Design, synthesis, and validation of a β-turn mimetic library targeting protein - Protein and peptide - Receptor interactions

Article abstract of DOI:10.1021/ja201878v

The design and synthesis of a β-turn mimetic library as a key component of a small-molecule library targeting the major recognition motifs involved in protein - protein interactions is described. Analysis of a geometric characterization of 10?245 β-turns in the protein data bank (PDB) suggested that trans-pyrrolidine-3,4-dicarboxamide could serve as an effective and synthetically accessible library template. This was confirmed by initially screening select compounds against a series of peptide-activated GPCRs that recognize a β-turn structure in their endogenous ligands. This validation study was highlighted by identification of both nonbasic and basic small molecules with high affinities K_i = 390 and 23 nM, respectively) for the ?-opioid receptor (KOR). Consistent with the screening capabilities of collaborators and following the design validation, the complete library was assembled as 210 mixtures of 20 compounds, providing a total of 4200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally occurring β-turn. Unique to the design and because of the C ₂ symmetry of the template, a typical 20 × 20 × 20-mix (8000 compounds prepared as 400 mixtures of 20 compounds) needed to represent 20 variations in the side chains of three amino acid residues reduces to a 210 × 20-mix, thereby simplifying the library synthesis and subsequent screening. The library was prepared using a solution-phase synthetic protocol with liquid - liquid or liquid - solid extractions for purification and conducted on a scale that insures its long-term availability for screening campaigns. Screening the library against the human opioid receptors (KOR, MOR, and DOR) identified not only the activity of library members expected to mimic the opioid receptor peptide ligands but also additional side-chain combinations that provided enhanced receptor binding selectivities (>100-fold) and affinities (as low as K_i = 80 nM for KOR). A key insight to emerge from the studies is that the phenol of Tyr in endogenous ligands bearing the H-Tyr-Pro-Trp/Phe-Phe-NH₂ β-turn is important for MOR binding but may not be important for KOR (accommodated, but not preferred) and that the resulting selectivity for KOR observed with its removal can be increased by replacing the phenol OH with a chlorine substituent, further enhancing KOR affinity.

Full text of DOI:10.1021/ja201878v

ARTICLE
pubs.acs.org/JACS
Design, Synthesis, and Validation of a β-Turn Mimetic Library
Targeting ProteinꢀProtein and PeptideꢀReceptor Interactions
Landon R. Whitby,† Yoshio Ando,† Vincent Setola,‡ Peter K. Vogt,† Bryan L. Roth,‡ and Dale L. Boger*,†
†Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, California 92037, United States
‡NIMH Psychoactive Drug Screening Program, University of North Carolina Chapel Hill, Genetic Medicine Building, Chapel Hill,
North Carolina 27514, United States
S Supporting Information
b
ABSTRACT: The design and synthesis of a β-turn mimetic
library as a key component of a small-molecule library targeting
the major recognition motifs involved in proteinꢀprotein
interactions is described. Analysis of a geometric characteriza-
tion of 10 245 β-turns in the protein data bank (PDB) suggested
that trans-pyrrolidine-3,4-dicarboxamide could serve as an
effective and synthetically accessible library template. This
was confirmed by initially screening select compounds against
a series of peptide-activated GPCRs that recognize a β-turn
structure in their endogenous ligands. This validation study was highlighted by identification of both nonbasic and basic small
molecules with high affinities (Ki = 390 and 23 nM, respectively) for the k-opioid receptor (KOR). Consistent with the screening
capabilities of collaborators and following the design validation, the complete library was assembled as 210 mixtures of 20
compounds, providing a total of 4200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally
occurring β-turn. Unique to the design and because of the C2 symmetry of the template, a typical 20 ꢁ 20 ꢁ 20-mix (8000
compounds prepared as 400 mixtures of 20 compounds) needed to represent 20 variations in the side chains of three amino acid
residues reduces to a 210 ꢁ 20-mix, thereby simplifying the library synthesis and subsequent screening. The library was prepared
using a solution-phase synthetic protocol with liquidꢀliquid or liquidꢀsolid extractions for purification and conducted on a scale
that insures its long-term availability for screening campaigns. Screening the library against the human opioid receptors (KOR,
MOR, and DOR) identified not only the activity of library members expected to mimic the opioid receptor peptide ligands but also
additional side-chain combinations that provided enhanced receptor binding selectivities (>100-fold) and affinities (as low as
Ki = 80 nM for KOR). A key insight to emerge from the studies is that the phenol of Tyr in endogenous ligands bearing the H-Tyr-
Pro-Trp/Phe-Phe-NH2 β-turn is important for MOR binding but may not be important for KOR (accommodated, but not
preferred) and that the resulting selectivity for KOR observed with its removal can be increased by replacing the phenol OH with a
chlorine substituent, further enhancing KOR affinity.
’ INTRODUCTION
majority of the recognition or binding affinity.3 Significantly and
although endogenous small-molecule ligands may not exist,
many if not most proteinꢀprotein interactions are mediated by
three main recognition motifs (R-helix, β-turn, and β-strand).4
Consequently, an attractive approach for the discovery of modu-
lators of proteinꢀprotein interactions is to mimic the key
interaction residues using small-molecule mimetics of these three
major recognition motifs.
