Synthesis and incorporation into cyclic peptides of tolan amino acids and their hydrogenated congeners: Construction of an array of A-B-loop mimetics of the Cε3 domain of human IgE

Article abstract of DOI:10.1021/jo202604q

The disruption of the human immunolobulin E-high affinity receptor I (IgE-FcεRI) protein-protein interaction (PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of conformationally constrained cyclic peptides based on an epitope of the A-B loop within the Cε3 domain of IgE. The peptides contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational constraints. Modest antagonist activity (IC₅₀ ～660 μM) is displayed by the peptide containing a 2,2′-tolan, which is the one predicted by molecular modeling to best mimic the conformation of the native A-B loop epitope in IgE.

Full text of DOI:10.1021/jo202604q

Article
pubs.acs.org/joc
Synthesis and Incorporation into Cyclic Peptides of Tolan Amino
Acids and Their Hydrogenated Congeners: Construction of an Array
of A−B-loop Mimetics of the Cε3 Domain of Human IgE
Daniel A. Offermann,† John E. McKendrick,‡ Jimmy J. P. Sejberg,† Bingli Mo,† Mary D. Holdom,§
Birgit A. Helm,∥ Robin J. Leatherbarrow,† Andrew J. Beavil,§ Brian J. Sutton,§ and Alan C. Spivey*,†
†Department of Chemistry, South Kensington Campus, Imperial College, London SW7 2AZ, U.K.
‡Department of Chemistry, University of Reading, Reading RG6 6A, U.K.
§King’s College London, The Randall Division of Cell & Molecular Biophysics, New Hunt’s House, Guy’s Hospital Campus, London
SE1 1UL, U.K.
∥Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western
Bank, Sheffield S10 2UH, U.K.
S
* Supporting Information
ABSTRACT: The disruption of the human immunolobulin E−high affinity receptor I (IgE−FcεRI) protein−protein interaction
(PPI) is a validated strategy for the development of anti asthma therapeutics. Here, we describe the synthesis of an array of
conformationally constrained cyclic peptides based on an epitope of the A−B loop within the Cε3 domain of IgE. The peptides
contain various tolan (i.e., 1,2-biarylethyne) amino acids and their fully and partially hydrogenated congeners as conformational
constraints. Modest antagonist activity (IC50 ∼660 μM) is displayed by the peptide containing a 2,2′-tolan, which is the one
predicted by molecular modeling to best mimic the conformation of the native A−B loop epitope in IgE.
class of compounds that has been evaluated for this purpose is
peptides,9,13−15,21 initially as tools for mapping the binding
interface,22−34 as synthetic vaccines,33,34 as biochemical
tools,35−43 and as affinity purification aids,44−54 and more
recently as potential leads for therapeutic development.55−71
Linear and disulfide-bond constrained loop peptides have been
described, and several patents72−74 have been filed relating to
the use of epitope sequences of both the protein partners (i.e.,
the Cε3 domain of IgE and the extracellular α-chain of FcεRI)
in potential peptide-based therapeutics.
Previously, we have described the solid-phase synthesis of a
cyclic peptide containing a 19-residue epitope (Leu-340 to Cys-
358) found in the A−B loop of the Cε3 domain of human IgE
via an on-resin Sonogashira macrocyclization which concom-
itantly installed a 2,2′-tolan (i.e., 1,2-biarylethyne) amino acid
conformational constraint to mimic the Tyr-339/Leu-359 loop
entry/exit residues (see Figure 1).75 This compound, like its
INTRODUCTION
■
Incidence of asthma and related allergic manifestations is
increasing in the Western world.1 The symptoms associated
with these conditions are invariably debilitating, and the
resulting financial burden on national healthcare systems2 has
focused attention on the development of improved therapeutic
strategies to combat these afflictions.3,4 One strategy that offers
the prospect of a preventative rather than ameliorative
intervention is to administer molecules that can block the
interaction of human immunolobulin E (IgE) with its high
affinity receptor FcεRI; a protein−protein interaction (PPI)
that is central to the allergic signal transduction cascade.5−17
Proof of principle that this strategy can be effective, and does
not suffer from unacceptable side effects, comes from the
successful clinical use of the monoclonal antibody omalizumab
(Xolair), which is indicated for severe persistent asthma and
operates by sequestering IgE in the blood and preventing
binding to FcεRI.18,19 However, its high cost and non oral
mode of delivery has fueled interest in the development of
alternative antagonists of this PPI.20 The most well-studied
Received: December 22, 2011
Published: March 7, 2012
© 2012 American Chemical Society
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Figure 1. Schematic representation of the antiparallel A−B β-strand/Ω-turn region of the Cε3 domain of IgE showing the site of our previous 2,2′-
tolan amino acid constraint (Tyr-339/Leu-359) and the proposed site of introduction of this and related constraints in this work (Ser-344/Pro-354).
Figure 2. Amino acid conformational constraints employed in this sudy.
disulfide-constrained “parent” peptide, showed inhibition of
IgE-triggered 5-hydroxytryptamine secretion in a rat basophilic
leukemia cell line transfected with human FcεRI α-chain with
an IC50 of ∼12 μM.15 The Cε3 A−B loop epitope of human
IgE is not part of the extensive contact surface between IgE and
FcεRI as revealed in the X-ray crystal structure of a complex
between human IgE−Fc and the extracellular, soluble α-chain
of FcεRI (Brookhaven PDB code 1F6A, 3.5 Å resolution).76−78
Consequently, we were motivated to prepare further con-
strained analogues of this peptide to aid elucidation of the
mechanism of action of these mimetics. In particular, we were
keen to reduce the number of amino acid residues contained
within the loop as this was anticipated to increase conforma-
tional rigidity and enhance proteolytic stability. It was envisaged
to achieve this by removing the residues within the original
sequence which comprise an occluded β-strand leading to the
surface exposed Ω-turn; the only individual residues that have
been demonstrated to be important for activity are Phe-349 and
Arg-351 (as determined by Phe-349-Ala and Arg-351-Lys site-
directed mutagenesis on native human IgE) which are located
within the Ω-turn region.66 Consequently, the plan was to
prepare a small array of cyclic peptides containing the 9-residue
Pro-345 to Ser-353 epitope in which the Ser-344/Pro-354 loop
entry/exit residues would be mimicked by a set of tolan amino
acid conformational constraints and their fully and partially
hydrogenated congeners. We envisioned that the synthesis and
biological evaluation of this array of compounds as potential
antagonists of the IgE−FcεRI PPI using an enzyme-linked
immunosorbent assay (ELISA) and comparison with appro-
priate controls might provide important insight into the
conformational requirements for activity and a lead peptidomi-
metic suitable for NMR conformation studies and cocrystalliza-
tion experiments with the soluble FcεRI α-chain protein
(Figure 1).
generic suite of linkages that could be introduced as cyclizing
elements for the construction of peptidomimetics for many
peptide epitopes having a loop conformation in their native
protein environment (Figure 2).75
The 2,2′-tolan unit II is an established conformational
constraint introduced by Kemp,79,80 which can induce a β-turn-
like structure via a 10-membered H-bonded ring,81 but the 2,3′-,
3,2′-, and 3,3′-regioisomers III−V as well as the partially
reduced 2,2′-(E)- and 2,2′-(Z)-congeners VI and VII and the
fully reduced 2,2′-bibenzyl analogue VIII have not previously
been described. Related suites of biphenyl82 and 2-butynyl/2-
butenyl/butyl83 amino acid families have been described, the
latter as stable replacements for disulfide linkages in protein
stapling applications.
Inspection of the A−B loop region of IgE protein within the
X-ray crystal structure of the IgE−FcεRI complex reveals that
the Ser-344 carbonyl carbon and Pro-354 amine atoms are
separated by ∼8.0 Å and deliver/receive the intervening loop
residues with a vectorial disposition that can be mimicked
closely with the 2,2′-tolan amino acid II (Figure 3).75
Here, we describe the synthesis of this A−B loop mimetic
peptide containing the 2,2′-tolan amino acid II and also
analogous looped peptides containing the same 9-residue A-B
loop epitope but constrained by each of the other amino acids
III−VIII inserted at the same position as well as the screening
of these and two control peptidomimetics as potential
antagonists of the IgE−FcεRI PPI using ELISA.
RESULTS AND DISCUSSION
■
The general approach we envisaged for the construction of our
library of A−B-loop mimics is shown below (Scheme 1).
It was expected that the linear resin-bound peptide IX would
be constructed via standard84,85 automated solid-phase peptide
synthesis. The various tolans and hydrogenated analogues X
would then be coupled to give the linear solid-supported
peptide XI. Finally, cleavage from the resin and macro-
The four-tolan and three-tolan-derived amino acid con-
straints (II−VIII) prepared in this work were designed as a
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a
Scheme 2. Tolan Synthesis
Figure 3. Portion of the X-ray crystal structure of IgE−Fc
(Brookhaven PDB code 1F6A) showing the A−B loop and the
position for synthetic insertion of tolan and related conformational
constraints.
cyclization would yield the target peptidomimetics I. This
strategy was selected in preference to our previous on-resin
Sonogashira cyclization strategy75 because despite extensive
experimentation we were unable to obtain comparable yields
using this latter approach.
We began with the preparation of the tolan intermediates
and related analogues. At the outset, we were concerned that
the restricted flexibility of the tolan moiety might conforma-
tionally restrict opportunities for cyclization and that the low
nucleophilicity of the aniline nitrogen would also render
cyclization via intramolecular peptide coupling challenging.86
To improve the cyclization potential of our peptides, we
therefore decided to attach the first amino acid (Ser-344) to the
tolan prior to cyclization (Scheme 2).
To this end, 2- and 3-iodobenzoic acids (1a and 1b) were
first esterified under acidic conditions. The resulting methyl
esters 2a and 2b were treated with TMS-acetylene under Pd/
Cu catalyzed Sonogashira coupling conditions to afford alkynes
3a and 3b. Each of these was coupled separately to 2- and 3-
iodoanilines (4a and 4b) via a second Sonogashira coupling to
give tolan amino esters 5a−d after an in situ TMS-
deprotection. The order of addition of reagents for the
Sonogashira couplings was found to be crucial and yields
were also improved by the addition of further portions of
palladium and copper catalysts after 12 h. These optimized
conditions were only employed for the construction of tolan 5a
a
Reagents and conditions: (i) H2SO4, MeOH; (ii) 2-TMS-acetylene,
PPh3, Pd(PPh3)2Cl2, Et3N, CuI, THF; (iii) iodoaniline, PPh3,
Pd(PPh3)2Cl2, piperidine, CuI, THF, K2CO3, MeOH; (iv) oxalyl
chloride, cat. DMF, CH2Cl2; (v) AgCN, CH2Cl2; (vi) LiAlH4, THF;
(vii) 1 M Jones' reagent, acetone.
and this is reflected in the improved isolated yield of 5a (80%
from 1a) compared to the related compounds 5b−d (31−
40%). Moreover, the two Sonogashira coupling steps and
intermediate TMS-deprotection could be performed in a single
operation in a manner similar to that described by Doye and
co-workers.87 Thus, intitial Pd/Cu-catalyzed Sonogashira
coupling of 2-iodoaniline (4a) with TMS-acetylene followed
by treatment with KOH to affect TMS-deprotection, and Pd/
Cu catalyzed Sonogashira coupling of the crude product with 2-
Scheme 1. Overview of Synthetic Approach to Peptidomimetics
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a
Scheme 3. Enantiomeric Dipeptides ent-8a and ent-9a for Verifying Stereochemical Integrity
a
Reagents and conditions: (i) LiAlH4, THF; (ii) 1 M Jones' reagent, acetone.
a
Scheme 4. Synthesis of Fully Saturated 2,2′-Tolan Analogue
a
Reagents and conditions: (i) Pd/C, H2, EtOAc; (ii) 7, AgCN, CH2Cl2; (iii) LiAlH4, THF; (iv) 1 M Jones’ reagent, acetone.
a
Scheme 5. Synthesis of Partially Hydrogenated 2,2′-Tolan Analogues
a
Reagents and conditions: (i) Lindlar’s catalyst, H2, EtOAc; (ii) I2, CHCl3; (iii) Pd(CN)2Cl2, silica gel, CHCl3; (iv) Lindlar’s catalyst, PhCO2H, H2,
EtOAc; (v) LiAlH4, THF; (vi) 1 M Jones' reagent, acetone.
iodobenzoic acid methyl ester (2a) gave tolan amino ester 5a
directly in excellent yield (85%).
In the light of the need to convert (S)-Fmoc-Ser(t-Bu)−OH
(>99.5% ee) to its acid chloride and subsequently expose the
resulting dipeptide to LiAlH4, we considered that it would be
prudent to verify the stereochemical integrity of the resulting
Ser-containg tolans 9a−d. Therefore, following an analogous
synthetic sequence, (R)-Fmoc-Ser(t-Bu)-OH (>97% ee, ent-6)
was coupled with 2,2′-tolan methyl ester 5a to construct the
antipodal dipeptides ent-8a and ent-9a (Scheme 3).
Chiral HPLC analysis showed no detectable erosion in
stereochemistry of dipeptide 9a (>99.8% ee) or its unnatural
enantiomer ent-9a (>97.2% ee) when compared to the starting
commercial amino acids. Reassured as to the stereochemical
integrity of our synthetic approach, we continued with the
Treatment of commercially available (S)-Fmoc-Ser(t-Bu)-
OH (6) with oxalyl chloride and catalytic DMF afforded acid
chloride 7, which was in turn coupled to each tolan amino ester
5a−d to yield the corresponding amides 8a−d. Not
surprisingly, initial attempts to saponify the methyl ester 8a
while retaining the Fmoc-protecting group failed.88 Pleasingly,
however, a two-step procedure involving reduction of the
methyl ester with LiAlH4, followed by oxidation of the resultant
alcohol using Jones’ reagent, afforded the serine-containing
tolan acids 9a−d ready for peptide incorporation.
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a
Scheme 6. Solid-Phase Synthesis of Linear Peptides
a
Reagents and conditions: (i) protected amino acid, HOBt/HBTU, i-Pr2EtN, DMF; (ii) 20% piperidine/DMF; (iii) TFA/i-Pr3SiH/H2O
(95:2.5:2.5); (iv) PyBOP, tolan or hydrogenated analogue 9a−d/12/13/16, i-Pr2EtN, DMF; (v) 20% piperidine/DMF; (vi) 1% TFA/CH2Cl2.
construction of the remaining tolan analogues. Starting with the
fully saturated analogue of the 2,2′-tolan (Scheme 4).
allowing the final cyclization step to be performed in solution.
An on-resin cyclization method, which employed a sulfamylbu-
tyryl safety-catch linker, was also investigated.92 However, a
comparison of cost, product yields, and ease of handling
favored the use of 2CT resin.93−95
Tolan amino ester 5a was hydrogenated to provide bibenzyl
amino ester 10 in near quantitative yield. Commercially
available (S)-Fmoc-Ser(t-Bu)-OH (6) was again converted to
its acid chloride 7 and then coupled to aniline 10 to afford
serine-containing dipeptide 11 in excellent yield. Treatment of
ester 11 with LiAlH4, followed by oxidation using Jones’
reagent, afforded carboxylic acid 12 ready for incorporation
into a peptide. Alternatively, bibenzyl dipeptide 12 could be
accessed directly by hydrogenating the previously prepared
tolan dipeptide acid 9a in similar overall yield. Stereoselective
partial hydrogenation of this dipeptide derivative was also
envisaged to give access to the (E) and (Z) stilbene-containing
congeners (Scheme 5).
