FULL PAPER

DOI: 10.1002/ejoc.201301765

A Quaternary Nitronyl Nitroxide α-Amino Acid: Synthesis, Configurationaland 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] andClaudio Toniolo[c,d]

Keywords: Sensors / EPR spectroscopy / Magnetic properties / Amino acids / Peptides / Protein modifications / Proteinfolding

To expand the presently extremely limited repertoire ofknown nitronyl nitroxide α-amino acids, we report here thesynthesis of a novel, conformationally constrained, quater-nary, chiral residue, Aic(CN), and its subsequent conversioninto a blue-colored, imidazolinyl nitronyl nitroxide-bearingα-amino acid, Aic(NN). Deprotection and peptide couplingreactions were examined. The configurational assignment ofAic(NN) was achieved by X-ray crystallographic analysis of

Introduction

Nitronyl nitroxides are members of a class of stable freeradicals that have attracted considerable interest, in particu-lar as spin labels and organic magnetic materials. In thiscontext, the sub-class of the Ullman imidazolinyl nitronylnitroxide monoradicals[1] has been the most extensively in-vestigated. The properties of these latter compounds havebeen 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 inwhich the chiral center is located either in the imidazolinylring 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 complexesof nitronyl nitroxides with transition metals have been pre-

[a] ILV, UMR 8180, University of Versailles,78035 Versailles, FranceE-mail: karen.wright@.uvsq.frwww.ilv.uvsq.fr

[b] GEMAC, UMR 8635, University of Versailles,78035 Versailles, France

[c] Department of Chemistry, University of Padova,35131 Padova, Italy

[d] Institute of Biomolecular Chemistry, Padova Unit, CNR,35131 Padova, ItalySupporting information for this article is available on theWWW under http://dx.doi.org/10.1002/ejoc.201301765.

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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 andmagnetic properties of its derivatives and the tripeptide arealso described. Of particular relevance for future applicationsare its UV/Vis absorption, NMR, and EPR signatures.

pared in attempts to increase ferromagnetic ordering bycreating extended structures.[13]

In the field of peptide chemistry, free radical spin labelsare used in conjunction with EPR spectroscopy particularlyin conformational studies. The nitronyl nitroxide moietyhas not, as yet, been widely employed for this applicationcompared with the more popular nitroxides, for examplesidechain 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 labeledwith nitronyl nitroxides through N-terminal or sidechainmodification. Weinkam and Jorgensen[5] prepared nitronylnitroxyl 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 theEPR spectrum of this analogue was examined. Peng andco-workers[6] used nitronyl nitroxides derived from salicylal-dehyde to spin label the N-terminal or the sidechains ofLys, Arg, Asp and Glu in short peptides, and employedthese compounds to study free radical (e.g., nitric oxide)scavenging and bioactivity. Both groups of authors wereable to show that these free radicals could survive most ofthe experimental conditions typically used in peptide syn-thesis, such as acidic N-Boc deprotection, saponification,and coupling reactions.

Our groups have investigated extensively Ciα�Ci

α-cyclized, Cα,α-disubstituted glycines [in particular 1-amino-cyclopentane-1-carboxylic acid, Ac5c[16] (Figure 1)], whichare sterically restricted α-amino acids that promote the for-

K. Wright et al.FULL PAPERmation of β-turns[17] and helical structures[16,18] when incor-porated into peptides. More recently, we were interested inthe possibility of creating a rigid, chiral, nitronyl nitroxidebearing, Cα-tetrasubstituted (quaternary) α-amino acid,Aic(NN) (Figure 1) as a new spin label. We have previouslysynthesized 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 isitself interesting as a Cα-tetrasubstituted analogue of p-cyanophenylalanine, which has been used as an IR absorp-tion and fluorescence spectroscopic label to study proteinfolding, environment, and binding.[21] The nitrile functionof Aic(CN) serves as a precursor of the aldehyde group,which is necessary for the installation of the imidazolinylring. A preliminary report of a limited part of this workhas been published.[22]

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)gave dibromide 2, which was used to form the racemic Cα-tetrasubstituted α-amino ethyl ester 3 by bis(alkylation) ofethyl isocyanoacetate under phase-transfer conditions.[23]

Acidic hydrolysis of the intermediate isocyanide, followedby Nα-Boc protection with Boc anhydride and saponifica-tion of the ester function, gave the desired, racemic, Nα-protected α-amino acid 4 (Scheme 1). A method for the op-tical resolution of 4 was sought that would not requireharsh acidic conditions to remove the chiral auxiliary,which could be incompatible with the nitrile group.

We have previously used the easily hydrolyzed dia-stereomeric amides of (S)-phenylglycinol (Phg-ol) to sepa-rate acid-sensitive quaternary azetidines.[24] When racemic4 was coupled with (S)-Phg-ol, in the presence of N-eth-yl,N�-[3-(dimethylamino)propyl)]carbodiimide (EDC)/ethyl2-cyano-2-(hydroxyimino)acetate (Oxyma),[25] and N-meth-

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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 5bwere obtained, which could be separated by columnchromatography. Hydrolysis of the two separate amides byaqueous 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 fromhydrolysis of the nitrile function, were detected in the reac-tion mixtures under these conditions. Nα-Boc protectiongave the enantiomeric, fully protected α-amino acids 6a and6b.

