A Quaternary Nitronyl Nitroxide α-Amino Acid: Synthesis, Configurational and Conformational Assignments, and Physicochemical Properties

Article abstract of DOI:10.1002/ejoc.201301765

To expand the presently extremely limited repertoire of known nitronyl nitroxide α-amino acids, we report here the synthesis of a novel, conformationally constrained, quaternary, chiral residue, Aic(CN), and its subsequent conversion into a blue-colored, imidazolinyl nitronyl nitroxide-bearing α-amino acid, Aic(NN). Deprotection and peptide coupling reactions were examined. The configurational assignment of Aic(NN) was achieved by X-ray crystallographic analysis of an appropriate tripeptide. This latter investigation, accompanied by an IR absorption conformational analysis, strongly supports the view that the Aic(NN) residue is an efficient peptide turn former. Numerous spectroscopic and magnetic properties of its derivatives and the tripeptide are also described. Of particular relevance for future applications are its UV/Vis absorption, NMR, and EPR signatures.

Full text of DOI:10.1002/ejoc.201301765

FULL PAPER
DOI: 10.1002/ejoc.201301765
A Quaternary Nitronyl Nitroxide α-Amino Acid: Synthesis, Configurational
and Conformational Assignments, and Physicochemical Properties
Karen Wright,*[a] Edouard d’Aboville,[a] Joseph Scola,[b] Tommaso Margola,[c]
Antonio Toffoletti,[c] Marta De Zotti,[c] Marco Crisma,[d] Fernando Formaggio,[c,d] and
Claudio Toniolo[c,d]
Keywords: Sensors / EPR spectroscopy / Magnetic properties / Amino acids / Peptides / Protein modifications / Protein
folding
To expand the presently extremely limited repertoire of
known nitronyl nitroxide α-amino acids, we report here the
synthesis of a novel, conformationally constrained, quater-
nary, chiral residue, Aic(CN), and its subsequent conversion
into a blue-colored, imidazolinyl nitronyl nitroxide-bearing
α-amino acid, Aic(NN). Deprotection and peptide coupling
reactions were examined. The configurational assignment of
Aic(NN) was achieved by X-ray crystallographic analysis of
an appropriate tripeptide. This latter investigation, ac-
companied by an IR absorption conformational analysis,
strongly supports the view that the Aic(NN) residue is an ef-
ficient peptide turn former. Numerous spectroscopic and
magnetic properties of its derivatives and the tripeptide are
also described. Of particular relevance for future applications
are its UV/Vis absorption, NMR, and EPR signatures.
pared in attempts to increase ferromagnetic ordering by
creating extended structures.[13]
Introduction
Nitronyl nitroxides are members of a class of stable free
radicals that have attracted considerable interest, in particu-
lar as spin labels and organic magnetic materials. In this
context, the sub-class of the Ullman imidazolinyl nitronyl
nitroxide monoradicals[1] has been the most extensively in-
vestigated. The properties of these latter compounds have
been carefully examined by using UV/Vis-IR absorp-
tion,[1b,2,3a] Raman,[2a] circular dichroism (CD),[2c,4] fluores-
cence quenching,[3] electron paramagnetic resonance
(EPR),[1,5–7] and nuclear magnetic resonance[1e,5b,7d,8] spec-
troscopy, and by X-ray diffraction analysis.[2c,4b,7d,7e,8b,9]
Optically active nitronyl nitroxides have been synthesized in
which the chiral center is located either in the imidazolinyl
ring itself or at a site in the 2-substituent part of the mole-
cule.[10] These compounds, which combine optical and mag-
netic properties,[11] may exhibit interesting physical effects,
such as magnetochiral dichroism.[12] Numerous complexes
of nitronyl nitroxides with transition metals have been pre-
In the field of peptide chemistry, free radical spin labels
are used in conjunction with EPR spectroscopy particularly
in conformational studies. The nitronyl nitroxide moiety
has not, as yet, been widely employed for this application
compared with the more popular nitroxides, for example
sidechain Cys derivatives[14] and 4-amino-1-oxyl-2,2,6,6-
tetramethylpiperidine-4-carboxylic acid (TOAC).[15] A lim-
ited number of peptides have been previously spin labeled
with nitronyl nitroxides through N-terminal or sidechain
modification. Weinkam and Jorgensen[5] prepared nitronyl
nitroxyl alanine [Ala(NN)] from the known Asp β-semi-al-
dehyde. The sidechain of Ala(NN) resembles that of His.
This residue was incorporated into the C-terminal tripep-
tide of the hormone angiotensin II replacing His, and the
EPR spectrum of this analogue was examined. Peng and
co-workers[6] used nitronyl nitroxides derived from salicylal-
dehyde to spin label the N-terminal or the sidechains of
Lys, Arg, Asp and Glu in short peptides, and employed
these compounds to study free radical (e.g., nitric oxide)
scavenging and bioactivity. Both groups of authors were
able to show that these free radicals could survive most of
the experimental conditions typically used in peptide syn-
thesis, such as acidic N-Boc deprotection, saponification,
and coupling reactions.
[a] ILV, UMR 8180, University of Versailles,
78035 Versailles, France
E-mail: karen.wright@.uvsq.fr
www.ilv.uvsq.fr
[b] GEMAC, UMR 8635, University of Versailles,
78035 Versailles, France
[c] Department of Chemistry, University of Padova,
35131 Padova, Italy
Our groups have investigated extensively CiαǞCiα-
cyclized, Cα,α-disubstituted glycines [in particular 1-amino-
cyclopentane-1-carboxylic acid, Ac5c[16] (Figure 1)], which
are sterically restricted α-amino acids that promote the for-
[d] Institute of Biomolecular Chemistry, Padova Unit, CNR,
35131 Padova, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301765.
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K. Wright et al.
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mation of β-turns[17] and helical structures[16,18] when incor-
porated into peptides. More recently, we were interested in
the possibility of creating a rigid, chiral, nitronyl nitroxide
bearing, Cα-tetrasubstituted (quaternary) α-amino acid,
Aic(NN) (Figure 1) as a new spin label. We have previously
synthesized derivatives of 2-amino-indan-2-carboxylic acid
(Aic)[19] (Figure 1), a constrained Phe analogue, as fluores-
cence markers or photoaffinity labels.[20] The Aic frame-
work could be exploited to carry a nitronyl nitroxide func-
tion by derivatization of racemic 2-amino-5-cyanoindan-2-
carboxylic acid [Aic(CN)] (Figure 1). This amino acid is
itself interesting as a Cα-tetrasubstituted analogue of p-
cyanophenylalanine, which has been used as an IR absorp-
tion and fluorescence spectroscopic label to study protein
folding, environment, and binding.[21] The nitrile function
of Aic(CN) serves as a precursor of the aldehyde group,
which is necessary for the installation of the imidazolinyl
ring. A preliminary report of a limited part of this work
has been published.[22]
Scheme 1. Reagents and conditions: (i) NBS, benzoyl peroxide,
CCl4, 90 °C; (ii) a. CNCH2COOEt, K2CO3, 18-crown-6, CH3CN,
room temp.; b. HCl aq. 37%, CH2Cl2/EtOH, 0 °C; (iii) a. Boc2O,
CH3CN, room temp.; b. NaOH, THF/MeOH/H2O, room temp.;
(iv) (S)-Phg-ol, EDC, Oxyma, NMM, CH2Cl2, room temp.; (v) a.
3 m H2SO4 aq., dioxane, 110 °C; b. EtOH; (vi) Boc2O, NaHCO3,
CH3CN/H2O, room temp.
ylmorpholine (NMM), diastereomeric amides 5a and 5b
were obtained, which could be separated by column
chromatography. Hydrolysis of the two separate amides by
aqueous H2SO4, followed by in situ esterification with eth-
anol (EtOH), gave the enantiomeric, Nα-deprotected α-
amino ethyl esters 3a and 3b in 75 and 83% yields, respec-
tively. Less than 10% of the diethyl esters, resulting from
hydrolysis of the nitrile function, were detected in the reac-
tion mixtures under these conditions. Nα-Boc protection
gave the enantiomeric, fully protected α-amino acids 6a and
6b.
Figure 1. Chemical structures of Ac5c, Aic, Aic(CN), and Aic(NN).