A related class of interactions in which small-molecule mi-
metics of peptide secondary structure have already proven
valuable is the peptideꢀreceptor interactions, the best character-
ized example of which is the interaction of the peptide-activated
G protein-coupled receptors (GPCRs) with their endogenous
The interaction of proteins to form higher-order complexes is
critical to nearly all biological processes, including cellular
signaling.1 Despite their central role in cellular signaling, such
proteinꢀprotein interactions have emerged relatively slowly as
viable small-molecule therapeutic targets.2 This can be attributed
to the fact that proteinꢀprotein interfaces do not typically bind
endogenous small-molecule ligands that could provide lead
structures for drug discovery programs and that the interfaces
often also present physical challenges to small-molecule binding.
These challenges are generally thought to come in the form of the
noncontiguous binding regions within the interacting proteins as
well as an often relatively large and/or flat binding interface.
However, systematic case studies of selected proteinꢀprotein
interactions have revealed that there is typically a small cluster of
key residues near the center of the interface that contributes the
Received: March 7, 2011
Published: May 24, 2011
r
2011 American Chemical Society
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peptide ligands. Significantly, GPCRs comprise the targets of
over one-third of the currently registered pharmaceuticals,
although only a subset of GPCRs are peptide-activated. Although
the peptide ligands of this subset of GPCRs are usually flexible
and able to adopt variable secondary structures, the active
conformation recognized by the receptor typically involves a
turn structure (β- or γ-turn).5 Due to this pervasive recognition
pattern, small-molecule mimetics of turn structures (typically
β-turn)5 and peptides with turn constraints have been successful
in targeting this class of receptors.5,6 As the roles of these
receptors have emerged, small-molecule scaffolds capable of
mimicking peptide turn structures continue to be valuable in
defining the residues and secondary structure responsible for the
binding recognition and affinity, useful in validating new drug
targets, and central to the subsequent development of new
therapeutics.6b
Figure 1. Schematic diagram of a β-turn.
Over the past decade, we have enlisted a simple solution-phase
library synthesis protocol, complementary to more conventional
solid-phase techniques, for the generation of libraries capable of
targeting proteinꢀprotein or proteinꢀDNA interactions.7 The
protocol employs acid/base liquidꢀliquid or liquidꢀsolid ex-
tractions for the purification of products (>95% pure irrespective
of the reaction efficiency) and offers the advantages of a less
limiting scale, expanded repertoire of chemical reactions (use of
heterogeneous catalysts and reagents), direct production of
soluble intermediates and final products for assay, and the lack
of required linking, attachment and detachment, or capping
steps. The approach is amenable to convergent synthetic strate-
gies, the synthesis of mixture libraries, or the use of dynamic
libraries. Notably, a number of effective small-molecule modula-
tors of proteinꢀprotein7ꢀ12 or proteinꢀDNA interactions13
have been identified by screening the libraries prepared to date.
Recently, we initiated a program to expand our current library
of 95 000 compounds2f,7 with the preparation of a comprehen-
sive small-molecule library designed to mimic the three major
recognition motifs that mediate proteinꢀprotein interactions,
namely, the R-helix, the β-turn, and the β-strand. Three libraries
built around templates designed to mimic each secondary
structure and substituted with all triplet combinations of groups
representing the 20 natural amino acid side chains would contain
a member capable of mimicking the key interaction residues of
many, if not most, proteinꢀprotein interactions.4 Such a library
would provide a powerful tool to interrogate proteinꢀprotein
interaction networks in order to validate new therapeutic targets,
to provide therapeutic lead compounds, and to afford modulators
of biological processes for study. The screening of the compre-
hensive library would not only provide lead structures for many
proteinꢀprotein interaction targets even if the nature of the
interaction is unknown, but it can also be expected to yield key
insights into the recognition motif and key residues mediating
the interaction.14 Screening of the library would also provide an
initial comprehensive structureꢀactivity relationship (SAR)
study for subsequent iterative lead optimization.14 Notably, the
β-turn component of this library would be particularly valuable
for the protein- and peptide-activated GPCRs that recognize turn
structures in their endogenous ligands.4,5 To date, we have
disclosed the design, synthesis, and validation of an 8000-
membered R-helix mimetic component of this library,15 and we
disclose herein the second component of this comprehensive
library, a β-turn mimetic library targeting proteinꢀprotein and
peptideꢀreceptor interactions.
Figure 2. Mean and standard deviations of the CR distances taken from
a set of 10 245 β-turns in the PDB.
’ RESULTS AND DISCUSSION
β-Turn Mimetic Design. The β-turn is one of the three main
secondary structural motifs found in proteins and peptides and
occurs where the polypeptide strand reverses direction. As
illustrated in Figure 1, the β-turn consists of four amino acid
residues designated i to i þ 4 in which the distance between CRi
and CRiþ3 is e7 Å. Several different types of β-turns exist,
depending on the dihedral angles ψ and j of the i þ 1 and i þ 2
residues.
