Synthesis of new conformationally rigid parametric α-amino acids

Article abstract of DOI:10.1016/j.tetlet.2003.10.020

A pyrroline nitroxide based cyclic tetrasubstituted α-amino acid and paramagnetic homoproline and their derivatives are described. Introduction of a new paramagnetic protecting group to follow the incorporation of an amino acid into peptides by EPR is also suggested.

Full text of DOI:10.1016/j.tetlet.2003.10.020

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 9213–9217
Synthesis of new conformationally rigid paramagnetic
a-amino acids
Ma´ria Balog,a Tama´s Ka´lai,a Jo´zsef Jeko,b Zolta´n Berente,c Heinz-Ju¨rgen Steinhoff,d
3
Martin Engelharde and Ka´lma´n Hidega,*
aInstitute of Organic and Medicinal Chemistry, University of Pe´cs, H-7602 Pe´cs, PO Box 99, Hungary
bICN Hungary Ltd., H-4440 Tiszavasva´ri, PO Box 1, Hungary
cInstitute of Biochemistry and Medical Chemistry, University of Pe´cs, H-7602 Pe´cs, PO Box 99, Hungary
dDepartment of Physics, University of Osnabru¨ck, Barbara st. 7, D-49069 Osnabru¨ck, Germany
eMax Planck Institute of Molecular Physiology, PO Box 50 02 47, D-44202 Dortmund, Germany
Received 26 August 2003; revised 26 September 2003; accepted 3 October 2003
Abstract—A pyrroline nitroxide based cyclic tetrasubstituted a-amino acid and paramagnetic homoproline and their derivatives
are described. Introduction of a new paramagnetic protecting group to follow the incorporation of an amino acid into peptides
by EPR is also suggested.
© 2003 Elsevier Ltd. All rights reserved.
serves as a good reporter of backbone flexibility.13
TOAC was incorporated into a-melanocyte stimulating
hormone without loss of biological activity.14 TOAC
was used in the synthesis of paramagnetic fullerenes15
as well as of nitroxyl peptides to catalyze enantioselec-
tive oxidation.16 However, the six-membered ring of
TOAC is sensitive to reduction/oxidation and acidic
media, the latter required for Merrifield solid phase
synthesis.3 It is known that pyrroline nitroxides are less
sensitive toward these experimental conditions.17 In our
Electron spin resonance (ESR) is one of the most
important tools for studying structure and function of
proteins and biomolecules.1 Nowadays there are two
fundamental methods to attach a paramagnetic label in
a site-specific fashion to a protein. One possibility is
making cysteine mutants followed by labeling with a
methanethiosulfonate spin label.2 This method is
referred to as site directed spin labeling (SDSL).
Another approach is solid phase protein synthesis,
where natural and unnatural amino acids3 are incorpo-
rated in a step by step process.4 A new trend with
increasing significance is the nonsense suppression tech-
nology to build in unnatural amino acids.5 For ESR
studies of proteins, a variety of paramagnetic a-amino
acids (TOAC,6 I,7,8 II9), b-amino-acids (POAC,6 b-
TOAC10) and g-amino acids (III)11 have been synthe-
sized (Fig. 1). In several cases naturally occurring
amino acids were modified by alkylation or acylation
with functionalized nitroxides to obtain a paramagnetic
protein building block.12 TOAC (4-amino-1-oxyl-
2,2,6,6-tetramethyl-piperidine-4-carboxylic acid) by far
the most popular among the above mentioned amino
acids, is easily available from 1-oxyl-4-oxo-2,2,6,6-tetra-
methyl-piperidine. This paramagnetic amino acid is
considered to be a b-turn and 310-helix inducer and
Keywords: amino acids; nitroxides; protecting group; TOAC.
* Corresponding
author.
Fax:
+(36)-72-536-219;
e-mail:
kalman.hideg@aok.pte.hu
Figure 1. Chemical structure of paramagnetic amino acids.
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.020

