Fmoc solid phase synthesis of polyamides containing pyrrole and imidazole amino acids

Article abstract of DOI:10.1021/ol0156796

Matrix presented Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available peptide synthesizers.

Full text of DOI:10.1021/ol0156796

ORGANIC
LETTERS
2001
Vol. 3, No. 8
1201-1203
Fmoc Solid Phase Synthesis of
Polyamides Containing Pyrrole and
Imidazole Amino Acids
Nicholas R. Wurtz, James M. Turner, Eldon E. Baird, and Peter B. Dervan*
DiVision of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, California 91125
derVan@its.caltech.edu
Received February 7, 2001
ABSTRACT
Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity
for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides
has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were
prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities
and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and
complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available
peptide synthesizers.
Small molecules specifically targeted to any predetermined
DNA sequence would be useful tools in molecular biology
and potentially in human medicine. Polyamides containing
N-methylpyrrole (Py), 3-hydroxylpyrrole (Hp), and N-
methylimidazole (Im) are synthetic ligands that have an
affinity and specificity for DNA comparable to those of
naturally occurring DNA binding proteins. DNA recognition
depends on side-by-side aromatic amino acid pairings
oriented N f C with respect to the 5′ f 3′ direction of the
DNA helix in the minor groove. An antiparallel pairing of
Im opposite Py (Im/Py pair) recognizes a G‚C base pair,
while a Py/Im combination recognizes C‚G. A Hp/Py pair
prefers T‚A over A‚T. A Py/Py pair is degenerate and
recognizes either an A‚T or T‚A base pair.1
synthesizers, we set out to develop an alternative solid phase
synthesis incorporating a different protecting group that
would provide a route to polyamides with comparable purity
and recovery to Boc chemistry. The synthetic schemes made
available by this new chemistry could make available a
greater variety of bifunctional polyamide conjugates and
derivatives. As a minimum first step, we report here
monomer synthesis and general protocols for machine-
assisted Fmoc-chemistry solid phase synthesis of pyrrole-
imidazole polyamides, ImPyPyPy-γ-PyPyPyPy-â-Dp 1 and
ImPyPyPy-γ-ImPyPyPy-â-Dp 2 (Figure 1), which are identi-
cal or superior in recovery, purity, and coupling times to
Boc-chemistry protocols.
The synthesis of Fmoc-Py acid has previously been
reported.3 We report a convenient multigram scalable
synthesis of Fmoc-Py acid with a higher overall yield without
the use of column chromatography, as well as the first-
reported synthesis of Fmoc-Im acid.
The large repertoire of pyrrole-imidazole polyamides
synthesized hallmarks the development of Boc-chemistry
solid phase polyamide synthesis.2 However, to ensure
compatibility with most commercially available peptide
(1) Bu¨rli, R. W.; Dervan, P. B. Curr. Opin. Chem. Biol. 1999, and
references therein.
(2) Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141-
6146.
(3) (a) Va´zquez, E.; Caaman˜o, A. M.; Castedo, L.; Mascaren˜as, J. L.
Tetrahedron Lett. 1999, 40, 3621. (b) Ko¨nig, B.; Ro¨del, M. Synth. Commun.
1999, 29, 943.
10.1021/ol0156796 CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/28/2001

butoxide in refluxing tert-butyl alcohol yielding 5a and 5b
in 95% and 84% yields, respectively. Reduction of 5a and
5b in DMF in the presence of 10% Pd/C under 500 psi of
hydrogen proceeds to completion in 3 h. After filtering the
reaction mixture through a pad of Celite, Fmoc-Cl and
diisopropylethylamine were added to provide 6a and 6b in
66% and 35% yields, respectively. The tert-butyl esters were
deprotected with TiCl4 in dichloromethane at 0 °C to prevent
the decarboxylation, which was observed with standard
trifluoroacetic acid deprotection. Precipitation with 1.0 M
HCl afforded the final products 7a and 7b in 88% and 86%
yields, respectively. The overall yields from commercially
available starting materials for the Fmoc-Py acid and Fmoc-
Im acid are 33% and 11%, respectively.
Additionally, the first two steps of the Fmoc-Im acid
synthesis provide a safer, higher yielding route to the Boc-
Im acid monomer. The original published synthesis2 of the
Boc-Im acid required acylation of N-methyl imidazole with
ethyl chloroformate, followed by a highly exothermic nitra-
tion at the 4- and 5- positions in sulfuric acid and fuming
nitric acid. The isomers were separated by a difficult
recrystallizaton to provide the desired ethyl 1-methyl-4-
nitroimidazole-2-carboxylate 8. In the new synthetic route,
nitration of trichloroketone 3b in acetic anhydride provides
the desired isomer, 4-nitro-2-(trichloroacetyl)-1-methylimi-
dazole 4b, by milder nitration conditions. The product was
esterified with sodium ethoxide in ethanol to yield ethyl
1-methyl-4-nitroimidazole-2-carboxylate 8 in 95% yield
(Figure 3).
Figure 1. ImPyPyPy-γ-PyPyPyPy-â-Dp (top) and ImPyPyPy-γ-
PyPyPyPy-â-Dp (bottom) prepared by multistep solid phase
synthesis.
Pyrrole monomer precursors 3a and 4a were synthesized
using literature protocols.2 Previously reported 2-(trichloro-
acetyl)-1-methylimidazole 3b was converted to tert-butyl
1-methylimidazole-2-carboxylate 4b using modifications of
previously published procedures.4 Compound 3b in acetic
anhydride was cooled to 0 °C followed by the addition of
fuming nitric acid and sulfuric acid over 2 h (Figure 2).
Figure 3. (i) NaOEt/EtOH.
This intermediate was converted in three steps into the
desired Boc-Im acid 9 using previously reported reactions2
in an improved overall yield of 18%, compared to the 8%
overall yield in the original synthesis.
For Fmoc solid phase synthesis, the polyamide was
attached to an insoluble matrix by a linkage, which is cleaved
in a single step, introducing a positive charge into the
polyamide. Fmoc-â-alanine-Wang resin is commercially
available in appropriate substitution levels (0.86 mmol/g).
Manual solid phase synthesis uses HBTU/DIEA activation
protocols as previously reported.2
Figure 2. (i) Fuming nitric acid, sulfuric acid, Ac2O; (ii) NaOt-
Bu/t-BuOH, (iii) 500 psi of H2, 10% Pd/C, DMF; (iv) Fmoc-Cl,
DIEA; (v) TiCl4, CH2Cl2.
Fmoc-chemistry machine-assisted solid phase polyamide
synthesis protocols were modified from the Boc-chemistry
protocols reported for use on an ABI 430A peptide synthe-
sizer. Coupling cycles for machine-assisted synthesis are
rapid, 180 min per residue, and require no special precautions
beyond those used for ordinary solid phase peptide synthesis.
Machine-assisted solid phase synthesis of a pyrrole-imida-
Crystallization provided 528 g of 4b in 56% yield. tert-Butyl
esters 5a and 5b were synthesized from 4a and 4b with tert-
(4) Nishiwaki, E.; Tanaka, S.; Lee, H.; Shibuya, M. Heterocycles 1988,
27, 1945.
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Org. Lett., Vol. 3, No. 8, 2001