Nitronyl Nitroxide Amino Acid [Aic(NN)] Synthesis

Conditions for the selective conversion of the nitrilegroup into an aldehyde were sought that left the carbamateand ester protecting groups intact. When separate re-ductions of 6a and 6b were carried out with Raney nickelin the presence of sodium hypophosphite (Scheme 2),[26] thecorresponding aldehydes 7a and 7b were obtained in 73 and91% yields, respectively. Two possible routes to convert al-dehydes into nitronyl nitroxides have been previously re-ported. The first method, as originally proposed by Ull-man,[1] relies on the condensation of 2,3-bis(hydroxy-amino)-2,3-dimethylbutane with the aldehyde followed byoxidation of the intermediate. In our case, the preparationof 2,3-bis(hydroxyamino)-2,3-dimethylbutane proved to befrustratingly difficult. This method was finally abandonedin favor of the route proposed by Rey and co-workers,[27] inwhich the aldehyde is first condensed with 2,3-diamino-2,3-dimethylbutane to give the corresponding tetramethylimid-

A Quaternary Nitronyl Nitroxide α-Amino Acid

azolidine. This procedure was successfully applied to alde-hydes 7a and 7b. The imidazolidine intermediates 8a and 8bwere oxidized with 3-chloroperbenzoic acid (mCPBA) thenwith NaIO4, to afford the desired nitronyl nitroxides Boc-(R)- and Boc-(S)-Aic(NN)-OEt 9a and 9b in 58 and 64%yields, respectively. The corresponding imino nitroxides[5]

were isolated from these oxidations in less than 5 % yield.

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-tronyl nitroxide amino acid in deprotection and peptidecoupling reactions were carried out by using Boc-(S)-Aic-(NN)-OEt 9b (Scheme 3). Saponification of the ester func-tion and reaction of the resulting carboxylic acid withH-Ala-OMe in the presence of O-(7-azabenzo-1,2,3-triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HATU)[28] and diisopropyl-ethylamine (DIEA) affordeddipeptide 10 in good yield. It was also possible to removethe Nα-Boc group of 9b with dilute trifluoroacetic acid(TFA) at 0 °C, without extensive decomposition of the ni-tronyl nitroxide moiety, to give 11 in reasonable yield. How-ever, the acid- and light-sensitive nature of the radicals indi-cated that it would be more straightforward to build a pept-ide chain by using the Aic(CN) precursor amino acid, andto introduce the nitronyl nitroxide moiety as a final step. Atripeptide, Boc-Ala-(R)-Aic(CN)-Ala-OMe 13 was synthe-

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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sized to demonstrate the feasibility of this strategy. Cou-pling of Boc-Ala-OH with 3a in the presence of HATUgave dipeptide 12 in 90% yield. Saponification of the ethylester function of 12 and coupling of the resulting carboxylicacid to H-Ala-OMe afforded tripeptide 13. Reduction ofthe nitrile function with Raney nickel/sodium hypophos-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 ofthis 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 wasknown to be (S), because it is that of the amino acids usedin our synthetic procedures. Based on this information, weassigned the configuration of the Aic(NN) residue presentin the crystalline tripeptide 15 as (R).

The corresponding backbone torsion angles are similarin the two molecules, and their difference does not exceed7°. All urethane, peptide, and ester (ω) torsion angles arein the common trans disposition. However, in both mole-cules the torsion angle (ω1) of the peptide bond betweenAla1 and Aic(NN)2 is strongly distorted, by as much as 16–19° from planarity.

Preliminary information on the 3D-structure adopted bythe tripeptide was obtained from the critical N–Cα–C� (τ)bond angles of the three residues in each molecule. Theirvalues range between 109.7(6)° and 112.7(6)°, typical of asemi-extended or a helical conformation, but not of a fully-extended structure.[18a,18d,18e,29] We found that the con-formations of the Ala1 and Ala3 residues are semi-extended,whereas that of Aic(NN)2 is helical. In particular, the φ2

torsion angle of the latter residue is closer to that expectedfor an α-helix (�63°) than for a 310-helix (�57°).[18a] How-

K. Wright et al.FULL PAPER

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 –172.5(6) –172.3(6)φ1 –59.3(8) –64.8(8)ψ1 136.8(5) 142.3(5)ω1 163.8(5) 161.3(5)φ2 66.9(7) 66.3(7)ψ2 26.0(8) 24.1(8)ω2 175.7(5) –179.2(6)φ3 –61.6(8) –68.5(8)“ψ”3 140.7(7)[a] 140.7(7)[c]

“ω”3 178.9(8)[b] 178.4(8)[d]

[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 (�30°for a 310-helix, �42° for an α-helix). Interestingly, the rela-tionship between chirality and helix screw sense of Aic(NN)in this structure is the same as that of common Cα-trisubsti-tuted α-amino acids, i.e., an (R) residue tends to fold in aleft-handed helix (its φ and ψ torsion angles are both posi-tive). As a consequence, the molecules exhibit a type-II β-turn conformation.[17] However, in these molecules this typeof folding is “open”, i.e., it is not stabilized by the classical

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i � i+3 intramolecular C=O···H–N H-bond, in that theO0A···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 theβ-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 thequaternary α-amino acids,[16,18] is a strong turn-promotingresidue.

