First access to the spin-labelled β-amino acid POAC in an enantiopure state by resolution through its binaphthyl esters

Article abstract of DOI:10.1016/S0040-4039(03)00920-1

Resolution of trans 3-(9-fluorenylmethyloxycarbonylamino)-1-oxyl-2,2,5,5- tetramethylpyrrolidine-4-carboxylic acid (Fmoc-POAC-OH) was quickly achieved upon esterification with (aR)-1,1′-binaphthyl-2,2′-diol, chromatographic separation of the obtained diastereomers, and facile saponification of the aryl ester function with removal of the chiral auxiliary.

Full text of DOI:10.1016/S0040-4039(03)00920-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 4183–4186
First access to the spin-labelled b-amino acid POAC in an
enantiopure state by resolution through its binaphthyl esters
Karen Wright,a,* Fernando Formaggio,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 20 March 2003; revised 8 April 2003; accepted 8 April 2003
Abstract—Resolution of trans 3-(9-fluorenylmethyloxycarbonylamino)-1-oxyl-2,2,5,5-tetramethylpyrrolidine-4-carboxylic acid
(Fmoc-POAC-OH) was quickly achieved upon esterification with (aR)-1,1%-binaphthyl-2,2%-diol, chromatographic separation of
the obtained diastereomers, and facile saponification of the aryl ester function with removal of the chiral auxiliary. © 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 Optically active nitroxides have also been
developed as enantioselective oxidizing agents, and for
stereoselective coupling with prochiral radicals.2 As far
as amino acids are concerned, the nitroxide bearing,
achiral Ca,a-disubstituted glycine 4-amino-1-oxyl-
2,2,6,6-tetramethylpiperidine-4-carboxylic acid residue
(TOAC) (Fig. 1),3 has been widely used to label pep-
tides at N-terminal and internal positions for biological
studies and conformational analysis by ESR methods.4
The TOAC residue has also been previously employed
in our groups as a probe for structural investigations
involving intramolecular energy transfer (fluorescence
quenching) and intramolecular spin polarization
(CIDEP) effects in designed, rigid, peptide-based sys-
tems.5 However, the TOAC structure presents a few
disadvantages: its achiral character may hinder access
to stereochemical information, and its tetrasubstituted
a-carbon induces a reduced reactivity of the amino
function which may be problematic if the residue is to
be placed at an internal position of a peptide. Accord-
ingly, we have been interested by the prospect of creat-
ing spin-labelled cyclic i-amino acids that could be
obtained in enantiopure form. The cyclic structure was
designed to provide molecular rigidity, allowing a topo-
logically well defined placement of the nitroxide func-
tion, while the choice of a b-amino acid structure was
expected to allow easier peptide couplings than the
TOAC structure. With this aim, we have recently
reported the synthesis of enantiopure 4-amino-1-oxyl-
2,2,6,6-tetramethylpiperidine-3-carboxylic acid (cis-b-
Figure 1. Chemical structures of the spin-labelled amino acids TOAC, b-TOAC and POAC.
Keywords: binaphthol esters; chiral nitroxides; modified b-amino acids; spin-labelled amino acids; POAC; resolution.
* Corresponding author. Fax: (33) 01 39 25 44 52; 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)00920-1