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M. Balog et al. / Tetrahedron Letters 44 (2003) 9213–9217
Compound 3 can be regarded as a paramagnetic deriva-
tive of DL-pipecolic acid, a homolog of proline which is
a widespread non-proteinogenic amino acid in plants,
moreover its incorporation into a biologically active
peptide in place of L
-proline was successful.22 Consider-
ing this achievement, we decided to explore the chem-
istry of this paramagnetic proline-like building block,
therefore its N-Boc derivatives 8 and 9 were synthesized
in processes analogous to these leading to compounds 6
and 7.
Figure 2. Conformationally rigid amino acids.
laboratory several carbocycle and heterocycle anellated
pyrroline nitroxides have been synthesized,18 so it was
obvious that we should aim to make a new conforma-
tionally rigid paramagnetic amino acid which combines
the advantages of TOAC and pyrroline nitroxides (Fig.
2).
The carboxycyclohexyloxy amine-masking group was
introduced earlier,23 but has not found wide applica-
tion. We believe that its paramagnetic derivative
2,2,6,6-tetramethyl-piperidine could be useful to moni-
tor the solid phase amino acid synthesis by EPR. The
paramagnetic protecting group TmpcCl can be synthe-
sized from the easily available 4-hydroxy-2,2,6,6-tetra-
methylpiperidine (TEMPOL) and triphosgene.24 The
acid chloride was converted to acid azide TmpcN3,25
which is considered to be a milder reagent, with NaN3
under phase transfer conditions. Acylation of amino
acid ester 3 either with TmpcCl or TmpcN3 gave birad-
ical 10, which could be hydrolyzed by aqueous NaOH
to acid 11 without hydrolysis of the carbamate. The
principle behind the use of Tmpc is that this group can
be observed by its EPR spectrum when it is attached to
an amino acid introduced at the N-terminal of a pep-
tide. Upon hydrolytic cleavage of the Tmpc protecting
group its increased mobility is revealed by the changes
in triplet line shape (or, in the case of a paramagnetic
amino acid, the quintet disappears) indicating the suc-
cessful deprotection and binding of the amino acid in
question, which may be the critical issue in the case of
unnatural amino acids. The TmpcCl itself can be con-
sidered as a cheap acylating spin label reagent.26 To
fulfil the requirements of Nvoc and Fmoc chemistry, we
also synthesized the photo-cleavable 3,4-dimethoxy-6-
In this paper we report the synthesis of a pyrroline
TOAC abbreviated as ‘PTOAC’ by us. Twofold alkyla-
tion of ethyl N-diphenylmethylene glycine with para-
magnetic bis(allylic bromide) 118a under phase transfer
conditions19 gave the monoalkylated product 2, which
could be readily hydrolyzed under acidic conditions to
the corresponding amine, alkaline treatment of which
yielded the cyclic six-membered a-aminoester 3.20 Oxi-
dation of compound 3 with activated MnO2 gave the
pyrroline nitroxide anellated pyridine 2-carboxylic acid
ethyl ester 4, synthesized earlier in our laboratory.18b
The benzylidene derivative of glycine ethyl ester was
treated with 2.2 equivalent of NaHDMS in THF at
−78°C followed by twofold alkylation with compound 1
and acidic hydrolysis21 giving a,a-disubstituted a-amino
acid ester 5 in moderate yield. It was found in prelimi-
nary experiments with compound 7 that the N-Boc
group can be removed without reduction of nitroxide to
diamagnetic hydroxylamine. Treatment of compound 5
with di-tert-butyldicarbonate gave the acid sensitive
N-Boc derivative 6 which could be hydrolyzed with
NaOH in EtOH to the N-Boc protected amino acid 7
(Scheme 1).
Scheme 1. Reagents and conditions: (a) Ph2CꢀNCH2CO2Et (1.0 equiv.), 10% aq. NaOH (2.0 equiv.), CH2Cl2, Bu4NHSO4 (0.1
equiv.), rt, 2 h, 78%; (b) PhCHꢀNCH2CO2Et, THF, −78°C, NaHMDS (2.2 equiv., 1.0 M in THF) 45 min, then 1, −78°C“rt, 2
h; (c) 5% aq. H2SO4 (1.1 equiv.), EtOH, 30 min rt, then NaOH to pH 8, 15–84%; (d) MnO2, CHCl3, reflux, 1 h, 53%; (e) Boc2O
(1.0 equiv.), THF, 40°C, 30 min, 36%; (f) 10% aq. NaOH (1.1 equiv.), EtOH, 78°C, 30 min, then EtOH evaporated, aq. H2SO4
to pH 3, 48%.

