γ-AApeptides bind to RNA by mimicking RNA-binding proteins

Article abstract of DOI:10.1039/c1ob05738c

The interactions between proteins and RNAs are of vital importance for many cellular processes, including transcription and processing of RNA, translation, and viral infections. Here we report an γ-AApeptide that can mimic HIV-1 Tat protein and bind to TA

Full text of DOI:10.1039/c1ob05738c

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PAPER
c-AApeptides bind to RNA by mimicking RNA-binding proteins†
Youhong Niu,‡a Alisha “Jonesy” Jones,‡b Haifan Wu,a Gabriele Varani*b,c and Jianfeng Cai*a
Received 11th May 2011, Accepted 1st July 2011
DOI: 10.1039/c1ob05738c
The interactions between proteins and RNAs are of vital importance for many cellular processes,
including transcription and processing of RNA, translation, and viral infections. Here we report an
g-AApeptide that can mimic HIV-1 Tat protein and bind to TAR RNAs of HIV and BIV with
nanomolar affinity, comparable to that of the RNA-binding fragment of Tat (amino acids 49–58). The
interaction is resistant to the presence of a large excess of tRNA. With resistance to proteolytic
hydrolysis and limitless potential for diversification, g-AApeptides may emerge as a new class of
peptidomimetics to modulate RNA-protein interactions.
likely to compete with a protein for binding to RNA. Additionally,
Introduction
they are also much more amenable to clinical development
than oligonucleotides. In the past, a variety of peptide mimetics
of RNA-binding proteins have been introduced,4–11 but none
has reached even the pre-clinical stage of development as drug
candidates. Here we report the discovery of g-AApeptides, mimics
of a well-known RNA-binding protein.
The discovery of novel molecules that can bind strongly to RNA
and regulate RNA-protein interactions could have very broad
application in chemical and molecular biology, since RNA-protein
interactions are important in many aspects of cellular functions.1–3
These RNA-binding molecules could be very promising tools
or novel therapeutics in the biological sciences and medicine.
A well-known approach to discovering such molecules is to
develop oligonucleotides to directly target RNA or to regulate gene
expression by antisense or RNAi mechanisms.4 However, there
are considerable limitations for clinical development, including
cost, delivery and metabolic stability.4 Another attractive approach
is to develop small molecules which can mimic RNA binding
proteins, since many RNAs are highly structured and create unique
binding sites to specific proteins. In contrast to oligonucleotides,
small molecules are more easily adaptable to clinical therapeutic
development. Nonetheless, the development of small molecules
that can bind to RNAs with high affinity is very challenging.
This is because RNA-protein interactions are similar to protein-
protein interactions, involving a large surface area for recognition
and tight binding. Thus, competition of small molecular weight
drugs with large macromolecular complexes has so far been a very
difficult task to achieve. An alternative approach that relies instead
of using larger molecular weight peptides has been advocated. This
approach could be more effective because peptides are much larger
than traditional small molecular weight drugs, and therefore more
The HIV TAR RNA-Tat complex is one of the best studied
protein-RNA interactions because of its involvement in transcrip-
tional activation and essential role for viral replication of human
immunodeficiency virus type 1 (HIV-1).6 The Tat (transactivator)
viral protein specifically binds to the transactivator response
element (TAR) RNA and stimulates the transcription of the viral
genome.12 TAR has been found to be extremely conserved among
viral isolates, and the Tat-TAR interaction is unique and essential
to the virus,5 without which HIV would fail to replicate. Therefore,
the TAR-Tat complex is a promising target for the development
of new antiviral agents through the disruption of the TAR-Tat
interaction, which would inhibit viral replication at both latent and
active stages of infected cells.5 Therefore, the TAR-Tat interaction
is an excellent testing ground as well as a promising therapeutic
target for the development of novel peptidomimetics to disrupt
RNA-protein interactions.