Automated solid-phase peptide synthesis96 was employed to
construct resin-bound linear peptide 18 from commercially
available H2N-Lys(Boc)-2CT resin 17. An iterative process
involving amino acid coupling (using HOBt/HBTU) followed
by Fmoc-deprotection was used to construct the desired
sequence in seven cycles. At this stage, a small quantity of resin-
bound peptide 18 was treated with concentrated TFA to
provide linear peptide 19, the identity of which was confirmed
by MALDI-MS (MH+ m/z 1036). The remaining resin-bound
peptide 18 was coupled (using PyBOP) as separate aliquots to
each of the Ser-containing tolan dipeptides 9a−d or hydro-
genated analogues 12, 13, and 16 to yield resin-bound peptides
20a−g. For analytical purposes, a small quantity of each of
these resin-bound peptides was treated with concentrated TFA
to provide linear peptides 21a−g, the identity of which was
confirmed by MALDI-MS. The remaining resin-bound peptides
20a−g were treated with 1% TFA/CH2Cl2 for just 2 min to
reveal linear peptides 22a−g with side-chain protecting groups
intact.
Tolan 9a was partially hydrogenated to (Z)-stilbene 13 in
modest yield, using Lindlar’s catalyst (Scheme 4). Unfortu-
nately, attempts to isomerize to (E)-stilbene 13 using either
90
iodine89 or catalytic Pd(CN)2Cl2 failed. Increasing the
palladium catalyst loading (to 50 mol %) and reaction time
(to 48 h) led to the formation of an unwanted byproduct,
presumed to be the lactone resulting from cyclization of the
carboxylic acid onto the tolan triple bond.91 To avoid this
unwanted transformation, it was decided to perform the
isomerization on the corresponding methyl ester 8a. Surpris-
ingly, the use of Lindlar’s catalyst under an atmosphere of
dihydrogen failed to hydrogenate tolan 8a. It was found
however, that the addition of benzoic acid allowed for almost
quantitative conversion to (Z)-stilbene methyl ester 14. To our
delight, isomerization of this substrate using catalytic Pd-
(CN)2Cl2 afforded (E)-stilbene methyl ester 15 cleanly. The
usual procedure of reduction using LiAlH4, followed by
oxidation with Jones’ reagent finally provided carboxylic acid
16 ready for peptide incorporation.
We were pleased to note that addition of PyBOP to a DMF
solution of linear peptide 22a and i-Pr2EtN afforded the desired
cyclization product 23a, albeit in only 6% yield after side-chain
removal. Spurred on by this result, we investigated a variety of
conditions in an attempt to improve this key step including
increased dilution, slow addition of reagents, and the use of
different coupling agents. Our best cyclization results were
achieved by slow addition (0.5 mL h−1) of a DMF solution of
linear peptides 22a−g into a DMF solution of HATU (Scheme
7).
Additional coupling agent (6 equiv total) was added portion-
wise over the course of the 32 h reactions. Side-chain
deprotection was then achieved by treatment with neat TFA
(and scavengers) for 3 h to afford our target peptidomimetics
23a−g in fair yields.
With the completion of the Ser-containing tolan dipeptides
9a−d and hydrogenated analogues 12, 13 and 16, work began
on the synthesis of the A−B epitope peptide (Scheme 6).
All peptides were constructed on the solid phase using 2-
chlorotrityl (2CT) resin preloaded with the first amino acid
(Lys). This resin is cleaved under mildly acidic conditions,
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a
Scheme 7. Solution-Phase Peptide Cyclization
Table 1. Evaluation of the Binding Affinity of
Peptidomimetics 23a−g, 24, and 25 Using IgE-FceRIα
ELISA
d
constraint
(Axx)
IC50 std error
entry compd
sequence
(μM)
a
1
2
3
4
5
6
23a
23b
23c
23d
23e
23f
2,2′-tolan
A−B loop
660 70
a
a
a
a
a
e
2,3′-tolan
3,2′-tolan
3,3′-tolan
2,2′-bibenzyl
A−B loop
A−B loop
A−B loop
A−B loop
A−B loop
5300 1800
e
3700 600
f
no inhibition
f
no inhibition
f
2,2′-(Z)-
stilbene
no inhibition
a
f
7
23g
2,2′-(E)-
stilbene
A−B loop
no inhibition
b
a
8
9
24
25
2,2′-tolan
2,2′-tolan
scrambled
2500 150
Reagents and conditions: (i) HATU, i-Pr2EtN, DMF; (ii) TFA/i-
f
Phe-349-Ala
no inhibition
Pr3SiH/H2O (95:2.5:2.5).
c
mutant
a
b
Cyclic --Ser-Axx-Pro-Phe-Asp-Leu-Phe-Ile-Arg-Lys--. Cyclic --Ser-
Two peptidomimetics containing the 2,2′-tolan constraint
but alternative peptides sequences were also prepared as
controls. The first, cyclic peptide 24, was a scrambled analogue
of 23a which was designed using GenScript’s scrambling tool,97
keeping the positions of the Ser residue at the N-terminus of
the 2,2′-tolan amino acid and the Lys residue at the C-terminus
constant. The second, cyclic peptide 25, was a Phe-349-Ala
mutant of peptidomimetic 23a, designed to correspond to the
Phe-349-Ala mutant of human IgE reported by Presta et al.66
which displayed just 10% binding to FcεRI α-chain cf.
wildtype98(Figure 4).
The ability of the seven A−B loop peptidomimetics
analogues 23a−g and the two control peptidomimetics 24
and 25 to inhibit the IgE−FcεRI PPI was assessed using an
ELISA we have described previously (Table 1).20,99
The ELISA binding assay was performed as described in the
Supporting Information. All the titrations were performed twice
in duplicate. The IC50 values were obtained using GraFit100 and
calculated as described in the Supporting Information.
Rather disappointingly, none of the peptidomimetics
displayed comparably potent antagonism to the parent 19-
mer disulfide- and 2,2′-tolan-constrained peptides (IC50∼12
μM) from which they were designed. Indeed, the only member
of the array that displayed submillimolar activity was the 2,2′-
tolan peptidomimetic 23a (IC50 ∼660 μM, entry 1). The data
does, however, indicate that, as expected, the nature of the
conformational constraint strongly influences the ability of the
A−B loop epitope peptide to act as an antagonist of the IgE−
FcεRI PPI: the IC50 values for the other peptidomimetics
(23b−g, entries 2−7) are at least ∼6 fold less potent. The level
of activity diminishes significantly for the 2,3′- and the 3,2′-
tolan-constrained peptides 23b and 23c (IC50 ∼5.3 and ∼3.7
mM, respectively, entries 2 and 3) and the 3,3′-tolan-, 2,2′-
bibenzyl-, and both 2,2-stilbene-constrained peptides display no
discernible inhibition. That the 2,2′-tolan peptidomimetic 23a
shows the greatest level of activity is consistent with our
hypothesis that the 2,2′-tolan constraint II is best able to span
the separation between the Ser-344 N- and the Pro-345 C-
c
Axx-Phe-Arg-Pro-Phe-Ile-Leu-Asp-Lys--. Cyclic --Ser-Axx-Pro-Phe-
Asp-Leu-Ala-Ile-Arg-Lys--. For details for the ELISA protocol, see
the Supporting Information. Inhibition less than 50% at highest
d
e
concentration tested and so the IC50 value is only indicative of
potency. Inhibition less than 20% at highest concentration tested.
f
termini of the A−B peptide epitope so as to present the
intervening residues in an active conformation. This correlates
with our molecular modeling based on the conformation of this
epitope as found within the crystal structure for IgE (cf. Figure
3), although the drop in activity of this truncated sequence
relative to the parent 19-mer peptidomimetic suggests that the
conformation presented and/or its ability to flex appropriately
during a dynamic binding event is suboptimal. The absence of
measurable activity for the 2,2′-tolan-constrained peptide
analogue containing the Phe-349-Ala mutation (peptidomi-
metic 25, entry 9) is consistent with published mutagenesis
experiments on IgE itself vide infra and is indicative that the
interaction is sequence dependent. The weak inhibition
observed for the 2,2′-tolan-constrained peptide analogue
containing the scrambled A−B loop sequence (peptidomimetic
24, IC50 ∼2.5 mM, entry 8) is difficult to interpret in the
absence of additional structural data but could conceivably be
attributed to this sequence, by chance, displaying some key
residues in an appropriate manner for productive interaction;
e.g., both Phe residues are only slightly displaced relative to the
wildtype sequence.
CONCLUSIONS
■
We have prepared a range of Ser-containing tolan dipeptides
and their hydrogenated analogues 9a−g which were incorpo-
rated into an array of cyclic peptidomimetics 23a−g. The use of
solid-phase peptide techniques ensures that our approach is
amenable to automation and hence incorporation into larger
libraries. In principle, incorporation into virtually any peptide
sequence is possible using the strategy outlined herein. It is also
anticipated that the hydrogenation of tolan regioisomers, other
Figure 4. Structures of control peptidomimetics: 24 (scrambled) and 25 (Phe-349-Ala mutant).
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(100 MHz, CDCl3) δ 0.1, 52.0, 99.7, 103.3, 123.2, 128.2, 130.3, 131.5,
132.5, 134.5, 166.9; MS (EI+) m/z 232 (M+, 19), 217 (54), 187
(100); HRMS (EI+) m/z calcd for C13H16O2Si (M+) 232.0920, found
232.0916.
than those demonstrated, would be possible using the protocols
described. These tolans and their congeners have been
designed to be deployed as, e.g., redox-inert, conformation-
restricted replacements for Cys-Cys disulfide links in
peptidomimetics or as hydrocarbon “staples” for protein
secondary structural motifs.101
Although the level of binding displayed by the 2,2′-tolan-
constrained peptidomimetic 23a, which was designed to
present the IgE A−B loop epitope Pro-345 to Ser-353 in a
close-to-native conformation, was significantly lower (IC50
∼660 μM) than the parent 2,2′-tolan-constrained 19-mer
peptidomimetic that we employed as a starting point (IC50
∼12 μM) it is hoped that the reduced conformational flexibility
of the compound may aid cocrystal formation with one of the
protein partners of this PPI in order to throw light on the mode
of action of these noninterface mimetics. Further experiments
toward this end are underway in our laboratories.
3-Trimethylsilanylethynylbenzoic Acid Methyl Ester (3b).
Compound 2b (4.00 g, 15.3 mmol), 2-TMS-acetylene (1.64 g, 16.7
mmol), PPh3 (102 mg, 0.389 mmol), Pd(PPh3)2Cl2 (545 mg, 0.776
mmol), and Et3N (13.0 mL, 93.5 mmol) were dissolved in THF (50
mL). The reaction mixture was degassed and stirred under an
atmosphere of nitrogen for 20 min before the addition of CuI (62 mg,
0.326 mmol). The reaction was then stirred at rt for 12 h, after which
time additional Pd(PPh3)2Cl2 (110 mg, 0.157 mmol) and CuI (15 mg,
0.079 mmol) were added and stirring continued for 6 h. The reaction
mixture was diluted with diethyl ether (100 mL) and then washed with
0.1 M HCl solution (2 × 100 mL), H2O (100 mL) and brine (100
mL). The organic layer was dried (Na2SO4) and concentrated in vacuo
and the resulting residue purified by FC (10% v/v, EtOAc/hexane) to
afford 3b as an orange oil (3.41 g, 96%): IR (neat) 2957, 2159, 1729,
1
1292, 1104, and 848 cm−1; H NMR (400 MHz, CDCl3) δ 0.23 (s,
9H), 3.90 (s, 3H), 7.36 (t, J = 7.8 Hz, 1H), 7.61 (dt, J = 7.5 and 1.2
Hz, 1H), 7.95 (dt, J = 7.8 and 1.3 Hz, 1H), 8.11 (t, J = 1.5 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 0.1, 52.3, 95.4, 103.9, 123.6, 128.4,
EXPERIMENTAL SECTION
■
2-Iodobenzoic Acid Methyl Ester (2a). Commercially available
2-iodobenzoic acid (1a, 12.98 g, 52.33 mmol) was dissolved in
methanol (300 mL), and then concentrated H2SO4 (30.0 mL) was
added cautiously and the resulting solution refluxed under an
atmosphere of nitrogen for 2.5 h and allowed to cool to rt. The
reaction mixture was diluted with diethyl ether (300 mL) and then
washed with H2O (2 × 300 mL), saturated NaHCO3 solution (300
mL), and brine (300 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuo to afford 2a as a pale yellow oil (12.17 g, 89%):
IR (neat) 2950, 1730, 1431, 1291, 1253, and 739 cm−1; 1H NMR (400
MHz, CDCl3) δ 3.91 (s, 3H), 7.13 (td, J = 7.7 and 1.7 Hz, 1H), 7.38
(td, J = 7.7 and 1.1 Hz, 1H), 7.78 (dd, J = 7.8 and 1.6 Hz, 1H), 7.97
(dd, J = 8.1 and 0.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 52.5,
94.1, 127.9, 130.9, 132.7, 135.1, 141.3, 167.0; MS (EI+) m/z 262 (M+,
97), 231 (100), 203 (44); HRMS (EI+) m/z calcd for C8H7O2I (M+)
261.9491, found 261.9492.
3-Iodobenzoic Acid Methyl Ester (2b). Commercially available
3-iodobenzoic acid (1b, 4.00 g, 16.13 mmol) was dissolved in
methanol (100 mL), concentrated H2SO4 (10.0 mL) was added
cautiously, and the resulting solution refluxed under an atmosphere of
nitrogen for 2.5 h and allowed to cool to rt. The reaction mixture was
then diluted with diethyl ether (100 mL) and washed with H2O (2 ×
100 mL), saturated NaHCO3 solution (100 mL), and then brine (100
mL). The organic layer was dried (Na2SO4) and concentrated in vacuo
to afford 2b as a white solid (4.11 g, 97%): mp 54−56 °C (lit.102 mp
54−55 °C); IR (Nujol mull) 2923, 1731, 1436, 1259, 1116, and 744
cm−1; 1H NMR (400 MHz, CDCl3) δ 3.89 (s, 3H), 7.16 (t, J = 7.8 Hz,
1H), 7.86 (m, 1H), 7.98 (m, 1H), 8.35 (t, J = 1.6 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 52.4, 93.8, 128.8, 130.1, 132.0, 138.5, 141.8,
165.6; MS (EI+) m/z 262 (M+, 100), 231 (89), 203 (44); HRMS (EI
+) m/z calcd for C8H7O2I (M+) 261.9491, found 261.9503.
129.4, 130.3, 133.1, 136.0, 166.4; MS (EI+) m/z 232 (M+, 30), 217
(100); HRMS (EI+) m/z calcd for C13H16O2Si (M+) 232.0920, found
232.0917.
2-(2-Aminophenylethynyl)benzoic Acid Methyl Ester (5a).