Figure 4. Fmoc solid phase synthetic scheme for polyamides 1 and 2 starting from commercially available Fmoc-â-alanine-Wang resin:
(i) 20% piperidine/NMP; (ii) Fmoc-Py acid, HBTU, DIEA; (iii) 20% piperidine/NMP; (iv) Fmoc-Py acid, HBTU, DIEA; (v) 20% piperidine/
NMP; (vi) Fmoc-Py acid, HBTU, DIEA; (vii) 20% piperidine/NMP; (viii) Fmoc-Py acid (for 1) or Fmoc-Im acid (for 2), HBTU, DIEA;
(ix) 20% piperidine/NMP; (x) Fmoc-γ-aminobutyric acid, HBTU, DIEA; (xi) 20% piperidine/NMP; (xii) Fmoc-Py acid, HBTU, DIEA;
(xiii) 20% piperidine/NMP; (xiv) Fmoc-Py acid, HBTU, DIEA; (xv) 20% piperidine/NMP; (xvi) Fmoc-Py acid, HBTU, DIEA; (xvii) 20%
piperidine/NMP; (xviii) Im-acid, HBTU, DIEA; (xix) N,N-dimethylaminopropylamine, 55 °C.
zole polyamide consists of a DCM wash, an NMP wash,
removal of the Fmoc group with piperidine/NMP, an NMP
wash, a DCM wash, an NMP wash, addition of activated
monomer, coupling for 180 min, capping with acetic
anhydride, taking a resin sample for analysis, and a final
NMP wash.
amide was sufficient to obtain a recovery of 38% and 9%
for 1 and 2, respectively, and a final purity for both
compounds greater than 98% as determined by a combination
1
of analytical HPLC, H NMR, and mass spectroscopy.
Pyrrole-imidazole polyamides are a promising class of
compounds used to target a predetermined DNA sequences.
The synthetic schemes made available by Fmoc-chemistry
solid phase synthesis will allow a greater variety of bifunc-
tional conjugates and derivatives to be synthesized.
Because the aromatic amine of the pyrrole and imidazole
do not react in the quantitative ninhydrin test, stepwise
cleavage of a sample of resin and analysis by HPLC are used
to indicate that high stepwise yields (>99%) are routinely
achieved. We note that coupling of the imidazole amine with
Fmoc-Py-OBt ester was not satisfactory. However, acylation
with Boc-Py anhydride ester (synthesized in situ using DCC,
DMAP in DCM) proceeds to completion within 3 h.2
ImPyPyPy-γ-PyPyPyPy-â-Dp 1 and ImPyPyPy-γ-ImPy-
PyPy-â-Dp 2 were prepared in 19 steps using the protocols
described in the Experimental Section (Figure 4). The yield
of each individual coupling step was established as >99%
by HPLC analysis. The resin was cleaved in high yield
(>90%) by aminolysis with (N,N-dimethylamino)propyl-
amine. A single HPLC purification of the eight-ring poly-
Acknowledgment. We are grateful to the National
Institute of Health for research support, the Ralph M. Parsons
Foundation for a predoctoral fellowship for N.R.W., J.
Edward Richter for an undergraduate fellowship to J.M.T.,
and the Howard Hughes Medical Institute for a predoctoral
fellowship to E.E.B.
Supporting Information Available: Complete experi-
mental details and product characterization. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0156796
Org. Lett., Vol. 3, No. 8, 2001
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