"@-Tides": The 1,2-dihydro-3(6h)-pyridinone unit as a β-strand mimic

Article abstract of DOI:10.1021/ja0168460

The cyclic amino acid surrogate 1 was designed to mimic the extended conformation of a peptide unit and to provide hydrogen bond donor and acceptor functions conducive to β-sheet formation. A convenient synthesis of this unit and solution and solid-phase methods for its incorporation into an oligomer alternating with peptide units have been devised. The resulting "@-tides", as these oligomers have been designated, show a high propensity for self-association in comparison to oligopeptides; insights into the structure and dynamical properties of their antiparallel dimers have been obtained by NMR.

Full text of DOI:10.1021/ja0168460

Published on Web 12/04/2001
“@-Tides”: The 1,2-Dihydro-3(6H)-pyridinone Unit as a
â-Strand Mimic
Scott T. Phillips, Miroslav Rezac, Ulrich Abel, Michael Kossenjans, and
Paul A. Bartlett*
Contribution from the Center for New Directions in Organic Synthesis,† Department of
Chemistry, UniVersity of California, Berkeley, California 94720-1460
Received August 14, 2001
Abstract: The cyclic amino acid surrogate 1 was designed to mimic the extended conformation of a peptide
unit and to provide hydrogen bond donor and acceptor functions conducive to â-sheet formation. A
convenient synthesis of this unit and solution and solid-phase methods for its incorporation into an oligomer
alternating with peptide units have been devised. The resulting “@-tides”, as these oligomers have been
designated, show a high propensity for self-association in comparison to oligopeptides; insights into the
structure and dynamical properties of their antiparallel dimers have been obtained by NMR.
stabilizing template.5,6 Short â-sheets are known only as
Introduction
insoluble aggregates, and peptides designed to exist in mono-
Local conformation within proteins and peptides is largely
described by secondary structural elements, such as R-helices,
â-turns, and â-strands, which determine the three-dimensional
orientation of the amino acid side chains and thereby the longer
range interstrand and intermolecular interactions. â-Strands, and
â-sheets derived from them, play important roles in protein-
protein interactions and in the association of proteins with other
biopolymers such as nucleic acids.1,2 The â-sheetlike association
and precipitation of hydrophobic protein fragments in amyloid
plaques is strongly implicated in neurodegenerative diseases.3,4
Despite its ubiquity, the fully extended â-strand conformation
is typically a minor component of the dynamic equilibrium for
an oligopeptide outside the context of a folded protein structure,
in which the hydrogen-bonded network of a â-sheet provides a
meric, all-â-sheet form contain at least 20 amino acids.5b
A
variety of small-molecule â-sheet templates have been described
which either juxtapose two peptide strands in the antiparallel
orientation7 or provide an extended array of hydrogen-bonding
sites via a â-turn linkage.8 A single-strand mimic was introduced
by Hirschmann, Smith, and their co-workers, based on the
pyrrolinone scaffold, which mimics both the functionality and
the side chain orientation of a peptide in the extended
conformation.9
â-Sheet templates that could act intermolecularly would be
useful tools for studying â-sheet structure and dynamics, in
devising peptide host-guest systems based on a â-sheet motif,
or for disrupting macromolecular interactions that involve
â-strands at the recognition domain. Oligomeric structures
† The Center for New Directions in Organic Synthesis is supported by
Bristol-Myers Squibb as a Sponsoring Member.
(5) (a) Nesloney, C. L.; Kelly, J. W. Bioorg. Med. Chem. 1996, 4, 739. (b)
Kortemme, T.; Ramirez-Alvarado, M.; Serrano, L. Science 1998, 281, 253.
(6) (a) Nesloney, C. L.; Kelly, J. W. Bioorg. Med. Chem. 1996, 4, 739. (b)
Schenck, H. L.; Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 4869. (c)
Haque, T. S.; Little, J. C.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118,
6975. (d) Kim, C. A.; Berg, J. M. Nature 1993, 362, 267. (e) Smith, C. K.;
Regan, L. Science 1995, 270, 980. (f) Venkatraman, J.; Shankaramma, C.
S.; Balaram, P. Chem. ReV. 2001, 101, 3131.
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Sun, Y. J. Am. Chem. Soc. 2000, 122, 7654. (b) Gong, B.; Yan, Y.; Zeng,
H.; Skrzypczak-Jankunn, E.; Kim, Y. W.; Zhu, J.; Ickes, H. J. Am. Chem.
Soc. 1999, 121, 5607.
(8) (a) Nowick, J. S. Chem. Ber. 1997, 33, 336. (b) Nowick, J. S.; Smith, E.
M.; Pairish, M. Chem. Soc. ReV. 1996, 25, 401. (c) Brandmeier, V.; Sauer,
W. H. B.; Feigel, M. HelV. Chim. Acta 1994, 77, 70. (d) Kemp, D. S.;
Bowen, B. R. Tetrahedron Lett. 1988, 29, 5077 and 5081. (e) Kemp, D.
S.; Li, Z. Q. Tetrahedron Lett. 1995, 36, 4179. (f) Nowick, J. S.; Smith, E.
M.; Pairish, M. Chem. Soc. ReV. 1996, 25, 401. (g) Chitnumsub, P.; Fiori,
W. R.; Lashuel, H. A.; Diaz, H.; Kelly, J. W. Bioorg. Med. Chem. 1999,
7, 30. (h) Schneider, J. P.; Kelly, J. W. J. Am. Chem. Soc. 1995, 117, 2533.
(i) Tsai, J. H.; Waldman, A. S.; Nowick, J. S. Bioorg. Med. Chem. 1999,
7, 29. (j) Nowick, J. S.; Tsai, J. H.; Bui, Q. D.; Maitra, S. J. Am. Chem.
Soc. 1999, 121, 8409.
(9) (a) Smith, A. B.; Keenan, T. P.; Holcomb, R. C.; Sprengeler, P. A.; Guzman,
M. C.; Wood, J. L.; Carroll, P. J.; Hirschmann, R. J. Am. Chem. Soc. 1992,
114, 10672. (b) Smith, A. B.; Knight, S. D.; Sprengeler, P. A.; Hirschmann,
R. Bioorg. Med. Chem. 1996, 4, 1021. (c) Smith, A. B.; Liu, H.;
Hirschmann, R. Org. Lett. 2000, 2, 2037. (d) Smith, A. B.; Liu, H.;
Okumura, H.; Favor, D. A.; Hirschmann, R. Org. Lett. 2000, 2, 2041.
(1) (a) Fitzgerald, F. M. D.; McKeever, G. M.; VanMiddlesworth, J. F.;
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Darke, P. L. J. Biol. Chem. 1990, 265, 14209. (b) Zutshi, R.; Franciscovich,
J.; Shultz, M.; Schweitzer, B.; Bishop, P.; Wilson, M.; Chmielewski, J. J.
Am. Chem. Soc. 1997, 119, 4841. (c) Babe, L. M.; Rose, J.; Craik, C. S.
Protein Sci. 1992, 1, 1244. (d) Maitra, S.; Nowick, J. S. Beta-Sheet
Interactions between Proteins. In The Amide Linkage: Selected Structural
Aspects in Chemistry, Biochemistry, and Materials Science; Greenberg, A.,
Breneman, C. M., Liebman, J. F., Eds.; John Wiley & Sons: New York,
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(2) (a) Siligardi, G.; Drake, A. F. Biopolymers (peptide science) 1995, 37, 281.
(b) Stanfield, R. L.; Wilson, I. A. Curr. Opin. Struct. Biol. 1995, 5, 103.
(c) Buckle, A. M.; Zahn, R.; Fersht, A. R. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 3571. (d) Taneja, B. C.; Mande, S. Protein Eng. 1999, 12, 815.
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Strominger, J. L.; Wiley: D. C. Nature 1994, 368, 215. (f) Moss, N.;
Beaulieu, P.; Duceppe, J.; Ferland, J.; Gauthier, J.; Ghiro, E.; Goulet, S.;
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Med. Chem. 1996, 39, 2178. (g) Sauer, F. G.; Futterer, K.; Pinkner, J. S.;
Dodson, K. W.; Hultgren, S. J.; Waksman, G. Science 1999, 285, 1058.
