A selectively deprotectable triazacyclophane scaffold for the construction of artificial receptors.

Article abstract of DOI:10.1021/ol0101741

[reaction: see text]. The synthesis of a triazacylclophane scaffold bearing a set of selectively removable protecting groups is described. This versatile scaffold, which can be linked to a solid support, allows the attachment of three different side chain

Full text of DOI:10.1021/ol0101741

ORGANIC
LETTERS
2001
Vol. 3, No. 22
3499-3502
A Selectively Deprotectable
Triazacyclophane Scaffold for the
Construction of Artificial Receptors
Till Opatz and Rob M. J. Liskamp*
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences,
Utrecht UniVersity, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
r.m.j.liskamp@pharm.uu.nl
Received August 10, 2001
ABSTRACT
The synthesis of a triazacylclophane scaffold bearing a set of selectively removable protecting groups is described. This versatile scaffold,
which can be linked to a solid support, allows the attachment of three different side chains and can therefore be used for the combinatorial
synthesis of libraries of artificial receptor molecules of high structural diversity.
The construction of artificial receptors capable of selectively
binding small peptides or other biologically relevant mol-
ecules is an interesting approach for uncovering new
molecules with antibacterial or antifungal properties. Espe-
cially the use of combinatorial methods for the generation
of large libraries of synthetic receptor molecules and
subsequent screening of the binding affinity to a particular
ligand may be a powerful strategy. This approach somewhat
resembles the unsurpassed way the immune system fights
infections by screening antigenic structures with the antibod-
ies present on lymphocytes which constantly roam the body.
In a recent publication we demonstrated the use of a
triazacyclophane scaffold for the combinatorial construction
of vancomycin mimetics.1 These artificial receptors, like the
glycopeptide antibiotics vancomycin and teicoplanin, are able
to bind the C-terminal D-Ala-D-Ala sequence of bacterial
peptidoglycan.2 In addition to mimicking the binding proper-
ties of the naturally occurring glycopeptides, their artificial
counterparts can be designed to bind the mutant D-Ala-D-
Lac sequence found in bacteria that have become resistant
to these valuable drugs which are often referred to as the
last resort of current antibacterial chemotherapy.3
In a library of our recently described tripodal receptor
molecules, compound 1 (Figure 1) was able to bind both
the D-Ala-D-Ala and the D-Ala-D-Lac sequences. In this
(1) Monnee, M. C. F.; Brouwer, A. J.; Verbeek, L. M.; van Wageningen,
A. M. A.; Liskamp, R. M. J. Bioorg. Med. Chem. Lett. 2001, 11, 1521.
(2) (a) Perkins, H. R. Biochem. J. 1968, 106, 35P. (b) Williams, D. H.;
Williamson, M. P.; Butcher, D. W.; Hammond, S. J. J. Am. Chem. Soc.
1983, 105, 1332.
Figure 1. Triazacyclophanes.
10.1021/ol0101741 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/04/2001

