High-loading resin beads for solid phase synthesis using triple branching symmetrical dendrimers

Article abstract of DOI:10.1039/a906515f

Resin beads with a loading of 200 nmol per bead have been prepared using a Generation 2.0 tris-based dendrimer to amplify resin loading sites.

Full text of DOI:10.1039/a906515f

High-loading resin beads for solid phase synthesis using triple branching
symmetrical dendrimers
Christophe Fromont and Mark Bradley*
Department of Chemistry, University of Southampton, Highfield, Southampton, UK SO17 1BJ
Received (in Liverpool, UK) 9th August 1999, Accepted 11th January 2000
Resin beads with a loading of 200 nmol per bead have been
prepared using a Generation 2.0 tris-based dendrimer to
amplify resin loading sites.
observed. The average diameters of the resin beads in DMF
were: aminomethyl resin, 310 mm, PS-Gen 1.0, 400 mm and PS
Gen. 2.0, 500 mm. These beads were extremely stable to
solvation changes and extended manipulations and behaved in
all respects as normal resin beads. They proved to be ideal for
a broad range of chemistries. In addition, with ca. 27 000
The process of split and mix by its very nature rapidly generates
large libraries of compounds in such a manner that a single resin
bead carries a single compound. It is one of the most efficient
and cost effective chemical methods for the generation of large
compound numbers,1 with compound numbers increasing
exponentially with only a linear increase in the number of
synthetic steps. The single bead/single compound concept
opens up the option of screening compounds released from
single beads as a powerful tool for high throughput screening
and thus several major pharmaceutical companies now use
single bead screening in anger in the area of lead generation.
The drawbacks to this process are the tiny amounts of
compound found on single beads, usually in the order of a few
hundred picomoles, and the identification of the compound on
the bead. Compound quantity maybe sufficient for one or two
biological assays but it is usually insufficient for IC50
determinations and chemical analysis/determination of the
structure of the compound. These analysis issues have led to a
number of elegant tagging solutions that encode the chemical
sequence to which a bead has been exposed, although
complicating the synthetic process.2 However the need for
encoding and resolution of the screening problems could be
overcome if the loading of the solid support could be increased
to a more practical level. We previously reported the synthesis
of polyamine based Generation 3.0 PAMAM dendrimers on
resins beads as a means of enhancing loading.3
Here we describe a practical approach to the synthesis of a tri-
branching symmetrical dendrimer on the solid phase with an
1
8-fold amplification of resin loading per bead. Beads with a
loading of 200 nmol per bead were prepared and used in a
number of synthetic applications, with single beads containing
more than sufficient compound for conventional analysis,
including NMR!
Scheme 1 Reagents and conditions: i, acrylonitrile, 1,4-dioxane, 40% KOH,
4
The trifunctional dendrimer monomers were prepared in
room temperature, 3 days; ii, HCl, MeOH, 4–6 h, reflux; iii, Boc O, DMAP
2
, room temp., 30 min; iv, 4 or 5a,b, DMAP (cat), Pri
2
NEt
2
Cl or DMF, 12 h; v, propane-1,3-diamine, MeOH, 3 days or
bulk ( > 50 g) by alkylation of tris 1 with acrylonitrile followed
by nitrile hydrolysis in a saturated solution of HCl in dry MeOH
to give the methyl ester 2. Alternatively, the triacid could be
obtained by direct hydrolysis. The hindered amine 2 was
(cat), CH
(2 equiv.), CH
2
Cl
2
2
i
2
propane-1,3-diamine, DMSO, 3 days; vi, 3, DMAP (cat), Pr NEt (2 equiv.),
DMF, 12 h.
converted to the isocyanate 3 by treatment with Boc
DMAP as described by Knölker to give the stable symmetrical
monomer 3. Aminomethylpolystyrene resin (0.7 mmols g
2
O and
5
2
1
,
2
50–300 mm, Polymer Laboratories) was directly derivatised
with 3 as shown in Scheme 1. Displacement of the methyl ester
by propane-1,3-diamine was followed by on-bead IR (ATR)
and NMR analysis of cleaved material.† Repeating the process
gave Generation 2.0 dendrimer beads with a loading of 85% of
the theoretical maximum (36 nmol per bead). Swelling studies
(
Fig. 1) showed that the dendrimer beads swelled to a much
greater degree in more polar solvents than the starting
aminomethyl resin, as expected due to the nature of the
dendrimer itself. It should however also be noted that swelling
was measured in units of ml g . Since the dendrimer beads
have a density that is different to those of polystyrene (the mass
of the generation 2.0 dendrimer is 2111 Da), trends rather than
absolute swelling ratios in terms of bead sizes are being
2
1
Fig. 1 Swelling studies: (5) aminomethyl resin; (:) PS Gen 1.0; (-) PS
Gen 2.0.
Chem. Commun., 2000, 283–284
This journal is © The Royal Society of Chemistry 2000
283