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M.; Aksenova, M.; Aksenov, M.; Markesbery, W. R.; Butterfield, D. A. J.
Mol. Neurosci. 1998, 11, 183.
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337.
9
58 VOL. 124, NO. 1, 2002 J. AM. CHEM. SOC.
10.1021/ja0168460 CCC: $22.00 © 2002 American Chemical Society

@-Tides
A R T I C L E S
Scheme 1
represented by 1 were devised for this purpose, with the cyclic
amino acid replacement providing some conformational restric-
tion and the tertiary amide limiting hydrogen bonding to a single
edge of the strand. In its simplest form, the design of 1 suffers
from the lack of an R-substituent, and from the possibility of
cis-trans isomerism about the tertiary amide bond. However,
the substitution of only one amino acid as well as the opportunity
for ready incorporation of such a unit in a modified peptide
synthesis made it an attractive target for investigation. For
convenience, we use “Ach” as a 3-letter abbreviation (from the
trivial “azacyclohexenone”) and the “@” symbol as a 1-letter
code for the cyclic unit; alternating oligomers with amino acids
we refer to as “@-tides”.
Synthesis of the Ach Building Block and Incorporation
in Oligomeric @-Tides
at the tertiary amide linkage. As the chain is elongated, this
multiplicity disappears because dimerization shifts the confor-
mational equilibrium to the extended form. We have not carried
out an exhaustive investigation of the stereochemical course of
the amino acid acylation reactions. However, a sample of D-Phe-
Ach-L-Ile prepared from Boc-D-Phe and Ach-L-Ile-OtBu under
the standard coupling conditions (PyBroP, DIEA, and DMAP
in methylene chloride for 24 h) was shown to be contaminated
with less than 1% of the L-L diastereomer, which is readily
resolved on HPLC.
Alternatively, the ester can be deprotected (e.g., to 6) and
the acid then coupled as a unit for more rapid chain elongation.
The convergent synthetic strategy was employed in the synthesis
of compound 9 (Scheme 2). The Alloc group was removed from
adduct 4a and the amine was coupled to acid 6 to give tetra-
@-tide 7, which was converted in a straightforward fashion to
penta-@-tide 9.
We envisaged a synthetic approach to @-tides involving the
incorporation of an N-protected, O-activated intermediate in
analogy to normal peptide synthesis. Our initial attempts to
prepare such a synthon began with N-benzylpiperidine-3,5-
dione, reported by Ziegler and Bennett.10 However, an alterna-
tive, shorter synthesis leading directly to a suitably N-protected
precursor was developed from 3,5-dimethoxypyridine (Scheme
1). Addition of sodium borohydride to an acetonitrile solution
of this material at -45 °C, followed by addition of allyl
chloroformate, affords an intermediate N-acyl dihydropyridine.
This material is not isolated but is hydrolyzed directly to the
protected enolic dione 2. This one-pot sequence affords the key
intermediate in multigram quantities in good yields (75-85%).
Activation of the vinylogous acid 2 for coupling to a peptide
proved to be more of a challenge than we had anticipated;
traditional coupling methods led to complex mixtures apparently
resulting from initial reaction of the amine nucleophile in a 1,2-
rather than a 1,4-manner. However, these problems were
overcome by activation of the hydroxyl group as the mixed
anhydride 3, formed with mesitylenesulfonyl chloride, and
catalysis of the coupling reaction with either ytterbium triflate
or tin triflate in THF.11,12
Solid-Phase Synthesis
This coupling process can be translated to solid phase as
demonstrated by the synthesis of tri-@-tide 13 and penta-@-
tide 15 in 75% and 43% overall yields, respectively (Scheme
3). The solid-phase procedure is similar to that in solution, but
it requires a few significant modifications. The mixed solvent
DMF/methylene chloride is more effective than CH2Cl2 alone
in promoting complete reaction of the resin-bound intermediates.
As a consequence, we found that tin triflate is more effective
than ytterbium triflate in coupling the activated Ach unit, 3, to
the support-bound substrate. For example, the ytterbium-
catalyzed reaction between 3 and isoleucine on resin resulted
in only a 78% yield of di-@-tide 11, while better than 95%
conversion was achieved with tin triflate. We attribute this
difference in reactivity to the greater solubility of Sn(OTf)2 in
the mixed solvent system.
The adduct 4 can be N-deprotected and coupled to an amino
acid to afford the tertiary amide (e.g., 5) in good yield (75-
93%) following normal protocols.13,14 Low molecular weight
@-tides such as 5 typically show multiple resonances in the
NMR spectrum in chloroform as a result of cis-trans isomerism
(10) Ziegler, F. E.; Bennett, G. B. J. Am. Chem. Soc. 1973, 95, 7458.
(11) (a) Pe´rez, M.; Pleixats, R. Tetrahedron 1995, 51, 8355. (b) Laszlo, P.
Tetrahedron Lett. 1989, 30, 3969. (c) Matsubara, S.; Yoshioka, M.; Utimoto,
K. Chem. Lett. 1994, 827.
(12) The corresponding chloroenone (3, with Cl in place of OSO2Mes) can also
be coupled to an amino acid ester with catalysis by ytterbium; however, a
major byproduct arises from loss of the Alloc protecting group. This side
reaction is not seen with the mesitylene sulfonate 3 or in the absence of
ytterbium, so it presumably reflects an Yb-catalyzed nucleophilic dealky-
lation of the Alloc group by chloride ion.
It also proved to be necessary to modify the scavenging
reagent for the palladium-catalyzed Alloc deprotection of the
resin-bound @-tides. A significant quantity of the N-allylated
(13) Deziel, R. Tetrahedron Lett. 1987, 28, 4371.
(14) Coste, J.; Dufour, M. N.; Pantaloni, A.; Castro, B. Tetrahedron Lett. 1990,
31, 669.
9
J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002 59

A R T I C L E S
Phillips et al.
Scheme 2
Figure 1. NH chemical shifts of @-tide 9 and peptide 16 in 1% CD3OH/
CDCl3 at 20 °C. The superscript a indicates the resonance for amide rotamer
observed at δ 8.12 ppm.
1
ments of the 13C and H spectra were obtained as follows.
Broadband 1H-decoupled 13C spectra were assigned via DEPT
subspectra and comparison of observed chemical shifts with
those predicted by an NMR simulation program.17 Two-
dimensional HMQC experiments then led directly from the
1
assigned 13C chemical shifts to the corresponding H signals.
The methylene hydrogens of the two Ach units in 9, which show
almost identical shifts, could be distinguished by their short-
range NOE cross-peaks to nearby aliphatic side chains.18 Amide
hydrogens were assigned from 2D TOCSY spectra acquired in
1% CD3OH/CDCl3.19 The TOCSY spectra also confirmed the
Scheme 3
1
other H assignments.
The NH chemical shifts of 9 in CDCl3 provided the first
indication that the @-tide hydrogen-bonds like a â-sheet.
Hydrogen-bonded NH protons in peptides typically resonate
around 8 ppm, which is ca. 2 ppm downfield of their chemical
shifts when not hydrogen-bonded.8b In penta-@-tide 9, NH
protons resonate from 7.5 to 8.7 ppm (Figure 1), significantly
downfield from the corresponding resonances (6.7-7.2 ppm)
observed for a control pentapeptide, 16, in which the Ach units
have been replaced with sarcosine. These data suggest that
@-tide 9 participates in hydrogen-bonding interactions more
extensively than does peptide 16. However, since two of the
NH resonances in @-tide 9 are vinylogous amides, the downfield
shifts should be considered in the context of other experimental
data supporting a â-sheet model of dimerization.