Method 1. Compound 3a (9.01 g, 38.78 mmol), 2-iodoaniline
(4a, 9.34 g, 42.64 mmol), PPh3 (2.00 g, 7.62 mmol),
Pd(PPh3)2Cl2 (2.70 g, 3.85 mmol), and piperidine (56.0 mL,
566 mmol) were dissolved in THF (500 mL). The reaction
mixture was degassed and stirred under an atmosphere of
nitrogen for 20 min before the addition of CuI (1.50 g, 7.88
mmol) and K2CO3 (6.40 g, 46.31 mmol). The reaction mixture
was refluxed, MeOH (50 mL) was added dropwise, and heating
was continued for 12 h. Additional Pd(PPh3)2Cl2 (270 mg,
0.385 mmol) and CuI (150 mg, 0.788 mmol) were then added,
and stirring was continued for 6 h at reflux. The reaction
mixture was cooled to rt, diluted with ethyl acetate (500 mL),
and then washed with 0.1 M HCl solution (2 × 500 mL), H2O
(500 mL), and brine (500 mL). The organic layer was dried
(Na2SO4) and concentrated in vacuo and the resulting residue
purified by FC (0−20% v/v, EtOAc/hexane) to afford 5a as a
bright yellow solid (7.77 g, 80%): mp 77−78 °C; IR (neat)
1
3471, 3363, 2207, 1718, 1255, and 1081 cm−1; H NMR (400
MHz, CDCl3) δ 3.91 (s, 3H), 5.02 (br s, 2H), 6.66 (m, 1H),
6.70 (d, J = 8.1 Hz, 1H), 7.13 (m, 1H), 7.39−7.30 (m, 2H),
7.49 (td, J = 7.6 and 1.2 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 8.01
(dd, J = 7.9 and 1.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
52.3, 92.6, 93.7, 107.2, 114.1, 117.1, 124.7, 127.4, 130.2, 130.3,
130.6, 132.1, 133.7, 149.8, 166.3; MS (ES+) m/z 252, 253
(MH+, 100, 19); HRMS (ES+) m/z calcd for C16H14NO2
(MH+) 252.1025, found 252.1032.
Method 2. A solution of 2-iodoaniline (4a, 200 mg, 0.91 mmol),
Pd(PPh3)2Cl2 (13 mg, 0.019 mmol), PPh3 (10 mg, 0.038 mmol), and
CuI (3.6 mg, 0.019 mmol) in toluene (1.5 mL) was evacuated and
purged with N2 repeatedly (×3). i-Pr2NH (0.31 mL, 2.21 mmol) was
then added and the mixture evacuated and purged with N2 repeatedly
(×3) and then stirred for 20 min at rt before the addition of TMS-
acetylene (0.26 mL, 1.84 mmol) under a flow of N2. The reaction
mixture was stirred for 18 h at rt, and a solution of KOH (0.25 g, 0.49
mmol) in H2O/MeOH (1:2, 0.6 mL) was added. The reaction mixture
was stirred for 2 h at rt and then quenched with a saturated solution of
NH4Cl (10 mL) and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were washed with H2O (10 mL) and brine
(10 mL), dried (MgSO4), and filtered, and the solvents were removed
in vacuo to give the crude coupling product. A solution of methyl 2-
iodobenzoate (2a, 0.28 mL, 1.91 mmol), Pd(PPh3)2Cl2 (13 mg, 0.019
mmol), PPh3 (10 mg, 0.038 mmol), and CuI (3.6 mg, 0.019 mmol) in
toluene (0.5 mL) was evacuated and purged with N2 repeatedly (×3).
i-Pr2NH (0.31 mL, 2.21 mmol) was then added and the mixture
2-Trimethylsilanylethynylbenzoic Acid Methyl Ester (3a).
Compound 2a (11.00 g, 41.98 mmol), 2-TMS-acetylene (4.50 g, 45.82
mmol), PPh3 (280 mg, 1.07 mmol), Pd(PPh3)2Cl2 (1.50 g, 2.14
mmol), and Et3N (35.0 mL, 252 mmol) were dissolved in THF (150
mL). The reaction mixture was degassed and stirred under an
atmosphere of nitrogen for 20 min before the addition of CuI (170
mg, 0.89 mmol). The reaction was then stirred at rt for 12 h, after
which time additional Pd(PPh3)2Cl2 (300 mg, 0.43 mmol) and CuI
(40 mg, 0.21 mmol) were added and stirring continued for 6 h. The
reaction mixture was diluted with diethyl ether (200 mL) and then
washed with 0.1 M HCl solution (2 × 200 mL), H2O (200 mL), and
brine (200 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuo and the resulting residue purified by FC (0−
25% v/v, EtOAc/hexane) to afford 3a as a pale yellow oil (9.01 g,
1
92%): IR (neat) 2957, 2159, 1730, 1297, 1252, and 1081 cm−1; H
NMR (400 MHz, CDCl3) δ 0.25 (s, 9H), 3.90 (s, 3H), 7.34 (td, J =
7.6 and 1.3 Hz, 1H), 7.42 (td, J = 7.6 and 1.4 Hz, 1H), 7.56 (dd, J =
7.7 and 1.0 Hz, 1H), 7.88 (dd, J = 7.8 and 1.2 Hz, 1H); 13C NMR
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Article
evacuated and purged with N2 repeatedly (×3) and then stirred for 20
min at rt before the addition of the crude coupling product in toluene
(1.5 mL) under a flow of N2. The reaction mixture was stirred for 23 h
at rt and then quenched with a saturated solution of NH4Cl (10 mL)
and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers
were washed with H2O (10 mL) and brine (10 mL), dried (MgSO4),
and filtered, and the solvents were removed in vacuo. Purification of
the crude product by FC (10% v/v, EtOAc/hexane) gave the title
compound as a yellow oil (196 mg, 85%).
The organic layer was dried (Na2SO4) and concentrated in vacuo and
the resulting residue purified by FC (10−40% v/v, EtOAc/hexane) to
afford 5d as a yellow solid (572 mg, 31%): mp 92−95 °C; IR (Nujol
1
mull) 3466, 2924, 1712, 1457, 1293, and 752 cm−1; H NMR (400
MHz, CDCl3) δ 3.75 (br s, 2H), 3.96 (s, 3H), 6.70 (dd, J = 7.9 and 2.0
Hz, 1H), 6.89 (m, 1H), 8.22 (m, 1H), 6.97 (d, J = 7.6 Hz, 1H), 7.17 (t,
J = 7.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.71 (m, 1H), 8.01 (m, 1H);
13C NMR (100 MHz, CDCl3) δ 52.3, 87.7, 90.6, 115.6, 117.8, 122.1,
123.5, 123.9, 128.5, 129.1, 129.4, 130.4, 132.8, 135.7, 146.4, 166.5; MS
(EI+) m/z 251, 252 (M+, 100, 17); HRMS (ES+) m/z calcd for
C16H14NO2 (MH+) 252.1025, found 252.1037.
2 - { 2 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid Methyl Ester (8a). To a stirred solution of (S)-Fmoc-
Ser(tBu)-OH (6, 3.52 g, 9.18 mmol) and oxalyl chloride (1.35 mL,
15.95 mmol) in CH2Cl2 (100 mL) was added DMF (5 drops) at 0 °C
under an atmosphere of nitrogen. After 20 min, the reaction mixture
was allowed to warm to rt, and stirring was continued for 1 h. The
reaction mixture was then concentrated in vacuo to afford (S)-Fmoc-
Ser(tBu)-Cl (7) as a yellow oil which was used immediately without
further purification.
3-(2-Aminophenylethynyl)benzoic Acid Methyl Ester (5b).
Compound 3b (1.70 g, 7.32 mmol), 2-iodoaniline (4a, 1.77 g, 8.08
mmol), PPh3 (380 mg, 1.45 mmol), Pd(PPh3)2Cl2 (510 mg, 0.727
mmol), and piperidine (10.5 mL, 106 mmol) were dissolved in THF
(100 mL). The reaction mixture was degassed and stirred under an
atmosphere of nitrogen for 20 min before the addition of CuI (285
mg, 1.50 mmol) and K2CO3 (1.21 g, 8.75 mmol). The reaction
mixture was refluxed, MeOH (9.0 mL) was added dropwise, and
heating was continued for 12 h. Additional Pd(PPh3)2Cl2 (51 mg,
0.073 mmol) and CuI (29 mg, 0.15 mmol) were then added, and
stirring was continued for 6 h at reflux. The reaction mixture was
cooled to rt, diluted with CH2Cl2 (150 mL), and then washed with 0.1
M HCl solution (2 × 100 mL), H2O (100 mL) and brine (100 mL).
The organic layer was dried (Na2SO4) and concentrated in vacuo, and
the resulting residue was purified by FC (20−33% v/v, EtOAc/
hexane) to afford 5b as a yellow oil (744 mg, 40%): IR (neat) 3375,
2198, 1721, 1616, 1261, and 752 cm−1; 1H NMR (400 MHz, CDCl3)
δ 3.92 (s, 3H), 4.28 (br s, 2H), 6.74−6.67 (m, 2H), 7.14 (td, J = 8.0
and 1.4 Hz, 1H), 7.35 (dd, J = 8.1 and 1.2 Hz, 1H), 7.41 (t, J = 7.8 Hz,
1H), 7.68 (m, 1H), 7.97 (d, J = 7.9 Hz, 1H), 8.18 (m, 1H); 13C NMR
(100 MHz, CDCl3) δ 52.4, 87.0, 93.7, 107.4, 114.4, 118.0, 123.8,
128.6, 129.1, 130.1, 130.5, 132.3, 132.5, 135.5, 147.9, 166.5; MS (ES+)
m/z 252, 253 (MH+, 100, 21); HRMS (ES+) m/z calcd for
C16H14NO2 (MH+) 252.1025, found 252.1025.
2-(3-Aminophenylethynyl)benzoic Acid Methyl Ester (5c).
Compound 3a (2.60 g, 11.19 mmol), 3-iodoaniline (4b, 2.70 g, 12.33
mmol), PPh3 (580 mg, 2.21 mmol), Pd(PPh3)2Cl2 (780 mg, 1.11
mmol), and piperidine (16.0 mL, 162 mmol) were dissolved in THF
(150 mL). The reaction mixture was degassed and stirred under an
atmosphere of nitrogen for 20 min before the addition of CuI (433
mg, 2.27 mmol) and K2CO3 (1.85 g, 13.39 mmol). The reaction
mixture was refluxed, MeOH (14.0 mL) was added dropwise, and
heating was continued for 12 h. Additional Pd(PPh3)2Cl2 (78 mg, 0.11
mmol) and CuI (43 mg, 0.23 mmol) were then added, and stirring was
continued for 6 h at reflux. The reaction mixture was cooled to rt,
diluted with CH2Cl2 (200 mL), then washed with 0.1 M HCl solution
(2 × 200 mL), H2O (200 mL) and brine (200 mL). The organic layer
was dried (Na2SO4) and concentrated in vacuo and the resulting
residue purified by FC (10−50% v/v, EtOAc/hexane) to afford 5c as
an orange oil (1.12 g, 40%): IR (neat) 3371, 1720, 1598, 1297, 1253,
and 1079 cm−1; 1H NMR (400 MHz, CDCl3) δ 3.69 (br s, 2H), 3.94
(s, 3H), 6.65 (m, 1H), 6.88 (m, 1H), 6.96 (d, J = 7.7 Hz, 1H), 7.12 (t,
J = 7.8 Hz, 1H), 7.35 (td, J = 7.7 and 1.2 Hz, 1H), 7.46 (td, J = 7.6 and
1.3 Hz, 1H), 7.61 (dd, J = 7.8 and 0.7 Hz, 1H), 7.94 (dd, J = 7.9 and
1.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 52.2, 87.6, 94.7, 115.6,
117.9, 122.2, 123.8, 124.0, 127.8, 129.3, 130.4, 131.7, 131.9, 134.0,
146.3, 166.8; MS (ES+) m/z 252, 253 (MH+, 100, 19); HRMS (ES+)
m/z calcd for C16H14NO2 (MH+) 252.1025, found 252.1020.
To a stirred solution of the above prepared (S)-Fmoc-Ser(tBu)-Cl
(7) and 5a (1.92 g, 7.66 mmol) in CH2Cl2 (100 mL) was added
AgCN (1.36 g, 10.12 mmol) under an atmosphere of nitrogen. Stirring
was continued at rt with the exclusion of light for 2 h. The reaction
mixture was then filtered and concentrated in vacuo, and the resulting
residue purified by FC (20% v/v, EtOAc/hexane) to afford 8a as a
white foam (4.49 g, 95%): mp 135−138 °C; IR (neat) 3320, 1696,
1
1528, 1450, 1271, and 1081 cm−1; H NMR (400 MHz, CDCl3) δ
1.06 (s, 9H), 3.64 (dd, J = 9.1 and 5.2 Hz, 1H), 3.84 (dd, J = 9.0 and
4.1 Hz, 1H), 4.01 (s, 3H), 4.18 (t, J = 7.1 Hz, 1H), 4.33 (d, J = 7.0 Hz,
2H), 5.08 (m, 1H), 6.20 (d, J = 8.1 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H),
7.30−7.24 (m, 2H), 7.41−7.30 (m, 4H), 7.55−7.51 (m, 2H), 7.62−
7.56 (m, 2H), 7.68 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 8.07
(d, J = 7.6 Hz, 1H), 8.55 (d, J = 8.2 Hz, 1H), 9.45 (s, 1H); 13C NMR
(100 MHz, CDCl3) δ 27.3, 47.2, 53.1, 55.7, 63.1, 66.9, 73.3, 90.7, 95.1,
112.2, 119.8, 119.9, 123.4, 124.2, 125.2, 127.0, 127.6, 128.2, 129.9,
130.1, 130.8, 132.1, 132.4, 134.0, 140.2, 141.3, 143.9, 156.0, 166.3,
169.7; MS (ES+) m/z 639, 640, 641 (MNa+, 100, 42, 10), 617 (MH+,
13); HRMS (ES+) m/z calcd for C38H37N2O6 (MH+) 617.2652, found
617.2659.
3 - { 2 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid Methyl Ester (8b). To a stirred solution of (S)-Fmoc-
Ser(tBu)-OH (6, 861 mg, 2.25 mmol) and oxalyl chloride (395 μL,
4.53 mmol) in CH2Cl2 (30 mL) was added DMF (2 drops) at 0 °C
under an atmosphere of nitrogen. After 10 min, the reaction mixture
was allowed to warm to rt, and stirring was continued for 1 h. The
reaction mixture was then concentrated in vacuo to afford (S)-Fmoc-
Ser(tBu)-Cl (7) as a yellow oil which was used immediately without
further purification.
To a stirred solution of the above prepared (S)-Fmoc-Ser(tBu)-Cl
(7) and 5b (583 mg, 2.32 mmol) in CH2Cl2 (50 mL) was added
AgCN (324 mg, 2.42 mmol) under an atmosphere of nitrogen. Stirring
was continued at rt with the exclusion of light for 2 h. The reaction
mixture was then filtered and concentrated in vacuo and the resulting
residue purified by FC (10−50% v/v, EtOAc/hexane) to afford 8b as a
white solid (810 mg, 57%): mp 158−160 °C; IR (neat) 3367, 1725,
1524, 1449, 1261, and 756 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.11
(s, 9H), 3.53 (dd, J = 8.8 and 6.7 Hz, 1H), 3.86 (s, 3H), 3.91 (m, 1H),
4.10 (m, 1H), 4.28 (m, 1H), 4.49−4.34 (m, 2H), 5.84 (s, 1H), 7.11 (t,
J = 7.5 Hz, 1H), 7.22 (br s, 2H), 7.43−7.28 (m, 4H), 7.56−7.45 (m,
3H), 7.70 (dd, J = 7.6 and 1.0 Hz, 3H), 7.96 (d, J = 7.7 Hz, 1H), 8.21
(s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.89 (s, 1H); 13C NMR (100 MHz,
CDCl3) δ 27.5, 47.2, 52.5, 56.3, 61.9, 67.4, 74.4, 85.5, 95.5, 112.6,
120.2, 123.1, 124.1, 125.1, 125.2, 127.2, 127.9, 128.8, 129.9, 130.1,
130.7, 132.2, 133.0, 135.8, 138.7, 141.4, 143.8, 143.8, 156.4, 166.4,
168.8; MS (ES+) m/z 639, 640, 641 (MNa+, 100, 45, 11), 617, 618
(MH+, 34, 14); HRMS (ES+) m/z calcd for C38H37N2O6 (MH+)
617.2652, found 617.2667.