NMR data shown in Fig. 2. It should be noted that these very
large beads were not as robust as the smaller beads used earlier,
with solvent shock often causing the beads to shatter, necessitat-
ing a gradual change from one solvent to another and gentle
handling, thereby limiting their utility.
We have thus devised a fast and efficient method of
generating symmetrical tri-branched resin linked dendrimers in
a very efficient manner. The smaller, robust Gen 2.0 beads with
2
1
a loading of 36 nmol bead (which has been extended to Gen
2
1
3
.0, beads with a loading of 100 nmol bead ) are we believe
ideally practical for single bead screening applications.
We acknowledge the Royal Society for a University Research
Fellowship to M. B. and the BBSRC for generous support of this
research (ROPA grant).
Notes and references
†
Using cleavable linkers dendrimers were cleaved at the 0.5n and n
generations and fully analysed, and clearly showed that dendrimer synthesis
had proceeded cleanly and as anticipated.
‡
By Fmoc determination.
1
A. Furka, F. Sebestyen, M. Asgedom and G. Dibo, Int. J. Pept. Protein
Res., 1991, 37, 487; R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R.
Appel, C. T. Dooley and J. H. Cuervo, Nature, 1991, 354, 84; K. S. Lam,
S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski and R. J.
Knapp, Nature, 1991, 354, 82.
1
Fig. 2 (a) Crude H NMR spectrum (5 mm NMR tube, 256 scans, 400 MHz)
and (b) HPLC trace (10% of sample injected, 3.6 mm 3 18 cm, C-18
column) for the Suzuki biaryl product cleaved from a single resin bead.
2 H. P. Nestler, P. A. Bartlett and W. C. Still, J. Org. Chem., 1994, 59,
723; J. J. Baldwin, J. J. Burbaum, I. Henderson and M. H. J. Ohlmeyer,
4
beads g21 a split and mix synthesis on a gram of resin beads
J. Am. Chem. Soc., 1995, 117, 5588; J. J. Burbaum, M. H. J. Ohlmeyer,
J. C. Reader, I. Henderson, L. W. Dillard, G. Li, T. L. Randle, N. H. Sigal,
D. Chelsky and J. J. Baldwin, Proc. Natl. Acad. Sci. U.S.A., 1995, 92,
clearly provides the numbers of compounds and the amounts
necessary for a serious single bead screening approach.
Dendrimer synthesis was also carried out on 400–500 mm
aminomethyl polystyrene resin beads, loaded with the poly-
6
027; M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, J. C. Reader, G.
Asouline, R. Kobayashi, M. Wigler and W. C. Still, Proc. Natl. Acad. Sci.
U.S.A., 1993, 90, 10922.
3
amine scaffolds 5a,b, as shown in Scheme 1. Generation 1.0
3
4
N. J. Wells, M. Davies and M. Bradley, J. Org. Chem., 1998, 63, 6430;
N. J. Wells, A. Basso and M. Bradley, Biopolymers, 1998, 47, 381; V.
Swali, N. J. Wells, G. J. Langley and M. Bradley, J. Org. Chem., 1997,
62, 4902; V. Swali and M. Bradley, Anal. Commun., 1997, 34, 15.
G. R. Newkome, C. N. Moorefield and G. R. Baker, Aldrichim. Acta,
2
1
21
(
8000 beads g ) had a loading of 116 nmol bead , while
2
1
Generation 2.0 (4300 beads g ) had a loading of 230
2
1
nmol bead .‡ The dendrimer beads were used in a number of
synthetic applications attaching a linker onto the free amino
groups of the dendrimer to allow cleavage of the desired
compound. This included peptide chemistry (synthesis of
1
992, 25, 31; G. R. Newkome, C. D. Weis and B. J. Childs, Designed
Monomers and Polymers, 1998, 1, 3.
H.-J. Knölker, T. Braxmeier and G. Schlechtingen, Angew. Chem., Int.
Ed. Engl., 1995, 34, 2497.
5
6
6
Fmoc-Val-Phe-Ala-OH) and a Suzuki coupling. Thus 4-iodo-
benzoic acid was esterified onto the HMPB linker and
B. Ruhland, A. Bombrun and M. A. Gallop, J. Org. Chem., 1997, 62,
4
-methylbenzene boronic acid coupled under standard condi-
tions [K CO , Pd(PPh , DMF, 110 °C, 16 h]. Cleavage of the
biaryl compound from a single resin bead gave the HPLC and
7
820.
2
3
3 4
)
Communication a906515f
284
Chem. Commun., 2000, 283–284