The CRΗ chemical shifts for penta-@-tide 9 provided
additional evidence for the â-sheet conformation. Relative to
the chemical shifts observed for the R-hydrogens of a peptide
in an unstructured, random-coil conformation, those of an
R-helix are shifted upfield and those of a â-strand (or extended)
conformation are downfield.20 The chemical shifts for the
R-hydrogens of penta-@-tide 9 are well downfield of those
expected for a random-coil model (Figure 2), which provides
further evidence for the extended conformation expected in a
hydrogen-bonded dimer.
@-tides was observed with N-methylmorpholine (NMM) in
acetic acid/chloroform (37:1:2 CHCl3/NMM/AcOH) for the
deprotection.15 However, when Me3SiN(Me)2 is employed as
the scavenger, formation of this byproduct is completely
suppressed.16
3JHNr Coupling Constants
3
The magnitude of the JHNR coupling constant for a peptide
residue is dependent on the φ-angle and therefore on the local
(15) Kates, S. A.; Daniels, S. B.; Sole, N. A.; Barany, G.; Albericio, F. Anal.
Biochem. 1993, 212, 303.
Characterization of @-Tide Association
(16) Merzouk, A.; Guibe´, F. Tetrahedron Lett. 1992, 33, 477.
(17) ACD-Labs online version. Web address: www.acdlabs.com.
(18) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons:
New York, 1986.
We anticipated that penta-@-tide 9 would be self-comple-
mentary, so we looked for evidence of dimerization as an
indication of its ability to mimic a â-strand. Complete assign-
(19) Hwang, T. L.; Shaka, A. J. J. Am. Chem. Soc. 1992, 114, 3157.
(20) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry 1992, 31, 1647.
9
60 J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002

@-Tides
A R T I C L E S
Figure 2. CR-H chemical shifts of 26 mM @-tide 9 in 1% CD3OH/CDCl3
at 20 °C (random coil values in parentheses).
Figure 5. Concentration dependence of NH chemical shifts for @-tide 9
in CDCl3 at 20 °C.
3
Figure 3. JHNR coupling constants of 20 mM @-tide 9 in 5% CD3OH/
differences among the various NH groups of the penta-@-tide,
with lower values for those in the center of the strand than those
at the ends. This behavior is consistent with an antiparallel dimer
structure in which the least solvent exposed NH exhibits the
smallest ∆δ/∆T value.
CDCl3 at 20 °C (random coil values in parentheses).
Concentration Dependence of NH Chemical Shifts
@-Tide dimerization can be detected by observing changes
in NH chemical shifts as a function of concentration. For a
dimerization process with dissociation constant Kd and NMR
chemical shifts δmono and δdi, respectively, the observed chemical
shift, δobs, as a function of concentration is expressed by eq 1.
2
-Kd
2
Kd
1
2c
δobs ) δdi + (δmono - δdi)
+
+ 2K c
d
(
)
x
4
(c ) concentration) (1)
Experimental data were obtained for several @-tides and peptide
16 in CDCl3 at 25 °C and were fitted to this equation, as shown
for penta-@-tide 9 in Figure 5, to give the dissociation constants
listed in Table 1. Whereas the dimerization constant determined
for the peptide 16 is greater than 150 mM, that for penta-@-
tide 9 is 0.4 mM in pure CDCl3,24 demonstrating quantitatively
the profound effects that the Ach unit has on the conformation
and hydrogen-bonding ability of the @-tide. Increasing the
length of the @-tides dramatically increases the affinity of the
homodimer, so much so that the dissociation constants for
related tri-, penta-, and hepta-@-tides cannot be measured by
NMR under the same conditions. Methanol promotes dissocia-
tion, so the @-tides 17, 18, and 19 were measured at increasing
methanol concentrations (Table 1). Although direct comparison
under identical conditions is not possible, the trend is quite
apparent. Interestingly, the data suggest that the C-terminal
carboxylic acid moiety promotes dimerization more strongly
than the N-methyl amide (compare Entry 2 with Entries 4 and
5).
Figure 4. Temperature dependence of NH chemical shifts (∆δ/∆T, ppb/
K) for @-tide 9 and peptide 16 in 1% CD3OH/CDCl3 (16-22 mM).
conformation of the polypeptide backbone.21 3JHNR values for
â-sheet conformations fall in the range from 8 to 10 Hz, while
3JHNR values for an unstructured random coil range from 5.8 to
7.3 Hz. NH-CRH coupling constants for the Leu and Ile
residues of penta-@-tide 9, shown in Figure 3, are within the
range for a â-sheet structure and are significantly higher than
those predicted for a random coil. Although the differences in
coupling constants give an indication of â-sheet conformation
for the mimics, they do not provide an indication of the φ-angle
directly, since the Karplus equation was derived for peptide
amides. Direct comparison of @-tide 9 with peptide 16 was
not possible, since an NH-CRH coupling constant could only
be resolved for the Phe residue in the peptide, which in turn
was not resolved for the @-tide.
Temperature Dependence of NH Chemical Shifts
Whether an NH group is hydrogen bonded intermolecularly
or is exposed to solvent can be revealed by the temperature
dependence of the chemical shift: low values for ∆δ/∆T reflect
persistent, intermolecular hydrogen bonds, and high values
indicate an equilibrium between hydrogen-bonded and non-
bonded states.22,23 In 1% CD3OH/CDCl3, dramatic differences
are observed between the peptide 16 and the penta-@-tide 9
(Figure 4), with the former exhibiting much higher ∆δ/∆T
values than the latter. More revealingly, there are significant
The dissociative effect of methanol was explored with penta-
@-tide 18 (Figure 6). The dependence of Kd on methanol
(21) Smith, L. J.; Bolin, K. A.; Schwalbe, H.; MacArthur, M. W.; Thornton, J.
M.; Dobson, C. M. J. Mol. Biol. 1996, 255, 494.
(22) Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512.
(23) Dyson, H. J.; Cross, K. J.; Houghten, R. A.; Wilson, I. A.; Lerner, R. A.
Nature 1985, 318, 480.
(24) The Kd for @-tide 9 was estimated to be 0.4 ( 0.1 mM based on multiple
independent titrations. δfree and δbound determined for the NH protons were
respectively as follows: Phe, 7.90 & 8.84 ppm; Ile, 7.21 & 7.97 ppm;
Leu, 7.03 & 7.55 ppm.
9
J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002 61

A R T I C L E S
Phillips et al.
Table 1. @-Tide Dimerization in CD3OH/CDCl3 Mixtures
solvent
entry
oligomer
(% CD OH/CDCl3)
K (mM)
d
3
1
2
3
4
5
6
Ac-Phe-Sar-Leu-Sar-Ile-NHMe (16)
Ac-Phe-Ach-Leu-Ach-Ile-NHMe (9)
Ac-Leu-Ach-Val-OH (17)
Ac-Phe-Ach-Leu-Ach-Val-OH (18)
Ac-Phe-Ach-Leu-Ach-Val-OH (18)
Ac-Leu-Ach-Val-Ach-Leu-Ach-Phe-OH (19)
0
0
1
>150
0.4
35, 71a
0.09
8
2.5
5
15b
1.5
a Amide rotamers with different Kd values were observed for tri-@-tide 17. b 15% CD3OH in CDCl3 was necessary to observe changes in chemical shift
of 19 with concentration; at lower percentages of CD3OH, no change was observed down to 0.2 mM.
Table 2. Intermolecular NOE Crosspeaks Observed for
Penta-@-tide 9 at Varying Concentrations of CD3OH/CDCl3
a,b
NOE no.
protons involved
1% CD OH
2.5% CD OH
3
3
1
2
3
4
5
6
7
8
9
Phe-arylsIle-δ
Phe-arylsIle-γ
Ac-MesNH-CH3
Phe-âsIle-NH
Ach-I-γsAch-II-γ
Ach-II-γsLeu-NH
Ach-II-γsPhe-aryl
Ile-δsPhe-â
S
W
W
M
W
M
W
W
S
S
S
S
M
n/o
n/o
W
M
n/o
n/o
n/o
M
M
n/o
n/o
Ile-âsPhe-â
10
11
12
NH-CH3sAc
Ile-γsAc
Ile-âsPhe-aryl
a Spectra were recorded at 20-35 mM concentrations at 20 °C; mixing
times were optimized to minimize spin-diffusion.25, 26 bS ) strong, M )
medium, W ) weak, n/o ) not observed.
