Synthesis of enantiomerically pure cis- and trans-4-amino-1-oxyl-2,2,6,6- tetramethylpiperidine-3-carboxylic acid: A spin-labelled, cyclic, chiral β-amino acid, and 3D-structural analysis of a doubly spin-labelled β-hexapeptide

Article abstract of DOI:10.1002/ejoc.200700153

Animation of 3-carboxymethyl-1-oxyl-2,2,6,6-tetramethyl-4-piperidone (1) with either (R)- or (S)-α-methylbenzylamine gave corresponding enamines 2. Whereas the reduction with NaBH₃CN/CH₃COOH afforded predominantly a mixture of two po

Full text of DOI:10.1002/ejoc.200700153

FULL PAPER
DOI: 10.1002/ejoc.200700153
Synthesis of Enantiomerically Pure cis- and trans-4-Amino-1-oxyl-2,2,6,6-
tetramethylpiperidine-3-carboxylic Acid: A Spin-Labelled, Cyclic,
Chiral β-Amino Acid, and 3D-Structural Analysis of a Doubly Spin-Labelled
β-Hexapeptide
Karen Wright,*[a] Matthieu Sarciaux,[a] Augustin de Castries,[a] Michel Wakselman,[a]
Jean-Paul Mazaleyrat,[a] Antonio Toffoletti,[b] Carlo Corvaja,[b] Marco Crisma,[b]
Cristina Peggion,[b] Fernando Formaggio,[b] and Claudio Toniolo*[b]
Keywords: Circular dichroism / Conformation analysis / EPR spectroscopy / β-Peptides / Nitroxide radicals
Amination of 3-carboxymethyl-1-oxyl-2,2,6,6-tetramethyl-4-
piperidone (1) with either (R)- or (S)-α-methylbenzylamine
gave corresponding enamines 2. Whereas the reduction with
NaBH3CN/CH3COOH afforded predominantly a mixture of
two possible cis diastereomers of 3, (1ЈR,3S,4S)/(1ЈR,3R,4R)
or (1ЈS,3R,4R)/(1ЈS,3S,4S), which could be separated by crys-
tallisation of their HCl salts, the use of NaBH4/(CH3)2-
CHCOOH as the reducing agent resulted in a mixture of one
trans- and one cis diastereomer of 3 (1ЈR,3S,4R)/(1ЈR,3R,4R)
or (1ЈS,3R,4S)/(1ЈS,3S,4S) in varying proportions depending
upon the conditions used. The stereochemistry of the four
diastereomers of 3 was clearly established by X-ray diffrac-
tion analysis of one of them, combined with 1H NMR spectro-
scopic studies after nitroxide reduction. Removal of the chiral
auxiliary from the separated diastereomers of 3 by hydrogen-
ation and regeneration of the nitroxide radical gave expected
amino esters 4. A model β-hexapeptide containing (3R,4S)-
β-TOAC combined with (1S,2S)-2-aminocyclohexane car-
boxylic acid was synthesised by solution methods and its pre-
ferred conformation (314-helix) was assessed by FTIR absorp-
tion, CD, and EPR spectroscopy.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
proteins, or introduced into model peptide systems for
studies of physical effects such as intramolecular energy
transfer (fluorescence quenching) and spin polarisation
(CIDEP).[6,8]
Introduction
Nitroxide free radicals derived from TEMPO (2,2,6,6-tet-
ramethyl-1-piperidinyloxy) have found applications in
chemistry and medicine as spin labels,[1] spin traps,[2] oxidis-
ing agents,[3] antioxidants[4] and MRI contrast agents.[5] To
date, their most common use is in the study of conforma-
tion and structural mobility of peptides and proteins by
EPR spectroscopy. Spin labels can be introduced either by
functionalisation of the side chain of protein amino acids[6]
(for example, the thiol group of cysteine with methanethio-
sulfonate spin labels) or by the insertion of synthetic ni-
troxide-bearing amino acids into the peptide chain. A vari-
ety of such novel amino acids have been prepared where the
spin label has been placed either in the side chain of α-
amino acids or incorporated into cyclic structures of α,α-
disubstituted-, β- or γ-amino acids.[7] These amino acids
have been used to spin label biologically active peptides and
The TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperi-
dine-4-carboxylic acid) α-amino acid residue[9–11] (Figure 1)
has been widely used for these purposes. The tetrasubsti-
tuted α carbon of TOAC is responsible for its ability to
induce β-turn or 310/α-helical structures in peptides, but
also for the reduced reactivity of its amino group. We
wished to synthesise chiral, spin-labelled amino acids which
retained the conformationally rigid character of TOAC,
while allowing milder peptide coupling conditions. The ra-
cemic trans-β-amino acid POAC (3-amino-1-oxyl-2,2,5,5-
tetramethylpyrrolidine-4-carboxylic acid) (Figure 1), first
described by Rassat and Rey,[11a] attracted our attention[12]
[a] ILV, UMR 8180, University of Versailles, CNRS,
45 Avenue des Etats-Unis, 78035 Versailles, France
Fax: +33-1-3925-4452
E-mail: wright@chimie.uvsq.fr
[b] Institute of Biomolecular Chemistry, Padova Unit, CNR, De-
partment of Chemistry, University of Padova,
via Marzolo 1, 35131 Padova, Italy
Figure 1. Chemical structures of the spin-labelled amino acids
TOAC, POAC and β-TOAC.
Fax: +39-049-827-5239
E-mail: claudio.toniolo@unipd.it
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K. Wright, C. Toniolo et al.
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as its easy inclusion in a peptide synthesised by solid-phase Gellman and coworkers[20] synthesised trans-3-amino-pyr-
methods had been demonstrated,[13] whereas similar synthe- rolidine-4-carboxylic acid and trans-2-amino-cyclopen-
ses with the TOAC residue proved problematic.[13,14] A β- tanecarboxylic acid by reductive amination of a β-keto ester
amino acid form of TOAC (4-amino-1-oxyl-2,2,6,6-tet- with either (R)- or (S)-α-methylbenzylamine in the presence
ramethylpiperidine-3-carboxylic acid, β-TOAC) was de- of NaBH3CN, and subsequent selective crystallisation of
signed that could be obtained in enantiopure form.[15,16]
the hydrochloride salts of the obtained β-amino esters to
The β-amino acids have been synthesised and studied in provide either trans enantiomer. Cimarelli and Palmieri[21]
the last ten years since it was demonstrated that their oligo- showed that cyclic β-enamino esters, derived from α-methyl-
mers may fold into helical conformations (stable in organic benzylamine and a cyclic β-keto ester, could be selectively
and aqueous solvents) and are resistant to enzymatic hy- reduced by NaHB(OAc)3 to give a predominant cis dia-
drolysis.[17] Gellman and coworkers[18,19] investigated the stereomer. This method was developed further by Xu et
synthesis and conformation of peptides containing trans- al.[22] for the preparation of cis-ACHC, who performed the
ACHC (2-amino-cyclohexane-1-carboxylic acid) and trans- reduction step using NaBH4 and a bulky organic acid, and
ApiC (4-aminopiperidine-3-carboxylic acid) and found that isolated the major diastereomer formed as its hydrobromide
they form 314-helical structures. As the trans-β-TOAC resi- salt. This pathway was also used recently by Gellman and
[19]
due has a similar structure to trans-ApiC, it was particularly coworkers
to obtain trans-2-amino-cyclohexanecarbox-
interesting to assess if it could be used as a spin probe to ylic acid by epimerisation of the cis isomer. Similar methods
study β-peptide secondary structures in the same way as applied to 3-carboxymethyl-1-oxyl-2,2,6,6-tetramethyl-4-pi-
TOAC had been exploited in the case of α-peptides. In this peridone (1) (Scheme 1) were attempted as a route to
paper, full experimental details of the synthesis of cis- and enantiopure β-TOAC.
trans-β-TOAC and the synthesis of peptides containing
Commercially available 2,2,6,6-tetramethyl-4-piperidone
trans-β-TOAC and trans-ACHC up to the hexamer level was oxidised by a described procedure to its 1-oxyl deriva-
with their conformational analyses by infrared, circular di- tive.[23] Carboxylation of this compound with carbon diox-
chroism and EPR spectroscopy are reported.
ide in the presence of potassium phenoxide following a
[24]
known method
did not give reproducible results in our
hands, as the yield of keto ester 1 obtained after the reac-
tion with diazomethane varied between 15–50% over dif-
ferent runs (35% yield in ref.[24]). Amination of 1 with (R)-
or (S)-α-methylbenzylamine in the presence of acetic acid
proceeded smoothly to provide desired products (R)-2 or
(S)-2 in 61 and 66% yields, respectively. The enamine struc-
Results and Discussion
Synthesis and Characterisation
The syntheses of cis- and trans-β-TOAC were based on
methods developed to prepare other cyclic β-amino acids.
