:1), 16 h, 64% over two
bromoacetate (1 equiv), Et
3
N (3 equiv), THF, 24 h, 55%; (iii) NaOH (1 equiv), CH
pH 8.5–9.0, 45 min, 40%; over two steps; (v) TFA/DCM (1:1), 2 h; (vi) Fmoc–OSu (1.2 equiv), DIPEA (2 equiv), THF/H
steps.
3
OH/H O/dioxane (3:1:8); (iv) Fmoc–OSu (1 equiv) H
2
2
3
2
1
1
DIC chemistry and microwave irradiation (Scheme 2).
More generally, microwave irradiation offers huge promise
in dendrimer synthesis, allowing reactions to be readily
forced. To ensure coupling completion two colorimetric
to the final compounds in crude yields of 85% (see Fig. 1)
displaying 4, 8 and 16 primary and secondary amines on
their surface just after 1, 2 and 3 coupling steps. MS anal-
ysis of the peptoid dendrimers gave the following data:
positive ES G1 calcd (C H N O ) 485.7, found m/z:
1
3
tests were used: (i) The Kaiser test for primary amines
and (ii) a chloranil test for detecting secondary amines.
2
4
51
2+
7
3
1
4
+
486.3 [M+H] , 243.7 [M+2H] ; MALDI-TOF G2 calcd
+
The first generation dendrimer gave, not unexpectedly,
negative tests after a single coupling, but subsequent gener-
ations required second couplings to ensure 100% reaction.
Following final Fmoc removal the polyamidoamines
were cleaved from the resin using TFA/TIS/DCM
(C H N O ), 1109.91, found m/z: 1110.64 [M+H] ;
5
6
115 15
7
MALDI-TOF G3 calcd (C120H243N O ), 2358.92, found
31
15
+
m/z: 2359.94 [M+H] .
To evaluate the properties of these compounds as trans-
fection agents, their complexes with pEGFP-N1 (a plasmid
which carries the gene that encodes green fluorescence
(
90:5:5) and precipitated with cold diethyl ether to give rise
(
(
(
(
i)
ii)
iii)
iv)
Peptoid dendrimers
G1, G2 and G3
H N Rink PS
2
6
O
H
N
X
NH
H
2
N
N
X
O
X
O
HN
NH
2
X
HN
NH
NH
2
X
O
X
NH
NH
N
N
NH
2
NH
2
X
X
O O
O
X
HN
X
NH
2
NH2
O
N
N
X
NH
N
NH
O
X
HN
H
NH
2
O
X
N
O
O
O
NH
2
NH2
X
NH
N
X
NH
O
X
HN
O
N
O
O
N
X
NH2
2
H N
H
N
X
NH2
N
H
N
N
H
X
O
X
HN
N
O
HN
X
NH
O
O
O
O
NH
2
O
X
HN
HN
HN
X
O
X
X
2
H N
2
H N
NH
Peptoid dendrimer G1
Peptoid dendrimer G2
Peptoid dendrimer G3
NH
2
1
1
Scheme 2. Solid-phase synthesis of peptoid dendrimers using monomer 5. Reagents and conditions: (i) acid 5 (3 equiv), DIC (3 equiv) and HOBt
(
(
3 equiv) in DMF at 0.1 M; microwave irradiation at 60 °C for 20 min; (ii) Fmoc deprotection: 20% piperidine in DMF (2 ꢀ 10 min) at room temperature;
iii) repeat (i) and (ii); (iv) TFA/TIS/DCM (90:5:5) for 2 h. X = (CH
2 6
) .

J. J. Diaz-Mochon et al. / Tetrahedron Letters 49 (2008) 923–926
925
ADC1 A, ADC1 (ELSD 2007-08-21 HPLC NO 223\072-0501.D)
PMP1 , Solvent B
1
1
20
00
Norm
5
:1
5
:1
10:1
40:1
1
4000
2000
0000
10:1
20:1
4
0:1
1
1
20:1
8
0
0
0
0
8
6
4
2
000
000
000
000
0
6
4
2
min
16
0
0
2
4
6
8
10
12
14
Untreated cells
DNA
G2 PAMAM
G3 Peptoiddendrimer
Fig. 1. HPLC trace of crude peptoid dendrimer G3. HPLC conditions:
Gradient from 5% CH CN to 95% CH CN over 12 min on a C18 prodigy
Fig. 3. Cytotoxicity of G3 peptoid dendrimer with HEK293T cells
3
3
1
9
compared to PAMAM G2 as measured by the MTT assay. Viability was
expressed as the percentage of the untreated control cells. Each bar
represents the mean ± SD; n = 3.
column (150 ꢀ 4.60 mm; 5 lm) with evaporative light scattering (ELS)
detection.
