Asymmetric synthesis of a new helix-forming β-amino acid: trans-4-aminopiperidine-3-carboxylic acid

Article abstract of DOI:10.1002/ejoc.200390112

We report a synthesis of a protected derivative of trans-4aminopiperidine-3-carboxylic acid (APiC). The route provides either enantiomer. All intermediates are purified by crystallization, and large-scale preparation is therefore possible. An analogous ro

Full text of DOI:10.1002/ejoc.200390112

FULL PAPER
Asymmetric Synthesis of a New Helix-Forming β-Amino Acid:
trans-4-Aminopiperidine-3-carboxylic Acid
Marina Schinnerl,[a] Justin K. Murray,[a] Joseph M. Langenhan,[a] and
Samuel H. Gellman*[a]
Keywords: Asymmetric synthesis / Amino acids / Helical structures
We report a synthesis of a protected derivative of trans-4-
aminopiperidine-3-carboxylic acid (APiC). The route pro-
vides either enantiomer. All intermediates are purified by
crystallization, and large-scale preparation is therefore pos-
sible. An analogous route provides either enantiomer of
trans-2-aminocyclohexanecarboxylic acid (ACHC). We have
previously shown that β-peptide oligomers containing ACHC
adopt a helical conformation defined by 14-membered C=
O(i)···H−N(i − 2) hydrogen bonds (‘‘14-helix’’). Here we show
that APiC residues can be incorporated into the 14-helix.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2003)
Introduction
amenable to large-scale preparations. A similar synthetic
route provides access to either enantiomer of ACHC, again
without chromatography. We show that a hexa-β-peptide
containing both ACHC and APiC forms a 14-helix in aque-
ous solution.
Unnatural oligomers with a strong tendency to adopt
specific and predictable conformations in solution (‘‘folda-
mers’’) have been the subject of extensive investigation in
recent years.[1] β-Amino acid oligomers (‘‘β-peptides’’) have
been particularly well studied in this regard as β-peptides
can adopt several distinct folding patterns if residue substi-
tution patterns are chosen appropriately.[2] Constrained β-
amino acid residues, such as those in which the CαϪCβ
bond is part of a small ring, lead to extremely stable con-
formations. We have shown, for example, that β-peptides
containing as few as six residues adopt either of two helical
conformations in aqueous solution depending on the type
of residue constraint.[3,4] A five-membered ring constraint
[as in trans-2-aminocyclopentanecarboxylic acid (ACPC)]
gives rise to a 12-helix, defined by 12-membered ring Cϭ
O(i)···HϪN(i ϩ 3) hydrogen bonds,[3] while a six-membered
ring constraint [as in trans-2-aminocyclohexanecarboxylic
acid (ACHC)] leads to a 14-helix, defined by 14-membered
ring CϭO(i)···HϪN(i Ϫ 2) hydrogen bonds.[4] In contrast,
conventional peptides (α-amino acid residues) do not form
helices at such short lengths, nor do most β-peptides com-
prised of unconstrained β-amino acid residues.[5]
Results and Discussion
Synthesis
Our synthetic approach (Scheme 1) follows the strategy
we have previously employed for protected β-amino acids
with five-membered ring constraints, including ACPC[6]
and a pyrrolidine analogue.[7] This approach was inspired
by the asymmetric synthesis of cis-2-aminocyclohexanecar-
boxylic acid reported by Wu et al.[8] The route to Fmoc-
APiC(Boc) (4) starts with commercially available β-oxo es-
ter 1, which bears a benzyl group on the ring nitrogen atom.
Hydrogenolytic removal of the benzyl group and reaction
with di-tert-butyl dicarbonate provided the Boc-protected
β-oxo ester, which was allowed to react with (R)-(ϩ)-α-
methylbenzylamine[9] in refluxing benzene containing a
catalytic amount of p-toluenesulfonic acid to form enamine
2. The enamine was then allowed to react with NaBH4 in
isobutyric acid. 1H NMR analysis indicated that this reduc-
tion generated a 4:1 mixture of the two cis diastereomers of
the expected β-amino ester. This mixture was treated with
sodium ethoxide in ethanol, causing epimerization to the
trans-β-amino esters. 1H NMR analysis of the product mix-
ture suggested a 2.5:1 preference for trans vs. cis diastereo-
mers. A single trans diastereomer was isolated from this
mixture following a two-stage crystallization protocol. The
crude diastereomeric mixture was dissolved in diethyl ether,
and 4  HCl in dioxane was added slowly. Adding hexanes
and storing this solution at 0 °C led to the precipitation of
Here we report a short and enantioselective synthesis of
trans-4-aminopiperidine-3-carboxylic acid (APiC), a new β-
amino acid with a six-membered ring constraint. The route
employs α-methylbenzylamine as a chiral auxiliary and ni-
trogen source, which allows one to generate either enanti-
omer of APiC. No chromatography is required; interme-
diates are purified by crystallization. Thus, this method is
[a]
Department of Chemistry, University of Wisconsin,
Madison, WI 53706-1396, USA
E-mail: gellman@chem.wisc.edu
Eur. J. Org. Chem. 2003, 721Ϫ726  2003 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1434Ϫ193X/03/0204Ϫ0721 $ 20.00ϩ.50/0
721

M. Schinnerl, J. K. Murray, J. M. Langenhan, S. H. Gellman
FULL PAPER
Scheme 1
a white crystalline solid. Recrystallization of the precipit- 9 was isolated by crystallization of the HBr salt, recrystal-
ated solid from acetonitrile provided the pure diastereomer lization from acetonitrile, and a base wash.[8] Base-cata-
3 (Ն 99% de according to NMR analysis) in 16% yield from lyzed epimerization provided a diastereomeric mixture with
crude 2. The absolute configuration of 3 prepared using (S)- a 4:1 trans/cis ratio. Pure diastereomer 10 was obtained in
(Ϫ)-α-methylbenzylamine was established by removal of the greater than 99% de by a two-stage crystallization protocol;
Boc group and conversion into the bis(HCl) salt 5, for the overall yield from crude 8 was 25%. The absolute con-
which a crystal structure was obtained (Figure 1). The de- figuration of Fmoc-ACHC (11) obtained from (R)-(ϩ)-α-
sired Fmoc-β-amino acid 4, obtained from 3 by the final methylbenzylamine was established as (R,R) by comparing
three steps indicated in Scheme 1, was crystallized with the optical rotation with (R,R)-Fmoc-ACHC obtained by a
more than 99% ee, as demonstrated by chiral HPLC ana- route that involves an enzymatic desymmetrization.[10] The
lysis of the corresponding dinitrophenyl derivative 6.
