Fluorous synthesis of minor groove binding agents related to distamycin

Article abstract of DOI:10.1016/j.tetlet.2006.08.058

Minor groove binding agents related to distamycin have been shown to target specific DNA sequences with high affinity. We report a new method for the preparation of these agents using fluorous synthesis in which the fluorous tag is located on what will become the cationic tail of the molecule. We demonstrate that fluorous synthesis yields both simple and complex polyamides in good yields and in high purity.

Full text of DOI:10.1016/j.tetlet.2006.08.058

Tetrahedron Letters 47 (2006) 7431–7434
Fluorous synthesis of minor groove binding agents
related to distamycin
Sreeman K. Mamidyala and Steven M. Firestine*
Department of Pharmaceutical Sciences, Eugene Apllebaum College of Pharmacy and Health Sciences,
Wayne State University, Detroit, MI 48201, USA
Received 11 July 2006; revised 16 August 2006; accepted 17 August 2006
Abstract—Minor groove binding agents related to distamycin have been shown to target specific DNA sequences with high affinity.
We report a new method for the preparation of these agents using fluorous synthesis in which the fluorous tag is located on what will
become the cationic tail of the molecule. We demonstrate that fluorous synthesis yields both simple and complex polyamides in good
yields and in high purity.
Ó 2006 Elsevier Ltd. All rights reserved.
Small molecules that can bind to a predefined target
within DNA are important in a number of fields ranging
from molecular biology to drug development. Of the
compounds that target DNA, agents that bind to minor
groove display the greatest ability to read the sequence
of DNA. Naturally occurring minor groove binding
agents, like distamycin or netropsin, possess limited
sequence selectivity; however, researchers have modified
these agents to create new molecules that have the abil-
ity to target any sequence combination.1–4 These agents,
termed polyamides or lexitropsins,5 are composed of a
variety of heterocycles joined by an amide bond to gen-
erate a polymer in which the presence of various hydr-
ogen bond donors and acceptors is complementary to
that found within the minor groove of DNA. These
molecules also contain a positive charge which results
in a favorable electrostatic interaction with DNA.
Polyamides can bind to DNA in either a 1:1 or 2:1
stoichiometry with the 2:1 binding mode possessing
the greatest sequence selectivity.2 Polyamides and lexi-
tropsins have found utility as gene regulators and as
antimicrobial or antiviral agents.2
boxylate derivative of the next heterocycle. Both solid-
and solution-phase methods have been published and
not surprisingly both methods borrow heavily from
the literature on the synthesis of peptides.6–9 For the
solid-phase synthesis, both Boc- and Fmoc-protected
heterocycles have been utilized and solid-phase
synthesis can be automated and used in the synthesis of
a wide range of polyamides.6,9 Solution-phase methods
utilizing either standard peptide coupling conditions or
a haloform reaction have also been developed.7,8
To complement these methods, we have developed a
fluorous synthesis of polyamides. Fluorous synthesis
is compatible with a wide variety of chemistries and
because molecules containing multiple fluorines
preferentially bind to other polyfluorinated molecules,
purification can be accomplished simply by passing the
reaction mixture through a column containing fluorous
silica.10,11 Non-fluorous materials are washed away
while polyfluorinated products are retained on the
column. Elution of the products under specific solvent
conditions provides a rapid and effective method for
purification. Fluorous synthesis has been utilized in
the synthesis of a wide range of molecules, including
oligonucleotides,12 peptides,13 and carbohydrates.14
Given the utility of these agents, it is not surprising that
a wide variety of methods exists for their synthesis. All
methods rely upon amide bond formation between the
amine of one heterocycle and the carboxylic acid or car-
To conduct fluorous synthesis, a polyfluorinated tag
must be present within the molecule of interest. We
chose to incorporate the tag into what would eventually
become the cationic tail that is present in all minor
groove binding agents. Synthesis would then proceed
*
Corresponding author. Tel.: +1 313 577 0455; fax: +1 313 577
2033; e-mail: sfirestine@wayne.edu
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.08.058