Results and Discussion
Synthesis of 2-Amino-5-cyanoindan-2-carboxylic Acid
[Aic(CN)]
Racemic Boc-Aic(CN)-OH (Boc = tert-butoxycarbonyl)
4 was prepared in five steps (Scheme 1). Bromination of 3,4-
dimethyl benzonitrile 1 with N-bromosuccinimide (NBS)
Nitronyl Nitroxide Amino Acid [Aic(NN)] Synthesis
Conditions for the selective conversion of the nitrile
gave dibromide 2, which was used to form the racemic Cα- group into an aldehyde were sought that left the carbamate
tetrasubstituted α-amino ethyl ester 3 by bis(alkylation) of and ester protecting groups intact. When separate re-
ethyl isocyanoacetate under phase-transfer conditions.[23] ductions of 6a and 6b were carried out with Raney nickel
Acidic hydrolysis of the intermediate isocyanide, followed in the presence of sodium hypophosphite (Scheme 2),[26] the
by Nα-Boc protection with Boc anhydride and saponifica- corresponding aldehydes 7a and 7b were obtained in 73 and
tion of the ester function, gave the desired, racemic, Nα- 91% yields, respectively. Two possible routes to convert al-
protected α-amino acid 4 (Scheme 1). A method for the op- dehydes into nitronyl nitroxides have been previously re-
tical resolution of 4 was sought that would not require ported. The first method, as originally proposed by Ull-
harsh acidic conditions to remove the chiral auxiliary, man,[1] relies on the condensation of 2,3-bis(hydroxy-
which could be incompatible with the nitrile group.
amino)-2,3-dimethylbutane with the aldehyde followed by
We have previously used the easily hydrolyzed dia- oxidation of the intermediate. In our case, the preparation
stereomeric amides of (S)-phenylglycinol (Phg-ol) to sepa- of 2,3-bis(hydroxyamino)-2,3-dimethylbutane proved to be
rate acid-sensitive quaternary azetidines.[24] When racemic frustratingly difficult. This method was finally abandoned
4 was coupled with (S)-Phg-ol, in the presence of N-eth- in favor of the route proposed by Rey and co-workers,[27] in
yl,NЈ-[3-(dimethylamino)propyl)]carbodiimide (EDC)/ethyl which the aldehyde is first condensed with 2,3-diamino-2,3-
2-cyano-2-(hydroxyimino)acetate (Oxyma),[25] and N-meth- dimethylbutane to give the corresponding tetramethylimid-
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A Quaternary Nitronyl Nitroxide α-Amino Acid
azolidine. This procedure was successfully applied to alde- sized to demonstrate the feasibility of this strategy. Cou-
hydes 7a and 7b. The imidazolidine intermediates 8a and 8b pling of Boc-Ala-OH with 3a in the presence of HATU
were oxidized with 3-chloroperbenzoic acid (mCPBA) then gave dipeptide 12 in 90% yield. Saponification of the ethyl
with NaIO4, to afford the desired nitronyl nitroxides Boc- ester function of 12 and coupling of the resulting carboxylic
(R)- and Boc-(S)-Aic(NN)-OEt 9a and 9b in 58 and 64% acid to H-Ala-OMe afforded tripeptide 13. Reduction of
yields, respectively. The corresponding imino nitroxides[5] the nitrile function with Raney nickel/sodium hypophos-
were isolated from these oxidations in less than 5% yield.
phite, followed by condensation with 2,3-diamino-2,3-di-
methylbutane and finally oxidation gave the desired, ter-
minally protected tripeptide nitronyl nitroxide Boc-Ala-(R)-
Aic(NN)-Ala-OMe (15).
X-ray Diffraction
We succeeded in growing a blue crystal of Boc-Ala-(R)-
Aic(NN)-Ala-OMe (15) by recrystallisation from EtOH.
The 3D-structures of the two, conformationally distinct, in-
dependent molecules (A and B) in the asymmetric unit of
this terminally-protected tripeptide are illustrated in Fig-
ure 2. Their backbone torsion angles are listed in Table 1.
The configuration of the two chiral Ala residues was
known to be (S), because it is that of the amino acids used
in our synthetic procedures. Based on this information, we
assigned the configuration of the Aic(NN) residue present
in the crystalline tripeptide 15 as (R).
Scheme 2. Reagents and conditions: (i) Raney-Ni, NaH2PO2, pyr-
idine/AcOH/H2O, 40 °C; (ii) 2,3-diamino-2,3-dimethylbutane,
CHCl3, 60 °C; (iii) a. mCPBA, CH2Cl2/NaHCO3 aq. 0 °C; b.
NaIO4 aq., 0 °C.
Peptide Synthesis
Preliminary experiments to test the resistance of the ni-
The corresponding backbone torsion angles are similar
tronyl nitroxide amino acid in deprotection and peptide in the two molecules, and their difference does not exceed
coupling reactions were carried out by using Boc-(S)-Aic- 7°. All urethane, peptide, and ester (ω) torsion angles are
(NN)-OEt 9b (Scheme 3). Saponification of the ester func- in the common trans disposition. However, in both mole-
tion and reaction of the resulting carboxylic acid with cules the torsion angle (ω1) of the peptide bond between
H-Ala-OMe in the presence of O-(7-azabenzo-1,2,3-triazol- Ala1 and Aic(NN)2 is strongly distorted, by as much as 16–
1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate 19° from planarity.
Preliminary information on the 3D-structure adopted by
(HATU)[28] and diisopropyl-ethylamine (DIEA) afforded
dipeptide 10 in good yield. It was also possible to remove the tripeptide was obtained from the critical N–Cα–CЈ (τ)
the Nα-Boc group of 9b with dilute trifluoroacetic acid bond angles of the three residues in each molecule. Their
(TFA) at 0 °C, without extensive decomposition of the ni- values range between 109.7(6)° and 112.7(6)°, typical of a
tronyl nitroxide moiety, to give 11 in reasonable yield. How- semi-extended or a helical conformation, but not of a fully-
ever, the acid- and light-sensitive nature of the radicals indi- extended structure.[18a,18d,18e,29] We found that the con-
cated that it would be more straightforward to build a pept- formations of the Ala1 and Ala3 residues are semi-extended,
ide chain by using the Aic(CN) precursor amino acid, and whereas that of Aic(NN)2 is helical. In particular, the φ2
to introduce the nitronyl nitroxide moiety as a final step. A torsion angle of the latter residue is closer to that expected
tripeptide, Boc-Ala-(R)-Aic(CN)-Ala-OMe 13 was synthe- for an α-helix (Ϯ63°) than for a 310-helix (Ϯ57°).[18a] How-
Scheme 3. Reagents and conditions: (i) NaOH, THF/MeOH/H2O, room temp.; (ii) H-Ala-OMe, HATU, DIEA, THF, room temp.; (iii)
TFA/CH2Cl2 (9:1), 0 °C; (iv) Boc-Ala-OH, HATU, DIEA, THF, room temp.; (v) Raney-Ni, NaH2PO2, pyridine/AcOH/H2O, 40 °C; (vi)
2,3-diamino-2,3-dimethylbutane, CHCl3, 60 °C; (vii) a. mCPBA, CH2Cl2/NaHCO3 aq., 0 °C; b. NaIO4 aq., 0 °C.
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K. Wright et al.
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i ǟ i+3 intramolecular C=O···H–N H-bond, in that the
O0A···N3 and O0B···N7 distances are slightly too long
(3.545(8) Å in molecule A and 3.853(8) Å in molecule B).[30]
In any case, since short Ala homo-peptides (either ter-
minally protected or unprotected) are known to prefer the
β-pleated sheet conformation in the solid state,[31,32] the re-
sult of the present X-ray diffraction analysis strongly sup-
ports the view that the bulky Aic(NN), like most of the
quaternary α-amino acids,[16,18] is a strong turn-promoting
residue.
