Synthesis and spectroscopic characterization of enantiopure protected trans-4-amino-1-oxyl-2,2,6,6-tetramethyl piperidine-3-carboxylic acid (trans β-TOAC)

Article abstract of DOI:10.1016/j.tetlet.2005.06.049

Enantiomerically pure (3R,4S) and (3S,4R) protected 4-amino-1-oxyl-2,2,6,6- tetramethylpiperidine-3-carboxylic acids were synthesized by reduction of the enamines resulting from the condensation of 3-carboxymethyl-1-oxyl-2,2,6,6- tetramethyl-4-piperidone

Full text of DOI:10.1016/j.tetlet.2005.06.049

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 5573–5576
Synthesis and spectroscopic characterization of
enantiopure protected trans-4-amino-1-oxyl-2,2,6,6-tetramethyl
piperidine-3-carboxylic acid (trans b-TOAC)
Karen Wright,a,* Augustin de Castries,a Matthieu Sarciaux,a Fernando Formaggio,b
Claudio Toniolo,b Antonio Toffoletti,b Michel Wakselmana and Jean-Paul Mazaleyrata
a
ˆ
SIRCOB, UMR CNRS 8086, Bat. Lavoisier, University of Versailles, F-78000 Versailles, France
bDepartment of Chemistry, University of Padova, 35131 Padova, Italy
Received 12 May 2005; revised 6 June 2005; accepted 6 June 2005
Available online 5 July 2005
Abstract—Enantiomerically pure (3R,4S) and (3S,4R) protected 4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acids
were synthesized by reduction of the enamines resulting from the condensation of 3-carboxymethyl-1-oxyl-2,2,6,6-tetramethyl-4-pip-
eridone with (R) or (S)-a-methylbenzylamine. While NaBH3CN/CH3COOH reduction gave predominantly a mixture of the two
possible cis-diastereomers, the use of NaBH4/(CH3)2CHCOOH resulted in a mixture of only one trans- and one cis-diastereomer.
Removal of the chiral auxiliary from the separated diastereoisomers by hydrogenolysis and regeneration of the nitroxide radical
gave the desired b-amino esters. The ESR spectrum of the (3R,4S)-enantiomer is also reported.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Nitroxide free radicals have found applications in a vari-
ety of fields because of their significant stability, for
example, (i) as spin labels in the study of conformation
and structural mobility of biological systems,1 including
previous work from our groups with the nitroxide-
based, achiral, Ca-tetrasubstituted a-amino acid TOAC
(4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carbox-
ylic acid)2 (Fig. 1), (ii) as spin traps of other radical spe-
cies,3 and (iii) as oxidizing agents.4 Optically active
nitroxides have been employed as enantioselective oxi-
dizing agents and for stereoselective coupling with pro-
chiral radicals.5 We recently reported the synthesis of
both enantiomers of cis-b-TOAC (cis-4-amino-1-oxyl-
2,2,6,6-tetramethylpiperidine-3-carboxylic acid), a cyclic
b-amino acid bearing a nitroxide group.6 However, we
were drawn towards the synthesis of the trans-enantio-
mers of these compounds, given their similarity to the
b-amino acids trans-ACHC (2-aminocyclohexane-1-car-
boxylic acid),7 and trans-APiC (4-aminopiperidine-3-
carboxylic acid).8 Gellman and co-workers had demon-
strated that oligomers of trans-ACHC fold to preferen-
tially form a helical secondary structure, the 314-helix,7
O
N
O
O
N
H
N
N
COOH
COOH
COOH
COOH
H2N
TOAC
Figure 1. Chemical structures of the amino acids TOAC, b-TOAC, ACHC and APiC.
COOH
NH2
NH2
NH2
trans-(1S,2S)-ACHC
NH2
trans-(3S,4S)-APiC
β
cis-(3S,4S)- -TOAC
trans-(3R,4S)- -TOAC
β
Keywords: Chiral nitroxides; Modified b-amino acids; Spin-labelled amino acids; trans-b-TOAC.
