Highly efficient synthesis of DNA-binding hairpin polyamides via the use of a new triphosgene coupling strategy

Article abstract of DOI:10.1021/ol9015139

Figure Presented A facile and highly efficient solid phase synthesis method is reported for the preparation of hairpin DNA-binding polyamides using the costeffective triphosgene (BTC) activating agent. Difficult polyamide sequences were prepared from N-me

Full text of DOI:10.1021/ol9015139

ORGANIC
LETTERS
2009
Vol. 11, No. 17
3910-3913
Highly Efficient Synthesis of
DNA-Binding Hairpin Polyamides via the
Use of a New Triphosgene Coupling
Strategy
Wu Su, Stephen J. Gray, Ruggero Dondi, and Glenn A. Burley*
Department of Chemistry, UniVersity of Leicester, UniVersity Road,
Leicester, U.K., LE1 7RH
gab13@le.ac.uk
Received July 2, 2009
ABSTRACT
A facile and highly efficient solid phase synthesis method is reported for the preparation of hairpin DNA-binding polyamides using the cost-
effective triphosgene (BTC) activating agent. Difficult polyamide sequences were prepared from N-methylimidazole (Im) and N-methylpyrrole
(Py) building blocks with high stepwise yields (>98%) using Boc chemistry. The versatility of the triphosgene coupling approach was also
demonstrated for the first time on aryl hydrazine resins to afford biomedically relevant tail-truncated polyamides in excellent isolated yields.
The ability to modulate the expression of any gene is one of
the key goals in molecular medicine. Genetic-based methods
such as RNA interference (RNAi) provide the means to silence
specific gene expression; however, RNAi still poses challenges
both as a research tool1 and as a therapeutic strategy.2,3 Pyrrole-
Imidazole (Py-Im) polyamides4-6 are cell-permeable7-12
synthetic ligands which can be programmed to bind to
predetermined sequences of DNA with nanomolar binding
affinity and with specificities that equal or exceed natural
eukaryotic transcriptional regulatory proteins.4,6,13-15
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10.1021/ol9015139 CCC: $40.75
Published on Web 08/11/2009
 2009 American Chemical Society