new route provides access to either enantiomer of Fmoc-
ACHC, since either enantiomer of α-methylbenzylamine
can be used. The enantiomeric excess of 11 was shown to be
greater 99% by chiral HPLC analysis of the corresponding
dinitrophenyl derivative 12. This synthetic protocol repres-
ents a substantial improvement in terms of efficiency relat-
ive to earlier routes to enantiomerically pure ACHC deriv-
atives.[10,11]
Oligomer Conformational Analysis
The ability of the APiC residue to support 14-helix
formation was evaluated by preparing a hexamer (13) in
which (R,R)-ACHC and (R,R)-APiC residues alternate.
Circular dichroism (CD) in the far-UV region has been
used for low-resolution characterization of β-peptide sec-
ondary structure,[2b] and we applied this method to 13. Fig-
ure 2 compares the CD spectra of the homochiral hexa-β-
peptide 13 in methanol and in aqueous buffer. Methanol
strongly promotes helix formation in β-peptides con-
structed from flexible residues (no ring constraints), while
folding in water is generally more difficult to achieve.[5a]
The β-peptide 13 displays a maximum near 217 nm in both
solvents, consistent with at least partial population of the
14-helical conformation. The maxima in these two solvents
have very similar intensities, which suggests that the 14-he-
lix is populated to similar extents in water and methanol.
This similarity indicates that APiC is a strong promoter of
14-helicity in aqueous solution.
An analogous route (Scheme 2) provided enantiomer-
ically pure Fmoc-ACHC (11) from the commercially avail-
able β-oxo ester 7. Reduction of enamine 8 yielded predom-
inantly the cis-β-amino esters, and the desired diastereomer
Figure 1. Ball-and-stick representation of the conformation deter-
mined crystallographically for 5 in the solid state (H atoms not
shown)
722
Eur. J. Org. Chem. 2003, 721Ϫ726

Asymmetric Synthesis of a New Helix-Forming β-Amino Acid
FULL PAPER
Scheme 2
Experimental Section
General Procedures: Melting points were determined with a capil-
lary melting point apparatus and are uncorrected. Optical rotations
were measured using sodium light (D line, 589.3 nm). Benzene and
toluene were distilled from sodium/benzophenone ketyl under N2
and ethanol from sodium/diethyl phthalate. Unless otherwise
noted, all other commercially available reagents and solvents were
purchased from Aldrich and used without further purification, ex-
cept for 4  HCl in dioxane, which was purchased from Pierce,
and Fmoc-OSu, which was purchased from Advanced ChemTech.
Analytical thin-layer chromatography (TLC) was carried out on
Whatman TLC plates precoated with silica gel 60 (250 µm layer
thickness). Visualization was accomplished using either a UV lamp
or phosphomolybdic acid (PMA) stain (10% phosphomolybdic
acid in ethanol). Column chromatography was performed on EM
Science silica gel 60 (230Ϫ400 mesh). Solvent mixtures used for
TLC and column chromatography are reported in v/v ratios. Dias-
Figure 2. Circular dichroism spectra of hexamer 13 in 10 m aque-
ous Tris, pH ϭ 7 (solid line) and in methanol (dashed line)
1
tereomeric excesses were determined using H NMR spectroscopy.
Enantiomeric excesses were determined using chiral HPLC.
Enamine 2: Pd/C (10%, 2.40 g) and ammonium formate (15.7 g,
249 mmol) were added to a clear solution of ethyl N-benzyl-4-oxo-
piperidine-3-carboxylate hydrochloride (15.3 g, 51.4 mmol) in abso-
lute ethanol (480 mL) under nitrogen. The mixture was refluxed
for 1 h. The cooled solution was filtered through Celite and washed
with ethanol. The filtrate was concentrated to obtain a white solid
Conclusion
Cyclically constrained β-amino acids are very important
building blocks for the creation of short β-peptides that ad-
opt defined conformations in aqueous solution. Here we (9.85 g, 92%). This solid was taken up in chloroform (85 mL), and
a solution of NaHCO3 (4.17 g, 49.6 mmol) in water (80 mL) and
NaCl (8.33 g, 143 mmol) were added. A solution of di-tert-butyl
dicarbonate (10.4 g, 47.5 mmol) in chloroform (30 mL) was slowly
added at room temperature (15 min), and the mixture was then
refluxed for 15 h. The organic layer was separated, and the aqueous
phase was extracted with chloroform (3 ϫ 50 mL). The combined
organic layers were dried with Na2SO4, and the solvent was evapor-
ated to obtain a yellow solid (Rf ϭ 0.29, hexane/ethyl acetate, 7:1).