As for the geometry of the heterocyclic moiety, which is
common to all Ullman imidazolinyl nitronyl nitroxides,
bond lengths and bond angles are in general agreement
with the corresponding mean values extracted from a statis-
tical analysis of published X-ray diffraction structures.[2c] In
particular, the N–O bond lengths range from 1.294(9) to
1.309(10) Å. Within each molecule (A and B) the two N–O
bond lengths differ by less than 1.5 σ (0.015 Å).[7d]
The twist angle APNN between the plane of the phenyl
ring and the nitronyl nitroxide NCN plane[2c] is 25.8(3)° in
molecule A and 27.6(4)° in molecule B. This parameter is
of great relevance in governing the magnetic characteristics
of a phenyl nitronyl nitroxide, in that the delocalization of
the unpaired electron is dependent on the conjugation be-
tween its two cyclic moieties. The co-planar arrangement is
the most stable in vacuo.[2c] In the majority of the solid 3D-
structures, however, the phenyl nitronyl nitroxides differ to
a greater or lesser extent from this conformation, with the
average value of the statistical distribution of APNN being
27.5Ϯ8.5°.[2c] Therefore, the optimal packing of the mole-
cules in the crystal apparently plays a major role. In any
case, this out-of-planarity arrangement does not completely
prevent the formation of intramolecular (ortho aromatic)
Csp2-H···O–N H-bonds through the generation of six-mem-
bered pseudo-cyclic structures. These interactions are weak
since the C–H···O angles (117–121°) are very far from the
ideal values (about 150°), despite the acceptable C···O dis-
tances [in the range 2.883(9)–2.922(8) Å].
Figure 2. X-ray diffraction structures of the two independent mole-
cules (A and B) in the asymmetric unit of Boc-Ala-(R)-Aic(NN)-
Ala-OMe (15) with atom numberings.
Table 1. Backbone torsion angles [°] for tripeptide 15.
Torsion angle
Molecule A
Molecule B
ω0
φ1
ψ1
ω1
φ2
ψ2
–172.5(6)
–59.3(8)
136.8(5)
163.8(5)
66.9(7)
–172.3(6)
–64.8(8)
142.3(5)
161.3(5)
66.3(7)
Additional interesting conformational features are the
puckering motifs of the two five-membered rings (cyclopen-
tene and imidazoline) of the Aic(NN) residue in molecules
A and B. The cyclopentenyl ring in molecule A, with pa-
rameters q2 = 0.334(8) Å and φ2 = 182.6(9)°,[33] shows a
26.0(8)
24.1(8)
ω2
φ3
“ψ”3
“ω”3
175.7(5)
–61.6(8)
140.7(7)[a]
178.9(8)[b]
–179.2(6)
–68.5(8)
140.7(7)[c]
178.4(8)[d]
puckering similar to that seen in molecule B, with q2
=
0.350(9) Å and φ2 = 179.4(9)°. Both rings have an E1 enve-
lope conformation with an axial Nα atom and an equatorial
carbonyl C atom on the α-carbon. Overall, the cyclopent-
enyl ring is only slightly out of the plane of the fused phenyl
ring in each Aic moiety. The angle between the normals to
[a] N3–C3A–C3–OTA. [b] C3A–C3–OTA-CTA. [c] N7–C7A–C7–
OTB. [d] C7A–C7–OTB-CTB.
ever, the opposite is observed for the ψ2 torsion angle (Ϯ30° the cyclopentenyl and phenyl average planes is 10.97(18)°
for a 310-helix, Ϯ42° for an α-helix). Interestingly, the rela- in molecule A and 8.77(19)° in molecule B. The imid-
tionship between chirality and helix screw sense of Aic(NN) azolinyl ring of the Aic(NN) residue in molecule A shows
in this structure is the same as that of common Cα-trisubsti- puckering parameters q2 = 0.288(7) Å and φ2 = 119.4(16)°
tuted α-amino acids, i.e., an (R) residue tends to fold in a (relative to the atom sequence N21–C2Z–N22–C24–C23).
left-handed helix (its φ and ψ torsion angles are both posi- Similar values characterize the imidazolinyl ring puckering
tive). As a consequence, the molecules exhibit a type-II β- in molecule B, namely q2 = 0.247(8) Å and φ2 = 118(2)°.
5
turn conformation.[17] However, in these molecules this type Both rings adopt a T4 twist conformation, as commonly
of folding is “open”, i.e., it is not stabilized by the classical observed in Ullman imidazolinyl nitronyl nitroxides.[2c]
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A Quaternary Nitronyl Nitroxide α-Amino Acid
IR Absorption
the participation of the N–O groups of our compounds in
weak H-bonds.
The FTIR absorption spectrum of the terminally pro-
tected tripeptide 15 at 1.0 mm concentration in CDCl3 solu-
tion is reported in Figure 3 A. Remarkably, the ratio be-
tween the areas under the peaks at 3355 and
3427 cm–1[32a,32b] is approximately 1.1 and does not change
with dilution. Since this ratio is indicative of the amount of
intramolecularly C=O···H–N H-bonded species, we con-
clude that in this low-polarity solvent the sequence -Ala-
(R)-Aic(NN)-Ala- is significantly more folded than the cor-
responding Ala homopeptide sequence, in which the ratio
is less than 0.15.[32a] This result is in good accordance with
our aforementioned X-ray diffraction data.
UV/Vis Absorption and Circular Dichroism (CD)
In the near UV/Vis regions of the electronic spectrum in
methanol (MeOH) solution, the Aic(NN) derivatives 9a
and 9b, and tripeptide 15 show three well-separated absorp-
tions centered near 280, 370, and 590 nm (Figure 4). The
last, very broad and weak band[2,3a,4b] is associated with
the nǞπ* transition of the ONCNO chromophore of the
nitronyl nitroxide moiety[4b] and is responsible for the typi-
cal blue color of these compounds. The 370 nm
band[2c,2d,4b] is assigned to the πǞπ* transitions of the
same chromophore from a common ground state to excited
states with two orbital contributions.[4b]
Figure 4. UV/Vis absorption spectrum of tripeptide 15 in MeOH
solution.
Figure 5 illustrates the CD spectra of derivatives 9a and
9b and tripeptide 15 in MeOH solution in the near-UV (a)
and visible (b) regions. Not surprisingly, the signs of the
strong Cotton effects between 250 and 600 nm are related
to the absolute configuration of the α-carbon of the
Aic(NN) residue, as both compounds 9a and 15, where the
Aic(NN) chirality is (R), exhibit the same signs. However,
the band intensities are always higher in tripeptide 15. In-
terpretation of these CD spectra is difficult because the
wavelengths of the maxima of most of the numerous CD
bands do not correspond to any UV/Vis absorption maxi-
mum.[2c,4]
Figure 3. FTIR absorption spectra of tripeptide 15 in CDCl3 solu-
tion in the amide N–H (a) and N–O (b) stretching regions. Peptide
concentration: 1 mm.
Combinations of the component chromophores (nitronyl
nitroxide and the linked phenyl), including exciton coupling
and charge transfer effects, and the twist angle between
The N–O stretching band of the nitronyl nitroxide moi- them, are known to contribute heavily to the observed spec-
ety, which usually appears at 1361–1370 cm–1 in the solid tra. In addition, the CD contributions arising from the ni-
state,[2b,2c] is positioned at 1358–1360 cm–1 in tripeptide 15 tronyl nitroxide chromophore below 250 nm might interfere
(Figure 3 B) and in the Aic(NN) derivatives 9a and 9b in with the use of this chirospectroscopic technique to deter-
CDCl3. This modest shift to lower frequency might suggest mine the secondary structure of peptides.
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K. Wright et al.
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Figure 5. CD spectra of the two enantiomers 9a (full line) and 9b (dashed line) of Boc-Aic(NN) OEt and tripeptide 15 (dotted line) in
MeOH solution in the UV (a) and visible (b) regions.
Nuclear Magnetic Resonance
tons of Ala1 and Ala3 resonate at δ = 1.37 and 1.23 ppm,
whereas those of their αCH protons resonate at δ = 3.93
and 4.43 ppm, respectively. As for the protecting groups,
the resonance of the Boc methyl groups is at δ = 1.37 ppm
(overlapping that of the Ala1 βCH3) and that of the methyl
ester OMe is at δ = 3.67 ppm.
The 400 MHz spectrum of the Boc/OMe-protected tri-
peptide 15 in deuterated acetone (D6) is reported in Fig-
ure 6. We clearly detected all proton resonances of the pept-
ide backbone and Ala sidechains, without any significant
line broadening, despite the presence of a nearby paramag-
netic nitronyl nitroxide moiety. This result indicates that the
spin densities at these protons are low-to-moderate,[7d,8] at
variance with those obtained with nitroxyl-containing pept-
ides,[34] and suggests the very important point that the con-
formation of longer nitronyl nitroxide-based peptides might
be readily accessible by using 2D-NMR spectroscopy.