Scheme 1. (i) (S)- [or (R)-]-α-methylbenzylamine, AcOH, EtOH, 4-Å MS, room temp., 48 h. (ii) NaBH3CN, AcOH, EtOH, 75 °C, 2 h;
then HCl/EtOAc, 0 °C, filtration and recrystallisation from MeCN. (iii) NaOMe, MeOH, 70 °C, 18 h.
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Enantiomerically Pure β-TOAC
FULL PAPER
1
ture of 2 was determined from its H NMR spectrum after tures, which allowed the assignment of the absolute config-
nitroxide reduction.[25,26]
uration of the resulting trans isomers (1ЈS,3R,4S)-3/
Isolated enamine (R)-2 was first reduced in the presence (1ЈS,3S,4R)-3 and (1ЈR,3S,4R)-3/(1ЈR,3R,4S)-3. Interest-
1
of NaBH3CN and acetic acid to give a mixture of reduced ingly, the H NMR spectra (Figure 3) of the four isomers
1
products 3. The H NMR spectra of this mixture, after ni- of 3·HCl after nitroxide reduction show different chemical
troxide reduction, showed that it contained the two cis dia- shifts for H3, H4 and H5–H5Јin both pairs of isomers cis-
stereomers in a proportion of 1:1, with trace quantities of (1ЈR,3S,4S)-3/trans-(1ЈR,3R,4S)-3 and cis-(1ЈR,3R,4R)-3/
the two trans diastereomers. The hydrochloride salts were trans-(1ЈR,3S,4R)-3, whereas the coupling constant of the
prepared by adding a solution of HCl in EtOAc to a cold H3 proton is significantly higher for the trans isomers (11.4
solution of 3 in EtOAc. The precipitate formed was col- and 11.8 Hz) than for the cis isomers (4.0 and 4.0 Hz) as
lected and recrystallised from MeCN to give (1ЈR,3S,4S)-3 expected. These differences allowed easy analysis of the
1
as a sole diastereomer as judged from its H NMR spec- crude cis/trans mixtures.
trum. Diastereomer (1ЈR,3R,4R)-3 was obtained from the
EtOAc mother liquor. The same reaction sequence was fol-
lowed starting from enamine (S)-2 to give (1ЈS,3R,4R)-3 as
crystals and (1ЈS,3S,4S)-3 from the EtOAc mother liquor
[Scheme 1; products obtained from (R)-2 are shown in
brackets]. Derivative (1ЈS,3R,4R)-3·HCl was obtained after
recrystallisation from MeOH/MeCN as large orange crys-
tals suitable for X-ray diffraction analysis,[15a,15c] which al-
lowed the assignment of the configurations at C3 and C4 on
the basis of the known configuration at C1Ј (Figure 2).
Figure 3. 1H NMR spectra (D2O) of the diastereomers 3·HCl after
nitroxide reduction.
Separation of the cis/trans isomers was troublesome:
whereas 42% of (1ЈS,3R,4S)-3 could be isolated from epi-
merisation of (1ЈS,3S,4S)-3, diastereomer (1ЈS,3S,4R)-3
Figure 2. X-ray diffraction structure of cis-3(1ЈS,3R,4R) hydrochlo-
could not be separated by column chromatography from
ride with selected atom numbering. Displacement ellipsoids for
(1ЈS,3R,4R)-3. Repeated preparative thin-layer chromatog-
non-hydrogen atoms are drawn at the 30% probability level.
raphy of the reaction mixture resulting from the epimer-
The X-ray diffraction structure shows that the piperidine isation of (1ЈR,3S,4S)-3 allowed the recovery of only a mix-
ring adopts a slightly distorted chair conformation with ture of (1ЈR,3R,4S)-3 and (1ЈR,3S,4S)-3 in a ratio of 9:1.
puckering parameters QT = 0.529(4) Å, θ2 = 154.8(4)° and
To obtain greater quantities of a single diastereomer of
φ2 = 338.5(12)°. The amino and carboxyl substituents oc- trans-3, the reduction of enamine 2 was examined again.
cupy the equatorial and axial positions, respectively. The Xu et al.[22] developed an asymmetric reductive amination
values of the torsion angles θ [N–C4–C3–C1: 70.7(5)°] and method, first described by Cimarelli and Palmieri,[21] to al-
ψ [C4–C3–C1–OT: –117.1(4)°] are close to those reported low large-scale synthesis of ethyl 2-amino-1-cyclohex-
for the β-peptide 314-helix (60° and –140°, respec- anecarboxylate from the corresponding α-methylbenzyla-
tively).[17d,18] No significant comparison can be made be- mine-derived enamines. Whereas Cimarelli and Palmieri[21]
tween the value of the C1Ј–N–C4–C3 torsion angle, used NaHB(OAc)3 for the reduction of similar cyclic β-en-
172.2(3)°, with that of the φ torsion angle in β-peptides ow- amino esters, Xu et al.[22] improved the stereoselectivity of
ing to the different nature (amino vs. amide) of the nitrogen the reduction step by using NaBH4 in the presence of a
atom.
bulky organic acid (e.g. isobutyric acid or pivalic acid).
Epimerisation of cis isomers (1ЈS,3S,4S)-3/(1ЈS,3R,4R)-3 They achieved an optimal cis/trans selectivity of 60:1, with
and (1ЈR,3R,4R)-3/(1ЈR,3S,4S)-3 of known absolute config- a de of 84% when using excess isobutyric acid and toluene
uration in the presence of NaOMe gave 1:1 cis/trans mix- as a cosolvent.
Eur. J. Org. Chem. 2007, 3133–3144
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K. Wright, C. Toniolo et al.
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These optimised reaction conditions were applied to the product open to the air in the presence of Cu(OAc)2 for
reduction of enamine (S)-2 (Scheme 2), with surprising re- 7 d.[27] In this way, compounds (3S,4S)-4, (3R,4R)-4,
sults: NMR spectroscopic analysis of the crude reaction (3S,4R)-4 and (3R,4S)-4 (Scheme 3) were obtained in 53,
product revealed the presence of only the cis (1ЈS,3S,4S)
and trans (1ЈS,3R,4S) diastereomers of 3 in a ratio of 1:1,
with no trace of the other two diastereomers. After
chromatography, 29% (1ЈS,3S,4S)-3 and 30% (1ЈS,3R,4S)-
3 were obtained. The reduction was repeated with enamine
(R)-2 with similar results; 38% cis-(1ЈR,3R,4R)-3 and 39%
trans-(1ЈR,3S,4R)-3 were isolated, whereas the other two
diastereomers could not be detected in the NMR spectra of
the crude product. Intrigued by this unexpected outcome,
we returned to the reduction conditions proposed by Cima-
relli and Palmieri;[21] reduction of (S)-2 with NaBH4 in the
Scheme 3. Removal of the chiral auxiliary from diastereomers 3,
followed by C-deprotection/N-protection of resulting β-amino ester
enantiomer 4. (i) H2, Pd/C 10%, 95% EtOH, room temp., 30 min.
(ii) Cu(OAc)2, MeOH, air, room temp., 7 d. (iii) NaOH (aq),
MeOH, reflux, 5 h. (iv) Fmoc-succinimidyl carbonate; NaHCO3;
acetone/water, 2:1; room temp.; 18 h.
presence of AcOH gave only the two cis diastereomers
(1ЈS,3S,4S)-3 and (1ЈS,3R,4R)-3 in a ratio of 7:1, with no
trace of the trans diastereomers in the NMR spectra of the
crude product. Investigation of this reduction step was pur-
sued by changing the acid or the ratio of acid to NaBH4
used. The results are summarised in Table 1. Whereas tri-
fluoroacetic acid and phenylacetic acid were unsuitable, piv-
aloic acid gave a result similar to that of isobutyric acid
with only (1ЈS,3S,4S)-3 and (1ЈS,3R,4S)-3 isolated from the
reaction mixture, although in low yields. A lower yield was
also observed with isobutyric acid when the excess of acid
used was reduced, with the starting enamine recovered from
the reaction mixture. A difference in cis/trans selectivity was
noted when the relative proportion of NaBH4:isobutyric
acid was reduced; cis diastereomer (1ЈS,3S,4S)-3 was fav-
oured with a concomitant fall in the yield of the reduced
product.