Figure 6. Dependence of dimer dissociation constant for penta-@-tide 18
on the percentage of CD3OH in CDCl3 (R2 for the exponential line fit is
0.96).
concentration is dramatic, increasing more than 3 orders of
magnitude between 3% and 6% methanol. The effect is roughly
exponential, as would be expected at low concentrations of the
dissociating agent, where incremental effects on the free energy
of association are additive. Because of sensitivity limitations
in the NMR method used to determine the dissociation constants,
Kd values below 100 µM cannot be determined accurately;
however, extrapolation of the line in Figure 6 suggests that the
dissociation constant of 18 in pure chloroform could be as low
as 0.13 µM.
NOE Spectroscopy
In an antiparallel â-sheet structure, interstrand NOE effects
are generally observed between the side chains and between
the amide hydrogens of opposing residues. Additional evidence
for @-tide dimer formation was thus sought by acquiring
NOESY spectra in 1% CD3OH/CDCl3. At a concentration of
20 mM, the spectrum of the penta-@-tide 9 shows cross-peaks
between hydrogens at opposite ends of the molecule, which
would not be expected to arise intramolecularly (Table 2). These
NOE interactions are also identified in Figure 7. Spectra
obtained at increasing CD3OH concentrations demonstrated that
these cross-peaks are intermolecular; they are weaker in 2.5%
CD3OH/CDCl3 and absent entirely in 10% CD3OH/CDCl3. For
comparison, the NOESY spectrum for peptide 16 was obtained
with the same parameters, solvent, and concentration as those
for penta-@-tide 9; however, no cross-peaks between hydrogens
at opposite ends of the molecule were observed for peptide 16.
Further evidence for the â-strand conformation of mimic 9
is provided by the intramolecular cross-peaks in the NOE
spectrum (Figure 8). Cross-peaks between the CR hydrogens of
the amino acids and the C2 methylene and C4 vinyl hydrogens
of the Ach units are consistent with a conformation in which
Figure 7. Intermolecular NOE cross-peaks observed for penta-@-tide 9
in 1% CD3OH/CDCl3 at 20 mM concentration: (a) side chain-side chain
cross-peaks; (b) cross-peaks involving backbone hydrogens.
these atoms lie close to each other in the pleated conformation.
Similarly, cross-peaks are observed between the C6 methylene
hydrogens of the Ach units and the Leu and Ile amide
hydrogens. Equally telling are the cross-peaks that are not
observed, for example between the amide hydrogens and the
C2 and C4 positions, or the CR hydrogens and the C6 position.27
(25) Derome, A. E. Modern NMR Techniques for Chemistry Research; Pergamon
Press: New York, 1987.
9
62 J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002

@-Tides
A R T I C L E S
1, J ) 25), 5.63 (s, 1), 5.87-6.00 (m, 1), 9.90 (br s, 1); 13C NMR δ
46.56, 47.44, 66.89, 102.84, 118.31, 131.95, 154.73, 184.93, 186.86;
HRMS (FAB, m/z) Calcd. for C9H11NO4 (M + H+), 198.0766; found,
198.0767.
Prop-2-enyl 3-Oxo-5-[(2,4,6-trimethylphenyl)sulfonyloxy]-1,2,6-
trihydropyridine-1-carboxylate (3). To a stirring solution of dione 2
(9.5 g, 48 mmol) in anhydrous CH2Cl2 (150 mL) under a nitrogen
atmosphere was added powdered anhydrous K2CO3 (10.97 g, 79.50
mmol) and mesitylenesulfonyl chloride (15.8 g, 72.3 mmol). After 4
h, excess reagent was quenched by addition of saturated NH4Cl (100
mL). The aqueous phase was washed three times with CH2Cl2 (100
mL), and the combined organic phases were washed with brine, dried
over (Na2SO4), and concentrated under vacuum. The crude product was
chromatographed (EtOAc/hexanes 1:2) to yield the mixed anhydride 3
(9.1 g, 24 mmol, 69%) as a pale yellow oil. Anhydride 3 was found to
be stable at room temperature in a 0.1 M CH2Cl2 solution, but for
prolonged storage the compound was dissolved in CH2Cl2 (1 M) and
kept at -78 °C. 1H NMR δ 2.35 (s, 3), 2.63 (s, 6), 4.09 (s, 2), 4.32 (s,
2), 4.62 (d, 2, J ) 5.5), 5.24 (d,1, J ) 10.6), 5.30 (d, 1, J ) 17.5),
5.83-5.98 (m, 2), 7.05 (s, 2); 13C NMR δ 20.92, 22.49, 44.02, 50.30,
66.69, 113.78, 118.12, 129.93, 131.86, 132.03, 139.94, 144.94, 154.15,
191.96; MS (FAB) m/z (%) 144 (100), 323 (70), 380 (30, M + H+).
Figure 8. Intramolecular NOE interactions consistent with a â-strand
conformation for penta-@-tide 9. NOE measurements were obtained in 1%
CD3OH/CDCl3 at 20 mM and 20 °C.
Conclusion
It is apparent that replacing amino acids at alternate positions
in a peptide with the 1,2-dihydro-3(6H)-pyridinone (“Ach”) unit
affords an oligomeric molecule that exhibits many of the NMR
and hydrogen-bonding characteristics of a peptide in the
extended, â-strand conformation in chloroform and chloroform/
methanol. This behavior is revealed by an enhanced propensity
to dimerize in comparison to a related peptide, by reduced
exposure of the central NH groups to solvent, and by a pattern
of solvent-dependent NOE interactions that are consistent with
an antiparallel hydrogen-bonded dimer. This amino acid sur-
rogate thus offers promise as a â-strand mimetic and in model
studies of â-sheet formation.
tert-Butyl (2S,3S)-3-Methyl-2-{[5-oxo-1-(prop-2-enyloxycarbo-
nyl)-1,2,6-trihydropyridyl]amino}pentanoate (Alloc-Ach-Ile t-Butyl
Ester, 4a). To a solution of anhydride 3 (1.0 g, 2.6 mmol) in dry THF
(11 mL) were added isoleucine tert-butyl ester hydrochloride (0.5 g,
2.7 mmol), anhydrous ytterbium(III) triflate (1.64 g, 2.65 mmol), and
DIEA (1.38 mL, 7.92 mmol) under a nitrogen atmosphere. After 24 h,
saturated NH4Cl was added (10 mL), and the mixture was extracted
with EtOAc (3 × 10 mL). The combined organic extracts were washed
with brine, dried over MgSO4, and evaporated. Purification of the crude
product by flash chromatography (hexanes/EtOAc, 1:1) gave vinylogous
amide 4a (0.70 g, 1.9 mmol, 73%) as a light yellow oil. Proton and
Experimental Section
General. Reagents were obtained from commercial suppliers and
used as received. Solvents were purchased from commercial suppliers
in the anhydrous form or were dried via distillation. Flash chromatog-
raphy was performed according to the method of Still28 with 60-mesh
silica gel from E. Merck & Co.
Abbreviations: @ and Ach, the 1,2-dihydro-3(6H)-pyridinyl unit;
PyBroP, bromotris(pyrrolidino)phosphonium hexafluorophosphate; DIEA,
diisopropylethylamine; 4-DMAP, 4-(dimethylamino)pyridine.