A stirred solution of this solid, (R)-(ϩ)-α-methylbenzylamine
(6.05 g, 49.9 mmol) and a catalytic amount of p-toluenesulfonic
acid (451 mg, 2.37 mmol, 5 mol%) in 150 mL of dry benzene was
identify a new preorganized residue, APiC, that can confer
aqueous solubility and we provide a short, practical and
scalable synthetic route that leads to either enantiomer. In
addition, we have extended this route to generate a practical
synthesis of enantiomerically pure ACHC. (After our work
was completed Berkessel et al. reported an alternative and
very practical synthesis of ACHC, in either enantiomeric
form.[12]) These building blocks should further the explora-
tion of 14-helical β-peptides, and they should be useful for
a variety of other applications.
Eur. J. Org. Chem. 2003, 721Ϫ726
723

M. Schinnerl, J. K. Murray, J. M. Langenhan, S. H. Gellman
FULL PAPER
refluxed under nitrogen with continuous removal of water by using
a DeanϪStark trap for 7 h. The cooled reaction mixture was
washed twice with saturated aqueous NaHCO3 (2 ϫ 100 mL). The
organic layer was dried with Na2SO4. After removal of the solvent,
the yellow oily residue was filtered through a pad of silica gel
get a second crop. The combined crops were further dried under
vacuum to give 2.07 g of 3 as a white crystalline solid (16% yield
from 2). 1H NMR of the corresponding free amine indicated the
diastereomeric excess to be greater than 99%. M.p. 197Ϫ198 °C.
[α]rD.t. ϭ ϩ11.7 (c ϭ 1.03, MeOH). 1H NMR (300 MHz, CDCl3):
(CH2Cl2, washing until the filtrate became colorless; a yellow color δ ϭ 10.20 (br. s, 1 H), 9.92 (br. s, 1 H), 7.79Ϫ7.76 (m, 2 H),
remained on the silica gel). The filtrate was concentrated to obtain
7.44Ϫ7.40 (m, 3 H), 4.60 (br. s, 1 H), 4.27 (q, J ϭ 7.2 Hz, 2 H),
3.87 (br. d, J ϭ 11.8 Hz, 1 H), 3.28Ϫ3.19 (m, 2 H), 3.00Ϫ2.78 (m,
13.7 g of enamine 2 as a pale yellow oil (71% over three steps):
1
Rf ϭ 0.31, hexane/ethyl acetate, 6:1. H NMR (300 MHz, CDCl3): 1 H), 2.54 (br. s, 1 H), 2.06Ϫ1.93 (m, 4 H), 1.74Ϫ1.60 (m, 1 H),
δ ϭ 9.25 (d, J ϭ 7.3 Hz, 1 H), 7.35Ϫ7.22 (m, 5 H), 4.60 (quint,
J ϭ 6.7 Hz, 1 H), 4.19 (q, J ϭ 7.0 Hz, 2 H), 4.07 (s, 2 H), 3.46Ϫ3.38 171.30 (C), 153.67 (C), 136.10 (C), 129.24 (CH), 129.02 (CH),
1.44Ϫ1.30 (m, 13 H) ppm. 13C NMR (75 MHz, CDCl3): δ ϭ
(m, 1 H), 3.33Ϫ3.26 (m, 1 H), 2.43Ϫ2.35 (m, 1 H), 2.09Ϫ1.99 (m,
1 H), 1.50 (d, J ϭ 6.7 Hz, 3 H), 1.43 (s, 9 H), 1.29 (t, J ϭ 7.0 Hz,
128.34 (CH), 80.05 (C), 61.63 (CH2), 59.78 (CH), 54.86 (CH), 44.74
(CH), 44.12 (CH2), 41.70 (CH2), 28.56 (CH2), 27.99 (CH3), 20.46
3 H) ppm. 13C NMR (75 MHz, CDCl3): δ ϭ 168.77 (C), 156.91 (CH3), 13.88 (CH3) ppm. MS-ESI: m/z ϭ 377.2 [M Ϫ Cl]ϩ, 399.2
(C), 154.33 (C), 145.00 (C), 128.28 (CH), 126.85 (CH), 125.62
(CH), 88.35 (C), 79.37 (C), 58.73 (CH2), 52.02 (CH), 41.21 (CH2),
39.67 (CH2), 28.16 (CH3), 26.02 (CH2), 24.98 (CH3), 14.34 (CH3)
ppm. MS-ESI: m/z ϭ 375.2 [M ϩ H]ϩ, 397.2 [M ϩ Na]ϩ, 771.4 [2
M ϩ Na]ϩ.
[M Ϫ HCl ϩ Na]ϩ, 775.4 [2 M Ϫ HCl ϩ Na]ϩ.
