Novel amphiphilic α-helix mimetics based on a bis-benzamide scaffold

Article abstract of DOI:10.1021/ol901785v

To mimic amphophilic a-helices, a new scaffold was designed based on a bis-benzamide that places four side-chain functional groups found at the i, i+2, i+5, and i+7 positions of a helix. Its two hydrogen bonds fix the conformation and provide accurate bif

Full text of DOI:10.1021/ol901785v

ORGANIC
LETTERS
2009
Vol. 11, No. 19
4418-4421
Novel Amphiphilic r-Helix Mimetics
Based on a Bis-benzamide Scaffold
Srinivasa Marimganti, Murthy N. Cheemala, and Jung-Mo Ahn*
Department of Chemistry, UniVersity of Texas at Dallas, 800 West Campbell Road,
Richardson, Texas 75080
jungmo@utdallas.edu
Received August 3, 2009
ABSTRACT
To mimic amphiphilic r-helices, a new scaffold was designed based on a bis-benzamide that places four side-chain functional groups found
at the i, i+2, i+5, and i+7 positions of a helix. Its two hydrogen bonds fix the conformation and provide accurate bifacial arrangement of the
four substituents, simultaneously representing two opposing helical sides. An efficient synthetic route was achieved for the construction of
bis-benzamides, and their superior r-helix mimicry was confirmed by X-ray crystallography.
As one of the most abundant protein secondary structures,1
R-helices are often found to play an important role in protein
complex formations through which diverse regulatory pro-
cesses like gene expression, enzyme activity, signal trans-
duction, immune response, and apoptosis are modulated.2
This makes R-helical peptides a versatile tool to investigate
protein-protein and peptide hormone-receptor interactions
as well as to ultimately develop potent therapeutic candidates.
However, short peptide fragments are prone to have a random
coil or less characterized structure in solution when taken
out of proteins.3 In addition, short peptides generally suffer
from serious limitations, such as high susceptibility to
enzymatic degradation, poor bioavailability, and difficulty
in penetrating membranes, which must be improved for
effective use in biomedical applications.
bridge,4 a salt bridge,5 and a disulfide bridge6 between amino
acid residues. Hydrocarbon stapling by olefin metathesis was
also found to effectively stabilize R-helices.7 In addition, a
variety of foldamers like ꢀ-peptides were developed to mimic
helical structures.8 These conformational restrictions not only
increase binding affinity to target proteins by stabilizing the
required helical structure but also improve enzymatic stability
and bioavailability.
Another approach is de novo design of nonpeptide R-helix
mimetics. Using rigid and preorganized scaffolds, R-helix
mimetics place side-chain functional groups in a proper
(4) (a) Ospay, G.; Taylor, J. W. J. Am. Chem. Soc. 1992, 114, 6966. (b)
Phelan, J. C.; Skelton, N. J.; Braisted, A. C.; McDowell, R. S. J. Am. Chem.
Soc. 1997, 119, 455.
(5) Marqusee, S.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1987,
84, 8898.
To preserve R-helicity in short peptides, several strategies
have been developed including the formation of a lactam
(6) Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz,
P. G. J. Am. Chem. Soc. 1991, 113, 9391.
(7) (a) Blackwell, H. E.; Grubbs, R. H. Angew. Chem., Int. Ed. 1998,
37, 3281. (b) Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc.
2000, 122, 5891. (c) Patgiri, A.; Jochim, A. L.; Arora, A. S. Acc. Chem.
Res. 2008, 41, 1289.
(1) Barlow, D. J.; Thornton, J. M. J. Mol. Biol. 1988, 201, 601.
(2) (a) Cochran, A. G. Chem. Biol. 2000, 7, R85–R94. (b) Peczuh, M,
W.; Hamilton, A. D. Chem. ReV 2000, 100, 2479.
(3) (a) Scholtz, J. M.; Baldwin, R. L. Annu. ReV. Biophys. Biomol. Struct.
1992, 21, 95. (b) Schneider, J. P.; Kelly, J. W. Chem. ReV. 1995, 95, 2169.
(8) (a) Horne, W. S.; Gellman, S. H. Acc. Chem. Res. 2008, 41, 1399.
(b) Seebach, D.; Gardiner, J. Acc. Chem. Res. 2008, 41, 1366.
10.1021/ol901785v CCC: $40.75
Published on Web 08/31/2009
 2009 American Chemical Society

