Stereoselective synthesis of 3-substituted 2-aminocyclopentanecarboxylic acid derivatives and their incorporation into short 12-helical β-peptides that fold in water

Article abstract of DOI:10.1021/ja0258778

A stereoselective synthetic route is reported for the introduction of side chains at the 3-position of trans-2-aminocyclopentanecarboxylic acid (ACPC). Ring opening of the aziridine 2-benzyloxymethyl-6-azabicyclo[3.1.0]hexane with selected nucleophiles occurs in a regioselective manner and provides ACPC precursors with functional groups at the 3-position, trans to the 2-amino group. Oligomers composed of the 3-substituted ACPC residues maintain the 12-helical conformation displayed by the nonsubstituted analogues, as shown by their similar circular dichroism signatures. The added diversity of the new residues provides good dispersion of NMR signals, allowing the assignment of nearly all the NOE signals of a selected hexamer in aqueous solution. The NOEs between protons on nonadjacent residues are characteristic of the 12-helix. 3-Substituted ACPC residues allow one to arrange specific functional groups in a geometrically defined fashion, which should facilitate the design of β-peptides for biological applications.

Full text of DOI:10.1021/ja0258778

Published on Web 09/28/2002
Stereoselective Synthesis of 3-Substituted
2-Aminocyclopentanecarboxylic Acid Derivatives and Their
Incorporation into Short 12-Helical â-Peptides That Fold in
Water
Matthew G. Woll,† John D. Fisk,‡ Paul R. LePlae,† and Samuel H. Gellman*,†,‡
Contribution from the Department of Chemistry and Graduate Program in Biophysics,
UniVersity of Wisconsin, Madison, Wisconsin 53706
Received February 8, 2002
Abstract: A stereoselective synthetic route is reported for the introduction of side chains at the 3-position
of trans-2-aminocyclopentanecarboxylic acid (ACPC). Ring opening of the aziridine 2-benzyloxymethyl-6-
azabicyclo[3.1.0]hexane with selected nucleophiles occurs in a regioselective manner and provides ACPC
precursors with functional groups at the 3-position, trans to the 2-amino group. Oligomers composed of
the 3-substituted ACPC residues maintain the 12-helical conformation displayed by the nonsubstituted
analogues, as shown by their similar circular dichroism signatures. The added diversity of the new residues
provides good dispersion of NMR signals, allowing the assignment of nearly all the NOE signals of a selected
hexamer in aqueous solution. The NOEs between protons on nonadjacent residues are characteristic of
the 12-helix. 3-Substituted ACPC residues allow one to arrange specific functional groups in a geometrically
defined fashion, which should facilitate the design of â-peptides for biological applications.
Introduction
Unnatural oligomers with discrete folding propensities (“fol-
damers”) have received much attention in recent years.1,2
â-Peptide foldamers constructed from â-amino acids constrained
with five-membered rings (Figure 1), like trans-2-aminocyclo-
pentanecarboxylic acid (ACPC) and trans-3-aminopyrrolidine-
4-carboxylic acid (APC), adopt a robust helical conformation
defined by 12-membered-ring hydrogen bonds, CdO(i) f
N-H(i + 3) (“12-helix”).3 Helical structure can be observed
with as few as six residues in aqueous solution,4 which makes
these oligomers attractive as scaffolds for biological applications.
Antimicrobial activity paralleling that of natural host-defense
peptides has been reported for a 12-helical â-peptide composed
of ACPC and APC.5 Several groups have reported biological
applications for non-12-helical â-peptides composed of acyclic
â-amino acids.6-10
Figure 1. â-Amino acids, with a five-membered constraint.
To enhance the utility of 12-helical â-peptides, we are
developing strategies for appending functionality at specific sites
along the helix. Recently we showed that diversity can be
introduced into 12-helical â-peptides by sulfonylation of the
ring nitrogen of APC11 or by introduction of a few acyclic
â-amino acids among ACPC and/or APC residues (the latter
approach is limited by diminution of 12-helical stability).12 Here
we demonstrate a complementary strategy, side chain introduc-
tion at the 3-position of ACPC.
At the outset it was not clear that side chain introduction at
the 3-position would be tolerated by the 12-helix, given the
proximity of the substitution site to the backbone nitrogen. We
were motivated to explore this possibility because success would
provide a geometrically defined way to place functional groups
* To whom correspondence should be addressed. E-mail: gellman@
chem.wisc.edu.
† Department of Chemistry.
‡ Graduate Program in Biophysics.
(1) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001, 101,
3219-3232.
(2) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem.
ReV. 2001, 101, 3893-4011.
(3) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.; Huang,
X.; Barchi, J. J., Jr.; Gellman, S. H. Nature 1997, 387, 381-384.
(4) Wang, X.; Espinosa, J. F.; Gellman, S. H. J. Am. Chem. Soc. 2000, 122,
4821-4822.
(5) Porter, E. A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gellman, S. H. Nature
2000, 404, 565.
(9) Gademann, K.; Kimmerlin, T.; Hoyer, D.; Seebach, D. J. Med. Chem. 2001,
44, 2460-2468.
(6) Werder, M.; Hauser, H.; Abele, S.; Seebach, D. HelV. Chim. Acta 1999,
(10) Umezawa, N.; Gelman, M. A.; Haigis, M. C.; Raines, R. T.; Gellman, S.
H. J. Am. Chem. Soc. 2002, 124, 368-369.
82, 1774-1783.
(7) Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999,
121, 12200-12201.
(11) Lee, H.-S.; Syud, F. A.; Wang, X.; Gellman, S. H. J. Am. Chem. Soc. 2001,
123, 7721-7722.
(8) Liu, D.; DeGrado, W. F. J. Am. Chem. Soc. 2001, 123, 7553-7559.
(12) LePlae, P. R.; Fisk, J. D.; Porter, E. A.; Weisblum, B.; Gellman, S. H.
9
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J. AM. CHEM. SOC. 2002, 124, 12447-12452
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A R T I C L E S
Woll et al.