*
Corresponding author. Tel.: +33 01 39 25 43 66; fax: +33 01 39 25 44 52; e-mail: wright@chimie.uvsq.fr
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.06.049

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K. Wright et al. / Tetrahedron Letters 46 (2005) 5573–5576
in which trans-APiC residues can be incorporated.8 Simi-
lar b-peptide helical structures were later shown to be
stable in both organic and aqueous solvents, and resis-
tant to enzymic hydrolysis and microbial digestion.9
(R)-2 or (S)-2 was reduced in the presence of NaBH3CN
and acetic acid to give a 1:1 mixture of the cis-diastereo-
mers 3. Under these conditions, only traces of each of
the two trans-diastereomers could be detected by 1H
NMR in the crude reduction product.10,11 The cis-dia-
stereomers 3 were separated, as previously described,
by formation of their HCl salts and selective crystalliza-
tion from MeCN.6a Selective epimerization at carbon-3
of these products in the presence of NaOMe gave 1:1
We were interested in the incorporation of the trans-b-
TOAC residue into a b-peptide, and the examination
of the resulting secondary structure using ESR spectro-
scopy in the same way that we had used TOAC to probe
310-helical forms in a-peptides.2 With this aim in mind,
we needed a synthetic route to trans-b-TOAC that
would furnish us with a sufficient quantity of nitroxide
b-amino acid to allow subsequent peptide synthesis.
1
cis/trans mixtures as determined by H NMR analysis
of the crude products.10,11 However, while trans
(10S,3R,4S)-3 could be isolated in 43% yield from the
epimerization of cis (10S,3S,4S)-3, the diastereomer
trans (10S,3S,4R)-3 could not be separated by column
chromatography from cis (10S,3R,4R)-3.
Our initial intention was to use the synthetic pathway
that we had already developed to obtain the cis-b-
TOAC diastereomers 3 (Fig. 2),6a and then to epimerize
these compounds under basic conditions to obtain the
corresponding trans-diastereomers, in the same manner
as the route reported by Gellman and co-workers for
the synthesis of trans-ACHC and trans-APiC.8 Amina-
tion of 3-carboxymethyl-1-oxyl-2,2,6,6-tetramethyl-4-
piperidone 1 with either (R)- or (S)-a-methyl-benzyl-
amine gave the corresponding enamine (R)-2 or
(S)-2 in 87% and 82% yield, respectively. The enamine
To obtain greater quantities of a single diastereomer of
trans-3 we reinvestigated the reduction of enamine (S)-2,
applying the optimized reaction conditions (NaBH4 in
the presence of a bulky organic acid) previously devel-
oped by Xu and et al.12 for the synthesis of ethyl 2-ami-
no-1-cyclohexanecarboxylates by asymmetric reduction
of the corresponding a-methylbenzylamine derived en-
amine. However, when using NaBH4 in the presence
of excess isobutyric acid and toluene as co-solvent, we
1
were surprised by the results obtained: H NMR analy-
sis of the crude reaction product revealed the presence of
only one cis (10S,3S,4S)-3 and one trans (10S,3R,4S)-3
diastereomer in a ratio of ca. 1:1 (Fig. 3), with no trace
O
N
O
N
(i)
of the other two diastereomers.10,11 Chromatographic
separation afforded cis (10S,3S,4S)-3
in 29% yield
6a
COOMe
COOMe
and trans (10S,3R,4S)-313,14 in 30% yield.