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K. Wright et al. / Tetrahedron Letters 44 (2003) 4183–4186
TOAC),6 and we now wish to present our preliminary
results relative to the practical resolution of 3-amino-1-
asymmetric addition on chiral a,b-ethylenic esters,
failed when performed under the same experimental
conditions as those employed in the reaction of 3c.
oxyl-2,2,5,5-tetramethylpyrrolidine-4-carboxylic
acid
(trans-POAC) (Fig. 1).
Having met with failure in our attempts at direct asym-
metric synthesis, we switched to the search for a practi-
cal resolution procedure of racemic POAC, that we
The interest in the trans POAC residue, first described
by Rassat and Rey,3a was recently highlighted by
Nakaie and co-workers,7 who repeated Rassat’s synthe-
sis in order to obtain trans-5 (Fig. 2), and then pre-
pared its Na-Fmoc (9-fluorenylmethyloxycarbonyl)
protected derivative trans-6 for use in solid-phase pep-
tide synthesis in view of its incorporation into an
angiotensin II analogue. In that study 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 Another poten-
tial utility of POAC concerns the field of b-peptides,
which has been the subject of increasing interest in
recent years, after it was demonstrated that oligomers
of b-amino acids may fold into new stable helical
conformations.9 Therefore, as it represents a valuable
additional spin-labelled b-amino acid, POAC is likely
to be used as a tool for conformational studies of
b-peptides by ESR methods, in the same way that
TOAC has been used in the a-amino acid series.
To our knowledge, POAC was synthetically obtained
only as a mixture of the two trans enantiomers and
used as such.3a,7 The access to both its enantiomers in
enantiopure form appeared to us to be highly desirable:
(i) in order to avoid separation problems related to the
formation of diastereomers in the couplings with chiral
a- or b-amino acids, and (ii) in view of investigations
into b-peptide helix conformations, as it has been
shown that absolute configuration at Ca and Cb in
b-amino acids influences helical screw sense.9,10
Figure 2. Synthetic path for the preparation of racemic trans-
POAC 53a,7 and trans-Fmoc-POAC-OH 67 (the trans spatial
relationship between the C4ꢀN bond and the C3ꢀCN or
C3ꢀCOOH bond of compounds 4, 5 and 6 is not represented
to avoid confusion with enantiomerically pure compounds).
(i) NaWO4·2H2O; H2O2 35%; NaHCO3; MeOH/MeCN; 0°C
to rt; 24 h. (ii) TsCl; pyridine; rt; 36 h. (iii) NH3 (aq); rt; 5
days. (iv) Ba(OH)2; H2O; reflux; 48 h. (v) Fmoc-OSu (Fmoc-
succinimidyl carbonate); NaHCO3; acetone:H2O 2:1; rt; 18 h.
We began by exploring alternative pathways for the
asymmetric synthesis of trans-POAC by Michael addi-
tion of (R)-(+)-a-methylbenzylamine to 3-car-
bomethoxy-2,2,5,5-tetramethylpyrroline 3a11 (Fig. 3),
its N-Boc protected derivative 3b,12 3-cyano-1-oxyl-
2,2,5,5-tetramethylpyrroline 3c,13 a synthetic intermedi-
ate in the preparation of trans-5 (Fig. 2), and its
3-carbomethoxy-1-oxyl
2,2,5,5-tetramethylpyrroline
analogue 3d,13 both readily obtained from the carbox-
amide 1.13 We had confidence in this strategy, as not
only was it the key step in the synthesis of racemic
trans-POAC 5 via Michael addition of ammonia to the
a,b-unsaturated nitrile 3c,3a,7 but also because Gellman
and co-workers have recently achieved an asymmetric
synthesis of trans-3-aminopyrrolidine-4-carboxylic acid
by Michael addition of (R)-a-methylbenzylamine to the
corresponding a,b-unsaturated ester.14
Figure 3. Attempts at the synthesis of chiral trans-POAC by
asymmetric Michael addition to the a,b-unsaturated esters or
nitrile 3a–d. (i) H2O; 55°C; 8 days. (ii) Yb(OTf)3; toluene;
50°C; 5 days. (iii) Microwave activation, (a) no solvent;
150°C; 10 min (1.6 bar), or (b) KF/alumina; no solvent;
150°C; 10 min, or (c) H2O; 150°C; 10 min (6 bar), or (d)
H2O:THF 2:1; 100°C; 10 min (3 bar), or (e) xylene; 150°C; 10
min (1.6 bar).
However, in our hands, none of the substrates 3a–d
provided the corresponding Michael adduct, whatever
the applied activation modes (Fig. 3):15 use of water,14
or ytterbium triflate catalysis,16 or combined use of
pressure and microwave activation.17,18 Even Michael
addition of aqueous ammonia on the methyl ester 3d,
which was attempted in anticipation of a considered