Scheme 1 a
Cbz-Cl, followed by aziridine ring opening with cyanide.
Reduction of the nitrile to the amine, followed by Boc
protection, yielded 15. Removal of the benzyl and Cbz groups
with Na/NH3,19 followed by Fmoc protection of the resulting
free amine and oxidation of the alcohol, produced 16. The routes
in Scheme 2 should provide access to a wide range of
3-substituted ACPC residues.
a Reagents and conditions: (a)Bom-Cl, DMF, -40 °C, 15 min. (b) (+)-
IPC2BH, THF, -78 to -10 °C, 48 h. (c) NaOH/H2O2, room temperature,
12 h, 73% (over three steps). (d) TsCl, pyridine, 0 °C to room temperature,
83%. (e) LAH, Et2O, reflux, 20 h, 77%.
To determine whether 3-substituted ACPC residues would
be compatible with the 12-helical conformation, we first
prepared two hexamers, Ac-(APC-1)3-NH2 and Ac-(3-ACPC)3-
NH2. The CD signatures of these two hexamers in water (Figure
2) were similar to that of Ac-(APC-ACPC)3-NH2, which is
known to be 12-helical.4,20
along the 12-helical framework without diminishing the back-
bone’s intrinsic folding preference.
Results and Discussion
Monomers 1-3 were prepared enantioselectively, starting
with the alkylation of sodium cyclopentadienide by benzylchlo-
romethyl ether (Scheme 1). This step is generally plagued by
the formation of a significant amount of the double-bond-
isomerized side product. Previous work has shown that isomer-
ization can be circumvented by using thallium(II) cyclopenta-
dienide instead of sodium cyclopentadienide.13 We found that
isomerization could be avoided by simply switching from the
traditional THF alkylation solvent to DMF, which removes the
need to work with the toxic thallium compounds. The resulting
diene was then converted to alcohol 4 in 97% ee via asymmetric
hydroboration-oxidation with (+)-Ipc2BH, according to the
procedure of Biggadike et al.14 Use of (-)-Ipc2BH provided
the enantiomer of 4. Tosylation of the alcohol followed by
recrystallization from heptane gave the tosylate in >99% ee.
The tosylate was reduced to the highly enantiopure alkene 5
with LiAlH4.
Conversion to cis-epoxide 6 (Scheme 2), previously per-
formed by Colombini et al.15 from racemic 5, employs a
Sharpless chlorohydroxylation.16 Epoxide ring opening with
NaN3 followed by alcohol activation with MsCl yielded a 2:3
regioisomeric mixture of azido-mesylates, which was converted
to 7 by azide reduction and concomitant ring-closing mesylate
displacement.
Conversion of 7 to the tert-butyl carbamate allowed facile
ring opening with BF3 and an alcohol17 (Scheme 2). This
reaction gave only one product, the regiochemistry of which
was established crystallographically in the case of methanol as
the nucleophile. Crystals were obtained by slow evaporation of
CHCl3 from racemic 8. Analysis of the X-ray diffraction data
confirmed the positions of the substituents on the ring as well
as their cis/trans relationships.
The ring-opened intermediates 8 and 9 were converted to their
respective N-Boc-â-amino acids by removal of the benzyl
protecting group followed by oxidation.18 A protecting group
change from Boc to Fmoc afforded â-amino acid derivatives
(e.g., 12 and 13) suitable for solid-phase synthesis. Access to
an alternative type of side chain began with reaction of 7 with
We prepared hexamer 17 (Figure 3), which contains residues
1-3, for NMR analysis. We anticipated that the high chemical
diversity of 17 (five different residues) would lead to dispersion
in the proton NMR spectrum, which is critical for structural
analysis. Indeed, nearly all proton resonances could be resolved
in the NMR spectrum of 17 in 9:1 H2O:D2O (6 mM peptide,
100 mM acetate buffer (pH 3), 4 °C). Two-dimensional NMR
data, including NOESY21 and ROESY,22 provided information
about the three-dimensional structure of 17. NOEs between
protons on residues that are not adjacent in sequence are
particularly informative. All possible CâHi f NHi+2, CâHi f
NHi+3, and CâHi f CRHi+2 NOEs were observed (a few were
ambiguous due to NMR resonance overlap). These three sets
of NOEs are characteristic of the 12-helical conformation.3,4,11
No NOEs inconsistent with the 12-helical conformation were
observed. It is very likely that â-peptide 17 interconverts
between 12-helical and unfolded states rapidly on the spectro-
scopic time scale under the conditions used for the NMR studies.
We cannot rule out the possibility that the conformational
ensemble includes states that are partially 12-helical and partially
unfolded, along with the fully folded state and a completely
unfolded state. The abundance of NOEs, particularly at the
terminal residues, suggests that 17 is very well structured in
aqueous solution.
Conclusions
We have shown that diversity can be conveniently introduced
into 12-helical â-peptides in a geometrically defined way, while
retaining the conformational preorganization provided by the
five-membered ring, by placing side chains at the 3-position of
the ring, trans to the 2-amino group. Employing aziridine 7 as
a divergence point allows for various nucleophiles to be
introduced. The capacity to produce a large pool of diverse
monomers will prove important in preparing libraries of
â-peptides to be screened for biological activities.
Experimental Section
General Procedures. Reagents were obtained from Aldrich Chemi-
cal Co. and were used without further purification, except 4 N HCl in
dioxane, which was purchased from Pierce. Melting points (mp) were
obtained on a Thomas-Hoover capillary melting point apparatus and
are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker AC-300 (300 MHz) spectrometer. Chemical
(13) Corey, E. J.; Koelliker, U.; Neuffer, J. J. Am. Chem. Soc. 1971, 93, 1489-
1490.
(14) Biggadike, K.; Borthwick, A. D.; Evans, D.; Exall, A. M.; Kirk, B. E.;
Roberts, S. M.; Stephenson, L.; Youds, P. J. Chem. Soc., Perkin Trans. 1
1988, 549-554.
(15) Colombini, M.; Crotti, P.; Di Bussolo, V.; Favero, L.; Gardelli, C.; Macchia,
F.; Pineschi, M. Tetrahedron 1995, 51, 8089-8112.