In order to develop inhibitors of Tat-TAR interaction, sig-
nificant effort has been dedicated to synthesize and evaluate
short peptides that can mimic Tat protein and disrupt Tat
binding to TAR.4–11 Among them, oligopeptidomimetics such
as oligocarbamates,13 oligoureas,14 b-peptides,15 peptoids16 and
templated cyclic peptides5 were considered, since these structures
are resistant to proteolytic degradation. However, more than a
decade’s exploration has not led to any clinical drugs, in part
because a structure of the HIV-1 TAR/Tat complex remains
to be determined, due to its highly conformational dynamics.17
Recently, our group has developed a new class of peptide mimics
– g-AApeptides,18 based on the g-PNA backbone.19 These g-
AApeptides can project the same number of functional groups
as peptides of equivalent length, suggesting that they could
aDepartment of Chemistry, University of South Florida, 4202 E. Fowler Ave.,
Tampa, FL, 33620, USA. E-mail: jianfengcai@usf.edu
bDepartment of Chemistry, University of Washington, Box 351700, Seattle,
WA, 98195, USA
cDepartment of Biochemistry, University of Washington, Box 357350,
Seattle, WA, 98195, USA. E-mail: varani@chem.washington.edu
† Electronic supplementary information (ESI) available: NMR and MS
spectra of building blocks, HPLC traces and MALDI of oligomeric
sequences. See DOI: 10.1039/c1ob05738c
‡ These authors contributed equally to this work.
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structurally mimic an RNA-binding protein. They can be modified
with virtually limitless potential by introducing a wide variety of
functional groups and are resistant to proteolytic degradation.18
Their potential biomedical application has been demonstrated
by their capability to disrupt the p53-MDM2 protein-protein
interaction.18 To further explore the applications of g-AApeptides,
we demonstrate here that a g-AApeptide analogue of Tat 48–57
can bind to HIV TAR RNA with nanomolar affinity. The results
indicate that g-AApeptides are valid peptide mimics of RNA
binding proteins, and they can potentially be further developed
to modulate RNA-protein interactions in the future.
The arginine-rich segment of HIV-1 Tat (residues 48–57) makes
direct contacts with the TAR trinucleotide bulge region, and
is the key determinant of the Tat-TAR interaction.17 Many
oligopeptidomimetic inhibitors were designed based on this
fragment of Tat.5,8,12–16 Since HIV Tat 48–57 adopts an extended
conformation,20 we hypothesized that g-AApeptide c-AA1 (Fig. 1)
would be able to mimic HIV Tat 48–57. c-AA1 and Tat 48–57 have
identical molecular weight and project exactly the same functional
groups; the relative positions of these functional groups are similar
to each other when the peptide conformation is extended. To
test this hypothesis, we synthesized g-AApeptide c-AA1 and g-
AApeptide c-AA2 (a truncated sequence mimicking Tat 48–53).
The control HIV Tat 48–57 peptide P1 was also prepared for
comparison.
Fig. 2 Synthesis of g-AApeptide building blocks and of g-AApeptides.
hydrogenation provided Fmoc protected g-AApeptide building
blocks 4. These g-AApeptide building blocks were assembled on
solid phase, and the desired sequences were cleaved from the solid
support, purified by HPLC and characterized by MALDI (see ESI
for details†).
To investigate whether g-AApeptides could mimic the Tat 48–
57 peptide P1, we tested their binding to HIV-1 TAR RNA by
measuring Kd’s using EMSA (electrophoretic mobility shift assay);
the closely related BIV TAR RNA (bovine immunodeficiency
virus) was used as a control for specific binding. Both HIV-1
and BIV are lentiviruses, and the functions of Tat and TAR
are conserved; the sequences and secondary structures of HIV-
1 and BIV TAR are also highly similar (Fig. 3). Because of these
similarities, we anticipated that these Tat g-AApeptide mimetics
should bind tightly to both HIV-1 and BIV TAR by recognizing
their TAR-Tat binding regions.
The EMSA experiments demonstrated that g-AApeptide c-AA1
(Fig. 4) can bind to both HIV-1 TAR and BIV TAR RNAs with
nanomolar affinity. Furthermore, binding in the low mM regime
is retained even in the presence of 25,000-fold excess of tRNA
(Fig. 4), suggesting that the interaction is partially specific for the
TAR RNA structures, in that even a huge excess of tRNA fails
to completely abolish complex formation. Binding affinities are
listed in Table 1 and compared with those of g-AApeptide c-AA2
and Tat 48–57 peptide P1, which were also obtained by EMSA.
Kd’ values shown in Table 1 were calculated from data as shown
in Fig. 4, B and D, where a 250-fold excess of tRNA was present. In
the absence of tRNA, band smearing was observed. This smearing
may occur due to partial dissociation of complexes during gel
electrophoresis,15 as well as to the likely presence of non-specific,
lower affinity complexes between the peptides and the TAR RNAs,
Fig. 1 g-AApeptides g-AA1 and g-AA2 and control Tat 48-57 peptide
P1.