M. Balog et al. / Tetrahedron Letters 44 (2003) 9213–9217
9215
nitrobenzyl (Nvoc) carbamate and base sensitive N-
protected 9-fluorenylmethoxycarbonyl (Fmoc) deriva-
tive. Treatment of compound 3 with Ba(OH)2 followed
by saturation with CO2 yielded compound DL-12.
Acylation of DL-12 with Fmoc-succinate in the pres-
ence of KHCO3 gave amino acid 13 with a base (e.g.
piperidine) sensitive N-protecting group and acylation
of 12 with 6-nitroveratryl-chloroformate in aqueous
dioxane in the presence of K2CO3 yielded compound 14
with a photo-cleavable protecting group (Scheme 2).27
their use in biological systems. We suggest the introduc-
tion of a paramagnetic protecting group, Tmpc, to
follow the amino acid incorporation process by EPR.
The applications of the spin labels described above are
in progress and will be reported separately.
Acknowledgements
In conclusion, we have synthesized two new conforma-
tionally rigid amino acids for use in the synthesis of
paramagnetic peptides: a new TOAC-like pyrroline
nitroxide based achiral paramagnetic amino acid, which
can be directly used in peptide labeling, and a paramag-
netic racemic homoproline and their N-protected
derivatives.28 These last ones should be resolved before
This work was supported by grant from Hungarian
National Research Foundations (OTKA T34307) and
Deutsche Forschungsgemeinschaft (EN87/12-1 for M.
E., Hi 823/1-1 for K.H. and STE 640/4-3 for H.-J.S.).
The authors thank Viola H. Csokona for elemental
analyses and Ma´ria Szabo´ for mass spectral measure-
ments (ICN, Hungary).
Scheme 2. Reagents and conditions: (a) Boc2O (1.0 equiv.), THF, 40°C, 30 min, 68%; (b) 10% aq. NaOH (1.1 equiv.), EtOH, 78°C,
30 min, then EtOH evaporated, aq. H2SO4 to pH 3, 61–68%; (c) TmpcCl (1.0 equiv.), Et3N (1.0 equiv.), THF, 65°C, 3 h, 55%;
(d) Ba(OH)2·8 H2O (1.1 equiv.), MeOH/H2O (1:1), 65°C, 30 min, then excess dry ice, filtration, evaporation, 48%; (e) Fmoc-O-Su
(1.0 equiv.), dioxane/H2O (2:1), KHCO3 (2.0 equiv.), rt, 4 h, then 5% aq. H2SO4 to pH 3, 75%; (f) 6-nitroveratryl chloroformate
(1.1 equiv.), K2CO3 (2.0 equiv.), dioxane, 18-crown-6 (0.05 equiv.), rt, 6 h, then evaporated, 5% aq. H2SO4 to pH 3, 55%; (g)
triphosgene (0.5 equiv.), pyridine (1.0 equiv.), CH2Cl2, −20°C, 15 min, then rt, 30 min, 59%; (h) NaN3, 18-crown-6, dioxane, rt,
3 h, 47%.