orientation as found in R-helices to interact with target
proteins. The absence of peptide structure also results in high
enzymatic stability and enhances bioavailability. In the past
decade, several R-helix mimetics9 were developed by using
scaffolds like indans,10 terphenyls,11 tris-pyridylamides,12
ladder-like polycyclic ethers,13 tris-benzamides,14 and py-
ridazines.15 High structural rigidity in these scaffolds easily
places three functional groups corresponding to the i, i+3
(or i+4), and i+7 positions in an R-helix that organizes one
helical face.
Model (version 9, Schro¨dinger) demonstrated high structural
rigidity resulting from two hydrogen bonds between a
benzamide proton and two adjacent alkoxy substituents (R2
and R3). These hydrogen bonds lock the conformation of
the bis-benzamide and arrange the two functional groups (R2
and R3) on the same side, with the other two (R1 and R4)
positioned on the opposite side. Superimposition of its lowest
energy conformation on an R-helix shows that the four
functional groups of a bis-benzamide are well overlaid on
the corresponding side chains of a helix, properly mimicking
helical amphiphilicity.
However, most of the R-helices found in proteins and
biologically active peptides are amphiphilic, possessing a
hydrophilic surface on the opposite side of a hydrophobic
one. In particular, R-helical segments in peptide hormones
highly rely on amphiphilicty for maximal interaction with
target receptor proteins. This clearly suggests that presenting
only three functional groups found on a single side of a helix,
as observed in the existing R-helix mimetics, does not model
essential helical amphiphilicity. The lack of functional groups
found on the opposite side of a helix in the structure of “one-
sided” R-helix mimetics may result in not only suboptimal
affinity to target proteins but also lowered selectivity that
would significantly limit their utility by potential promiscuity.
In an effort to achieve superior R-helix mimicry with
higher affinity and selectivity to taget proteins, we report
herein novel amphiphilic R-helix mimetics based on a bis-
benzamide scaffold (Figure 1). A suitable scaffold for
amphiphilic R-helix mimetics should facilitate the installation
of functional groups on both sides of the scaffold and also
ensure rigid conformation by its preorganized framework.
Satisfying these criteria, the bis-benzamide scaffold easily
places four functional groups corresponding to the i, i+2,
i+5, and i+7 positions in a helix by employing its four
hydroxyl groups.
Scheme 1. Synthesis of an Amphiphilic Subunit 5
Since a bis-benzamide comprises of two identical subunits,
a protected form of a 4-amino-2,5-dihydroxybenzoic acid 5
was first synthesized (Scheme 1). Starting with 2-hydroxy-
4-nitrobenzoic acid, an Elbs persulfate oxidation reaction16
was carried out to introduce a 5-hydroxy group. The
regioselectivity of the oxidation at the 5-position was
confirmed by 1H NMR with two singlets in aromatic region.
Then, the two hydroxyl groups at the 2- and 5-positions of
the hydroxyquinone 2 were easily differentiated by the
formation of a ketal between 1-carboxylate and 2-hydroxy
groups. Reduction of a nitro group in the ketal 3 and
subsequent protection of the resulting amine with a Boc
group produced the amphiphilic builidng block 5 for the
construction of bis-benzamides.
Having the protected benzoate 5 in hand, we sought to
mimic R-helical regions of peptide hormones to demonstrate
proof-of-concept. Since numerous R-helical peptide hor-
mones utilize both hydrophobic and hydrophilic surfaces for
maximal interaction with their receptors, amphiphilic R-helix
mimetics based on the bis-benzamide scaffold would be
effective in simultaneously emulating both helical faces and
result in high affinity and selectivity. This approach would
Figure 1. (a) Amphiphilic R-helix mimetic 1 based on a bis-
benzamide scaffold; (b) its lowest-energy conformation; (c) super-
imposition of 1 (orange) on an R-helix (green).
(11) (a) Yin, H.; Lee, G.-I.; Park, H. S.; Payne, G. A.; Rodriguez, J. M.;
Sebti, S. M.; Hamilton, A. D. Angew. Chem., Int. Ed. 2005, 44, 2704. (b)
Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem. Soc. 2001, 123,
5382.
A Monte Carlo conformational search and subsequent
energy minimization of a bis-benzamide by using Macro-
(12) Ernst, J. T.; Becerril, J.; Park, H. S.; Yin, H.; Hamilton, A. D.
Angew. Chem., Int. Ed. 2003, 42, 535.
(9) For reviews on R-helix mimetics, see: (a) Davis, J. M.; Tsou, L. K;
Hamilton, A. D. Chem. Soc. ReV. 2007, 36, 326. (b) Garner, J.; Harding,
M. M. Org. Biomol. Chem. 2007, 5, 3577. (c) Che, Y.; Brooks, B. R.;
Marshall, G. R. J. Comput.-Aided Mol. Des. 2006, 20, 109.
(10) Horwell, D. C.; Howson, W.; Ratcliffe, G. S.; Willems, H. M. G.
Bioorg. Med. Chem. 1996, 4, 33.
(13) Oguri, H.; Oomura, A.; Tanabe, S.; Hirama, M. Tetrahedron Lett.
2005, 46, 2179.
(14) Ahn, J.-M.; Han, S.-Y. Tetrahedron Lett. 2007, 48, 3543.
(15) Volonterio, A.; Moisan, L.; Rebek, J. Org. Lett. 2007, 9, 3733.
(16) Bhattachryya, S. C.; Seymour, D. E. J. Chem. Soc. 1950, 1139.
Org. Lett., Vol. 11, No. 19, 2009
4419