Results and discussion
Table 1 Summary of the affinity of different peptide and peptide mimetic
sequences for their interaction with HIV-1 and BIV TAR, as determined
by EMSA
The synthesis of g-AApeptides was carried out by manual
solid-phase synthesis from Fmoc-protected g-AApeptide building
blocks, a method developed by our group recently to synthesize
AApeptide sequences.18,21 Typically (Fig. 2), a Fmoc protected
amino aldehyde 1 reacted with benzyl glycinate to form secondary
amine 2, which was acylated by either g-Boc-amino butyric
acid or di-Boc-guanidinopropionic acid to give 3. Subsequent
Sequences
Kd ’ (HIV), nM
Kd ’ (BIV), nM
AA1
AA2
P1
166
>33000
166
300
>33000
333
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Fig. 3 Secondary structures of HIV-1 and BIV TAR RNA, and partial
sequences of HIV-1 Tat protein (48–47) and BIV Tat protein (65–81); these
fragments are largely responsible for the interaction with their respective
TAR RNAs.
Fig. 4 Binding of g-AApeptide c-AA1 to HIV-1 and BIV TAR RNAs
assayed by EMSA. A, Binding of g-AApeptide 1 to HIV-1 (0.4 nM) and
BIV TAR (0.4 nM); the buffer contains a 25,000-fold excess of tRNA to
reduce non-specific binding. B, Binding of g-AApeptide c-AA1 to HIV-1
(0.4 nM) and BIV TAR (0.4 nM) RNAs; the buffer contains a smaller
excess of tRNA (250-fold) to measure Kd’s more accurately. C, Binding
of Tat peptide P1 to HIV-1 (0.4 nM) and BIV TAR (0.4 nM); the buffer
contains a 25,000-fold excess of tRNA to reduce non-specific binding. D,
Binding of Tat peptide P1 to HIV-1 (0.4 nM) and BIV TAR (0.4 nM)
RNAs; the buffer contains a smaller excess of tRNA (250-fold) to measure
Kd’s more accurately.
which were reduced or eliminated in the presence of the tRNA,
resulting only in the presence of a specific peptide-TAR complex.
The EMSA results show that g-AApeptide c-AA1 binds to HIV
TAR with Kd of 166 nM, 2-fold more tighly than binding to BIV
TAR (Kd’ = 300 nM). As expected, Tat peptide P1 can bind to
HIV and BIV TAR tightly with the same Kd’ of 166 nM and
333 nM, respectively. Interestingly, g-AApeptide c-AA2, although
carrying a few postively charged side functional groups, failed
to provide any binding capability to both HIV and BIV TAR
under our experimental conditions. The EMSA results show that
g-AApeptide c-AA1 binds to HIV-1 and BIV TAR RNAs as tightly
as the Tat-derived P1 peptide 48–57, even if the g-AApeptide
backbone is more flexible than the conventional peptide backbone.
Although structural information is not yet available for the
complete HIV TAR/Tat complex, we have previously used NMR
to investigate the conformational change of HIV TAR when
binding to Tat and other small molecules and peptides.12,22,23 In
the presence of Tat and other ligands, the bulge region of TAR
undergoes a local conformational rearrangement and forms a
more stable structure (Fig. 5). This folding process can be induced
by any ligand containing a guanidinium group and even by the
single amino acid analogue argininamide. However, the interaction
of this guanidinium group with TAR is not the only source of
binding affinity and specificity for Tat recognition. NMR studies
demonstrated that there are multiple points of contacts between
base functional groups and phosphate groups of HIV TAR and
amino acid residues of Tat. These interactions contribute not only
to the affinity of the interaction but also to its specificity. Therefore,
based on the experimental results, we postulate that c-AA1 binds
to HIV TAR by mimicking the Tat peptide P1, because they posses
the same side functional groups. Following the mechanism of
Tat-TAR interaction, c-AA1 could recognize the bulge of HIV
TAR using one of the guanidinium groups, which would re-fold
Fig. 5 Schematic representation of the mechanism of HIV TAR/Tat
recognition (adapted from reference22). Binding of Tat re-folds the bulge
of TAR into a locally different conformation.
the HIV TAR bulge and define the precise positioning of critical
functional groups in the major groove. Compared to a-peptides,
half of the C O groups have been relocated to side chains, which
leads to increased comformational freedom for the g-AApeptide
backbone. As a result, c-AA1 may be able to more easily adjust its
conformation in the TAR- g-AApeptide complex by mimicking
that of the Tat peptide so as to achieve optimal binding.17
Satisfactorily, the truncated g-AApeptide c-AA2 has completely
lost its binding capability to both HIV-1 and BIV TAR RNAs,
strongly supporting the vitally important presence of three
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neighboring guanidino functional groups for binding. This result
also further suggests that the TAR- g-AApeptide interaction is
not purely driven by electrostatic interactions, since the truncated
g-AApeptide c-AA2 retains as many guanidino functional groups
as small molecules that were shown to bind to TAR with nM
affinity.9 The sequence is highly positively charged, yet it shows no
interaction with TAR RNA in the presence or absence of tRNA.
