Synthesis of new 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radicals and 2-substituted-2,5,5-trimethylpyrrolidin-1-yloxyl radicals based α-amino acids

Article abstract of DOI:10.1055/s-2004-834806

Unnatural paramagnetic α-amino acids with 2,2,5,5-tetramethyl-2,5- dihydro-1H-pyrrol-1-yloxyl-3-yl radical or 2,5,5-trimethylpyrrolidin-1-yloxyl-2- yl radical side-chains, including a lysine mimic azido precursor and their derivatives, are described. The

Full text of DOI:10.1055/s-2004-834806

LETTER
2591
Synthesis of New 2,2,5,5-Tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl
Radicals and 2-Substituted-2,5,5-trimethylpyrrolidin-1-yloxyl Radicals
Based a-Amino Acids
S
a
ynthesis of
N
ew 2,2,5
á
,5-Tetrame
r
thyl-2, 5-
i
dihydr
a
o-1
H
-pyrrol-1-ylo
B
xyl
R
adicals alog,a Tamás Kálai,a József Jekő,b Heinz-Jürgen Steinhoff,c Martin Engelhard,d Kálmán Hideg*a
b
c
d
ICN Hungary Ltd., P. O. Box 1, 4440 Tiszavasvári, Hungary
Department of Physics, University of Osnabrück, Barbara Str. 7, 49069 Osnabrück, Germany
Max Planck Institute of Molecular Physiology, P.O. Box 50 02 47, 44202 Dortmund, Germany
Received 17 August 2004
strained amino acids have been reported.8 Until now,
mainly 3-substituted 2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-1-yloxyl radicals have been used for the synthesis
of paramagnetic amino acids. In this paper, we report the
Abstract: Unnatural paramagnetic a-amino acids with 2,2,5,5-tet-
ramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl-3-yl radical or 2,5,5-tri-
methylpyrrolidin-1-yloxyl-2-yl radical side-chains, including a
lysine mimic azido precursor and their derivatives, are described.
The new set of paramagnetic amino acids presented in this work extension of the above procedure for 3,4-disubstituted
with different (polar, nonpolar, aliphatic, aromatic, etc.) side-chains
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radi-
offers a useful tool for the ESR study of the protein structure and
cals and 2-substituted 2,5,5-trimethylpyrrolidine-1-yloxyl
function after incorporation, fulfilling diverse structural require-
radicals with different alkyl and aromatic substituents and
ments.
spacers, leading to second generation of paramagnetic a-
amino acids. The introduction of 2-substituted 2,5,5-tri-
methylpyrrolidine-1-yloxyl radicals generates a new a-
amino acid series with a proline-like side chain with an
orientation different from amino acids containing 3-sub-
Key words: amino acids, azides, free-radicals, O’Donnell synthe-
sis, protecting groups
Unnatural amino acids have been the focus of biophysical,
biochemical, and synthetic and medicinal chemical stud-
1-yloxyl radicals (Figure 1).
stituted 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-
ies, particularly as they are applied to design of novel pep-
tides.1,2 One main group of unnatural amino acids have
fluorophores or paramagnetic labels in the side chain,
which allows to follow them by biophysical methods.3–6
There are two main approaches to modifying peptides
with spin labels. One approach is site-directed spin label-
ing, which requires synthesis of cysteine mutants which
can be modified afterwards with paramagnetic meth-
Figure 1 Chemical structure of paramagnetic amino acids.
anethiosulfonates.7 The other approach includes the incor-
poration of a paramagnetic amino acid in a step-by-step
Alkylation of ethyl N-diphenylmethylene glycine with
synthesis, e. g. Merrifield synthesis or nonsense suppres-
paramagnetic allylic bromide 1a,14 1b obtained from the
sion methodology.2 For the ESR studies of proteins, a va-
corresponding alcohol15 1c,16 1d17 obtained from 3,4-
9
riety of paramagnetic a-amino acids,3–5,8 b-amino acids
bis(bromomethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-
and g-amino acids10 have been synthesized. In several cas-
pyrrol-1-yloxyl radical,18 1e19 and benzylic bromide 1f20
es naturally occurring amino acids were modified by alky-
under phase transfer conditions21 gave the monoalkylated
lation or acylation with functionalized pyrrol-1-yloxyl
product 2a–f, which could be readily hydrolyzed under
radicals to obtain a paramagnetic protein building block11
acidic conditions to the corresponding amine 3a–f, with-
and very recently paramagnetically modified cysteine and
out affecting the N-oxyl radical moiety. The treatment of
tyrosine were inserted using nonsense incorporation in
DL-amino acid esters with t-butoxycarbonyl anhydride
Xenopus Oocytes.12 TOAC,3 (4-amino-1-oxyl-2,2,6,6-tet-
gave the corresponding protected N-Boc amino acid ethyl
ramethyl-piperidine-4-carboxylic acid) by far the most
esters 4a–f, which can be hydrolyzed to acids 5a–f allow-
ing utilization in Merrifield synthesis (Scheme 1). Incor-
poration of amino acid 5c as a bromine containing
popular among the above mentioned a-amino acids, was
incorporated into a-melanocyte stimulating hormone
without loss of biological activity.13 Very recently, from
compound is not only a spin label but may support pro-
our laboratory, paramagnetic amino acids obtained by
teomic analysis by mass spectrometry with the diagnostic
O’Donnell synthesis,4 including conformationally con-
unique twin peaks arising from the bromine isotopes.22
Compound 5d is designed to convert e-azidobutyl side-
SYNLETT 2004, No. 14, pp 2591–2593
Advanced online publication: 20.10.2004
DOI: 10.1055/s-2004-834806; Art ID: G32104ST
© Georg Thieme Verlag Stuttgart · New York
2
2
.1
1
.2
0
0
4
chain to e-aminobutyl side-chain after incorporation into
a protein or it can be used for protein immobilization or
aiding cross-links by Staudinger ligation,23 while synthe-