(16) Klunder, J. M.; Caron, M.; Uchiyama, M.; Sharpless, K. B. J. Org. Chem.
1985, 50, 912-915.
(19) Li, Y. L.; Wu, Y. L. Liebigs Ann. 1996, 2079-2082.
(20) Applequist, J.; Bode, K. A.; Appella, D. H.; Christianson, L. A.; Gellman,
S. H. J. Am. Chem. Soc. 1998, 120, 4891-4892.
(21) Macura, S.; Ernst, R. R. Mol. Phys. 1980, 41, 95-117.
(22) Bothner-By, A. A.; Stephens, R. L.; Lee, J.; Warren, C. D.; Jeanloz, R. W.
J. Am. Chem. Soc. 1984, 106, 811-813.
(17) Dauban, P.; Dubois, L.; Tran Huu Dau, M. E.; Dodd, R. H. J. Org. Chem.
1995, 60, 2035-2043.
(18) Timmers, C. M.; Turner, J. J.; Ward, C. M.; van der Marel, G. A.;
Kouwijzer, M. L. C. E.; Grootenhuis, P. D. J.; van Boom, J. H. Chem.-
Eur. J. 1997, 3, 920-929.
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Diverse 12-Helical â-Peptides
A R T I C L E S
Scheme 2 a
a Reagents and conditions: (a) TiCl4/TBHP, CH2Cl2, 30 min, -78 °C. (b) KOtBu, benzene, room temperature, 3 h, 85% (over two steps). (c) NaN3/
NH4Cl, MeOH/H2O, reflux, 12 h, 85%. (d) MsCl, pyridine, 0 °C, 2 h. (e) LAH, THF, 0 °C to room temperature, 2 h, 84% (over two steps). (f) Boc2O/NEt3,
MeOH, room temperature, 2 h, 84%. (g) BF3‚OEt2/ROH, CH2Cl2, -78 °C, 15 min, 71-87%. (h) 10% Pd/C/NH4HCO2, MeOH, reflux, 10 h, 93%. (i) Jones
reagent, acetone, 0 °C, 2 h, 73-89%. (j) 4 N HCl, dioxane, room temperature, 1 h. (k) Fmoc-OSu/NaHCO3, acetone/H2O, room temperature, 12 h, 61-74%
(over two steps). (l) Cbz-Cl/NEt3, CH2Cl2, 0 °C, 2 h, 93%. (m) KCN/18-crown-6, DMSO, 80 °C, 2 h, 90%. (n) BH3‚THF, THF, room temperature, 4 h. (o)
Boc2O/NEt3, MeOH, room temperature, 2 h, 75% (over two steps). (p) Na/NH3, -78 °C, 30 min. (q) Fmoc-OSu/NaHCO3, acetone/H2O, room temperature,
12 h, 47% (over two steps). (r) TEMPO/NaClO, CH2Cl2/H2O, 0 °C, 30 min, 57%.
Figure 2. Circular dichroism of Ac-(APC-ACPC)3-NH2, Ac-(APC-1)3-NH2, and Ac-(3-ACPC)3-NH2, 0.07 mM in H2O (25 °C). Data were obtained on an
Aviv instrument with 1 mm path length cells. Data were normalized for peptide concentration and number of residues.
shifts were reported in parts per million (ppm, δ) relative to tetram-
ethylsilane (δ 0.00). 1H NMR splitting patterns are designated as singlet
(s), doublet (d), triplet (t), or quartet (q). All first-order splitting patterns
were assigned on the basis of the appearance of the multiplet. Splitting
patterns that could not be easily interpreted are designated as multiplet
(m) or broad (br). Carbon nuclear magnetic resonance (13C NMR)
spectra were recorded on a Bruker AC-300 (300 MHZ) spectrometer.
Carbon resonances were assigned using distortionless enhancement by
polarization transfer (DEPT) spectra obtained with phase angles of 90°
and 135°. Mass spectra (MS) were obtained using an electrospray
ionization (ESI) mass spectrometer. Molecular ions are reported as the
sodium salt with molecular weights 22.9898 greater than the actual
compound molecular weight. Analytical thin-layer chromatography was
carried out on Whatman TLC plates precoated with silica gel 60 (250
µm thickness). Visualization was performed using a UV lamp or
potassium permanganate stain. Column chromatography was performed
on EM Science silica gel 60 (230-400 mesh). Solvent mixtures for
chromatography are reported as v/v ratios. THF, benzene, and Et2O,
used as reaction solvents, were distilled from sodium/benzophenone
under nitrogen immediately prior to use. CH2Cl2, NEt3, and pyridine
used as reaction solvents were distilled from CaH over nitrogen
immediately prior to use.
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A R T I C L E S
Woll et al.
combined filtrate at reduced pressure, leaving a clear oil (3.23 g, 77%).
HRMS (ESI): [MNa]+ calculated 211.1099, found 211.1097. 1H NMR
(CDCl3, 300 MHz, room temperature): δ 1.57 (1H, m), 2.03 (1H, m),
2.33 (2H, m), 2.99 (1H, br), 3.36 (2H, m), 4.53 (2H, s), 5.72 (1H, m),
5.80 (1H, m), 7.25-7.35 (5H, m). 13C NMR: δ 26.5 (CH2), 31.7 (CH2),
46.0 (CH), 72.9 (CH2), 74.3 (CH2), 127.3 (CH), 127.4 (CH), 128.1
(CH), 131.9 (CH), 138.5 (C) (note: in the aromatic region, some signals
may represent more than one carbon).