Specifically, the NH protons of urethane-protected Ala1,
Aic(NN)2 and Ala3 are seen at δ = 6.21, 7.65, and 7.59 ppm,
respectively. The signals corresponding to the βCH3 pro-
Electron Paramagnetic Resonance
The complete experimental EPR spectra of compounds
9a/9b in a 1 mm CDCl3 solution at 293 K are shown in the
inset of Figure 7 (a), while that of the tripeptide 15 is re-
ported in the inset of Figure 7 (b). The EPR spectra of the
sample solutions diluted to 0.1 mm concentration do not
show any difference except for a lower signal-to-noise ratio.
All spectra are characterized by a quintet of hyperfine lines
with relative intensity ratios 1:2:3:2:1, as expected for the
isotropic coupling of the unpaired electron spin with two
equivalent 14N nuclear spins (I = 1). The hyperfine coupling
constant is aN = 0.761 mT for all compounds examined
(Table 2). In Figure 7 an expansion of the central lines (hy-
perfine line with MI = MN1 + MN2 = 0) in the quintets
are also shown. These expansions reveal further hyperfine
couplings of the electron spin with the 1H nuclei of the mo-
lecule (two ortho protons and one meta proton of the phenyl
ring, in addition to twelve protons of the four methyl
groups). Figure 7 also shows the spectra calculated with the
routines of the EasySpin software package[35] running in the
MATLAB environment, and employing the g factor and
hyperfine coupling constants ai as variable parameters. The
simulations based on the best fit values of the g factor and
the ai constants, reported in Table 2, reproduce quite well
the experimental EPR signals. The magnetic parameters ob-
tained are in line with those reported for similar imid-
1
Figure 6. H NMR spectrum of tripeptide 15 in [D6]acetone. The
peak at δ = 2.04 ppm is associated with the methyl protons of the
solvent.[8a]
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A Quaternary Nitronyl Nitroxide α-Amino Acid
azolinyl nitronyl nitroxides in organic solvents of low po-
larity.[1b–1e,7c]
Figure 8. Magnetic susceptibility (χM) (bullets) of rac-Boc-
Aic(NN)-OEt (9) plotted against (T–θ)–1 (bottom axis). The corre-
sponding temperatures T, from 8 to 180 K, are shown in the top
axis. The solid line is the Curie–Weiss law fit.
Conclusions
The novel, constrained, enantiomeric imidazolinyl ni-
tronyl nitroxide α-amino acid derivatives Boc-(R)- and Boc-
(S)-Aic(NN)-OEt 9a and 9b were prepared from the inter-
mediates Boc-(R)- and (S)-Aic(CN)-OEt. The precursor
amino acid, Aic(CN), was resolved by separation of its dia-
stereomeric amides of (S)-phenylglycinol. Short Aic(NN)-
containing peptides could be synthesized either directly by
using the nitronyl nitroxide amino acids, or by derivati-
zation of the Aic(CN) peptide precursors. The Aic(NN)
peptides are the second compounds of this type (after those
Figure 7. Experimental and simulated EPR spectra. (a) The solid
line is the MI = 0 component of the experimental EPR spectrum
of a 1 mm CDCl3 solution of compounds 9a/9b at T = 293 K. The
dotted line is the simulated spectrum. Inset: complete EPR spec-
trum with 1:2:3:2:1 intensity ratio of the main components, due to
the hyperfine coupling with two equivalent 14N nuclei. (b) The solid
line is the MI = 0 component of the experimental EPR spectrum
of a 1 mm CDCl3 solution of tripeptide 15 at T = 295 K. The dotted based on the azulene chromophore[36]) that are blue colored
line is the simulated spectrum. Inset: complete EPR spectrum with
in the absence of any metal cation.
1:2:3:2:1 intensity ratio of the main components, due to the hyper-
An X-ray diffraction analysis and an FTIR absorption
fine coupling with two equivalent 14N nuclei. For the parameters
study of the terminally protected tripeptide 15 unambigu-
of the simulations, see text and Table 2.
ously established the configuration of the central Aic(NN)
residue and the remarkable propensity of its peptides to fold
into a turn conformation. This latter property suggests that,
to avoid unwanted conformational modifications, Aic(NN)
should preferentially be incorporated into peptides already
known to fold.
Table 2. The g factors and hyperfine coupling constants ai (Mt)[a]
used to reproduce the EPR spectra of compounds 9a/9b and 15 in
CDCl3 at T = 295 K.
[b]
[b]
[b]
g factor
aN
aH–ortho
aH–neta
aH–methyl
(2 nuclei) (2 nuclei)
(1 nucleus) (12 nuclei)
The IR-UV/Vis absorption and CD spectroscopic prop-
erties, and the NMR/EPR and magnetic susceptibility sig-
natures of the Aic(NN) compounds were also investigated.
It is foreseen that this turn-supporting residue will prove to
be useful as a rigid, paramagnetic probe in peptide chemis-
try.
9a/9b
15
2.0066
2.0062
0.761
0.761
0.650
0.675
0.359
0.380
0.215
0.210
[a] Estimated errors: Ϯ0.0002 for the g factor, Ϯ0.002 for the hy-
perfine constants ai. [b] The ortho and meta are positions in the
phenyl ring relative to the nitronyl nitroxide moiety. Methyl refers
to the H-nuclei in the four methyl group substituents of the five-
membered heterocycle.
1
Experimental Section
Magnetic Susceptibility
Synthesis: Melting points were determined with a capillary tube
immersed in an oil bath (Tottoli apparatus, Büchi). 1H and 13C
NMR spectra were recorded with a Bruker WM300 spectrometer
operating at 300 and 77 MHz, respectively. The solvent used as the
internal standard was CDCl3 (1H: δ = 7.27 ppm; 13C: δ =
77.00 ppm). Splitting patterns are abbreviated as follows: (s) singlet,
(d) doublet, (t) triplet, (q) quartet, (m) multiplet. High-resolution
mass spectra (electrospray mode) were performed by Dr. Estelle
Galmiche (ILV) with a Xevo Q-TOF (Waters) mass spectrometer.
Elemental analyses were performed by the C.N.R.S. Service of
The magnetic susceptibility χM [χM = C/(T – θ)] of rac-
Boc-Aic(NN)-OEt (9), measured under a 90 kOe magnetic
field, is plotted against (T – θ)–1 in Figure 8. The data are
well-fitted by a Curie–Weiss law, revealing paramagnetic be-
havior between 8 and 180 K, with the Curie constant C =
0.35 emuK/mol consistent with values reported for similar
compounds.[4b,41] The negative value of θ (–2.7 K) indicates
a small antiferromagnetic interaction.
Eur. J. Org. Chem. 2014, 1741–1752
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1747

K. Wright et al.
FULL PAPER
Microanalyses in Gif-sur-Yvette (France). Optical rotations were
measured, with an accuracy of 0.3%, in a 1-dm thermostatted cell.
Analytical thin-layer chromatography (TLC) and column
chromatography were carried out on silica gel F 254 (Merck) and
Kieselgel gel 60 (0.040–0.063 mm) (Merck), respectively.
[D5]DMF, 25 °C): δ = 7.70–7.64 (m, 2 H, ArH), 7.48–7.46 (m, 2 H,
ArH, NH), 3.68–3.59 (m, 2 H, CH2), 3.48–3.41 (m, 2 H, CH2),
1.39 (br. s, 9 H, CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ
= 176.1, 156.6 (CO), 148.4, 143.9, 132.0, 129.1, 126.7 (ArC), 120.3
(CN), 110.9 (ArC), 79.3 (C(CH3)3), 66.7 (Cα), 44.6, 44.0 (CH2),
28.9 (CH3) ppm. HRMS (ESI): m/z calcd. for C16H18N2O4 + H+
[M + H]+ 303.1345; found 303.1345.