Small-molecule mimetics of β-turns have been extensively
investigated and utilized to discover compounds that can mimic
or disrupt β-turn-mediated recognition events.16,17 An ideal
β-turn mimetic scaffold around which to build a screening library
would be constrained to approximate the correct geometric
display of the amino acid side-chain functionality found within
a β-turn, be sufficiently flexible to allow the side chains to
approximate the side-chain vectors of the many turn types, and
be amenable to robust library synthesis.
We envisioned a β-turn mimetic scaffold that could be
substituted with all triplet combinations of groups representing
the 20 natural amino acid side chains and that was amenable to
substitution using our solution-phase library synthesis protocol.
This would enable production of a library of pure compounds
(>95%) on a scale that would permit virtually unlimited screen-
ing opportunities and enable efficient optimization of lead
compounds through the rapid synthesis of subsequent lead
optimization libraries. In order to identify a template that would
allow the appropriate geometric display of the amino acid side-
chain functionality, we utilized a geometric analysis of the mean
distances found between R-carbon centers in a set of 10 245
β-turns in the protein data bank (PDB, Figure 2).18,19
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Figure 3. (A) The trans-pyrrolidine-3,4-dicarboxamide template.
(B) Low-energy conformation and measured distances between sub-
stituted centers of a trans-pyrrolidine-3,4-dicarboxamide with ethyl
substituents. (C) A trans-pyrrolidine-3,4-dicarboxamide substituted
with groups representing the side chains of Leu, Phe, and His.
Figure 4. (A) Overlay of a potential mode of β-turn mimicry by a
trisubstituted trans-pyrrolidine-3,4-dicarboxamide. (B) Overlay and
calculated rmsd value for attachment of the three substituted centers
of the template with three R-carbons in a type-I β-turn.
Recognizing that one of the turn amino acids (i þ 1 or i þ 2)
often serves a structural rather than a recognition role (e.g., Pro
or Gly),4,18 we sought to mimic CR triplets in which either CRiþ1
Figure 5. Screening data of compounds against μ-opioid receptor
(MOR) and k-opioid receptor (KOR).
or CRiþ2 is omitted (i.e., CRi, CRiþ2, CRiþ3 or CRi, CRiþ1
,
CRiþ3). From this analysis, we found that trans-pyrrolidine-3,
4-dicarboxamide could serve as a synthetically accessible tem-
plate upon which to display the amino acid side-chain groups
(Figure 3A). Rigidified by an intramolecular H-bond, the lowest-
energy conformation of the trans-pyrrolidine-3,4-dicarboxamide
has substituted centers that conform closely to the triangle
geometries of the CR triplets found in Figure 2 in which either
CRiþ1 or CRiþ2 is omitted (Figure 3B). An overlay of the low-
energy conformation with the peptide backbone of a type-I
β-turn (Figure 4A and B) demonstrates a potential mode of
mimicry and yielded a rmsd value of 0.26 Å for ethyl substituted
centers versus the CR triplet CRi, CRiþ2, CRiþ3. In line with the
design, the noncovalent constraints that stabilize the lowest-
energy conformer also permit a degree of flexibility to allow the
compound to adopt variable H-bond donor/acceptor patterns
(e.g., which the carbonyl is exo or endo in the pseudo-7-member
ring) and permit the attached side chains to approximate the
correct vector display of amino acid side chains in a wide range of
β-turn structures.18 Significantly, the trans-pyrrolidine-3,4-dicar-
boxamide can be substituted using amide coupling reactions and
possesses a simplifying C2 symmetry axis that allows all triplet
combinations of 20 different side chains to be accomplished
with only 4200 compounds (versus 8000). Thus, in addition to
potentially serving as a β-turn mimetic accurately matching the
triangle geometries of the CR triplets of a β-turn while flexibly
accommodating the vector displays of side chains in a wide range
of β-turn structures,18 it also possesses a unique simplifying C2
symmetry and is functionalized in a manner amenable to library
synthesis.
Evaluation and Validation of the Design against Peptide-
Activated GPCRs: The Opioid Receptors. In order to establish
the ability of substituted trans-pyrrolidine-3,4-dicarboxamides to
modulate β-turn-mediated recognition events and before em-
barking on a comprehensive library synthesis, the evaluation of a
select series of compounds against a representative set of peptide-
activated GPCRs that are thought to recognize β-turns in their
endogenous ligands was conducted. The μ-opioid receptor
(MOR) is a well-known clinical target for the treatment of pain.