2592
M. Balog et al.
LETTER
Scheme 1 Reagents and conditions: (a) Ph2C=NCH2CO2Et (1.0 equiv), 10% aq NaOH, CH2Cl2, Bu4NHSO4 (0.5 equiv), r.t., 2 h, 50–78%;
(b) 5% aq H2SO4, EtOH, 30 min., r.t., then solid K2CO3 to pH = 8, 15–84%; (c) Boc2O (1.1 equiv), THF, 40 °C, 30 min, 36–70%; (d) 10% aq
NaOH, EtOH, 1 h, then aq H2SO4 to pH = 3, 36–53%.
sis of compounds 5e–f were intended to mimic the natural In conclusion, new N-protected a-amino acids27 with
amino acids with aromatic side chain such as phenylala- paramagnetic side chains with different length, orienta-
nine.
tion, shape and polarity have been synthesized. The reso-
lution of these new, second-generation paramagnetic
amino acids with chiral chromatography as well as their
incorporation into peptides are in progress as part of an-
other ongoing project.
The other approach to the synthesis of paramagnetic ami-
no acids by the O’Donnell method uses 2-substituted
2,5,5-trimethylpyrrolidine-1-yloxyl radicals as alkylating
agents.
These alkylating agents are readily available from 2,5,5-
trimethyl-1-pyrroline N-oxide (TMPO)24 by Grignard
reaction of propargyl alcohol25 or 4-(dimethoxymeth-
yl)phenyl bromide26 followed by functional group trans-
formations. Although quite simple, this method has the
disadvantage that a second chiral center is introduced into
the molecule, necessitating a final purification step to re-
solve the two diastereomers. Alkylation of ethyl N-diphe-
nylmethylene glycine with paramagnetic propargylic
6a,25 allylic 6b25 and benzylic bromide 6c26 under phase-
transfer conditions gave the monoalkylated product 7a–c,
which could be readily hydrolyzed under acidic condi-
tions to the corresponding amine 8a–c. Treatment of race-
mic amino acid esters with t-butoxycarbonyl anhydride
gave the protected N-Boc amino acid ethyl esters 9a–c
which can be hydrolyzed to the corresponding N-protect-
ed amino acids 10a–c as described above (Scheme 2). The
paramagnetic 10a propargyl glycine, 10b allyl glycine
and 10c phenylalanine with different orientation, spacer
rigidity and saturation forms a novel paramagnetic amino
acid series.
Acknowledgment
This work was supported by grant from Hungarian National
Research Foundations (OTKA T34307) and Deutsche Forschungs-
gemeinschaft (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 N. Lazsányi for elemental
analyses and Mária Szabó for mass spectral measurements (ICN,
Hungary).
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Scheme 2 Reagents and conditions: (a) Ph2C=NCH2CO2Et (1.0 equiv), 10% aq NaOH, CH2Cl2, Bu4NHSO4 (0.5 equiv), r.t., 2 h, 39–70%;
(b) 5% aq H2SO4, EtOH, 30 min, r.t., then solid K2CO3 to pH = 8, 34–56%; (c) Boc2O (1.1 equiv), THF, 40 ºC, 30 min, 44–61%; (d) 10% aq
NaOH, EtOH, 1 h, then aq H2SO4 to pH = 3, 49–59%.
Synlett 2004, No. 14, 2591–2593 © Thieme Stuttgart · New York