1
carbon spectra show peak doubling due to amide bond rotamers. H
NMR δ 0.89-0.98 (m, 6), 1.47-1.49 (s, 9, rot), 1.49-1.63 (m, 1);
1.65-1.78 (m, 1); 1.83-1.93 (m, 1); 3.88 (dd, 1, J ) 4.9, 7.7), 4.02
(d, 1, J ) 17.9), 4.10 (d, 1), 4.27 (d, 1, J ) 16.1), 4.38 (d, 1, J )
16.6); 4.63 (d, 2, J ) 5.5), 5.18 (s, 1), 5.23 (d, 1, J ) 10.4), 5.31 (m,
1, J ) 17.3, 1.6, 3.1), 5.84 (d, 1, J ) 6.9), 5.83-5.99 (m, 1); 13C
NMR δ 11.55, 11.67, 14.06, 14.83, 15.53, 24.69, 25.96, 27.94, 27.97,
37.31, 39.18, 44.22, 50.57, 59.28, 59.46, 66.53, 80.67, 82.95, 95.64,
117.83, 174.71; MS (FAB) m/z (%) 450 (100), 367 (M+ + H), 338
(42), 311 (84), 292 (22), 265 (20), 244 (28), 225 (20), 198 (32), 179
(10), 154 (18); HRMS (FAB, m/z) Calcd. for C19H30N2O5 (MH+),
367.2233; found, 367.2232.
Prop-2-enyl 5-Hydroxy-3-oxo-1,2,6-trihydropyridine-1-carboxy-
late (2). To a solution of 3,5-dimethoxypyridine29 (8.5 g, 61 mmol) in
dry MeCN (230 mL) at -45 °C was added NaBH4 (4.16 g, 110 mmol)
in portions over 10 min, and the resulting mixture was stirred for an
additional 10 min. Allyl chloroformate (7.79 mL, 73.4 mmol) was added
over 45 min while the temperature (internal thermometer) was kept at
-45 to -40 °C. The reaction was allowed to proceed for an additional
15 min at -40 °C, and then 1 N HCl (150 mL) was added at -40 °C.
The HCl addition was followed immediately by addition of saturated
NaHCO3 (100 mL) until the pH was basic. The aqueous layer was
extracted with EtOAc (3 × 50 mL) and the organic layer was dried
over Na2SO4 and evaporated in vacuo. The crude product was dissolved
in THF (200 mL) and 1 N HCl (200 mL). The reaction mixture was
stirred for 30 min at room temperature and then made basic with solid
NaOH at 0 °C. The aqueous layer was washed once with EtOAc (50
mL), and the organic layer was subsequently washed with 1 N NaOH
until the aqueous layer was no longer yellow. The combined aqueous
layers were acidified with 6 N HCl at 0 °C, saturated with NaCl, and
extracted three times with EtOAc (50 mL). The combined organic layer
was dried over Na2SO4 and concentrated to a thick oil. The enolic
diketone tended to decompose on standing, so the crude product (9.5
g, 48 mmol, ca. 79%) was used immediately in the following step. An
analytical sample was purified by flash chromatography using a gradient
tert-Butyl (2S,3S)-3-Methyl-2-[(5-oxo-1,2,6-trihydro-3-pyridyl)-
amino]pentanoate (Ach-Ile t-Butyl Ester). To a solution of Alloc-
protected dimer 4a (0.46 g, 1.3 mmol) in a 1:1 mixture of THF/
diethylamine (4.8 mL) at room temperature was added tetrakis-
(triphenylphosphine)palladium(0) (Pd(PPh3)4; 0.12 g, 0.11 mmol), and
the mixture was stirred for 1 h. The solvent was evaporated, 1 N HCl
(25 mL) was added, and the solution was washed three times with
EtOAc. The aqueous solution was brought to pH >14 with solid NaOH
and extracted with three portions of EtOAc. The pH was readjusted to
>14 and the extraction was repeated. The combined organic extracts
were washed with brine and with brine containing diethyl dithiocar-
bamic acid, dried over Na2SO4, and evaporated to afford the crude
amine, which was used immediately in the next step. An analytical
sample was purified by flash chromatography with CH2Cl2/MeOH (9:
1) containing 3% Et3N. 1H NMR δ 0.91 (d, 3, J ) 6.6), 0.96 (t, 3, J )
7.5), 1.31-1.38 (m, 1H), 1.49 (s, 9), 1.51-1.61 (m, 1), 1.81-1.91 (m,
1), 3.39 (s, 2), 3.60 (s, 2), 3.86 (dd, 1, J ) 4.6, J ) 7.5), 5.11 (s, 1),
5.59 (d, 1, J ) 7.7); 13C NMR δ 11.54, 14.84, 25.94, 27.94, 37.28,
47.20, 53.23, 59.20, 82.93, 95.39, 162.73, 170.33, 195.98; MS (FAB)
m/z (%) 338 (48), 292 (30), 283 (74, M+H+), 227 (66); the mass
spectrum also showed aggregates with masses higher than M+.
1
of petroleum ether/EtOAc to give the enolic diketone 2 as an oil. H
NMR δ 4.20 (s, 4), 4.64 (d, 2, J ) 5.3), 5.27 (d, 1, J ) 19), 5.31 (d,
(26) A very weak interaction was observed between the Leu-NH and the Ach
vinyl hydrogen, which may reflect some conformational heterogeneity.
(27) Sanders, J. K. M.; Hunter, B. K. Modern NMR Spectroscopy; Oxford
University Press: Oxford, 1994.
(28) Still, W. C.; Kahn, M.; Mitra, S. J. Org. Chem. 1978, 43, 2923.
(29) Testaferri, L.; Tiecco, M.; Tingoli, M.; Bartoli, D.; Massoli, A. Tetrahedron
1985, 41, 1373-84.
9
J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002 63

A R T I C L E S
Phillips et al.
Fmoc-Ile-Ach-Phe tert-Butyl Ester (5). To a solution of the Alloc-
protected Ach-Phe ester 4d (100 mg, 0.25 mmol) in a 1:1 mixture of
THF/diethylamine (2 mL) at room temperature was added Pd(PPh3)4
(28 mg, 0.03 mmol), and the mixture was stirred for 1 h. The solvent
was evaporated under reduced pressure, then coevaporated under
reduced pressure from dioxane (2 × 2 mL) to afford the crude amine,
which was used immediately in the next step. To this product (80 mg,
0.25 mmol) in CH2Cl2 (3.5 mL) was added Fmoc-isoleucine (0.18 g,
0.51 mmol), PyBroP (0.24 g, 0.52 mmol), and DIEA (0.44 mL, 2.5
mmol). The reaction mixture was stirred at room temperature under a
nitrogen atmosphere for 26 h, then evaporated under reduced pressure.
The residue was redissolved in EtOAc and the solution was washed
with 1 M HCl (3 × 3 mL), NaHCO3 (3 mL), and brine (3 mL), dried
over MgSO4, and concentrated. The crude product was purified by flash
chromatography (EtOAc/hexanes (2:1)) to afford tri-@-tide 5 (0.13 g,
0.21 mmol, 82%) as a light yellow oil. 1H NMR δ 0.59 (bs, 0.3), 0.68
(bs, 0.3), 0.88 (m, 6), 1.15 (m, 1), 1.37 (s, 9), 1.50 (bm, 1), 1.60 (bm,
1), 3.13 (m, 2), 4.06 (m, 2), 4.18 (m, 2), 4.32 (m, 3), 4.56 (m, 1), 5.06
(s, 0.5), 5.09 (s, 0.5), 5.23 (s, 0.2), 5.28 (s, 1), 5.62 (d, 0.2), 5.71 (d,
0.2), 6.23 (d, 1), 6.54 (bs, 1), 7.12 (m, 2), 7.24 (m, 6), 7.36 (m, 1),
7.46 (m, 1), 7.55 (m, 1), 7.67 (m, 1), 7.74 (d, 1); 13C NMR δ 11.19,
15.67, 24.25, 37.33, 37.60, 42.86, 47.11, 52.66, 54.87, 56.43, 83.64,
95.20, 119.92, 125.08, 125.14, 126.89, 127.08, 127.30, 127.63, 128.42,
128.47, 128.52, 129.40, 131.90, 131.92, 132.02, 132.10, 132.88, 135.10,
141.22, 143.76, 143.91, 156.44, 159.96, 169.76, 171.74, 189.53; MS
(FAB) m/z (%) 652 (55, M + H+), 596 (20), 400 (10); HRMS (FAB,
m/z) Calcd. for C39H46N3O6 (M + H+), 652.3387; found, 652.3394.