2-(Benzyloxymethyl)-6-oxabicyclo[3.1.0]hexane (6). To a -78 °C
solution of 5 (3.12 g, 16.6 mmol) and tert-butyl hydroperoxide (4 mL,
5-6 M in nonane) in CH2Cl2 (250 mL) was added TiCl4 (2.73 mL, 25
mmol) dropwise over 5 min.15 After 15 min, the reaction was quenched
by transfer via cannula into a mixture of vigorously stirred Et2O:
saturated aqueous Na2SO4. After the mixture was stirred for 15 min,
the Et2O layer was washed with saturated aqueous Na2SO3 and saturated
aqueous NaCl. The organic layer was dried over MgSO4. Removal of
the organic solvent at reduced pressure produced a yellow oil that was
dissolved in benzene (160 mL). To the solution was added potassium
tert-butoxide (3.92 g, 35 mmol). The suspension was stirred at room
temperature for 2.5 h and then quenched with H2O. The organic layer
was then washed with saturated aqueous NaCl and dried over MgSO4.
Upon removal of the solvent, a pale yellow oil (98:2 cis:trans mixture)
was obtained, which was carried on without further purification (2.88
g, 85%). HRMS (ESI): [MNa]+ calculated 227.1048, found 227.1050.
1H NMR (CDCl3, 300 MHz, room temperature): δ 0.99 (1H, m), 1.62
(2H, m), 2.03 (1H, m), 2.26 (1H, m), 3.46-3.59 (3H), 4.55 (2H, ab, J
) 11.7 Hz), 7.24-7.37 (5H). 13C NMR: δ 21.9 (CH2), 26.9 (CH2),
40.4 (CH), 57.2 (CH), 57.7 (CH), 70.7 (CH2), 73.0 (CH2), 127.4 (CH),
127.4 (CH), 128.2 (CH), 138.3 (C) (note: in the aromatic region, some
signals may represent more than one carbon).
Figure 3. NOEs between residues that are not adjacent in the sequence
observed for 17 in 9:1 H2O:D2O. Dashed lines indicate NOEs that are
ambiguous because of resonance overlap. Data were obtained on a Varian
600 MHz spectrometer.
2-(Benzyloxymethyl)cyclopent-3-enol (4). Sodium cyclopentadi-
enide (25 mL, 2 M in THF from Aldrich) was added dropwise to a
solution of benzylchloromethyl ether23 (13.9 mL, 75 mmol) in DMF
(125 mL) at -40 °C. After 15 min of vigorous stirring at -40 °C, the
reaction mixture was quenched by pouring it into pentane (400 mL):
ice water (200 mL). After shaking and allowing the phases to separate,
the organic layer was washed two times with 200 mL of ice water and
dried over MgSO4 with stirring, maintaining the temperature below 0
°C to avoid isomerization of the product. After removal of the drying
agent by filtration, the pentane was removed under reduced pressure
at 0 °C. The resulting yellow oil was diluted into THF (200 mL), cooled
to -78°C, and added via cannula to a suspension of (+)-IPC2BH24
(14.3 g, 50 mmol) in THF (300 mL) at -78 °C.14 The mixture was
allowed to warm slowly to -10 °C and stirred for 48 h at that
temperature. The reaction was then quenched by addition of MeOH
(20 mL), followed by 3 M NaOH (20 mL) and 30% H2O2 (20 mL).
After 12 h of vigorous stirring at room temperature, the THF was
removed under reduced pressure. The remaining aqueous suspension
was partitioned between EtOAc (350 mL) and saturated aqueous NaCl.
The organic layer was dried with MgSO4, followed by removal of the
solvent at reduced pressure. The remaining oil was chromatographed
on silica gel with 3:1 hexanes:EtOAc. A pale yellow oil was recovered
(7.41 g, 73%). Chiral HPLC analysis (Chiracel OD 4.6 mm, 95:5
hexanes:iPrOH, 1 mL/min) revealed a product of 97% ee (retention
time for the major enantiomer, 13.3 min, and for the minor enantiomer,
16.1 min). HRMS (ESI): [MNa]+ calculated 227.1048, found 227.1059.
1H NMR (CDCl3, 250 MHz, room temperature): δ 2.00 (1H, br), 2.31
(1H, m), 2.70 (1H, ddq, J ) 17.0, 7.0, 2.3 Hz), 2.88 (1H, m), 3.30
(1H, t, J ) 9.0 Hz), 3.57 (1H, dd, J ) 9.0, 5.3 Hz), 4.31 (1H, h, J )
2.8 Hz), 4.54 (2H, s), 5.56 (1H, m), 5.73 (1H, m), 7.28-7.38 (5H).
13C NMR: δ 40.9 (CH2), 55.0 (CH), 71.9 (CH2), 73.0 (CH2), 75.7 (CH),
127.5 (CH), 128.2 (CH), 129.1 (CH), 129.9 (CH), 138.1 (C) (note: in
the aromatic region, some signals may represent more than one carbon).
