Synthesis and resolution of new paramagnetic α-amino acids

Article abstract of DOI:10.1016/j.tet.2007.11.020

New, paramagnetic unnatural α-amino acids were synthesized by the O'Donnell method. In the new amino acids nitroxide is condensed with thiophene, benzene, and tetrahydroisoquinoline ring, or linked through a methylene, benzyl or propargyl spacer. Some of

Full text of DOI:10.1016/j.tet.2007.11.020

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 1094e1100
www.elsevier.com/locate/tet
Synthesis and resolution of new paramagnetic a-amino acids
a
Tamas Kalai , Jozsef Schindler , Maria Balog , Elemer Fogassy , Kalman Hideg
b
a
c
a,
*
´
´
´
´
´
´
´
a Institute of Organic and Medicinal Chemistry, University of Pe´cs, H-7602 Pe´cs, PO Box 99, Hungary
b Research Group of Department of Organic Chemistry and Technology, Budapest University of Technology and Economics,
PO Box 91, H-1521 Budapest, Hungary
c Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, PO Box 91, H-1521 Budapest, Hungary
Received 29 August 2007; received in revised form 16 October 2007; accepted 1 November 2007
Available online 7 November 2007
´
Dedicated to Professor Csaba Szantay on the occasion of his 80th birthday
Abstract
New, paramagnetic unnatural a-amino acids were synthesized by the O’Donnell method. In the new amino acids nitroxide is condensed with
thiophene, benzene, and tetrahydroisoquinoline ring, or linked through a methylene, benzyl or propargyl spacer. Some of the racemic paramag-
netic a-amino acid esters described earlier or in this work were resolved by fractional crystallization of diastereomeric salts. Another approach
for optically active paramagnetic amino acids is the modification of S-tyrosine derivatives with Pd-catalyzed cross-coupling reactions with para-
magnetic acetylene and with a paramagnetic boronic acid.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Amino acid; Nitroxides; O’Donnell reaction; Pd-catalyzed cross-coupling; Resolution
1. Introduction
unnatural amino acids into proteins in vivo opens up entirely
new pathways for determining protein function and structure.
The molecular engineering of the translational machinery that
consists of transfer RNA and aminoacyl tRNA synthetase to
recognize unnatural amino acids makes possible the incorpo-
ration of an unnatural amino acid into a specific protein site
during translation by applying amber codon as the genetic
codon for unnatural amino acids.4 This new method induced
a renaissance in synthesizing various unnatural amino acids.
A helicogenic paramagnetic amino acid, TOAC (1)5
(Scheme 1) has been widely used and incorporated into
many peptides such as alamethicin,6 phospholamban,7 and
melanocortin peptides8 just to mention a few recent examples.
Proteins, consisting of 20 natural amino acids are responsible
for the majority of functional attributes of living systems from
viruses to mammals. These biopolymers are formed by natural
biosynthetic pathways at the ribosome. The physical and chem-
ical properties of proteins are a reflection of the side chains of
each of the constituent amino acids. However, for studying func-
tion and structure of proteins by various spectroscopic methods
it is often necessary to have special groups on the amino acids
with various selected physical and chemical properties, such as
fluorescent-, NMR-, EPR-probe group and crosslinker.1
One of the most convenient and economical methods for
studying a protein is the chemical or biochemical modification
of it, mainly at the amino acid side chain,2 however, this
method requires high function- and regioselectivity. The other
approach is the incorporation of amino acids into the protein
by classical Merrifield amino acid synthesis or by solid-phase
peptide synthesis.3 Recent development in incorporating
CO2C2H5
CO2C2H5
H2N
HN
H2N CO2H
N
O
3
N
O
2
N
O
1
* Corresponding author. Tel.: þ36 72 536 220; fax: þ36 72 536 219.
E-mail address: kalman.hideg@aok.pte.hu (K. Hideg).
Scheme 1. Structures of TOAC (1) and PTOAC (2) and paramagnetic homo-
proline (rac)-(3).
0040-4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.11.020

T. Ka´lai et al. / Tetrahedron 64 (2008) 1094e1100
1095
From our laboratory a new pyrroline nitroxide based para-
magnetic achiral amino acid ester, so called PTOAC (2) with
a rigid side chain has been reported.9 However, faced with
difficulties of a many-step synthesis9,10 and low yield because
of the concurrent formation of compound (rac)-3, we returned
to the synthesis of a-amino acids with flexible chains by the
O’Donnell synthesis,11,12 alkylating N-dibenzylidene glycine
ester with various paramagnetic allylic bromides yielding
paramagnetic amino acids with different size, polarity, and
side-chain length.13
than TOAC, because as non-helicogenic amino acids can be
incorporated into b-strands. TOAC is capable of studying
the backbone dynamics, while newly synthesized amino acids
with paramagnetic side chains designed to mimic salient
features of the native side chains they replace, moreover a pyr-
roline nitroxide is more stable toward chemical manipulations
than a piperidine nitroxide.