As for the geometry of the heterocyclic moiety, which iscommon to all Ullman imidazolinyl nitronyl nitroxides,bond lengths and bond angles are in general agreementwith the corresponding mean values extracted from a statis-tical analysis of published X-ray diffraction structures.[2c] Inparticular, the N–O bond lengths range from 1.294(9) to1.309(10) Å. Within each molecule (A and B) the two N–Obond lengths differ by less than 1.5 σ (0.015 Å).[7d]

The twist angle APNN between the plane of the phenylring and the nitronyl nitroxide NCN plane[2c] is 25.8(3)° inmolecule A and 27.6(4)° in molecule B. This parameter isof great relevance in governing the magnetic characteristicsof a phenyl nitronyl nitroxide, in that the delocalization ofthe unpaired electron is dependent on the conjugation be-tween its two cyclic moieties. The co-planar arrangement isthe most stable in vacuo.[2c] In the majority of the solid 3D-structures, however, the phenyl nitronyl nitroxides differ toa greater or lesser extent from this conformation, with theaverage value of the statistical distribution of APNN being27.5� 8.5°.[2c] Therefore, the optimal packing of the mole-cules in the crystal apparently plays a major role. In anycase, this out-of-planarity arrangement does not completelyprevent 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 weaksince the C–H···O angles (117–121°) are very far from theideal values (about 150°), despite the acceptable C···O dis-tances [in the range 2.883(9)–2.922(8) Å].

Additional interesting conformational features are thepuckering motifs of the two five-membered rings (cyclopen-tene and imidazoline) of the Aic(NN) residue in moleculesA and B. The cyclopentenyl ring in molecule A, with pa-rameters q2 = 0.334(8) Å and φ2 = 182.6(9)°,[33] shows apuckering 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 equatorialcarbonyl C atom on the α-carbon. Overall, the cyclopent-enyl ring is only slightly out of the plane of the fused phenylring in each Aic moiety. The angle between the normals tothe cyclopentenyl and phenyl average planes is 10.97(18)°in molecule A and 8.77(19)° in molecule B. The imid-azolinyl ring of the Aic(NN) residue in molecule A showspuckering parameters q2 = 0.288(7) Å and φ2 = 119.4(16)°(relative to the atom sequence N21–C2Z–N22–C24–C23).Similar values characterize the imidazolinyl ring puckeringin molecule B, namely q2 = 0.247(8) Å and φ2 = 118(2)°.Both rings adopt a 5T4 twist conformation, as commonlyobserved in Ullman imidazolinyl nitronyl nitroxides.[2c]

A Quaternary Nitronyl Nitroxide α-Amino Acid

IR Absorption

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 and3427 cm–1[32a,32b] is approximately 1.1 and does not changewith dilution. Since this ratio is indicative of the amount ofintramolecularly 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 ratiois less than 0.15.[32a] This result is in good accordance withour aforementioned X-ray diffraction data.

Figure 3. FTIR absorption spectra of tripeptide 15 in CDCl3 solu-tion in the amide N–H (a) and N–O (b) stretching regions. Peptideconcentration: 1 mm.

The N–O stretching band of the nitronyl nitroxide moi-ety, which usually appears at 1361–1370 cm–1 in the solidstate,[2b,2c] is positioned at 1358–1360 cm–1 in tripeptide 15(Figure 3 B) and in the Aic(NN) derivatives 9a and 9b inCDCl3. This modest shift to lower frequency might suggest

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the participation of the N–O groups of our compounds inweak H-bonds.

UV/Vis Absorption and Circular Dichroism (CD)

In the near UV/Vis regions of the electronic spectrum inmethanol (MeOH) solution, the Aic(NN) derivatives 9aand 9b, and tripeptide 15 show three well-separated absorp-tions centered near 280, 370, and 590 nm (Figure 4). Thelast, very broad and weak band[2,3a,4b] is associated withthe n �π* transition of the ONCNO chromophore of thenitronyl nitroxide moiety[4b] and is responsible for the typi-cal blue color of these compounds. The 370 nmband[2c,2d,4b] is assigned to the π �π* transitions of thesame chromophore from a common ground state to excitedstates with two orbital contributions.[4b]

Figure 4. UV/Vis absorption spectrum of tripeptide 15 in MeOHsolution.

Figure 5 illustrates the CD spectra of derivatives 9a and9b and tripeptide 15 in MeOH solution in the near-UV (a)and visible (b) regions. Not surprisingly, the signs of thestrong Cotton effects between 250 and 600 nm are relatedto the absolute configuration of the α-carbon of theAic(NN) residue, as both compounds 9a and 15, where theAic(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 thewavelengths of the maxima of most of the numerous CDbands do not correspond to any UV/Vis absorption maxi-mum.[2c,4]

Combinations of the component chromophores (nitronylnitroxide and the linked phenyl), including exciton couplingand charge transfer effects, and the twist angle betweenthem, are known to contribute heavily to the observed spec-tra. In addition, the CD contributions arising from the ni-tronyl nitroxide chromophore below 250 nm might interferewith the use of this chirospectroscopic technique to deter-mine the secondary structure of peptides.

K. Wright et al.FULL PAPER

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) inMeOH solution in the UV (a) and visible (b) regions.

Nuclear Magnetic Resonance

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 significantline broadening, despite the presence of a nearby paramag-netic nitronyl nitroxide moiety. This result indicates that thespin densities at these protons are low-to-moderate,[7d,8] atvariance 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 mightbe 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-

Figure 6. 1H NMR spectrum of tripeptide 15 in [D6]acetone. Thepeak at δ = 2.04 ppm is associated with the methyl protons of thesolvent.[8a]

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tons of Ala1 and Ala3 resonate at δ = 1.37 and 1.23 ppm,whereas those of their αCH protons resonate at δ = 3.93and 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 methylester OMe is at δ = 3.67 ppm.

