Fluorescent Mu selective opioid ligands from a mixture based cyclic peptide library

Article abstract of DOI:10.1021/co300110t

A positional scanning cyclic peptide library was generated using a penta-peptide thioester scaffold. Glycine was fixed at position R¹. Diaminopropionic acid was fixed at position R³, with its γ-amino attaching to an anthraniloyl group. Positions R² and R⁴ contained 36 l- and d- amino acids and position R⁵ contained 19 l- amino acids. Cyclization was performed in a mixture of acetonitrile and 1.5 M aqueous imidazole solution (7:1 v/v) at room temperature for 5 days. No significant cross-oligomerization was detected under the cyclization conditions. The library was screened in a binding assay for mu opioid receptor, identifying the active amino acid mixture at each position. A total of 40 individual cyclic peptides were identified and synthesized by the combinations of the most active amino acid mixtures found at three positions 5 × 4 × 2. Two cyclic peptides exhibited high binding affinities to opioid receptor. The most active cyclic peptide in the library was yielded to have Tyr at R², d-Lys at R⁴, and Tyr at R⁵. Further investigation on this compound revealed the side chain-to-tail isomer to have greater binding affinity (14 nM) than the head-to-tail isomer (39 nM). Both isomers were selective for the mu-opioid receptor.

Full text of DOI:10.1021/co300110t

Research Article
pubs.acs.org/acscombsci
Fluorescent Mu Selective Opioid Ligands from a Mixture Based Cyclic
Peptide Library
Yangmei Li, Colette T. Dooley, Jaime A. Misler, Ginamarie Debevec, Marc A. Giulianotti,
Margaret E. Cazares, Laura Maida, and Richard A. Houghten*
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, Florida 34987, United States
S
* Supporting Information
ABSTRACT: A positional scanning cyclic peptide library was generated using a penta-peptide thioester scaffold. Glycine was
fixed at position R1. Diaminopropionic acid was fixed at position R3, with its γ-amino attaching to an anthraniloyl group. Positions
R2 and R4 contained 36 L- and D- amino acids and position R5 contained 19 L- amino acids. Cyclization was performed in a
mixture of acetonitrile and 1.5 M aqueous imidazole solution (7:1 v/v) at room temperature for 5 days. No significant cross-
oligomerization was detected under the cyclization conditions. The library was screened in a binding assay for mu opioid
receptor, identifying the active amino acid mixture at each position. A total of 40 individual cyclic peptides were identified and
synthesized by the combinations of the most active amino acid mixtures found at three positions 5 × 4 × 2. Two cyclic peptides
exhibited high binding affinities to opioid receptor. The most active cyclic peptide in the library was yielded to have Tyr at R2, D-
Lys at R4, and Tyr at R5. Further investigation on this compound revealed the side chain-to-tail isomer to have greater binding
affinity (14 nM) than the head-to-tail isomer (39 nM). Both isomers were selective for the mu-opioid receptor.
KEYWORDS: cyclic peptide, positional scanning library, synthetic cyclic peptide library, opioid ligand, mu selective ligand,
fluorescent label
chemical synthesis.4 The need for a facile synthetic strategy for
INTRODUCTION
■
the synthesis of cyclic peptide library is clear.
We have recently developed an imidazole-promoted
Cyclic peptides are widely recognized as potential therapeutic
compounds. Compared to their linear counterparts, cyclic
peptides have enhanced binding affinity and specificity to G-
coupled receptors,1 but more importantly they have increased
stability in vivo owing to the constrained structure imposed by
the cyclization. Therapeutic agents such as daptomycin,
cyclosporine A, polymyxin, and octreotide are cyclic peptides.
A potential limitation of cyclic peptides is that they can be
difficult to synthesize. The ground-state E geometry of the
peptide bond prevents the peptides from attaining the ring-like
conformation conducive to cyclization.2 Furthermore, oligome-
rization is a common side reaction in macrocyclization. Such
synthetic barriers have hindered the discovery of new
therapeutically promising molecules and limited construction
of cyclic peptide-based libraries. To date, there are only a
handful of studies where cyclic peptide libraries have been
applied to the identification of potent ligands in drug
discovery.3 Many of the cyclic peptide libraries reported were
constructed by phage display strategies rather than de novo
cyclization approach to synthesize cyclic peptides from their
fully unprotected linear peptide thioesters.5 This method is
highly efficient for the synthesis of cyclic peptides ranging from
5 to 11 amino acid residues. Our studies also demonstrate that
oligomerization was minimal using the imidazole-promoted
cyclization. In our continuing efforts to develop new potent
ligand tools for opioid receptors, we designed, synthesized, and
screened an anthraniloyl labeled cyclic peptide library in the
positional scanning format using the imidazole-promoted
cyclization method.