O
NH
At this stage we are unable to explain the selectivity of
the reduction of enamine (S)-2 in the presence of isobu-
tyric acid. In any case, we were able to take advantage of
this finding to shorten the synthetic path leading to
trans-b-TOAC. Hydrogenation of the hydrochloride salt
of trans (10S,3R,4S)-3 over Pd/C for 30 min, followed by
regeneration of the nitroxide radical from the resulting
hydroxylamine intermediate with a catalytic amount of
Cu(OAc)2 in the air for 7 days,15,16 gave the desired
trans H-(3R,4S)-b-TOAC-OMe 4 diastereomer13 in
41% yield (Fig. 4). This compound is indeed ESR-active
and its spectrum in MeOH solution shows a three line
pattern typical of mono-nitroxide radicals (Fig. 5). Sim-
Ph
Me
(S)-2
1
(ii)
O
O
N
N
+
COOMe
COOMe
NH
NH
Me
1 : 1
Ph
Me
Ph
cis-3 (1’S,3S,4S)
cis-3 (1’S,3R,4R)
(iii)
(iii)
O
N
O
N
O
N
O
N
O
N
+
COOMe
NH
COOMe
COOMe
COOMe
COOMe
NH
Me
NH
NH
NH
Ph
Me
Ph
Ph
Me
(S)-2
Ph
Me
Ph
Me
trans-3 (1’S,3S,4R)
trans-3 (1’S,3R,4S)
cis-3 (1'S,3S,4S)
trans-3 (1'S,3R,4S)
˚
Figure 2. (i) (R)-a-Methyl-benzylamine; AcOH; EtOH; 4 A-MS; rt;
48 h, 87%; (ii) NaBH3CN; AcOH; EtOH; 75 ꢁC; 2 h; then HCl/EtOAc;
0 ꢁC; filtration and recrystallization from MeCN, (10S,3S,4S)-3 31%,
(10S,3R,4R)-3 26%; (iii) NaOMe; MeOH; 70 ꢁC; 18 h, trans (10S,3R,4S)-3
43%, cis (10S,3S,4S)-3 47%.
1 : 1
Figure 3. Reduction of the enamine (S)-2 with NaBH4, (CH3)2-
CHCOOH, toluene, 0 ꢁC to rt, 24 h; cis (10S,3S,4S)-3 29%, trans
(10S,3R,4S)-3 30%.

K. Wright et al. / Tetrahedron Letters 46 (2005) 5573–5576
5575
O
N
O
O
N
(i) (ii)
(iii) (iv)
N
COOMe
NH.HCl
COOMe
COOH
Ph
NH2
Fmoc-NH
Me
trans-3 (1'S,3R,4S)
trans H-(3R,4S)-β-TOAC-OMe 4
trans Fmoc-(3R,4S)-β-TOAC-OH 5
Figure 4. Removal of the chiral auxiliary from 3, followed by C-deprotection/N-protection of the resulting b-amino ester enantiomer 4. (i) Pd/C 10%;
95% EtOH; rt; 30 min. (ii) Cu(OAc)2; MeOH; air; rt; 7 days, 41%; (iii) NaOH (aq); MeOH; reflux; 5 h. (iv) Fmoc-succinimidyl carbonate; NaHCO3;
acetone/water 2:1; rt; 18 h, 40%.
2. (a) Hanson, P.; Martinez, G.; Milhauser, G.; Formaggio,
F.; Crisma, M.; Toniolo, C.; Vita, C. J. Am. Chem. Soc.
1996, 118, 271–272; (b) Toniolo, C.; Crisma, M.; For-
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Figure 5. ESR spectrum of trans H-(3R,4S)–TOAC-OMe 4 in a
0.5 mM MeOH solution at room temperature.
ilar treatment of trans (10R,3S,4R)-313 obtained by a
similar NaBH4 reduction of enamine (R)-2 in an isobu-
tyric acid/toluene solution, gave the trans H-(3S,4R)-b-
TOAC-OMe 413 enantiomer (not shown). Saponifica-
tion of the methyl ester function of trans H-(3R,4S)-b-
TOAC-OMe 4 and trans H-(3S,4R)-b-TOAC-OMe 4
followed by protection of the amino function with the
Fmoc group gave the b-amino acid derivatives trans
Fmoc-(3R,4S)-b-TOAC-OH 5 and its (3S,4R) 5 enantio-
mer (Fig. 4), suitable for use in peptide synthesis.
4. (a) Adam, W.; Saha-Mo¨ller, C. R.; Ganeshpure, P. A.