Electron Paramagnetic Resonance

The complete experimental EPR spectra of compounds9a/9b in a 1 mm CDCl3 solution at 293 K are shown in theinset of Figure 7 (a), while that of the tripeptide 15 is re-ported in the inset of Figure 7 (b). The EPR spectra of thesample solutions diluted to 0.1 mm concentration do notshow any difference except for a lower signal-to-noise ratio.All spectra are characterized by a quintet of hyperfine lineswith relative intensity ratios 1:2:3:2:1, as expected for theisotropic coupling of the unpaired electron spin with twoequivalent 14N nuclear spins (I = 1). The hyperfine couplingconstant 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 quintetsare also shown. These expansions reveal further hyperfinecouplings of the electron spin with the 1H nuclei of the mo-lecule (two ortho protons and one meta proton of the phenylring, in addition to twelve protons of the four methylgroups). Figure 7 also shows the spectra calculated with theroutines of the EasySpin software package[35] running in theMATLAB environment, and employing the g factor andhyperfine coupling constants ai as variable parameters. Thesimulations based on the best fit values of the g factor andthe ai constants, reported in Table 2, reproduce quite wellthe experimental EPR signals. The magnetic parameters ob-tained are in line with those reported for similar imid-

A Quaternary Nitronyl Nitroxide α-Amino Acid

azolinyl nitronyl nitroxides in organic solvents of low po-larity.[1b–1e,7c]

Figure 7. Experimental and simulated EPR spectra. (a) The solidline is the MI = 0 component of the experimental EPR spectrumof a 1 mm CDCl3 solution of compounds 9a/9b at T = 293 K. Thedotted line is the simulated spectrum. Inset: complete EPR spec-trum with 1:2:3:2:1 intensity ratio of the main components, due tothe hyperfine coupling with two equivalent 14N nuclei. (b) The solidline is the MI = 0 component of the experimental EPR spectrumof a 1 mm CDCl3 solution of tripeptide 15 at T = 295 K. The dottedline is the simulated spectrum. Inset: complete EPR spectrum with1:2:3:2:1 intensity ratio of the main components, due to the hyper-fine coupling with two equivalent 14N nuclei. For the parametersof the simulations, see text and Table 2.

Table 2. The g factors and hyperfine coupling constants ai (Mt)[a]

used to reproduce the EPR spectra of compounds 9a/9b and 15 inCDCl3 at T = 295 K.

g factor aN aH–ortho[b] aH–neta

[b] aH–methyl[b]

(2 nuclei) (2 nuclei) (1 nucleus) (12 nuclei)

9a/9b 2.0066 0.761 0.650 0.359 0.21515 2.0062 0.761 0.675 0.380 0.210

[a] Estimated errors: �0.0002 for the g factor, �0.002 for the hy-perfine constants ai. [b] The ortho and meta are positions in thephenyl ring relative to the nitronyl nitroxide moiety. Methyl refersto the 1H-nuclei in the four methyl group substituents of the five-membered heterocycle.

Magnetic Susceptibility

The magnetic susceptibility χM [χM = C/(T – θ)] of rac-Boc-Aic(NN)-OEt (9), measured under a 90 kOe magneticfield, is plotted against (T – θ)–1 in Figure 8. The data arewell-fitted by a Curie–Weiss law, revealing paramagnetic be-havior between 8 and 180 K, with the Curie constant C =0.35 emu K/mol consistent with values reported for similarcompounds.[4b,41] The negative value of θ (–2.7 K) indicatesa small antiferromagnetic interaction.

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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 topaxis. 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 precursoramino 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 byusing 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 thosebased on the azulene chromophore[36]) that are blue coloredin the absence of any metal cation.

An X-ray diffraction analysis and an FTIR absorptionstudy of the terminally protected tripeptide 15 unambigu-ously established the configuration of the central Aic(NN)residue and the remarkable propensity of its peptides to foldinto a turn conformation. This latter property suggests that,to avoid unwanted conformational modifications, Aic(NN)should preferentially be incorporated into peptides alreadyknown to fold.

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 tobe useful as a rigid, paramagnetic probe in peptide chemis-try.

Experimental SectionSynthesis: Melting points were determined with a capillary tubeimmersed in an oil bath (Tottoli apparatus, Büchi). 1H and 13CNMR spectra were recorded with a Bruker WM300 spectrometeroperating at 300 and 77 MHz, respectively. The solvent used as theinternal 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-resolutionmass spectra (electrospray mode) were performed by Dr. EstelleGalmiche (ILV) with a Xevo Q-TOF (Waters) mass spectrometer.Elemental analyses were performed by the C.N.R.S. Service of

K. Wright et al.FULL PAPERMicroanalyses in Gif-sur-Yvette (France). Optical rotations weremeasured, with an accuracy of 0.3%, in a 1-dm thermostatted cell.Analytical thin-layer chromatography (TLC) and columnchromatography were carried out on silica gel F 254 (Merck) andKieselgel gel 60 (0.040–0.063 mm) (Merck), respectively.