synthetic receptor, the central triazacyclophane ring carries
three peptide chains that may form a cavity into which the
cognate ligand fits.
Scheme 1. Synthesis of 2a
Previous investigations have shown the amino acid
sequence of the receptor arms to be crucial for the selective
recognition of the ligand molecules. Consequently, combi-
natorial solid phase synthesis and on-bead screening tech-
niques are the methods of choice in the search for receptors
with superior affinity and/or selectivity.1,4
Although having a large number of potential applications,
only a limited number of selectively deprotectable scaffolds
have been described so far. In particular, templates that in
addition allow a parallel alignment of attached side chains
are scarce.5,6 Herein, we report the synthesis of 2, a protected
triazacyclophane scaffold whichsupon attachment to a solid
supportsallows for selective introduction of a wide variety
of substituents and elongation of three different peptidic or
peptidomimetic chains, thus giving access to libraries of
artificial receptors with a very high structural diversity. For
the protection of the three secondary amines in the triaza-
cyclophane, a combination of the Fmoc, the Aloc, and the
o-nitrobenzenesulfonyl (oNBS) group was chosen. In contrast
to the Boc-/Fmoc-/Aloc triad employed by Savage et al. for
the protection of a steroid-derived triamine scaffold,5 our
protective group pattern allows the introduction of amino
acids with acid labile side chain protection at any position
as well as their elongation to form oligopeptide side chains
following the protocols of standard Fmoc peptide chemistry.
The synthesis of 2 started with the reaction of bis(3-
aminopropyl)amine (10 equiv) with o-nitrobenzenesulfonyl
chloride. The crude product was then trifluoroacetylated
using ethyl trifluoroacetate (3 equiv) in the presence of water
(1 equiv) leading to trifluoroacetate salt 3.7,8
This salt can be crystallized from the reaction mixture
(yield: 52% over two steps), but analysis by mass spec-
trometry still revealed the presence of small amounts of the
bis(o-nitrobenzenesulfonyl)amine as an impurity which was
preferrably removed after introduction of the Aloc-group in
the next step. This furnished the triply protected triamine 4
which was easily purified by column chromatography and
obtained in 85% yield.
a Reagents and conditions: (i) oNBS-Cl, CH2Cl2, rt; then (ii)
CF3CO2Et, H2O, MeCN, reflux; 52% over two steps; (iii) Aloc-
Cl, NaHCO3, H2O/dioxane, rt; 85%; (iv) Cs2CO3, Bu4NBr, MeCN,
reflux; (v) Tesser’s base (neat), then (vi) Fmoc-OSu, DIPEA, H2O/
MeCN; 95% over two steps.
Subsequently, compound 4 was reacted with 3,5-bis-
(bromomethyl)benzoic acid methyl ester9 under basic condi-
tions to give the crystalline triazacyclophane 5 in an
acceptable yield of 47%.8,10 The yield of this macrocycliza-
tion reaction was significantly higher when both termini of
the linear triamine precursor carried the same protecting
group. Thus, the symmetrical bis(o-nitrobenzenesulfonyl)s
as well as bis(trifluoroacetyl)sderivatives cyclized smoothly
under similar conditions. The lower yield of 5 was caused
by formation of considerable amounts of the dimeric byprod-
uct 6. Dilution of the heterogeneous reaction mixture or
changing solvents or base did not prevent the formation of
this remarkable 28-membered hexaazacycle. Unfortunately,
desymmetrization of the above-mentioned more readily
accessible symmetrical triazacyclophanes by means of double
deprotection followed by mono(re)protection was unsatisfac-
tory so that desymmetrization in the first synthetic step was
preferable.
(3) (a) Arthur, M.; Molinas, C.; Bugg, T. D. H.; Wright, G. D.; Walsh,
C. T.; Courvalin, P. Antimicrob. Agents Chemother. 1992, 36, 867. (b)
Wright, G. D.; Walsh, C. T. Acc. Chem. Res. 1992, 25, 468.
(4) For example, see: Lo¨wik, D. W. P. M.; Weingarten, M. D.;
Broekema, M.; Brouwer, A. J.; Still, W. C.; Liskamp, R. M. J. Angew.
Chem., Int. Ed. Engl. 1998, 37, 1846.
(5) Zhu, X.-T.; Rehman, A.; Li, C.; Savage, P. B. Org. Lett. 2000, 2,
3015.
(6) (a) Barry, J. F.; Davis, A. P.; Pe´rez-Paya´n, M. N.; Elsegood, M. R.
J.; Jackson, R. F. W.; Gennari, C.; Piarulli, U.; Gude, M. Tetrahedron Lett.
1999, 40, 2849. (b) Davis, A. P.; Lawless, L. J. J. Chem. Soc., Chem.
Commun. 1999, 9. (c) Wess, G.; Bock, K.; Kleine, H.; Kurz, M.; Guba,
W.; Hemmerle, H.; Lopez-Calle, E.; Baringhaus, K.-H.; Glombik, H.;
Ehnsen, A.; Kramer, W. Angew. Chem., Int. Ed. Engl. 1996, 35, 2222. (d)
Hioki, H.; Yamada, T.; Fujioka, C.; Kodama, M. Tetrahedron Lett. 1999,
40, 6821.
(7) Floch, V.; Le Bolch, G.; Gable-Guillaume, C.; Le Bris, N.; Yaouanc,
J.-J.; et al. Eur. J. Med. Chem. Chim. Ther. 1998, 33, 923.
(8) (a) An, H.; Cummins, L. C.; Giffrey, R. H.; Bharadwaj, R.; Haly, B.
D.; Fraser, A. S.; Wilson-Lingardo, L.; Rinsen, L. M.; Wyatt, J. R.; Cook,
P. D. J. Am. Chem. Soc. 1997, 119, 3696. (b) For an orthogonally protected
piperazinyl polyazaphane, see: Sprankle, K.; Swayze, E. Book of abstracts,
218th National Meeting of the American Chemical Society, New Orleans;
American Chemical Society: Washington, DC, 1999; ORGN-334.
3500
Org. Lett., Vol. 3, No. 22, 2001