Figure 4. β-Hexapeptide 11 containing two (3R,4S)-β-TOAC resi-
dues at positions 2 and 5 (Boc is tert-butyloxycarbonyl).
Scheme 2. Reduction of enamine (S)-2. (i) NaBH4, AcOH, EtOH,
0 °C to room temp., 7 h. (ii) NaBH4, (CH3)2CHCOOH, toluene,
0 °C to room temp., 24 h.
Removal of the chiral auxiliary from 3 was achieved by
hydrogenation over Pd/C for only a short time. Under these
conditions, the nitroxide group was reduced to an interme-
diate N-hydroxy compound, from which the nitroxide func-
Scheme 4. Synthesis of dipeptides 6 and 7. (i) EDC, HOAt, DIEA,
CH2Cl2, 0 °C to room temp., 18 h {EDC, N-ethyl,NЈ-[3Ј-(dimeth-
ylamino)propyl]carbodiimide; HOAt, 7-aza-1-hydroxy-1,2,3-
tion was regenerated by stirring the crude hydrogenated benzotriazole}.
Table 1. Reduction of (S)-2 by NaBH4 in the presence of organic acids.
Entry
Acid
Substrate/NaBH4/acid Cosolvent
Temp. [°C]
Yield [%]
Ratio cis/trans[a]
1
2
3
4
5
6
trifluoroacetic
phenylacetic
pivaloic
isobutyric
isobutyric
isobutyric
1:5:20
1:10:20
1:5:50
1:10:20
1:10:60
1:15:60
toluene
toluene/acetonitrile
toluene
toluene
toluene
20
20
20
20
20
20
decomposition
no reaction
44
35
42
77
–
–
2.3:1
1.2:1
10:1
1:1
toluene
[a] (1ЈS,3S,4S)-3 and (1ЈS,3R,4S)-3. Ratio determined from yields of isolated products after column chromatography.
3136 Eur. J. Org. Chem. 2007, 3133–3144
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Enantiomerically Pure β-TOAC
FULL PAPER
Scheme 5. Synthesis of hexapeptide 11. (i) CH3CN/Et2NH, 9:1; room temp.; 2 h. (ii) NaOH (aq), MeOH, reflux. (iii) H-(1S,2S)-ACHC-
OMe, EDC, HOAt, DIEA, CH2Cl2, 0 °C to room temp. (iv) EDC, HOAt, DIEA, CH2Cl2, 0 °C to room temp., 18 h.
62, 57 and 41% yield, respectively. A derivative suitable for
Figure 5 shows the FTIR absorption curves in the amide
peptide synthesis was prepared from (3R,4S)-4 by saponifi- N–H stretching (amide A) region of the N-terminal di-, tri-
cation of the methyl ester and Fmoc (9-fluorenylmethyloxy- and tetrapeptides of β-hexamer 11 in CDCl3 solution. Our
carbonyl) protection of the amino group to provide build- results clearly indicate that intense bands below 3400 cm–1,
ing block (3R,4S)-5 in 40% yield.
typically exhibited by H-bonded -CONH- groups,[29] are
We wished to incorporate the (3R,4S)-β-TOAC residue first seen at the level of tetramer. The curves of the dimer
into a model β-peptide to study in particular the resulting and trimer are dominated by the strong absorption above
structure by EPR spectroscopy.[28] We envisaged peptide 11, 3400 cm–1, which is characteristic of free -CONH- groups.
shown in Figure 4, incorporating two (3R,4S)-β-TOAC resi- The spectrum of the hexapeptide was not recorded owing
dues combined with four (1S,2S)-ACHC residues, the to its very poor solubility in this halohydrocarbon. Because
homooligomers of which are known to fold into 314-heli- the spectrum of the tetramer does not remarkably change
ces.[17] In this case, the β-TOAC residues at positions i and upon dilution of the concentration from 1.0 to 0.1 mm, it
i+3 should be positioned on the same face of the ternary is safe to conclude that the H-bonding observed in this con-
helix, which should allow an interaction between their two centration range is of the intramolecular type.
radical groups. With this aim in mind, we examined initially
the coupling reaction between (3R,4S)-4 and (3R,4S)-5 with
derivatives of (1S,2S)-ACHC (Scheme 4). Whereas the reac-
tion of (3R,4S)-4 with Boc-(1S,2S)-ACHC-OH in the pres-
ence of EDC/HOAt gave desired dipeptide 6 in 90% yield,
the coupling of H-(1S,2S)-ACHC-OMe with (3R,4S)-5 un-
der the same conditions was less satisfactory, with product
7 recovered in only 40% yield.
Following these results, we decided to use a segment
coupling approach to form the hexapeptide by reaction of
a C-deprotected tetrapeptide with an N-deprotected dipep-
tide (Scheme 5). Dipeptide 7 was treated with diethylamine
in MeCN to give N-deprotected dipeptide 8. Elongation of
dipeptide 6 by two cycles of saponification and reaction
with H-(1S,2S)-ACHC-OMe in the presence of EDC/HOAt
Figure 5. FTIR absorption spectra in the amide N–H stretching
gave firstly tripeptide 9 in 49% yield, and secondly tetrapep-
region of the Boc/OMe terminally protected, N-terminal, synthetic
intermediates of hexapeptide 11: dipeptide 6, tripeptide 9 and tetra-
peptide 10 in CDCl3 solution (peptide concentration: 1 mm).
tide 10 in 63% yield. Saponification of 10 and reaction with
8 in the presence of EDC/HOAt gave the desired hexa-
peptide Boc-{(1S,2S)-ACHC-(3R,4S)-β-TOAC-(1S,2S)-
ACHC}2-OMe (11) in 65% yield.
CD spectroscopy in the far-UV region (190–260 nm),
where the nǞπ* and πǞπ* transitions of chiral amide com-
pounds are observed, is extensively used to analyse the sec-
ondary structure of polypeptides.[30] Hexamer 11 displays
Conformational Analysis
We investigated the preferred conformations of the ter- an intense, negative Cotton effect centred at 218 nm in
minally protected, (1S, 2S)-ACHC/(3R, 4S)-β-TOAC β- MeOH solution (Figure 6), which closely resembles that of
peptides in structure-supporting solvents (CDCl3, MeOH) 314-helical β-peptides.[18b,31] A strictly related spectrum was
by using FTIR absorption, CD and EPR spectroscopic published for the (1S, 2S)-ACHC homohexamer in the
techniques.
same solvent.[18b] In addition, it is worth noting that in the
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K. Wright, C. Toniolo et al.
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near UV and visible regions (between 350 and 600 nm) di-
For comparison, Figure 7d shows the EPR spectrum of
chroic absorptions associated with the aminoxyl nǞπ* monolabelled tetrapeptide 10 in methanol solution at the
transition[32] (not shown) stand out clearly, which make this same temperature (aiso = 1.63 mT, g = 2.0065Ϯ0.0004). We
chromophoric group of β-TOAC potentially useful as a chi- have chosen this peptide as the reference in that it repre-
rospectroscopic probe for the assignment of the 3D-struc- sents a nitroxide system with rotational diffusion character-
ture of β-polypeptides and detection of the environment po- istics as close as possible to those of 11. The good agree-
larity. Interestingly, an induced CD in the 350–550 nm re- ment between the difference spectrum (c) and spectrum (d)
gion was already reported for TOAC-containing, chiral suggests the attribution of the sharp lines to a monoradical
peptides.[33]
impurity. The broad component (b) is that expected for
peptide conformations where the two nitroxides are at a
close distance and the electron–electron dipolar interaction
is large and not fully averaged out by the molecular rota-
tional diffusion motion. In frozen (120 K) MeOH solution,
the spectrum shows indeed a well-resolved pattern (Fig-
ure 8b), typical of a two unpaired electron system in a trip-
let state. Here, again a narrow feature is present in the cen-
tre of the spectrum, consistent with a monoradical EPR
spectrum (Figure 8c). The dipolar interaction parameter D,
obtained by comparing the experimental spectrum with a
computer simulated spectrum (Figure 8a), is 12.0 mT. The
simulation was performed by assuming an isotropic hyper-
fine coupling of 0.8 mT between the two 14N nuclei, which
is one half the typical value for nitroxide radicals (1.65 mT
in this solvent). This is expected for the case of a biradical
with a large exchange interaction J compared with the hy-
perfine interaction.[34,35] The same is true for the aniso-
tropic tensor components. Moreover, the hyperfine tensor
principal axes of the two 14N nuclei were assumed to be
coincident according to the expected peptide 314-helix con-
formation.[18] The resulting, corresponding distance be-
tween the two radical centres is R = 6.1 Å, which was calcu-
lated with the assumption that the spin density was local-
ised on the middle of the nitroxide N–O bond by the point
dipole approximation D = (3/2)g2β2/R3. According to a re-
cent paper,[36] the point dipole approximation applied to a
pair of nitroxide groups at a distance R Ͻ 9 Å gives an
overestimate of the R value (of the order of 5–10% de-
pending on the relative nitroxide orientation). This result
implies that the experimental 12.0 mT value should be asso-
ciated to a lower nitroxide···nitroxide distance (R = 5.5–
5.7 Å), which compares very well with the O···O distance
(5.43 Å) extracted from our computer modelling (Figure 9)
Figure 6. Far-UV CD spectrum of hexapeptide 11 in MeOH solu-
tion (peptide concentration: 1 mm).