3,4-Bis(bromomethyl)benzonitrile (2): 3,4-Dimethylbenzonitrile(9.17 g, 70 mmol) was dissolved in CCl4 (150 mL), the solution wasplaced under argon, and NBS (24.9 g, 140 mmol) and benzoyl per-oxide (700 mg) were added. The reaction mixture was heated atreflux 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, N4.76.

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 filteredthrough 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 °Cfor 4 h. Excess saturated aqueous NaHCO3 solution was added andthe mixture was stirred for 15 min, then the mixture was extractedthree times with CH2Cl2. The combined organic extracts werewashed with saturated aqueous NaCl solution, dried with MgSO4,filtered, and concentrated under reduced pressure. The residue waspurified by column chromatography (CH2Cl2/MeOH, 95:5) to give3 (3.35 g, 55%) as an oil. Rf = 0.47 (CH2Cl2/MeOH, 95:5). 1HNMR (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),1.29 (t, J = 7.1 Hz, 3 H, CH3) ppm. 13C NMR (75 MHz, CDCl3,25 °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.

2-(tert-Butyloxycarbonylamino)-5-cyanoindan-2-carboxylic Acid[Boc-(R,S)-Aic(CN)-OH; 4]: Compound 3 (4.88 g, 21.2 mmol) wasdissolved in CH3CN (250 mL) and Boc2O (11.5 g, 53 mmol) wasadded. The reaction mixture was stirred at room temp. for 42 h andconcentrated under reduced pressure. The residue was purified bycolumn 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 for45 min, cooled, and diluted with water. Volatiles were removed un-der reduced pressure and the resulting solution was cooled on anice bath and acidified by addition of 2 m aqueous HCl. The mixturewas extracted three times with CH2Cl2 and the combined organicextracts were dried with MgSO4, filtered, and concentrated underreduced pressure. The residue was purified by column chromatog-raphy (CH2Cl2/MeOH, 90:10) to give 4 (5.58 g, 87 %) as a foam.Rf = 0.37 (CH2Cl2/MeOH/AcOH, 95:5:1). 1H NMR (300 MHz,

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[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.

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 wascooled 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 twicewith 0.5 m aqueous HCl, then with water, and finally with saturatedaqueous NaHCO3 solution. The organic phase was dried withMgSO4, filtered, and concentrated under reduced pressure. The re-sulting diastereomeric mixture was separated by columnchromatography (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]D25 = +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. 13CNMR (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.

Boc-(S)-Aic(CN)-(S)-Phg-ol (5b): Rf = 0.40 (THF/pentane/CH2Cl2,45:55:20); m.p. 209 °C; [α]D25 = +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.

H-(R)-Aic(CN)-OEt (3a): Compound 5a (1.23 g, 2.92 mmol) wasdissolved in 1,4-dioxane (100 mL) and a solution of 3 m aqueousH2SO4 (4.8 mL) was added. The reaction mixture was heated atreflux for 24 h, then cooled. EtOH (50 mL) was added and volatileswere removed under reduced pressure. EtOH (100 mL) was addedto the residue, and the mixture was heated at reflux for 72 h. Themixture 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 withCH2Cl2 and washed with saturated aqueous NaCl solution. Theorganic phase was dried with MgSO4, filtered, and concentratedunder reduced pressure. The residue was purified by columnchromatography (CH2Cl2/MeOH, 97:3) to give 3a (505 mg, 75%)as an oil. [α]D25 = +8 (c = 0.25, CH2Cl2). HRMS (ESI): m/z calcd.for C13H14N2O2 + H+ [M + H]+ 231.1134; found 231.1131.

H-(S)-Aic(CN)-OEt (3b): The (S)-enantiomer was prepared in thesame way from 5b (1.00 g, 2.37 mmol) to give 3b (453 mg, 83%) asan oil. [α]D25 = –8 (c = 0.27, CH2Cl2). HRMS (ESI): m/z calcd. forC13H14N2O2 + H+ [M + H]+ 231.1134; found 231.1130.

Boc-(R)-Aic(CN)-OEt (6a): Compound 3a (308 mg, 1.34 mmol)was dissolved in CH3CN (6 mL) and Boc2O (585 mg, 2.68 mmol)

A Quaternary Nitronyl Nitroxide α-Amino Acid

was added. The reaction mixture was stirred at room temp. for24 h, then concentrated under reduced pressure. The residue waspurified by column chromatography (pentane/EtOAc, 8:2) to give6a (295 mg, 67%) as a foam. Rf = 0.55 (pentane/EtOAc, 75:25);[α]D25 = –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, 2H, 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. forC18H22N2O4 + H+ [M + H]+ 331.1658; found 331.1658.

Boc-(S)-Aic(CN)-OEt (6b): The (S)-enantiomer was prepared in thesame way from 3b (380 mg, 1.65 mmol) to give 6b (387 mg, 71%)as a foam. [α]D25 = +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) wasdissolved in pyridine (1.5 mL), and AcOH (1.5 mL) and sodiumhypophosphite hydrate (370 mg) were added. A suspension ofRaney nickel 2800 (ca. 200 mg) in water (2 mL) was added and thereaction mixture was stirred vigorously at 45 °C for 1 h and thencooled. The supernatant was decanted and the remaining nickelwas washed twice with 95% EtOH. The combined organic phaseswere diluted with diethyl ether and washed with saturated aqueousNaCl solution. The organic phase was dried with MgSO4, filtered,and concentrated under reduced pressure. The residue was purifiedby column chromatography (pentane/EtOAc, 8:2) to give 7a(152 mg, 73%) as a syrup. Rf = 0.59 (pentane/EtOAc, 7:3); [α]D25 =–28 (c = 0.54, CH2Cl2). 1H NMR (300 MHz, CDCl3, 25 °C): δ =9.96 (s, 1 H, CHO), 7.72–7.70 (m, 2 H, ArH), 7.37–7.27 (m, 1 H,ArH), 5.20 (br. s, 1 H, NH), 4.21 (q, J = 7.1 Hz, 2 H, CH2), 3.71–3.60 (m, 2 H, CH2), 3.37–3.27 (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 = 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.