3-(3-Aminophenylethynyl)benzoic Acid Methyl Ester (5d).
Compound 3b (1.70 g, 7.32 mmol), 3-iodoaniline (4b, 1.77 g, 8.08
mmol), PPh3 (381 mg, 1.45 mmol), Pd(PPh3)2Cl2 (510 mg, 0.727
mmol), and piperidine (10.5 mL, 106 mmol) were dissolved in THF
(100 mL). The reaction mixture was degassed and stirred under an
atmosphere of nitrogen for 20 min before the addition of CuI (285
mg, 1.50 mmol) and K2CO3 (1.21 g, 8.75 mmol). The reaction
mixture was refluxed, MeOH (9.0 mL) was added dropwise, and
heating was continued for 12 h. Additional Pd(PPh3)2Cl2 (52 mg,
0.074 mmol) and CuI (28 mg, 0.147 mmol) were then added, and
stirring was continued for 6 h at reflux. The reaction mixture was
cooled to rt, diluted with CH2Cl2 (150 mL), and then washed with 0.1
M HCl solution (2 × 100 mL), H2O (100 mL) and brine (100 mL).
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Article
2 - { 3 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid Methyl Ester (8c). To a stirred solution of (S)-Fmoc-
Ser(tBu)-OH (6, 820 mg, 2.14 mmol) and oxalyl chloride (375 μL,
4.30 mmol) in CH2Cl2 (30 mL) was added DMF (2 drops) at 0 °C
under an atmosphere of nitrogen. After 10 min, the reaction mixture
was allowed to warm to rt, and stirring was continued for 1 h. The
reaction mixture was then concentrated in vacuo to afford (S)-Fmoc-
Ser(tBu)-Cl (7) as a yellow oil which was used immediately without
further purification.
(200 mL) and extracted with ethyl acetate (3 × 100 mL). The
combined organic fractions were dried (MgSO4) and concentrated in
vacuo, and the resulting residue was purified by FC (10−50% v/v,
EtOAc/hexane) to afford 9a as an off-white foam (1.35 g, 62%,
20
>99.8% ee by HPLC): mp 90−93 °C; [α]D +10.2 (c 1.01, CHCl3);
HPLC [Chiralpak AS-H, isocratic TFA/iPrOH/hexane (0.5:9.5:90),
40 °C, 0.25 mL/min] Rt = 19.8 min; IR (neat) 3303, 1688, 1526,
1
1450, 1261, and 757 cm−1; H NMR (400 MHz, CDCl3) δ 1.13 (s,
9H), 3.62 (dd, J = 9.1 and 6.7 Hz, 1H), 3.76 (dd, J = 9.2 and 5.6 Hz,
1H), 4.19 (t, J = 7.0 Hz, 1H), 4.44−4.31 (m, 2H), 5.30 (m, 1H), 5.93
(d, J = 9.0 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.3 Hz, 2H),
7.36 (dd, J = 13.8 and 6.8 Hz, 4H), 7.51 (t, J = 7.2 Hz, 2H), 7.60−7.54
(m, 2H), 7.63 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 7.5 Hz, 2H), 8.09 (d, J
= 7.8 Hz, 1H), 8.52 (d, J = 8.3 Hz, 1H), 9.62 (s, 1H); 13C NMR (100
MHz, CDCl3) δ 27.3, 47.1, 55.4, 60.4, 63.9, 65.9, 67.5, 74.4, 90.3, 95.6,
112.5, 119.7, 120.0, 123.6, 123.9, 125.1, 127.1, 127.8, 128.2, 130.0,
131.1, 132.0, 132.2, 133.4, 140.3, 141.3, 143.7, 143.8, 156.8, 168.1,
169.3; MS (ES+) m/z 625, 626, (MNa+, 100, 45), 603, 604 (MH+, 90,
36); HRMS (ES+) m/z calcd for C37H35N2O6 (MH+) 603.2495, found
603.2510.
To a stirred solution of the above prepared (S)-Fmoc-Ser(tBu)-Cl
(7) and 5c (555 mg, 2.21 mmol) in CH2Cl2 (50 mL) was added
AgCN (308 mg, 2.30 mmol) under an atmosphere of nitrogen. Stirring
was continued at rt with the exclusion of light for 2 h. The reaction
mixture was then filtered and concentrated in vacuo and the resulting
residue purified by FC (10−40% v/v, EtOAc/hexane) to afford 8c as
an off-white foam (929 mg, 68%). The product was obtained as a 5:1
mixture of rotamers as observed by 1H NMR spectroscopy: mp 137−
1
138 °C; IR (neat) 3317, 1715, 1547, 1490, 1253, and 1080 cm−1; H
NMR (400 MHz, CDCl3) δ 1.26 (s, 9H), 3.45 (m, 1H), 3.92 (m, 1H),
3.95 (s, 3H), 4.23 (t, J = 6.9 Hz, 1H), 4.35 (br s, 1H), 4.49−4.38 (m,
2H), 5.84 (br s, 1H), 7.41−7.26 (m, 7H), 7.53−7.45 (m, 2H), 7.65−
7.57 (m, 3H), 7.79−7.69 (m, 3H), 7.97 (d, J = 7.9 Hz, 1H), 8.91 and
8.74 (each br s, minor and major, total 1H); 13C NMR (100 MHz,
CDCl3) δ 27.7, 47.3, 52.4, 55.0, 61.9, 67.4, 75.1, 88.8, 94.0, 120.1,
120.2, 122.8, 123.7, 124.4, 125.3, 127.3, 128.0, 128.2, 129.4, 130.7,
131.9, 132.1, 134.2, 137.8, 141.5, 144.0, 156.3, 166.9, 168.7; MS (ES+)
m/z 639, 640 (MNa+, 100, 42), 617, 618 (MH+, 39, 16); HRMS (ES
+) m/z calcd for C38H37N2O6 (MH+) 617.2652, found 617.2646.
3 - { 3 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid Methyl Ester (8d). To a stirred solution of (S)-Fmoc-
Ser(tBu)−OH (6, 680 mg, 1.77 mmol) and oxalyl chloride (310 μL,
3.55 mmol) in CH2Cl2 (25 mL) was added DMF (1 drop) at 0 °C
under an atmosphere of nitrogen. After 10 min, the reaction mixture
was allowed to warm to rt, and stirring was continued for 1 h. The
reaction mixture was then concentrated in vacuo to afford (S)-Fmoc-
Ser(tBu)-Cl (7) as a yellow oil which was used immediately without
further purification.
3 - { 2 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid (9b). To an ice-cold solution of 8b (780 mg, 1.62
mmol) in THF (40 mL) was added LiAlH4 (145 mg, 3.82 mmol) and
stirring continued under an atmosphere of nitrogen for 20 min. The
reaction mixture was then diluted with diethyl ether (100 mL) and
cautiously quenched with a saturated solution of Rochelle’s salt (50
mL). The organic phase was then separated, washed with H2O (2 ×
100 mL), dried (MgSO4), and concentrated in vacuo. The resulting
residue was dissolved in acetone (40 mL), a solution of 1 M Jones’
reagent in acetone (4.10 mL) was added dropwise, and stirring was
continued at rt for 2 h. The reaction mixture was diluted with H2O
(100 mL) and extracted with ethyl acetate (3 × 50 mL). The
combined organic fractions were dried (MgSO4) and concentrated in
vacuo, and the resulting residue was purified by FC (50% v/v, EtOAc/
hexane) to afford 9b as an off-white foam (335 mg, 44%): mp 121−
27
124 °C; [α]D +4.3 (c 1.10, EtOAc); IR (neat) 3303, 1685, 1525,
1
1260, 1078, and 738 cm−1; H NMR (400 MHz, CDCl3) δ 1.12 (s,
9H), 3.56 (t, J = 7.6 Hz, 1H), 3.90 (m, 1H), 4.11 (t, J = 7.1 Hz, 1H),
4.29 (m, 1H), 4.41 (m, 1H), 4.49 (m, 1H), 5.91 (br s, 1H), 7.11 (t, J =
7.5 Hz, 1H), 7.25−7.15 (m, 2H), 7.31 (t, J = 7.3 Hz, 2H), 7.42−7.35
(m, 2H), 7.55−7.46 (m, 3H), 7.68 (d, J = 7.3 Hz, 2H), 7.73 (d, J = 7.4
Hz, 1H), 7.97 (d, J = 7.5 Hz, 1H), 8.24 (br s, 1H), 8.41 (m, 1H), 8.90
(s, 1H); 13C NMR (100 MHz, CDCl3) δ 27.5, 47.3, 56.2, 61.9, 66.1,
67.5, 74.5, 95.3, 112.6, 120.2, 123.2, 124.1, 125.2, 127.2, 127.9, 128.9,
130.2, 130.4, 132.3, 133.5, 136.5, 138.7, 141.5, 143.8, 156.5, 169.0,
170.3; MS (ES+) m/z 625, 626, (MNa+, 100, 42), 603, 604 (MH+, 97,
42); HRMS (ES+) m/z calcd for C37H35N2O6 (MH+) 603.2495, found
603.2518.
To a stirred solution of the above prepared (S)-Fmoc-Ser(tBu)-Cl
(7) and 5d (460 mg, 1.83 mmol) in CH2Cl2 (30 mL) was added
AgCN (255 mg, 1.90 mmol) under an atmosphere of nitrogen. Stirring
was continued at rt with the exclusion of light for 2 h. The reaction
mixture was then filtered and concentrated in vacuo and the resulting
residue purified by FC (10−40% v/v, EtOAc/hexane) to afford 8d as
an off-white foam (892 mg, 79%): mp 179−182 °C; IR (neat) 3317,
1
1723, 1263, 910, and 739 cm−1; H NMR (400 MHz, CDCl3) δ 1.26
(s, 9H), 3.46 (m, 1H), 3.91 (m, 1H), 3.92 (s, 3H), 4.23 (t, J = 6.9 Hz,
1H), 4.36 (br s, 1H), 4.47−4.40 (m, 2H), 5.85 (br s, 1H), 7.33−7.26
(m, 4H), 7.41−7.36 (m, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.46 (dt, J = 7.3
and 2.1 Hz, 1H), 7.60 (d, J = 7.2 Hz, 2H), 7.68 (m, 1H), 7.78−7.71
(m, 3H), 7.99 (m, 1H), 8.20 (t, J = 1.3 Hz, 1H), 8.75 (s, 1H); 13C
NMR (100 MHz, CDCl3) δ 27.7, 47.3, 52.5, 55.0, 60.6, 61.9, 67.4,
75.07, 88.8, 90.0, 120.1, 120.2, 122.8, 123.7, 123.9, 125.3, 127.3, 127.9,
128.0, 128.7, 129.4, 129.5, 130.7, 133.0, 135.9, 137.8, 141.5, 143.9,
156.3, 166.6, 168.7; MS (ES+) m/z 639, 640 (MNa+, 100, 48), 617,
618 (MH+, 73, 30); HRMS (ES+) m/z calcd for C38H37N2O6 (MH+)
617.2652, found 617.2643.
2 - { 2 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid (9a). To an ice-cold solution of 8a (2.24 g, 3.63 mmol)
in THF (120 mL) was added LiAlH4 (413 mg, 10.88 mmol) and
stirring continued under an atmosphere of nitrogen for 20 min. The
reaction mixture was then diluted with diethyl ether (200 mL) and
cautiously quenched with a saturated solution of Rochelle’s salt (100
mL). The organic phase was then separated, washed with H2O (2 ×
200 mL), dried (MgSO4), and concentrated in vacuo. The resulting
residue was dissolved in acetone (120 mL), a solution of 1 M Jones’
reagent in acetone (12.0 mL) was added dropwise, and stirring was
continued at rt for 2 h. The reaction mixture was diluted with H2O
2 - { 3 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid (9c). To an ice-cold solution of 8c (892 mg, 1.45
mmol) in THF (50 mL) was added LiAlH4 (166 mg, 4.37 mmol) and
stirring continued under an atmosphere of nitrogen for 20 min. The
reaction mixture was then diluted with diethyl ether (100 mL) and
cautiously quenched with a saturated solution of Rochelle’s salt (50
mL). The organic phase was then separated, washed with H2O (2 ×
100 mL), dried (MgSO4), and concentrated in vacuo. The resulting
residue was dissolved in acetone (50 mL), a solution of 1 M Jones’
reagent in acetone (4.70 mL) was added dropwise, and stirring was
continued at rt for 2 h. The reaction mixture was diluted with H2O
(100 mL) and extracted with ethyl acetate (3 × 50 mL). The
combined organic fractions were dried (MgSO4) and concentrated in
vacuo, and the resulting residue was purified by FC (50−75% v/v,
EtOAc/hexane) to afford 9c as an off-white foam (505 mg, 58%): mp
27
102−103 °C; [α]D −8.6 (c 0.92, CHCl3); IR (neat) 3410, 1683,
1557, 1238, 1079, and 737 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.20
(s, 9H), 3.46 (t, J = 8.5 Hz, 1H), 3.85 (m, 1H), 4.19 (t, J = 6.9 Hz,
1H), 4.48−4.32 (m, 3H), 6.00 (m, 1H), 7.18 (t, J = 7.8 Hz, 1H),
7.30−7.24 (m, 3H), 7.41−7.33 (m, 3H), 7.45 (d, J = 7.8 Hz, 1H), 7.50
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Article
(t, J = 7.6 Hz 1H), 7.58 (d, J = 5.9 Hz, 2H), 7.63 (d, J = 7.6 Hz, 1H),
7.75−7.68 (m, 3H), 8.08 (d, J = 7.9 Hz, 1H), 8.79 (s, 1H); 13C NMR
(100 MHz, CDCl3) δ 27.6, 47.3, 55.1, 62.0, 67.5, 75.1, 88.6, 95.0,
120.2, 120.3, 122.9, 124.1, 124.3, 125.3, 127.3, 128.0, 128.3, 129.3,
131.1, 131.5, 132.6, 134.4, 137.7, 141.5, 144.0, 156.5, 168.9, 170.3; MS
(ES+) m/z 625, 626, (MNa+, 100, 42), 603, 604 (MH+, 57, 23);
HRMS (ES+) m/z calcd for C37H35N2O6 (MH+) 603.2495, found
603.2501.
0.688 mmol) in THF (30 mL) was added LiAlH4 (80 mg, 2.11 mmol)
and stirring continued under an atmosphere of nitrogen for 20 min.
