Double exponential growth of aliphatic polyamide dendrimers via AB 2 hypermonomer strategy

Article abstract of DOI:10.1039/b903948a

We report for the first time a novel strategy for the synthesis of aliphatic polyamide dendrimers based on a hypermonomer (1) starting from allylamine. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b903948a

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Double exponential growth of aliphatic polyamide dendrimers
via AB2 hypermonomer strategyw
Baghavathy Subramani Balajia and Michael Robert Lewis*ab
Received (in Cambridge, UK) 25th February 2009, Accepted 1st June 2009
First published as an Advance Article on the web 17th June 2009
DOI: 10.1039/b903948a
We report for the first time a novel strategy for the synthesis
of aliphatic polyamide dendrimers based on a hypermonomer (1)
starting from allylamine.
to probe or detect the actions of enzymes, proteins, and
other biomolecules are gaining attention. The avidin/
streptavidin–biotin system has become a universal tool in
many biotechnological applications13 owing to the stability
of these complexes over a wide range of temperature and pH
conditions. Unlike avidin, streptavidin can bind four biotin
units for enhanced signal intensity; however, the signal
intensity obtained with many systems leaves much to be
improved. In order to enhance signal-to-noise ratios for
diverse and advanced applications, we envisioned building a
new biotin–dendrimer complex for general use in biochemical
detection. The use of multiple end groups per biotin molecule
has the potential to increase the level of sensitivity for various
biosensing applications. Since biotin can be efficiently coupled
with various amino acids using standard solid-phase peptide
chemistry, we chose polyamide units as building blocks. Thus
the target molecule will have biotin at the core and extend its
dendritic arms that terminate with markers or labels. Since
biotin is sensitive to various conditions required for exponential
polyamide dendrimer synthesis, we choose to introduce the
biotin in the last step of our approach. Thus our strategy is
broken into a three-part synthesis consisting of building the
dendrimer scaffold, attaching the biotin unit and adding the
end groups. The last two components can be interchanged.
A survey of the literature provided very little information on
compounds similar to our target polyamide dendrimers.
Initially one of the related methods15,16 was followed, which
used 6-aminohexanoic acid as the starting point for the
polyamide compound. However, this method was found to
be not only lengthy but also associated with poor yields and
long reaction times. Moreover, the alkylation step proved very
difficult to achieve. It required 3 days of reflux and resulted in
a very poor yield. Hence it was decided to abandon this
method.
Interest in dendrimers,1,2 since their introduction, has grown
considerably owing to their uniqueness in synthesis and
applications. It is attractive for researchers to exploit the
distinctive and invaluable properties of dendrimers, as many
of these compounds find use in medicine,3 semiconductor,4
and other fields.5 Whether it is molecular architecture6 or
complex supramolecular assembly,7 multi-fold strategies for
their synthesis have been reported. Two main synthetic
strategies remained unchallenged, namely convergent synthesis
developed by Frechet and Hawker8 and Moore and Xu9 and
divergent synthesis developed by Tomalia et al.,10 Newkome
and Shreiner1. The main drawbacks associated with both
methods are iterative steps, lengthy syntheses requiring
substantial amounts of time and their regular purifications
of most of the intermediates, thus leading to typically poor
overall yields.
Polyamido amine (PAMAM) dendrimers10 are one of the
important classes of dendrimers. The presence of multiple
arms in PAMAM dendrimers enhances the availability and
use of terminal functionalities.11 Owing to multiple synthetic
steps, it is often difficult to synthesize monodisperse higher
generation dendrimers without truncated arms. In order to
improve the efficiency of dendrimer synthesis and reduce the
time taken to prepare higher generations, several methods to
shorten these syntheses have been reported, including double
stage, double exponential growth, hypermonomer, and
orthogonal coupling strategies. We are particularly interested
in the double exponential growth strategy. In this method
hyperbranched monomers12,13 (AB2) can be utilized to make
higher generation products in a fewer number of steps. Use of
orthogonal functionalities for AB2 type monomers can
improve many of the shortcomings imposed by conventional
dendrimer synthesis. However, an easy and convenient
synthetic route to aliphatic polyamide dendrimers using AB2
hypermonomer has not yet been reported.