The EPR spectrum of hexamer 11 in liquid MeOH solu-
tion at 258 K (Figure 7) consists of three sharp lines sepa-
rated by 1.65 mT and centred at g = 2.0058Ϯ0.0004 and
superimposed on a broad line spectrum. The broad and
sharp components (Figure 7b and c, respectively) can be
separated by fast Fourier transforming (FFT) the spectrum,
filtering the short time components in the time domain and
transforming them back into the frequency (magnetic field)
domain (FFT procedure). The double integrated intensity
of the sharp component corresponds to about 5% of the
total intensity.
Figure 7. Analysis of the EPR spectrum (a) of dilabelled hexapep-
tide 11. The sharp component (c) was obtained by subtracting the
broad component (b) from spectrum (a). Trace (d) is the EPR spec-
trum of monolabelled tetrapeptide 10. The two experimental spec-
tra were obtained in liquid MeOH solution at 258 K (peptide con-
centration: 1 mm).
Figure 8. Simulated (a) and experimental (b) EPR spectra of dilab-
elled hexapeptide 11. Trace (c) is the spectrum of monolabelled
tetrapeptide 10. The two experimental spectra were obtained in fro-
zen MeOH solution at 120 K (peptide concentration: 1 mm).
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Enantiomerically Pure β-TOAC
FULL PAPER
for a regular 314-helix structure.[18] The asymmetry param-
eter E is zero, as expected for interacting electron spins
localised on N–O groups placed at a long distance.
Hewlett–Packard HP5989MS spectrometer by Vincent Steinmetz
(ILV). High-resolution mass spectra were performed by the CNRS
Central Analytical Service in Vernaison (France). Analytical TLC
and preparative column chromatography were performed on Kie-
selgel F254 and Kieselgel 60 (0.040–0.063 mm) (Merck), respec-
tively. UV light (λ = 254 nm) allowed visualisation of the spots
after TLC runs for all compounds. Except when noted, all starting
materials and solvents were obtained from commercial suppliers
and were used as received. Boc-(1S,2S)-ACHC-OH was obtained
from NeoMPS SA, Strasbourg, France.
Preparation of NMR Samples: Well-resolved 1H and 13C NMR
spectra of the nitroxide compounds described in this study could
only be obtained by preliminary use of the sodium dithionite re-
duction procedure described in ref.[26] The nitroxide sample was
dissolved in [D6]acetone or D2O and sodium dithionite (2 equiv.)
dissolved in D2O was added.
Figure 9. Model of hexapeptide 11 in the 314-helical structure,
which highlights the distance between the oxygen atoms of the ni-
troxyl probes of the two (3R, 4S) β-TOAC residues.
Enamines (R)-2 and (S)-2: Keto ester 1[23,24] (5.24 g, 23 mmol) was
dissolved in absolute EtOH (85 mL) and 4-Å MS were added. The
mixture was placed under an Ar atmosphere and (R)-α-methylben-
zylamine (20.7 mL, 160.9 mmol) and glacial acetic acid (9.2 mL,
160.9 mmol) were added. The mixture was stirred at room temp.
for 48 h. The mixture was concentrated, and the residue taken up
in CH2Cl2. The mixture was filtered, and the filtrate was washed
successively with saturated aqueous NaHCO3 and brine. The or-
ganic phase was dried with MgSO4, filtered and concentrated. The
residue was purified by chromatography (cyclohexane/EtOAc, 9:1)
to give enamine (R)-2 (6.63 mg, 87%) as an orange oil. [α]25 = –345
(589), –367 (578), –441 (546), –914 (436), abs.(365), (c = 0.25,
MeOH). 1H NMR (300 MHz, D2O/[D6]acetone): δ = 7.48–7.63 (m,
5 H, ArH), 4.98 (m, 1 H, CH), 3.96 (s, 3 H, OCH3), 2.75, 2.39 (2
d, J = 16.9 Hz, 2 H, CH2), 1.71 (d, J = 6.6 Hz, 3 H, CHCH3), 1.68,
1.64, 1.34, 1.10 (4s, 12 H, 4CH3) ppm. 13C NMR (77 MHz, D2O/
[D6]acetone): δ = 172.1 (C=O), 158.1 (C4), 147.0, 130.3, 126.9,
116.7 (ArC), 98.9 (C3), 61.8 (C2), 56.8 (C6), 53.3 (NCH), 51.4
(OCH3), 41.4 (C5), 28.4, 27.6, 26.0, 25.3 (CH3) ppm. ES-MS: m/z
The narrow triplet of lines in the solution spectrum (Fig-
ure 7c) and the central feature in the frozen spectrum (Fig-
ure 8b) can be attributed to a monoradical impurity in the
sample, possibly a modified peptide with one of the two
nitroxide groups reduced to a hydroxylamine functionality.
However, it is not excluded that the observed spectral prop-
erties would be due, at least to a small extent, to peptide
conformations where the distance between the nitroxides is
large and both electron exchange and electron dipolar inter-
actions are small relative to the nitrogen hyperfine coupling.
Conclusions
The cis and trans enantiomers of the β-TOAC residue
bearing a nitroxide function were obtained in an enantio-
pure form by selective reduction of the corresponding en- (%) = 685.4 (55) [2M + Na]+, 354.2 (72) [M + Na]+, 332.2 (64) [M
+ H]+. C19H27N2O3 (331.4): calcd. C 68.85, H 8.21, N 8.45; found
amines. Derivatives suitable for peptide synthesis were also
C 68.61, H 8.13, N 8.24. Enamine (S)-2 was prepared in the same
prepared. The trans-(3R,4S)-β-TOAC enantiomer was in-
way from keto ester 1 and (S)-α-methylbenzylamine in 82% yield.
corporated into a β-hexapeptide in combination with
(1S,2S)-ACHC. Conformational analysis of this peptide by
a combination of optical spectroscopies showed that it
largely populates the classical 314-helical structure of β-
[α]25 = +357 (589), +380 (578), +456 (546), +954 (436), abs.(365),
1
(c = 0.26, MeOH). H and 13C NMR (D2O/[D6]acetone): identical
to (R)-2. C19H27N2O3 (331.4): calcd. C 68.85, H 8.21, N 8.45;
found C 68.58, H 8.41, N 8.25.
polypeptides.
We believe that these spin-labelled, rigid and chiral, β-
amino acids will find interesting applications in peptidomi-
metic conformational and biological studies.