protein (GFP)) were incubated with human embryonic kid-
ney (HEK293T) cells. PAMAM 2.0 was used as a control
as it also possesses 16 amino groups.
were also non-toxic. This can be rationalised by the fact that
a combination of primary and secondary amines is known
to generate a proton sponge effect which can facilitate the
DNA transfection process, by facilitating the release of
1
5
Cells were incubated for 48 h with the dendrimer mixed
1
6,17
with the plasmid at various molar ratios
(5:1, 10:1, 20:1
20
the plasmid from the cytoplasmic lysosome. Finally, in
and 40:1). Analysis showed that while generations 1 and 2
had no noticeable effect, G3 was able to transfect
HEK293T cells with high efficiency (Fig. 2).
areas outside transfection, peptoid dendrimers could also
offer a solution to one of the known problems of peptide
21
dendrimers, namely their very rapid biodegradation.
Figure 3 shows the viability of HEK293T cells when
treated with peptoid dendrimers G3 and PAMAM 2.0
Acknowledgements
1
9
complexes with the plasmid. The peptoid dendrimers
were found to be non-toxic at all of the ratios tested, rein-
forcing the use of these molecules as gene delivery agents.
In conclusion, peptoid dendrimers containing primary
and secondary amines on their periphery have been success-
fully synthesised by solid phase and microwave mediated
methods using a ‘lysine-type’ peptoid monomer. The higher
generation peptoid dendrimers were able to transfect cells
with higher efficiency than the PAMAM counterpart and
J.J.D.-M. (EPSRC) R.M.S.-M. thank the Royal Society
for the award of a Dorothy Hodgkin Fellowship.
References and notes
1
. Buhler, E.; Wehner, W.; V o¨ gtle, F. Synthesis 1978, 155–158.
2. Hawker, C. J.; Fr e´ chet, J. M. J. J. Am. Chem. Soc. 1990, 112, 7638.
3
4
. Sanclimens, G.; Shen, H.; Giralt, E.; Albericio, F.; Saltzman, M. W.;
Royo, M. Biopolymers 2005, 80, 800–814.
. (a) Kobayashi, H.; Brechbiel, M. W. Adv. Drug Deliv. Rev. 2005, 57,
2
271–2286; (b) Venditto, V. J.; Regino, C. A. S.; Brechbiel, M. W.
Mol. Pharm. 2005, 2, 302–311.
. Stiriba, S.-E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41,
4
3
3
2
2
1
1
000
500
000
500
000
500
000
5
6
1
329–1334.
. (a) Zhou, J.; Wu, J.; Hafdi, N.; Behr, J.; Erbacher, P.; Peng, L. Chem.
Commun. 2006, 2362–2364; (b) Lesniak, W. G.; Kariapper, M. S. T.;
Nair, B. M.; Tan, W.; Hutson, A.; Balogh, L. P.; Khan, M. K.
Bioconjugate Chem. 2007, 18, 1148–1154.
. Sadler, K.; Tam, J. P. Rev. Mol. Biotechnol. 2002, 90, 195–229.
. Tam, J. P. Proc. Natl. Acad. Nat. Sci. U.S.A. 1988, 85, 5409–5413.
7
8
9. (a) Basso, A.; Bradley, M.; Evans, B.; Pegg, N. Chem. Commun. 2001,
97–698; (b) Cummins, W. J.; Hamilton, A. L.; Bradley, M.; Ellard,
J.; Zollitsch, T.; Briggs, M. S. J.; WO 03014743; 2003; Chem. Abstract
003, 138, 166223; (c) Lebreton, S.; How, S.-E.; Buchholz, M.;
6
2
5
00
Yingyongnarongkul, B.-E.; Bradley, M. Tetrahedron 2003, 59, 3945–
3953; (d) Swali, V.; Wells, N. J.; Langley, G. J.; Bradley, M. J. Org.
Chem. 1997, 62, 4902–4903; (e) Ternon, M.; Diaz-Mochon, J. J.;
Belsom, A.; Bradley, M. Tetrahedron 2004, 60, 8721–8728; (f) Wells,
N. J.; Basso, A.; Bradley, M. Biopolymers 1999, 47, 381–396.
0. Peretto, I.; Sanchez-Martin, R. M.; Wang, X.; Ellard, J.; Mittoo, S.;
Bradley, M. Chem. Commun. 2003, 2312–2313.
11. (a) Fara, M. A.; Diaz-Mochon, J. J.; Bradley, M. Tetrahedron Lett.
2006, 47, 1011–1014; (b) Collins, J. M.; Leadbeater, N. E. Org.
Biomol. Chem. 2007, 5, 1141.