2. Results and discussion
Our intention on to continue this research was inspired by
the fact that cysteine and tyrosine modified with a spin label
were incorporated into Xenopus oocytes using the nonsense
suppression technique14 and compound (rac)-25 was incorpo-
rated into Ras-binding domain of c-Raf 1 protein by using an
expressed protein ligation.15 The drawback of the O’Donnell
synthesis is that racemic amino acids are formed, so we
thought that their resolution would be desirable as well as
the synthesis of new paramagnetic amino acids, which mimic
more precisely the natural amino acids. Several methods have
been used for the resolution of amino acids.16 In the present
study we used the chiral recognition between N-acetyl amino
acids and amino acid esters.17 We proposed that amino acid
esters, similarly to phenylalanine, could be resolved by enan-
tiomers of N-acetylphenylglycine.18
2.1. Synthesis of new paramagnetic amino acids
For the synthesis of a paramagnetic amino acid containing
an isoindoline side chain, ester (4)10 was reduced to alcohol
(5) by refluxing with a suspension of NaBH4 in tert-BuOH,
while MeOH was added dropwise19 (Scheme 2). Treatment
of alcohol (5) with methanesulfonyl chloride in the presence
of Et3N gave the mesylate, which was converted to bromo
compound (6) with LiBr in acetone.20 Alkylation of N-diphe-
nylmethylene glycine ethyl ester under phase transfer condi-
tions followed by hydrolysis of the Schiff base resulted in
the formation of racemic amino acid ethyl ester (rac)-(7).12
To get an amino acid with a rigid spacer between the nitro-
xide ring and a-carbon atom we introduced a propargylic
spacer. Sonogashira reaction21 seemed the most reasonable
solution for this problem, therefore treatment of vinylic iodide
822 with a tert-butyldimethyl-(2-propynyloxy)silane in the
presence of CuI and PdCl2(Ph3P)2 catalysts in Et3N as a
solvent under N2, yielded the protected propargyl alcohol
derivative, which was deprotected with tetrabutylammonium
In this paper we report the synthesis of paramagnetic amino
acids and the resolution of new and known compounds as well
as modification of natural, amino acids with nitroxides by
Pd-catalyzed CeC bond forming reactions. These new amino
acids with a flexible side chain can find different applications
CO2C2H5
Br
OH
CO2CH3
NH2
b
c
a
N
O
N
O
N
O
5
N
O
4
(rac)-7
6
CO2C2H5
NH2
Br
OH
I
b
c
d
N
O
N
O
N
O
8
N
O
9
(rac)-11
10
CO2C2H5
CO2C2H5
NH2
HN
Br
Br
Br
S
S
e
c
N
O
N
O
N
O
N
O
(rac)-13
(rac)-15
12
14
Scheme 2. (a) NaBH4 (7.0 equiv), MeOH (excess), tert-BuOH, 3 h, reflux, 61%; (b) MsCl (1.1 equiv), Et3N (1.1 equiv) CH2Cl2, 0 ꢀC, 1 h, then LiBr (2.0 equiv),
acetone, reflux, 30 min (45e69%); (c) Ph2C]NCH2CO2Et (1.0 equiv), 10% aq NaOH, CH2Cl2, Bu4NHSO4 (0.5 equiv), rt, 2 h, then 5% aq H2SO4, EtOH, 30 min,
rt, then solid K2CO3 to pH¼8, 15e52%; (d) HC^CHCH2OTBDMS (2.0 equiv), CuI (0.05 equiv), PdCl2(PPh3)2 (0.1 equiv), Et3N, under N2, rt, 5 h, then Bu4NF
(1 equiv), THF, rt, 30 min, 58%.

1096
T. Ka´lai et al. / Tetrahedron 64 (2008) 1094e1100
CO2CH3
NHBoc
fluoride to yield compound 9. This compound was then
converted into propargylic bromide (10) derivative via the
mesylate. By utilizing the O’Donnell procedure as above,
compound (rac)-11 was achieved after hydrolysis of the imine.
The same O’Donnell method was applied for synthesis of
amino acid ester (rac)-(13) with a thieno[2,3-c]pyrrole moiety
containing side chain starting from bromo compound23 (12) as
well as for the synthesis of tetrahydroisoquinoline amino acid
(rac)-15 starting from dibromo compound (14).24 Under these
conditions the dialkylation of a-carbon atom does not occur,
while monoalkylation of a-carbon atom and nitrogen alkyl-
ation under work-up conditions has been observed instead.10
1,2,3,4-Tetrahydroisoqunoline-3-carboxylic acid is a widely
used unnatural amino acid building block occurring in ACE
inhibitor Quinapril25 and in opioid peptide antagonists,26
therefore its paramagnetic analogue may find important
applications.
CO2R
CO2CH3
NHBoc
NHBoc
a
O
c
I
N
O
N
OH
OH
(S)-16
O
N
O
(S)-20
17
(S)-18, (S)-19
R
18
19
CH3
H
b
CO2C2H5
NHBoc
CO2C2H5
O
NHBoc
B
O
e
d
(+)-24
+
N
O
N
O
O
SO2CF3
2.2. Synthesis of new paramagnetic amino acids from
S-tyrosine derivatives
(S)-21
22
(S)-23
Scheme 3. (a) 17 (1.5 equiv), CuI (0.05 equiv), PdCl2(PPh3)2 (0.1 equiv),
Et3N, under N2, rt, 5 h, 39%; (b) 1.1 equiv NaOH, MeOH/water, rt, 2 h,
then Hþ, 78%; (c) Bu4NF, THF, reflux, 1 h, 39%; (d) 21 (1.0 equiv), dioxane,
Pd(PPh3)4, 10% aq Na2CO3, reflux, 2 h, 31%; (e) Boc2O (1.25 equiv), Et3N
(1.25 equiv), CH2Cl2, rt, 2 h, 82%.