(Cyclopent-2-enylmethoxymethyl)benzene (5). To a solution of 4
(0.98 g, 4.8 mmol) in pyridine (5 mL) was added TsCl (0.95 g, 5.0
mmol) at 0 °C. The reaction was allowed to warm to room temperature
over 30 min and then stirred at room temperature for 12 h. Addition of
20 mL of H2O, followed by vigorous stirring, resulted in precipitation
of the product, which could be filtered from the solvent. The solid was
chromatographed on silica gel with 5:1 hexanes:EtOAc. A white solid
was obtained, which was further purified by recrystallization from
n-heptane to yield a white crystalline material (1.42 g, 83%). Mp: 68-
69 °C. The enantiomers of this tosylate could not be resolved by chiral
HPLC, but chiral HPLC of intermediate 14 (Cbz aziridine) suggested
that the tosylate was 99.7% enantiomerically pure. The tosylate (8.0 g,
22.3 mmol) was suspended in 100 mL of Et2O and then added to a
suspension of LAH (1.52 g, 40 mmol) in Et2O (100 mL). The mixture
was heated to reflux for 20 h and then quenched by slow addition of
1 M NaOH. When the suspension appeared white, it was filtered over
Celite. The filter cake was broken up and triturated with ether, which
was then removed by filtration. All solvents were removed from the
2-(Benzyloxymethyl)-6-azabicyclo[3.1.0]hexane (7). To a solution
of compound 6 (9.79 g, 48 mmol) in 8:1 MeOH:H2O (200 mL) were
added NaN3 (15.0 g, 230 mmol) and NH4Cl (6.0 g, 112 mmol).15 The
mixture was heated to reflux for 15 h. The MeOH was then removed
under reduced pressure, leaving a yellow oil, which was partitioned
between EtOAc and H2O. The organic layer was separated, washed
with saturated aqueous NaCl, and dried over MgSO4. Removal of the
solvents, followed by chromatography of the azido alcohols with 4:1
hexanes:EtOAc, gave as a clear oil a 3:2 mixture of diastereomers (10.1
g, 41 mmol, 85%). The mixture of azido alcohols (7.13 g, 29 mmol)
was dissolved in pyridine (25 mL) and cooled to 0 °C. Methane sulfonyl
chloride (2.7 mL, 35 mmol) was added dropwise to the solution, after
which the reaction solution was allowed to warm slowly to room
temperature. After being stirred for 2 h at room temperature, the mixture
was diluted into 200 mL of EtOAc and extracted three times with
saturated aqueous CuSO4 solution. The organic layer was then washed
with saturated aqueous NaCl and dried over MgSO4. Removal of the
solvents gave an oil that was taken up in THF (50 mL) and added
dropwise to a suspension of LAH (4.26 g, 112 mmol) in THF (150
mL) at 0 °C. The reaction mixture was stirred at room temperature for
5 h. Excess LAH was slowly quenched with 1 M NaOH at 0 °C until
the suspension appeared completely white. The suspension was filtered
through Celite, and the isolated solid was washed with ether. The filtrate
solvents were removed under reduced pressure, and the resulting oil
was chromatographed on silica gel with EtOAc and then 5:1 EtOAc:
MeOH, yielding a clear oil, which contains a 2% cis impurity carried
over from the epoxide formation (4.8 g, 84%). HRMS (ESI): [MNa]+
calculated 226.1208, found 226.1218. 1H NMR (CDCl3, 300 MHz, room
temperature): δ 0.40 (1H, br), 1.39-1.47 (2H), 1.63-1.85 (2H), 2.42-
2.48 (2H, m), 3.28-3.38 (2H, abx, Jab ) 9.3 Hz, Jax ) 7.5 Hz, Jbx
)
6.9 Hz), 4.52 (2H, s), 7.26-7.36 (5H). 13C NMR: δ 21.8 (CH2), 25.8
(CH2), 35.1 (CH), 37.8 (CH), 40.0 (CH), 71.9 (CH2), 72.9 (CH2), 127.3
(CH), 128.2 (CH), 138.3 (C) (note: in the aromatic region, some signals
may represent more than one carbon).
[2-(Benzyloxymethyl)-5-methoxycyclopentyl]carbamic Acid tert-
Butyl Ester (8). To a solution of 7 (1.90 g, 9.4 mmol) in MeOH (50
(23) Connor, D. S.; Klein, G. W.; Taylor, G. N. Org. Synth. 1972, 52, 16-19.
(24) Brown, H. C.; Joshi, N. N. J. Org. Chem. 1988, 53, 4059-4062.
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Diverse 12-Helical â-Peptides
A R T I C L E S
mL) were added NEt3 (1.31 mL, 9.4 mmol) and di-tert-butyl dicarbonate
(2.18 g, 10.0 mmol). The reaction stirred at room temperature for 2 h.
The MeOH was then removed under reduced pressure, and the
remaining oil was taken up in EtOAc, washed with saturated aqueous
NaCl, and dried over MgSO4. The solvent was removed under reduced
pressure, leaving a yellow oil, which was chromatographed on silica
gel with 5:1 hexanes:EtOAc to give the Boc-aziridine intermediate as
a clear oil (2.38 g, 84%). A solution of CH2Cl2 (50 mL) and the Boc-
aziridine intermediate (1.00 g, 3.3 mmol) was cooled to -78 °C. MeOH
(1.34 mL, 33 mmol) was then added to the solution, followed by slow
addition of BF3‚Et2O (2.10 mL, 16.5 mmol).5 After 5 min, the reaction
was quenched with addition of saturated aqueous NaHCO3 addition.
The mixture was allowed to warm to room temperature with stirring.
The organic layer was separated, washed with saturated aqueous NaCl,
and dried over MgSO4. Removal of the organic solvent left a yellow
oil, which was chromatographed on silica gel with 3:1 hexane:EtOAc,
yielding a clear oil (0.96 g, 87%). HRMS (ESI): [MNa]+ calculated
temperature): δ 1.46 (9H, s), 1.67-1.98 (3H), 2.28 (1H, br), 2.82 (1H,
br), 3.36 (3H, s), 3.62 (1H, q, J ) 5.4 Hz), 3.88 (1H, br), 5.03 (1H,
br). 13C NMR: δ 26.8 (CH2), 28.9 (CH3), 29.1 (CH2), 50.2 (CH), 57.2
(CH), 61.5 (CH), 79.5 (CH), 87.5 (CH3), 156.9 (C), 175.8 (C) (note:
in the aromatic region, some signals may represent more than one
carbon).
2-tert-Butoxycarbonylamino-3-phenoxycyclopentanecarboxylic Acid
(11). This compound was prepared by a procedure similar to that used
to prepare 10. Purification was performed by chromatography on silica
gel with 100:1 CHCl3:AcOH, yielding a white solid (83%), which was
recrystallized from heptane:EtOAc. Mp: 135-136 °C. HRMS (ESI):
1
[MNa]+ calculated 344.1474, found 344.1482. H NMR (CDCl3, 300
MHz, room temperature): δ 1.45 (9H, s), 1.89-2.28 (4H), 3.04 (1H,
br), 4.06 (1H, br), 4.67 (1H, br), 5.05 (1H, br), 6.80 (2H, m), 6.95
(1H, m), 7.27 (2H, m). 13C NMR: δ 26.6 (CH2), 28.6 (CH3), 29.7
(CH2), 49.3 (CH), 61.7 (CH), 79.4 (CH), 83.2 (CH), 116.5 (CH), 121.5
(CH), 130.3 (CH), 156.5 (C), 159.0 (C), 175.3 (C) (note: in the aromatic
region, some signals may represent more than one carbon).