(3S,4S)-4-[(1ЈR)-Phenylethyl]amino-3-methoxycarbonyl-2,2,6,6-
tetramethyl-piperidine-1-oxyl Hydrochloride [(1ЈR,3S,4S)-3·HCl]
and (3R,4R)-4-[(1ЈR)-Phenylethyl]amino-3-methoxycarbonyl-
2,2,6,6-tetramethyl-piperidine-1-oxyl Hydrochloride [(1ЈR,3R,4R)-3·
HCl]: Enamine (R)-2 (543 mg, 1.64 mmol) was dissolved in abso-
lute EtOH (10 mL) and glacial acetic acid (0.19 mL, 3.28 mmol)
was added. The mixture was placed under an Ar atmosphere and
sodium cyanoborohydride (310 mg, 4.92 mmol) was added. The
mixture was heated at 70 °C for 1 h. The mixture was cooled and
then concentrated. The residue was taken up in ether and washed
Experimental Section
General: Melting points were measured by means of a capillary
tube immersed in an oil bath (Tottoli apparatus, Büchi) and are
uncorrected. 1H and 13C NMR spectra were recorded with a
Bruker WM300 spectrometer operating at 300 MHz and 77 MHz, with saturated aqueous NaHCO3. The aqueous phase was ex-
respectively, the solvent being used as the internal standard. Split-
ting patterns are abbreviated as follows: (s) singlet, (d) doublet, (t)
triplet, (q) quartet, (m) multiplet. The optical rotations were mea-
sured in a 1-dm thermostatted cell with a Perkin–Elmer 241 polari-
meter, with an accuracy of 0.3%. Elemental analyses were per-
formed by the C.N.R.S. Service of Microanalyses in Gif-sur-Yvette
tracted with diethyl ether. The combined ether phases were washed
with brine, then dried with MgSO4, filtered and concentrated. The
residue was purified by chromatography (cyclohexane/EtOAc, 2:1)
to give 3 (371 mg, 68%) as an orange oil. The residue was dissolved
in EtOAc (5 mL) and cooled to 0 °C. A solution of HCl in EtOAc
(approx. 3.4 , 0.35 mL) was added. The mixture was kept at 4 °C
(France). Mass spectra (electrospray mode) were recorded with a overnight. The resulting precipitate was filtered off. The collected
Eur. J. Org. Chem. 2007, 3133–3144
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3139

K. Wright, C. Toniolo et al.
solid was recrystallised from MeCN to give (1ЈR,3S,4S)-3·HCl saturated NaHCO3 solution and once with brine. The organic
FULL PAPER
[157 mg, 26 % from (R)-2] as light orange crystals. M.p. 202–
phase was dried with MgSO4, filtered and concentrated. Column
204 °C. [α]25 = +29 (589), +32 (578), +48 (546), –39 (436), –115 chromatography (cyclohexane/EtOAc, 2:1) of the residue success-
1
(365), (c = 0.25, MeOH). H NMR (300 MHz, D2O): δ = 7.55 (s, ively gave enamine (S)-2 (165 mg, 33 %) and a mixture of
5 H, ArH), 4.67 (m, 1 H, CH), 3.87 (s, 3 H, OCH3), 3.65 (m, 1 H, (1ЈS,3R,4R)-3 and (1ЈS,3S,4S)-3 (298 mg, 59 %). The cis dia-
H4), 3.31 (d, J = 4.0 Hz, 1 H, H3), 2.23 (m, 2 H, H5), 1.73 (d, J = stereomers 3 were converted into their HCl salts by the addition of
1
6.6 Hz, 3 H, CHCH3), 1.51, 1.44, 1.36, 1.32 (4 s, 12 H, 4 CH3) a solution of HCl in EtOAc for H NMR characterisation, which
ppm. 13C NMR (77 MHz, D2O): δ = 173.1 (C=O), 137.6, 133.1,
132.7, 130.5 (ArC), 60.7, 60.0 (C2, C6), 59.4 (CHPh), 56.3 (OCH3),
50.5 (C3, C4), 34.7 (C5), 32.8, 29.3, 27.1, 27.0, (CH3), 20.9 (CH3)
ppm. ES-MS: m/z (%) = 335.2 (100) [M + 2H]+, 334.2 (98) [M + H]
·. C19H29N2O3·HCl (369.9): calcd. C 61.69, H 8.17, N 7.57; found C
61.75, H 8.22, N 7.41. The EtOAc filtrate was evaporated to give
(1ЈR,3R,4R)-3·HCl [127 mg, 21% from (R)-2] as an orange foam.
[α]25 = +41 (589), +41 (578), +41 (546), +105 (436) (c = 0.25,
MeOH). 1H NMR (300 MHz, D2O): δ = 7.54–7.60 (m, 5 H, ArH),
4.14 (m, 1 H, CH), 3.92 (s, 3 H, OCH3), 3.58 (m, 1 H, H4), 3.40
(d, J = 4.0 Hz, 1 H, H3), 2.15, 1.82 (2 m, 2 H, H5), 1.73 (d, J =
6.9 Hz, 3 H, CHCH3), 1.45, 1.44, 1.31, 1.26 (4 s, 12 H, 4 CH3)
ppm. 13C NMR (77 MHz, D2O): δ = 173.1 (C=O), 137.4, 133.1,
132.7, 130.6 (ArC), 60.6, 59.9 (C2, C6), 59.7 (CHPh), 56.6 (OCH3),
50.8, 49.6 (C3, C4), 36.0 (C5), 32.8, 29.4, 27.1, 26.9, (CH3), 21.9
(CH3) ppm. ES-MS: m/z (%) = 335.2 (100) [M + 2H]+, 334.2 (61)
[M + H]+. C19H29N2O3·HCl (369.9): calcd. C 61.69, H 8.17, N
7.57; found C 61.11, H 8.41, N 7.05.
showed the ratio of (1ЈS,3R,4R)-3·HCl/(1ЈS,3S,4S)-3·HCl to be 1:7.
(3R,4S)-4-[(1ЈS)-Phenylethyl]amino-3-methoxycarbonyl-2,2,6,6-
tetramethyl-piperidine-1-oxyl [(1ЈS,3R,4S)-3·HCl]: Method (i): So-
dium (55 mg, 2.4 mmol) was added slowly in portions to MeOH
(3 mL), and a solution of (1ЈS,3S,4S)-3 (125 mg, 0.37 mmol) dis-
solved in MeOH (1 mL) and added. The mixture was heated at
reflux for 24 h. The resulting solution was cooled to 0 °C, and the
pH of the mixture was adjusted to 6 by the addition of 1  HCl.
The mixture was concentrated under reduced pressure, diluted with
CH2Cl2, and washed with a saturated NaHCO3 solution. The or-
ganic phase was dried with MgSO4, filtered and concentrated. Col-
umn chromatography (cyclohexane/EtOAc, 2:1) of the residue gave
(1ЈS,3R,4S)-3 (51 mg, 42 %) and (1ЈS,3S,4S)-3 (70 mg, 57 %).
Method (ii): Isobutyric acid (3.5 mL, 30 mmol) was cooled to 0 °C
and NaBH4 (288 mg, 7.6 mmol) was added slowly in portions over
40 min. The mixture was then stirred at room temp. for 30 min.
Enamine (S)-2 (497 mg, 1.5 mmol) was dissolved in toluene (4 mL)
and added dropwise to the mixture. Further portions of NaBH4
(57 mg, 1.5 mmol) were added to the mixture after 2, 4 and 12 h.
After 20 h, the mixture was diluted with CH2Cl2 (40 mL) and
washed with a saturated NaHCO3 solution (2ϫ30 mL) and with
brine (30 mL). The organic phase was dried with MgSO4, filtered
and concentrated. Column chromatography (cyclohexane/EtOAc,
2:1) of the residue successively gave enamine (S)-2 (67 mg, 13%),
(1ЈS,3R,4S)-3 (151 mg, 30 %) and (1ЈS,3S,4S)-3 (149 mg, 29%).
trans Diastereomer (1ЈS,3R,4S)-3 was converted into its HCl salt
by the addition of a solution of HCl in EtOAc for characterisation.