3,4-Bis(bromomethyl)benzonitrile (2): 3,4-Dimethylbenzonitrile
(9.17 g, 70 mmol) was dissolved in CCl4 (150 mL), the solution was
placed under argon, and NBS (24.9 g, 140 mmol) and benzoyl per-
oxide (700 mg) were added. The reaction mixture was heated at
reflux for 4 h and filtered through Celite. The filtrate was concen-
trated under reduced pressure, then the residue was purified by col-
umn chromatography (diethyl ether/petroleum ether, 1:9) to give 2
(14.3 g, 71%) as a solid. Rf = 0.26 [pentane/ethyl acetate (EtOAc),
95:5]; m.p. 68–70 °C. 1H NMR (300 MHz, CDCl3, 25 °C): δ = 7.68
(s, 1 H, ArH), 7.61 (d, J = 8.1 Hz, 1 H, ArH), 7.49 (d, J = 7.9 Hz,
1 H, ArH), 4.63 (s, 2 H, CH2), 4.62 (s, 2 H, CH2) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ = 141.5, 137.9, 134.3, 132.6, 131.7
(ArC), 117.6 (CN), 113.1 (ArC), 28.0, 27.8 (CH2) ppm. C9H7Br2N
(288.97): calcd. C 37.41, H 2.44, N 4.85; found C 37.26, H 2.43, N
4.76.
Boc-(R,S)-Aic(CN)-(S)-Phg-ol (5a/5b): Compound
4
(5.58 g,
17.21 mmol) was dissolved in CH2Cl2 (200 mL), the solution was
cooled to 0 °C, and EDC (3.54 g, 18.5 mmol), Oxyma (2.63 g,
18.5 mmol), and NMM (2 mL, 18.5 mmol) were added. The reac-
tion mixture was stirred for 5 min, then (S)-phenylglycinol (2.53 g,
18.5 mmol) was added. The mixture was warmed to room temp.,
stirred for 72 h, diluted with further CH2Cl2, and washed twice
with 0.5 m aqueous HCl, then with water, and finally with saturated
aqueous NaHCO3 solution. The organic phase was dried with
MgSO4, filtered, and concentrated under reduced pressure. The re-
sulting diastereomeric mixture was separated by column
chromatography (EtOAc/pentane/CH2Cl2, 55:45:20) to give 5a
(3.29 g, 45%) and 5b (2.96 g, 41%) as solids.
Boc-(R)-Aic(CN)-(S)-Phg-ol (5a): Rf = 0.45 (THF/pentane/CH2Cl2,
45:55:20); m.p. 216 °C; [α]2D5 = +29 (c = 1.0, CH2Cl2). 1H NMR
(300 MHz, CD3OD, 25 °C): δ = 7.57–7.53 (m, 2 H, ArH), 7.38–
7.24 (m, 6 H, ArH), 5.02 (t, J = 6.1 Hz, 1 H, CH), 3.88–3.62 (m,
4 H, CH2), 3.34–3.27 (m, 2 H, CH2), 1.45 (br, 9 H, CH3) ppm. 13C
NMR (75 MHz, CD3OD, 25 °C): δ = 175.4, 157.1 (CO), 148.3,
143.3, 141.2, 132.3, 129.5, 129.3, 128.5, 128.2, 126.8 (ArC), 120.3
(CN), 111.6 (ArC), 81.2 [C(CH3)3], 68.4 (Cα), 66.1 (CH2), 57.1
(CH), 44.4, 44.1 (CH2), 28.8 (CH3) ppm. HRMS (ESI): m/z calcd.
for C24H27N3O4 + H+ [M + H]+ 422.2080; found 422.2082.
Ethyl 2-Amino-5-cyanoindan-2-carboxylate [H-(R,S)-Aic(CN)-OEt;
3]: Compound 2 (7.63 g, 26.4 mmol) was dissolved in CH3CN
(600 mL) and the solution was placed under argon and 18-crown-
6 (696 mg, 2.6 mmol) and K2CO3 (36.4 g, 264 mmol) were added.
Ethyl isocyanoacetate (2.88 mL, 26.4 mmol) was added and the re-
action mixture was stirred at room temp. for 42 h and filtered
through Celite. The filtrate was concentrated under reduced pres-
sure, the residue was taken up in CH2Cl2 (180 mL) and EtOH
(30 mL), and the mixture was cooled on an ice bath. Aqueous 37%
HCl solution (2 mL) was added and the mixture was stirred at 0 °C
for 4 h. Excess saturated aqueous NaHCO3 solution was added and
the mixture was stirred for 15 min, then the mixture was extracted
three times with CH2Cl2. The combined organic extracts were
washed with saturated aqueous NaCl solution, dried with MgSO4,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography (CH2Cl2/MeOH, 95:5) to give
3 (3.35 g, 55%) as an oil. Rf = 0.47 (CH2Cl2/MeOH, 95:5). 1H
NMR (300 MHz, CDCl3, 25 °C): δ = 7.50–7.45 (m, 2 H, ArH),
7.32–7.27 (m, 1 H, ArH), 4.22 (q, J = 7.1 Hz, 2 H, CH2), 3.61–
3.52 (m, 2 H, CH2), 2.96–2.89 (m, 2 H, CH2), 1.77 (s, 2 H, NH2),
Boc-(S)-Aic(CN)-(S)-Phg-ol (5b): Rf = 0.40 (THF/pentane/CH2Cl2,
45:55:20); m.p. 209 °C; [α]2D5 = +85 (c = 1.0, CH2Cl2). 1H NMR
(300 MHz, CD3OD, 25 °C): δ = 7.56–7.52 (m, 2 H, ArH), 7.38–
7.24 (m, 6 H, ArH), 5.02 (t, J = 6.1 Hz, 1 H, CH), 3.87–3.61 (m,
4 H, CH2), 3.35–3.24 (m, 2 H, CH2), 1.45 (br. s, 9 H, CH3) ppm.
13C NMR (75 MHz, CD3OD, 25 °C): δ = 175.4, 157.2 (CO), 147.9,
143.6, 141.3, 132.3, 129.6, 129.4, 128.5, 128.2, 126.8 (ArC), 120.3
(CN), 111.6 (ArC), 81.3 [C(CH3)3], 68.4 (Cα), 66.2 (CH2), 57.1
(CH), 44.7, 43.7 (CH2), 28.8 (CH3) ppm. HRMS (ESI): m/z calcd.
for C24H27N3O4 + H+ [M + H]+ 422.2080; found 422.2083.
1.29 (t, J = 7.1 Hz, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3, H-(R)-Aic(CN)-OEt (3a): Compound 5a (1.23 g, 2.92 mmol) was
25 °C): δ = 175.8 (CO), 146.4, 141.9, 131.0, 128.3, 125.5 (ArC),
119.1 (CN), 110.5 (ArC), 64.8 (Cα), 61.6 (OCH2), 46.0, 45.5 (CH2),
14.1 (CH3) ppm. HRMS (ESI): m/z calcd. for C13H14N2O2 + H+
[M + H]+ 231.1134; found 231.1129.
dissolved in 1,4-dioxane (100 mL) and a solution of 3 m aqueous
H2SO4 (4.8 mL) was added. The reaction mixture was heated at
reflux for 24 h, then cooled. EtOH (50 mL) was added and volatiles
were removed under reduced pressure. EtOH (100 mL) was added
to the residue, and the mixture was heated at reflux for 72 h. The
mixture was cooled, and sufficient saturated aqueous NaHCO3
solution was added to adjust the pH of the solution to ca. pH 8.
The mixture was filtered through Celite and the filtrate was concen-
trated under reduced pressure. The residue was diluted with
CH2Cl2 and washed with saturated aqueous NaCl solution. The
organic phase was dried with MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by column
chromatography (CH2Cl2/MeOH, 97:3) to give 3a (505 mg, 75%)
as an oil. [α]2D5 = +8 (c = 0.25, CH2Cl2). HRMS (ESI): m/z calcd.
for C13H14N2O2 + H+ [M + H]+ 231.1134; found 231.1131.