A convenient approach for the synthesis of chiral paramag-
netic amino acid is the modification (alkylation) of the side
chain of a natural amino acid.13 The disadvantage of this ap-
proach is that modification of the heteroatom may lead to
the loss of bioactive properties. It was obvious that the car-
bonecarbon bond is required, however, under mild conditions,
which are compatible with ester and protected nitrogen. After
considering the possibilities of amino acid modifications27,28
we decided a utilization of the Pd-catalyzed cross-coupling
reactions.29 The Sonogashira reaction of N-protected S-3-iodo-
tyrosine ester30 (S )-16 with 17 paramagnetic acetylene10 un-
der conditions mentioned above yielded (S )-18 paramagnetic
amino acid ester, which could be hydrolyzed to (S )-19 carb-
oxylic acid with NaOH in aq methanol (Scheme 3). It was
obvious that compound (S )-18 as a 2-alkynylphenol offers
the construction of a benzo[b]furan side chain. Of the possible
cyclization techiques31 we used the treatment with tetrabutyl-
ammonium fluoride in THF32 to get the benzofuran side chain
containing amino acid (S )-20. The Suzuki reaction was also
a useful tool in the synthesis of paramagnetic phenylalanine
(S )-23 of which racemic form was reported earlier from our
laboratory.13 Treatment of N-protected S-tyrosine ethyl ester
O-triflate24 (S )-21 with paramagnetic boronic acid ester 2233
in dioxane/aq Na2CO3 solution in the presence of Pd(PPh3)4
catalyst afforded paramagnetic phenylalanine (S )-23, with
a pyrroline nitroxide ring in the 4-position of the aromatic
ring. The specific rotation of (S )-23 was þ3.3 with 0.2 less
than the resolved authentic sample (with ee 99%) indicating
that minimal racemization had occurred during the Suzuki
reaction conditions.
four cases amino acid esters were treated with half an equiva-
lent resolving agent in ethyl acetate. The precipitated diaste-
reomer salts were resuspended in ethyl acetate and heated to
reflux. The diastereomeric salts of (rac)-7, (rac)-13, and
(rac)-24 were recrystallized in the presence of triethylamine
equivalent with the less stable diastereomer, while (rac)-25
was filtered after cooling and 30 min stirring. This procedure
resulted in high enantiomer excess (ee>96%) for amino acids
24 and 25, in the case of compound 7 limited enantiomer
excess (ee 48%) and low enantiomer excess (16%) for com-
pound 13 were achieved. The absolute configuration in case
of (þ)-24 was found to be (S ), in other cases only the direction
of rotation is indicated (Scheme 3).
3. Conclusion
In conclusion, we have reported the synthesis of a new se-
ries of paramagnetic amino acids. In the case of the O’Donnell
synthesis racemic amino acids were formed. Some of them
could be resolved by fractional crystallization of diastereo-
meric salts from excellent to moderate enantiomeric excess.
The other method for achieving paramagnetic chiral amino
acids is further derivatization of natural amino acid derivatives
with Pd-catalyzed cross-coupling reactions, although this
method was limited to amino acids with an aromatic side
chain. The improvement of resolution techniques for paramag-
netic amino acids containing annellated pyrroline ring as well
as incorporation of optically active amino acids into proteins is
in progress.
2.3. Resolution of some racemic, paramagnetic amino
acids
Four amino acid esters ((rac)-7, (rac)-13, (rac)-24, (rac)-
25) were resolved with (R)-N-acetylphenylglycine. In all

T. Ka´lai et al. / Tetrahedron 64 (2008) 1094e1100
1097
4. Experimental
was separated, dried (MgSO4), filtered, and evaporated. The
residue was dissolved in anhyd acetone (20 mL), LiBr
(1.74 g, 20.0 mmol) was added and the mixture stirred and re-
fluxed for 30 min. After cooling, the acetone was evaporated
off and the residue was partitioned between water (10 mL)
and EtOAc (30 mL). The organic phase was separated, dried
(MgSO4), filtered, and evaporated. The residue was purified
by flash column chromatography (hexane/Et2O) to give
compounds 6 and 10.
4.1. General
Melting points were determined with a Boetius micro melt-
ing point apparatus. Elemental analyses (C, H, N, S) were per-
formed on Carlo Erba EA 1110 CHNS elemental analyzer.
Mass spectra were recorded on an Automass Multi instrument
in the EI mode (70 eV, direct inlet). ESI-TOF MS measure-
ments were performed with a BioTOF II instrument (Bruker
Daltonics, Billerica, MA). IR spectra were taken on Specord
M85 instrument. ESR spectra were obtained from 10ꢁ5 M so-
lutions (CHCl3), using a Magnettech MS200 spectrometer.
Preparative flash column chromatography was performed on
Merck Kieselgel 60 (0.040e0.063 mm). Optical purity was
determined by HPLC (Jasco UV-1575 detector, Jasco
PU-1580 pump) containing Chiralpack AD column at
T¼20 ꢀC with 0.8 mL/min flow rate. The mobile phase con-
sisted of 75% hexane and 25% EtOH and UV detection was
performed at 256 nm. Optical rotations were recorded at
20 ꢀC, with a PerkineElmer 343 polarimeter. Qualitative
TLC was carried out on commercially prepared plates (20ꢂ
20ꢂ0.02 cm) coated with Merck Kieselgel GF254. Dibenz-
ylidene glycine ethyl ester11 and compounds 1,5 2,9 3,9 4,10
8,22 12,23 14,24 1630 (the same as the reported R isomer),
17,10 21,27 22,33 23,13 and 2412 were prepared as described
earlier. tert-Butyldimethyl(2-propynyloxy)silane and all other
reagents and compounds were purchased from Aldrich or
Fluka. Triethylamine (Et3N) was distilled from CaH2 prior
to use.
4.3.1. 5-Bromomethyl-1,1,3,3-tetramethyl-1,3-dihydro-
2H-isoindol-2-yloxyl radical (6)
Yield 1.95 g (69%). Mp 108e110 ꢀC, Rf 0.30 (hexane/Et2O
2:1). IR Nujol (n): 1690, 1550 (C]C). EA calcd for
C13H17BrNO: C, 55.14; H, 6.05; N, 4.95. Found: C, 55.20;
H, 6.00; N, 5.86. MS (EI) (m/z): 284/282 (Mþ, 91/91), 269/
267 (38) 254/252 (17/17), 188 (100).
4.3.2. 3-(3-Bromoprop-1-ynyl)-2,2,5,5-tetramethyl-
2,5-dihydro-1H-pyrrol-1-yloxyl radical (10)
Yield 1.15 g (45%). Mp 82e83 ꢀC, Rf 0.39 (hexane/Et2O
2:1). EA calcd for C11H15BrNO: C, 51.38; H, 5.88; N, 5.45.