(R,R)-Fmoc-APiC(Boc) (4): Compound 3 (1.89 g, 4.58 mmol) was
dissolved in THF/EtOH/H2O, 2:1:1 (100 mL), and this clear solu-
tion was cooled to 0 °C. LiOH·H2O (1.02 g, 24.3 mmol), dissolved
in 10 mL of H2O, was added. The mixture was stirred at 0 °C for
16 h. The solvent was removed under reduced pressure to give a
white solid (Rf ϭ 0.30, CH2Cl2/MeOH, 10:1). Pd/C (10%, 1.1 g)
and ammonium formate (2.38 g, 37.7 mmol) were added under N2
at room temperature to a turbid solution of this white solid in 200
mL of MeOH. The mixture was refluxed for 2 h. After the reaction
was complete (disappearance of starting material, as monitored by
TLC), the cooled solution was filtered through Celite, and the fil-
trate was concentrated to obtain a white solid. This solid was dis-
solved in acetone/H2O, 2:1 (200 mL), cooled to 0 °C, and Fmoc-
OSu (1.53 g, 4.53 mmol) and NaHCO3 (3.60 g, 42.8 mmol) were
added. The turbid reaction mixture was stirred at 0 °C for 1 h and
was then stirred at room temperature overnight. The acetone was
removed under reduced pressure. The aqueous residue was diluted
with H2O (50 mL), stirred for 1 h at room temperature with diethyl
ether (200 mL), and the layers were separated. The organic phase
was washed with saturated aqueous NaHCO3 (3 ϫ 100 mL). All
the aqueous phases were combined, acidified with 1  aqueous
HCl, and extracted with ethyl acetate (3 ϫ 100 mL). The combined
organic layers were dried with MgSO4 and concentrated to give a
white solid. The crude product was purified by crystallization from
refluxing chloroform (100 mL) after careful addition of MeOH (ca.
3 mL) until all the solid had dissolved. For complete precipitation,
hexane was added to the cold reaction mixture until the solution
became turbid. After storage at 0 °C overnight, 1.92 g (90%) of 4
was obtained as a white solid. M.p. 202Ϫ203 °C. Rf ϭ 0.32,
CH2Cl2/MeOH, 8:1. [α]rD.t. ϭ Ϫ4.9 (c ϭ 0.51, MeOH). 1H NMR
(300 MHz, CDCl3/CD3OD): δ ϭ 7.77 (d, J ϭ 7.4 Hz, 2 H), 7.61
(d, J ϭ 7.5 Hz, 2 H), 7.42Ϫ7.29 (m, 4 H), 4.36Ϫ4.34 (m, 2 H),
4.24Ϫ4.19 (m, 2 H), 4.06Ϫ4.02 (m, 1 H), 3.92Ϫ3.85 (m, 1 H),
3.03Ϫ2.85 (m, 2 H), 2.47Ϫ2.41 (m, 1 H), 2.01Ϫ1.97 (m, 1 H), 1.47
(s, 10 H) ppm. 13C NMR (75 MHz, CDCl3/CD3OD): δ ϭ 173.44
(C), 156.04 (C), 154.46 (C), 143.49 (C), 140.95 (C), 127.33 (CH),
126.72 (CH), 124.68 (CH), 119.53 (CH), 80.25 (C), 66.35 (CH2),
49.96 (CH), 47.26 (CH), 46.81 (CH), 44.59 (CH2), 41.87 (CH2),
30.87 (CH2), 27.79 (CH3) ppm. MS-ESI: m/z ϭ 243.1 [M Ϫ
Fmoc]Ϫ, 465.2 [M Ϫ H]Ϫ, 931.3 [2 M Ϫ H]Ϫ. (S,S)-Fmoc-APiC
was prepared by starting with (S)-(Ϫ)-α-methylbenzylamine.
[α]rD.t. ϭ ϩ5.0 (c ϭ 0.51, MeOH).
HCl Salt 3: Sodium borohydride (3.60 g, 95.2 mmol) was added
portionwise under N2 at 0 °C to isobutyric acid (60.0 mL,
647 mmol). This mixture was further stirred at room temperature
for 0.5 h and then 10 mL of absolute toluene was added and the
mixture was cooled to 0 °C. A solution of enamine 2 (11.8 g,
31.6 mmol) in dry toluene (40 mL) was added dropwise under N2
at 0 °C. The mixture was stirred at 0 °C for 1 h, and then additional
sodium borohydride (0.7 g, 18.5 mmol) was added in four portions
over a 4 h period. When the reaction was complete (7 h), 100 mL
of water was added carefully, and the reaction mixture was stirred
for 10 min at room temperature. Afterwards the mixture was
brought to pH ϭ 10 with 3  NaOH and extracted with EtOAc (3
ϫ 150 mL). The combined organic layers were dried with MgSO4
and concentrated under reduced pressure. The resulting yellow oil
was applied to a plug of silica gel and washed with hexane/ethyl
acetate, 2:1. The filtrate was concentrated to obtain a colorless oil
(11.3 g, 30.1 mmol, 95%; Rf ϭ 0.35, hexane/ethyl acetate, 2:1). This
oil (dried under vacuum overnight) was dissolved in dry ethanol
(50 mL) under N2. In a separate flame-dried Schlenk flask was
placed dry ethanol (250 mL), and sodium (2.06 g, 89.6 mmol) was
added portionwise under N2. The mixture was kept under N2 and
vented to remove evolved gases until all of the sodium had dis-
solved. The clear solution of the carboxylate was then transferred
to the NaOEt solution, and the mixture was stirred at 50 °C under
N2 for 15 h. The solvent was removed in vacuo, and after addition
of brine (150 mL) the mixture was brought to pH ϭ 10 with 1 
NaOH and extracted with ethyl acetate (3 ϫ 100 mL). The com-
bined organic layers were dried with MgSO4 and concentrated un-
der reduced pressure. The resulting oil was applied to a plug of
silica gel and washed with hexane/ethyl acetate, 2:1. The filtrate
was concentrated and dried under vacuum overnight to obtain a
pale yellow oil (9.51 g, 25.3 mmol, 84%). This oil was dissolved in
diethyl ether (25 mL), and 4  HCl in dioxane (6.2 mL, 24.8 mmol)
was added dropwise. The solution was stirred for 1 h, and a precip-
itate formed during this time. The precipitation was completed by
adding hexanes (125 mL) and storing the mixture at 0 °C for 1 h.