also faciliate a rational design of peptidomimetics for
biologically important helical peptides. Compared to a
random screening campaign, a rational design approach has
many advantages including facile optimization of initial leads
by taking hints from structure-activity studies of target
peptides.
phobic face by using Phe12 and Tyr19 and a hydrophilic one
with Ser14,17. On the other hand, the other N-terminal R-helix
mimetic 1b includes side chains of Ser14, Val16, Tyr19, and
Glu21,whereas the C-terminal R-helix mimetic 1c contains
Ala24, Lys26, Ile29, and Trp31. Due to synthetic difficulties
previously observed,13 4-fluorobenzyl and 2-naphthylmethyl
groups were used as surrogates for the side chain functional
groups of Tyr and Trp, respectively.
Scheme 2. Synthesis of Amphiphilic R-Helix Mimetics (9a-c)
Figure 2. (a) Sequence of GLP-1; (b) amphiphilic R-helix mimetics
1a-c designed for N- and C-terminal helices in GLP-1.
To evaluate the bis-benzamide scaffold, we have focused
on helical segements in glucagon-like peptide-1 (GLP-1).
GLP-1 is a 30 amino acid-containing peptide hormone and
plays a critical role in glucose homeostasis by stimulating
insulin secretion and restoring pancreatic ꢀ-cell mass, which
are highly beneficial to treat type 2 diabetes.17 Structural
analysis by 2D-NMR,18 CD spectroscopy,19 and X-ray
crystallography20 showed that GLP-1 has two R-helical
regions between residues 13-20 and 24-34, and their
importance in receptor interaction was confirmed by our
recent cyclization scanning study.21 Since amino acids on
both sides of helices, such as Phe12, Ser14, Tyr19, Ile29, and
Trp31, are found to be important for receptor binding and
activation,22 the two R-helical segments of GLP-1 appear
to be suitable targets for amphiphilic R-helix mimetics.
Three amphiphilic R-helix mimetics 1a-c were designed
on the basis of the bis-benzamide scaffold to represent the
two helical segments in GLP-1 (Figure 2). One of the
N-terminal R-helix mimetics 1a presents side-chain functional
groups of Phe12, Ser14,17, and Tyr19 and organizes a hydro-
As described in Scheme 2, the first O-alkylation was
carried out by treating the protected amphiphilic subunit 5
with an alkyl halide and NaH to install a side-chain functional
group (R1) at the i position. After quantitative removal of
the ketal and the formation of an allyl ester 6a-c, the
released 2-hydroxy group was alkylated to introduce a
functional group (R2) at the i+2 position. Then removal of
the allyl ester with Pd0 and a coupling reaction with the
amphiphilic subunit 4 produced a bis-benzamide 7a-c. Two
functional groups (R3 and R4) for the i+5 and i+7 positions
were added by repeating O-alkylation and ketal removal steps
to complete the synthesis of the amphiphilic R-helix mimetics
9a-c. The side chain protecting groups in 9a-c (TBS, TMS,
MOM, Aloc) were removed with tetrabutylammonium
fluoride, TFA, or Pd0, respectively, and the resulting final
compounds 1a-c will be examined for biological activity.
To evaluate R-helix mimicry, we have crystallized the bis-
benzamide 1a. As shown in Figure 3, the crystal structure
of 1a confirms two hydrogen bonds between the benzamide
proton and two adjacent 2-hydroxyethoxy groups, which
arranges two substituents (R2 and R3) on the same side. The
hydrogen bonds also drive the other two functional groups
(R1 and R4) to the opposite side as designed. Well overlaid
on the corresponding R-helical peptide segment in GLP-1,
(17) (a) Baggio, L. L.; Drucker, D. J. Gastroenterology 2007, 132, 2131.
(b) Drucker, D. J.; Nauck, M. A. Lancet 2006, 368, 1696. (c) Aaboe, K.;
Krarup, T.; Madsbad, S.; Holst, J. J. Diabetes Obes. Metab. 2008, 1.
(18) (a) Thornton, K.; Gorenstein, D. G. Biochemistry 1994, 33, 3532.
(b) Neidigh, J. W.; Fesinmeyer, R. M.; Prickett, K. S.; Andersen, N. H.
Biochemistry 2001, 40, 13188.
(19) Runge, S.; Schimmer, S.; Oschmann, J.; Schiødt, C. B.; Knudsen,
S. M.; Jeppesen, C. B.; Madsen, K.; Lau, J.; Thøgersen, H.; Rudolph, R.
Biochemistry 2007, 46, 5830.
(20) Runge, S.; Thøgersen, H.; Madsen, K.; Lau, J.; Rudolph, R. J. Biol.
Chem. 2008, 283, 11340.
(21) Murage, E. N.; Schroeder, J. C.; Beinborn, M.; Ahn, J.-M. Bioorg.
Med. Chem. 2008, 16, 10106.
(22) Adelhorst, K.; Hedegaard, B. B.; Knudsen, L. B.; Kirks, O. J. Biol.
Chem. 1994, 269, 6275.
4420
Org. Lett., Vol. 11, No. 19, 2009

