4-Amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid (β-TOAC), the first spin-labelled, cyclic, chiral β-amino acid resolved in an enantiomerically pure state

Article abstract of DOI:10.1016/S0040-4039(03)00572-0

Amination of 3-carboxymethyl-1-oxyl-2,2,6,6-tetramethyl-4-piperidone with (R)-α-methylbenzylamine, NaBH₃CN reduction of the resulting enamine and removal of the chiral auxiliary from the separated diastereoisomers, led to enantiomerically pure

Full text of DOI:10.1016/S0040-4039(03)00572-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 3381–3384
4-Amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid
(b-TOAC), the first spin-labelled, cyclic, chiral b-amino acid
resolved in an enantiomerically pure state
Karen Wright,a,* Marco Crisma,b Claudio Toniolo,b Roland To¨ro¨k,c Antal Pe´ter,c
Michel Wakselmana and Jean-Paul Mazaleyrata
aSIRCOB, UMR CNRS 8086, Baˆt. Lavoisier, University of Versailles, F-78000 Versailles, France
bInstitute of Biomolecular Chemistry, CNR, Department of Organic Chemistry, University of Padova, I-35131 Padova, Italy
cDepartment of Inorganic and Analytical Chemistry, University of Szeged, PO Box 440, H-6701 Szeged, Hungary
Received 10 February 2003; revised 21 February 2003; accepted 27 February 2003
Abstract—Amination of 3-carboxymethyl-1-oxyl-2,2,6,6-tetramethyl-4-piperidone with (R)-a-methylbenzylamine, NaBH3CN
reduction of the resulting enamine and removal of the chiral auxiliary from the separated diastereoisomers, led to enantiomerically
pure (3S,4S) and (3R,4R) methyl 4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylates. © 2003 Elsevier Science Ltd. All
rights reserved.
Stable nitroxide free radicals are of continuing interest
for use as spin labels in the study of conformation and
structural mobility of biological systems,1a–d as spin
traps of other radical species1e–h and as oxidizing
agents.1i–k Moreover, optically active nitroxides were
developed as enantioselective oxidizing agents and for
stereoselective coupling with prochiral radicals.2 Fol-
lowing on from previous work in our groups with the
nitroxide-based, achiral, Ca-tetrasubstituted a-amino
and biological studies.5 The tetrasubstituted a-carbon
of TOAC is responsible for its ability to induce b-turn
or 310/a-helical structures in peptides, but also for the
reduced reactivity of its amino group, which may be
problematic if the residue is to be placed at an internal
position of a peptide.6 The b-amino acid POAC (3-
amino-1-oxyl-2,2,5,5-tetramethylpyrrolidine-4-carboxy-
lic acid), first described by Rassat and Rey,4a was
recently incorporated by solid phase synthesis into an
angiotensin-II analogue.7 It was noted that coupling of
the next amino acid after the POAC residue proceeded
smoothly, whereas the equivalent coupling after the
TOAC residue required a large excess of reagent and
repeated coupling steps.7,8 However, to our knowledge,
the POAC residue was synthesized as a mixture of the
two trans enantiomers, and used as such.9 It appeared
acid
TOAC
(4-amino-1-oxyl-2,2,6,6-tetramethyl-
piperidine-4-carboxylic acid),3 we were interested by the
prospect of creating a new chiral, spin-labelled amino
acid.
The TOAC residue4 (Fig. 1) was used to label peptides
at N-terminal and internal positions for conformational
Figure 1. Chemical structures of the spin-labelled amino acids TOAC, POAC and b-TOAC.
Keywords: chiral nitroxides; modified b-amino acids; spin-labelled amino acids.
* Corresponding author. Fax: +(33)-01-3925-4452; e-mail: wright@chimie.uvsq.fr
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00572-0