The white solid was isolated by filtration and washed with hexanes.
This crude product was purified by recrystallization from acetonitr-
ile. The solid was suspended in acetonitrile (20 mL) and heated to
reflux until the solid had dissolved completely. The solution was
then cooled to 0 °C overnight. The resulting precipitate was isol-
Chiral HPLC Assay of 6 and ent-6: Compound 4 was converted
into 6 by esterification, Fmoc deprotection, and reaction with 2,4-
ated by filtration and washed three times with 5-mL portions of dinitrofluorobenzene. Similarly, (S,S)-Fmoc-APiC was derivatized
cold acetonitrile. The mother liquor and the washings were com-
to obtain ent-6. Racemic 6 in hexanes/2-propanol, 9:1 (15 µL of a
bined and condensed to about half volume and cooled to 0 °C to
1.0 mg/mL solution) was injected onto a CHIRALCEL OD col-
724
Eur. J. Org. Chem. 2003, 721Ϫ726

Asymmetric Synthesis of a New Helix-Forming β-Amino Acid
FULL PAPER
umn (4.6 ϫ 250 mm; Daicel Chemical Ind., Ltd.). Elution with
hexanes/2-propanol, 1:1 at 0.6 mL/min resulted in separation of the
enantiomers (6: tr ϭ 19.7 min; ent-6: tr ϭ 32.4 min). Solutions of 6
and ent-6 (1.0 mg/mL) were similarly analyzed, and integration of
peak areas showed greater than 99% ee for both enantiomers.
H) ppm. 13C NMR (75 MHz, CDCl3) d 174.62 (C), 146.70 (C),
128.47 (2CH), 126.88 (CH), 126.75 (2 CH), 60.09 (CH2), 55.16
(CH), 53.55 (CH), 44.71 (CH), 30.06 (CH2), 25.67 (CH2), 24.79
(CH3), 23.46 (CH2), 22.99 (CH2), 14.53 (CH3) ppm. MS-ESI:
m/z ϭ 276.1 [M ϩ H]ϩ, 298.1 [M ϩ Na]ϩ, 573.2 [2 M ϩ Na]ϩ.
Enamine 8: A stirred solution of (R)-(ϩ)-α-methylbenzylamine
HCl Salt 10: Compound 9 (5.92 g, 21.5 mmol) was dissolved in dry
(8.01 g, 66.1 mmol), ethyl 2-oxocyclohexanecarboxylate (11.0 g, ethanol (50 mL) under N2. In a separate flame-dried Schlenk flask
64.7 mmol) and a catalytic amount of p-toluenesulfonic acid
(618 mg, 3.25 mmol, 5 mol %) in 100 mL of dry benzene was re-
fluxed under nitrogen with continuous removal of water by using
a DeanϪStark trap for 4 h. The cooled reaction mixture was
was placed dry ethanol (200 mL), and sodium (2.47 g, 107 mmol)
was added portionwise under N2. The mixture was kept under N2
and vented to remove evolved gases until all of the sodium had
dissolved. The clear solution of 9 was then transferred to the Na-
washed twice with saturated aqueous NaHCO3 (2 ϫ 50 mL). After OEt solution, and the mixture was stirred at 80 °C under N2 for
drying with Na2SO4 and removal of the solvent, the resulting yel-
15 h. The solvent was removed under vacuum, and after addition
low oily residue was fractionally distilled to give 15.4 g (87%) of 8
of brine (150 mL) the mixture was brought to pH ϭ 10 with 1 
as a pale yellow oil. B.p. 150Ϫ155 °C, 0.6 Torr. Rf ϭ 0.31, hexane/ NaOH and extracted with ethyl acetate (4 ϫ 100 mL). The com-
1
ethyl acetate, 20:1. H NMR (300 MHz, CDCl3): δ ϭ 9.42 (d, J ϭ bined organic layers were dried with MgSO4 and concentrated un-
7.5 Hz, 1 H), 7.36Ϫ7.22 (m, 5 H), 4.64 (quint, J ϭ 7.2 Hz, 1 H),
der reduced pressure. The resulting oil was applied to a plug of
4.18 (dq, J ϭ 1.0 Hz, 7.2 Hz, 2 H), 2.30Ϫ2.25 (m, 3 H), 1.98Ϫ1.91 silica gel and washed with hexane/ethyl acetate, 3:1. The filtrate
(m, 1 H), 1.54Ϫ1.47 (m, 7 H), 1.31 (t, J ϭ 7.2 Hz, 3 H) ppm. 13C
NMR (75 MHz, CDCl3): δ ϭ 170.76 (C), 158.88 (C), 145.65 (C),
was concentrated to obtain a pale yellow oil (4.85 g, 17.6 mmol,
82%). This oil was dissolved in ethyl acetate (20 mL), and 4 
128.48 (CH), 126.59 (CH), 125.23 (CH), 90.34 (C), 58.50 (CH2), HCl in dioxane (5.3 mL, 21.2 mmol) was added dropwise at room
51.79 (CH), 26.43 (CH2), 25.17 (CH3), 23.61 (CH2), 22.39 (CH2), temperature. The resulting solution was cooled to 0 °C and allowed
22.00 (CH2), 14.49 (CH3) ppm. MS-ESI: m/z ϭ 296.1 [M ϩ Na]ϩ, to stand at 0 °C overnight. A precipitate formed during this time.