(2S)-4-Methyl-2-[[5-oxo-1-(prop-2-enyloxycarbonyl)(3-oxo-1,2,6-
trihydropyridyl)]amino]pentanoic Acid (Alloc-Ach-Leu, 6). tert-
Butyl ester 4c (2.75 g, 7.48 mmol) was dissolved in neat TFA (25
mL) under argon and stirred for 2 h. After evaporation of the solvent,
EtOAc was added, and the solution was washed with two portions of
saturated NaH2PO4 and brine, dried over Na2SO4, and evaporated. The
residue was purified by flash chromatography using a gradient of
petroleum ether/EtOAc/AcOH (79:20:1, then 0:99:1); traces of acetic
acid were removed by coevaporation with three portions of toluene to
give the pure acid 6 as a yellow oil in quantitative yield (2.32 g, 7.48
mol). 1H NMR δ 0.92 (d, 3, J ) 5.0), 0.96 (d, 3, J ) 5.2), 1.68-1.79
(m, 3), 4.01-4.20 (m, 2), 4.12 (dd, 1, J ) 7.2, 7.2), 4.34 (d, 1, J )
17.4), 4.40 (d, 1, J ) 16.8), 4.61 (s, 2), 5.23 (d, 1, J ) 10.5), 5.30 (d,
1, J ) 16.9), 5.37 (s, 1), 5.86-5.94 (m, 1), 6.80 (bs, 1); 13C NMR δ
21.74, 22.57, 24.86, 40.46, 44.01, 54.52, 60.49, 67.01, 94.42, 118.35,
131.91, 154.88, 164.02, 171.40, 174.21; MS (FAB) m/z (%) 311 (M+,
100), 265 (20), 225 (33), 154 (86), 136 (74), 107 (34); HRMS (FAB,
m/z) Calcd. for C15H22N2O5 (M + H+), 311.1607; found, 311.1615.
158.28, 170.39, 188.52; MS (FAB) m/z (%) no M+, 480 (78), 424 (100),
323 (22), 265 (30), 225 (14), 179 (19).
Fmoc-Phe-Ach-Leu-Ach-Ile tert-Butyl Ester (8). To a solution of
tetramer 7 (1.10 g, 1.91 mmol, contaminated with tris(pyrrolidino)-
phosphoramide) in a 1:1 mixture of dry THF/Et2NH (20 mL) was added
Pd(PPh3)4 (20 µmol, 23 mg) under argon. After 4 h, 1 N HCl was
added to pH <1, and the mixture was extracted with three portions of
EtOAc. The aqueous layer was brought to pH >14 with 5 N NaOH
and extracted with four portions of EtOAc. Washing with brine
(containing ca. 200 mg of sodium diethyldithiocarbamate), drying over
Na2SO4, and evaporation of the solvent gave a crude product, which
was purified by flash chromatography (gradient of CH2Cl2/MeOH/Et3N
90:10:0 f 78:19:3) to give the N-deprotected Ach-Leu-Ach-Ile tert-
1
butyl ester (721 mg, 1.74 mmol, 77%) as a yellow solid. H NMR δ
0.87 (d, 3, J ) 5.6), 0.91 (d, 3, J ) 2.8), 0.92 (d, 3, J ) 3.2), 0.95 (t,
3, J ) 7.6), 1.30-1.34 (m, 1), 1.50 (s, 9), 1.52-1.69 (3), 1.71-1.79
(m 1), 1.84-1.92 (m, 1), 3.35 (s, 2), 3.58 (d, 1, J ) 16.9), 3.64 (d, 1,
J ) 16.6), 3.91 (dd, 1, J ) 5.0, 7.8), 4.00 (d, 1, J ) 17.1), 4.10 (d, 1,
J ) 16.9), 4.26 (d, 1, J ) 17.1), 4.46-4.52 (m, 1), 5.14-5.16 (s, 1),
5.19-5.22 (s, 1), 7.02 (d, 1, J ) 8.1), 7.18 (d, 1, J ) 7.3); 13C NMR
δ 12.23, 15.70, 22.05, 23.84, 25.37, 26.67, 28.65, 38.40, 41.63, 43.54,
46.56, 47.81, 51.17, 52.83, 54.15, 60.61, 83.68, 94.93, 95.11, 162.04,
165.38, 170.54, 171.78, 189.71, 196.36; MS (FAB) m/z (%) 491 (M+,
100), 435 (44), 340 (6), 319 (10), 281 (8), 225 (22), 179 (30), 154
(18), 136 (14), 111 (20); HRMS (FAB, m/z) Calcd. for C26H42N4O5
(M+ + H), 491.3233; found, 491.3233.
To a solution of the N-deprotected compound (510 mg, 1.04 mmol)
in dry CH2Cl2 (5 mL) were added Fmoc-phenylalanine (603 mg, 1.56
mmol), PyBroP (726 mg, 1.56 mmol), 4-DMAP (6 mg, 52 µmol), and
DIEA (723 µl, 4.16 mmol). The reaction mixture was stirred under
argon at room temperature for 16 h; EtOAc was added, and the solution
was washed with 1 N HCl, saturated NH4Cl, and brine, dried over Na2-
SO4, and evaporated. Purification by flash chromatography (EtOAc/
MeOH 95:5) gave pentamer 8 (812 mg, 944 µmol, 91%) as a white
1
solid. H NMR δ 0.80-0.92 (m, 12), 1.23-1.32 (m, 1), 1.40 (s, 9),
1.44-1.63 (m, 4), 1.77-1.86 (m, 1), 2.85-2.93 (m, 2), 2.99 (s, 2),
3.69-4.48 (m, 10), 4.74-5.18 (m, 4), 6.96-7.24 (m, 7), 7.25-7.35
(m, 2), 7.38-7.51 (m, 2), 7.60-7.73 (m, 2); 13C NMR δ 11.50, 13.94,
14.91, 20.82, 22.10, 23.06, 24.57, 26.11, 27.81, 27.86, 37.93, 38.83,
41.14, 42.06, 46.86, 51.68, 51.85, 59.64, 60.41, 67.02, 83.58, 93.70,
94.16, 119.80, 124.91, 124.99, 126.77, 127.09, 127.55, 128.53, 129.00,
135.60, 141.16, 143.53, 143.68, 156.15, 160.95, 161.17, 169.93, 170.50,
171.31, 171.47, 188.97, 189.65; MS (FAB) m/z (%) 861 (M+, 48), 179
(100), 154 (84), 137 (58); HRMS (FAB, m/z) Calcd. for C50H61N5O8
(M+ + H), 860.4598; found, 860.4611.
Alloc-Ach-Leu-Ach-Ile tert-Butyl Ester (7). To solution of acid 6
(1.66 g, 5.35 mmol) and Ach-Ile tert-butyl ester 4a (1.51 g, 5.35 mmol)
in dry CH2Cl2 (40 mL) at 0 °C was added DIEA (1.67 mL, 9.63 mmol),
4-DMAP (63 mg, 535 µmol), and PyBroP (3.24 g, 6.96 mmol). After
30 min, the ice bath was removed, and the mixture was stirred for 14
h at room temperature. After dilution with CH2Cl2, the solution was
extracted with four portions of 1 N HCl, saturated NaHCO3, and brine,
dried (MgSO4), and evaporated. The crude product was purified by
flash chromatography using a gradient of CH2Cl2/MeOH (97:3, 95:5)
to give a fraction of pure peptide 7 (1.28 g, 2.33 mmol, 42%) and a
fraction (2.65 g) contaminated with tris(pyrrolidino)phosphoramide. The
NMR spectra are complicated by peak doubling due to amide rotamers.