Found: C, 51.23; H, 5.72; N, 5.43. IR Nujol (n): 1600, 1555
(C]C). MS (EI) (m/z): 258/256 (Mþ, 20/20), 243/241 (26/
26), 228/226 (50), 162 (65), 41 (100).
4.4. 3-(3-Hydroxyprop-1-ynyl)-2,2,5,5-tetramethyl-2,5-
dihydro-1H-pyrrol-1-yloxyl radical (9)
The mixture of 8 iodo compound (1.33 g, 5.0 mmol),
PdCl2(PPh3)2 (350 mg, 0.5 mmol), and CuI (48 mg,
0.25 mmol) in anhyd Et3N (15 mL) was stirred and deoxygen-
ated with N2 for 15 min. Then tert-butyldimethyl(2-propynyl-
oxy)silane (1.73 g, 10.0 mmol) dissolved in anhyd Et3N
(5 mL) was added dropwise causing an immediate brown
precipitation from the yellow solution. Within 10 min the
mixture became a thick slurry and was vigorously stirred for
5 h at ambient temperature. The mixture was diluted with
EtOAc (20 mL), filtered through a Celite pad, washed with
EtOAc (5 mL) then the solvents were evaporated off. The
residue was partitioned between Et2O (30 mL) and
water (10 mL), the organic phase was separated, dried
(MgSO4), filtered, and evaporated. The crude product was
dissolved in anhyd THF (20 mL) and tetrabutylammonium
fluoride (5 mL, 5.0 mmol) in THF (1.0 M stock solution)
was added and the mixture was stirred at ambient temperature
for 30 min. Then THF was evaporated off, the crude product
was dissolved in CHCl3 (20 mL), washed with water
(10 mL), the organic phase was separated, dried (MgSO4), fil-
tered, and evaporated. The residue was purified by flash col-
umn chromatography (hexane/EtOAc) to yield 9 alcohol as
a yellow solid (970 mg, 58%). Mp 82e84 ꢀC, Rf 0.26 (hex-
ane/EtOAc 2:1). EA calcd for C11H16NO2: C, 68.01; H,
8.30; N, 7.21. Found: C, 68.12; H, 8.28; N, 7.15. IR Nujol
(n): 3320 (OH), 1600, 1540 (C]C). MS (EI) (m/z): 194
(Mþ, 32), 179 (71), 164 (100).
4.2. 5-Hydroxymethyl-1,1,3,3-tetramethyl-1,3-dihydro-
2H-isoindol-2-yloxyl radical (5)
To a stirred refluxing suspension of 4 ester (2.48 g,
10.0 mmol) and NaBH4 (2.64 g, 70.0 mmol) in anhyd tert-
BuOH (20 mL) a mixture of anhyd tert-BuOH/MeOH
(12:8 mL) was added dropwise over 3 h under N2. After
cooling, the mixture was concentrated in vacuo, quenched
with water (30 mL), extracted with CHCl3 (2ꢂ20 mL), dried
(MgSO4), filtered, and evaporated. The crude alcohol was pu-
rified by flash chromatography (hexane/EtOAc) to yield com-
pound 5 (1.34 g, 61%) as a yellow solid. Mp 108e110 ꢀC, Rf
0.20 (hexane/EtOAc 2:1). EA calcd for C13H18NO2: C, 70.88;
H, 8.24; N, 6.36. Found: C, 70.73; H, 8.15; N, 6.26. IR Nujol
(n): 3420 (OH), 1600, 1550 (C]C). MS (EI) (m/z): 220 (Mþ,
61), 205 (81), 190 (72), 105 (100).
4.3. General procedure for synthesis of bromo
compounds (6,10)
To a solution of alcohol 5 or 9 (10.0 mmol) and Et3N
(1.11 g, 11.0 mmol) in CH2Cl2 (20 mL) methanesulfonyl chlo-
ride (1.25 g, 11.0 mmol) was added dropwise at 0 ꢀC and the
mixture was stirred at ambient temperature for 60 min. Then
the mixture was washed with water (15 mL), the organic phase

1098
T. Ka´lai et al. / Tetrahedron 64 (2008) 1094e1100
4.5. General procedure for synthesis of amino acid esters
(rac)-7, -11, -13, -15
4.6. (S )-2-tert-Butoxycarbonylamino-3-[4-hydroxy-3-
(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-
ylethynyl)phenyl]propionic acid methyl ester radical (18)
To stirred solution of N-diphenylmethylene glycine ethyl
ester (801 mg, 3.0 mmol) and compound 6 or 10 or 12 or 14
(3.0 mmol) in CH2Cl2 (20 mL) and aq 10% NaOH (3 mL or
6 mL in case of 14) was added followed by addition of
Bu4NHSO4 (508 mg, 1.5 mmol) and the mixture was stirred
at rt for 2 h. The organic phase was separated, dried
(MgSO4), filtered, and evaporated to give the crude imine,
which was immediately subjected to acidic hydrolysis. The
residue was dissolved in EtOH (20 mL) and 5% aq H2SO4
(5 mL) was added and the mixture was allowed to stand at am-
bient temperature and the mixture was monitored by TLC. Af-
ter consumption of Schiff base (ca. 30 min) water (10 mL) was
added and the pH¼8 was adjusted by adding solid K2CO3. The
mixture was extracted with CHCl3 (2ꢂ20 mL), then organic
phase was separated, dried (MgSO4), filtered, evaporated,
and the residue was purified by flash column chromatography
(CHCl3/MeOH) to give racemates of 7, 11, 13, 15 amino acid
esters (15e52%) as yellow oils.
A solution of (S )-N-tert-butoxycarbonyl-3-iodo-tyrozine
methyl ester (842 mg, 2.0 mmol), PdCl2(PPh3)2 (140 mg,
0.2 mmol), and CuI (19 mg, 0.1 mmol) in anhyd Et3N
(10 mL) was stirred and deoxygenated with N2 for 15 min.