The reaction mixture was then diluted with diethyl ether (60 mL) and
cautiously quenched with a saturated solution of Rochelle’s salt (30
mL). The organic phase was then separated, washed with H2O (2 × 60
mL), dried (MgSO4), and concentrated in vacuo. The resulting residue
was dissolved in acetone (30 mL), a solution of 1 M Jones’ reagent in
acetone (2.10 mL) was added dropwise, and stirring was continued at
rt for 2 h. The reaction mixture was diluted with H2O (60 mL) and
extracted with ethyl acetate (3 × 30 mL). The combined organic
fractions were dried (MgSO4) and concentrated in vacuo, and the
resulting residue was purified by FC (10−50% v/v, EtOAc/hexane) to
afford ent-9a as a white foam (315 mg, 76%, >97.2% ee by HPLC):
3 - { 3 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid (9d). To an ice-cold solution of 8d (860 mg, 1.40
mmol) in THF (50 mL) was added LiAlH4 (160 mg, 4.22 mmol) and
stirring continued under an atmosphere of nitrogen for 20 min. The
reaction mixture was then diluted with diethyl ether (100 mL) and
cautiously quenched with a saturated solution of Rochelle’s salt (50
mL). The organic phase was then separated, washed with H2O (2 ×
100 mL), dried (MgSO4), and concentrated in vacuo. The resulting
residue was dissolved in acetone (50 mL), a solution of 1 M Jones’
reagent in acetone (4.50 mL) was added dropwise, and stirring was
continued at rt for 2 h. The reaction mixture was diluted with H2O
(100 mL) and extracted with ethyl acetate (3 × 50 mL). The
combined organic fractions were dried (MgSO4) and concentrated in
vacuo, and the resulting residue was purified by FC (50−75% v/v,
EtOAc/hexane) to afford 9d as a pale yellow solid (360 mg, 43%): mp
20
mp 88−91 °C; [α]D −10.5 (c 1.11, CHCl3); HPLC [Chiralpak AS-
H, isocratic TFA/iPrOH/hexane (0.5:9.5:90), 40 °C, 0.25 mL/min] tR
= 23.9 min; IR (neat) 3303, 1687, 1526, 1450, 1260, and 1078 cm−1;
1H NMR (400 MHz, CDCl3) δ 1.14 (s, 9H), 3.58 (dd, J = 9.1 and 7.3
Hz, 1H), 3.77 (dd, J = 9.3 and 5.2 Hz, 1H), 4.20 (t, J = 7.1 Hz, 1H),
4.44−4.33 (m, 2H), 5.34 (m, 1H), 5.95 (d, J = 9.1 Hz, 1H), 7.08 (t, J =
7.5 Hz, 1H), 7.28 (t, J = 7.3 Hz, 2H), 7.40−7.34 (m, 4H), 7.59−7.50
(m, 4H), 7.65 (d, J = 7.2 Hz, 1H), 7.74 (d, J = 7.5 Hz, 2H), 8.13 (d, J
= 7.8 Hz, 1H), 8.54 (d, J = 8.4 Hz, 1H), 9.68 (s, 1H); 13C NMR NMR
(100 MHz, CDCl3) δ 27.2, 46.9, 55.2, 63.9, 65.8, 67.3, 74.4, 90.2, 95.4,
112.3, 119.5, 119.9, 123.5, 123.9, 125.0, 127.0, 127.6, 128.1, 129.9,
130.5, 131.1, 131.9, 132.1, 133.4, 140.2, 141.2, 143.5, 143.6, 156.7,
167.8, 169.1; MS (ES+) m/z 625, 626, (MNa+, 100, 43), 603, 604
(MH+, 65, 27); HRMS (ES+) m/z calcd for C37H35N2O6 (MH+)
603.2495, found 603.2484.
2-[2-(2-Aminophenyl)ethyl]benzoic Acid Methyl Ester (10).
To a solution of 5a (500 mg, 1.99 mmol) in ethyl acetate (50 mL) was
added 10% palladium on charcoal (50 mg). The mixture was degassed,
flushed with dihydrogen, and stirred at rt for 16 h under an
atmosphere of dihydrogen. The reaction mixture was then filtered and
concentrated in vacuo to afford bibenzyl 10 as an off-white solid (505
mg, 99%): mp 51−53 °C; IR (neat) 3383, 1715, 1497, 1271, 1082, and
748 cm−1; 1H NMR (400 MHz, CDCl3) δ 2.85−2.74 (m, 2H), 3.22−
3.13 (m, 2H), 3.90 (s, 3H), 4.27 (br s, 2H), 6.75−6.66 (m, 2H), 7.12−
7.00 (m, 2H), 7.35−7.26 (m, 2H), 7.47 (td, J = 7.5 and 1.3 Hz, 1H),
7.96 (dd, J = 7.9 and 1.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
35.0, 35.3, 52.2, 115.5, 118.2, 125.9, 126.3, 127.3, 128.7, 130.2, 131.0,
131.6, 132.5, 144.4, 145.1, 167.9; MS (ES+) m/z 256, 257 (MH+, 100,
20), 224 (51); HRMS (ES+) m/z calcd for C16H18NO2 (MH+)
256.1338, found 256.1350.
27
158−161 °C; [α]D −14.1 (c 1.00 EtOAc); IR (Nujol mull) 3283,
1
1695, 1661, 1463, 1082, and 740 cm−1; H NMR (400 MHz, d6-
Acetone) δ 1.18 (s, 9H), 3.69 (dd, J = 9.1 and 6.1 Hz, 1H), 3.80 (dd, J
= 9.1 and 4.9 Hz, 1H), 4.26 (t, J = 7.0 Hz, 1H), 4.46−4.34 (m, 3H),
7.35−7.28 (m, 3H), 7.44−7.35 (m, 3H), 7.58 (t, J = 7.8 Hz, 1H),
7.76−7.65 (m, 3H), 7.80 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 7.5 Hz, 2H),
8.02 (br s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 8.19 (m, 1H); 13C NMR
(100 MHz, d6-Acetone) δ 28.1, 48.4, 57.5, 63.3, 67.8, 74.6, 89.3, 91.2,
121.3, 123.5, 124.4, 124.8, 126.6, 128.2, 128.4, 129.0, 130.3, 130.5,
130.8, 132.5, 133.7, 136.9, 140.4, 142.5, 145.4, 145.5, 157.4, 167.3,
170.4; MS (ES+) m/z 625, 626, (MNa+, 52, 22), 603, 604 (MH+, 100,
42); HRMS (ES+) m/z calcd for C37H35N2O6 (MH+) 603.2495, found
603.2503.
2 - { 2 - [ ( R ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid Methyl Ester (ent-8a). To a stirred solution of (R)-
Fmoc-Ser(tBu)-OH (ent-6, 533 mg, 1.39 mmol) and oxalyl chloride
(210 μL, 2.41 mmol) in CH2Cl2 (25 mL) was added DMF (1 drop) at
0 °C under an atmosphere of nitrogen. After 10 min the reaction
mixture was allowed to warm to rt and stirring was continued for 1 h.
The reaction mixture was then concentrated in vacuo to afford (R)-
Fmoc-Ser(tBu)-Cl (ent-7) as a yellow oil which was used immediately
without further purification.
2 - ( 2 - { 2 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenyl}ethyl)-
benzoic Acid Methyl Ester (11). To a stirred solution of (S)-Fmoc-
Ser(tBu)-OH (6, 370 mg, 0.965 mmol) and oxalyl chloride (170 μL,
1.95 mmol) in CH2Cl2 (20 mL) was added DMF (1 drop) at 0 °C
under an atmosphere of nitrogen. After 10 min, the reaction mixture
was allowed to warm to rt and stirring was continued for 1 h. The
reaction mixture was then concentrated in vacuo to afford (S)-Fmoc-
Ser(tBu)-Cl (7) as a yellow oil which was used immediately without
further purification.
To a stirred solution of the above prepared (R)-Fmoc-Ser(tBu)-Cl
(ent-7) and 2,2′-tolan methyl ester 5a (315 mg, 1.25 mmol) in CH2Cl2
(25 mL) was added AgCN (185 mg, 1.57 mmol) under an atmosphere
of nitrogen. Stirring was continued at rt with the exclusion of light for
2 h. The reaction mixture was then filtered and concentrated in vacuo
and the resulting residue purified by FC (20% v/v, EtOAc/hexane) to
afford dipeptide methyl ester ent-8a as a white foam (686 mg, 89%):
mp 133−136 °C; IR (neat) 3320, 1701, 1527, 1450, 1271, and 1081
To a stirred solution of the above prepared (S)-Fmoc-Ser(tBu)-Cl
(7) and aniline 10 (250 mg, 0.979 mmol) in CH2Cl2 (25 mL) was
added AgCN (140 mg, 1.05 mmol) under an atmosphere of nitrogen.
Stirring was continued at rt with the exclusion of light for 2 h. The
reaction mixture was then filtered and concentrated in vacuo and the
resulting residue purified by FC (25−50% v/v, EtOAc/hexane) to
afford bibenzyl dipeptide 11 as a white solid (540 mg, 90%): mp 137−
1
cm−1; H NMR (400 MHz, CDCl3) δ 1.05 (s, 9H), 3.64 (dd, J = 9.1
and 5.1 Hz, 1H), 3.84 (dd, J = 9.1 and 4.1 Hz, 1H), 4.01 (s, 3H), 4.17
(t, J = 7.1 Hz, 1H), 4.32 (d, J = 7.1 Hz, 2H), 5.08 (m, 1H), 6.20 (d, J =
8.2 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.29−7.25 (m, 2H), 7.41−7.31
(m, 4H), 7.55−7.51 (m, 2H), 7.61−7.56 (m, 2H), 7.68 (d, J = 7.7 Hz,
1H), 7.72 (d, J = 7.5 Hz, 2H), 8.07 (d, J = 7.8 Hz, 1H), 8.54 (d, J = 8.3
Hz, 1H), 9.45 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 27.3, 47.2,
53.1, 55.7, 63.1, 66.9, 73.3, 90.7, 95.1, 169.7, 112.2, 119.8, 119.9, 123.4,
124.2, 125.2, 125.2, 127.0, 127.7, 128.2, 129.9, 130.1, 130.8, 132.1,
132.4, 134.0, 140.2, 141.3, 143.9, 144.0, 156.0, 166.3; MS (ES+) m/z
639, 640, 641 (MNa+, 100, 44, 8), 617 (MH+, 13); HRMS (ES+) m/z
calcd for C38H37N2O6 (MH+) 617.2652, found 617.2658.
1
139 °C; IR (neat) 3356, 1692, 1534, 1450, 1252, and 1079 cm−1; H
NMR (400 MHz, CDCl3) δ 1.06 (s, 9H), 2.88 (dd, J = 9.4 and 8.0 Hz,
2H), 3.21−3.01 (m, 2H), 3.57 (m, 1H), 3.85 (dd, J = 8.8 and 3.9 Hz,
1H), 3.90 (s, 3H), 4.22 (t, J = 6.8 Hz, 1H), 4.40 (dd, J = 10.4 and 6.8
Hz, 1H), 4.48 (m, 1H), 4.85 (s, 1H), 6.37 (d, J = 8.1 Hz, 1H), 7.11 (t,
J = 7.3 Hz, 1H), 7.39−7.20 (m, 8H), 7.49 (t, J = 7.4 Hz, 1H), 7.62−
7.57 (m, J = 4.0, 7.1, 2H), 7.68 (d, J = 7.4 Hz, 1H), 7.72 (d, J = 7.5 Hz,
1H), 8.02 (d, J = 7.7 Hz, 1H), 8.08 (d, J = 7.9 Hz, 1H), 8.84 (s, 1H);
13C NMR (100 MHz, CDCl3) δ 27.5, 34.9, 37.2, 47.4, 52.9, 55.8, 63.0,
66.9, 73.7, 120.1, 123.2, 125.1, 125.2, 126.7, 127.2, 127.8, 128.2, 130.3,
2 - { 2 - [ ( R ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenylethynyl}-
benzoic Acid (ent-9a). To an ice-cold solution of ent-8a (424 mg,
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1
1
131.5, 131.9, 132.2, 133.2, 135.8, 141.5, 141.5, 144.0, 144.1, 144.5,
156.2, 168.1, 169.7; MS (ES+) m/z 643, 644 (MNa+, 97, 49), 621, 622
(MH+, 100, 42); HRMS (ES+) m/z calcd for C38H41N2O6 (MH+)
621.2965, found 621.2952.
rotamers as observed by H NMR spectroscopy: mp 53−56 °C; H
NMR (400 MHz, CDCl3) δ 1.22 (s, 9H), 3.48 (m, 1H), 3.84 (dd, J =
8.7 and 3.7 Hz, 1H), 3.88 (s, 3H), 4.23 (t, J = 7.1 Hz, 1H), 4.33 (m,
1H), 4.40 (d, J = 7.2 Hz, 2H), 5.90 (br d, J = 4.9 Hz, 1H), 6.62 (d, J =
11.9 Hz, 1H), 6.89 (t, J = 7.4 Hz, 1H), 6.95 (d, J = 6.5 Hz, 1H), 7.03
(m, 1H), 7.32−7.14 (m, 7H), 7.38 (t, J = 7.5 Hz, 2H), 7.61 (d, J = 7.1
Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.87 (m, 1H), 8.02 (d, J = 8.2 Hz,
1H), 8.83 and 8.43 (each br s, minor and major, total 1H); 13C NMR
(100 MHz, CDCl3) δ 27.5, 27.7, 47.3, 52.4, 55.6, 62.2, 67.3, 74.7,
120.2, 122.0, 124.5, 125.3, 125.6, 127.3, 127.6, 127.9, 128.2, 129.4,
130.4, 130.6, 131.1, 132.1, 133.9, 135.3, 138.4, 141.5, 143.9, 144.1,
156.2, 167.9, 168.8; MS (ES+) m/z 641, 642 (MNa+, 100, 42); HRMS
(ES+) m/z calcd for C38H39N2O6 (MH+) 619.2808, found 619.2802.
2-((E)-2-{2-[( S)-3-tert-Butoxy-2-(9H-fluoren-9-
ylmethoxycarbonylamino)propionylamino]phenyl}vinyl)-
benzoic Acid Methyl Ester (15). To a solution of (Z)-stilbene
dipeptide methyl ester 14 (875 mg, 1.42 mmol) in CHCl3 (10 mL)
was added silica gel (200 mg) and Pd(MeCN)2Cl2 (40 mg, 0.154
mmol). The reaction mixture was degassed and then stirred at rt under
an atmosphere of nitrogen for 16 h. The mixture was filtered and
concentrated in vacuo and the resulting residue purified by FC (10−
50% v/v, EtOAc/hexane) to afford (E)-stilbene dipeptide methyl ester
2 - ( 2 - { 2 - [ ( S ) - 3 - t e r t - B u t o x y - 2 - ( 9 H - f l u o r e n - 9 -
ylmethoxycarbonylamino)propionylamino]phenyl}ethyl)-
benzoic Acid (12). To an ice-cold solution of ester 11 (510 mg,
0.823 mmol) in THF (30 mL) was added LiAlH4 (94 mg, 2.48 mmol)
and stirring continued under an atmosphere of nitrogen for 20 min.
The reaction mixture was then diluted with diethyl ether (50 mL) and
cautiously quenched with a saturated solution of Rochelle’s salt (50
mL). The organic phase was then separated, washed with H2O (2 ×
100 mL), dried (MgSO4), and concentrated in vacuo. The resulting
residue was dissolved in acetone (30 mL), a solution of 1 M Jones’
reagent in acetone (2.50 mL) was added dropwise, and stirring was
continued at rt for 2 h. The reaction mixture was diluted with H2O
(100 mL) and extracted with ethyl acetate (3 × 50 mL). The
combined organic fractions were dried (MgSO4) and concentrated in
vacuo, and the resulting residue was purified by FC (100% CH2Cl2,
then 50% v/v, EtOAc/hexane) to afford acid 12 as a white solid (241
mg, 48%).