At this juncture a totally different approach to this problem
was intriguing. A simple retrosynthetic analysis yielded an
AB2 type monomer precursor. It could be envisioned that, if
both A and B are orthogonal functionalities, then selective
manipulation of either side will lead to the desired product in
fewer steps. In theory any amine can be sourced back to
ammonia. Thus, an ammonia equivalent to make an AB2
monomer became the strategic focus. Various ammonia
equivalents are reported in the literature.17 Most of them,18
including electrophilic amination reagents,19 employ strong
bases that are incompatible with esters. Some of them require
very expensive catalysts.20 In searching for an easy alternative
for the ammonia source, allylamine appeared promising. The
allyl group can be orthogonally employed with various acid
sensitive and/or base sensitive groups. Esters were chosen as
A variety of biological functions depend on host–guest
interactions.14 Applications of various synthetic molecules
a Department of Veterinary Medicine and Surgery, University of
Missouri-Columbia, Columbia, MO, USA
b Research Service, Harry S. Truman Memorial Veterans’ Hospital,
Columbia, MO, USA. E-mail: lewismic@missouri.edu;
Fax: +1 573-814-6551; Tel: +1 573-814-6000, Ext. 53703
w Electronic supplementary information (ESI) available: Experimental
details, NMR and mass data of compounds 1–10. See DOI:
10.1039/b903948a
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Scheme 1 Reaction conditions: (a) 2 eq. Br(CH2)5CO2Et, K2CO3–DMF; (b) Pd(PPh3)4; (c) NaOH–MeOH–H2O; (d) 2 eq. (2) + (3),
HBTU–DMF; (e) 2 eq. (5) + (6), HBTU–DMF.
counterparts to the allyl group, owing to their ease of removal.
The first alkylation of allylamine was attempted using ethyl
bromohexanoate under various conditions. It was found that
K2CO3–DMF gave the highest yield and the most pure
product. Pure product was obtained either by column
chromatography (35–40% EtOAc in hexane) or by vacuum
distillation. This AB2 monomer was obtained in a single step
in 93% yield (Scheme 1).
two products, 5 and 6, led to the hexadecamer dendrimer
(7, yield 60%). Thus, by using simple reactions, the hexadecamer
was obtained in five steps. Extension of this exponential
synthesis to larger dendrimers, such as a 256-mer, might be
hindered by steric effects, observed in dendrimers,24 and may
require additional modifications of the AB2 hypermonomer
strategy, in order to achieve optimal synthesis.
After successful synthesis of the dendrimer core, attention
shifted to preparation and attachment of a biotin unit as the
basis for a chemical probe or sensor.25 Even though biotin can
be added in a single step to the dendrimer unit, doing so will not
make the target compound stable under in vivo or ex vivo
conditions.15 Biotinidase selectively cleaves the secondary
amide bond connecting biotin to other molecules. This
enzymatic degradation will lead to the loss of biotin from the
dendritic unit. In order to avoid biotinidase-mediated
decomposition, the secondary amide nitrogen has to be protected.
A potentially simple step to protect the amide is to use alkyl-
substituted amino acids. Owing to very poor solubility in
many organic solvents, a solution-based amide coupling using
unmodified biotin never proceeded to completion. Moreover,
removing excess biotin from the desired product was not
practical. Next, solid-phase techniques for making the biotin
moiety were explored in order to circumvent the removal of
excess of biotin. A C5 or C6 linker would position the biotin
further from the dendrimer unit, thereby alleviating steric
hindrance and making biotin available for complexation with
avidin or streptavidin. However, a suitable C5 or C6 N-alkyl
amino acid could not be obtained easily. In order to overcome
this problem the simplest N-methyl amino acid, sarcosine, was
incorporated between the linker and biotin. The present solid-
phase synthesis began by linking N-fmoc 6-aminohexanoic
acid to 2-chlorotrityl chloride resin, using standard techniques
and standard, sequential coupling of sarcosine and biotin
(Scheme 2). Final cleavage and purification gave the required
product in high yield (77%). Using this solid-phase technique
we have successfully attached commercially available under-
ivatized biotin.
The next task was to remove selectively either of the
protecting groups. For removal of the ester group, NaOH-
based hydrolysis gave the best results (yield 92%).z For the
allyl group removal, Pd-catalyzed deallylation was chosen.21
However, purification was problematic after the deallylation
reaction. Neither the reported purification method nor column
chromatography was successful. However, the issue was
circumvented by selectively choosing the proper purification
method22 (which relies on subtle changes in basicity). This
finding not only gave very pure material (yield 89%) but also
significantly reduced the purification time. The purification
was achieved in less than 30 min. The newly developed method
for the AB2 monomer synthesis has various advantages over
the existing methods: (a) it involves only one step to make the
monomer, (b) manipulation of orthogonal functionalities are
straightforward and simple, (c) the yields are higher, and
(d) purifications can be achieved in a relatively short period
of time.