Then compound 17 (492 mg, 3.0 mmol) dissolved in anhyd
Et3N (5 mL) was added dropwise causing an immediate brown
precipitation from the yellow solution. Within 10 min the mix-
ture became a thick slurry and was vigorously stirred for 5 h at
ambient temperature. The mixture was diluted with EtOAc
(20 mL), filtered through a Celite pad, washed with EtOAc
(5 mL) then the solvents were evaporated off. The residue
was partitioned between Et2O (30 mL) and water (10 mL),
the organic phase was separated, dried (MgSO4), filtered,
and evaporated. The residue was purified by flash column
chromatography (hexane/EtOAc) to give the title compound
18 (356 mg, 39%) as a beige solid. Mp 126e128 ꢀC, Rf 0.18
(hexane/EtOAc 2:1), [a]D þ5 (c 0.21, MeOH). EA calcd for
C25H33N2O6: C, 65.63; H, 7.27; N, 6.12. Found: C, 65.59;
H, 7.22; N, 6.05. IR Nujol (n): 3300, 1730, 1680 (C]O),
1580, 1540 (C]C). MS (EI) (m/z): 457 (Mþ, 3), 427 (3),
371 (4), 57 (100).
4.5.1. (rac)-2-Amino-3-(2-oxyl-1,1,3,3-tetramethyl-1,3-
dihydro-2H-isoindol-5-yl)propionic acid ethyl ester
radical (7)
Yield 476 mg (52%), yellow oil, Rf 0.08 (CHCl3/Et2O 2:1).
EA calcd for C17H25N2O3: C, 66.86; H, 8.25; N, 9.17. Found:
C, 66.80; H, 8.14; N 9.01. IR neat (n): 3380, 3310 (NH2), 1730
(C]O), 1610 (C]C). MS (EI) (m/z): 305 (Mþ, 33), 290 (4),
232 (37), 189 (100).
4.7. (S )-2-tert-Butoxycarbonylamino-3-[4-hydroxy-3-
(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-
ylethynyl)phenyl]propionic acid radical (19)
To a solution of 18 ester (228 mg, 0.5 mmol) in MeOH
(5 mL) and NaOH (22 mg, 0.55 mmol) dissolved in water
(5 mL) was added in one portion, the mixture was allowed
to stand at rt and monitored by TLC. After consumption of
the ester (w2 h), water (10 mL) was added, the MeOH was
evaporated off. The aqueous phase was cautiously acidified
to pH¼5 with 5% aq H2SO4. The turbid solution was extracted
with CHCl3 (2ꢂ10 mL), the organic phase was separated,
dried (MgSO4), filtered, and evaporated. The residue was pu-
rified further with flash chromatography to give 19 acid as
a pale yellow solid (172 mg, 78%). Mp 178e180 ꢀC, Rf 0.68
(CHCl3/MeOH, 4:1), [a]D þ21 (c 0.21, MeOH). EA calcd
for C24H31N2O6: C, 64.99; H, 7.05; N, 6.32. Found: C,
65.02; H, 7.01; N, 6.25. IR neat (n): 3300 (OH), 1670
(C]O), 1570, 1550 (C]C). MS (ESI) (m/z): 442 (MꢁH)ꢁ.
4.5.2. (rac)-2-Amino-5-(1-oxyl-2,2,5,5-tetramethyl-2,5-
dihydro-1H-pyrrol-3-yl)-pent-4-ynoic acid ethyl
ester radical (11)
Yield 335 mg (40%), yellow oil, Rf 0.10 (CHCl3/Et2O 2:1).
EA calcd for C15H23N2O3: C, 64.49; H, 8.30; N, 10.03. Found:
C, 64.44; H, 8.32; N, 10.00. IR neat (n): 3380, 3300 (NH2),
2210 (C^C), 1730 (C]O), 1610 (C]C). MS (EI) (m/z):
279 (Mþ, 28), 264 (3), 249 (1), 234 (7), 102 (100).
4.5.3. (rac)-2-Amino-3-(5-oxyl-4,4,6,6-tetramethyl-4,6-
dihydro-5H-thieno[2,3-c]pyrrol-2-yl)propionic acid
ethyl ester radical (13)
Yield 354 mg (38%), yellow oil, Rf 0.11 (CHCl3/Et2O 2:1).
EA calcd for C15H23N2O3S: C, 57.85; H, 7.44; N, 9.00; S,
10.30. Found: C, 57.70; H, 7.28; N, 8.98; S, 10.21. IR neat
(n): 3390, 3310 (NH2), 1730 (C]O), 1600 (C]C). MS (EI)
(m/z): 311 (Mþ, 2), 281 (63), 238 (6), 179 (100).
4.8. (S )-2-tert-Butoxycarbonylamino-3-[2-(1-oxyl-
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-
benzofuran-5-yl]propionic acid methyl ester radical (20)
4.5.4. (rac)-2-Oxyl-1,1,3,3-tetramethyl-1,3,5,6,7,8-
hexahydro-2H-pyrrolo[3,4-g]isoquinoline-7-carboxylic
acid ethyl ester radical (15)
Yield 142 mg (15%), yellow oil, Rf 0.38 (CHCl3/Et2O 2:1).
EA calcd for C18H25N2O3: C, 68.11; H, 7.94; N, 8.83. Found:
C, 68.01; H, 7.92; N, 8.85. IR neat (n): 3410 (NH), 1740
(C]O), 1620, 1555 (C]C). MS (EI) (m/z): 317 (Mþ, 30),
287 (6), 244 (74), 229 (100).