MOR selective opiate analgesics such as morphine remain the
drugs of choice for the treatment of severe pain, but their use
is limited by the well-characterized side effects of respiratory
depression, desensitization with chronic use (tolerance), and
development of dependence. The additional two members of the
opioid receptor family, the k-opioid and δ-opioid receptors
(KOR and DOR), have also been investigated as analgesic
targets. DOR agonists produce analgesia but may also exhibit
side effects including convulsions. Activation of the KOR in the
central nervous system produces analgesia, though it is generally
accompanied by dysphoria, hallucinations, and sedation. Target-
ing of peripheral KOR, however, has emerged as a potentially
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Figure 6. (A,B) Structures of high-affinity KOR-selective compounds 8
and 9. The tyramine pharmacophore found in the “message” structure of
opioid compounds and peptides is highlighted in (B).
promising treatment for inflammatory and visceral pain as well as
arthritis.20
Two highly potent and selective endogenous peptide ligands
of the MOR are endomorphin-1 (H-Tyr-Pro-Trp-Phe-NH2) and
endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2).21 Additional endo-
genous agonists include Met-enkephalin (H-Tyr-Gly-Gly-Phe-
Met-OH) and Leu-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH),
which show high affinity for MOR and DOR, their longer peptide
precursors β-endorphin and dynorphin, as well as their C-termi-
nus-truncated neoendorphin peptides that display high affinity
for the KOR.20 Although some uncertainty remains, there is
considerable evidence that the active conformation of the
endomorphins is a β-turn, including activities observed by
analogues that incorporate turn constraints and appropriately
substituted β-turn mimetics.22 Therefore, we prepared a small
series of pyrrolidine-3,4-dicarboxamide-derived compounds de-
signed to mimic the three side-chain residues in the pharmaco-
phores of the endomorphins and enkephalins along with several
Ala negative controls (17ꢀ22) and measured their binding
(Ki values) to MOR, KOR and DOR (Figure 5). The highest
affinity for the MOR was exhibited by compound 10 with the
R1/R2 combination of Trp/HoPhe and R3 of Tyr (Ki = 820 nM)
followed by compound 8 with R1/R2 of Trp/Phe and R3 of
HoTyr (Ki = 930 nM). In addition to exhibiting submicromolar
activity using a simple template, the activity reflected a combina-
tion of side-chain residues found in the endogenous endorphin
ligands. Moreover, comparisons among the compounds exam-
ined demonstrated that the best affinity to MOR was observed
when single carbon extensions of key side chains (HoPhe or
HoTyr) were incorporated into the structure, and this influenced
our selection of side chains incorporated into the full library
design. Interestingly, the compounds showed much higher
affinities for the KOR than for the MOR, highlighted by 8 and
9 with Ki values measured at 390 and 23 nM, respectively
(Figure 6). The identification of 9 as the most active compound
is easy to appreciate considering that the R3 Tyr side chain is
attached to the pyrrolidine nitrogen via an alkyl rather than acyl
linkage, giving rise to the familiar tyramine group bearing a free
amine found in the “message” structure of nearly all opioid
analgesics and peptides (Figure 6B).20a However, even its N-acyl
variant lacking the basic nitrogen (8, Ki = 390 nM) proved
remarkably effective. Selectivity for KOR binding was observed
Figure 7. The 20 groups used in the synthesis of the β-turn mimetic
library.
with 9 (∼100 fold), and this trend was observed with nearly all
compounds tested. The origin of this selectivity is currently
undefined, but its consistency across the range of compounds
tested suggests that there are intrinsic properties to the trans-
pyrrolidine-3,4-dicarboxamide template, including its three-di-
mensional projection of side chains or interactions made by the
template itself, that favor KOR binding. Nevertheless, the high
nanomolar affinities of compounds 8 and 10 indicate that the
trans-pyrrolidine-3,4-dicarboxamide template can effectively mi-
mic the active turn structure of the endomorphins.
Synthesis of the β-Turn Mimetic Library. Convinced that
the pyrrolidine-3,4-dicarboxamide template would prove to be a
valuable β-turn mimetic and having established subtle design
features from the exploratory studies, the synthesis of the com-
prehensive β-turn mimetic library was initiated. The 20 amino
acid side-chain groups utilized to construct the library along with
corresponding protecting groups are provided in Figure 7. By
substituting each position of the trans-pyrrolidine-3,4-dicarbox-
amide template with all combinations of these 20 groups, a total
of 4200 compounds are produced representing all possible
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Scheme 1. Retrosynthetic Plan for Construction of the
Scheme 2
β-Turn Mimetic Library
permutations of 3 of the 4 residues in a naturally occurring β-turn.
Due to the perceived degeneracy of incorporating the side chains of
both aspartic and glutamic acid as well as asparigine and glutamine,
only the side chains of glutamic acid (Glu) and glutamine (Gln)
were used in the library. Similarly, an arginine side chain was
omitted, but the side chain of lysine (Lys) was incorporated. No
attempt to incorporate a cysteine side chain was made due to
anticipated stability and storage problems. Finally, the side chains of
glycine and proline were omitted due to their expected minor role in
recognition events. These six natural amino acid side-chain omis-
sions were replaced with additional unnatural aromatic side-chain
groups that often dominate proteinꢀprotein and proteinꢀpeptide
interactions or with groups that represent a one carbon extension of
such residues. Thus, O-methyl tyrosine (TyrMe), naphthyl (Nap),
and 4-chlorophenylalanine [Phe(4Cl)] were included in the library
as wellasthesidechainsoftheonecarbon extension residues homo-
phenylalanine (HoPhe), homotyrosine (HoTyr), and 4-chloroho-
mophenylalanine [HoPhe(4Cl)]. Having selected the 20 groups to
incorporate into the library, they were used in their terminal amine
form (for incorporation into the R1/R2 positions) or terminal
carboxylic acid form (for incorporation into the R3 position) for
the library synthesis.