2-(tert-Butyloxycarbonylamino)-5-cyanoindan-2-carboxylic
Acid
[Boc-(R,S)-Aic(CN)-OH; 4]: Compound 3 (4.88 g, 21.2 mmol) was
dissolved in CH3CN (250 mL) and Boc2O (11.5 g, 53 mmol) was
added. The reaction mixture was stirred at room temp. for 42 h and
concentrated under reduced pressure. The residue was purified by
column chromatography (pentane/EtOAc, 8:2) to give the interme-
diate Boc-(R,S)-Aic(CN)-OEt. The residue was dissolved in THF
(100 mL), and MeOH (50 mL), water (10 mL) and NaOH (860 mg,
21.5 mmol) were added. The mixture was stirred at 65 °C for
45 min, cooled, and diluted with water. Volatiles were removed un-
der reduced pressure and the resulting solution was cooled on an
ice bath and acidified by addition of 2 m aqueous HCl. The mixture
was extracted three times with CH2Cl2 and the combined organic
extracts were dried with MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatog-
H-(S)-Aic(CN)-OEt (3b): The (S)-enantiomer was prepared in the
same way from 5b (1.00 g, 2.37 mmol) to give 3b (453 mg, 83%) as
an oil. [α]2D5 = –8 (c = 0.27, CH2Cl2). HRMS (ESI): m/z calcd. for
C13H14N2O2 + H+ [M + H]+ 231.1134; found 231.1130.
raphy (CH2Cl2/MeOH, 90:10) to give 4 (5.58 g, 87%) as a foam. Boc-(R)-Aic(CN)-OEt (6a): Compound 3a (308 mg, 1.34 mmol)
Rf = 0.37 (CH2Cl2/MeOH/AcOH, 95:5:1). 1H NMR (300 MHz,
was dissolved in CH3CN (6 mL) and Boc2O (585 mg, 2.68 mmol)
1748
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1741–1752

A Quaternary Nitronyl Nitroxide α-Amino Acid
was added. The reaction mixture was stirred at room temp. for
24 h, then concentrated under reduced pressure. The residue was
purified by column chromatography (pentane/EtOAc, 8:2) to give
6a (295 mg, 67%) as a foam. Rf = 0.55 (pentane/EtOAc, 75:25);
[α]2D5 = –17 (c = 1.0, CH2Cl2). 1H NMR (300 MHz, CDCl3, 25 °C):
δ = 7.50–7.47 (m, 2 H, ArH), 7.30 (d, J = 8.1 Hz, 1 H, ArH), 5.20
(br. s, 1 H, NH), 4.22 (q, J = 7.1 Hz, 2 H, CH2), 3.68–3.58 (m, 2
H, CH2), 3.36–3.27 (m, 2 H, CH2), 1.42 (s, 9 H, CH3), 1.25 (t, J =
7.1 Hz, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3, 25 °C): δ =
173.0, 154.8 (CO), 145.9, 141.4, 1312, 128.0, 125.3 (ArC), 119.1
(CN), 110.7 (ArC), 80.4 [C(CH3)3], 65.6 (Cα), 61.9 (OCH2), 43.9,
43.4 (CH2), 28.2, 14.0 (CH3) ppm. HRMS (ESI): m/z calcd. for
C18H22N2O4 + H+ [M + H]+ 331.1658; found 331.1658.
der reduced pressure. The residue was purified by column
chromatography (pentane/EtOAc, 1:1) to give 9a (80 mg, 58%) as
25
a blue solid. Rf = 0.39 (pentane/EtOAc, 1:1); m.p. 88 °C; [α]
=
436
–50 (c = 0.062, CH2Cl2). HRMS (ESI): m/z calcd. for C24H34N3O6
+ H+ [M + H+] 460.2454; found 460.2448.
Boc-(S)-Aic(NN)-OEt (9b): The (S)-enantiomer was prepared in
the same way from aldehyde 7b (300 mg, 0.90 mmol) to give 9b
25
(187 mg, 64%). [α]
= +50 (c = 0.061, CH2Cl2). HRMS (ESI):
436
m/z calcd. for C24H34N3O6 + H+ [M + H+] 460.2454; found
460.2455.
Boc-(S)-Aic(NN)-Ala-OMe (10): Compound 9b (23 mg,
0.05 mmol) was dissolved in THF/MeOH/H2O (10:5:1, 1.6 mL), a
0.2 m aqueous solution of NaOH (0.4 mL) was added, and the reac-
tion mixture was stirred at room temp. for 8 h. The mixture was
diluted with water and volatiles were removed under reduced pres-
sure. The resulting solution was acidified by careful addition of
aqueous 0.5 m HCl solution and the mixture was extracted twice
Boc-(S)-Aic(CN)-OEt (6b): The (S)-enantiomer was prepared in the
same way from 3b (380 mg, 1.65 mmol) to give 6b (387 mg, 71%)
as a foam. [α]2D5 = +17 (c = 1.0, CH2Cl2). HRMS (ESI): m/z calcd.
for C18H22N2O4 + Na+ [M + Na]+ 353.1477; found 353.1481.
Boc-(R)-Aic(CHO)-OEt (7a): Ester 6a (204 mg, 0.62 mmol) was with EtOAc. The combined organic extracts were dried with
dissolved in pyridine (1.5 mL), and AcOH (1.5 mL) and sodium
hypophosphite hydrate (370 mg) were added. A suspension of
Raney nickel 2800 (ca. 200 mg) in water (2 mL) was added and the
reaction mixture was stirred vigorously at 45 °C for 1 h and then
cooled. The supernatant was decanted and the remaining nickel
was washed twice with 95% EtOH. The combined organic phases
were diluted with diethyl ether and washed with saturated aqueous
NaCl solution. The organic phase was dried with MgSO4, filtered,
and concentrated under reduced pressure. The residue was purified
by column chromatography (pentane/EtOAc, 8:2) to give 7a
MgSO4, filtered, and concentrated under reduced pressure. The res-
idue was dissolved in THF (1 mL) and HCl·H-Ala-OMe (6 mg,
0.04 mmol) was added. The mixture was cooled on an ice bath and
HATU (16 mg, 0.04 mmol) and DIEA (0.014 mL, 0.08 mmol) were
added. The mixture was stirred at room temp. for 6 h, then concen-
trated under reduced pressure and the residue was dissolved in
CH2Cl2. The solution was washed with 0.5 m aqueous HCl, then
with saturated aqueous NaCl solution, and finally with saturated
aqueous NaHCO3 solution. The organic phase was dried with
MgSO4, filtered, and concentrated under reduced pressure. The res-
idue was purified by column chromatography (CH2Cl2/MeOH,
(152 mg, 73%) as a syrup. Rf = 0.59 (pentane/EtOAc, 7:3); [α]2D5
=
1
–28 (c = 0.54, CH2Cl2). H NMR (300 MHz, CDCl3, 25 °C): δ = 95:5) to give 10 (18 mg, 69%) as a blue solid. Rf = 0.22 (CH2Cl2/
25
9.96 (s, 1 H, CHO), 7.72–7.70 (m, 2 H, ArH), 7.37–7.27 (m, 1 H, MeOH, 95:5); m.p. 96–98 °C (dec.); [α] = –20 (c = 0.06, CH2Cl2).
ArH), 5.20 (br. s, 1 H, NH), 4.21 (q, J = 7.1 Hz, 2 H, CH2), 3.71–
436
HRMS (ESI): m/z calcd. for C26H37N4O7 + H+ [M + H+] 518.2741;
3.60 (m, 2 H, CH2), 3.37–3.27 (m, 2 H, CH2), 1.42 (s, 9 H, CH3), found 518.2739.
1.25 (t, J = 7.1 Hz, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3,
H-(S)-Aic(NN)-OEt (11): Compound 9b (23 mg, 0.05 mmol) was
25 °C): δ = 191.9 (CHO), 173.1, 154.9 (CO), 147.7, 141.1, 135.9,
129.7, 125.1, 125.0 (ArC), 80.3 [C(CH3)3], 65.8 (Cα), 61.8 (OCH2),
43.9, 43.4 (CH2), 28.2, 14.1 (CH3) ppm. HRMS (ESI): m/z calcd.
for C18H23NO5 + H+ [M + H]+ 334.1654; found 334.1658.
dissolved in ice-cold CH2Cl2/TFA (9:1, 0.5 mL) and stirred at 0 °C
for 4 h. The reaction mixture was diluted with CH2Cl2 and washed
with 0.02 m aqueous ammonium acetate solution. The aqueous
phase was extracted twice with CH2Cl2, then the combined organic
phases were dried with Na2SO4, filtered, and concentrated under
Boc-(S)-Aic(CHO)-OEt (7b): The (S)-enantiomer was prepared in
the same way from ester 6b (350 mg, 1.06 mmol) to give 7b reduced pressure. The residue was purified by column chromatog-
(322 mg, 91%). [α]2D5 = +27 (c = 0.52, CH2Cl2). HRMS (ESI): m/z
raphy (CH2Cl2/MeOH, 95:5) to give 11 (14 mg, 77%) as a blue oil.