After successful preparation of these hypermonomers, steps
to make the dendrimer could be pursued. Solid-phase methods
were attempted first. Various attempts to couple the deallylamino
ester (2) to 2-chlorotrityl chloride resin were not successful.
Even though the resin-based amino ester could be made by
changing the starting material to 6-bromohexanoic ester on
Rink amide, the resin-based hydrolysis was not successful. In
order to perform hydrolysis under mild conditions, synthesis
of TBDMS ester or trichloroacetyl ester, instead of ethyl ester
for the AB2 monomer, was attempted. However, neither
change afforded the desired AB2 monomer.
Next solution-based methods for building the dendrimer
were investigated. The deallylamino ester (2) and allylic diacid
(3) were subjected to amide coupling conditions23 and the
tetrameric dendrimer (4) was obtained in high yield (86%).
The product was split into two fractions. Deallylation was
carried out on one to obtain the deallyl tetra ester (5, yield
84%), and the other was hydrolyzed to obtain the allyl tetra
acid (6, yield 71%). Repetition of the amide coupling of these
After synthesizing the dendrimer unit and biotin moiety,
methods to join them were investigated next. The tetramer unit
was chosen for proof-of-principle. Owing to poor solubility of
biotin in DMF the amide coupling was carried out in NMP.
After the reaction was over (TLC analysis) NMP was removed
using high vacuum, and the residue was purified using RP-HPLC.
The resulting biotin tetra ester (9) was hydrolyzed using a
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Scheme 2 Reaction conditions: (a) 2-Cl trityl chloride resin, DIEA, DCM; (b) piperidine–DMF; (c) fmoc-sarcosine, HBTU–DIEA, DMF;
(d) biotin, HBTU–DIEA, NMP–DMF; (e) 2% TFA in DCM; (f) 5, HBTU–DIEA, NMP; (g) NaOH–H2O–MeOH.
NaOH–MeOH–H2O mixture. Following the work-up method
mentioned above the biotin tetra acid was obtained as a white
solid (10). Work is currently under way to attach various
markers and radiolabels to this product, with the future
goal of comparing various signal-to-noise enhancements,
e.g. streptavidin immunohistochemistry, with respect to singly
modified biotin derivatives.
9 Z. Xu and J. S. Moore, Angew. Chem., 1993, 105, 261–264
(Angew. Chem., Int. Ed. Engl., 1993, 32, 246–248).
10 D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos,
S. Martin, J. Roeck, J. Ryder and P. Smith, Polymer, 1985, 17,
117–132; D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos,
S. Martin, J. Roeck, J. Ryder and P. Smith, Macromolecules, 1986,
19, 2466–2468.
11 W. Zhu, B. Okollie, Z. M. Bhujwalla and D. Artemov, Magn.
Reson. Med., 2008, 59, 679–685; V. J. Venditto, C. A. S. Regino
and M. W. Brechbiel, Mol. Pharm., 2005, 2, 302–311.
12 T. Kawaguchi, K. L. Walker, C. L. Wilkins and J. S. Moore,
J. Am. Chem. Soc., 1995, 117, 2159–2165.
13 S. Nara, V. Tripathi, S. K. Chaube, K. Rangari, H. Singh,
K. P. Kariya and T. G. Shrivastav, Talanta, 2008, 77, 210–216.
14 S. M. Sullivan and T. Holyoak, Proc. Natl. Acad. Sci. U. S. A.,
2008, 105, 13829–13834.
15 D. S. Wilbur, D. K. Hamlin, M.-K. Chyan, B. B. Kegley and
P. M. Pathare, Bioconjugate Chem., 2001, 12, 616–623.
16 D. S. Wilbur, D. K. Hamlin, P. M. Pathare and S. A. Weerawarna,
Bioconjugate Chem., 1997, 8, 572–584.