A solution of 18 (457 mg, 1.0 mmol) and tetrabutylammo-
nium fluoride (1.0 M stock solution, 2 mL, 2.0 mmol) in THF
(10 mL) was refluxed for 1 h. The solvent was evaporated off,
the residue was dissolved in EtOAc (10 mL), washed with
water (10 mL), the organic phase was dried over MgSO4, fil-
tered, evaporated, and the residue was purified by flash column
chromatography to give the title compound (178 mg, 39%) as

T. Ka´lai et al. / Tetrahedron 64 (2008) 1094e1100
1099
a pale brown solid. Mp 44e47 ꢀC, Rf 0.37 (hexane/EtOAc,
2:1), [a]D þ2 (c 0.20, MeOH). EA calcd for C25H33N2O6:
C, 65.63; H, 7.27; N, 6.12. Found: C, 65.59; H, 7.22; N,
6.05. IR neat (n): 3320 (NH), 1740, 1780 (C]O), 1610
(C]C). MS (EI) (m/z): 457 (Mþ, 2), 427 (3), 371 (15), 304
(85), 57 (100).
(5 mL and 5 mLþ30 mL Et3N, respectively) to give yellowish
powder (0.289 g, 0.54 mmol, 44.1%), [a]2D0 ꢁ59.4 (c 0.5,
MeOH). To this salt CH2Cl2 (5 mL) and saturated aq sodium
bicarbonate and sodium chloride solution (1 mL) were added
and it was stirred for 5 min. After separation of the aq phase,
it was further extracted by CH2Cl2 (2ꢂ5 mL). The organic
phase was dried over Na2SO4 and solvent was evaporated to
give (þ)-2-amino-3-[4-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-
1H-pyrrol-3-yl)phenyl]propionic acid ethyl ester radical
(0.179 g 43.9%), [a]2D0 þ12.7 (c 0.5, methanol), ee 99%. Reten-
tion time for (þ)-24: 11.33 min and (ꢁ)-24: 12.57 min.
4.9. Synthesis of (S )-2-tert-butoxycarbonylamino-3-
[4-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-
3-yl)phenyl]propionic acid ethyl ester (23)
4.9.1. Method A
To a stirred solution of (S)-2-tert-butoxycarbonylamino-4-
[[(trifluoromethyl)sulfonyl]oxy]benzenepropionic acid ethyl
ester (882 mg, 2.0 mmol) in dioxane (10 mL) and after
deoxygenation with N2 for 10 min. Pd(PPh3)4 (100 mg,
0.086 mmol) was added, followed by addition of compound
22 (532 mg, 2.0 mmol), aq 10% Na2CO3 (5 mL) and the mix-
ture was stirred and refluxed for 2 h. After cooling, the
solvents were evaporated off, the residue was partitioned be-
tween EtOAc (20 mL) and water (10 mL). The organic phase
was washed with saturated aq sodium bicarbonate solution
(10 mL), aq 10% citric acid (10 mL), and saturated sodium
chloride solution (10 mL). The organic phase was separated,
dried (MgSO4), filtered, and evaporated. The residue was pu-
rified by flash column chromatography (hexane/EtOAc) to
give (267 mg, 31%) yellow semisolid. Rf 0.41 (hexane/EtOAc,
2:1), [a]D þ3.3 (c 0.5, methanol). EA calcd for C24H35N2O5:
C, 66.80; H, 8.17; N, 6.49. Found: C, 66.75; H, 8.19; N, 6.42.
IR neat (n): 3320 (NH), 1730, 1680 (C]O), 1610 (C]C).
MS (EI) (m/z): 431 (Mþ, 28), 401 (3), 360 (22) 199 (40),
57 (100).
4.11. (þ)-2-Amino-3-(1-oxyl-2,2,5,5-tetramethyl-2,5-
dihydro-1H-pyrrol-3-yl)propionic acid ethyl ester
radical (25)
(rac)-2-Amino-3-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-
1H-pyrrol-3-yl)propionic acid ethyl ester radical (25)
(1.600 g, 6.27 mmol) and (R)-N-acetylphenylglycine (0.605 g,
3.13 mmol) were dissolved in hot EtOAc (11 mL). After cool-
ing and seeding it was left at rt overnight. It was then filtered
and suspended and stirred twice with hot EtOAc (10 and 9 mL,
respectively) to give yellowish powder (0.500 g, 1.1 mmol,
35.0%), [a]2D0 ꢁ69.3 (c 0.5, MeOH). To this salt CH2Cl2
(5 mL) and saturated aq sodium bicarbonate and sodium chlo-
ride solution (1 mL) were added and it was stirred for 5 min.
After separation of the aq phase, it was further extracted
by CH2Cl2 (2ꢂ5 mL). The organic phase was dried over
Na2SO4 and solvent was evaporated to give (þ)-2-amino-
3-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)pro-
pionic acid ethyl ester radical (0.273 g, 34.1%), [a]2D0 þ11.5 (c
0.5, methanol), ee 96%. Retention time for (þ)-25: 6.11 min
and (ꢁ)-25: 7.03 min.
4.9.2. Method B
To a stirred solution of (þ)-24 amino acid ester (66 mg,
0.2 mmol) and Et3N (25 mL, 0.25 mmol) in CH2Cl2 (5 mL)
di-tert-butoxycarbonate (53 mg, 0.25 mmol) was added in
one portion and the mixture was stirred for 2 h at rt. Then
the solvent was washed with saturated aq NaHCO3 (5 mL),
water (5 mL), dried (MgSO4), filtered, and evaporated. The
residue was purified by flash column chromatography (hex-
ane/EtOAc) to give (71 mg, 82%) yellow semisolid. Rf 0.41
(hexane/EtOAc, 2:1), [a]D þ3.5 (c 0.51, methanol). EA calcd
for C24H35N2O5: C, 66.80; H, 8.17; N, 6.49. Found: C, 66.74;
H, 8.10; N, 6.39. All the spectroscopic data were identical with
the sample obtained by method A.