Boc-(S)-Aic(CHO)-OEt (7b): The (S)-enantiomer was prepared inthe same way from ester 6b (350 mg, 1.06 mmol) to give 7b(322 mg, 91%). [α]D25 = +27 (c = 0.52, CH2Cl2). HRMS (ESI): m/zcalcd. for C18H23NO5 + Na+ [M + Na]+ 356.1474; found 356.1476.

Boc-(R)-Aic(NN)-OEt (9a): Aldehyde 7a (132 mg, 0.39 mmol) wasdissolved in CHCl3 (5 mL) and 2,3-dimethyl-butane-2,3-diamine(92 mg, 0.80 mmol) was added. The reaction mixture was heatedto reflux and stirred for 42 h, then cooled and dried with Na2SO4.The mixture was filtered and concentrated under reduced pressureand the residue was taken up in CH2Cl2 and filtered through a plugof silica gel, eluting with CH2Cl2/MeOH/triethylamine (90:10:1).The filtrate was evaporated to give the intermediate imidazolidine8a (152 mg) as a foam, which was used directly in the next stepwithout further purification. Imidazolidine 8a was dissolved inCH2Cl2 (7.5 mL) and a saturated aqueous NaHCO3 solution(3.7 mL) was added. The mixture was cooled on an ice bath, thena solution of m-CPBA (70%, 184 mg, 0.75 mmol) in CH2Cl2(2 mL) was added dropwise. The reaction mixture was stirred for1 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 for1 h at 0 °C, then diluted with CH2Cl2 and the phases were sepa-rated. The aqueous phase was extracted once with CH2Cl2 and thecombined organic phases were washed with saturated aqueousNaCl solution, dried with Na2SO4, filtered, and concentrated un-

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der reduced pressure. The residue was purified by columnchromatography (pentane/EtOAc, 1:1) to give 9a (80 mg, 58%) asa blue solid. Rf = 0.39 (pentane/EtOAc, 1:1); m.p. 88 °C; [α]436

25 =–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 inthe same way from aldehyde 7b (300 mg, 0.90 mmol) to give 9b(187 mg, 64 %). [α]436

25 = +50 (c = 0.061, CH2Cl2). HRMS (ESI):m/z calcd. for C24H34N3O6 + H+ [M + H+] 460.2454; found460.2455.

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), a0.2 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 wasdiluted with water and volatiles were removed under reduced pres-sure. The resulting solution was acidified by careful addition ofaqueous 0.5 m HCl solution and the mixture was extracted twicewith EtOAc. The combined organic extracts were dried withMgSO4, 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 andHATU (16 mg, 0.04 mmol) and DIEA (0.014 mL, 0.08 mmol) wereadded. The mixture was stirred at room temp. for 6 h, then concen-trated under reduced pressure and the residue was dissolved inCH2Cl2. The solution was washed with 0.5 m aqueous HCl, thenwith saturated aqueous NaCl solution, and finally with saturatedaqueous NaHCO3 solution. The organic phase was dried withMgSO4, filtered, and concentrated under reduced pressure. The res-idue was purified by column chromatography (CH2Cl2/MeOH,95:5) to give 10 (18 mg, 69%) as a blue solid. Rf = 0.22 (CH2Cl2/MeOH, 95:5); m.p. 96–98 °C (dec.); [α]436

25 = –20 (c = 0.06, CH2Cl2).HRMS (ESI): m/z calcd. for C26H37N4O7 + H+ [M + H+] 518.2741;found 518.2739.

H-(S)-Aic(NN)-OEt (11): Compound 9b (23 mg, 0.05 mmol) wasdissolved in ice-cold CH2Cl2/TFA (9:1, 0.5 mL) and stirred at 0 °Cfor 4 h. The reaction mixture was diluted with CH2Cl2 and washedwith 0.02 m aqueous ammonium acetate solution. The aqueousphase was extracted twice with CH2Cl2, then the combined organicphases were dried with Na2SO4, filtered, and concentrated underreduced pressure. The residue was purified by column chromatog-raphy (CH2Cl2/MeOH, 95:5) to give 11 (14 mg, 77%) as a blue oil.Rf = 0.22 (CH2Cl2/MeOH, 95:5); [α]436

25 = +15 (c = 0.06, CH2Cl2).HRMS (ESI): m/z calcd. for C19H26N3O4 + H+ [M + H+] 361.2002;found 361.2004.

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 cooledon 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 atroom temp. for 18 h. The mixture was concentrated under reducedpressure and the residue was dissolved in CH2Cl2. The solution waswashed with 0.5 m aqueous HCl, then with saturated aqueous NaClsolution, 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 columnchromatography (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]D25 =–48 (c = 0.53, CH2Cl2). 1H 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,

K. Wright et al.FULL PAPER25 °C): δ = 172.3 (CO), 145.9, 141.2, 131.2, 127.9, 125.3 (ArC),119.0 (CN), 110.7 (ArC), 80.4 [C(CH3)3], 65.2 (Cα), 61.9 (CH2),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.