During the course of our research program into the
utilization of polyamide ligands, we were unable to obtain
suitable quantities of polyamides such as 1, using conven-
tional methods (Figure 1).18-20 These synthetic protocols
utilize activated benzotriazole esters as a means to couple
the hetereocyclic Boc/Fmoc-protected amino acids.18,20-23
These methods proceed well for resin-bound aliphatic and
Py amine couplings with 4-[(tert-butoxycarbonyl)amino]-1-
methylpyrrole-2-carboxylic acid [BocPyOH]; however, as a
consequence of its lower inherent nucleophilicity, the
coupling efficiencies of resin bound Im amines [4-[(tert-
butoxycarbonyl)amino]-1-methylimidazole-2-carboxylic acid
(BocImOH)] are rather poor.20 As a consequence, the
preparation of polyamides in high yield and purity, which
contain Im building blocks located within the core of the
polyamide such as in the case of polyamide 1, has proved
challenging.
The most common reagents used for the formation of
activated benzotriazole esters for polyamide synthesis have
been uronium salts (HBTU/HATU) or DCC/HOAt. Both
reagent sets form the activated benzotriazole ester which can
be either isolated or utilized directly in situ. Quantification
of these coupling efficiencies by HPLC analysis revealed
that the HATU coupling agent was only efficient in effecting
Py-Py couplings under the typical HATU activation condi-
tions, whereas the DCC/HOAt method is efficient in effecting
amide bond formation for both Py-Py and Im-Im couplings
(Table 1). When these reagent sets were utilized for the
Figure 1. Hairpin polyamides 1-4 prepared in this study. (A)
Polyamide 1 was prepared using the ꢀ-Ala-PAM resin. (B)
Polyamides 2-4 were prepared using the aryl hydrazine resin.
DNA-binding polyamides provide an alternative small
molecule strategy to the RNAi-based gene silencing
approach by binding in the minor groove of DNA and
blocking gene transcription.4,6 Specificity is achieved
according to a series of base pairing rules4 where an
antiparallel arrangement of Py-Py building blocks binds
preferentially to A•T or T•A base pairs, whereas an Im-Py
arrangement preferentially targets G•C over C•G, A•T, or
T•A base pairs. Over 250 analogues of polyamides16 have
been prepared by both solid and solution phase synthesis
methodologies over the past 20 years,17,17-21 which has
enabled their utilization in areas ranging from biology4,6
through to nanotechnology,22-24 yet despite their growing
utility there is still no generally applicable method for the
facile, efficient, and modular preparation of combinatorial
collections of polyamides in high yield and purity, an
essential requirement for their widespread utility. In this
communication, we report such a method for the preparation
of hairpin polyamides bearing Py, Im, and aliphatic amino
acid building blocks which overcomes the low yields of
existing methods.
Table 1. Comparative Coupling Yields of Polyamide Building
Blocksa
HATU
(%)c
DCC/HOAt
(%)d
BTC
(%)e
amide bondb
BocPyOH f H2NIm-Resin
BocPyOH f H2NPy-Resin
BocImOH f H2NIm-Resin
5
95
12
8
95
>98
>98
>98
>98
a Yields were based on HPLC peak integration. b Resin ) ꢀ-Ala PAM.
c Activation in 1:1 DMF/NMP, Boc-monomer/HATU/DIEA, 3-5 min;
coupling for 20 min. d Activation in 1:1 DMF/NMP, Boc-monomer/DCC/
HOAt, 2 h, DIEA; coupling for 20 min. e Activation in THF, Boc-monomer/
BTC/Collidine, 1 min, DIEA; coupling for 20 min.
(16) Edelson, B. S.; Best, T. P.; Olenyuk, B.; Nickols, N. G.; Doss,
R. M.; Foister, S.; Heckel, A.; Dervan, P. B. Nucleic Acids Res. 2004, 32,
2802.
(17) Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141
.
(18) Wurtz, N. R.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Org. Lett.
2001, 3, 1201
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formation of the difficult Resin-Im-NH2/BocPyOH coupling,
both HATU and DCC/HOAt methods afforded only 5-8%
of the coupled product after 20 min (Table 1).
Although the solution-based preparation of BocPy-ImOH
dimers provides a potential route to circumvent this prob-
lematic Im-Py coupling,18 we found their utility in polya-
mide synthesis compromised by their inherent insolubility
in typical coupling solvents [e.g., DMF, NMP, DMSO],
resulting in the formation of polyamide products in low yield
(19) Krutzik, P. O.; Chamberlin, A. R. Bioorg. Med. Chem. Lett. 2002,
12, 2129
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(20) Chenoweth, D. M.; Harki, D. A.; Dervan, P. B. J. Am. Chem. Soc.
2009, 131, 7175
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P. B. J. Am. Chem. Soc. 2009, 131, 7182
(22) Cohen, J. D.; Sadowski, J. P.; Dervan, P. B. Angew. Chem., Int.
Ed. 2007, 46, 8384
(23) Cohen, J. D.; Sadowski, J. P.; Dervan, P. B. J. Am. Chem. Soc.
2008, 130, 402
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Angew. Chem., Int. Ed. 2007, 46, 8384
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Org. Lett., Vol. 11, No. 17, 2009
3911