1H NMR δ 0.86-0.98 (m, 12), 1.45-1.49 (s, 9), 1.64-2.08 (m, 4),
3.88 (dd, 1, J ) 4.9, 7.6), 4.00-4.38 (m, 6), 4.44 (dd, 1, J ) 4.9, 8.2),
4.46 (d, 1, J ) 16.9), 4.57 (d, 1, J ) 16.9), 4.63 (d, 2, J ) 5.2), 5.20
(s, 1), 5.22 (ddd, 1, J ) 1.4, 2.5, 10.5), 5.30 (ddd, 1, J ) 1.5, 3.1,
17.2), 5.39 (s, 1), 5.91 (ddt, 1, J ) 10.5, 17.2, 5.5), 6.08-6.17 (d, 1,
J ) 7.8), 6.43 (s, 1); 13C NMR δ 12.31, 12.35, 15.67, 16.09, 23.45,
25.40, 25.93, 26.73, 28.68, 28.73, 38.12, 38.54, 42.34, 44.82, 52.40,
55.83, 57.73, 60.55, 61.07, 67.38, 82.65, 83.97, 95.80, 96.23, 118.52,
Ac-Phe-Ach-Leu-Ach-Ile N-Methylamide (9). A solution of Fmoc-
pentamer 8 (749 mg, 871 µmol) in dry CH2Cl2 (5 mL) was treated
with Et2NH (5 mL) at room temperature under argon for 3 h. The
solution was evaporated under reduced pressure and the residue was
coevaporated with three portions of dichloroethane (5 mL) and dried
under high vacuum. The crude amine was redissolved in dry CH2Cl2
(5 mL), and dry pyridine (1.41 mL, 17.5 mmol) and acetic anhydride
(831 µL, 8.71 mmol) were added. After 50 min, the volatile materials
were removed under vacuum and the residue was coevaporated with
three portions of C2H4Cl2 (5 mL). Purification of the crude product by
flash chromatography (gradient of CH2Cl2/MeOH 95:5-9:1) as eluant
gave the acetyl derivative (505 mg, 743 µmol, 85%) as a yellowish
1
solid. H NMR (300 MHz, CDCl3) δ 0.85-1.02 (m, 12), 1.28-1.43
(m, 1), 1.49-1.53 (s, 9), 1.64-1.74 (m, 1), 1.84-1.95 (m, 1), 2.01-
2.06 (s, 3), 2.93 (d, 2, J ) 6.6), 3.80-4.72 (m, 10), 5.09-5.47 (m, 3),
6.94-7.29 (m, 5); 13C NMR (75 MHz, CDCl3) δ 11.38, 14.96, 20.80,
22.02, 22.56, 23.11, 24.43, 25.92, 27.81, 38.02, 39.01, 41.95, 42.49,
42.69, 49.68, 50.37, 51.58, 51.78, 59.63, 82.50, 83.13, 93.64, 94.04,
126.89, 128.29, 128.89, 135.38, 161.06, 161.26, 169.91, 170.12, 170.47,
170.90, 188.91, 189.59; MS (FAB) m/z (%) 680 (M+, 100), 624 (30),
9
64 J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002

@-Tides
A R T I C L E S
435 (30), 225 (36), 179 (54), 120 (62); HRMS (FAB, m/z) Calcd. for
C37H53N5O7 (M+ + H), 680.4023; found, 680.4012.
Alloc Deprotection. The resin (0.1 g, 0.91 mmol/g) was prewashed
once with dry CH2Cl2, and then suspended in 3 mL of dry CH2Cl2.
Me3SiN(Me)2 (0.29 mL, 1.8 mmol) was added to the resin followed
by Pd(PPh3)4 (0.11 g, 0.09 mmol). The resin was quickly shaken for
even mixing, followed by rotating for 40 min. The resin was washed
and then dried in vacuo for 2 h.
Fmoc-Amino Acid Addition. The resin (0.1 g, 0.91 mmol/g) was
prewashed once with dry CH2Cl2, and then suspended in 3 mL of dry
CH2Cl2. The desired Fmoc-protected amino acid (5 equiv in relation
to the resin) was added to the resin, followed by PyBroP (5 equiv) and
DIEA (10 equiv). The reaction vial was vigorously shaken, followed
by rotating at room temperature for 24 h. The resin was washed and
immediately Fmoc-deprotected as previously described.
Cleavage from Resin. The product was cleaved from resin after
Fmoc-deprotection without drying the resin prior to cleavage. The resin
was suspended in 1:1 CH2Cl2/TFA (3 mL) and rotated in a glass vial
for 2 h. The solvent was removed under reduced pressure, and the
residue was redissolved in MeOH, filtered, and washed (4 × 2 mL
MeOH). This solution was combined and the solvent was removed
under reduced pressure; the crude product was immediately purified
by preparative HPLC.
The above material (388 mg, 571 µmol) was dissolved in dichlo-
roethane (3.5 mL) and treated with TFA (1.5 mL) for 5 h. The volatile
materials were evaporated under reduced pressure, and the residue was
coevaporated with three portions of dichloroethane (5 mL) and dissolved
in CH2Cl2. The solution was washed with saturated NaH2PO4, dried
over Na2SO4, and evaporated to yield the crude acid (347 mg, 556
µmol, 97%) as a yellowish foam.
A solution of the crude acid and 1-hydroxy-7-azabenzotriazole (108
mg, 799 µmol), EDC (137 mg, 714 µmol), 4-DMAP (3.5 mg, 29 µmol),
and methylamine (2.0 M in THF, 570 µL, 1.14 mmol) in dry CH2Cl2
(5 mL) was stirred under argon at 0 °C for 20 h at room temperature.
CH2Cl2 was added, the solution was washed twice with 10% KHSO4
and saturated NaHCO3, and with brine, dried over Na2SO4, and
evaporated to give 240 mg of crude methylamide. Purification by flash
chromatography (gradient of CH2Cl2/MeOH 9:1-8:2) gave 154 mg
(242 µmol, 42% over 2 steps) of the amide 9 as a colorless solid: mp
1
210-215 °C dec. H NMR (CDCl3-CD3OH 10:1) δ 0.80-0.86 (m,
6), 0.87-0.94 (m, 6), 1.06-1.16 (m, 1), 1.54 (bs, 2), 1.57-1.64 (m,
1), 1.75-1.87 (m, 1), 1.89 (d, 0.5, J ) 6.4, rot), 1.95 (s, 3, rot), 2.71
(s, 3), 2.89 (dd, 1, J ) 8.9, 12.5); 2.96 (dd, 1, J ) 6.9, 13.3), 3.71 (d,
1, J ) 16.8), 3.82 (d, 1, J ) 16.8), 3.82-3.86 (m, 1), 3.98-4.07 (m,
1), 4.07-4.11 (m, 2), 4.27-4.40 (m, 1), 4.53 (dd, 1, J ) 7.6, 2.2);
4.82 (d, 1, J ) 17.6), 4.83 (d, 1, J ) 17.6), 5.01-5.09 (m, 0.3, rot),
5.21-5.25 (m, 1, rot), 5.19 (s, 1), 5.37 (s, 1), 7.08 (d, 2, J ) 6.4),
7.11-7.18 (m, 3), 7.35 (d, 1, J ) 9.0), 7.91 (d, 1, J ) 8.4), 8.05 (d, 1,
J ) 4.4), 8.35 (bs, 1); 13C NMR δ 10.89, 14.93, 22.19, 22.47, 23.18,
24.34, 25.01, 25.78, 37.89, 39.19, 42.08, 42.45, 42.63, 49.58, 50.32,
51.29, 51.60, 60.40, 93.66, 126.79, 128.31, 128.86, 135.76, 160.60,
161.61, 170.79, 170.82, 170.93, 171.00, 189.13, 189.38; MS (FAB)
m/z (%) 637 (M+, 100), 179 (64), 154 (32), 137 (28), 120 (68); HRMS
(FAB, m/z) Calcd. for C54H48N6O6 (M+ + H), 637.3714; found,
637.3723.