The retrosynthetic analysis for the construction of the library is
shown in Scheme 1. We anticipated that the final 4200 com-
pound library would be obtained by acylation of 210 individual
trans-pyrrolidine-3,4-dicarboxamides at the pyrrolidine nitro-
gen using an equimolar mixture of the carboxylic acids of the
20 selected side chains (210 ꢁ 20-mix). The order of the side-
chain introductions (amide couplings; R1, R2, and R3) and the
location of the 20-mix functionalization (R3) were dictated by
the C2 symmetry of the template and the simplifying opportu-
nity that it presented for the number of compounds required
to represent all 20 ꢁ 20 ꢁ 20-mix combinations (210 individual
R1/R2 combinations ꢁ 20-mix for R3). The 210 individual
amines were anticipated to be derived from their 2,2,2-tri-
chloroethoxycarbonyl (Troc) protected precursors, which
were individually synthesized starting from the mono methyl ester
Figure 8. Synthesized 20 monoamides with coupling yields and pro-
duct amounts.
of trans-N-Troc-pyrrolidine-3,4-dicarboxylic acid utilizing a straight-
forward amide coupling, methyl ester hydrolysis, and amide cou-
pling reaction sequence. In turn, the mono methyl ester was to be
prepared from the N-benzylpyrrolidine mixed benzyl methyl ester
using hydrogenolysis of the benzyl group followed by Troc protec-
tion of the free amine. Finally, the trans-N-benzylpyrrolidine-3,
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4-dicarboxylic acid benzyl methyl ester could be accessed using a [3
þ 2] dipolar cycloaddition between the mixed fumarate ester
and the azomethine ylide precursor N-benzyl-N-methoxy-
methyl-N-(trimethylsilyl)methylamine.23
The synthesis of the mono methyl ester of trans-N-Troc-
pyrrolidine-3,4-dicarboxylic acid (26) is shown in Scheme 2.
The azomethine ylide precursor N-benzyl-N-methoxymethyl-
N-(trimethylsilyl)methylamine (23) was prepared as described23
by condensing benzylamine with chloromethyltrimethylsilane to
yield N-benzyl-N-(trimethylsilyl)methylamine, followed by treat-
ment with formaldehyde/H2O/MeOH in the presence of K2CO3.
This material was employed in a [3 þ 2] dipolar cycloaddition
with the mixed fumarate ester in the presence of LiF and under
ultrasound conditions to give 24 in multigram quantities.23 This
material was further elaborated to the amino acid 25 by hydro-
genolysis removal of the benzyl groups [H2, Pd(OH)2] followed
by Troc protection of the pyrrolidine nitrogen using 2,2,2-
trichloroethyl chloroformate to provide 26.
With the starting template in hand, the library diversification
began with the first amide coupling using 20 primary amines
under EDCI/HOAt-mediated coupling conditions to provide
the monoamides (eq 1 and Figure 8), which were fully char-
1
acterized by H and 13C NMR, IR, and HRMS (Supporting
Information). Methyl ester saponification (LiOH) provided the
individual 20 monosubstituted carboxylic acids in high yields.
The next stage in the library synthesis entailed preparation of
the 210 trans-pyrrolidine-3,4-dicarboxamides (eq 2). The Troc-
protected dicarboxamides were prepared by amide coupling
(EDCI, HOAt, 2,6-lutidine, DMF, 25 °C, 14 h), and this enabled
isolation and purification of the products (62ꢀ100% yield) using
acid/base liquidꢀliquid extractions in which all reagents, reagent
byproducts, and any unreacted starting materials were removed.
A diagonal set of 20 diamides of the full 210 matrix, representing
a member of each of the monamides and each of the R2 amine
coupling reactions, was fully characterized (1H and 13C NMR, IR,
and HRMS), confirming both the compound structure and
purity (>95%) (Supporting Information). The subsequent Troc
deprotection was conducted using activated zinc nanopowder
(20 equiv) in 2:1 THF/AcOH (25 °C, 8 h).24 Following zinc
treatment, the compounds were isolated by filtration through Celite
to remove the zinc and removal of the solvent in vacuo with a
toluene azeotrope. Residual AcOH was completely removed by
dissolving the compound in MeOH and passing it through a small
column of basic silica gel (Chromatorex NH, Fuji Silysia Ltd.). This
procedure provided highly pure amines (>95%) in good yields
(product yields and amounts are in the Supporting Information)
and avoided the use of an aqueous base extraction, which was found
to result in low product recovery in certain cases due to the aqueous
solubility. As before, a representative but different diagonal set of 20
compounds of the full 210 matrix was fully characterized by 1H and
13C NMR, IR, and HRMS (Supporting Information).