17 E. Haak, H. Siebeneicher and S. Doye, Org. Lett., 2000, 2,
1935–1937; G. A. Artamkina, A. G. Sergeev, M. M. Stern and
I. P. Beletskaya, Synlett, 2006, 235–238; P. Anjanappa, D. Mullick,
K. Selvakumar and M. Sivakumar, TetrahedronLett., 2008, 49,
4585–4587; S. Jaime-Figueroa, Y. Liu, J. M. Muchowski and
D. G. Putman, Tetrahedron Lett., 1998, 39, 1313–1316.
18 X. Huang and S. L. Buchwald, Org. Lett., 2001, 3, 3417–3419;
S. Lee, M. Jorgensen and J. F. Hartwig, Org. Lett., 2001, 3,
2729–2732; R. B. Bedford and M. Betham, Tetrahedron Lett.,
2007, 48, 8947–8950.
19 Y. Shen and G. K. Friestad, J. Org. Chem., 2002, 67, 6236–6239.
20 C. Defieber, M. A. Ariger, P. Moriel and E. M. Carreira, Angew.
Chem., Int. Ed., 2007, 46, 3139–3143; R. Weihofen, O. Tverskoy
and G. Helmchen, Angew. Chem., Int. Ed., 2006, 45, 5546–5549.
21 F. Garro-Helion, A. Merzouk and F. Guibe, J. Org. Chem., 1993,
58, 6109–6113.
22 C. C. Tzschucke, C. Markert, W. Bannwarth, S. Roller, A. Hebel
and R. Haag, Angew. Chem., Int. Ed., 2002, 41, 3964–4000.
23 S. Aimoto, Curr. Org. Chem., 2001, 5, 45–87; Y. Okada, Curr. Org.
Chem., 2001, 5, 1–43.
24 K. M. Galie, A. Mollard and I. Zharov, Inorg. Chem., 2006, 45,
7815–7820; N. Ouali, S. Mery, A. Skoulios and L. Noirez,
Macromolecules, 2000, 33, 6185–6193.
In conclusion, for the first time successful application of
double exponential growth for the synthesis of aliphatic poly-
amide dendrimers has been achieved. Judicious choices of
purification methods not only afforded pure products but also
reduced purification times, thereby enabling faster assembly of
these dendrimer molecules. It was also shown that unactivated
biotin can be efficiently coupled to amino acids in high yields,
using solid-phase techniques. Further work is under way to
couple various markers and labels to the biotin–dendrimer
units, so that their use in various biotechnological applications
can be studied.
Notes and references
z Owing to trace amount of solubility of NaCl in methanol26 an
analytically pure sample could not be obtained. However, H, C and
mass analysis confirmed the product identity.
1 G. R. Newkome and C. D. Shreiner, Polymer, 2008, 49, 1–173.
2 D. K. Smith, Tetrahedron, 2003, 59, 3797–3798.
3 S. Svenson and A. Tomalia Donald, Adv. Drug Delivery Rev., 2005,
57, 2106–2129; C. Dufes, I. F. Uchegbu and A. G. Schaetzlein,
Adv. Drug Delivery Rev., 2005, 57, 2177–2202; H. Yang and
W. J. Kao, J. Biomater. Sci., Polym. Ed., 2006, 17, 3–19;
K. Sadler and J. P. Tam, Rev. Mol. Biotechnol., 2002, 90, 195–229.
4 S.-H. Hwang, C. N. Moorefield and G. R. Newkome, Chem. Soc.
Rev., 2008, 37, 2543–2557; M. Liu, G. Shen, W. Xu and G. Shen,
Polym. Int., 2007, 56, 1432–1439.
5 E. de Jesus and J. C. Flores, Ind. Eng. Chem. Res., 2008, 47,
7968–7981.
6 L. C. Palmer and S. I. Stupp, Acc. Chem. Res., 2008, 41,
1674–1684.
7 K. Yamamoto and K. Takanashi, Polymer, 2008, 49, 4033–4041.
8 C. Hawker and M. J. Frechet, J. Chem. Soc., Chem. Commun.,
1990, 1010–1013; S. M. Grayson and J. M. J. Frechet, Chem. Rev.,
2001, 101, 3819–3867.
25 D. S. Wilbur, P. M. Pathare, D. K. Hamlin, K. R. Buhler and
R. L. Vessella, Bioconjugate Chem., 1998, 9, 813–825.
26 S. P. Pinho and E. A. Macedo, J. Chem. Eng. Data, 2005, 50,
29–32.
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