569.3 [2 M ϩ Na]ϩ.
The white solid was filtered and washed three times with 20-mL
portions of cold ethyl acetate to provide the desired material. This
crude product could be purified by recrystallization from acetonitr-
ile. The solid was suspended in acetonitrile (50 mL) and heated to
reflux for 1 h. The mixture was then filtered through cotton wool
and cooled to 0 °C overnight. The resulting precipitate was isolated
by filtration and washed three times with 10-mL portions of cold
acetonitrile. The mother liquor and the washings were combined
and reduced to about half volume to obtain a second crop. The
combined crops were further dried under vacuum to give 3.44 g
of 10 as a white crystalline solid (51% yield from 9). 1H NMR
spectroscopy of the corresponding free amine indicated the diaster-
cis-β-Amino Ester 9: Sodium borohydride (2.45 g, 64.8 mmol) was
added portionwise under N2 at 0 °C to isobutyric acid (40.0 mL,
431 mmol). This mixture was further stirred at room temperature
for 0.5 h and then cooled to 0 °C. A solution of 8 (5.89 g,
21.6 mmol) in dry toluene (24.0 mL) was added dropwise under N2
at 0 °C. The mixture was stirred at 0 °C for 1 h, and then another
portion of sodium borohydride (0.50 g, 13.2 mmol) was added. The
reaction mixture was stirred at 0 °C for another 2 h. When the
reaction was complete, 100 mL of water was added carefully, and
the reaction mixture was stirred at room temperature for 10 min.
Afterwards, the mixture was brought to pH ϭ 10 with 3  NaOH
and extracted with EtOAc (3 ϫ 150 mL). The combined organic
layers were dried with MgSO4 and concentrated under reduced
pressure. The resulting oil was applied to a plug of silica gel and
washed with hexane/ethyl acetate, 1:1. The filtrate was concentrated
to obtain a colorless oil (5.92 g, 21.5 mmol, 99%; Rf ϭ 0.41, hex-
ane/ethyl acetate 4:1). This oil was dissolved in ethyl acetate (125
mL) and cooled to 0 °C. To this solution 30% (w/w) HBr in propi-
onic acid (5.3 mL, 25.8 mmol) was added dropwise with vigorous
stirring. A voluminous white precipitate formed during the addi-
tion. The mixture was stored at 0 °C overnight for complete precip-
itation. The white solid was isolated by filtration and washed with
three portions of cold ethyl acetate. The crude product was further
purified by recrystallization from acetonitrile. The solid was sus-
pended in acetonitrile (55 mL) and refluxed for 1 h. While hot, the
solution was filtered through a cotton wool plug, and the filtrate
was stored at 0 °C overnight. The resulting white crystals were isol-
eomeric excess to be greater than 99%. M.p. 211Ϫ212 °C. [α]rD.t.
ϭ
1
Ϫ37.5 (c ϭ 1.05, MeOH). H NMR (300 MHz, CDCl3): δ ϭ 9.76
(br. s, 1 H), 7.80Ϫ7.77 (m, 2 H), 7.47Ϫ7.38 (m, 3 H), 4.59 (q, J ϭ
7.0 Hz, 1 H), 4.34Ϫ4.18 (m, 2 H), 3.19Ϫ3.06 (m, 2 H), 2.22Ϫ2.18
(m, 1 H), 1.93Ϫ1.60 (m, 8 H), 1.34Ϫ1.24 (m, 5 H), 1.03Ϫ0.90 (m,
1 H) ppm. 13C NMR (75 MHz, CDCl3): δ ϭ 174.10 (C), 136.59
(C), 128.93 (CH), 128.83 (CH), 128.40 (CH), 61.13 (CH2), 59.44
(CH), 56.48 (CH), 45.87 (CH), 29.95 (CH2), 29.25 (CH2), 23.91
(CH2), 23.69 (CH2), 20.48 (CH3), 13.93 (CH3) ppm. MS-ESI:
m/z ϭ 276.1 [M Ϫ Cl]ϩ, 298.2 [M Ϫ HCl ϩ Na]ϩ.