Compond 5 was crystalline, only poorly soluble in
nonhalogenated solvents, and readily purified. Simultaneous
cleavage of the trifluoroacetyl group and the methyl ester
with Tesser’s base (4 N NaOH/methanol/dioxane 1:5:14 v/v)
yielded an amino carboxylic acid which without isolation
was reacted with Fmoc-O-hydroxysuccinimide. After chro-
matography, triprotected carboxylic acid 2 was obtained in
a yield of 95%.
As a test of its suitability for the eventual construction of
artificial receptors with three different binding arms, 2 was
attached via a Rink linker11 to Argogel resin (PEG-grafted
polystyrene devoid of PEG benzyl ether moieties for
improved chemical stability) and the remaining amino groups
on the resin were acetylated (Scheme 2). After cleavage of
Scheme 2. Solid Phase Synthesis of 8 as a Model for
Complete Functionalizationa
Figure 2. RP-HPLC of 8 (UV detection at 220 nm).
The second amino acid (Boc-L-valine) was then intro-
duced. Removal of the Aloc group was carried out by Pd0-
catalyzed allyl transfer to p-toluenesulfinate as the trapping
nucleophile.14 Provided that a sufficiently high concentration
and amount of this nucleophile (20 equiv) was used, the
undesired reallylation of the liberated amine by the cationic
allylpalladium intermediate could be almost completely
suppressed. In addition, N-formylation during Aloc cleavage
in DMF, which has recently been described by Ferna´ndez-
Forner and co-workers,15 could be prevented by using NMP
as the solvent. A mixture of p-toluenesulfinic acid and
morpholine and a solution of the stable and crystalline
anilinium p-toluenesulfinate performed equally well in this
step. Introduction of Fmoc-glycine as the third amino acid
was then accomplished followed by cleavage of the Rink
linker to furnish the product 8 in a yield of 98% (based on
determination of the resin loading by Fmoc quantification).
The RP-HPLC and ESI-MS analyses of the crude product
are shown in Figures 2 and 3.
a Reagents and conditions: (i) Argogel-Rink-NH2, HBTU, HOBt,
DIPEA, NMP; (ii) Ac2O, pyridine, dioxane; (iii) 20% piperidine,
NMP; (iv) Boc-L-Ala, HBTU, HOBt, DIPEA, DMF; (v) HS-
(CH2)2OH, DBU, NMP; (vi) Boc-L-Val, HBTU, HOBt, DIPEA,
DMF; (vii) p-TolSO2H, morpholine, cat. Pd(PPh3)4, NMP; (viii)
Fmoc-Gly, HBTU, HOBt, DIPEA, DMF; (ix) TFA, iPr3SiH, H2O.
the Fmoc-group, Boc-L-alanine was introduced using 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate12 (HBTU) as the coupling reagent in the presence
of 1-hydroxybenzotriazole (HOBt) as an additive. Despite
possible steric hindrance of the ring secondary amines, fast
and clean reactions were observed when a 5-fold excess of
amino acid, coupling reagent, and coupling additive was
used.
Cleavage of the oNBS group was achieved by treatment
with 2-mercaptoethanol and DBU in dry DMF.13 It proved
to be important that no dichloromethane was present during
this deprotection reaction which involves highly nucleophilic
thiolate anions.13c
Figure 3. ESI-MS of 8 (crude product).
Org. Lett., Vol. 3, No. 22, 2001
3501

In conclusion, the obtained results clearly demonstrate that
2 is a convenient, selectively deprotectable scaffold which
is well suited for the combinatorial synthesis of tripodal
receptor molecules on the solid phase. In addition, the use
of a sufficiently acid stable linker should allow the prepara-
tion of resin-bound tripods in their protected as well as in
their fully deprotected form. Besides application as a scaffold
for artificial receptors, under present investigation is the use
of 2 as a template for construction of enzyme mimics or
discontinuos epitopes.
Acknowledgment. This work was supported by the
European Commission (Marie Curie Individual Fellowship,
Contract No. HPMFCT-2000-00609).
Supporting Information Available: Experimental pro-
cedures, spectral data of compounds 2 to 5, spectra of
compound 8 (crude product and a purified sample) as well
as combustion analysis of compound 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
(9) Kurz, K.; Go¨bel, M. W. HelV. Chim. Acta 1996, 79, 1967.
(10) For a similar alkylation, see: Landini, D.; Penso, M. Synth. Commun.
1988, 18, 791.
(11) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
(12) Dourtoglu, V.; Ziegler, J. C.; Gross, B. Tetrahedron Lett. 1978, 15,
1269.
(13) (a) Miller, J. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301.
(b) Reichwein, J. F.; Liskamp, R. M. J. Tetrahedron Lett. 1998, 39, 1243.
(c) Reichwein, J. F. Ph.D. Dissertation, Utrecht University, Utrecht, The
Netherlands, 2000.
OL0101741
(14) (a) Honda, M.; Morita, H.; Nagakura, I. J. Org. Chem. 1997, 62,
8932. (b) Opatz, T.; Kunz, H. Tetrahedron Lett. 2000, 41, 10185.
(15) Ferna´ndez-Forner, D.; Casals, G.; Navarro, E.; Ryder, H.; Albericio,
F. Tetrahedron Lett. 2001, 42, 4471.
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Org. Lett., Vol. 3, No. 22, 2001