4.12. (þ)-2-Amino-3-(2-oxyl-1,1,3,3-tetramethyl-1,3-
dihydro-2H-isoindol-5-yl)propionic acid ethyl ester
radical (7)
(rac)-2-Amino-3-(2-oxyl-1,1,3,3-tetramethyl-1,3-dihydro-2H-
isoindol-5-yl)propionic acid ethyl ester radical (7) (0.747 g,
2.45 mmol)
and
(R)-N-acetylphenylglycine
(0.236 g,
1.22 mmol) were dissolved in hot EtOAc (5 mL). After cool-
ing and seeding it was left at rt overnight. To this suspension
giving diethyl ether (5 mL) and it was then filtered. Then
it was suspended and stirred twice with hot EtOAc
(5 mLþ50 mL Et3N and 5 mLþ10 mL Et3N, respectively) to
give yellowish powder (0.353 g, 0.71 mmol, 57.7%), [a]D20
ꢁ66.4 (c 0.5, MeOH). To this salt CH2Cl2 (5 mL) and satu-
rated aq sodium bicarbonate and sodium chloride solution
(1 mL) were added and it was stirred for 5 min. After separa-
tion of the aq phase, it was further extracted by CH2Cl2
(2ꢂ5 mL). The organic phase was dried over Na2SO4 and sol-
vent was evaporated to give (þ)-2-amino-3-(2-oxyl-1,1,3,3-
tetramethyl-1,3-dihydro-2H-isoindol-5-yl)propionic acid ethyl
ester radical (0.216 g, 57.7%), [a]2D0 þ7.7 (c 0.5, methanol).
Retention time for (þ)-7: 5.91 min and (ꢁ)-7 6.45 min.
4.10. (þ)-2-Amino-3-[4-(1-oxyl-2,2,5,5-tetramethyl-
2,5-dihydro-1H-pyrrol-3-yl)phenyl]propionic acid
ethyl ester radical (24)
(rac)-2-Amino-3-[4-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-
1H-pyrrol-3-yl)phenyl]propionic acid ethyl ester radical
(24) (0.815 g, 2.46 mmol) and (R)-N-acetylphenylglycine
(0.240 g, 1.23 mmol) were dissolved in hot EtOAc (5.6 mL).
After cooling and seeding it was left at rt overnight. It was
then filtered and suspended and stirred twice with hot EtOAc

1100
T. Ka´lai et al. / Tetrahedron 64 (2008) 1094e1100
3. Merrifield, B. Methods Enzymol. 1997, 289, 3e13.
4. (a) Rodrigez, E. A.; Lester, H. A.; Dougherty, D. A. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 8650e8655; (b) England, P. M. Biochemistry 2004, 43,
11623e11629; (c) Liao, J. Biotechnol. Prog. 2007, 23, 28e31.
5. Rassat, A.; Rey, P. Bull. Soc. Chim. Fr. 1967, 815e817.
6. Marsh, D.; Jost, M.; Peggion, C.; Toniolo, C. Biophys. J. 2007, 92, 473e
481.
4.13. (ꢁ)-2-Amino-3-(5-oxyl-4,4,6,6-tetramethyl-4,6-
dihydro-5H-thieno[2,3-c]pyrrol-2-yl)propionic acid
ethyl ester radical (13)
(rac)-2-Amino-3-(5-oxyl-4,4,6,6-tetramethyl-4,6-dihydro-
5H-thieno[2,3-c]pyrrol-2-yl)propionic acid ethyl ester radical
13 (103 mg, 0.33 mmol) and (R)-N-acetylphenylglycine
(31.9 mg, 0.16 mmol) were dissolved in hot EtOAc (1.3 mL).
After cooling and seeding it was left at rt overnight. To this
suspension giving diethyl ether (1 mL) and it was then filtered.
Then it was suspended and stirred twice with hot EtOAc
(1.3 mLþ4 mL Et3N and 1.3 mLþ4 mL Et3N, respectively) to
give yellowish powder (28.4 mg, 0.06 mmol, 33.6%), [a]D20
ꢁ72.3 (c 0.5, MeOH). To this salt CH2Cl2 (5 mL) and satu-
rated aq sodium bicarbonate and sodium chloride solution
(1 mL) were added and it was stirred for 5 min. After separa-
tion of the aq phase, it was further extracted by CH2Cl2
(2ꢂ5 mL). The organic phase was dried over Na2SO4 and sol-
vent was evaporated to give (ꢁ)-2-amino-3-(5-oxyl-4,4,6,6-
tetramethyl-4,6-dihydro-5H-thieno[2,3-c]pyrrol-2-yl)propionic
acid ethyl ester radical (15.6 mg, 30.3%), [a]2D0 ꢁ0.7 (c 0.5,
methanol), ee 16%. Retention time for (þ)-13: 8.28 min and
(ꢁ)-13: 14.64 min.
7. Zhang, Z. W.; Remmer, H. A.; Thomas, D. D.; Karim, C. B. Biopolymers
2007, 88, 29e35.
8. Fernandez, M.; Vieira, R. F. F.; Nakaie, C. B.; Ito, C. A.; Lamy, A. T.
Peptides 2005, 26, 1825e1834.
´
9. Balog, M.; Kalai, T.; JekT, J.; Berente, Z.; Steinhoff, H.-J.; Engelhard, M.;
Hideg, K. Tetrahedron Lett. 2003, 44, 9213e9217.
´
10. Kalai, T.; Balog, M.; JekT, J.; Hideg, K. Synthesis 1999, 973e980.
11. O’Donnell, M. J.; Boniece, J. M.; Earp, S. E. Tetrahedron Lett. 1978, 30,
2641e2644.
12. Lex, L.; Hideg, K.; Hankovszky, H. O. Can. J. Chem. 1982, 60, 1448e
1451.
´
13. Balog, M.; Kalai, T.; JekT, J.; Steinhoff, H. J.; Engelhard, M.; Hideg, K.
Synlett 2004, 2591e2593.