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) wasadded. The reaction mixture was stirred at room temp. for 1 h andthen 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 extractedthree times with EtOAc and the combined organic extracts weredried with MgSO4, filtered, and concentrated under reduced pres-sure. The residue obtained was dissolved in THF (15 mL) andHCl·H-Ala-OMe (120 mg, 0.88 mmol) was added. The mixture wascooled 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 roomtemp. for 48 h, then the mixture was concentrated under reducedpressure and the residue was dissolved in CH2Cl2. The solution waswashed with 0.5 m aqueous HCl, then with saturated aqueous NaClsolution, 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 columnchromatography (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]D25 = –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.

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) wasadded and the reaction mixture was vigorously stirred at 45 °C for2 h and cooled. The supernatant was decanted and the remainingnickel was washed twice with 95% EtOH. The combined organicphases were diluted with diethyl ether and washed with saturatedaqueous NaCl solution. The organic phase was dried with MgSO4,filtered, and concentrated under reduced pressure. The residue waspurified by column chromatography (CH2Cl2/MeOH, 95:5) to give14 (163 mg, 66%) as a solid. Rf = 0.25 (CH2Cl2/MeOH, 95:5); m.p.138–140 °C; [α]D25 = –58 (c = 0.52, CH2Cl2). 1H 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/zcalcd. 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

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with Na2SO4. The mixture was filtered and concentrated under re-duced pressure and the residue was taken up in CH2Cl2 and filteredthrough 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 inthe next step without further purification. The imidazolidine wasdissolved in CH2Cl2 (6 mL) and saturated aqueous NaHCO3 solu-tion (3.6 mL) was added. The mixture was cooled on an ice bathand a solution of m-CPBA (70%, 118 mg, 0.49 mmol) in CH2Cl2(3 mL) was added dropwise. The reaction mixture was stirred for1 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 for1 h at 0 °C, then diluted with CH2Cl2 and the phases were sepa-rated. The aqueous phase was extracted once with CH2Cl2 and thecombined organic phases were washed with saturated aqueousNaCl solution, dried with Na2SO4, filtered, and concentrated un-der reduced pressure. The residue was purified by columnchromatography (pentane/EtOAc, 9:1) to give 15 (65 mg, 55%) asa blue solid. Rf = 0.23 (pentane/EtOAc, 9:1); m.p. 210 °C; [α]436

25 =–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 ashighly elongated, but extremely thin plates. A specimen (ca. 1.00�

0.07� 0.02 mm3 in size) was glued on the tip of a glass capillary.X-ray diffraction data were collected at room temperature with anAgilent Technologies Gemini E four-circle kappa diffractometerequipped with a 92 mm EOS CCD detector, using graphite mono-chromated Cu Kα radiation (λ = 1.54178 Å). A total of 1880 frameswere collected by 0.5° ω oscillation with exposure times of 35 or70 s, depending on the θ values, in the 2.12° to 51.36° θ range. Thecrystal did not significantly diffract beyond 1.0 Å resolution. Datacollection and reduction were performed with the CrysAlisPro soft-ware (version 1.171.35.19, Agilent Technologies). A semiempiricalabsorption correction based on the multiscan technique usingspherical harmonics, implemented in the SCALE3 ABSPACK scal-ing algorithm, was applied. The structure was solved by ab initioprocedures of the SIR2002 program.[37] The trial solution with thehighest combined figure of merit allowed the location of 63 (out of84) atoms in two independent peptide molecules. The model wascompleted by recovering the positions of the remaining atoms fromsubsequent 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-97program.[38] In each peptide molecule, the aromatic ring of theAic(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 nottake place. In addition, the displacement ellipsoids of most of theatoms became unacceptably oblate. Even after the incorporation ofall H-atoms in the model, the R1 factor did not decrease below thevalue of 0.177. These findings, combined with the observation thatin the list of the “most disagreeable reflections” the intensities ofthe 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 withinthe PLATON suite of programs[39] suggested the occurrence of asecond twin domain, the orientation of which being given by thematrix (–1 0 0; 0 –1 0; 1 0 1). Application of such a twin law allowedthe refinement to converge smoothly in a few cycles. The batchscale for the second twin domain refined to the value of 0.4788(19).Restraints were applied to a number of bond lengths and bond

A Quaternary Nitronyl Nitroxide α-Amino Acid

angles. H-atoms were calculated at idealized positions and refinedby 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 viawww.ccdc.cam.ac.uk/data_request/cif

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; crystalsize: 1.00� 0.07 � 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 [I�2σ(I)]; wR2 = 0.130 (on F2, all data); goodness of fit onF2 = 1.017; largest peak and hole in the final difference Fouriermap: 0.199 and –0.168 eÅ–3.

Infrared Absorption: The FTIR absorption spectra were recordedwith a Perkin–Elmer 1720 X FTIR spectrophotometer, nitrogen-flushed, with a sample shuttle device and at 2 cm–1 nominal resolu-tion, averaging 100 scans. Solvent (baseline) spectra were recordedunder the same conditions. Cells with CaF2 windows andpathlengths of 0.1, 1.0, and 10 mm were used. Spectrograde deuter-iochloroform (99.8% D) was purchased from Aldrich. For spectralelaboration, the software SPECTRACALC, provided by Galactic,was employed.