This result indirectly shows the importance of multiple points of
interactions between HIV TAR and c-AA1, similar to the HIV
TAR-Tat interaction. It is also noteworthy that there is a small
binding preference of c-AA1 for HIV-1 compared to BIV TAR
(2-fold), which may be due to c-AA1 being the mimic of HIV-1
Tat protein and not of BIV Tat. Altogether, these observations
provide a starting point for the rational design of more potent and
selective RNA-binding g-AApeptides in the future.
added, followed by 2 equivalents of NaBH3CN. The solution was
allowed to stir at 0 ◦C for 1 h and continue at room temperature
overnight. The solvent was evaporated and 100 ml ethyl acetate
and 100 ml saturated sodium bicarbonate solution were added
to the residue. The organic layer was separated and washed with
100 ml brine, dried over anhydrous sodium sulfate, and removed
in vacuo. Flash chromatography using ethyl acetate/hexane 1 : 1
gave 2 as a colorless oil.
Typical synthesis of 3. Compound 2, 1.2 equiv. of DIC,
Oxohydroxybenzotriazole, and R2COOH were stirred in 20 ml
DMF overnight. The solution was then partitioned in 100 ml
ethyl acetate and 100 ml water. The organic layer was separated
and washed with water (3 ¥100 ml) and Brine (2 ¥100 ml), dried
over anhydrous sodium sulfate, and then concentrated in vacuo.
Flash chromatography using ethyl acetate/hexane 1 : 3 gave 3 as a
colorless oil.
Conclusions
Typical synthesis of4. 3 in 20 ml ethyl acetatewas added to 10%
Pd/C and hydrogenated at atmospheric pressure and room tem-
perature overnight. The solution was evaporated and the residue
was purified by flash chromatography 5–7% MeOH/CH2Cl2 to
give 4 as a white foam solid.
We have developed a new peptide mimetic structure, the g-
AApeptides, which mimics the Tat peptide and binds to TAR
RNAs with nanomolar affinity. Our findings suggest that g-
AApeptide structures are capable of binding to RNAs by mim-
icking RNA-binding proteins. These structures can be developed
further to probe or disrupt RNA-protein interactions in the future.
This is a further demonstration of the promising biological activity
of g-AApeptides, that have already been shown to disrupt protein-
protein interactions.18,21 Due to their resistance to proteolysis,
convenient synthesis and limitless diversification, there is great
potential for g-AApeptide AA1 to be further optimized to identify
new anti-viral leads. A challenge will be increasing the specificity of
these flexible structures, which could be achieved by restraining the
conformation of g-AApeptide by forming cyclic ring structures.
NMR studies of TAR- g-AApeptide complex are currently under
investigation.
Experimental
Yield was 41% in three steps. 1H NMR (400 MHz, DMSO-d6) d
(two rotamers) 11.39 (m, 1H), 10.71 (d, J = 4.0 Hz, 1H), 8.66–8.60
(m, 1H), 8.53–8.50 (m, 1H), 8.29–8.23 (m, 1H), 7.85 (d, J = 8.0 Hz,
2H), 7.65–7.62 (m, 2H), 7.41–7.35 (m, 2H), 7.29–7.09 (m, 15H),
4.30–4.14 (m, 3H), 4.05–4.03 (m, 1H), 3.98–3.75 (m, 2H) 3.46–
3.40 (m, 2H), 3.30–3.17 (m, 2H), 3.02–2.56 (m, 2H), 2.35–2.22 (m,
2H), 1.65–1.19 (m, 20H). 13C NMR (100 MHz, DMSO-d6) d (two
rotamers) 171.8, 170.8, 156.5, 155.3, 153.5, 152.1, 151.9, 145.3,
144.3, 144.2, 141.2, 128.9, 128.0, 127.8, 127.4, 126.7, 126.7, 125.5,
125.4, 120.6, 120.5, 84.0, 83.3, 69.6, 65.8, 56.4, 52.3, 49.9, 47.3,
47.2, 37.1, 33.0, 31.7, 28.4, 28.0, 27.97, 27.90, 18.97. HRMS for
[M+Na]+ Calc: 989.4420, found: 989.4428.