1
358.1994, found 358.1980. H NMR (CDCl3, 300 MHz, room tem-
perature): δ 1.44 (9H, s), 1.5-2.05 (5H), 3.35 (3H, s), 3.41 (1H, m),
3.59 (3H), 4.51 (2H, s), 7.25-7.35 (5H). 13C NMR: δ 25.7 (CH2),
28.3 (CH3), 29.2 (CH2), 45.2 (CH), 56.7 (CH3), 59.6 (CH), 72.8 (CH),
73.0 (CH), 86.7 (CH), 127.3 (CH), 127.4 (CH), 128.2 (CH), 138.4 (C),
155.1 (C) (note: in the aromatic region, some signals may represent
more than one carbon).
2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-methoxycyclopen-
tanecarboxylic Acid (12). A solution of 4 N HCl in dioxane was added
to 10 (176 mg, 0.68 mmol), and the resulting solution was stirred for
1 h. The HCl/dioxane was then blown off with a stream of N2. The
white powdery residue was taken up in 2:1 acetone:H2O (20 mL). To
this solution were added NaHCO3 (118 mg, 1.4 mmol) and Fmoc-OSu
(229 mg, 0.68 mmol). The reaction mixture was stirred at room
temperature for 16 h, and the acetone was then removed under reduced
pressure. The remaining aqueous suspension was extracted with CHCl3
(20 mL). The organic layer was washed with saturated aqueous NaCl
and dried over MgSO4. Removal of the solvents under reduced pressure,
followed by chromatography with 100:1 CHCl3:AcOH, yielded a white
solid (232 mg, 61%). Mp: 189-190 °C (recrystallized from MeOH:
H2O). HRMS (ESI): [MNa]+ calculated 404.1474, found 404.1464.
1H NMR (pyridine-d5, 300 MHz, room temperature): δ 1.89-2.16 (3H),
2.38 (1H, m), 3.36 (1H, m), 3.41 (3H, s), 4.04, (1H, q, J ) 6.6 Hz),
4.30 (1H, t, J ) 8.1 Hz), 4.63 (2H, d, J ) 8.4 Hz), 4.94 (1H, m), 7.26
(2H, d, J ) 8.7 Hz), 7.39 (2H, t, J ) 9 Hz), 7.65 (2H, t, J ) 7.8 Hz),
7.84 (2H, d, J ) 8.7 Hz), 8.84 (1H, d, J ) 9.6 Hz). 13C NMR: δ 27.3
(CH2), 30.9 (CH2), 48.4 (CH), 50.2 (CH), 57.4 (CH3), 62.1 (CH), 66.8
(CH2), 87.5 (CH), 120.9 (CH), 126.2 (CH), 128.0 (CH), 128.5 (CH),
142.2 (CH), 145.1 (CH), 145.3 (C), 157.3, (C), 177.5 (C) (note: in the
aromatic region, some signals may represent more than one carbon).
2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-phenoxycyclopen-
tanecarboxylic Acid (13). This compound was prepared by a procedure
similar to that used to prepare 12. Instead of partitioning the Fmoc-
protected product into CHCl3:H2O, however, the product was precipi-
tated by addition of H2O to the acetone:H2O reaction solution. The
precipitate was then chromatographed with CHCl3:AcOH 100:1,
yielding a white powdery solid (74%). Mp: 183-185 °C (recrystallized
from EtOH:H2O). HRMS (ESI): [MNa]+ calculated 466.1630, found
466.1646. 1H NMR (pyridine-d5, 300 MHz, room temperature): δ 2.03
(1H, m), 2.27 (1H, m), 2.50 (1H, m), 3.58 (1H, q, J ) 7.8 Hz), 4.30
(1H, t, J ) 6.6 Hz), 4.62 (2H, m), 5.10 (1H, m), 5.17 (1H, br), 6.97
(1H, t, J ) 7.2 Hz), 7.19-7.34 (5H), 7.39 (2H, t, J ) 7.5 Hz), 7.65
(2H, t, J ) 6.0 Hz), 7.83 (2H, d, J ) 7.5 Hz), 9.11 (2H, d, J ) 7.5
Hz). 13C NMR: δ 27.3 (CH2), 31.1 (CH2), 48.3 (CH), 49.6 (CH), 62.6
(CH), 66.9 (CH2), 83.5 (CH), 116.8 (CH), 120.9 (CH), 121.6 (CH),
123.7 (CH), 126.1 (CH), 127.9 (CH), 128.5 (CH), 130.4 (CH), 142.2
(C), 145.1 (C), 145.2 (C), 157.4 (C), 159.2 (C), 177.1 (C) (note: in
the aromatic region, some signals may represent more than one carbon).
(2-Benzyloxymethyl-5-cyanocyclopentyl)carbamic Acid Benzyl
Ester (14). To a 0 °C solution of CH2Cl2 (65 mL), 7 (4.9 g, 24 mmol),
and NEt3 (5.0 mL, 36 mmol) was added dropwise benzyl chloroformate
(2.92 mL, 26 mmol). The reaction mixture was allowed to warm
gradually to room temperature with stirring. After 2 h at room
temperature, the reaction mixture was washed with 1 M HCl and
saturated aqueous NaCl and then dried over MgSO4. Removal of the
[2-(Benzyloxymethyl)-5-phenoxycyclopentyl]carbamic Acid tert-
Butyl Ester (9). CH2Cl2 (50 mL) and phenol (3.10 g, 33 mmol) were
cooled to -78 °C. BF3‚OEt2 (16.5 mmol) was added, followed by slow
addition of the Boc-aziridine intermediate (prepared as described in
the procedure for 8) (0.9 g, 3.0 mmol) dissolved in CH2Cl2 (10 mL).