Received: September 12, 2012
Revised: October 18, 2012
Published: October 30, 2012
© 2012 American Chemical Society
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Research Article
Scheme 1. Synthesis Approach for Anthraniloyl-Tagged Peptide
a
Scheme 2. Synthesis of Anthraniloyl-Labeled Cyclic Pentapeptide
a
(1) Boc-AA−OH/PyBOP/DIEA; 55% TFA. (2) Pd(PPh3)4/PhSiH3; 2-nitrobenzoic acid/DIC. (3) SnCl2/DMF; 55% TFA. (4) HF (anhydrous)/
anisole, 0 °C, 2 h. (5) 1.5 M imidazole (aq)/acetonitrile (1:7 v/v), r.t. 72 h.
Figure 1. LC-MS of crude cyclic product cyclo[Asp-Leu-Orn(Ant)-Phe-Gly].
peptide is illustrated in Scheme 1. The anthraniloyl group is
generated by coupling of an o-nitrobenzoic acid to an amino
group of a peptide, followed by reduction with tin chloride(II).
The mild reduction in nearly neutral conditions is compatible
with both Boc- and Fmoc- chemistry.8
A model cyclic peptide was used to optimize the synthetic
approach. Synthesis of anthraniloyl-labeled cyclo[Asp-Leu-
Orn(Ant)-Phe-Gly] is presented in Scheme 2. The anthraniloyl
group was designed to attach to the third amino acid residue to
limit its possible effect on cyclization. Boc-Gly, Phe, Orn-
(Alloc), Leu, and Asp(OBzl) were subsequently coupled to the
mercaptomethylphenyl-functionalized silica gel 1 developed in
our lab as “volatilizable” support to obtain the resin bound
RESULTS AND DISCUSSION
■
Synthetic Approach to Anthraniloyl (Ant) Labeled
Cyclic Penta-Peptide. The anthraniloyl group is a well
characterized extrinsic fluorescent probe which has some
important features such as a relatively high quantum yield at
415 nm (excitation at 330 nm) for its small size and
hydrophilicity.6 The anthraniloyl fluorophore is less likely to
change the overall structural characteristics of a peptide and
interfere with biological activity. Indeed in our previous studies
we studied an N-terminal rhodamine-labeled tetrapeptide
library. We found that the fluorescent moiety was an intrinsic
component of the binding activity of the active compounds
identified.7 The synthetic strategy for the anthraniloyl-labeled
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Chart 1. Screening of the Cyclic Peptide PS-SCL for the Ability to Inhibit the Binding of Selective Radiolabeled [3H]DAMGO
a
to the Mu Receptor
a
Each panel represents one of the three positional SCLs. Each bar within a panel represents percent inhibition by a cyclic peptide mixture defined in
the O position. (a). Position 2; (b). Position 4; (c). Position 5 in a competitive radio receptor binding assay for the mu opioid receptor. (d). Cyclic
peptide PS-SCL.
pentapeptide 2.9 After removal of the Alloc protecting group, 2-
nitrobenzoic acid was coupled to the resin bound peptide 2 to
yield the resin bound peptide 3. The resin bound peptide 3 was
then reduced in 2 M SnCl2 to yield the resin bound
anthraniloyl peptide 4. The linear anthraniloyl peptide thioester
5 was obtained by the treatment of anhydrous HF at 0 °C for
1.5 h. Following removal of the HF with nitrogen stream and
lypholization, the crude linear peptide was cyclized at a
concentration of 1 mM in a mixed solution made up of
acetonitrile and 1.5 M imidazole in water plus (7:1 v/v),
forming the anthraniloyl-labeled fluorescent cyclic peptide 6.
Results from the LC-MS of the crude cyclization product are
shown in Figure 1. The yield of the desired cyclic peptide was
over 80%. There was less than 8% of the linear hydrolysis
byproduct, 7% of the linear precursor, and 5% of the cyclic
dimer.