(3R,4R)-4-[(1ЈS)-Phenylethyl]amino-3-methoxycarbonyl-2,2,6,6-
tetramethyl-piperidine-1-oxyl Hydrochloride [(1ЈS,3R,4R)-3·HCl]
and (3S,4S)-4-[(1ЈS)-Phenylethyl]amino-3-methoxycarbonyl-2,2,6,6-
tetramethyl-piperidine-1-oxyl Hydrochloride [(1ЈS,3S,4S)-3·HCl]:
Method (i): Enamine (S)-2 (1533 mg, 4.63 mmol) was dissolved in
absolute EtOH (30 mL) and glacial acetic acid (0.53 mL,
9.26 mmol) was added. The mixture was placed under an Ar atmo-
sphere and sodium cyanoborohydride (872 mg, 13.89 mmol) was
added. The mixture was heated at 70 °C for 2 h. The mixture was
cooled and then concentrated. The residue was taken up in CH2Cl2
and washed with saturated aqueous NaHCO3. The aqueous phase
was extracted with CH2Cl2. The combined CH2Cl2 phases were
washed with brine, then dried with MgSO4, filtered and concen-
trated. The residue was purified by chromatography (cyclohexane/
EtOAc, 2:1) to give 3 (1078 mg, 70%) as an orange oil. The residue
was dissolved in EtOAc (15 mL) and cooled to 0 °C. A solution of
HCl in EtOAc (approx. 3.4 , 0.95 mL) was added. The mixture
was kept at 0 °C for 30 min. The resulting precipitate was filtered
off. The collected solid was recrystallised from MeCN to give
(1ЈS,3R,4R)-3·HCl [157 mg, 26% from (S)-2] as light orange crys-
1
[α]25 = –39 (589), –44 (578), –52 (546) (c 0.25, MeOH). H NMR
(300 MHz, D2O): δ = 7.42 (m, 5 H, ArH), 4.52 (q, J = 11.7 Hz, 1
H, PhCHCH3), 3.78 (m, 1 H, H4), 3.72 (s, 3 H, OCH3), 2.97 (d,
J3,4 = 11.8 Hz, 1 H, H3), 1.70, 1.40 (2 m, 2 H, H5), 1.58 (d, J =
6.7 Hz, 3 H, PhCHCH3), 1.46, 1.25, 1.16, 1.04 (4 s, 12 H, 4 CH3)
ppm. 13C NMR (77 MHz, D2O): δ = 173.1 (C=O), 151.9, 137.9,
133.1, 132.5, 130.9 (ArC), 62.7, 61.2 (C2, C6), 58.8 (CHPh), 56.4
(OCH3), 55.1 (C4), 53.3 (C3), 39.8 (C5), 31.5, 31.3, 25.6, 24.5,
(CH3), 21.0 (PhCH3) ppm. ES-MS: m/z (%) = 335.2 (100) [M +
2H]+, 334.2 (61) [M + H]+. C19H29N2O3·HCl (369.9): calcd. C
61.69, H 8.17, N 7.57; found C 61.55, H 8.29, N 6.95.
(3S,4R)-4-[(1ЈR)-Phenylethyl]amino-3-methoxycarbonyl-2,2,6,6-
tals. M.p. 209–211 °C (dec.). [α]25 = –29 (589), –36 (578), –52 (546), tetramethyl-piperidine-1-oxyl Hydrochloride [(1ЈR,3S,4R)-3·HCl]:
1
+40 (436) (c = 0.26, MeOH). H and 13C NMR (300 MHz, D2O):
identical to (1ЈR,3S,4S)-3. C19H29N2O3·HCl (369.9): calcd. C
61.69, H 8.17, N 7.57; found C 61.49, H 8.34, N 7.41. Recrystalli-
sation from MeOH/MeCN (1:10) at 0 °C gave large orange crystals
suitable for X-ray diffraction analysis. The EtOAc filtrate was evap-
Isobutyric acid (3.7 mL, 40 mmol) was cooled to 0 °C and NaBH4
(380 mg, 10 mmol) was added slowly in portions over 40 min. The
mixture was then stirred at room temp. for 30 min. Enamine (R)-
2 (497 mg, 1.5 mmol) was dissolved in toluene (4 mL) and added
dropwise to the mixture. Further portions of NaBH4 (76 mg,
orated to give (1ЈS,3S,4S)-3·HCl [533 mg, 31% from (S)-2] as an 2 mmol) were added to the mixture after 2 and 12 h. After 20 h,
orange oil. [α]25 = –45 (589), –45 (578), –47 (546), –130 (436), –248
(365) (c = 0.25, MeOH). 1H and 13C NMR (D2O): identical to
(1ЈR,3R,4R)-3. C19H29N2O3·HCl (369.9): calcd. C 61.69, H 8.17,
N 7.57; found C 61.11, H 8.41, N 7.05. Method (ii): A solution of
glacial acetic acid (4.3 mL, 75.5 mmol) in MeCN (3 mL) was co-
oled to 0 °C. Sodium borohydride (283 mg, 7.5 mmol) was added
slowly in portions over 40 min. A solution of the enamine (S)-2
(503 mg, 1.5 mmol) in MeCN (3 mL) was added dropwise. The
mixture was stirred at 0 °C for 3 h, then at room temp. for 4 h. The
mixture was diluted with CH2Cl2, and then washed twice with a
the mixture was diluted with CH2Cl2 (40 mL) and washed with a
saturated NaHCO3 solution (2ϫ30 mL) and with brine (30 mL).
The organic phase was dried with MgSO4, filtered and concen-
trated. Column chromatography (cyclohexane/EtOAc, 2:1) of the
residue successively gave enamine (R)-2 (193 mg, 29 %),
(1ЈR,3S,4R)-3 (92 mg, 14 %) and (1ЈR,3R,4R)-3 (186 mg, 28%).
trans Diastereomer (1ЈR,3S,4R)-3 was converted into its HCl salt
by the addition of a solution of HCl in EtOAc for characterisation.
Orange foam. [α]25 = +37 (589), +40 (578), +47 (546) (c = 0.26,
MeOH); 1H and 13C NMR (D2O): identical to (1ЈS,3R,4S)-3.
3140
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Eur. J. Org. Chem. 2007, 3133–3144

Enantiomerically Pure β-TOAC
FULL PAPER
C19H29N2O3·HCl (369.9): calcd. C 61.69, H 8.17, N 7.57; found C
62.01, H 8.34, N 7.12.
salt]: δ = 4.19 (m, 1 H, H4), 3.84 (s, 3 H, OCH3), 3.04 (d, J3,4
=
11.4 Hz, 1 H, H3), 2.31, 1.93 (2 m, 2 H, H5), 1.61, 1.53, 1.48 (3 s,
12 H, 4 CH3) ppm. 13C NMR (77 MHz, D2O) [HCl salt]: δ = 170.1
(C=O), 58.7, 56.8 (C2, C6), 54.3 (C4), 52.9 (OCH3), 44.5 (C3), 37.9
(C5), 29.5, 28.8, 24.1, 22.2 (CH3) ppm. ES-MS: m/z (%) = 252.5
(41) [M + Na]+, 230.6 (100) [M + H]+. C11H21N2O3·2H2O
(265.326): calcd. C 49.79, H 9.49, N 10.56; found C 49.64, H 9.67,
N 10.00.
General Method for the Reduction of (S)-2 by NaBH4 in the Pres-
ence of Organic Acids: A solution of the organic acid in toluene
(and additionally MeCN in the case of phenylacetic acid) was co-
oled to 0 °C and NaBH4 was added slowly in portions over 40 min.
Enamine (S)-2 was dissolved in toluene and added dropwise to the
mixture. The temperature of the mixture was allowed to rise to
room temp., and then stirred for 3 h. The mixture was diluted with
CH2Cl2 and washed with a saturated NaHCO3 solution and with
brine. The organic phase was dried with MgSO4, filtered and con-
centrated. The residue was purified by column chromatography (cy-
clohexane/EtOAc, 2:1) to successively give enamine (S)-2,
(1ЈS,3R,4S)-3 and (1ЈS,3S,4S)-3.
(3R,4S)-4-Amino-3-methoxycarbonyl-2,2,6,6-tetramethylpiperidine-
1-oxyl [(3R,4S)-4]: The hydrochloride salt of (1ЈS,3R,4S)-3
(1025 mg, 2.77 mmol) was dissolved in 95% EtOH (100 mL). The
solution was placed under an Ar atmosphere and 10 % Pd/C
(660 mg) was added. The mixture was then stirred under H2 for
20 min. The mixture was filtered and the filtrate concentrated. The
residue was dissolved in MeOH (100 mL) and copper (II) acetate
(10 mg) was added. The mixture was stirred rapidly open to air for
7 d. The mixture was concentrated, and the residue was purified
by chromatography (CH2Cl2/MeOH, 9:1) to give amine (3R,4S)-4
(258 mg, 41%) as an orange oil. [α]25 = –21 (589), –22 (578), –24
(3S,4S)-4-Amino-3-methoxycarbonyl-2,2,6,6-tetramethylpiperidine-
1-oxyl [(3S,4S)-4]: The hydrochloride salt of (1ЈR,3S,4S)-3 (400 mg,
1.08 mmol) was dissolved in 95% EtOH (40 mL). The solution was
placed under an Ar atmosphere and 10 % Pd/C (400 mg) was
added. The mixture was stirred under H2 for 30 min. The mixture
was filtered and the filtrate concentrated. The residue was dissolved
in MeOH (30 mL) and copper (II) acetate (20 mg) was added. The
mixture was stirred rapidly open to air for 24 h. The mixture was
concentrated, and the residue was purified by chromatography
(CH2Cl2/MeOH, 9:1) to give amine (3S,4S)-4 (131 mg, 53%) as an
amorphous orange solid. M.p. 94–96 °C. [α]25 = +10 (589), +11
(578), +23 (546), –56 (436), –197 (365) (c = 0.105, MeOH). 1H
NMR (300 MHz, D2O) [HCl salt]: δ = 4.28 (m, 1 H, H4), 3.87 (s,
3 H, OCH3), 3.28 (d, J3,4 = 4.8 Hz, 1 H, H3), 2.15, 2.07 (2 m, 2 H,
H5), 1.62, 1.57, 1.53, 1.48 (4 s, 12 H, 4 CH3) ppm. 13C NMR
(77 MHz, D2O) [HCl salt]: δ = 173.5 (C=O), 60.6, 60.0 (C2, C6),
56.3 (OCH3), 51.5 (C4), 46.1 (C3), 36.7 (C5), 32.9, 29.5, 27.5, 27.2,
(CH3) ppm. ES-MS: m/z (%) = 231.1 (70) [M + 2H]+, 230.1 (100)
[M + H]+, 215.1 (100), 213.1 (34). C11H21N2O3 (229.3): calcd. C
57.61, H 9.23, N 12.22; found C 57.85, H 9.28, N 12.08.