25
calcd. for C18H23NO5 + Na+ [M + Na]+ 356.1474; found 356.1476. Rf = 0.22 (CH2Cl2/MeOH, 95:5); [α] = +15 (c = 0.06, CH2Cl2).
436
HRMS (ESI): m/z calcd. for C19H26N3O4 + H+ [M + H+] 361.2002;
Boc-(R)-Aic(NN)-OEt (9a): Aldehyde 7a (132 mg, 0.39 mmol) was
found 361.2004.
dissolved in CHCl3 (5 mL) and 2,3-dimethyl-butane-2,3-diamine
(92 mg, 0.80 mmol) was added. The reaction mixture was heated
to reflux and stirred for 42 h, then cooled and dried with Na2SO4.
The mixture was filtered and concentrated under reduced pressure
and the residue was taken up in CH2Cl2 and filtered through a plug
of silica gel, eluting with CH2Cl2/MeOH/triethylamine (90:10:1).
The filtrate was evaporated to give the intermediate imidazolidine
8a (152 mg) as a foam, which was used directly in the next step
without further purification. Imidazolidine 8a was dissolved in
CH2Cl2 (7.5 mL) and a saturated aqueous NaHCO3 solution
(3.7 mL) was added. The mixture was cooled on an ice bath, then
Boc-Ala-(R)-Aic(CN)-OEt (12): Boc-Ala-OH (251 mg, 1.33 mmol)
was added to a solution of H-(R)-Aic(CN)-OEt 3a (256 mg,
1.11 mmol) in THF (20 mL) and the reaction mixture was cooled
on an ice bath. DIEA (0.58 mL, 3.33 mmol) and HATU (505 mg,
1.33 mmol) were added to the mixture, which was then stirred at
room temp. for 18 h. The mixture was concentrated under reduced
pressure and the residue was dissolved in CH2Cl2. The solution was
washed with 0.5 m aqueous HCl, then with saturated aqueous NaCl
solution, and finally with saturated aqueous NaHCO3 solution.
The organic phase was dried with MgSO4, filtered, and concen-
a solution of m-CPBA (70%, 184 mg, 0.75 mmol) in CH2Cl2 trated under reduced pressure. The residue was purified by column
(2 mL) was added dropwise. The reaction mixture was stirred for
1 h at 0 °C, then a solution of NaIO4 (96 mg, 0.45 mmol) in water
(2.5 mL) was added dropwise. The reaction mixture was stirred for
1 h at 0 °C, then diluted with CH2Cl2 and the phases were sepa-
rated. The aqueous phase was extracted once with CH2Cl2 and the
combined organic phases were washed with saturated aqueous
NaCl solution, dried with Na2SO4, filtered, and concentrated un-
chromatography (CH2Cl2/MeOH, 97:3) to give 12 (400 mg, 90%)
as a solid. Rf = 0.32 (CH2Cl2/MeOH, 95:5); m.p. 65–67 °C; [α]2D5
=
1
–48 (c = 0.53, CH2Cl2). H NMR (300 MHz, CDCl3, 25 °C): δ =
7.46–7.44 (m, 2 H, ArH), 7.27–7.23 (m, 1 H, ArH), 7.08 (s, 1 H,
NH), 4.84 (br. s, 1 H, NH), 4.20–4.05 (m, 3 H, CH Ala, CH2),
3.67–3.53 (m, 2 H, CH2), 3.38–3.24 (m, 2 H, CH2), 1.33 (s, 9 H,
CH3), 1.29–1.19 (m, 6 H, CH3) ppm. 13C NMR (75 MHz, CDCl3,
Eur. J. Org. Chem. 2014, 1741–1752
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1749

K. Wright et al.
FULL PAPER
25 °C): δ = 172.3 (CO), 145.9, 141.2, 131.2, 127.9, 125.3 (ArC),
with Na2SO4. The mixture was filtered and concentrated under re-
119.0 (CN), 110.7 (ArC), 80.4 [C(CH3)3], 65.2 (Cα), 61.9 (CH2), duced pressure and the residue was taken up in CH2Cl2 and filtered
49.6 (CH), 43.5, 43.2 (CH2), 28.1, 17.1, 14.0 (CH3) ppm. HRMS
(ESI): m/z calcd. for C21H27N3O5 + Na+ [M + Na]+ 424.1848;
found 424.1849.
through a plug of silica gel, eluting with CH2Cl2/MeOH/triethyl-
amine (90:10:1). The filtrate was evaporated to give the intermedi-
ate imidazolidine (132 mg) as a solid, which was used directly in
the next step without further purification. The imidazolidine was
dissolved in CH2Cl2 (6 mL) and saturated aqueous NaHCO3 solu-
tion (3.6 mL) was added. The mixture was cooled on an ice bath
and a solution of m-CPBA (70%, 118 mg, 0.49 mmol) in CH2Cl2
(3 mL) was added dropwise. The reaction mixture was stirred for
1 h at 0 °C, then a solution of NaIO4 (62 mg, 0.29 mmol) in water
(3 mL) was added dropwise. The reaction mixture was stirred for
1 h at 0 °C, then diluted with CH2Cl2 and the phases were sepa-
rated. The aqueous phase was extracted once with CH2Cl2 and the
combined organic phases were washed with saturated aqueous
NaCl solution, dried with Na2SO4, filtered, and concentrated un-
der reduced pressure. The residue was purified by column
Boc-Ala-(R)-Aic(CN)-Ala-(OMe) (13): Dipeptide 12 (362 mg,
0.90 mmol) was dissolved in a mixture of THF/MeOH/H2O
(4.5 mL/2.25 mL/0.25 mL) and NaOH (40 mg, 1.00 mmol) was
added. The reaction mixture was stirred at room temp. for 1 h and
then diluted with water. Volatiles were removed under reduced pres-
sure and the resulting solution was cooled on an ice bath and acidi-
fied by addition of 2 m aqueous HCl. The mixture was extracted
three times with EtOAc and the combined organic extracts were
dried with MgSO4, filtered, and concentrated under reduced pres-
sure. The residue obtained was dissolved in THF (15 mL) and
HCl·H-Ala-OMe (120 mg, 0.88 mmol) was added. The mixture was
cooled on an ice bath and HATU (334 mg, 0.88 mmol) and DIEA
(0.3 mL, 1.76 mmol) were added. The mixture was stirred at room
temp. for 48 h, then the mixture was concentrated under reduced
pressure and the residue was dissolved in CH2Cl2. The solution was
washed with 0.5 m aqueous HCl, then with saturated aqueous NaCl
solution, and finally with saturated aqueous NaHCO3 solution.
The organic phase was dried with MgSO4, filtered, and concen-
trated under reduced pressure. The residue was purified by column
chromatography (CH2Cl2/MeOH, 97:3) to give 13 (310 mg, 84%)
as a solid. Rf = 0.32 (CH2Cl2/MeOH, 95:5); m.p. 219–221 °C;
[α]2D5 = –54 (c = 0.51, CH2Cl2). 1H NMR (300 MHz, CDCl3, 25 °C):
δ = 7.46–7.44 (m, 2 H, ArH), 7.30–7.18 (m, 2 H, ArH, NH), 7.08
(s, 1 H, NH), 5.07 (d, J = 5.9 Hz, 1 H, NH), 4.54–4.49 (m, 1 H,
CH), 3.99–3.95 (m, 1 H, CH), 3.83–3.66 (m, 5 H, CH2, OCH3),
3.42–3.26 (m, 2 H, CH2), 1.42–1.28 (m, 15 H, CH3) ppm. 13C NMR
(75 MHz, CDCl3, 25 °C): δ = 173.2, 171.7 (CO), 146.3, 141.1,
131.2, 128.1, 125.5 (ArC), 119.0 (CN), 110.6 (ArC), 80.6
[C(CH3)3], 66.9 (Cα), 52.3 (OCH3), 48.5 (CH), 43.3, 42.8 (CH2),
28.1, 17.7 (CH3) ppm. HRMS (ESI): m/z calcd. for C23H30N4O6 +
H+ [M + H]+ 459.2244; found 459.2248.
chromatography (pentane/EtOAc, 9:1) to give 15 (65 mg, 55%) as
25
a blue solid. Rf = 0.23 (pentane/EtOAc, 9:1); m.p. 210 °C; [α]
=
436
–6 (c = 0.053, CH2Cl2). HRMS (ESI): m/z calcd. for C29H42N5O8
+ H+ [M + H+] 589.3111; found 589.3134.