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Table 1. Ki Values for Individual Compounds (Crude) from the Cyclic Peptide Library at Mu Receptor
a
b
c
a
b
c
#
R2
R4
R5
Ki /nM Ki/26 nM
STD /26
#
R2
R4
R5
Ki /nM Ki/26 /nM
STD /26
1
D-Phe
D-Phe
D-Phe
D-Phe
D-Phe
D-Phe
D-Phe
D-Phe
Trp
D-Lys
D-Lys
Arg
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
1552
84860
8803
21910
30730
n.a.
60
7
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
D-Met
D-Met
D-Met
D-Met
Tyr
Trp
Trp
Tyr
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
Tyr
Trp
60535
34145
40500
168150
463
2328
1313
1558
6467
18
289
94
2
3264
339
843
1182
n.a.
737
43
3
14
4
Arg
217
195
n.a.
477
n.a.
2
Tyr
2198
1
5
Trp
D-Lys
D-Lys
Arg
6
Trp
Tyr
5203
24745
n.a.
200
952
n.a.
22
7
Tyr
25360
n.a.
975
n.a.
Tyr
108
n.a.
n.a.
n.a.
.a.
8
Tyr
Tyr
Arg
9
D-Lys
D-Lys
Arg
673
26
Tyr
Trp
Trp
Tyr
n.a.
n.a.
10
11
12
13
14
15
16
17
18
19
20
Trp
5525
30995
186500
54055
n.a.
212
807
7173
2079
n.a.
14
Tyr
n.a.
n.a.
Trp
36
Tyr
n.a.
n.a.
Trp
Arg
1887
130
n.a.
578
253
0.3
260
108
231
Tyr
Tyr
n.a.
n.a.
n.a.
0.3
22
Trp
Trp
Phe
D-Lys
D-Lys
Arg
232
9
Trp
Trp
Phe
7582
89155
27425
n.a.
292
3429
1055
n.a.
Trp
Tyr
75970
46675
4059
49725
20755
2922
1795
156
1912
798
1367
Phe
1590
195
n.a.
n.a.
22
Trp
Tyr
Phe
Arg
D-Met
D-Met
D-Met
D-Met
D-Lys
D-Lys
Arg
Phe
Trp
Trp
Tyr
Phe
n.a.
n.a.
Phe
6700
25120
258
966
Arg
35540
Phe
Tyr
224
a
b
Measured Ki value of crude sample. Adjusted Ki value of crude sample by using apparent concentration of peptide in the mixture. The apparent
c
concentration of peptide was estimated using the mass ratio of peptide and imidazole in the mixture. Adjusted standard error.
Synthesis and Deconvolution of a Positional Scan-
ning Synthetic Combinatorial Library of Anthraniloyl-
Labeled Cyclic Peptide. The synthetic strategy described
above was applied to the construction of an anthraniloyl-labeled
cyclic pentapeptide library in the positional scanning format.10
Position 1 was fixed with glycine to ensure the efficient
cyclization as determined in our previously study.5 2,3-
Diaminopropionic acid was fixed at position 3. Its 3-amino
was coupled with the fluorescent anthraniloyl-group. The
library was composed of three sublibraries, in which each of the
three positions 2, 4, or 5 were defined with either a single
amino acid (O) or a mixture of amino acids (X). For each of
the three subset mixtures the two remaining positions were
made up of a mixture of amino acids (X). Position 2 and 4
contained 36 L- and D- amino acids; while position 5 contained
19 natural L- amino acids. Thus this positional scanning library
was composed of 91 mixtures. Each mixture contained 684
head-to-tail penta-cyclic peptides at position 2 and 4 and 1296
penta-cyclic peptides at position 5. The library contains a total
of 24,624 individual head-to-tail penta-cyclic peptides.
The anthraniloyl-labeled cyclic pentapeptide library was
screened in a competitive radio receptor binding assay for the
mu opioid receptor. Owing to the large excess of imidazole
contained in each mixture, the effect of imidazole on the mu
opioid receptor binding assay was tested before screening of the
library. The result demonstrated that 1 mg/mL (14.7 mM)
imidazole did not have a significant effect in the binding assay.
Thus imidazole at this concentration exhibited negligible
inhibition.