1. General experimental methods
Fmoc protected a-amino acids and Knorr resin were obtained
from Chem-Impex International, Inc. All other reagents and
solvents were provided by either Sigma-Aldrich or Fisher Scien-
tific. NMR spectra of intermediates and g-AApeptide building
blocks were obtained on a Varian Inova 400. g-AApeptide
sequences were prepared on a Knorr resin in peptide synthesis
vessels on a Burrell Wrist-Action shaker. The g-AApeptides were
analyzed and purified on a Waters HPLC with both analytical and
preparative modules, respectively, and the desired fractions were
lyophilized using a Labconco lyophilizer. Molecular weights of g-
AApeptides were identified on a Bruker AutoFlex MALDI-TOF
mass spectrometer.
2. Synthesis of c-AApeptide building blocks
General procedure. Typical synthesis of 2. To glycine benzyl
ester hydrochloride in 20 ml methanol in a 100 ml round bottom
flask was added 1.2 equiv. of triethylamine and stirred at 0 ◦C
for 15 min. Stoichiometric amount of a Fmoc protected amino
acid aldehyde24,25 was added and the solution mixture was stirred
for another 30 min. Catalytic amount of acetic acid was then
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1
Yield was 38% in three steps. H NMR (400 MHz, DMSO-
131.8, 127.98, 127.43, 125.6, 125.5, 124.7, 120.5, 116.7, 86.7, 83.2,
78.5, 65.7, 65.5, 55.3, 52.41, 52.39, 49.9, 47.9, 47.3, 47.2, 42.9,
36.96, 36.8, 32.5, 32.0, 29.8, 29.5, 28.7, 28.4, 28.0, 27.98, 26.2,
19.4, 18.0, 18.0, 12.7. HRMS for [M+H]+ Calc: 1004.4677, found:
1004.4677.
d6) d (two rotamers) 11.43 (s, 1H), 8.58–8.50 (m, 1H), 7.86–7.80
(m, 2H), 7.64–7.61 (m, 2H), 7.40–7.25 (m, 4H), 4.25 (m, 3H),
2.75–3.67 (m, 2H), 3.55–3.42 (m, 5H), 3.15–3.10 (m, 2H), 2.55–
2.54 (m, 1H), 2.45–2.42 (m, 1H), 1.42&1.40&1.34&1.33 (4 s, 18H)
13C NMR (100 MHz, DMSO-d6) d (two rotamers) 172.3, 163.6,
156.52, 156.5, 152.3, 144.3, 143.0, 141.1, 139.9, 137.9, 129.4, 127.7,
127.3, 125.7, 129.4, 127.7, 127.5, 125.7, 120.5, 83.2, 79.63, 78.5,
65.9, 56.5, 53.4, 47.2, 38.6, 36.8, 32.5, 28.4, 28.04. HRMS for
[M+H]+ Calc: 654.3137, found: 654.3138.
3. Solid phase synthesis, purification and characterization of
c-AApeptides.
The Tat 48–57 peptide P1 was synthesized and analyzed by the
USF peptide facility, and was used without further purification.
The two g-AApeptides were prepared on a Knorr resin in peptide
synthesis vessels on a Burrell Wrist-Action shaker following
the standard Fmoc chemistry of solid phase peptide synthesis
protocol. Each coupling cycle included an Fmoc deprotection
using 20% Piperidine in DMF, and 4 h coupling of 1.5 equiv of
g-AApeptide building blocks onto resin in the presence 2 equiv
of DIC (diisopropylcarbodiimide)/Oxohydroxybenzotriazole in
DMF. After the desired sequences were assembled, they were
transferred into a 4 ml vial and cleaved from solid support in
48 : 50 : 2 TFA/CH2Cl2/triisopropylsilane overnight. Then solvent
was evaporated and the residues were analyzed and purified on an
analytical (1 ml min-1) and a preparative Waters (20 ml min-1)
HPLC systems, respectively. The same methods were used by
running 5% to 100% linear gradient of solvent B (0.1% TFA in
acetonitrile) in A (0.1% TFA in water) over 40 min, followed
by 100% solvent B over 10 min. The desired fractions were
>70% in crude (as determined by HPLC) and eluted as single
peaks at > 95% purity. They were collected and lyophilized. The
molecular weights of g-AApeptides and Tat peptide were obtained
on Bruker AutoFlex MALDI-TOF mass spectrometer using a–
cyano-4-hydroxy-cinnamic acid as the matrix.