and purity. In light of these limitations, we surmised that if
one could increase the electrophilicity of the activated
carboxylic acid, one could subsequently increase the ef-
ficiency of these problematic Im-NH2/BocPyOH couplings.
In 2002, Jung et al. reported an Fmoc-based synthesis of
cyclic peptides containing sterically hindered secondary
amines on a solid support in which BTC [bis(trichlorom-
ethyl)carbonate or triphosgene] was used as the coupling
agent.25,26 The BTC reagent putatively forms acid chlorides
in situ in high yield and has shown application as a highly
efficient coupling agent for the Fmoc-mediated synthesis of
aromatic oligoamides using automated peptide synthesizers.27
It has also been shown that BTC performs better than TFFH28
and exclusively our BTC protocol, we were pleased to prepare
polyamide 1 in 33% yield after CBz deprotection of the γ-turn
motif; i.e. this is a 330-fold increase in isolated yield for 1 using
the BTC coupling protocol.
Encouraged by this result, we then investigated the
preparation of polyamides using solid supports that do not
install an A•T/T•A encoding ꢀ-Ala tail on the C-termini of
polyamides. This is an important requisite for biological
applications as the presence of ꢀ-Ala tails is known to
correlate with generally poor cellular uptake as well as
enforcing an A•T/T•A encoding end sequence.4,10,17 The
Dervan group recognized this limitation and developed a
polyamide synthetic protocol which utilized the Kaiser resin
to delete the ꢀ-Ala tail.32 As a consequence of their increased
stability in strongly acidic and basic conditions coupled with
a mild oxidative resin release protocol that enables the
installation of various tail functionalities, aryl hydrazide
resins offer potential advantages over Kaiser oxime resins
currently used for truncated polyamide synthesis.31 We
evaluated the compatibility of the BTC coupling strategy with
aryl hydrazide resins via the preparation of polyamide 2.
Polyamide 2 was chosen as it comprises a diverse range of
couplings not encountered in the synthesis of 1 such as an
Im-Im coupling, in addition to the known challenging Resin-
ImNH2-BocPyOH coupling late in the synthesis. The solid
phase synthesis of polyamide 2 proceeded smoothly using the
BTC protocol. Polyamide release from the hydrazine resin was
then achieved by oxidative activation of the resin in the presence
of 2.0 equiv of NBS in pyridine for 10 min at room temperature.
Cleavage of the polyamide from the activated diazene solid
support with dimethylamino propylamine (Dp) at 40 °C for 5 h
afforded the crude polyamide product 2 in 73% purity. After
semipreparative HPLC purification, the isolated yield of 2 was
29% yield and with a purity of 98% (Figure 2). The versatility
29
and POCl3 in the solid phase synthesis of difficult se-
quences.
The in situ formation of the putative acid chloride also enables
the utilization of acid-labile protecting groups such as tert-butyl
esters, which are reported to have a limited shelf life in the
presence of acid chlorides.30 Although there have been no
reports of its use in Boc-based solid phase synthesis, we
reasoned that the increased electrophilicity of the acid chloride
generated in situ by the reaction of BTC with the appropriate
aromatic carboxylic acid would considerably enhance coupling
efficiencies of polyamides and reduce reaction times. Indeed
the BTC method was found to be far superior to current
benzotriazole-based protocols in all couplings tested (Table 1).
Activation times of both the BocPyOH and BocImOH only
required 1 min when 0.33 equiv of BTC was used, compared
to 2 h for DCC/HOAt-mediated activations.20 Coupling times
of 20 min were typically required for quantitative conversion
to coupled products using BTC according to HPLC analysis,
which enabled each deprotection-coupling-wash cycle to be
effected well within 1 h. To the best of our knowledge, this is
the first demonstration of a BTC-mediated solid phase synthesis
protocol using Boc chemistry.
Encouraged by our model studies, we then investigated
whether a BTC-based coupling methodology could be applied
to the preparation of challenging hairpin polyamide sequences.
We chose to investigate the synthesis of polyamide 1 which
targets the DNA sequence 5′-WWGWGCW-3′ (where W is
either A or T) with nanomolar affinity.31 Compound 1 was
prepared in only 0.1% reported yield using the ꢀ-Ala PAM resin
via a traditional Boc-chemistry/benzotriazole-based ester pro-
tocol.30 The low yield is most likely attributed to a challenging
Resin-ImNH2/BocPyOH coupling late in the synthesis sequence,
which was confirmed in our laboratory using the conventional
coupling methodology. This polyamide sequence also comprises
other typical coupling sequences such as Py-Py, Py-Im, and
Im-aliphatic couplings which enable us to ascertain the scope
of the BTC coupling methodology. Using Boc-based chemistry
Figure 2. HPL chromatogram of (A) crude and (B) purified
polyamide 2. The retention time of 2 is 13.02 min.33
(25) Thern, B.; Rudolph, J.; Jung, G. Angew. Chem., Int. Ed. 2002, 41,
of the aryl hydrazine method was also demonstrated by the
NBS-mediated oxidative release of polyamides outfitted with
amino- (3) and alkyne-modified (4) tail structures in 22% and
12% yields, respectively.
According to the high purities of crude 3 and 4 by HPLC
analysis, the lower isolated yield of these polyamides
compared to polyamide 1 most likely correlates to their
2307
.
(26) Thern, B.; Rudolph, J.; Jung, G. Tetrahedron Lett. 2002, 43, 5013
.
(27) Konig, H. M.; Gorelik, T.; Kolb, U.; Kilbringer, A. F. M. J. Am.
Chem. Soc. 2007, 129, 704.
(28) Carpino, L. A.; Elfaham, A. J. Am. Chem. Soc. 1995, 117, 5401.
(29) Rijkers, D. T. S.; Adams, H. P. H. M.; Hemker, H. C.; Tesser,
G. I. Tetrahedron 1995, 51, 11235.
(30) Falb, E.; Yechezkel, T.; Salitra, Y.; Gilon, C. J. Pept. Res. 1999,
53, 507.
3912
Org. Lett., Vol. 11, No. 17, 2009