Figure 9. HPLC comparison of library (EXP 1) and authentic (EXP 2)
equimolar mixture of the final 20 compounds derived from 27. HPLC
conditions: linear gradient, 0ꢀ90% MeCN in H2O over 7 min and then
90% MeCN for 10 min at a flow rate of 0.75 mL/min.
equimolar mixture of the carboxylic acids of the 20 groups shown
in Figure 10 (eq 3). To ensure that each of the 20 carboxylic acids
was fully consumed to yield an equimolar mixture of 20 products,
1.2 equiv of the free amine was employed in each coupling
reaction. The excess starting amine was subsequently removed
upon completion of the reaction using aqueous acid extractions.
The final global deprotection of all side-chain protecting groups
was accomplished by treatment with 3:1:1 TFA/MeOH/H2O
(25 °C). Using this condition, complete removal of Boc, tert-
butylester, TIPS, and trityl groups was observed in 12ꢀ16 h. We
then verified that all final products in 20 representative final
mixtures were detected by MS using a diagonal 20/210 matrix
characterization (see Supporting Information).
To confirm the quality of the construction of the library as 20
compound mixtures, a representative final library mixture was
selected for comparison by HPLC to an authentic equimolar
mixture prepared from the individually synthesized compounds
(Figure 9). Although a separation of all 20 mixture components is
not possible on a single HPLC run, the nearly identical detection
The final library was obtained by coupling (EDCI, HOAt, 2,
6-lutidine, DMF, 25 °C, 12 h) each of the 210 amines with an
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Figure 10. (A) % inhibition of [3H]U69593 binding for the indicated R1/R2 side-chain combinations in the screening of the 210 mixtures against the
KOR. (B) % inhibition of [3H]DAMGO binding for the indicated R1/R2 side-chain combinations in the screening of the 210 mixtures against the MOR.
Figure 11. Mixtures selected for deconvolution.
profiles displayed by the two mixtures confirms that not only are
all 20 compounds present in the library mixture but that they
must be present in amounts that approach equimolar.
Screening the Library against the Opioid Receptors. The
entire library composed of 210 mixtures of 20 compounds (210
wells) was screened at 10 μM (total concentration, 0.5 μM per
compound) for activity at opioid receptors in radioligand
binding assays at human cloned receptors (KOR, MOR, and
DOR). The summary of the screening results for the KOR and
MOR is shown in Figure 10. The overall trends against the three
receptors proved consistent with our previous single-compound
results in exhibiting an intrinsic binding selectivity of the trans-
pyrrolidine-3,4-dicarboxamides for the KOR, followed by the
MOR and then DOR (data not shown). A clear SAR trend is
evident from the primary screening results even without the
examination of individual compounds, illustrating that hydro-
phobic aromatic side chains generally dominate the interaction
with the opioid receptors, with Phe(4Cl), Trp, and Nap showing
strong inhibition against the KOR and the HoTyr, Trp, and
HoPhe(4Cl) side chains ranking among the best against the
MOR. The exception to this trend with both the KOR and MOR
was the potent inhibition observed with a basic His side chain in
the R1/R2 position, with a particularly potent R1/R2 combination
being His and a bulky aliphatic residue (Val or Leu).
Deconvolution of Selected Mixtures and Screening of
Individual Compounds. On the basis of the primary screening
data for the library and with the recognition β-turn sequence of
the endomorphins (H-Tyr-Pro-Trp/Phe-Phe-NH2) in mind, three
related mixtures were selected for deconvolution (Figure 11).
The series containing R1/R2 side chains Trp and Phe is the
closest representative within the library of the endomorphin-1
sequence and contains compound 8, which exhibited the
measured Ki values of 390 and 930 nM for KOR and MOR,
Figure 12. Screening results (% inhibition at 10 μM) for the three series
of deconvolution compounds against MOR along with measured Ki
values (nM) (versus [3H]DAMGO).
respectively. The Trp/Phe(4Cl) series represents a closely
related series to the Trp/Phe series, but the addition of the
4-Cl substituent enhanced binding of the mixture to both the
KOR (86 versus 73% inhibition) and the MOR (66 versus 56%
inhibition). Its deconvolution could be expected to yield indivi-
dual compounds with enhanced affinity to both receptors while
also allowing a further evaluation of the β-turn mimicry of
the compounds. The final series chosen for deconvolution, the
Phe(4Cl)/Phe(4Cl) series, was the mixture that displayed
the highest affinity against the KOR (91% inhibition) and also the
greatest difference between KOR and MOR affinity (91 versus
48%). These three mixtures were deconvoluted by resynthesis of
the individual compounds from the three archived penultimate
intermediates using the conditions found in eq 3 to yield 60
individual compounds for screening.
The 60 individual compounds were screened at 10 μM for
activity at the KOR and MOR via the radioligand binding assays.
Because of the lower activity at the MOR, where the % inhibi-
tion was not pegged at levels approaching 90% for each series, the
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Figure 14. Chemical structures of compounds with the highest affinity
for KOR from the Trp/Phe(4Cl) series (28) and the Phe(4Cl)/
Phe(4Cl) series (29).