LETTER
Synthesis of New 2,2,5,5-Tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl Radicals
2593
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Representative Synthesis of Compound 5d: To stirred
solution of N-diphenylmethylene glycine (801 mg, 3.0
mmol) and compound 1d (864 mg, 3.0 mmol) in CH2Cl2 (20
mL), 10% aq NaOH (3 mL) was added followed by addition
of Bu4NHSO4 (508 mg, 1.5 mmol) and the mixture was
stirred at r.t. for 2 h. The organic phase was separated, dried
(MgSO4), filtered and evaporated to give compound 2d as a
yellow oil 740 mg (52%). The crude product was
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immediately subjected to acidic hydrolysis. Compound 2d
was dissolved in EtOH (20 mL), 5% aq H2SO4 (5 mL) was
added, the mixture was allowed to stand at r.t. and the
mixture was monitored by TLC. After consumption of
compound 2d (ca 30 min) H2O (10 mL) was added, and the
pH = 8 was adjusted by addition of solid K2CO3, extracted
with CHCl3 (2 × 20 mL). Then, the organic phase was
separated, dried (MgSO4), filtered, evaporated and the
residue was purified by flash column chromatography
(CHCl3–MeOH) to give compound 3d (203 mg, 42%) as a
yellow oil. IR (nujol): n = 3350, 3280, 2095, 1730 cm–1. MS
(EI): m/z (%) = 310 (3) [M+], 249 (43), 233 (31), 161 (100).
Anal. Cald for C14H24N5O3: C, 54.18; H, 7.79; N, 22.56.
Found: C, 54.01; H, 7.71; N, 22.40.
To a solution of compound 3d (310 mg, 1.0 mmol) in dry
THF (15 mL) t-butoxycarbonyl anhydride (240 mg, 1.1
mmol) was added and the mixture was stirred at 40 °C for 30
min. After cooling, Et2O (20 mL) was added and the organic
phase was washed with brine (10 mL). Then, the organic
phase was separated, dried (MgSO4), filtered and evaporated
to give crude 4d as a yellow solid 279 mg (68%). This crude
4d was dissolved in EtOH (10 mL), then H2O (3 mL) and
10% aq NaOH (1 mL) were added and the mixture was
allowed to stand at r.t. and monitored by TLC. After
consumption of compound 4d (ca 1 h) the solution was
acidified to pH = 3 by cautious addition of 5% aq H2SO4.
The aqueous phase was extracted with CHCl3 (2 × 20 mL),
the combined organic phase was dried (MgSO4), filtered and
evaporated. The residue was purified by flash column
chromatography (CHCl3–MeOH) to give compound 5d as a
yellow solid 101 mg (39%), mp 160–162 °C. Anal. Calcd for
C17H28N5O5: C, 53.39; H, 7.38; N, 18.31. Found: C, 53.43;
H, 7.35; N, 18.50. MS was taken with thermospray
technique (TSP): m/z = 383 [M + H]+.
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1476.
(17) Synthesis of 1d: To a stirred solution of 3,4-
bis(bromomethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-1-yloxyl radical (326 mg, 1.0 mmol) in acetone (10
mL) NaN3 (65 mg, 1.0 mmol) dissolved in H2O (2 mL) was
added and the mixture was stirred for 3 h at 40 °C. The
acetone was evaporated off and after adding of H2O (5 mL)
the mixture was extracted with CHCl3 (2 × 10 mL). The
organic layer was separated, dried (MgSO4), filtered and
evaporated. Purification of the residue by flash column
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(37%), mp 70–72 °C, Rf = 0.28 (hexane–Et2O, 2:1). IR
(nujol): n = 2095 cm–1. MS (EI): m/z (%) = 287/289 (10/10)
[M+], 193 (37), 152 (67), 41 (100). The side product is 3,4-
bis(azidomethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-1-yloxyl radical, 68 mg (27%), mp 90–92 °C, Rf =
0.24 (hexane–Et2O, 2:1).
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Synlett 2004, No. 14, 2591–2593 © Thieme Stuttgart · New York