When a cutoff value of 35% was used, the most active amino
acids for the mixture making up position 2 were D-Phe, L-Trp,
D-Met, L-Tyr, and L-Phe; at position 4 they were D-Lys, L-Arg, L-
Trp, and L-Tyr; while L-Tyr and L-Trp were the two most active
amino acids at position 5. Since the percent inhibitions
observed were on the linear part of the competition curve (i.e.,
between 20 and 80% inhibition) IC50 values were not required
in this instance to differentiate activities.
Individual Peptides from the Cyclic Library. A total of
40 individual cyclic peptides were synthesized in parallel by the
combination of the most active amino acids identified at the
three positions 5 × 4 × 2. The individual cyclic peptides were
tested before purification in the binding assay for the μ-opioid
receptor. The results of the 40 crude compounds are shown in
Table 1. The molar ratio of imidazole and peptide in solution is
187:1 by comparing the concentration of imidazole (final 0.187
M) and peptide mixture (1 mM in total). A mass ratio of 25:1
is obtained by using an average molecular weight of 500 Da of
the cyclic peptide. An adjusted Ki value by dividing by 26 is
then calculated and listed to reflect the apparent concentration
of cyclic peptide in the test samples.
Four individual cyclic peptides, #1, #9, #25, and # 33,
exhibited significant mu receptor activity were selected for
further purification. The four cyclic peptides have a similar
sequence with D-Lys at position 4 and L-Tyr at position 5,
differing only by the residue at position 2. Following removal of
imidazole and other possible byproducts by HPLC, each
purified cyclic peptide was tested in the binding assay for μ-
opioid receptor. The results are shown in Table 2. The most
active compound having L-Tyr at R2, D-Lys at R4, and Tyr at R5
shows a Ki of 16 nM at the μ-opioid receptor.
Determination of the Structure of the Most Active
Compound. The HPLC profile of the most active compounds
actually showed two peaks having an identical mass weight that
matched the desired cyclic product. This is caused by the head-
to-tail and side chain-to-tail cyclization when R4 is D-Lys
(Scheme 3). Our previous study indicated that the imidazole-
Each mixture in the library was then screened at a
concentration of 1 mg/mL. The screening results for the mu
selective binding assay are shown in Chart 1. Several mixtures
at each position exhibited percent inhibitions above that of the
all X mixture in which all cyclic peptides are present as a single
mixture (all X, White bar labeled X). One mg/mL Imidazole
was demonstrated not to have a significant effect in the binding
assay, exhibiting negligible inhibition (White bar labeled Imid).
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Table 2. Binding Affinities of Active Compounds at Mu
Receptor
Table 3. Binding Affinities of cyclo[YkDap(Ant)YG] and Y-
cyclo[kDap(Ant)YG] at Three Opioid Receptors
peptide
R1
R2
R3
R4
R5
Ki/ nM STD
Ki (nM)
#1
#9
#25
#33
Gly
Gly
Gly
Gly
D-Phe
Trp
Dap(Ant)
Dap(Ant)
Dap(Ant)
Dap(Ant)
D-Lys
D-Lys
D-Lys
D-Lys
Tyr
Tyr
Tyr
Tyr
144
505
16
37
25
1
cyclization pattern
MOR
KOR
DOR
side chain-to-tail
head-to-tail
14 0.54
39 1.06
3231 863
3197 129
865 424
Tyr
1221 278
Phe
20
1
promoted cyclization is not regioselective when Lys is an
internal residue. Besides the cyclic isomers, the main product
(impurity) was the linear peptide acid from hydrolytic side
reaction. To determine the structure of the two cyclic isomers,
they were isolated and reacted with 2,4-dinitro-1-fluoroben-
zene, separately. The two derivatives were hydrolyzed with 6 M
HCl at 110 °C overnight. Nε-(dinitrophenyl)-D-lysine was used
as control to determine the cyclization pattern of the parent
cyclic products. It was found that the side chain-to-tail cyclic
peptide was eluted earlier than the head-to-tail cyclic peptide.
These two isolated and structurally determined cyclic peptides
were tested in binding assays for all three opioid receptors, mu,
delta, and kappa. The results show that both cyclic isomers are
selective for the μ-opioid receptor (Table 3); however, the side
chain-to-tail cyclic isomer has a greater binding affinity (Ki = 14
nM).
Cross-Oligomerization Control for Library Construc-
tion. Effective deconvolution of positional scanning synthetic
combinatorial libraries relies on the precise construction of the
library and the quality of the library components. For a cyclic
peptide library, one of the major concerns is the possible side
reactions of dimerization or oligomerization during cyclization.