Alternatively, dipeptide tolan acid 9a (50 mg, 0.083 mmol) was
dissolved in ethyl acetate (20 mL) and 10% palladium on charcoal (5
mg) was added. The mixture was degassed, flushed with dihydrogen,
and stirred at rt for 16 h under an atmosphere of dihydrogen. The
reaction mixture was then filtered and concentrated in vacuo to afford
acid 12 as a white solid (42 mg, 84%): mp 139−141 °C; [α]D26 +10.2
(c 1.01, CHCl3); IR (neat) 3333, 1686, 1540, 1452, 1247, and 737
cm−1; 1H NMR (400 MHz, d6-Acetone) δ 1.11 (s, 9H), 2.94 (t, J = 8.4
Hz, 2H), 3.27−3.17 (m, 2H), 3.71 (dd, J = 9.0 and 5.9 Hz, 1H), 3.84
(dd, J = 9.0 and 4.8 Hz, 1H), 4.23 (t, J = 7.0 Hz, 1H), 4.36 (d, J = 6.7
Hz, 2H), 4.78 (m, 1H), 7.08 (t, J = 7.3 Hz, 1H), 7.21 (t, J = 7.5 Hz,
1H), 7.40−7.24 (m, 7H), 7.44 (d, J = 7.2 Hz, 1H), 7.49 (t, J = 7.2 Hz,
1H), 7.70 (t, J = 6.7 Hz, 2H), 7.82 (d, J = 7.5 Hz, 2H), 8.10−8.02 (m,
2H); 13C NMR (100 MHz, d6-Acetone) δ 28.0, 35.3, 37.5, 48.3, 55.3,
57.0, 63.7, 67.9, 74.3, 121.1, 124.3, 125.8, 126.5, 127.6, 127.7, 128.3,
128.9, 130.1, 131.2, 132.5, 132.9, 133.8, 137.4, 142.4, 145.2, 145.3,
157.5, 170.3, 170.4; MS (ES+) m/z 629, 630 (MNa+, 58, 28), 607, 608
(MH+, 100, 47); HRMS (ES+) m/z calcd for C37H39N2O6 (MH+)
607.2808, found 607.2800.
2-((Z)-2-{2-[(S)-3-tert-Butoxy-2-(9H-fluoren-9-
ylmethoxycarbonylamino)propionylamino]phenyl}vinyl)-
benzoic Acid (13). To a solution of tolan acid 9a (100 mg, 0.166
mmol) in ethyl acetate (25 mL) was added Lindlar’s catalyst (10 mg,
5% Pd loading). The mixture was degassed, flushed with dihydrogen,
and stirred at rt for 16 h under an atmosphere of dihydrogen. The
reaction mixture was then filtered and concentrated in vacuo and the
resulting residue purified by FC (50−75% v/v, EtOAc/hexane) to
afford (Z)-stilbene dipeptide acid 13 as a white solid (66 mg, 66%):
mp 147−150 °C; [α]D27 +10.5 (c 1.00, CHCl3); 1H NMR (400 MHz,
CD3OD) δ 1.21 (s, 9H), 3.66 (m, 1H), 3.72 (m, 1H), 4.23 (t, J = 6.7
Hz, 1H), 4.39 (m, 1H), 4.42 (d, J = 6.7 Hz, 2H), 6.63 (d, J = 12.0 Hz,
1H), 6.95−6.87 (m, 2H), 7.31−7.07 (m, 8H), 7.36 (t, J = 7.4 Hz, 2H),
7.60 (d, J = 7.9 Hz, 1H), 7.69−7.63 (m, J = 5.2 Hz, 2H), 7.78 (d, J =
7.5 Hz, 2H), 7.92 (d, J = 7.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
27.6, 47.3, 55.5, 62.4, 67.6, 74.8, 120.2, 121.8, 124.4, 125.3, 127.3,
127.6, 128.0, 128.1, 130.3, 131.2, 132.4, 134.9, 135.3, 139.1, 141.5,
144.0, 156.6, 168.7, 171.4; MS (ES+) m/z 627, 628 (MNa+, 100, 42),
605, 606 (MH+, 94, 39); HRMS (ES+) m/z calcd for C37H37N2O6
(MH+) 605.2652, found 605.2652.
2-((Z)-2-{2-[(S)-3-tert-Butoxy-2-(9H-fluoren-9-
ylmethoxycarbonylamino)propionylamino]phenyl}vinyl)-
benzoic Acid Methyl Ester (14). To a solution of tolan dipeptide
methyl ester 8a (987 mg, 1.60 mmol) and benzoic acid (290 mg, 2.37
mmol) in ethyl acetate (100 mL) was added Lindlar’s catalyst (100
mg, 5% Pd loading). The mixture was degassed, flushed with
dihydrogen, and stirred at rt for 16 h under an atmosphere of
dihydrogen. The reaction mixture was then filtered and concentrated
in vacuo to afford (Z)-stilbene dipeptide methyl ester 14 as a white
foam (953 mg, 97%). The product was obtained as a 5:1 mixture of
1
15 as a white solid (646 mg, 74%): mp 151−153 °C; H NMR (400
MHz, CDCl3) δ 1.09 (s, 9H), 3.53 (m, 1H), 3.88 (s, 3H), 3.94 (m,
1H), 4.16 (t, J = 7.0 Hz, 1H), 4.36 (d, J = 7.3 Hz, 2H), 4.47 (m, 1H),
6.00 (br s, 1H), 6.99 (d, J = 16.0 Hz, 1H), 7.39−7.16 (m, 9H), 7.44 (t,
J = 7.4 Hz, 1H), 7.54 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 7.2 Hz, 1H),
7.65 (d, J = 6.4 Hz, 1H), 7.72 (d, J = 7.5 Hz, 2H), 7.93−7.81 (m, 3H),
8.56 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 27.5, 47.3, 52.4, 55.5,
62.1, 67.3, 74.7, 76.9, 77.2, 77.6, 120.2, 124.1, 125.2, 125.3, 125.8,
126.6, 127.2, 127.8, 127.8, 127.9, 128.6, 130.4, 130.8, 131.8, 132.5,
134.6, 139.5, 141.5, 143.9, 156.4, 167.8, 169.1; MS (ES+) m/z 641,
642 (MNa+, 100, 42); HRMS (ES+) m/z calcd for C38H39N2O6
(MH+) 619.2808, found 619.2816.
2-((E)-2-{2-[( S)-3-tert-Butoxy-2-(9H-fluoren-9-
ylmethoxycarbonylamino)propionylamino]phenyl}vinyl)-
benzoic Acid (16). To an ice-cold solution of (E)-stilbene dipeptide
methyl ester 15 (530 mg, 0.857 mmol) in THF (30 mL) was added
LiAlH4 (100 mg, 2.64 mmol) and stirring continued under an
atmosphere of nitrogen for 20 min. The reaction mixture was then
diluted with diethyl ether (50 mL) and cautiously quenched with a
saturated solution of Rochelle’s salt (50 mL). The organic phase was
then separated, washed with H2O (2 × 100 mL), dried (MgSO4), and
concentrated in vacuo. The resulting residue was dissolved in acetone
(30 mL), a solution of 1 M Jones’ reagent in acetone (3.00 mL) was
added dropwise, and stirring was continued at rt for 2 h. The reaction
mixture was diluted with H2O (100 mL) and extracted with ethyl
acetate (3 × 50 mL). The combined organic fractions were dried
(MgSO4) and concentrated in vacuo, and the resulting residue was
purified by FC (50% v/v, EtOAc/hexane) to afford (E)-stilbene
dipeptide acid 16 as an off-white solid (207 mg, 40%): mp 159−162
°C; [α]D −4.4 (c 0.40, EtOAc); H NMR (400 MHz, CD3OD) δ
1.17 (s, 9H), 3.70 (m, 1H), 3.77 (dd, J = 9.0 and 5.1 Hz, 1H), 4.18 (t, J
= 6.8 Hz, 1H), 4.32 (m, 1H), 4.45−4.36 (m, J = 8.4, 2H), 7.17 (d, J =
16.0 Hz, 1H), 7.30−7.20 (m, 5H), 7.34 (t, J = 7.4 Hz, 4H), 7.42 (m,
1H), 7.61 (d, J = 7.3 Hz, 2H), 7.81−7.70 (m, 4H), 7.87 (d, J = 7.5 Hz,
1H), 7.93 (d, J = 16.0 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 27.9,
55.0, 57.8, 63.1, 68.2, 75.0, 121.1, 126.3, 127.5, 127.7, 127.9, 128.3,
128.5, 128.7, 128.9, 129.2, 131.2, 131.8, 133.2, 134.5, 135.9, 140.4,
142.7, 145.3, 158.6, 171.4, 172.5; MS (ES+) m/z 627, 628 (MNa+,
100, 39), 605, 606 (MH+, 82, 33); HRMS (ES+) m/z calcd for
C37H37N2O6 (MH+) 605.2652, found 605.2650.
27
1
Resin-Bound Peptide 18 and Linear Peptide 19. Commer-
cially available L-Lys(Boc)-2-chlorotrityl resin (17, 240 mg, nominal
loading level 0.83 mmol g−1) was swelled in DMF (4 mL) at rt for 1 h.
The resin was then subjected automated coupling/deprotection cycles
to build up the linear peptide sequence required. The amino acid
derivatives employed were Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Ile-OH,
Fmoc-L-Phe-OH, Fmoc-L-Leu-OH, Fmoc-L-Asp(tBu)-OH, and
Fmoc-L-Pro-OH. All residues were double coupled using standard
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HOBt/HBTU (5 equiv) coupling cycles at rt for 45 min. Fmoc
deprotection was achieved via treatment with 20% piperidine/DMF at
rt for 15 min. The resulting resin-bound peptide was then filtered,
washed with DMF (3 × 5 mL), CH2Cl2 (3 × 5 mL), MeOH (3 × 5
mL), diethyl ether (3 × 5 mL) and dried to afford resin-bound peptide
18 as an orange resin (420 mg, 0.66 mmol g−1 by Fmoc test). For
analytical purposes, a portion of the resin (3 mg) was treated with
TFA/TIS/H2O (95:2.5:2.5 v/v/v, 1 mL) and allowed to stand with
occasional swirling at rt for 3 h. The acidic solution was then decanted,
concentrated (∼100 μL) under a stream of nitrogen, and diluted with
diethyl ether (1.5 mL). The resulting precipitate was collected by
centrifugation and washed with diethyl ether (3 × 1.5 mL) to afford
crude peptide 19 as a white solid. Analysis by reversed-phase HPLC
(gradient 2−98% MeCN/H2O v/v with 0.1% TFA v/v), tR 11.1 min,
purity >95%; MS (MALDI-TOF+) m/z 1036, 1037 (MH+, 100, 63).
Resin-Bound Peptide 20a and Linear Peptides 21a and 22a.
2,2′-Tolan dipeptide 9a (360 mg, 0.600 mmol), PyBOP (315 mg,
0.605 mmol), and i-Pr2EtN (210 μL, 1.20 mmol) were dissolved in
DMF (12 mL) and allowed to stand at rt for 10 min. The solution was
then transferred to resin-bound peptide 18 (180 mg, 0.66 mmol g−1)
and the suspension shaken at rt for 16 h. The resulting resin was
filtered, washed with DMF (3 × 10 mL), and then treated with 20%
piperidine/DMF (12 mL) and shaken at rt for 3 h. The resin was again
filtered and washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL)
to afford resin-bound peptide 20a. For analytical purposes, a portion of
the resin (3 mg) was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 1
mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was then decanted, concentrated (∼100 μL) under a
stream of nitrogen, and diluted with diethyl ether (1.5 mL). The
resulting precipitate was collected by centrifugation and washed with
diethyl ether (3 × 1.5 mL) to afford crude peptide 21a as a white solid.
Analysis by reversed-phase HPLC (gradient 2−98% MeCN/H2O v/v
with 0.1% TFA v/v), tR 12.1 min, purity >95%; MS (MALDI-TOF+)
m/z 1342, 1343, 1344 (MH+, 100, 90, 42).
The remaining resin-bound peptide 20a was suspended in 1% TFA/
CH2Cl2 (12 mL), shaken at rt for 2 min, and then filtered (repeated
five times). The combined filtrate was neutralized with 10% pyridine/
MeOH (18 mL) and concentrated under reduced pressure to
approximately 5 mL. Water was then added, and the resulting
precipitate was collected by centrifugation, washed with H2O (3 × 10
mL), and lyophilized to afford crude peptide 22a as a white solid (200
mg): MS (ES+) m/z 1806, 1807, 1808, 1809 (MH+, 70, 79, 46, 20),
903, 904, 904, 905 (MH+/2, 91, 100, 61, 29).
Resin-Bound Peptide 20b and Linear Peptides 21b and 22b.
2,3′-Tolan dipeptide 9b (180 mg, 0.299 mmol), PyBOP (156 mg,
0.300 mmol), and i-Pr2EtN (105 μL, 0.601 mmol) were dissolved in
DMF (12 mL) and allowed to stand at rt for 10 min. The solution was
then transferred to resin-bound peptide 18 (180 mg, 0.66 mmol g−1)
and the suspension shaken at rt for 16 h. The resulting resin was
filtered, washed with DMF (3 × 10 mL), and then treated with 20%
piperidine/DMF (12 mL) and shaken at rt for 3 h. The resin was again
filtered and washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL)
to afford resin-bound peptide 20b. For analytical purposes, a portion
of the resin (3 mg) was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v,
1 mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was then decanted, concentrated (∼100 μL) under a
stream of nitrogen, and diluted with diethyl ether (1.5 mL). The
resulting precipitate was collected by centrifugation and washed with
diethyl ether (3 × 1.5 mL) to afford crude peptide 21b as a white
solid. Analysis by reversed-phase HPLC (gradient 2−98% MeCN/
H2O v/v with 0.1% TFA v/v), tR 12.2 min, purity >95%; MS (MALDI-
TOF+) m/z 1342 (MH+, 100).
mg): MS (ES+) m/z 1806, 1807 (MH+, 13, 7), 903, 904, 904, 905
(MH+/2, 93, 100, 64, 29).
Resin-Bound Peptide 20c and Linear Peptides 21c and 22c.
3,2′-Tolan dipeptide 9c (240 mg, 0.398 mmol), PyBOP (208 mg,
0.400 mmol), and i-Pr2EtN (140 μL, 0.802 mmol) were dissolved in
DMF (12 mL) and allowed to stand at rt for 10 min. The solution was
then transferred to resin-bound peptide 18 (180 mg, 0.66 mmol g−1)
and the suspension shaken at rt for 16 h. The resulting resin was
filtered, washed with DMF (3 × 10 mL), and then treated with 20%
piperidine/DMF (12 mL) and shaken at rt for 3 h. The resin was again
filtered and washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL)
to afford resin-bound peptide 20c. For analytical purposes, a portion of
the resin (3 mg) was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 1
mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was then decanted, concentrated (∼100 μL), under a
stream of nitrogen and diluted with diethyl ether (1.5 mL). The
resulting precipitate was collected by centrifugation and washed with
diethyl ether (3 × 1.5 mL) to afford crude peptide 21c as a white solid.