Chem. Rev. 2001, 101, 3499–3548; (b) Sheldon, R. A.;
Arends, I. W. C. E.; Ten Brink, G.-J.; Dijksman, A. Acc.
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5. (a) Naik, N.; Braslau, R. Tetrahedron 1998, 54, 667–696;
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Mezzato, S.; Polese, A.; Barazza, A.; Antonello, S.;
Maran, F.; Broxterman, Q. B.; Kaptein, B.; Kamphuis,
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A.; Wakselman, M.; Mazaleyrat, J.-P. Tetrahedron Lett.
In conclusion, the two enantiomers of the trans-b-
TOAC residue, bearing a nitroxide function, have been
synthesized in enantiopure form and in a reasonable
yield. Their synthesis was facilitated by an unexpected
selectivity in the reduction of an enamine intermediate.
Derivatives 4 and 5 of trans (3R,4S)-b-TOAC are cur-
rently being used, in combination with (1S,2S)-ACHC,
for the synthesis and 3D-structural analysis of a
designed b-hexapeptide.
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2003, 44, 3381–3384; (b) Peter, A.; To¨ro¨k, R.; Wright, K.;
Wakselman, M.; Mazaleyrat, J.-P. J. Chromatogr. A 2003,
1021, 1–10.
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K. Wright et al. / Tetrahedron Letters 46 (2005) 5573–5576
10. Well resolved 1H and 13C NMR spectra could only be
obtained by preliminary reduction of the nitroxide func-
tion with sodium dithionite.11
11. Ozinskas, A. J.; Bobst, A. M. Helv. Chim. Acta 1980, 63,
1407–1411.
CH2Cl2 (40 mL) and washed with saturated NaHCO3
solution (2 · 30 mL) and with brine (30 mL). The organic
phase was dried over MgSO4, filtered and concentrated.
Column chromatography using cHex/EtOAc 2:1 as eluent
gave successively the enamine (S)-2 (67 mg, 13%),
(10S,3R,4S)-3 (151 mg, 30%) and (10S,3S,4S)-3 (149 mg,
29%). The trans-diastereomer (10S,3R,4S)-3 was converted
to its HCl salt by addition of a solution of HCl in EtOAc
12. Xu, D.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetra-
hedron: Asymmetry 1997, 8, 1445–1451.
13. All new compounds gave satisfactory analytical data
(1H/13C NMR, C,H,N analysis and/or ESI/MS). Further
experimental details of syntheses will be given in a full
1
for characterization. H NMR (D2O) 7.42 (m, 5H, ArH),
4.52 (q, 1H, PhCHCH3), 3.78 (m, 1H, H-4), 3.72 (s, 3H,
OCH3), 2.97 (d, 1H, H-3, J3,4 = 11.8 Hz), 1.70, 1.40 (2m,
2H, H-5), 1.58 (d, 3H, PhCHCH3), 1.46, 1.25, 1.16, 1.04
(4s, 12H, 4CH3); 13C NMR 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); ES-MS m/z
(%) 335.2 (100) [M+2H]+, 334.2 (61) [M+H]+.
15. Dulog, L.; Wang, W. Liebigs Ann. Chem. 1992, 301–
303.
25
account of this study. Optical rotations ½aꢀ546 are as
follows: (10S,3R,4S)-3: ꢁ52 (c 0.27; MeOH). (10R,3S,4R)-
3: +48 (c 0.28; MeOH). (3R,4S)-4: ꢁ24 (c 0.1; MeOH).
(3S,4R)-4: +24 (c 0.1; MeOH).
14. Synthesis of (10S,3R,4S)-3: Isobutyric acid (3.5 mL,
30 mmol) was cooled to 0ꢁC and NaBH4 (288 mg,
7.6 mmol) was added slowly in portions over 40 min.
The mixture was then stirred at rt for 30 min. The 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
16. The extent of oxidation of the intermediate hydroxyl-
amine to the nitroxide was monitored by thin layer
chromatography.