To check the possible extent of dimerization/oligomerization, a
test sample containing two linear penta-peptide thioesters were
mixed at a final concentration of 1 mM each (2 mM in total) to
cyclize. Asp-Leu-Thr-Phe-Gly-SCH2Ph and Asp-Leu-Val-Phe-
Gly-SCH2Ph were found to produce on intramolecular
cyclization individually; no significant dimerization, oligomeri-
zation, and cross-oligomerization products could be identified
by HPLC. Since the total concentration is 2-fold higher in the
test sample than in each mixture in the library, we therefore
assume that the oligomerization should not happen to each
mixture in the library. Each linear peptide thioester in the
mixture based library reacts individually to form cyclic
monomer.
and it is fully responsible for the activity seen, then that side
reaction becomes an interesting/desired product. While the
goal is, of course, to cyclize 100% monomer, this is in realty
highly unlikely. Since we are tracking the activity not the purity
to make the individual compounds, as long as the side reaction
is reproducible, mixture libraries should be deconvoluted
successfully to identify the active compounds. It can be seen
from this cyclic peptide library that the most active compound
identified is the side-to-tail cyclic peptide, not the originally
designed head-to-tail version.
Effect of Imidazole on Binding Assay and Removal of
Imidazole. The cyclic peptide library contains a high ratio of
imidazole to cyclizable starting material. The mole ratio of
imidazole and peptide is 187:1 by comparing the concentration
of imidazole and peptide mixture in the solution. Though
imidazole is used as a biological buffer, imidazole at such high
concentration may damage membranes and interfere with the
binding assay. To explore the effect of high concentration of
imidazole that may impose on the assay, imidazole at a
concentration of 1 mg/mL has been tested in the binding assay.
No significant effect was found on the binding of 3H-DAMGO
to the mu opioid receptor. Thus, the high ratio of imidazole in
the mixtures will affect only the apparent concentration of each
mixture, but not the affinity of the library components to the
receptor.
Though imidazole did not appear to interfere with the
screening results in this assay, the removal of imidazole has
been investigated by sublimation under high vacuum
(approximately 100 mTorr). Imidazole itself was removed
quantitatively after sublimation for 3 days at 40 °C. A test
sample containing a mixture of imidazole and a peptide,
CH3CO-His-Phe-Arg-Trp-Gly-NH2, at a molar ratio of 80:1
was treated under the described sublimation conditions. Only
90% of imidazole in the mixture was readily removed (assuming
that the loss of mass was resulted only by sublimation of
imidazole). The remaining 10% of the imidazole could not be
sublimed, leading to a mixture of imidazole and peptide at a
molar ratio of 8:1. To check the structural consistency of the
Even if dimers (or other side reaction) form to varying
degrees, we test for activity in the mixtures and that information
is used to make individual compounds. If a side reaction occurs,
Scheme 3. Two Possible Isomers of the Most Active Cyclic Peptide
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1
peptide during sublimation, LC-MS and H NMR spectra of
the samples before and after sublimation were recorded. LC-
MS data showed the peptide in both samples had the same
retention time and mass weight, indicating that the peptide kept
unchanged during sublimation. Though the chemical shifts of
the amide protons of the peptide in the sublimated sample
slightly moved upfield in DMSO-d6 compared with pure
peptide, the sublimated sample and a fresh-mixed sample
containing a mixture of imidazole and the peptide at a molar
ratio of 8:1 had nearly identical 1H NMR spectra. Both LC-MS
and 1H NMR experiments supported that the peptide remained
unchanged under the sublimation conditions.
group. After removal of the solvent in vacuo, 34 μL of DIEA in
1 mL of DCM was added. To the solution was added 2,4-
dinitro-1-fluorobenzene (13 μL, 0.1 mmol). The mixture was
reacted at room temperature for 10 min. After removal of the
solvent in vacuo, the residue was treated with 2 mL of 20%
piperidine in DMF for 10 min. The product was extracted with
25% acetonitrile in water with 10% TFA and purified by
preparative HPLC and confirmed by ESI-MS (found 313.1,
calculated for [M + H]+ 313.1).