HoTyr or Tyr was the favored R3 substituent in all three series,
with HoTyr performing best in the Trp/Phe and Trp/Phe(4Cl)
series and Tyr yielding the most potent compound in the
Phe(4Cl)/Phe(4Cl) series. Beautifully, and if the turn recogni-
tion motif were unknown, these results would represent the
identification of the Tyr-XXX-Trp-Phe and Tyr-XXX-Phe-Phe
β-turn motifs. This result also reinforced our earlier decision to
incorporate single carbon extensions of key side chains such as
Tyr into the library. In general, other aromatic R3 side chains
were less active but roughly equal in potency, and all were
typically more active than compounds bearing the aliphatic or
charged side chains regardless of the series.
The data from each of the three deconvolution series are
shown in Figure 13. Clear from these comparisons is the greatest
activity against the KOR (KOR > MOR > DOR) for all three
series and that the activity of the three series generally follows the
order of Phe(4Cl)/Phe(4Cl) > Trp/Phe(4Cl) > Trp/Phe, as
observed in the original mixture screening results (Figure 11).
The excellent activity of 8 (Ki = 390 nM) used originally to test
the design and found in the Trp/Phe series was improved with
the replacement of Phe with Phe(4Cl) in the Trp/Phe(4Cl)
series providing 28 (Ki = 250 nM, Figure 14A), and this series
provided several related compounds that exhibited Ki’s of <300
nM. Finally, the most active Phe(4Cl)/Phe(4Cl) series not only
showed a greater activity for the KOR, but it also exhibited a
greater selectivity for the KOR versus the MOR or DOR, as
inferred from the original mixture screening results (Figure 11).
The most potent compound in this series for the KOR was 29
(Ki = 80 nM, Figure 14B), which was found to be >100-fold
selective versus the MOR or DOR (Ki > 10 000 nM).
As detailed earlier, the trends for MOR binding illustrated that
the HoTyr [Trp/Phe, Trp/Phe(4Cl)] or Tyr [Phe(4Cl)/Phe-
(4Cl)] were the clearly favored R3 substituents in all three series,
indicative of a prominent or productive role for the Tyr free
phenol. Other aromatic R3 side chains were less active and
roughly equal in potency (Figure 12). This unique preference
for HoTyr or Tyr and the presence of the free phenol were not
observed with KOR. While still preferring a third aryl R3 side-
chain substituent [Phe(4Cl), HoTyr > Tyr = Tyr(Me), Phe >
HoPhe(4Cl), > Trp, HoPhe > Nap] and accommodating the
free phenol of Tyr, it no longer exhibits a phenol preference
(Figure 15). Without over interpreting the minor differences
Figure 13. Screening results (% inhibition at 10 μM) for the three series
of deconvolution compounds against KOR (versus [3H]U69593),
MOR (versus [3H]DAMGO), and DOR (versus [3H]DADLE) along
with measured Ki values (nM) against KOR.
general trends were clearest in examining its results. The R3 side-
chain preferences for the individual compounds against the
MOR are shown in Figure 12. According to the proposed mode
of β-turn mimicry by the compounds and the sequence of the
MOR-selective endomorphins, Tyr or HoTyr would be ex-
pected to be the favored side chain at the R3 position if mim-
icry of an endomorphin β-turn is being achieved. Consistent
with this expectation, the screening demonstrated that either
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Figure 15. Screening results (% inhibition at 10 μM) for the three series of deconvolution compounds against KOR (versus [3H]U69593).
in the measured and sometimes pegged assay values, there was
virtually no difference in Tyr and Tyr(Me) for each of the three
series, indicating that protection of the phenol as a methyl ether
has no impact in binding affinity, and both were nearly identical
to Phe itself in each of the three series. Among the best of the R3
substituents in nearly each of the three series was Phe(4Cl) and
HoPhe(4Cl). In addition to suggesting that the Tyr free phenol of
the endomorphins and enkephalins may not be important to KOR
binding,25 it also suggests that a way to enhance KOR versus MOR
and DOR binding selectivity is to remove this pharmacophore
phenol OH. Like the behavior of 29, further enhancements in
KOR affinity may be achieved by utilizing Phe(4Cl), resulting in
higher affinity and even more selective KOR ligands.
proteinꢀprotein or peptideꢀreceptor interaction. Additionally,
the use of such a comprehensive library can take advantage of the
principles of selection to discover compounds with improved
properties (e.g., of affinity, selectivity) over those that mimic the
endogenous protein or peptide ligands. The screening of the
complete library against the opioid receptors demonstrated that
not only could the expected activity be observed with library
mixtures containing the compounds shown to mimic the opioid
receptor endogenous peptide ligands but that additional side-
chain combinations could be identified with even higher receptor
binding affinities and selectivity, providing new insights into the
β-turn recognition events.