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M. Balog et al. / Tetrahedron Letters 44 (2003) 9213–9217
23. McKay, F.; Albertson, F. N. J. Am. Chem. Soc. 1957, 79,
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26. Synthesis of TmpcCl: To a stirred solution of TEMPOL
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H, 6.19; N, 5.50; MS (EI) m/z: 512/514 (M+, 2/2), 266
(27), 193 (43), 167 (100). 3: oil; calcd for C14H23N2O3: C,
62.90; H, 8.67; N, 10.48, found: 63.02; H, 8.70; N, 10.55;
1
IR (neat) w: 3300, 1730 cm−1; H NMR (CDCl3) l: 4.21
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(2H, q, J=7.1 Hz, O-CH2), 3.57 (1H, dd, J1=8.4 Hz,
J2=5.0 Hz, CH), 3.42–3.30 (2H, m, NCH2), 2.30–2.14
(2H, m, CH2), 1.28 (t, 3H, J=7.1 Hz, Et-CH3), 1.23 (s,
3H, C-CH3), 1.21 (s, 3×3H, C-CH3); MS (EI) m/z: 267
(M+, 28), 253 (27), 237 (28), 194 (100). 5: mp 57–59°C;
calcd for C14H23N2O3: C, 62.90; H, 8.67; N, 10.48, found:
C, 63.10; H, 8.75; N, 10.39; IR (neat) w: 3350, 3300, 1720
cm−1 1H NMR (CDCl3) l: 4.24 (2H, q, J=7.2 Hz,
;
O-CH2), 2.96 (2H, br d, CHH ‘eq’), 2.37 (2H, br d, CHH
‘ax’), 1.54 (6H, s, C-CH3), 1.50 (6H, s, C-CH3), 1.31 (3H,
t, J=7.2 Hz, Et-CH3); 13C NMR (CDCl3) l: 175.8
(carboxy), 141.0 (br, bridgehead), 74.4 (br, CCH3), 62.0
(OCH2), 41.7 (br, CH2), 23.1 (br, CCH3), 22.8 (br,
CCH3), 14.2 (Et-CH3); MS (EI) m/z: 267 (M+, 15), 253
(30), 237 (35), 164 (100). 7: mp 169–171°C, calcd for
C17H27N2O5: C, 60.16; H, 8.02; N, 8.25, found: C, 60.14;
H, 8.06; N, 8.18; MS (EI) m/z: 339 (M+, 1), 251 (7), 205
(17), 57 (100). 9: mp 136–138°C; calcd for C17H27N2O5:
C, 60.16; H, 8.02; N, 8.25, found: C, 60.22; H, 8.10; N,
8.20; IR (nujol) w: 1720, 1600 cm−1; MS (EI) m/z: 339
(M+, 1), 325 (2), 269 (5), 57 (100). 10: mp 103–105°C;
calcd for C24H39N3O6: C, 61.69; H, 8.44; N, 9.03, found:
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C, 61.75; H, 8.40; N, 9.10; IR (nujol) w: 1730, 1600 cm−1
;
MS (EI) m/z: 465 (M+, 16), 435 (6), 311 (27), 124 (100).
11: mp 145–146°C; calcd for C22H35N3O6: C, 60.39; H,

M. Balog et al. / Tetrahedron Letters 44 (2003) 9213–9217
9217
8.06; N, 9.60, found: C, 60.35; H, 8.03; N, 9.51; IR
(nujol) w: 1720, 1605 cm−1; MS (EI) m/z: 437 (M+, 1),
235 (21), 221 (100), 205 (60). 12: mp 209–211°C,
decomp.; calcd for C12H19N2O3: C, 59.48; H, 9.15; N,
11.56, found: C, 59.51; H, 8.10; N, 8.07; IR (nujol) w:
3300, 1600 cm−1; MS (EI) m/z: 239 (M+, 14), 225 (30),
209 (18), 177 (100). 13: mp 110–112°C; calcd for
C27H29N2O5: C, 70.26; H, 6.33; N, 6.07, found: C,
70.15; H, 6.29; N, 5.95; IR (nujol) w: 1720, 1600 cm−1
;
MS (EI) m/z: 461 (M+, 1), 447 (1), 431 (2), 178 (100).
14: mp 124–126°C; calcd for C22H28N3O9: C, 55.23; H,
5.90; N, 8.78, found: C, 55.25; H, 6.01; N, 8.80; IR
(nujol) w: 1705, 1570, 1505 cm−1; MS (EI) m/z: 478 (M+,
4), 448 (3), 219 (44), 136 (100).