Analysis by reversed-phase HPLC (gradient 2−98% MeCN/H2O v/v
with 0.1% TFA v/v), tR 13.7 min, purity >95%; MS (MALDI-TOF+)
m/z 1342 (MH+, 100).
The remaining resin-bound peptide 20c was suspended in 1% TFA/
CH2Cl2 (12 mL), shaken at rt for 2 min, then filtered (repeated five
times). The combined filtrate were neutralized with 10% pyridine/
MeOH (18 mL) and concentrated under reduced pressure to
approximately 5 mL. Water was then added and the resulting
precipitate was collected by centrifugation, washed with H2O (3 × 10
mL), and lyophilized to afford crude peptide 22c as a white solid (88
mg): MS (ES+) m/z 1806, 1807 (MH+, 26, 22), 903, 904, 904, 905
(MH+/2, 92, 100, 61, 29).
Resin-Bound Peptide 20d and Linear Peptides 21d and 22d.
3,3′-Tolan dipeptide 9d (180 mg, 0.299 mmol), PyBOP (156 mg,
0.300 mmol), and i-Pr2EtN (105 μL, 0.601 mmol) were dissolved in
DMF (12 mL) and allowed to stand at rt for 10 min. The solution was
then transferred to resin-bound peptide 18 (180 mg, 0.66 mmol g−1)
and the suspension shaken at rt for 16 h. The resulting resin was
filtered, washed with DMF (3 × 10 mL), then treated with 20%
piperidine/DMF (12 mL) and shaken at rt for 3 h. The resin was again
filtered and washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL)
to afford resin-bound peptide 20d. For analytical purposes, a portion
of the resin (3 mg) was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v,
1 mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was then decanted, concentrated (∼100 μL) under a
stream of nitrogen and diluted with diethyl ether (1.5 mL). The
resulting precipitate was collected by centrifugation and washed with
diethyl ether (3 × 1.5 mL) to afford crude peptide 21d as a white
solid. Analysis by reversed-phase HPLC (gradient 2−98% MeCN/
H2O v/v with 0.1% TFA v/v), tR 13.3 min, purity >95%; MS (MALDI-
TOF+) m/z 1342 (MH+, 100).
The remaining resin-bound peptide 20d was suspended in 1%
TFA/CH2Cl2 (12 mL), shaken at rt for 2 min, then filtered (repeated
five times). The combined filtrate were neutralized with 10% pyridine/
MeOH (18 mL) and concentrated under reduced pressure to
approximately 5 mL. Water was then added and the resulting
precipitate was collected by centrifugation, washed with H2O (3 × 10
mL) and lyophilized to afford crude peptide 22d as a white solid (119
mg): MS (ES+) m/z 1806, 1807 (MH+, 19, 17), 903, 904, 904, 905
(MH+/2, 49, 53, 34, 13), 282 (100).
Resin-Bound Peptide 20e and Linear Peptides 21e and 22e.
2,2′-Bibenzyl dipeptide 12 (162 mg, 0.267 mmol), PyBOP (140 mg,
0.269 mmol) and i-Pr2EtN (92 μL, 0.527 mmol) were dissolved in
DMF (12 mL) and allowed to stand at rt for 10 min. The solution was
then transferred to resin-bound peptide 18 (180 mg, 0.66 mmol g−1)
and the suspension shaken at rt for 16 h. The resulting resin was
filtered, washed with DMF (3 × 10 mL), and then treated with 20%
piperidine/DMF (12 mL) and shaken at rt for 3 h. The resin was again
filtered and washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL)
to afford resin-bound peptide 20e. For analytical purposes, a portion of
the resin (3 mg) was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 1
mL) and allowed to stand with occasional swirling at rt for 3 h. The
The remaining resin-bound peptide 20b was suspended in 1%
TFA/CH2Cl2 (12 mL), shaken at rt for 2 min, then filtered (repeated
five times). The combined filtrate were neutralized with 10% pyridine/
MeOH (18 mL) and concentrated under reduced pressure to
approximately 5 mL. Water was then added and the resulting
precipitate was collected by centrifugation, washed with H2O (3 × 10
mL) and lyophilized to afford crude peptide 22b as a white solid (122
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acidic solution was then decanted, concentrated (∼100 μL) under a
stream of nitrogen and diluted with diethyl ether (1.5 mL). The
resulting precipitate was collected by centrifugation and washed with
diethyl ether (3 × 1.5 mL) to afford crude peptide 21e as a white solid.
Analysis by reversed-phase HPLC (gradient 2−98% MeCN/H2O v/v
with 0.1% TFA v/v), tR 11.9 min, purity >95%; MS (MALDI-TOF+)
m/z 1346 (MH+, 100).
The remaining resin-bound peptide 20e was suspended in 1% TFA/
CH2Cl2 (12 mL), shaken at rt for 2 min, then filtered (repeated 5
times). The combined filtrate were neutralized with 10% pyridine/
MeOH (18 mL) and concentrated under reduced pressure to
approximately 5 mL. Water was then added and the resulting
precipitate was collected by centrifugation, washed with H2O (3 × 10
mL), and lyophilized to afford crude peptide 22e as a white solid (110
mg): MS (ES+) m/z 905, 906, 907, 907 (MH+/2, 79, 90, 55, 25), 338
(100).
Resin-Bound Peptide 20f and Linear Peptides 21f and 22f.
2,2′-(Z)-Stilbene dipeptide 13 (98 mg, 0.162 mmol), PyBOP (80 mg,
0.154 mmol), and i-Pr2EtN (53 μL, 0.303 mmol) were dissolved in
DMF (6 mL) and allowed to stand at rt for 10 min. The solution was
then transferred to resin-bound peptide 18 (90 mg, 0.66 mmol g−1)
and the suspension shaken at rt for 16 h. The resulting resin was
filtered, washed with DMF (3 × 10 mL), and then treated with 20%
piperidine/DMF (8 mL) and shaken at rt for 3 h. The resin was again
filtered and washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL)
to afford resin-bound peptide 20f. For analytical purposes, a portion of
the resin (3 mg) was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 1
mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was then decanted, concentrated (∼100 μL) under a
stream of nitrogen and diluted with diethyl ether (1.5 mL). The
resulting precipitate was collected by centrifugation and washed with
diethyl ether (3 × 1.5 mL) to afford crude peptide 21f as a white solid.
Analysis by reversed-phase HPLC (gradient 2−98% MeCN/H2O v/v
with 0.1% TFA v/v), tR 12.2 min, purity >95%; MS (MALDI-TOF+)
m/z 1344 (MH+, 100).
The remaining resin-bound peptide 20f was suspended in 1% TFA/
CH2Cl2 (12 mL), shaken at rt for 2 min, then filtered (repeated 5
times). The combined filtrate were neutralized with 10% pyridine/
MeOH (18 mL) and concentrated under reduced pressure to
approximately 5 mL. Water was then added and the resulting
precipitate was collected by centrifugation, washed with H2O (3 × 10
mL) and lyophilized to afford crude peptide 22f as a white solid (76
mg): MS (ES+) m/z 1808, 1809, 1810, 1811 (MH+, 24, 27, 19, 5),
905, 906, 906 (MH+/2, 17, 12, 5), 208 (100).
Resin-Bound Peptide 20g and Linear Peptides 21g and 22g.
2,2′-(E)-Stilbene dipeptide 16 (100 mg, 0.165 mmol), PyBOP (80 mg,
0.154 mmol), and i-Pr2EtN (53 μL, 0.303 mmol) were dissolved in
DMF (6 mL) and allowed to stand at rt for 10 min. The solution was
then transferred to resin-bound peptide 18 (90 mg, 0.66 mmol g−1)
and the suspension shaken at rt for 16 h. The resulting resin was
filtered, washed with DMF (3 × 10 mL), then treated with 20%
piperidine/DMF (8 mL) and shaken at rt for 3 h. The resin was again
filtered and washed with DMF (3 × 10 mL) and CH2Cl2 (3 × 10 mL)
to afford resin-bound peptide 20g. For analytical purposes, a portion
of the resin (3 mg) was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v,
1 mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was then decanted, concentrated (∼100 μL) under a
stream of nitrogen and diluted with diethyl ether (1.5 mL). The
resulting precipitate was collected by centrifugation and washed with
diethyl ether (3 × 1.5 mL) to afford crude peptide 21g as a white solid.
Analysis by reversed-phase HPLC (gradient 2−98% MeCN/H2O v/v
with 0.1% TFA v/v), tR 12.1 min, purity >95%; MS (MALDI-TOF+)
m/z 1344 (MH+, 100).
mg): MS (ES+) m/z 1808, 1809, 1810 (MH+, 23, 32, 12), 905, 905,
906, 906 (MH+/2, 96, 100, 71, 34).
Cyclic Peptide 23a. 2,2′-Tolan peptide 22a (140 mg, 0.078 mmol)
and i-Pr2EtN (150 μL, 0.859 mmol) were dissolved in DMF (10 mL).
The resulting solution was added via syringe pump (0.5 mL h−1) into a
stirred solution of HATU (45 mg, 0.118 mmol) in DMF (10 mL).
Stirring was continued at rt under an atmosphere on nitrogen for 32 h.
Additional HATU (3 × 45 mg, 0.355 mmol) was added at 6 h
intervals. The reaction mixture was then concentrated (∼2 mL) under
a stream of nitrogen and then diluted with H2O (30 mL). The
resulting precipitate was collected by centrifugation, washed with H2O
(3 × 30 mL), and lyophilized to afford a white solid (153 mg). The
solid was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 5 mL) and
allowed to stand with occasional swirling at rt for 3 h. The acidic
solution was concentrated (∼1 mL) under a stream of nitrogen then
diluted with diethyl ether (10 mL). The resulting precipitate was
collected by centrifugation, washed with diethyl ether (3 × 10 mL),
and dried. Purification by preparative reversed-phase HPLC (gradient
2−98% MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic peptide
23a as a white solid after lyophilization (38 mg, 28%): tR 13.7 min; MS
(ES+) m/z 1324, 1325 (MH+, 32, 26), 662, 663, 663 (MH+/2, 100,
82, 34).
Cyclic Peptide 23b. 2,3′-Tolan peptide 22b (120 mg, 0.066
mmol) and i-Pr2EtN (130 μL, 0.744 mmol) were dissolved in DMF
(10 mL). The resulting solution was added via syringe pump (0.5 mL
h−1) into a stirred solution of HATU (39 mg, 0.103 mmol) in DMF
(10 mL). Stirring was continued at rt under an atmosphere on
nitrogen for 32 h. Additional HATU (3 × 39 mg, 0.308 mmol) was
added at 6 h intervals. The reaction mixture was then concentrated
(∼2 mL) under a stream of nitrogen and then diluted with H2O (30
mL). The resulting precipitate was collected by centrifugation, washed
with H2O (3 × 30 mL), and lyophilized to afford a white solid (96
mg). The solid was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 5
mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was concentrated (∼1 mL) under a stream of nitrogen
and then diluted with diethyl ether (10 mL). The resulting precipitate
was collected by centrifugation, washed with diethyl ether (3 × 10
mL) and dried. Purification by preparative reversed-phase HPLC
(gradient 2−98% MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic
peptide 23b as a white solid after lyophilization (29 mg, 33%): tR 13.4
min; MS (ES+) m/z 1324, 1325 (MH+, 25, 20), 662, 663, 663 (MH+/
2, 100, 83, 37).
Cyclic Peptide 23c. 3,2′-Tolan peptide 22c (86 mg, 0.048 mmol)
and i-Pr2EtN (92 μL, 0.527 mmol) were dissolved in DMF (10 mL).
The resulting solution was added via syringe pump (0.5 mL h−1) into a
stirred solution of HATU (28 mg, 0.074 mmol) in DMF (10 mL).
Stirring was continued at rt under an atmosphere on nitrogen for 32 h.
Additional HATU (3 × 28 mg, 0.221 mmol) was added at 6 h
intervals. The reaction mixture was then concentrated (∼2 mL) under
a stream of nitrogen then diluted with H2O (30 mL). The resulting
precipitate was collected by centrifugation, washed with H2O (3 × 30
mL), and lyophilized to afford a white solid (64 mg). The solid was
treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 5 mL) and allowed to
stand with occasional swirling at rt for 3 h. The acidic solution was
concentrated (∼1 mL) under a stream of nitrogen then diluted with
diethyl ether (10 mL). The resulting precipitate was collected by
centrifugation, washed with diethyl ether (3 × 10 mL), and dried.
Purification by preparative reversed-phase HPLC (gradient 2−98%
MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic peptide 23c as a
white solid after lyophilization (12 mg, 19%): tR 13.9 min; MS (ES+)
m/z 1324, 1325 (MH+, 47, 38), 662, 663, 663 (MH+/2, 100, 83).
Cyclic Peptide 23d. 3,3′-Tolan peptide 22d (110 mg, 0.061
mmol) and i-Pr2EtN (117 μL, 0.670 mmol) were dissolved in DMF
(10 mL). The resulting solution was added via syringe pump (0.5 mL
h−1) into a stirred solution of HATU (35 mg, 0.092 mmol) in DMF
(10 mL). Stirring was continued at rt under an atmosphere on
nitrogen for 32 h. Additional HATU (3 × 35 mg, 0.276 mmol) was
added at 6 h intervals. The reaction mixture was then concentrated
(∼2 mL) under a stream of nitrogen then diluted with H2O (30 mL).
The resulting precipitate was collected by centrifugation, washed with
The remaining resin-bound peptide 20g was suspended in 1%
TFA/CH2Cl2 (12 mL), shaken at rt for 2 min, and then filtered
(repeated five times). The combined filtrate were neutralized with 10%
pyridine/MeOH (18 mL) and concentrated under reduced pressure to
approximately 5 mL. Water was then added and the resulting
precipitate was collected by centrifugation, washed with H2O (3 × 10
mL), and lyophilized to afford crude peptide 22g as a white solid (59
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a
Scheme 8. Solid-Phase Synthesis and Solution-Phase Cyclization of Scrambled Control Peptidomimetic 24
a
Reagents and conditions: (i) protected amino acid, HOBt/HBTU, i-Pr2EtN, DMF; (ii) 20% piperidine/DMF; (iii) TFA/i-Pr3SiH/H2O
(95:2.5:2.5); (iv) PyBOP, tolan 9a, i-Pr2EtN, DMF; (v) 20% piperidine/DMF; (vi) 1% TFA/CH2Cl2; (vii) HATU, i-Pr2EtN, DMF.
H2O (3 × 30 mL), and lyophilized to afford a white solid (96 mg).
The solid was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 5 mL)
and allowed to stand with occasional swirling at rt for 3 h. The acidic
solution was concentrated (∼1 mL) under a stream of nitrogen and
then diluted with diethyl ether (10 mL). The resulting precipitate was
collected by centrifugation, washed with diethyl ether (3 × 10 mL),
and dried. Purification by preparative reversed-phase HPLC (gradient
2−98% MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic peptide
23d as a white solid after lyophilization (22 mg, 27%): tR 14.5 min; MS
(ES+) m/z 1324, 1325, 1326 (MH+, 65, 55, 22), 662, 663, 663 (MH+/
2, 68, 56, 25), 282 (100).