X-ray Diffraction: Crystals of tripeptide Boc-Ala-(R)-Aic(NN)-
Ala-OMe (15) were grown by slow evaporation from EtOH as
highly elongated, but extremely thin plates. A specimen (ca. 1.00ϫ
0.07ϫ 0.02 mm3 in size) was glued on the tip of a glass capillary.
X-ray diffraction data were collected at room temperature with an
Agilent Technologies Gemini E four-circle kappa diffractometer
equipped with a 92 mm EOS CCD detector, using graphite mono-
chromated Cu Kα radiation (λ = 1.54178 Å). A total of 1880 frames
were collected by 0.5° ω oscillation with exposure times of 35 or
70 s, depending on the θ values, in the 2.12° to 51.36° θ range. The
crystal did not significantly diffract beyond 1.0 Å resolution. Data
collection and reduction were performed with the CrysAlisPro soft-
ware (version 1.171.35.19, Agilent Technologies). A semiempirical
absorption correction based on the multiscan technique using
spherical harmonics, implemented in the SCALE3 ABSPACK scal-
ing algorithm, was applied. The structure was solved by ab initio
procedures of the SIR2002 program.[37] The trial solution with the
highest combined figure of merit allowed the location of 63 (out of
84) atoms in two independent peptide molecules. The model was
completed by recovering the positions of the remaining atoms from
subsequent difference Fourier maps interleaved by cycles of refine-
ment. Refinement was carried out by full-matrix least-squares pro-
cedures on F2, using all data, by application of the SHELXL-97
program.[38] In each peptide molecule, the aromatic ring of the
Aic(NN) side chain was constrained to the idealized geometry. Re-
straints were applied to a number of bond lengths and bond angles.
When anisotropic refinement of the non-H atoms was introduced,
the expected significant drop of the value of the R1 factor did not
take place. In addition, the displacement ellipsoids of most of the
atoms became unacceptably oblate. Even after the incorporation of
all H-atoms in the model, the R1 factor did not decrease below the
value of 0.177. These findings, combined with the observation that
in the list of the “most disagreeable reflections” the intensities of
the observed structure factors were invariably higher than the cal-
culated values, suggested the possibility of twinning of the crystal.
Data analysis performed with the TwinRotMat algorithm within
the PLATON suite of programs[39] suggested the occurrence of a
second twin domain, the orientation of which being given by the
matrix (–1 0 0; 0 –1 0; 1 0 1). Application of such a twin law allowed
the refinement to converge smoothly in a few cycles. The batch
scale for the second twin domain refined to the value of 0.4788(19).
Restraints were applied to a number of bond lengths and bond
Boc-Ala-(R)-Aic(CHO)-Ala-(OMe) (14): Tripeptide 13 (246 mg,
0.54 mmol) was dissolved in pyridine (5 mL), and AcOH (2.5 mL)
and sodium hypophosphite hydrate (510 mg) were added. A sus-
pension of Raney nickel 2800 (ca. 300 mg) in water (3 mL) was
added and the reaction mixture was vigorously stirred at 45 °C for
2 h and cooled. The supernatant was decanted and the remaining
nickel was washed twice with 95% EtOH. The combined organic
phases were diluted with diethyl ether and washed with saturated
aqueous NaCl solution. The organic phase was dried with MgSO4,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography (CH2Cl2/MeOH, 95:5) to give
14 (163 mg, 66%) as a solid. Rf = 0.25 (CH2Cl2/MeOH, 95:5); m.p.
1
138–140 °C; [α]2D5 = –58 (c = 0.52, CH2Cl2). H NMR (300 MHz,
CDCl3, 25 °C): δ = 9.94 (s, 1 H, CHO), 7.71–7.69 (m, 2 H, ArH),
7.37–7.27 (m, 2 H, ArH, NH), 6.98 (s, 1 H, NH), 4.96 (d, J =
5.4 Hz, 1 H, NH), 4.59–4.49 (m, 1 H, CH), 4.02–3.93 (m, 1 H,
CH), 3.84–3.67 (m, 5 H, CH2, OCH3), 3.47–3.31 (m, 2 H, CH2),
1.42–1.30 (m, 15 H, CH3) ppm. 13C NMR (75 MHz, CDCl3,
25 °C): δ = 191.9 (CHO), 173.2, 171.9 (CO), 148.0, 140.9, 135.9,
129.9, 125.3 (ArC), 80.7 [C(CH3)3], 67.1 (Cα), 52.3 (OCH3), 48.5
(CH), 43.2, 42.8 (CH2), 28.1, 17.8 (CH3) ppm. HRMS (ESI): m/z
calcd. for C23H31N3O7 + H+ [M + H]+ 462.2240; found 462.2242.
Boc-Ala-(R)-Aic(NN)-Ala-(OMe) (15): Aldehyde 14 (158 mg,
0.34 mmol) was dissolved in CHCl3 (3 mL) and 2,3-dimethylbut-
ane-2,3-diamine (78 mg, 0.68 mmol) was added. The reaction mix-
ture was heated to reflux and stirred for 24 h, cooled, and dried
1750
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1741–1752

A Quaternary Nitronyl Nitroxide α-Amino Acid
angles. H-atoms were calculated at idealized positions and refined
by using a riding model. CCDC-962381 contains the supplemen-
tary crystallographic data for this paper. These data can be ob-
tained from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif
2012). M. C. is grateful to Prof. A. Dolmella (Department of Phar-
maceutical Sciences, University of Padova) for granting access to
the Gemini diffractometer and for his help with X-ray data collec-
tion and processing.
Boc-(S)-Ala-(R)-Aic(NN)-(S)-Ala-OMe: Formula: C29H42N5O8;
formula weight: 588.7; monoclinic, space group P21; unit cell pa-
rameters: a = 6.0627(3), b = 25.1284(8), c = 21.0284(7) Å, β =
98.279(3)°; V = 3170.2(2) Å3; Z = 4; ρcalcd. = 1.233 Mgm–3; crystal
size: 1.00ϫ 0.07ϫ 0.02 mm3; Cu-Kα radiation (λ = 1.54178 Å); μ
= 0.748 mm–1; 10198 reflections collected; 5898 independent reflec-
tions (Rint = 0.028); data/restraints/parameters: 5898/67/734; R1 =
0.050 [IՆ2σ(I)]; wR2 = 0.130 (on F2, all data); goodness of fit on
F2 = 1.017; largest peak and hole in the final difference Fourier
map: 0.199 and –0.168 eÅ–3.
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Infrared Absorption: The FTIR absorption spectra were recorded
with a Perkin–Elmer 1720 X FTIR spectrophotometer, nitrogen-
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under the same conditions. Cells with CaF2 windows and
pathlengths of 0.1, 1.0, and 10 mm were used. Spectrograde deuter-
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elaboration, the software SPECTRACALC, provided by Galactic,
was employed.
Circular Dichroism: CD spectra were obtained with a Jasco J-715
spectropolarimeter equipped with a Haake thermostat. Cylindrical,
fused quartz cells (Hellma) of 0.1 mm path lengths were used. The
values are expressed in terms of [Θ]T, the total molar ellipticity
[degcm2 dmol–1]. Spectrograde MeOH (Acros Organics) was used
as solvent.
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400 MHz using the TOPSPIN software package. The compounds
were dissolved in [D6]acetone (99.96% D6, EURISO-TOP) at 5 mm
concentration. The spin systems of the Ala residues in tripeptide
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Acknowledgments
J. S. thanks the C’Nano NOVATECS (project number IF-08-1453/
R) for financial support. M. D. Z. acknowledges financial support
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Received: November 25, 2013
Published Online: January 14, 2014
1752
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