inherent resistance to precipitation in diethyl ether rather than
suboptimal coupling. Polyamides 3 and 4 comprise useful
conjugating amine (3) and alkyne (4) functionalities which
highlight the flexibility of the aryl hydrazine resin protocol
coupled with the high yielding BTC coupling methodology.
investigate the broader application of the BTC coupling
protocol for the preparation of polyamides comprising diverse
building blocks both in solution and by automated solid phase
methods.
Acknowledgment. GAB [Advanced Fellowship] and WS
[Postdoctoral Fellowship] thank the EPSRC for financial
support [EP/E055095/1]. SJG and RD thank the EPSRC for
PhD studentships. We thank Shairbanu Ibrahim, Dr. Andrew
Botterill, and Dr. Sharad Mistry of PNACL for their
assistance in the MALDI-ToF characterization.
In summary, we have described a new and highly effective
approach to solid phase polyamide synthesis using BTC as
the coupling agent. The BTC protocol was found to be
experimentally simple, fast, and highly- atom and step-
efficient for their preparation of polyamides on both known
(PAM resins) and novel (aryl hydrazine) solid supports
regardless of their sequence. Efforts are now underway to
Supporting Information Available: Information concern-
ing the comparative coupling efficiencies of HATU, DCC/
HOAt, and BTC, the preparative procedures and MALDI-
ToF data of polyamides 1-4. This material is available free
of charge via the Internet at http://pubs.acs.org.
(31) Hsu, C. F.; Phillips, J. W.; Trauger, J. W.; Farkas, M. E.; Belitsky,
J. M.; Heckel, A.; Olenyuk, B. Z.; Puckett, J. W.; Wang, C. C. C.; Dervan,
P. B. Tetrahedron 2007, 63, 6146.
(32) Belitsky, J. M.; Nguyen, D. H.; Wurtz, N. R.; Dervan, P. B. Bioorg.
Med. Chem. Lett. 2002, 10, 2767.
(33) See Supporting Information for further details.
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