(R,R)-Fmoc-ACHC (11): Compound 10 (2.80 g, 8.98 mmol) was
dissolved in THF/EtOH/H2O, 2:1:1 (100 mL), and the clear solu-
tion was cooled to 0 °C. LiOH·H2O (1.89 g, 45.1 mmol), dissolved
in 10 mL of H2O, was added. The mixture was stirred at 0 °C for
36 h. The solvent was removed under reduced pressure to obtain a
white solid (Rf ϭ 0.31, CH2Cl2/MeOH, 8:1). Pd/C (10%, 2.1 g) and
ated by filtration and dried under vacuum (3.82 g, 10.7 mmol, ammonium formate (2.83 g, 44.9 mmol) were added under N2 at
50%). This solid was mixed with an excess of saturated aqueous room temperature to a turbid solution of this white solid in 200
NaHCO3 (150 mL) then extracted with diethyl ether (3 ϫ 50 mL). mL of MeOH. The mixture was refluxed for 2 h. After the reaction
The combined organic extracts were dried with MgSO4, concen- was complete (disappearance of starting material, as monitored by
trated, and dried under vacuum overnight to give 9 as a clear oil TLC), the cooled solution was filtered through Celite, and the fil-
1
(2.91 g, 10.6 mmol, 99%). H NMR spectroscopy indicat the dias-
trate was concentrated to obtain a white solid. This solid was dis-
tereomeric excess to be greater than 99%. Rf ϭ 0.41, hexane/ethyl
solved in acetone/H2O, 2:1 (100 mL), cooled to 0 °C, and Fmoc-
acetate, 4:1. 1H NMR (300 MHz, CDCl3): δ ϭ 7.36Ϫ7.17 (m, 5 OSu (3.03 g, 8.98 mmol) and NaHCO3 (7.32 g, 87.1 mmol) were
H), 4.17 (q, J ϭ 7.1 Hz, 2 H), 3.86 (q, J ϭ 6.6 Hz, 1 H), 2.82 (dt, added. The turbid reaction mixture was stirred at 0 °C for 1 h and
J ϭ 7.8, 3.7 Hz, 1 H), 2.73 (dt, J ϭ 7.2, 3.7 Hz, 1 H), 1.86 (dtd,
was then stirred at room temperature overnight. The acetone was
J ϭ 9.6, 7.8, 3.5 Hz, 1 H), 1.75Ϫ1.38 (m, 6 H), 1.38Ϫ1.15 (m, 8 removed under reduced pressure. The aqueous residue was diluted
Eur. J. Org. Chem. 2003, 721Ϫ726
725

M. Schinnerl, J. K. Murray, J. M. Langenhan, S. H. Gellman
FULL PAPER
with H2O (50 mL), stirred for 1 h at room temperature with diethyl
ether (200 mL), and the layers were separated. The diethyl ether
layer was washed with saturated aqueous NaHCO3 (3 ϫ 100 mL).
The aqueous layers were combined, acidified with 1  aqueous
spectrometry (MALDI-TOF-MS: calcd. for C39H66N10O6 [M]
770.5, found 770.9 [M ϩ Hϩ], 792.9 [M ϩ Naϩ], 808.9 [M ϩ Kϩ]).
The β-peptide concentration for all experiments was determined
from the weight of the lyophilized 13 calculated as the TFA salt
HCl, and extracted with ethyl acetate (3 ϫ 100 mL). The combined (assuming association of one molecule of TFA per cationic res-
organic layers were dried with MgSO4 and concentrated to give a
white solid. This crude product was purified by crystallization from
refluxing chloroform (300 mL) with careful addition of MeOH (ca.
10 mL) until all solid had dissolved. For complete precipitation,
hexane was added to the cooled solution until the solution became
turbid. After storage at 0 °C overnight, 2.52 g (77%) of 11 was
obtained as a white solid. M.p. 206Ϫ207 °C. Rf ϭ 0.44, CH2Cl2/
MeOH, 10:1. [α]rD.t. ϭ Ϫ36.4 (c ϭ 0.50, acetone). 1H NMR
(300 MHz, CDCl3/CD3OD): δ ϭ 7.77 (d, J ϭ 7.3 Hz, 2 H), 7.62
(d, J ϭ 7.0 Hz, 2 H), 7.42Ϫ7.29 (m, 4 H), 4.33Ϫ4.19 (m, 3 H),
3.73Ϫ3.67 (m, 1 H), 2.35Ϫ2.28 (m, 1 H), 2.02Ϫ1.98 (m, 2 H),
1.78Ϫ1.73 (m, 2 H), 1.63Ϫ1.51 (m, 1 H), 1.41Ϫ1.22 (m, 3 H) ppm.
13C NMR (75 MHz, CDCl3/CD3OD): δ ϭ 180.61 (C), 160.16 (C),
idue).
CD Spectroscopy: Circular dichroism (CD) spectra were obtained
with an Aviv instrument at 25 °C using a quartz cell with a 1-mm
path length, between 190 and 260 nm. β-peptide 13 was analyzed
at 0.01 m in methanol or Tris buffer (10 m, pH ϭ 7.4). The
data were normalized for β-peptide concentration and number of
residues (i.e., the vertical axis in CD plots is mean residue ellipti-
city).
Acknowledgments
This research was supported by the NIH (GM56414). M. S. was
147.69 (C), 147.53 (C), 144.92 (C), 131.26 (CH), 130.67 (CH), supported by a postdoctoral fellowship from the DAAD (German
128.69 (CH), 123.47 (CH), 70.23 (CH2), 55.12 (CH), 52.45 (CH), Academic Exchange Service; D/01/05604), J. K. M. was supported
50.84 (CH), 36.27 (CH2), 32.73 (CH2), 28.35 (CH2), 28.11 (CH2) in part by a Wisconsin Alumni Research Foundation Fellowship
ppm. MS-ESI: m/z ϭ 388.1 [M ϩ Na]ϩ, 753.2 [2 M ϩ Na]ϩ. (S,S)-
Fmoc-ACHC was prepared by starting with (S)-(Ϫ)-α-methyl-
benzylamine. [α]rD.t. ϭ ϩ36.7 (c ϭ 0.52, acetone).
and an NIH Biotechnology Training Grant (NIH, 5 T32
GM08349), and J. M. L. was supported in part by a predoctoral
fellowship from the National Science Foundation. We thank Dr.
Ilia Guzei for crystallographic analysis.