Phe-Ach-Ile (13). Resin-bound tri-@-tide 12 (0.71 mmol/g) was
assembled from Fmoc-Ile resin (NovaBiochem) according to the general
procedures described above. This material (0.46 g resin) was deprotected
and cleaved from the resin and purified by preparative reverse-phase
HPLC to afford tri-@-tide 13 (0.09 g, 0.25 mmol, 75% overall) as a
light yellow foam. The NMR spectra are complicated due to the
presence of rotamers. 1H NMR ((CD3)2CO) δ 0.93 (m, 35.9), 1.00 (d,
15.3, J ) 6.5), 1.04 (d, 1.6, J ) 7.0), 1.24 (br m, 1.4), 1.34 (m, 7.6),
1.51 (m, 1.9), 1.64 (br m, 7), 1.95 (s, 4.8), 2.06-2.07 (m, 47.4), 2.08
(s, 3.8), 3.10 (t, 1.4, J ) 9.5), 3.21 (m, 6), 3.30 (m, 4.2), 3.38 (m, 5.6),
3.52 (m, 0.46), 3.92 (br m, 17.4), 4.23 (d, 3.9, J ) 17), 4.33 (m, 1.4),
4.36 (d, 0.4, J ) 5.5), 4.44 (d, 1, J ) 6.0), 4.49 (m, 1.6), 4.73-4.81
(m, 5.4), 5.06 (m, 3.8), 5.29 (q, 0.3, J ) 5.0), 5.68 (q, 1.2, J ) 5.5,
8.5), 5.82 (q, 1), 6.87 (m, 0.3), 7.30 (m, 28), 7.38 (m, 9), 7.83 (s, 0.2),
7.93 (s, 0.2); 13C NMR (CD3OD) δ 10.19, 10.29, 10.4 (rot), 14.17 (rot),
14.31, 14.59 (rot), 20.89 (rot), 24.87, 25.15, 25.21 (rot), 35.54 (rot),
36.69, 36.79 (rot), 36.88 (rot), 37.05, 41.69, 44.68 (rot), 48.42, 50.86,
50.99 (rot), 51.11 (rot), 60.08 (rot), 60.26, 127.6 (rot), 127.67, 128.45
(rot), 128.64 (rot), 128.94, 129.13 (rot), 129.24, 129.34 (rot), 129.46
(rot), 133.32 (rot), 133.52, 166.81 (rot), 167.01, 171.79 (rot), 171.99,
190.11, 191.37 (rot); IR (film) νmax 3264, 2956, 2916, 1672 cm-1; MS
(FAB) m/z (%) 374 (100, M + H+), 227 (45), 120 (85); HRMS (FAB,
m/z) Calcd. for C20H28N3O4 (M + H+), 374.2080; found, 374.2083.
Phe-Ach-Phe-Ach-Ile (15). In a similar manner, penta-@-tide resin
14 (0.91 mmol/g) was synthesized and a sample (0.06 g) was
deprotected and cleaved as described above. The crude material was
purified by preparative reverse-phase HPLC to afford penta-@-tide 15
(15 mg, 0.03 mmol, 45% overall) as a light yellow foam. The proton
General Procedures for Solid-Phase Synthesis. Merrifield poly-
styrene resins were obtained from NovaBiochem loaded with Fmoc-
amino acids (ca. 0.7-0.9 mmol/g). Solid-phase syntheses were carried
out in silylated glass reaction vessels fitted with a frit. The resin was
washed in the following manner: DMF (3×), alternating MeOH and
CH2Cl2 (3× each), and CH2Cl2 (3×). When palladium was used in the
reaction, the washing included MeOH (1×) prior to the normal washing
procedure. During washings, the resin was agitated with nitrogen
bubbling for 2 min before the solvent was removed. The presence or
absence of free amine was detected by the Kaiser test.31 Fmoc
quantitation analysis was performed by using the method of MilliGen32
with a Uvikon 860 spectrometer. Reactions were agitated either with
a Burrell Wrist Action Shaker or a Labquake rotator. Deprotection of
Fmoc was accomplished by shaking the resin in 20% piperidine in DMF
for 20 min, followed by the washing procedure and drying of the resin
in vacuo for 16-20 h. Resins were stored dry at 0 °C.
1
spectrum is complicated due to the presence of rotamers; H NMR
(CD3OD) δ 0.99 (br m, 5.57), 1.31 (br m, 1.6), 1.61 (br m, 0.8), 1.94
(br m, 1), 3.07 (br m, 3.3), 3.86 (br m, 1.5), 4.02 (br m, 0.7), 4.07-
4.12 (br m, 0.8), 4.22 (br m, 1.0), 4.36 (br m, 0.4), 4.47 (br m, 1), 4.54
(br m, 1), 4.70 (t, 0.5), 4.95 (s, 0.3), 4.99 (d, 0.2), 5.10 (br m, 0.9),
Ach Addition. Tin(II) triflate (0.04 g, 0.09 mmol) was added to
resin (0.1 g, 0.91 mmol/g) followed by DIEA (0.08 mL, 0.46 mmol),
activated Ach unit, 3 (1M in CH2Cl2, 0.36 mL, 0.36 mmol), and DMF
(2.5 mL). The reaction vessel was rotated for 16 h at room temperature,
and workup involved the washing procedure described above and drying
of the resin in vacuo for 2 h.
7.17-7.31 (br m, 8); IR (film) νmax 3318, 2952, 2915, 2847, 1648 cm-1
;
MS (FAB) m/z (%) 616 (70, M + H+), 340 (60), 312 (90), 284 (100);
HRMS (FAB, m/z) Calcd. for C34H42N5O6 (M + H+), 616.3135; found,
616.3118.
Capping. To cap free amines remaining after an acylation or
coupling procedure, the resin (0.1 g, 0.91 mmol/g) was prewashed once
with dry CH2Cl2. The drained resin (0.1 g, 0.91 mmol/g) was swollen
in 3:1:1 CH2Cl2/DIEA/Ac2O (5 mL total volume), and the reaction was
allowed to proceed for 2 h prior to washing and drying of the resin.
NMR Analysis. NMR spectra were obtained with a Bruker 500 MHz
spectrometer in CDCl3 solution unless otherwise indicated. Spectral
data are reported as chemical shifts (multiplicity, number of hydrogens,
1
coupling constants in Hz). H NMR chemical shifts are referenced to
TMS (0 ppm) in CDCl3, CD3OD (3.31 ppm), or (CD3)2CO (2.05 ppm);
13C NMR spectra were proton decoupled and referenced to CDCl3
(77.16 ppm) or CD3OD (49.00 ppm). Resonance assignments were
obtained by the method of Wu¨thrich18 using TOCSY and NOESY
(30) Stewart, J. M.; Young, J. D. Solid-Phase Peptide Synthesis, 2nd ed.; Pierce
Chemical Company: Rockford, 1984; p 105.
(31) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.
1970, 34, 595.
(32) Procedure given on p P4 of the NovaBiochem 2000 catalog.
9
J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002 65

A R T I C L E S
Phillips et al.
spectra. Samples were analyzed at approximately 20 mM in CD3OH/
CDCl3 solutions. Rigorous degassing was performed prior to the
NOESY experiments using three freeze-pump-thaw cycles.25 NOESY
experiments were performed by the method of Ananikov30 with mixing
times optimized to minimize spin-diffusion (0.7 s).26 NOESY data were
collected with 2048 data points in F2 and 512 data points in F1. For
VT experiments, the sample was allowed to adapt to the adjusted
temperature for at least 10 min prior to acquisition.
mische Austausch Dienst. We also thank Dr. Steven H. Olson
for his contributions at the outset of the program and Tab
Toochinda for his insight into the synthesis of enolic dione 2.
Supporting Information Available: Experimental procedures
and characterization of the compounds not described above; full
TOCSY and NOESY spectra, VT data, and data on concentra-
tion dependence of NH shifts for @-tide 9 as well as peptide
16 (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by a grant from
the National Institutes of Health (grant no. GM28965), a
fellowship (to U.A.) from the Deutsche Forschungs Gemeind-
schaft, and a fellowship (to M.K.) from the Deutsche Acade-
JA0168460
9
66 J. AM. CHEM. SOC. VOL. 124, NO. 1, 2002