After the addition was complete, saturated aqueous NaHCO3 was added
to the solution to quench the reaction. The mixture was allowed to
warm to room temperature with vigorous stirring. The organic layer
was separated, washed two times with 1 M NaOH and then saturated
aqueous NaCl, and dried over MgSO4. Solvents were removed under
reduced pressure. The recovered oil was chromatographed on silica
gel with 6:1 hexane:EtOAc, yielding a white crystalline solid (0.93 g,
71%). Mp: 86-88 °C (recrystallized from hexane:EtOAc). HRMS
(ESI): [MNa]+ calculated 420.2151, found 420.2131. 1H NMR (CDCl3,
300 MHz, room temperature): δ 1.42 (9H, s), 1.66 (1H, m), 1.79-
2.18 (4H), 3.49 (1H, dd, J ) 9, 7.2 Hz), 3.61 (1H, dd, J ) 9.3, 5.7
Hz), 3.79 (1H, td, J ) 7.5, 4.5 Hz), 4.51 (2H, ab, J ) 12.9 Hz), 4.61
(1H, br), 6.89-6.94 (3H), 7.22-7.36 (7H). 13C NMR: δ 25.7 (CH2),
28.2 (CH3), 29.4 (CH2), 44.2 (CH), 60.2 (CH), 72.5 (CH2), 73.0 (CH2),
82.5 (CH), 115.5 (CH), 120.5 (CH), 127.3 (CH), 127.4 (CH), 128.1
(CH), 129.2 (CH), 138.3 (C), 155.1 (C), 157.8 (C) (note: in the aromatic
region, some signals may represent more than one carbon).
2-tert-Butoxycarbonylamino-3-methoxycyclopentanecarboxylic Acid
(10). To 8 (390 mg, 1.16 mmol) in MeOH (12 mL) were added NH4-
HCO2 (731 mg, 11.6 mmol) and 10% Pd/C (250 mg). The suspension
refluxed for 12 h and then filtered over Celite. The solvents were
removed under reduced pressure, and the resulting oil was dissolved
in EtOAc (40 mL) and washed with H2O (40 mL) to remove any left
over salts. After drying over MgSO4, the organic solvent was removed
under reduced pressure, leaving a clear oil (264 mg, 93%). The oil
(118 mg, 0.48 mmol) was subsequently dissolved in acetone (5 mL)
and cooled to 0 °C. Jones reagent (0.5 mL, 2 M) was then added
dropwise to the solution. After being stirred for 2 h at 0 °C, the mixture
was partitioned between 50 mL of EtOAc and 20 mL of 0.5 M aqueous
NaHSO4. The organic layer was separated and extracted with saturated
aqueous NaHCO3 (10 mL). The aqueous extract was then acidified to
pH 2 with 0.5 M aqueous NaHSO4 and extracted twice with EtOAc.
The combined organic layers were washed with saturated aqueous NaCl
and dried over MgSO4. Removal of the solvents at reduced pressure
yielded 91 mg (73%) of a white crystalline solid. Mp: 114-115 °C
(recrystallized from diisopropyl ether). HRMS (ESI): [MNa]+ calcu-
1
lated 282.1317, found 282.1309. H NMR (CDCl3, 300 MHz, room
9
J. AM. CHEM. SOC. VOL. 124, NO. 42, 2002 12451

A R T I C L E S
Woll et al.
organic solvent produced a clear oil that was purified by chromatog-
raphy on silica gel, eluting with 6:1 hexanes:EtOAc. A clear oil product
was obtained (7.5 g, 93%). The oil was found to be 99.4% ee by chiral
HPLC (Chiracel OD column, 95:5 hexanes:iPrOH, 1 mL/min; retention
time for the major enantiomer, 18.7 min, and for the minor enantiomer,
16.8 min). The Cbz-aziridine intermediate (4.80 g, 14.2 mmol) was
dissolved in DMSO (120 mL). To the solution were added KCN (4.8
g, 74 mmol) and NH4Cl (0.85 g, 16 mmol). 18-Crown-6 (19.5 g, 74
mmol) was then added to the reaction mixture, and the resulting mixture
was heated to 80 °C for 6 h. The reaction mixture was then poured
into 300 mL of H2O. The aqueous layer was extracted with 300 mL of
Et2O. The organic layer was washed with saturated aqueous NaCl and
dried over MgSO4. Removal of the Et2O under reduced pressure gave
a tan solid (4.7 g, 90%). This material could be recrystallized from
heptane:EtOAc to give pure white crystals. Mp: 111-112 °C. HRMS
(ESI): [MNa]+ calculated 387.1763, found 387.1781. 1H NMR (CDCl3,
300 MHz, room temperature): δ 1.65 (1H, m), 1.91-2.03 (2H), 2.14
(1H, m), 2.23 (1H, br), 3.02 (1H, br), 3.48 (2H, d, J ) 5.4 Hz), 3.91
(1H, d, J ) 8.4 Hz), 4.48 (2H, s), 5.00 (1H, br), 5.10 (2H, s), 7.27-
7.35 (10H). 13C NMR: δ 26.1 (CH2), 27.8 (CH2), 34.1 (CH), 44.2 (CH),
59.4 (CH), 66.8 (CH2), 70.9 (CH2), 73.1 (CH2), 121.3 (C), 127.4 (CH),
127.5 (CH), 128.0 (CH), 128.1 (CH), 128.2 (CH), 128.4 (CH), 135.9
(C), 137.9 (C), 155.5 (C) (note: in the aromatic region, some signals
may represent more than one carbon).