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S. K. Mamidyala, S. M. Firestine / Tetrahedron Letters 47 (2006) 7431–7434
umn with 80% MeOH/H2O. The desired fluorous-con-
taining material could be obtained by simply washing
the column with 100% THF. This purification method
was rapid (ꢀ10 min per compound) and generated pure
material (>95%). This procedure was used for the
purification of fluorous containing compounds.
The conversation of the nitro group in 5 to the amine for
the next reaction could be accomplished by standard
hydrogenation conditions (H2, Pd/C); however, we
found that this method was not reproducible on a large
scale. Thus, we investigated other methods to reduce the
nitro group. We found that reduction of 5 with NaBH4,
Pd/C in ethyl acetate/MeOH at 0 °C gave quantitative
conversion to the amine within 1 h.16 The resulting
amine was immediately reacted with 4 to generate the
dipeptide NO2–Py–Py–NH(CH2)3NHFBoc (6) in 84%
purified yield. Reduction of 6 followed by coupling with
2 equiv of N-methylpyrrole 2-carboxylic acid under
standard peptide coupling conditions (EDCI, DMAP,
Scheme 1. Synthesis of FBoc 1,3-diaminopropane.
from the C- to the N-terminus of the molecule. Since the
fluorous tag would need to be removed before biological
studies could be conducted, we focused on fluorinated
protecting groups and choose to use fluorous Boc
(FBoc).15 Treatment of excess 1,3-diaminopropane (1)
F
with the commercially available Boc reagent 2 yielded
the desired mono-protected product 3 in 94% yield
(Scheme 1). We observed no di-addition in this reaction.
The free amine of 3 could then be attached to a hetero-
cycle via standard coupling conditions to start the
synthesis of the minor groove binding agent.
DMF)
generated
the
tripeptide,
Py–Py–Py–
NH(CH2)3NHFBoc (7), in 85% yield. Removal of the
FBoc under acidic conditions gave the final product,
Py–Py–Py–NH(CH2)3NH2 (8), in 93% yield.
We initially focused on the synthesis of a three ring poly-
amide composed of only N-methylpyrrole units. This
distamycin analogue was prepared as outlined in
Scheme 2. The formation of the amide bonds was done
using either the haloform reaction or standard peptide
coupling conditions. We primarily used the haloform
reaction for amide bond formation since the reaction re-
quires no additional reagents, is simple to conduct and
the required starting material is easy to acquire in large
amounts and in good yield.8 Reaction of 3 with 2 equiv
of 4-nitro-2(-trichloroacetyl)-N-methylpyrrole (4) in dry
DMF at room temperature gave the desired product 5 in
94% purified yield. Purification was accomplished using
a fluorous silica gel chromatography. The crude reaction
mixture was added to the fluorous silica column and the
non-fluorous materials were eluted by washing the col-
The fluorous synthesis of the tripeptide gave an overall
yield of 67% to 7 and 61% to 8. Furthermore, we were
able to recover unreacted heterocycle from the reaction
simply by evaporating the material which did not bind
to the fluorous silica column. In most cases, the recov-
ered heterocycle was pure enough to be used without
additional purification.
Previous studies have shown that the 2:1 binding mode
of distamycin and related analogues provides the great-
est degree of sequence selectivity.2 Using this principle,
Dervan and co-workers have developed a number of
agents in which two polyamides are covalently attached
to each other via a linker molecule. The location and
nature of the linkage has been widely explored, but the
most common is the hairpin motif in which the N-termi-
nus of one polyamide is connected to C-terminus of the
second.1 The most common attachment is through the
use of c-aminobutyric acid (c). Given the increased
sequence selectivity and binding affinity that hairpin
polyamides possess over single polyamides like 8, we
decided to examine the ability of fluorous synthesis to
prepare hairpin polyamides (Scheme 3). The dipeptide
6 was reduced to the amine which was reacted with 4
to generate the tripeptide NO2–Py–Py–Py–NH(CH2)3-
NHFBoc (9) in 89%. The tripeptide 9 was reduced
to the amine and reacted with Fmoc-c-aminobutyric
acid under peptide coupling conditions to yield the
product
FmocNH–c-Py–Py–Py–NH(CH2)3NHFBoc
(10) in 94% yield. Fmoc deprotection of 10 was con-
ducted using basic conditions to generate the free amine
11. Reaction of 11 with 4 gave the peptide NO2–Py–Py–
Py–NH(CH2)3NHFBoc (12) in 95% yield. However,
conversation of 12 into the necessary amine resulted in
decomposition. We believe that the decomposition was
due to the instability of the amine and we are currently
exploring alternative methods to produce and stabilize
the amine.
Scheme 2. Fluorous synthesis of tripeptide Py–Py–Py–NH2.

S. K. Mamidyala, S. M. Firestine / Tetrahedron Letters 47 (2006) 7431–7434
7433
Scheme 4. Fluorous synthesis of hairpin polyamide.
Scheme 3. Attempted stepwise fluorous synthesis of hairpin
polyamide.
in the synthetic scheme was advantageous when
compared to conventional purification methods. While
the fluorous synthesis of polyamides offers several
apparent advantages over conventional methods of syn-
thesis, we should point out two caveats to our work.
First, we have examined only the incorporation of N-
methylpyrrole into the polyamide. Previous investiga-
tors have noted that the insertion of N-methylimidazole
into the polyamide can be challenging.8 Second, the syn-
thetic method outlined here can only generate one spe-
cific cationic tail. However, it has been well established
that the presence of additional hydrophobic groups on
the cationic tail can enhance binding affinity and aid
in the transport of these molecules through the cell
wall.17 Thus, the method presented here would not pro-
vide a convenient route to the synthesis of numerous
derivatives of the tail portion of the molecule. We are
currently working on synthetic methods that address
these two issues and will report on our results in due
course.
Given this problem with the stepwise approach, we
examined an alternative method to the preparation of
the desired hairpin polyamide (Scheme 4). Reduction
of methyl 4-nitro-N-methylpyrrole-2-carboxylate (13)
using NaBH4 generated the amine which was reacted
with 4 to produce the dipeptide in modest yield.
Reduction of 14 followed by coupling with N-methyl-
pyrrole-2-carboxylic acid gave the protected tripeptide,
Py–Py–Py–OMe (15), which upon hydrolysis yielded
the acid 16 in 80% yield. The acid was reacted with 11
to generate the desired protected hairpin polyamide 17
in 85% yield. Removal of the FBoc protecting group
was accomplished under acidic conditions to generate
the final product 18 in 95% yield. NMR analysis of 18
indicated that the material was 90% pure after passage
through a fluorous silica column to remove the fluorous
residue. Normal silica gel column was used to purify the
material further. The synthesis of 18 could be accom-
plished in a 71% overall yield starting from 6 or in a
47% overall yield starting from 5.
In conclusion, we have demonstrated the utility of fluor-
ous synthesis for the preparation and isolation of minor
groove binding polyamides containing N-methylpyrrole.
We found that the simple isolation of the intermediates
Acknowledgements
The authors acknowledge the support of Wayne State
University for the funding of this project.

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S. K. Mamidyala, S. M. Firestine / Tetrahedron Letters 47 (2006) 7431–7434
10. Curran, D. P. In Handbook of Fluorous Chemistry;
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