Cyclic Peptide 23e. 2,2′-Bibenzyl peptide 22e (100 mg, 0.055
mmol) and i-Pr2EtN (106 μL, 0.607 mmol) were dissolved in DMF
(10 mL). The resulting solution was added via syringe pump (0.5 mL
h−1) into a stirred solution of HATU (32 mg, 0.084 mmol) in DMF
(10 mL). Stirring was continued at rt under an atmosphere on
nitrogen for 32 h. Additional HATU (3 × 32 mg, 0.252 mmol) was
added at 6 h intervals. The reaction mixture was then concentrated
(∼2 mL) under a stream of nitrogen and then diluted with H2O (30
mL). The resulting precipitate was collected by centrifugation, washed
with H2O (3 × 30 mL), and lyophilized to afford a white solid (108
mg). The solid was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 5
mL) and allowed to stand with occasional swirling at rt for 3 h. The
acidic solution was concentrated (∼1 mL) under a stream of nitrogen
then diluted with diethyl ether (10 mL). The resulting precipitate was
collected by centrifugation, washed with diethyl ether (3 × 10 mL),
and dried. Purification by preparative reversed-phase HPLC (gradient
2−98% MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic peptide
23e as a white solid after lyophilization (30 mg, 41%): tR 13.4 min; MS
(ES+) m/z 1328, 1329, 1330 (MH+, 55, 42, 18), 664, 665, 665 (MH+/
2, 78, 64, 27), 282 (100).
32 h. Additional HATU (3 × 25 mg, 0.197 mmol) was added at 6 h
intervals. The reaction mixture was then concentrated (∼2 mL) under
a stream of nitrogen and diluted with H2O (20 mL). The resulting
precipitate was collected by centrifugation, washed with H2O (3 × 20
mL), and lyophilized to afford a white solid (58 mg). The solid was
treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 4 mL) and allowed to
stand with occasional swirling at rt for 3 h. The acidic solution was
concentrated (∼1 mL) under a stream of nitrogen then diluted with
diethyl ether (10 mL). The resulting precipitate was collected by
centrifugation, washed with diethyl ether (3 × 10 mL), and dried.
Purification by preparative reversed-phase HPLC (gradient 2−98%
MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic peptide 23f as a
white solid after lyophilization (13 mg, 24%): tR 13.2 min; MS (ES+)
m/z 1326 (MH+, 7), 663, 664, 664 (MH+/2, 100, 79, 38).
Cyclic Peptide 23g. 2,2′-(E)-Stilbene peptide 22g (58 mg, 0.032
mmol) and i-Pr2EtN (70 μL, 0.401 mmol) were dissolved in DMF (7
mL). The resulting solution was added via syringe pump (0.5 mL h−1)
into a stirred solution of HATU (20 mg, 0.053 mmol) in DMF (7
mL). Stirring was continued at rt under an atmosphere on nitrogen for
32 h. Additional HATU (3 × 20 mg, 0.158 mmol) was added at 6 h
intervals. The reaction mixture was then concentrated (∼2 mL) under
a stream of nitrogen then diluted with H2O (20 mL). The resulting
precipitate was collected by centrifugation, washed with H2O (3 × 20
mL) and lyophilized to afford a white solid (52 mg). The solid was
treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 4 mL) and allowed to
stand with occasional swirling at rt for 3 h. The acidic solution was
concentrated (∼1 mL) under a stream of nitrogen and then diluted
with diethyl ether (10 mL). The resulting precipitate was collected by
centrifugation, washed with diethyl ether (3 × 10 mL), and dried.
Purification by preparative reversed-phase HPLC (gradient 2−98%
MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic peptide 23g as a
white solid after lyophilization (15 mg, 35%): tR 13.1 min; MS (ES+)
m/z 1326 (MH+, 5), 663, 664, 664, 665 (MH+/2, 100, 86, 43, 16).
Synthetic Procedures for Preparation of Control Peptidomi-
metics (Scheme 8). Resin-Bound Peptide SCR1 and Linear
Peptide SCR2. Commercially available L-Lys(Boc)-2-chlorotrityl
resin (17, 200 mg, nominal loading level 1.0 mmol g−1) was swelled
Cyclic Peptide 23f. 2,2′-(Z)-Stilbene peptide 22f (76 mg, 0.042
mmol) and i-Pr2EtN (85 μL, 0.487 mmol) were dissolved in DMF (8
mL). The resulting solution was added via syringe pump (0.5 mL h−1)
into a stirred solution of HATU (25 mg, 0.066 mmol) in DMF (8
mL). Stirring was continued at rt under an atmosphere on nitrogen for
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a
Scheme 9. Solid-Phase Synthesis and Solution-Phase Cyclization of Phe-349-Ala Mutant Control Peptidomimetic 25
a
Reagents and conditions: (i) protected amino acid, HOBt/HBTU, i-Pr2EtN, DMF; (ii) 20% piperidine/DMF; (iii) TFA/i-Pr3SiH/H2O
(95:2.5:2.5); (iv) PyBOP, tolan 9a, i-Pr2EtN, DMF; (v) 20% piperidine/DMF; (vi) 1% TFA/CH2Cl2; (vii) HATU, i-Pr2EtN, DMF.
in DMF (4 mL) at rt for 1 h. The resin was then subjected automated
coupling/deprotection cycles to build up the linear peptide sequence
required. The amino acid derivatives employed were Fmoc-L-
Arg(Pbf)-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Leu-OH,
Fmoc-L-Asp(tBu)-OH, and Fmoc-L-Pro-OH. All residues were double
coupled using standard HOBt/HBTU (5 equiv) coupling cycles at rt
for 35 min. Fmoc deprotection was achieved via treatment with 20%
piperidine/DMF at rt for 15 min. The resulting resin-bound peptide
was then filtered, washed with DMF (3 × 5 mL), CH2Cl2 (3 × 10
mL), MeOH (3 × 10 mL), diethyl ether (3 × 10 mL) and dried to
afford resin-bound peptide SCR1 as an orange resin. For analytical
purposes, a portion of the resin (2 mg) was treated with TFA/TIS/
H2O (95:2.5:2.5 v/v/v, 1 mL) and allowed to stand with occasional
swirling at rt for 1 h. The acidic solution was then decanted and
concentrated under a stream of nitrogen to afford crude peptide SCR2
as an off-white solid. Analysis by reversed-phase HPLC (gradient 5−
98% MeCN/H2O v/v with 0.1% TFA v/v), tR 3.98 min; MS (ES+) m/
z 1036 (MH+).
Linear Peptides SCR4 and SCR5. 2,2′-Tolan dipeptide 9a (420
mg, 0.700 mmol), PyBOP (375 mg, 0.720 mmol), and i-Pr2EtN (260
μL, 1.49 mmol) were dissolved in DMF (12 mL) and allowed to stand
at rt for 10 min. The solution was then transferred to resin-bound
peptide SCR1 and the suspension shaken at rt for 22 h. The resulting
resin was filtered, washed with DMF (3 × 20 mL), treated with 20%
piperidine/DMF (20 mL), and shaken at rt for 4 h. The resin was
again filtered and washed with DMF (4 × 20 mL) and CH2Cl2 (4 × 20
mL) to afford resin-bound peptide SCR3. For analytical purposes, a
portion of the resin (2 mg) was treated with TFA/TIS/H2O
(95:2.5:2.5 v/v/v, 1 mL) and allowed to stand with occasional
swirling at rt for 1 h. The acidic solution was then decanted and
concentrated under a stream of nitrogen to afford crude peptide SCR4
as an off-white solid. Analysis by reversed-phase HPLC (gradient 5−
98% MeCN/H2O v/v with 0.1% TFA v/v), tR 11.32 min; MS (ES+)
m/z 1342 (MH+).
(repeated five times). The combined filtrates were neutralized with
10% pyridine/MeOH (5 mL) and concentrated under reduced
pressure to approximately 5−6 mL. Water (25 mL) was then added,
and the resulting precipitate was collected by centrifugation, washed
with H2O (3 × 15 mL) and lyophilized to afford crude peptide SCR5
as a white solid (293 mg): MS (ES+) m/z 1806, 1807, 1808, 1809
(MH+, 43, 46, 29, 13), 903, 904, 904, 905 (MH+/2, 85, 100, 65, 30).
Cyclic Peptide 24. 2,2′-Tolan peptide SCR5 (200 mg, 0.11 mmol)
and i-Pr2EtN (70 μL, 0.40 mmol) were dissolved in DMF (15 mL).
The resulting solution was added via syringe pump (0.5 mL h−1) into a
stirred solution of HATU (65 mg, 0.17 mmol) and i-Pr2EtN (150 μL,
0.86 mmol) in DMF (15 mL). Stirring was continued at rt under an
atmosphere on nitrogen for 45 h. Additional HATU (3 × 65 mg, 0.51
mmol) was added at 8 h intervals. The reaction mixture was then
quenched with H2O (2 mL), concentrated (∼2.5−3 mL) under a
stream of nitrogen, and diluted with H2O (35 mL). The resulting
precipitate was collected by centrifugation, washed with H2O (3 × 40
mL), and lyophilized to afford a cream colored solid (172 mg). The
solid was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 7.5 mL) and
allowed to stand with occasional swirling at rt for 4 h. The acidic
solution was concentrated (∼1.5 mL) under a stream of nitrogen then
diluted with diethyl ether (15 mL). The resulting precipitate was
collected by centrifugation, washed with diethyl ether (3 × 15 mL),
and dried. Purification by preparative reversed-phase HPLC (gradient
2−98% MeCN/H2O v/v with 0.1% TFA v/v) afforded cyclic peptide
24 as a white solid after lyophilization (8 mg, 6%) (the yield of this
peptide was compromised by its precipitation on the HPLC column):
tR 12.25 min; MS (ES+) m/z 1324, 1325, 1326 (MH+, 45, 38, 15),
662, 663, 663, 664 (MH+/2, 100, 83, 36, 13).
Resin-bound Peptide MUT1 and Linear Peptide MUT2
(Scheme 9). Commercially available L-Lys(Boc)-2-chlorotrityl resin
(17, 200 mg, nominal loading level 1.0 mmol g−1) was swelled in DMF
(4 mL) at rt for 1 h. The resin was then subjected automated
coupling/deprotection cycles to build up the linear peptide sequence
required. The amino acid derivatives employed were Fmoc-L-Ala-OH,
Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Leu-
The remaining resin-bound peptide SCR3 was suspended in 1%
TFA/CH2Cl2 (14 mL), shaken at rt for 2 min, and then filtered
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OH, Fmoc-L-Asp(tBu)-OH, and Fmoc-L-Pro-OH. All residues were
double coupled using standard HOBt/HBTU (5 equiv) coupling
cycles at rt for 35 min. Fmoc deprotection was achieved via treatment
with 20% piperidine/DMF at rt for 15 min. The resulting resin-bound
peptide was then filtered, washed with DMF (3 × 5 mL), CH2Cl2 (3 ×
10 mL), MeOH (3 × 10 mL), and diethyl ether (3 × 10 mL), and
dried to afford resin-bound peptide MUT1 as an orange resin. For
analytical purposes, a portion of the resin (2 mg) was treated with
TFA/TIS/H2O (95:2.5:2.5 v/v/v, 1 mL) and allowed to stand with
occasional swirling at rt for 1 h. The acidic solution was then decanted
and diluted with tert-butylmethyl ether (1.0 mL). The resulting
precipitate was collected by centrifugation and washed with tert-
butylmethyl ether (3 × 1.5 mL) to afford crude peptide MUT2 as an
off-white solid. Analysis by reversed-phase HPLC (gradient 5−98%
MeCN/H2O v/v with 0.1% TFA v/v), tR 9.76 min; MS (ES+) m/z
960, 961 (MH+, 100, 53).
and plots for Table 1. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: a.c.spivey@Imperial.ac.uk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Asthma UK, the Wellcome Trust, the MRC,
and the European Commission (FP7Marie-Curie Fellowship
for J.J.P.S.) for financial support of this work.
Linear Peptides MUT4 and MUT5. 2,2′-Tolan dipeptide 9a (420
mg, 0.700 mmol), PyBOP (375 mg, 0.720 mmol), and i-Pr2EtN (260
μL, 1.49 mmol) were dissolved in DMF (12 mL) and allowed to stand
at rt for 10 min. The solution was then transferred to resin-bound
peptide MUT1 and the suspension shaken at rt for 22 h. The resulting
resin was filtered, washed with DMF (3 × 20 mL), treated with 20%
piperidine/DMF (20 mL), and shaken at rt for 4 h. The resin was
again filtered, washed with DMF (4 × 20 mL) and CH2Cl2 (4 × 20
mL) to afford resin-bound peptide MUT3. For analytical purposes, a
portion of the resin (2 mg) was treated with TFA/TIS/H2O
(95:2.5:2.5 v/v/v, 1 mL) and allowed to stand with occasional
swirling at rt for 1 h. The acidic solution was then decanted and
concentrated under a stream of nitrogen to afford crude peptide
MUT4 as an off-white solid. Analysis by reversed-phase HPLC
(gradient 5−98% MeCN/H2O v/v with 0.1% TFA v/v), tR 10.74 min;
MS (ES+) m/z 1266 (MH+).
The remaining resin-bound peptide MUT3 was suspended in 1%
TFA/CH2Cl2 (14 mL), shaken at rt for 2 min, and then filtered
(repeated five times). The combined filtrate were neutralized with 10%
pyridine/MeOH (5 mL) and concentrated under reduced pressure to
approximately 5−6 mL. Water (25 mL) was then added and the
resulting precipitate was collected by centrifugation, washed with H2O
(3 × 15 mL) and lyophilized to afford crude peptide MUT5 as a white
solid (283 mg): MS (ES+) m/z 1730, 1731, 1732 (MH+, 49, 53, 29),
865, 866, 866, 867 (MH+/2, 94, 100, 61, 28).
Cyclic Peptide 25. 2,2′-Tolan peptide MUT5 (190 mg, 0.11
mmol) and i-Pr2EtN (70 μL, 0.40 mmol) were dissolved in DMF (15
mL). The resulting solution was added via syringe pump (0.5 mL h−1)
into a stirred solution of HATU (65 mg, 0.17 mmol) and i-Pr2EtN
(150 μL, 0.859 mmol) in DMF (15 mL). Stirring was continued at rt
under an atmosphere on nitrogen for 45 h. Additional HATU (3 × 65
mg, 0.51 mmol) was added at 8 h intervals. The reaction mixture was
then quenched with H2O (2 mL) and concentrated (∼2.5−3 mL)
under a stream of nitrogen then diluted with H2O (35 mL). The
resulting precipitate was collected by centrifugation, washed with H2O
(3 × 40 mL) and lyophilized to afford a cream colored solid (180 mg).
The solid was treated with TFA/TIS/H2O (95:2.5:2.5 v/v/v, 7.5 mL)
and allowed to stand with occasional swirling at rt for 4 h. The acidic
solution was concentrated (∼1.5 mL) under a stream of nitrogen then
diluted with diethyl ether (15 mL). The resulting precipitate was
collected by centrifugation, washed with diethyl ether (3 × 15 mL),
and dried to afford a cream-colored solid (153 mg). Purification of a
smaller sample (45 mg) by preparative reversed-phase HPLC
(gradient 2−98% MeCN/H2O v/v with 0.1% TFA v/v) afforded
cyclic peptide 25 as a white solid after lyophilization (15 mg, 38%): tR
11.02 min; MS (ES+) m/z 1248, 1249 (MH+, 26, 19), 624, 625, 625,
626 (MH+/2, 100, 75, 31, 9).
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1
compounds 2a−16, CSP-HPLC chromatograms for 9a and ent-
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published on the Web on March 7, 2012, with
errors in Table 1. The corrected version was reposted on March
8, 2012.
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