1
(546) +99 (436) (c = 0.1, MeOH). H and 13C NMR (D2O): iden-
tical to (3S,4R)-4. ES-MS: m/z (%) = 230.5 (100) [M + H]+. FAB-
HRMS: calcd. for C11H21N2O3 + 2H 231.1709; found 231.1705.
(3R,4S)-4-(9H-Fluoren-9-ylmethoxycarbonylamino)-2,2,6,6-tetra-
methylpiperidine-1-oxyl-3-carboxylic Acid [(3R,4S)-5]: Amino ester
(3R, 4S)-4 (320 mg, 1.39 mmol) was dissolved in MeOH (15 mL)
and water (6 mL). A 1  solution of NaOH (6 mL) was added, and
the mixture was heated at 75 °C for 2 h. The mixture was cooled
in an ice bath and diluted with water (10 mL). The pH was adjusted
to approximately 6 by the addition of a 2  HCl solution. The
solution was cooled to 0 °C and acetone (40 mL), NaHCO3
(1170 mg, 13.9 mmol) and Fmoc-OSu (612 mg, 1.82 mmol) were
added. The mixture was stirred at room temp. for 18 h. The acetone
was removed under reduced pressure. The solution was diluted with
water and washed twice with diethyl ether. The aqueous phase was
acidified by the addition of a 0.5  HCl solution and extracted
twice with CH2Cl2. The combined organic extracts were washed
with water, dried with MgSO4, filtered and concentrated. The resi-
due was purified by chromatography (CH2Cl2/MeOH, 9:1) to give
acid (3R,4S)-5 (246 mg, 40%) as a light orange solid. [α]25 = –2
(589), –3 (578), –2 (546) (c = 0.2, MeOH). FAB-HRMS: calcd. for
C25H29N2O5 + 2H 439.2233; found 439.2231.
(3R,4R)-4-Amino-3-methoxycarbonyl-2,2,6,6-tetramethylpiperidine-
1-oxyl [(3R,4R)-4]: The hydrochloride salt of (1ЈR,3R,4R)-3
(452 mg, 1.08 mmol) was dissolved in 95 % EtOH (45 mL). The
solution was placed under an Ar atmosphere and 10 % Pd/C
(290 mg) was added. The mixture was stirred under H2 for 30 min.
The mixture was filtered and the filtrate concentrated. The residue
was dissolved in MeOH (20 mL) and copper (II) acetate (10 mg)
was added. The mixture was stirred rapidly open to air for 7 d. The
mixture was concentrated, and the residue was purified by
chromatography (CH2Cl2/MeOH, 9:1) to give amine (3R,4R)-4
(131 mg, 62 %) as an amorphous orange solid. M.p. 88–91 °C.
[α]25 = –10 (589), –11 (578), –22 (546), +38 (436), +140 (365) (c =
0.104, MeOH). 1H and 13C NMR (D2O): identical to (3S,4S)-4.
C11H21N2O3 (229.3): calcd. C 57.61, H 9.23, N 12.22; found C
57.39, H 9.23, N 11.64.
Boc-(1S,2S)-ACHC-(3R,4S)-β-TOAC-OMe (6): Boc-(1S,2S)-
ACHC-OH (294 mg, 1.21 mmol) and (3R,4S)-4 (213 mg,
0.93 mmol) were dissolved in CH2Cl2 (7 mL). HOAt (253 mg,
1.86 mmol) was added, and the mixture was cooled in an ice bath.
DIEA (0.16 mL, 0.93 mmol) was added, followed by EDC (268 mg,
1.40 mmol). The mixture was stirred at room temperature for 15 h.
The mixture was diluted with CH2Cl2 and washed successively with
0.5  HCl, a saturated NaHCO3 solution and water. The organic
phase was dried with MgSO4, filtered and concentrated. The resi-
due obtained was purified by preparative thin-layer chromatog-
raphy (CH2Cl2/MeOH, 9:1) to give dipeptide 6 (379 mg, 90%).
[α]25 = –4 (589), –4 (578), –4 (546), –+53 (436), +53 (365) (c = 0.1,
MeOH). ES-MS: m/z (%) = 477.7 (100) [M + H + Na]+.
C23H40N3O6 (454.574): calcd. C 60.77, H 8.87, N 9.24; found C
60.81, H 8.79, N 9.05.
(3S,4R)-4-Amino-3-methoxycarbonyl-2,2,6,6-tetramethylpiperidine-
1-oxyl [(3S,4R)-4]: The hydrochloride salt of (1ЈR,3S,4R)-3
(1035 mg, 2.80 mmol) was dissolved in 95% EtOH (100 mL). The
solution was placed under an Ar atmosphere and 10 % Pd/C
(489 mg) was added. The mixture was stirred under H2 for 30 min.
The mixture was filtered and the filtrate concentrated. The residue
was dissolved in MeOH (40 mL) and copper (II) acetate (5 mg) was
added. The mixture was stirred rapidly open to air for 5 d. The
Fmoc-(3R,4S)-β-TOAC-(1S,2S)-ACHC-OMe (7): Boc-(1S,2S)-
ACHC-OH (100 mg, 0.41 mmol) was dissolved in CH2Cl2 (5 mL)
mixture was concentrated, and the residue was purified by and cooled in an ice bath. Trifluoroacetic acid (1 mL) was added,
chromatography (CH2Cl2/MeOH, 95:5) to give amine (3S,4R)-4
and the mixture was stirred at 0 °C for 15 min and then at room
temperature for 1 h. The mixture was concentrated under reduced
(365 mg, 57%) as an orange oil. [α]25 = +23 (589), +24 (578), +32
(546), –89 (436) (c = 0.1, MeOH). 1H NMR (300 MHz, D2O) [HCl pressure. The resulting residue was dissolved in MeOH (3 mL) and
Eur. J. Org. Chem. 2007, 3133–3144
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K. Wright, C. Toniolo et al.
FULL PAPER
cooled in an ice bath. Thionyl chloride (0.7 mL) was added drop-
wise. The mixture was stirred from 0 °C to room temperature for
18 h. The mixture was concentrated under reduced pressure. Di-
ethyl ether was added to the residue, and the mixture was again
concentrated under reduced pressure. This procedure was repeated
three times to give the crude amino ester hydrochloride, which was
used as such without further purification. Thus, the obtained hy-
drochloride HCl·H-(1S,2S)-ACHC-OMe (72 mg, 0.37 mmol) and
aqueous HCl solution. The mixture was concentrated under re-
duced pressure to remove MeOH. The remaining solution was di-
luted with water, and extracted with CH2Cl2. The combined or-
ganic extracts were washed with water, then dried with MgSO4,
filtered and concentrated. The crude Boc-(1S,2S)-ACHC-(3R,4S)-
β-TOAC-(1S,2S)-ACHC-OH (101 mg, 0.179 mmol, 81%) obtained
was used as such in the next step without further purification. This
residue and the hydrochloride HCl·H-(1S,2S)-ACHC-OMe (69 mg,
(3R,4S)-5 (123 mg, 0.28 mmol) were dissolved in CH2Cl2 (5 mL) 0.359 mmol) were dissolved in CH2Cl2 (5 mL), and the mixture was
and THF (5 mL). HOAt (77 mg, 0.56 mmol) was added, and the
mixture was cooled in an ice bath. DIEA (0.14 mL, 0.84 mmol)
was added, followed by EDC (83 mg, 0.42 mmol). The mixture was
stirred at room temperature for 16 h. The mixture was diluted with
CH2Cl2 and washed successively with 0.5  HCl, a saturated
NaHCO3 solution and water. The organic phase was dried with
MgSO4, filtered and concentrated. The residue obtained was puri-
fied by preparative thin layer chromatography (CH2Cl2/MeOH,
92:8) to give dipeptide 7 (63 mg, 40%). [α]25 = –2 (589), –3 (578),
–3 (546), –+31 (436), +13 (365) (c = 0.09, MeOH). ES-MS: m/z
cooled in an ice bath. HOAt (49 mg, 0.359 mmol) and DIEA
(0.12 mL, 0.72 mmol) were added, followed by EDC (52 mg,
0.269 mmol). The mixture was stirred at room temperature for
18 h. The mixture was diluted with CH2Cl2 and washed success-
ively with 0.5  HCl, water and a saturated NaHCO3 solution. The
organic phase was dried with MgSO4, filtered and concentrated.