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K. Wright et al. / Tetrahedron Letters 44 (2003) 3381–3384
to us to be desirable to design a cyclic derivative that
could be obtained in enantiopure form, while keeping
the b-amino acid structure that allows easy peptide
coupling. The compound which we named b-TOAC
(Fig. 1) was the chosen target.
The b-amino acids have been the subject of much
synthetic effort in recent years, particularly after it was
demonstrated that their oligomers may fold into stable
helical conformations.10 Gellman and co-workers suc-
cessfully developed asymmetric routes to trans-3-
aminopyrrolidine-4-carboxylic acid and trans-2-amino-
cyclopentanecarboxylic acid.11 The key steps in their
syntheses were the reductive amination of a b-ketoester
with either (R)- or (S)-a-methylbenzylamine in the
presence of NaBH3CN, and subsequent selective crys-
tallisation of the HCl salts of the obtained b-amino
esters, to provide either trans enantiomer. A similar
method for the preparation of cis-2-aminocyclohexane-
carboxylic acid had previously been described by Xu et
al.12 who performed the reduction step using NaBH4
and an organic acid, and isolated the major
diastereomer formed as its HBr salt. This pathway was
used recently by Gellman and co-workers13 who eventu-
ally obtained trans-2-aminocyclohexanecarboxylic acid
by epimerization of the cis isomer. We decided to
attempt such a procedure applied to 3-carboxymethyl-
1-oxyl-2,2,6,6-tetramethyl-4-piperidone 2 (Fig. 2) as a
route to enantiopure b-TOAC.
Commercially available 2,2,6,6-tetramethyl-4-piperi-
done was oxidized by a described procedure to its
1-oxyl derivative 1 (Fig. 2).14 The efficiency of carboxy-
lation of this compound with carbon dioxide in the
presence of potassium phenoxide following the pub-
lished method15 proved to be variable in our hands; the
yield of esterified product 2 obtained after reaction with
diazomethane (lit. 35%) varied between 15 and 50%
over different runs performed under apparently the
same conditions. Amination of 2 with (R)-a-methylbenz-
ylamine in the presence of acetic acid proceeded
smoothly to provide the desired products (R)-316 in
61% yield. The reaction was also performed with (S)-a-
methylbenzylamine, to give (S)-316 in 66% yield (not
shown). The enamine structure of 3 was determined
Figure 2. Synthetic path for amination of 3-carboxymethyl-
2,2,6,6-tetramethyl-4-piperidone
amine, followed by reduction of the resulting enamine (R)-3.
2
by (R)-a-methylbenzyl-
(i) KOPh; CO2; DMF; rt; 2 h, then CH2N2; Et2O. (ii)
1
from its H NMR spectrum.17
,
(R)-a-methylbenzylamine; AcOH; EtOH; 4A MS; rt; 48 h.
(iii) NaBH3CN; AcOH; EtOH; 75°C; 2 h. (iv) HCl/EtOAc;
0°C; filtration and recrystallisation from MeCN.
The isolated enamine (R)-3 was reduced in the presence
of NaBH3CN and acetic acid. These conditions are
known not to affect the nitroxide group.19 The mixture
of reduced products 4 was purified by column chro-
matography. The desired HCl salts were prepared by
adding a solution of HCl in EtOAc to a cold solution
of 4 in EtOAc. The precipitate formed was collected
and recrystallised from MeCN to give (1%R,3S,4S)-416
as a sole diastereomer as judged from its 1H NMR
spectrum. The other diastereomer (1%R,3R,4R)-416 was
obtained from the EtOAc mother liquor. The same
reaction sequence was followed starting from the enam-
ine (S)-3, to give (1%S,3R,4R)-416 as crystals and
(1%S,3S,4S)-416 from the EtOAc mother liquor (not
shown).
The derivative (1%S,3R,4R)-4 was obtained after recrys-
tallisation from MeCN as large orange crystals suitable
for X-ray diffraction analysis (Fig. 3),20 allowing the
assignment of the absolute configuration at C3 and C4.
The crystal structure confirmed the cis configuration of
the b-amino ester, which had been suggested by the
NMR spectra. This result is in agreement with the
findings of Cimarelli and Palmieri,21 who obtained cis
products from NaHB(OAc)3 reduction of cyclic b-
enamino esters. It is also consistent with the already
mentioned results of Xu et al.12 and Gellman and
co-workers13 in the case of six-membered rings. How-