Chiral HPLC Assay of 12 and ent-12: Compound 11 was converted
into 12 by esterification, Fmoc deprotection, and reaction with 2,4-
dinitrofluorobenzene. Similarly, (S,S)-Fmoc-ACHC was derivat-
ized to obtain ent-12. Racemic 12 in hexanes/2-propanol, 9:1 (15
µL of a 1.0 mg/mL solution) was injected onto a CHIRALCEL
OD column (4.6 ϫ 250 mm; Daicel Chemical Ind., Ltd.). Elution
with hexanes/2-propanol, 1:1 at 1.0 mL/min resulted in separation
of the enantiomers (12: tr ϭ 26.0 min; ent-12: tr ϭ 29.3 min). Solu-
tions of 12 and ent-12 (1.0 mg/mL) were similarly analyzed, and
integration of peak areas showed greater than 99% ee for both en-
antiomers.
[1]
S. H. Gellman, Acc. Chem. Res. 1998, 31, 173Ϫ180. D. J. Hill,
M. J. Mio, R. B. Prince, T. S. Hughes, J. S. Moore, Chem. Rev.
2001, 101, 3893Ϫ4011.
[2] [2a]
D. Seebach, J. L. Matthews, Chem. Commun. 1997,
[2b]
2015Ϫ2022.
R. P. Cheng, S. H. Gellman, W. F. DeGrado,
Chem. Rev. 2001, 101, 3219Ϫ3232.
[3]
[4]
X. Wang, J. F. Espinosa, S. H. Gellman, J. Am. Chem. Soc.
2000, 122, 4821Ϫ4822. H.-S. Lee, F. A. Syud, X. Wang, S. H.
Gellman, J. Am. Chem. Soc. 2001, 123, 7721Ϫ7722.
D. H. Appella, J. J. Barchi, S. R. Durell, S. H. Gellman, J. Am.
Chem. Soc. 1999, 121, 2309Ϫ2310.
Synthesis and Purification of 13: β-Peptide 13 was synthesized from
Fmoc-ACHC and Fmoc-APiC(Boc) on a 25-µmol scale by stand-
ard methods on Rink amide AM resin (Applied Biosystems), using
a Synergy automated synthesizer (Applied Biosystems). Amino
acid (3 equiv.), HBTU (3 equiv.), and DIEA (6 equiv.) were used
in each coupling cycle. Coupling cycles were 120 min in duration,
and piperidine deprotection cycles were approximately 60 min in
duration (actual deprotection time was regulated automatically by
monitoring of the conductivity trace). Residues 3Ϫ6 were double
coupled. The resin-bound β-peptide was cleaved from the solid sup-
port and deprotected simultaneously by using 2 mL of trifluo-
roacetic acid/H2O, 19:5 and stirring for 4 h. β-Peptide 13 was pre-
cipitated by concentration of the deprotection solution followed
by addition of a large excess of cold anhydrous diethyl ether; the
precipitate was collected by centrifugation. The β-peptide was puri-
fied by reversed-phase HPLC on a C18-silica semipreparative col-
umn (5 µm; 10 mm ϫ 250 mm; Vydac). The column was eluted
with a gradient of acetonitrile in water (4Ϫ32%; 0.1% trifluo-
roacetic acid in each) at a flow rate of 3 mL/min. Purified 13
(15.5 mg, 51%) was shown to be greater than 96% homogeneous
by HPLC on a C18-silica reversed-phase analytical column (5 µm;
4 mm ϫ 250 mm; Vydac), and its mass was confirmed by mass
[5] [5a]
S. Abele, G. Guichard, D. Seebach, Helv. Chim. Acta 1998,
[5b]
81, 2141Ϫ2156.
B. W. Gung, D. Zou, A. M. Stalcup, C. E.
[5c]
Cottrell, J. Org. Chem. 1999, 64, 2176Ϫ2177.
R. P. Cheng,
[5d]
W. F. DeGrado, J. Am. Chem. Soc. 2001, 123, 5162Ϫ5163.
P. I. Arvidsson, M. Rueping, D. Seebach, Chem. Commun.
2001, 649Ϫ650.
[6]
[7]
H.-S. Lee, P. L. LePlae, E. A. Porter, S. H. Gellman, J. Org.
Chem. 2001, 66, 3597Ϫ3599.
P. L. LePlae, N. Umezawa, H.-S. Lee, S. H. Gellman, J. Org.
Chem. 2001, 66, 5629Ϫ5632.
[8]
D. Wu, K. Prasad, O. Repic, T. J. Blacklock, Tetrahedron:
Asymmetry 1997, 8, 1445Ϫ1451.
[9]
´
E. Juaristi, J. L. Leon-Romo, A. Reyes, J. Escalante, Tetrahed-
ron: Asymmetry 1999, 10, 2441Ϫ2495.
[10]
D. H. Appella, P. R. LePlae, T. L. Raguse, S. H. Gellman, J.
Org. Chem. 2000, 65, 4766Ϫ4769. T. L. Raguse, E. A. Porter,
B. Weisblum, S. H. Gellman, J. Am. Chem. Soc. 2002, 124,
12774Ϫ12785.
[11]
[12]
D. H. Appella, L. A. Christianson, I. L. Karle, D. R. Powell,
S. H. Gellman, J. Am. Chem. Soc. 1999, 121, 6206Ϫ6212.
A. Berkessel, K. Glaubitz, J. Lex, Eur. J. Org. Chem. 2002,
17, 2948Ϫ2952.
Received August 21, 2002
[O02475]
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