The residue obtained was purified by preparative thin layer
chromatography (CH2Cl2/MeOH, 9:1) to give tetrapeptide 10
(80 mg, 63%). [α]25 = –2 (589), –2 (578), 0 (546), –26 (436), –108
(365) (c = 0.1, MeOH). ES-MS: m/z (%) = 727.4 (100) [M + Na]+,
705.5 (42) [M + H]+. C37H62N5O8·2H2O (740.938): calcd. C 59.97,
H 8.97, N 9.45; found C 60.15, H 8.87, N 9.09.
( % ) = 5 9 9 . 3 ( 1 0 0 ) [ M + N a ] + , 5 7 7 . 9 ( 7 ) [ M + 2 H ] +
.
C33H42N3O6·0.5H2O (585.698): calcd. C 67.67, H 7.40, N 7.17;
found C 67.35, H 7.65, N 6.69.
Boc-(1S,2S)-ACHC-(3R,4S)-β-TOAC-(1S,2S)-ACHC-(1S,2S)-
ACHC-(3R,4S)-β-TOAC-(1S,2S)-ACHC OMe (11): Tetrapeptide
10 (70 mg, 0.221 mmol) was dissolved in MeOH (3 mL) and a 1 
aqueous NaOH solution (1 mL) was added. The mixture was
heated at 75 °C for 15 h. The mixture was cooled, and the pH was
adjusted to 5–6 by the addition of a 0.5  aqueous HCl solution.
The mixture was concentrated under reduced pressure to remove
MeOH. The remaining solution was diluted with water, and ex-
tracted with CH2Cl2. The combined organic extracts were washed
with water, then dried with MgSO4, filtered and concentrated. The
crude Boc-(1S,2S)-ACHC-(3R,4S)-β-TOAC-(1S,2S)-ACHC-
(1S,2S)-ACHC-OH (67 mg, 0.097 mmol, 97%) obtained was used
as such in the next step without further purification. This residue
and dipeptide 8 (37 mg, 0.105 mmol) were dissolved in CH2Cl2
(5 mL), and the mixture was cooled in an ice bath. HOAt (26 mg,
0.194 mmol) and DIEA (0.03 mL, 0.194 mmol) were added, fol-
lowed by EDC (28 mg, 0.146 mmol). The mixture was stirred at
room temperature for 18 h. The mixture was diluted with CH2Cl2
and washed successively with 0.5  HCl, water and a saturated
NaHCO3 solution. The organic phase was dried with MgSO4, fil-
tered and concentrated. The residue obtained was purified by pre-
parative thin layer chromatography (CH2Cl2/MeOH, 9:1) to give
hexapeptide 11 (64 mg, 65 %). [α]25 = –37 (589), –41 (578), –46
(546), –38 (436), –137 (365) (c 0.09, MeOH); ES-MS: m/z (%) =
1051.2 (100) [M + Na + 2H]+, 1029.3 (27) [M + 3H]+. FAB-
HRMS: calcd. for C54H90N8O11 + Na + 2H 1051.6783; found
1051.6779; calcd. for C54H90N8O11 + Na + H 1050.6705; found
1050.6709.
H-(3R,4S)-β-TOAC-(1S,2S)-ACHC-OMe (8): Dipeptide 7 (54 mg,
0.094 mmol) was dissolved in MeCN (9 mL) and diethylamine
(1 mL) was added. The mixture was stirred at room temperature
for 2 h. The mixture was concentrated, and the residue obtained
was purified by purified by preparative thin layer chromatography
(CH2Cl2/MeOH, 90:10) to give dipeptide 8 (31 mg, 91%). [α]25
=
+12 (589), +12 (578), +17 (546), –+45 (436), +27 (365) (c = 0.08,
MeOH). ES-MS: m/z (%) = 377.2 (11) [M + Na]+, 355.3 (100) [M
+ H]+. ES-HRMS: calcd. for C18H32N3O4 + H 355.2471; found
355.2452.
Boc-(1S,2S)-ACHC-(3R,4S)-β-TOAC-(1S,2S)-ACHC-OMe (9): Di-
peptide 6 (338 mg, 0.744 mmol) was dissolved in MeOH (15 mL)
and a 1  aqueous NaOH solution (6 mL) was added. The mixture
was heated at 75 °C for 24 h. The mixture was cooled, and the pH
was adjusted to 5–6 by the addition of a 0.5  aqueous HCl solu-
tion. The mixture was concentrated under reduced pressure to re-
move MeOH. The remaining solution was diluted with water, and
extracted with CH2Cl2. The combined organic extracts were
washed with water, then dried with MgSO4, filtered and concen-
trated. The crude Boc-(1S,2S)-ACHC-(3R,4S)-β-TOAC-OH
(208 mg, 0.472 mmol, 63%) obtained was used as such in the next
step without further purification. This residue and the hydrochlo-
ride HCl·H-(1S,2S)-ACHC-OMe (119 mg, 0.614 mmol) were dis-
solved in CH2Cl2 (5 mL), and the mixture was cooled in an ice
bath. HOAt (128 mg, 0.944 mmol) and DIEA (0.24 mL,
1.42 mmol) were added, followed by EDC (136 mg, 0.71 mmol).
The mixture was stirred at room temperature for 48 h. The mixture
was diluted with CH2Cl2 and washed successively with 0.5  HCl,
water and a saturated NaHCO3 solution. The organic phase was
dried with MgSO4, filtered and concentrated. The residue obtained
was purified by preparative thin layer chromatography (CH2Cl2/
MeOH, 9:1) to give tripeptide 9 (133 mg, 49%). [α]25 = –50 (589),
–51 (578), –48 (546), –24 (436), –52 (365) (c = 0.1, MeOH). ES-
MS: m/z (%) = 601.9 (100) [M + Na]+. ES-HRMS: calcd. for
C30H51N4O7 + H 580.3836; found 580.3867.
FTIR Absorption: The FTIR absorption spectra were recorded with
a Perkin–Elmer model 1720X spectrophotometer, nitrogen-flushed,
equipped with a sample shuttle device, at 2 cm–1 nominal resolu-
tion, averaging 100 scans. Cells with path lengths of 0.1, 1.0, and
10 mm (with CaF2 windows) were used. Spectrograde deuteriochlo-
roform (99.8% D) was purchased from Aldrich. Solvent (baseline)
spectra were recorded under the same conditions.
Circular Dichroism: The CD spectra were obtained with a Jasco J-
710 dichrograph. Cylindrical fused quartz cells of 10, 1.0, 0.2 and
0.1-mm path length (Hellma) were used. The values are expressed
in terms of [θ]T, the total molar ellipticity (degϫcm2 ϫdmol–1).
Spectrograde MeOH (Aldrich) was used as solvent.
Boc-(1S,2S)-ACHC-(3R,4S)-β-TOAC-(1S,2S)-ACHC-(1S,2S)-
ACHC-OMe [10]: Tripeptide 9 (128 mg, 0.221 mmol) was dissolved
in MeOH (5 mL) and a 1  aqueous NaOH solution (1 mL) was
added. The mixture was heated at 75 °C for 15 h. The mixture was
cooled, and the pH was adjusted to 5–6 by the addition of a 0.5 
Electron Paramagnetic Resonance: The EPR spectra were recorded
with an X-band (9.5 GHz) Bruker ER2000 spectrometer. Tempera-
3142
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Enantiomerically Pure β-TOAC
FULL PAPER
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quartz tube of 1 mm inner diameter. The solution in the tube was
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K. Wright, C. Toniolo et al.
FULL PAPER
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Received: February 20, 2007
Published Online: May 22, 2007
3144
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Eur. J. Org. Chem. 2007, 3133–3144