K. Wright et al. / Tetrahedron Letters 44 (2003) 3381–3384
3383
Figure 3. X-Ray diffraction structure of (1%S,3R,4R)-4·HCl.20
ever, it is at variance with that of Gellman and co-
workers,11 who had obtained the trans reduction prod-
ucts from a pyrrolidine substrate when NaBH3CN was
used as the reducing agent.
Removal of the chiral auxiliary from 4 by hydrogena-
tion over Pd/C proved to be a little delicate. While it is
known that the nitroxide group may be completely
reduced under these conditions to the secondary
amine,22 it is also possible to obtain an intermediate
N-hydroxy compound, from which the nitroxide func-
tion can be regenerated.23,24 Our initial hydrogenation
attempts carried out at a pressure of 50 psi for several
hours resulted in complex mixtures of products. We
found that hydrogenation at atmospheric pressure and
for only a short period (<30 min) was enough to allow
efficient cleavage of the a-methylbenzyl group, while
avoiding over-reduction of the nitroxide group to the
amine and subsequent decomposition. The nitroxide
function was regenerated by stirring the crude hydro-
genated product open to the air in the presence of
Cu(OAc)2 for 7 days. In this way compounds (3S,4S)-
516 and (3R,4R)-516 were obtained in 54 and 62% yield,
respectively (Fig. 4). A derivative suitable for peptide
synthesis was prepared from (3S,4S)-5 by saponifica-
tion of the methyl ester and Fmoc (9-fluorenylmethyl-
oxycarbonyl) protection of the amino group, to provide
the building block (3S,4S)-616 in 45% yield. The other
enantiomer (3R,4R)-616 was prepared in the same way
from (3R,4R)-5 in 83% yield.
Figure 4. Removal of the chiral auxiliary from diastereoiso-
mers 4, followed by C-deprotection/N-protection of the
resulting b-amino ester enantiomers 5. (i) Pd/C 10%; 95%
EtOH; rt; 30 min. (ii) Cu(OAc)2; MeOH; air; rt; 7 days. (iii)
NaOH (aq); MeOH; reflux; 5 h. (iv) Fmoc-succinimidyl car-
bonate; NaHCO3; acetone/water 2:1; rt; 18 h.
route to the trans b-TOAC enantiomers. We believe
that these novel spin-labelled, rigid and chiral b-amino
acids will find applications in peptidomimetic confor-
mational and biological studies.
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3384
K. Wright et al. / Tetrahedron Letters 44 (2003) 3381–3384
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25
546
are as
follows: (R)-3: −441 (c 0.25;MeOH). (S)-3: +456 (c 0.26;
MeOH). (1%R,3S,4S)-4: +48 (c 0.25; MeOH). (1%R,3R,4R)-
4: +41 (c 0.25; MeOH). (1%S,3R,4R)-4: −52 (c 0.26; MeOH).
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1
17. Well resolved H and 13C NMR spectra of the nitroxide
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contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge at
http://www.ccdc.cam.ac.uk/conts/retrieving.html [or from
the Cambridge Crystallographic Data Centre, 12, Union
Road, Cambridge CB2 1EZ, UK; fax: (internat.) +44-1223/
336-033; E-mail: deposit@ccdc.cam.ac.uk].
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25. Column Chiralcel OD-RH (Daicel, Tokyo, Japan), eluent
0.1 M KPF6 (aq.): MeCN 70:30, flow rate 0.5 mL/min,
detection 254 nm, column temperature 30°C.