Determination of the Structure of the Most Active
Compound. The two cyclization isomers of compound #25
were isolated by semipreparative RP-HPLC using H2O (0.1%
formic acid) and CH3CN (0.1% formic acid) with a gradient
ranging from 2 to 60% in 30 min. Two milligrams of each
purified cyclic isomers were dissolved in 1 mL of DMF and
then treated with 17 μL of DIEA and 13 μL of 2, 4-dinitro-1-
fluorobenzene at room temperature for 10 min. After removal
of the solvent in vacuo, the samples were hydrolyzed with 6 M
HCl at 110 °C overnight. The hydrolysates were analyzed by
LC-MS. Nε-(dinitrophenyl)-D-lysine was used as control to
determine its presence of the digestion product.
Opioid Binding Assay. Membrane suspensions were
prepared and used on the same day. Rat brains, minus the
cerebellum, were homogenized using 50 mM Tris-HCl, pH 7.4,
centrifuged and rewashed. Each assay tube contained 0.5 mL of
membrane suspension, 1.9 nM competitor [3H]-DAMGO, 1
mg/mL mixture, and 50 mM Tris-HCl in a final volume of 0.65
mL. The assay was incubated for 1.5 h at room temperature,
and the reactions were terminated by filtration through GF-B
filters. Bound radioactivity was counted on a betaplate
scintillation counter.
CONCLUSION
■
A method has been developed to construct the positional
scanning cyclic peptide combinatorial library by using our
“volatilizable” resin and imidazole-promoted cyclization strat-
egy. A positional scan formatted penta-cyclic peptide library has
been synthesized and screened. Deconvolution of the library
identified potent individual fluorescent ligands for the mu
opioid receptor. The most active compound has been found to
have Tyr at R2, D-Lys at R4, and Tyr at R5. Further study on this
compound revealed that the side chain-to-tail cyclic isomer had
greater binding affinity than the head-to-tail isomer. Because of
the minimal side reactions leading to oligomerization this
method is promising for the construction of larger cyclic
peptide libraries having greater structural diversities. The power
to synthesize and screen libraries of cyclic peptides will have a
dramatic impact on the identification of biologically active
cyclic peptides for drug discovery.
EXPERIMENTAL PROCEDURES
ASSOCIATED CONTENT
■
■
S
* Supporting Information
Cyclic Peptide Synthesis. All linear peptide thioesters
were synthesized by solid-phase synthetic method using the
“tea-bag” approach. Functionalized mercaptomethylphenyl-
silica gel was synthesized and used as “volatilizable” support
as reported elsewhere.9 A 100 mg portion of functionalized
silica gel was sealed in each polypropylene tea bag. Boc amino
acids activated with PyBOP/DIEA in dimethylformamide
(DMF) were used in the peptide couplings. X-positions were
coupled as mixtures of Nα-Boc protected amino acids using
concertration ratios to compensate for the relative reaction
rates in competitive couplings. Position 3 was coupled with a
Nα-Boc-Nβ-Alloc-Diaminoproponic acid. After peptide elonga-
tion on resin, the Boc-protected peptides were treated with
Pd(PPh3)4 (0.1 equiv) in the presence of PhSiH3 (20 equiv) in
dichloromethane to remove the Alloc protection group. The
resin bound peptides were coupled with 2-nitrobenzoic acid,
following by the treatment of 2 M SnCl2 in DMF overnight to
reduce the nitro group to an amino group to generate the
anthraniloyl-label. After removal of the Boc group with 55%
TFA, the resin bound anthraniloyl-labeled peptides were
treated with anhydrous HF in the presence of 5% anisole at
0 °C for 2 h. After removal of the HF with nitrogen stream and
lypholization, the linear peptide thioesters were cyclized in a
mixed solution of 1.5 M imidazole (aq.) and acetonitrile (1:7 v/
v) at a concentration of 1 mM for 72 h, forming the
anthraniloyl-labeled fluorescent cyclic peptides.
1
Experimental detail, compound characterization, copies of H
NMR, 13C NMR spectra and LC-MS for the most active
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (772)-345-4800. Fax: (772)-345-3649. E-mail:
houghten@tpims.org.
■
Funding
This work was supported by the State of Florida, Executive
Officer of the Governor’s Office of Tourism, Trade and
Economic Development, National Science Foundation (R.A.H.
CHE 0455072), the National Institute on Drug Abuse
(DA031370), and NIH (R03DA025850).
Notes
The authors declare no competing financial interest.
REFERENCES
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(2) White, C. J.; Yudin, A. K. Contemporary Strategies for Peptide
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