In summary, we have designed a bis-benzamide scaffold
to construct amphiphilic R-helix mimetics, and four func-
tional groups installed on bis-benzamides represent amino
acid side chains at the i, i+2, i+5, and i+7 positions
accounting for helical amphiphilicity. Comprising two in-
dentical amphiphilic subunits, an efficient synthetic route was
achieved by repetitive O-alkylations and amide bond forma-
tion reactions. To evaluate R-helix mimicry, three am-
phiphilic R-helix mimetics were designed and synthesized
on the basis of the sequences of an R-helical peptide hormone
GLP-1. An X-ray crystal structure confirms that the bis-
benzamide scaffold shows superior R-helix mimicry by
simultaneously representing both helical faces with the
preorganized framework, which will potentially provide high
affinity and selectivity to target proteins.
Acknowledgment. This work was supported by the
American Diabetes Association (7-07-JF-02), the Welch
Foundation (AT-1595), and the Texas Advanced Research
Program (009741-0031-2006).
Figure 3. (A) X-ray crystal structure of an amphiphilic R-helix
mimetic 1a (ellipsoids depited at the 30% probablility level.23) and
(B) superimposition of 1a (orange) over the corresponding helical
segment in GLP-1 (green).
Supporting Information Available: Experimetal proce-
dures and full spectroscopic data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
the crystal structure of 1a clearly shows superior R-helix
mimicry accounting for helical amphiphilicity (Figure 3B).
(23) CCDC 732238 contains the supplementary crystallographic data
that can be obtained free of charge from the Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
OL901785V
Org. Lett., Vol. 11, No. 19, 2009
4421