K. Wright et al. / Tetrahedron Letters 44 (2003) 4183–4186
4185
synthesized by Rassat’s method. The nitrile 3c was
reacted with aqueous ammonia at room temperature
and atmospheric pressure, to provide the trans-b-amino
nitrile 43a in 40% yield, accompanied by 32% of the
amide 2 which could be recycled in later runs (Fig. 2).
form the mono-esters (Fig. 4) allowed the easy separa-
tion of the two obtained diastereomers 7a12 and 7b,12
isolated in 40 and 41% yield (80 and 82% of theoretical
yield), respectively, by standard column chromatogra-
phy on silica gel. Advantageously, for resolution up to
a gram-scale, it was also possible, prior to chromatog-
raphy, to partially purify the mixture by crystallization,
as the diastereomer with a lower Rf (7b) had an appre-
ciably poorer solubility in non-polar solvents than that
with a higher Rf (7a). Unfortunately, the obtained
crystals were not suitable for X-ray diffraction analysis,
which prevented the determination of the absolute
configuration of 7a and 7b, either (aR,3R,4R) or
(aR,3S,4S) (Fig. 4).
1
The H NMR spectrum of 4, performed after sodium
dithionite reduction of the nitroxide group,19 confirmed
that only the trans enantiomers were formed under
these conditions, as previously demonstrated by X-ray
diffraction analysis.7 Basic hydrolysis of trans-4 pro-
vided the free amino acid trans-POAC 5,3a which was
immediately Na-protected upon treatment with Fmoc-
OSu, to afford racemic trans-Fmoc-POAC-OH 67 in
58% overall yield.
For resolution of trans-6 we discarded the methods
involving amide bond formation, with either amines or
amino acid derivatives as chiral auxiliaries, because of
the harsh acidic conditions required for removal of the
chiral auxiliary from the separated diastereomers,
incompatible with the presence of the nitroxide group
of POAC.4a Rather, we considered resolution through
formation of diastereomeric pairs of amino esters by
reaction with chiral alcohols, only a few examples of
which, notably using menthol as auxiliary, are known.20
For this purpose, we selected 2,2%-dihydroxy-1,1%-
binaphthyl (binaphthol) as a new and potentially
efficient chiral auxiliary, taking into account the known
resolution of related dihydroxy-1,1%-binaphthyl com-
pounds by means of their esterification with chiral
amino acids (the reverse as in the present case).21,22
Indeed, while parallel attempts of esterification with
Alkaline hydrolysis of the esters 7a and 7b resulted in
partial cleavage of the Fmoc protecting group, as
expected.23 Subsequent re-protection of the amine func-
tion gave the desired Fmoc-amino acids (+)-612 and
(−)-6,12 in 57 and 43% yield, respectively. The two
enantiomers were examined by chiral HPLC using a
Chiralcel OD-RH column,24 which demonstrated the ee
of each to be >99.5%. We are currently pursuing the
search for a crystalline derivative of (+)-6 and (−)-6 to
allow the assignment of their absolute configuration by
X-ray diffraction analysis.
Altogether, the present study provides two conclusive
results: (i) the access to both enantiomers of POAC in
an enantiomerically pure state is now open for the first
time, and (ii) the straightforward resolution through
separation of the diastereomeric binaphthyl monoesters
of Fmoc-POAC-OH, represents a new procedure which
in our view deserves to be further investigated as a
general method for the separation of b- or a-amino acid
enantiomers.
(−)-menthol, (−)-10-dicyclohexylsulfamoyl-(D)-isobor-
neol and 1,2:3,4-di-O-isopropylidene galactose either
did not work or did not provide separable
diastereomeric esters, the use of (aR)-binaphthol to
Figure 4. Resolution of trans Fmoc-POAC-OH through its (aR) binaphthyl mono-esters. (i) EDC [1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride]; DMAP (4-dimethylaminopyridine); CH2Cl2:MeCN 1:1; 0°C; h. (ii) Crystallization
2
CH2Cl2:Et2O 1:1 then chromatography cHex:EtOAc 7:3. (iii) NaOH; H2O; MeOH; 40°C; 6 h. (iv) Fmoc-OSu; NaHCO3;
acetone:H2O 2:1; rt; 18 h.

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K. Wright et al. / Tetrahedron Letters 44 (2003) 4183–4186
Gellman, S. H. Acc. Chem. Res. 1998, 31, 173–180; (c)
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