´
14. Shafer, A. M.; Kalai, T.; Liu, S. Q. B.; Hideg, K.; Voss, J. Biochemistry
2004, 43, 8470e8482.
15. Becker, C. F. W.; Lausecker, K.; Balog, M.; Kalai, T.; Hideg, K.;
´
Steinhoff, H.-J.; Engelhard, M. Magn. Reson. Chem. 2005, 46, S34eS39.
16. (a) Yoshioka, R. Top. Curr. Chem. 2007, 269, 83e132; (b) Zimmermann,
V.; Beller, M.; Kragl, U. Org. Process Res. Dev. 2006, 10, 622e627; (c)
ꢀ
´
´
´
Peter, A.; Arki, A.; Tourwe, D.; Forro, E.; Fulop, F.; Armstrong, D. W.
¨ ¨
J. Chromatogr., A 2004, 1031, 159e170; (d) Liljeblad, A.; Kiviniemi,
A.; Kanerva, L. T. Tetrahedron 2004, 60, 671e677.
ꢀ
17. Fogassy, E.; Faigl, F.; Acs, M. Hungarian Patent 193202, Hungary, 1984.
Chem. Abstr. 1986, 104, 168835.
Acknowledgements
´
18. Fogassy, E.; Nogradi, M.; Kozma, D.; Egri, G.; Palovics, E.; Kiss, V. Org.
´
´
This work was supported by a grant from the Hungarian
National Research Fund (OTKA T048334, T67597, T042725
and OMFB-00525/2001, M 045190). The authors wish to
Biomol. Chem. 2006, 4, 3011e3030.
19. Soai, K.; Oyamada, H.; Ookawa, A. Synth. Commun. 1982, 12, 463e467.
20. Hankovszky, H. O.; Hideg, K.; Lex, L. Synthesis 1980, 914e916.
21. Sonogashira, K. J. Organomet. Chem. 2002, 653, 46e49.
´
thank Krisztina Kis for elemental analysis, Dr. Jozsef JekT
´
´
22. Kalai, T.; Bognar, B.; JekT, J.; Hideg, K. Synthesis 2006, 2573e2579.
´
(Department of Chemistry, College of Nyıregyhaza) for MS
´
´
23. Kalai, T.; Balog, M.; JekT, J.; Hideg, K. Synthesis 1998, 1476e1482.
measurements.
´
´
24. Kulcsar, Gy.; Kalai, T.; JekT, J.; Hideg, K. Synthesis 2003, 1361e1366.
25. Klutchko, S.; Blankley, C. J.; Fleming, R. W.; Hinkley, J. M.; Werner,
A. E.; Nordin, I.; Holmes, A.; Hoefle, M. L.; Cohen, D. M.; Essenburg,
A. D.; Kaplan, H. R. J. Med. Chem. 1986, 29, 1953e1961.
References and notes
´
26. Toth, G.; Ioja, E.; Tomboly, C.; Ballet, S.; Tourwe, D.; Peter, A.; Martinek,
1. (a) Ellmann, J. A.; Volkman, B. F.; Mendel, D.; Shultz, P. G.; Wemmer,
D. E. J. Am. Chem. Soc. 1992, 114, 7959e7960; (b) Inbaraj, J. J.; Cardon,
T. B.; Lariukhin, M.; Lorigan, G. A. J. Am. Chem. Soc. 2006, 128, 9549e
¨
¨
T.; Chung, N. N.; Schiller, P. W.; Benyhe, S.; Borsodi, A. J. Med. Chem.
2007, 50, 328e333.
´
27. Sieh, W.-C.; Carlson, J. A. J. Org. Chem. 1992, 57, 379e381.
28. Walker, W. H.; Rokita, S. E. J. Org. Chem. 2003, 68, 1563e1566.
29. Tsuji, J. Palladium Reagents and Catalysts: New Perspectives for the 21st
Century; Wiley: Hoboken, NJ, 2004.
9554; (c) Kalai, T.; Hideg, K. Tetrahedron 2006, 62, 10352e10360.
2. (a) Berliner, L. J.; Grunwald, J.; Hankovszky, H. O.; Hideg, K. Anal. Bio-
¨
chem. 1982, 119, 450e455; (b) Cornish, V. W.; Benson, D. R.; Altenbach,
C. A.; Hideg, K.; Hubbell, W. L.; Schultz, P. G. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 2910e2914; (c) Hermanson, G. T. Bioconjugate
Techniques; Academic: San Diego, CA, 1996; (d) Hubbell, W. L.;
Altenbach, C.; Hubbell, C. M.; Khorana, H. G. Adv. Protein Chem.
30. Chiarello, J.; Joullie, M. M. Synth. Commun. 1988, 18, 2211e2223.
´
31. (a) Novak, Z.; Timari, G.; Kotschy, A. Tetrahedron 2003, 59, 7509e7513;
(b) Liang, Y.; Tang, S.; Zhang, X.-.D.; Mao, L.-.Q.; Xie, Y.-.X.; Li, J.-.H.
Org. Lett. 2006, 8, 3017e3020.
´
´
2003, 63, 243e290; (e) Balint, J.; Kiss, V.; Egri, G.; Kalai, T.; Demeter,
ꢀ
32. Hiroya, K.; Suzuki, N.; Yasuhara, A.; Egawa, Y.; Kasano, A.; Sakamoto,
T. J. Chem. Soc., Perkin Trans. 1 2000, 4339e4346.
A.; Balog, M.; Fogassy, E.; Hideg, K. Tetrahedron: Asymmetry 2004,
15, 671e679; (f) Fanucci, G. E.; Cafiso, D. S. Curr. Opin. Struct. Biol.
2006, 16, 644e653.
´
33. Kalai, T.; JekT, J.; Hideg, K. Tetrahedron Lett. 2004, 45, 8395e8398.