0
G2
G1
G2
G3
40:1
Peptoid-dendrimers
PAMAM
untreated cells
5:1 10:1 20:1
1
Fig. 2. Transfection activity of peptoid dendrimers G1–G3 and PAMAM
.0 with HEK293T. Ninety-six well plates were used, with each well
containing 0.2 lg of DNA plasmid with different molar ratios
transfection agents/DNA.
1
8
2
1
6,17
of
16

926
J. J. Diaz-Mochon et al. / Tetrahedron Letters 49 (2008) 923–926
1
2. Acid 4 (1 g, 2 mmol) was dissolved in TFA/DCM (1:1, 20 mL), and
stirred for 2 h to remove the Boc protecting group. The solution was
concentrated in vacuo and the crude compound precipitated with
diethyl ether to give a sticky oil. The compound was dissolved in
16. The molar ratio is equal to mols of transfection agent/number of
plasmid equivalents. Plasmid equivalents is equal to the mass of
plasmid used/average mass of a single nucleotide and is approxi-
mately equal to the number of moles of phosphate within the
plasmid.
2
THF/H O (9:1, 20 mL) before adding Fmoc-OSu (0.8 g, 2.4 mmol)
and DIPEA (0.64 mL, 4 mmol). The reaction mixture was stirred at
room temperature for 16 h. 1 N HCl (20 mL) was added to the
solution before removing the THF in vacuo. The compound was
extracted with DCM (3 ꢀ 30 mL), the organic phase was washed with
brine (3 ꢀ 20 mL), dried with magnesium sulfate and then concen-
trated in vacuo. The crude material was purified by column
17. N:P ratios used (and the correspond to molecular ratios); were: 5:1,
10:1, 20:1, 40:1; G1 (20:1, 40:1, 80:1, 160:1); G2 (40:1, 80:1, 160:1,
320:1); G3 (80:1, 160:1, 320:1, 640:1) PAMAM 2.0 (80:1, 160:1, 320:1,
640:1). We feel that N:P ratio should be treated with caution as not
all the external nitrogens (amines) are protonated at physiological
pH’s.
chromatography on silica gel, eluting with DCM to DCM/MeOH
18. HEK293T cells were seeded in a 96 well plate and incubated in
DMEM complete media, 10% FCS, until 70% confluence one day
prior to transfection. 10 lL of transfection cocktail [2.5 lL DNA
(4.7Kb plasmid pEGFP-N1) containing 0.20 lg of DNA (0.6 nequiv
1
(
9:1, R
NMR (CDCl
m, 4H), 3.86 and 3.95 (two br s, 2H), 4.17 (m, 2H), 4.39 and 4.50 (two
f
= 0.3) to give acid 5 as a white powder (800 mg, 64%).
H
3
, 250 MHz): 1.09–1.46 (m, 8H), 3.12 and 3.35 (two br
1
3
16
m, 4H), 7.23–7.34 (m, 8H), 7.52–7.60 (m, 4H), 7.70–7.76 (m, 4H).
NMR (CDCl , 62.9 MHz): two rotamers d 26.1 and 26.2, 27.7, 28.1,
9.7, 40.9, 47.3, 48.5, 67.8, 120.0, 124.8 and 125.0, 127.1, 127.7, 141.4,
C
in TE buffer) were mixed with different molar ratios of dendrimer
3
(5 mM stock solutions in PBS) for 30 min before addition to each well
containing 90 lL of DMEM complete media. Transfection activities
were measured following 48 h of incubation by FACS analysis to
measure relative levels of GFP expression.
2
1
+
43.9, 156.1, 156.8, 174.2; mp = 95–100 °C; ES MS C38
38 2 6
H N O
+
+
(618) m/z (%): 619 [M+H] (100), 641 [M+Na] (25); HRMS (ESI):
+
[
M+NH
4
]
calcd for C38
H
42
N
3
O
6
, 636.3068; found, 636.3067.
19. Mosman, T. J. Immunol. Meth. 1983, 65, 55–63.
1
3. (a) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595–598; (b) Egner, B. J.; Bradley, M. Drug
Discovery Today 1997, 2, 102–109.
20. (a) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A
1995, 92, 7297–7301; (b) Liu, Y.; Wu, D.; Ma, Y.; Tang, G.; Wang, S.;
He, C.; Chung, T.; Goh, S. Chem. Commun. 2003, 2630–2631.
21. Boyd, B. J.; Kaminskas, L. M.; Karellas, P.; Krippner, G.; Lessene,
R.; Porter, C. J. H. Mol. Pharm. 2006, 3, 614–627.
14. Vojkovsky, T. Peptide Res. 1995, 8, 236–237.
15. PAMAM dendrimer, 1,4-diaminobutane core, generation 2.0 solution
20% wt. in methanol was purchased from Sigma–Aldrich.