[2-Benzyloxymethyl-5-(tert-butoxycarbonylaminomethyl)cy-
clopentyl]carbamic Acid Benzyl Ester (15). THF (78 mL) was added
to 14 (4.7 g, 12.9 mmol), and the resulting solution was cooled to 0
°C. BH3‚THF (78 mL, 1 M in THF) was then added dropwise with
stirring. The reaction mixture was allowed to stir at room temperature
for 5 h and then quenched by slow addition of 6 M HCl (aqueous)
until all the excess borane was destroyed. The solution was then made
basic with 1 M NaOH and extracted three times with EtOAc. The
combined organic layers were washed with saturated aqueous NaCl
and dried over MgSO4. After removal of the solvent under reduced
pressure, the crude oil was dissolved in 50 mL of MeOH. NEt3 (2.7
mL, 19.5 mmol) was added, followed by di-tert-butyl dicarbonate (3.05
g, 14.0 mmol). The reaction mixture stirred for 24 h at room
temperature. The MeOH was then removed under reduced pressure,
and the remaining residue was taken up in EtOAc, washed with H2O
and then saturated aqueous NaCl, and dried over MgSO4. Removal of
the organic solvent under reduced pressure, followed by chromatography
on silica gel with 3:1 hexane:EtOAc, yielded a white solid (4.55 g,
75%). Mp: 109-111 °C (recrystallized from diisopropyl ether). HRMS
(ESI): [MNa]+ calculated 491.2522, found 491.2546. 1H NMR (CDCl3,
300 MHz, room temperature): δ 1.43 (9H, s), 1.36-1.51 (2H), 1.76-
2.04 (4H), 3.10 (1H, m), 3.26 (1H, m), 3.39-3.53 (3H), 4.48 (2H, ab,
J ) 12.3 Hz), 4.84 (1H, d, J ) 8.1 Hz), 5.09 (2H, ab), 5.35 (1H, br),
7.23-7.39 (10H). 13C NMR: δ 25.7 (CH2), 26.1 (CH2), 28.3 (CH3),
42.0 (CH2), 45.9 (CH), 46.8 (CH), 66.6 (CH2), 72.5 (CH2), 73.1 (CH2),
78.7 (C), 127.3 (CH), 127.8 (CH), 128.0 (CH), 128.2 (CH), 128.4 (CH),
136.3 (C), 138.3 (C), 156.3 (C), 156.6 (C) (note: in the aromatic region,
some signals may represent more than one carbon).
turned a deep blue color. Compound 15 (2.55 g, 5.45 mmol), dissolved
in THF (100 mL), was added dropwise to the NH3/Na solution. After
being stirred for 30 min at -78 °C, the reaction was quenched with
solid NH4Cl until the blue color disappeared.19 The ammonia was then
allowed to evaporate from the flask with stirring at room temperature.
The remaining suspension was taken up in EtOAc and filtered. Removal
of the EtOAc under reduced pressure yielded a yellow oil, which was
dissolved in 3:1 acetone:H2O (200 mL) containing NaHCO3 (0.84 g,
10 mmol). Fmoc-OSu (1.82 g, 5.4 mmol) was added, and the solution
was allowed to stir overnight at room temperature. The acetone was
then removed under reduced pressure, and the remaining aqueous
mixture was washed with EtOAc. The organic layer was washed with
saturated aqueous NaCl and dried over MgSO4. After removal of the
organic solvent at reduced pressure, the remaining yellow solid was
recrystallized from heptane:EtOAc to afford a white solid (1.2 g, 47%).
CH2Cl2 (40 mL) was added to the white solid (1.1 g, 2.36 mmol), and
the resulting solution was cooled to 0 °C. KBr (27 mg, 0.23 mmol),
(n-Bu)4NBr (32 mg, 0.1 mmol), and TEMPO (8 mg, 0.05 mmol) were
added to the solution. Saturated aqueous NaHCO3 was then added (10
mL), followed by dropwise addition of a combined solution of 5%
NaClO (10.5 mL), saturated aqueous NaHCO3 (10 mL), and saturated
aqueous NaCl (10 mL). The reaction mixture turned a light yellow
color. After 10 min, the reaction was quenched with 0.5 M HCl. The
resulting mixture was extracted with EtOAc. The organic layer was
washed with saturated aqueous NaCl and dried over MgSO4. Removal
of solvent under reduced pressure yielded a white solid, which was
chromatographed on silica gel with CHCl3 and then CHCl3 containing
1% AcOH, to give a white foamy solid (0.65 g, 57%). Recrystallization
from CHCl3:MeOH gave a white powdery solid. Mp: 150-152 °C.
HRMS (ESI): [MNa]+ calculated 503.2158, found 503.2141. 1H NMR
(pyridine-d5, 300 MHz, room temperature): δ 1.48 (9H, s), 1.78 (1H,
m), 1.95-2.23 (3H), 2.45 (1H, m), 3.33 (1H, m), 3.47 (1H, m), 3.78
(1H, m), 4.31 (1H, m), 4.57 (2H, m), 4.65 (1H, m), 7.29 (2H, d, J )
7.5 Hz), 7.39 (2H, t, J ) 6.6 Hz), 7.62 (2H, t, J ) 6.6 Hz), 7.82 (2H,
d, J ) 7.5 Hz), 8.86 (1H, d, J ) 7.8 Hz). 13C NMR: δ 26.5 (CH2),
27.2 (CH2), 27.9 (CH3), 40.9 (CH), 45.5 (CH), 46.6 (CH), 56.1 (CH2),
66.8 (CH), 69.3 (CH2), 82.9 (C), 119.8 (CH), 124.9 (CH), 127.0 (CH),
127.6 (CH), 141.1 (C), 143.2 (C), 154.2 (C), 155.1 (C), 178.5 (C)
(note: in the aromatic region, some signals may represent more than
one carbon).
Acknowledgment. This research was supported by the NIH
(GM56414). J.D.F. was supported in part by a Biophysics
Training Grant from NIGMS, and P.R.L. by a fellowship from
Kodak. NMR spectrometers were purchased with partial support
from NIH and NSF. CD spectra were obtained in the NSF-
supported Biophysics Instrumentation Facility. Crystallographic
work was performed by Dr. Ilia A. Guzei.
Supporting Information Available: 1H and 13C spectra of
all numbered intermediates in Schemes 1 and 2 (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
3-(tert-Butoxycarbonylaminomethyl)-2-(9H-fluoren-9-ylmethoxy-
carbonylamino)cyclopentanecarboxylic acid (16). Sodium metal (1.27
g, 55 mmol) was dissolved in NH3 (200 mL) at -78 °C. The solution
JA0258778
9
12452 J. AM. CHEM. SOC. VOL. 124, NO. 42, 2002