’ ASSOCIATED CONTENT
’ CONCLUSIONS
S
Supporting Information. Full experimental details on
b
The solution-phase synthesis of a β-turn mimetic library as the
second component of a general small-molecule library targeting
the key recognition motifs involved in proteinꢀprotein interac-
tions is described. Using a geometric characterization of β-turns
in the PDB, the trans-pyrrolidine-3,4-dicarboxamide was pro-
posed and subsequently found to serve as an effective and
synthetically accessible library β-turn mimetic template, and this
was initially validated by screening test compounds against a
series of peptide-activated GPCRs that recognize β-turn struc-
ture in their ligands. The screening of selected compounds
designed to mimic the β-turn pharmacophore of the peptide
ligands of the opioid receptors demonstrated that appropriately
substituted trans-pyrrolidine-3,4-dicarboxamides could replace
the peptide backbone for effective display of side-chain groups in
a β-turn. This validation was highlighted by identification of both
nonbasic and basic small molecules with high affinity (Ki = 390
and 23 nM, respectively) for the KOR. The library was assembled
using a solution-phase protocol to provide 210 mixtures of 20
compounds for a total of 4200 compounds designed to mimic all
possible permutations of 3 of the 4 residues in a naturally
occurring β-turn. Even if the recognition motif is unknown or
unrecognized, the library screening should provide lead struc-
tures, provide insights into the nature of the interaction (β-turn),
and identify the key amino acid residues responsible for the
GPCR screening conditions and the library synthesis along with
single-compound characterizations of all 20 monoamides (eq 1:
1H and 13C NMR, IR, and HRMS), a diagonal set of 20 Troc-
diamides of the full 210 matrix representing a member of each
of the monamides and each of the R2 amine coupling reactions
(eq 2: H and 13C NMR, IR, and HRMS), confirming the
1
compound structure and purity (>95%), a table summarizing
the LCMS characterization of an additional 50 Troc-diamides,
establishing their isolation (MS) and purity, compound character-
izations of a second but different diagonal set of 20 compounds in
the full 210 matrix of diamide amines (eq 2: 1H and 13C NMR, IR,
and HRMS), MS characterization of a diagonal set of 20
of the final 210 mixtures, confirming the presence of all 20
components, the characterization of 33 individual final triamides
constituting deconvolution samples (1H NMR, MS), and three
tables summarizing the isolated amounts and yields of each
component in the three stages of the library synthesis. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
boger@scripps.edu
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’ ACKNOWLEDGMENT
(11) (a) Ambroise, Y.; Yuspan, B.; Ginsberg, M. H.; Boger, D. L.
Chem. Biol. 2002, 9, 1219–1226. (b) Capps, K. J.; Humiston, J.;
Dominique, R.; Hwang, I.; Boger, D. L. Bioorg. Med. Chem. Lett. 2005,
15, 2840–2844. (c) Chang, M. W.; Giffin, M. J.; Muller, R.; Savage, J.;
Lin, Y. C.; Hong, S.; Jin, W.; Whitby, L. R.; Elder, J. H.; Boger, D. L.;
Torbett, B. E. Biochem. J. 2010, 429, 527–532. (d) Eubanks, L. M.;
Hixon, M. S.; Jin, W.; Hong, S.; Clancy, C. M.; Tepp, W. H.; Malizio,
C. J.; Goodnough, M. C.; Barbieri, J. T.; Johnson, E. A.; Boger, D. L.;
Dickerson, T. J.; Janda, K. D. Proc. Natl. Acad. Sci. U.S.A 2007,
104, 2602–2607.
(12) (a) Lee, A. M.; Rojek, J. M.; Spiropoulou, C. F.; Gunderson, A.;
Jin, W.; Shaginian, A.; York, J.; Nunberg, J. H.; Boger, D. L.; Oldstone,
M. B. A.; Kunz, S. J. Biol. Chem. 2008, 283, 18734–18742. (b) Whitby,
L. R.; Lee, A. M.; Kunz, S.; Oldstone, M. B. A.; Boger, D. L. Bioorg. Med.
Chem. Lett. 2009, 19, 3771–3774.
(13) (a) Stover, J. S.; Shi, J.; Jin, W.; Vogt, P. K.; Boger, D. L. J. Am.
Chem. Soc. 2009, 131, 3342–3348. (b) Boger, D. L.; Fink, B. E.; Hedrick,
M. P. J. Am. Chem. Soc. 2000, 122, 6382–6394. (c) Boger, D. L.; Schmitt,
H.; Fink, B. E.; Hedrick, M. P. J. Org. Chem. 2001, 66, 6654–6661.
(d) Boger, D. L.; Fink, B. F.; Brunette, S. R.; Tse, W.; Hedrick, M. P.
J. Am. Chem. Soc. 2001, 123, 5878–5891. (e) Tse, W.; Boger, D. L. Acc.
Chem. Res. 2004, 37, 61–69. (f) Hauschild, K. E.; Stover, J. S.; Boger,
D. L.; Ansari, A. Z. Bioorg. Med. Chem. Lett. 2009, 19, 3779–3782.
(14) Unlike a peptide library, such mimetic templates not only
identify key residues mediating the interaction, but they may be used
to define the binding recognition motif (R-helix, β-turn, β-strand) and
may serve as stable in vitro and in vivo probes of biological processes
needed for target validation and as lead compounds for optimization in
therapeutics.
We gratefully acknowledge the financial support of the
National Institutes of Health (CA078045, D.L.B. and P.K.V.;
R01DA017204, R01DA027170, the Michael Hooker Distin-
guished Professorship and the NIMH Psychoactive Drug Screen-
ing Program Contract, B.L.R.). L.R.W. is a Skaggs Fellow.
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