Yield was 69% in three steps. 1H NMR (400 MHz, DMSO-d6)
d (two rotamers) 7.84 (d, J = 8.0 Hz, 2H), 7.64–7.61 (dd, J =
4.0, 8.0 Hz, 2H), 7.37 (t, J = 8.0 Hz, 2H), 7.28 (t, J = 8.0 Hz,
2H), 7.14 & 6.98 (2d, J = 8.0 Hz, 1H), 6.74–6.69 (m, 1H), 4.29–
4.14 (m, 3H), 3.40–3.81 (m, 2H), 3.62–3.52 (m, 2H), 3.28–3.16
(m, 1H), 2.95–2.69 (m, 4H), 2.36–2.22 (m, 1H), 2.12–2.01 (m,
1H), 1.70–1.13 (m, 26H). 13C NMR (100 MHz, CD3OH) d (two
rotamers) 174.7, 174.4, 171.5, 171.2, 157.32, 157.3, 157.1, 143.9,
143.8, 141.23, 141.20, 127.3, 126.7, 124.7, 124.6, 119.5 78.5, 78.4,
66.1, 65.9, 53.1, 51.2, 50.1, 49.9, 49.8, 39.8, 39.5, 39.3, 31.6, 31.2,
29.7, 29.5, 29.2, 27.4, 25.2, 22.9, 22.8. HRMS for [M+H]+ Calc:
697.3807, found: 697.3796.
4. HIV-1 and BIV TAR RNA preparation26
Body-labeled RNA was prepared by in vitro transcription using
T7 RNA polymerase, a synthetic oligonucleotide template, and a
nucleotide mixture containing [R-32P]-CTP (3000 Ci/mmol). The
RNA oligonucleotides were purified by denaturing PAGE, and
concentrations were determined by UV at 260 nm. RNA was
annealed by heatingat90 ◦C and slow cooling to room temperature
in sterile H2O at a concentration of 20–100 nM. Binding assays
were performed at 4 ◦C.
5. EMSA (Electrophoretic mobility shift assay)4
g-AAPeptides or Tat 48–57 peptide and RNA were incubated in
a buffer (10 mL) containing Tris-HCl (50 mM, pH 8.0), KCl (50
mM), DTT (200 mM), tRNA (Escherichia coli) and Triton X-
100 (0.05%). The samples were fractionated by loading onto 12%
native polyacrylamide gels in 0.5% TB buffer and electrophoresed
at 15 W and 4 ◦C. Dried gels were exposed to a phosphor imaging
plate and scanned with a Molecular Dynamics phosphor imager.
Bands corresponding to free and bound RNA were quantified
using ImageQuant.
Yield was 43% in three steps. 1H NMR (400 MHz, DMSO-d6) d
(two rotamers) 11.37 (s, 1H), 10.80 (d, J = 8.0 Hz, 1H), 8.83–8.77
(m, 1H), 8.36–8.32 (m, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.64–7.59
(m, 2H), 7.38–7.34 (m, 2H), 7.30–7.25 (m, 2H), 7.20–7.16 (m, 1H),
7.04 (d, J = 8.0 Hz, 1H), 6.72 (br.s, 1H), 6.40 (br.s, 1H), 4.28–4.11
(m, 4H), 4.10–3.81 (m, 2H), 3.64–3.54 (m, 1H), 3.53–3.15 (m,
3H), 3.04–2.94 (m, 2H), 2.90 (s, 2H), 2.76–2.62 (m, 2H), 2.50–2.42
(m, 4H), 2.39 (s, 3H), 1.96 (s, 3H), 1.49–1.19 (m, 28H). 13C NMR
(100 MHz, DMSO-d6) d (two rotamers) 172.2, 171.8, 163.5, 157.9,
156.6, 152.3, 144.4, 144.25, 144.23, 144.2, 141.2, 137.7, 134.7,
Acknowledgements
This work is supported by USF start-up fund to JC and by a grant
from NIH-NIAID to GV.
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14 N. Tamilarasu, I. Huq and T. M. Rana, J. Am. Chem. Soc., 1999, 121,
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15 M. A. Gelman, S. Richter, H. Cao, N. Umezawa, S. H. Gellman and T.
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