Circular Dichroism: CD spectra were obtained with a Jasco J-715spectropolarimeter equipped with a Haake thermostat. Cylindrical,fused quartz cells (Hellma) of 0.1 mm path lengths were used. Thevalues are expressed in terms of [Θ]T, the total molar ellipticity[degcm2 dmol–1]. Spectrograde MeOH (Acros Organics) was usedas solvent.

Nuclear Magnetic Resonance of 15: NMR experiments were carriedout with a Bruker AVANCE DRX-400 spectrometer operating at400 MHz using the TOPSPIN software package. The compoundswere dissolved in [D6]acetone (99.96% D6, EURISO-TOP) at 5 mm

concentration. The spin systems of the Ala residues in tripeptide15 were identified by using a standard CLEAN-TOCSY[40] spec-trum (spin-lock pulse sequence of 70 ms).

Electron Paramagnetic Resonance: Solutions (1.0 and 0.1 mm) inspectrograde deuteriochloroform (99.8% D) (Aldrich) of the aminoacid derivative 9a/9b and tripeptide 15 were prepared, transferredinto EPR quartz tubes, and connected to a vacuum line. The sam-ples were then subjected to a number of “freeze, pump, thaw” cyclesto eliminate dissolved oxygen from the solutions. Finally, the tubeswere sealed. The EPR spectra were recorded with a Bruker (ER-200D; X-band; ν ≈ 9.4 GHz) spectrometer. The sample temperaturewas controlled by a nitrogen flow cryostat controlled by a BrukerBVT 2000 unit. The EPR spectra were acquired with ca. 0.01 mTfield modulation amplitude and 0.2–2 mW microwave power.

Magnetic Susceptibility: Magnetic susceptibility was measured witha Quantum Design vibrating sample magnetometer. The maximumapplied magnetic field was 90 kOe.

Supporting Information (see footnote on the first page of this arti-cle): 1H and 13C NMR spectra for compounds 2–4, 5a, 5b, 3a, 3b,6a, 6b, 7a, 7b, and 12–14.

Acknowledgments

J. S. thanks the C’Nano NOVATECS (project number IF-08-1453/R) for financial support. M. D. Z. acknowledges financial supportfrom the Fondazione CARIPARO (Progetti di Eccellenza 2011/

Eur. J. Org. Chem. 2014, 1741–1752 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org 1751

2012). M. C. is grateful to Prof. A. Dolmella (Department of Phar-maceutical Sciences, University of Padova) for granting access tothe Gemini diffractometer and for his help with X-ray data collec-tion and processing.

[1] a) J. H. Osiecki, E. F. Ullman, J. Am. Chem. Soc. 1968, 90,1078–1079; b) E. F. Ullman, J. H. Osiecki, D. G. B. Boocock,R. Darcy, J. Am. Chem. Soc. 1972, 94, 7049–7059; c) D. G. B.Boocock, R. Darcy, E. F. Ullman, J. Am. Chem. Soc. 1968, 90,5945–5946; d) D. G. B. Boocock, E. F. Ullman, J. Am. Chem.Soc. 1968, 90, 6873–6874; e) R. W. Kreilick, J. Becker, E. F.Ullman, J. Am. Chem. Soc. 1969, 91, 5121–5124.

[2] a) P. Wautelet, J. Le Moigne, V. Videva, P. Turek, J. Org. Chem.2003, 68, 8025–8036; b) F. M. Romero, R. Ziessel, TetrahedronLett. 1999, 40, 1895–1898; c) M. Minguet, D. B. Amabilino, J.Cirujeda, K. Wurst, I. Mata, E. Molins, J. J. Novoa, J. Veciana,Chem. Eur. J. 2000, 6, 2350–2361; d) U. Samuni, Y. Samuni, S.Goldstein, J. Am. Chem. Soc. 2010, 132, 8428–8432.

[3] a) R. Ziessel, C. Stroh, Org. Lett. 2003, 5, 2397–2400; b) T.Kalai, K. Hideg, Tetrahedron 2006, 62, 10352–10360; c) H.Wang, D. Zhang, X. Guo, L. Zhu, Z. Shuai, D. Zhu, Chem.Commun. 2004, 670–671; d) Y. Yu, D. Zhang, W. Tan, Z. Wang,D. Zhu, Bioorg. Med. Chem. Lett. 2007, 17, 94–96.

[4] a) C. Hirel, J. Pécaut, S. Choua, Ph. Turek, D. B. Amabilino,J. Veciana, P. Rey, Eur. J. Org. Chem. 2005, 348–359; b) M.Minguet, D. B. Amabilino, K. Wurst, J. Veciana, J. Chem. Soc.Perkin Trans. 2 2001, 670–676; c) S. Shimono, R. Tamura, N.Ikuma, T. Takimoto, N. Kawame, O. Tamada, N. Sakai, H.Matsumura, J. Yamauchi, J. Org. Chem. 2004, 69, 475–481.

[5] a) R. J. Weinkam, E. C. Jorgensen, J. Am. Chem. Soc. 1971, 93,7028–7033; b) R. J. Weinkam, E. C. Jorgensen, J. Am. Chem.Soc. 1971, 93, 7033–7038.

[6] a) M. Zhao, J. Liu, C. Wang, L. Wang, H. Liu, S. Peng, J. Med.Chem. 2005, 48, 4285–4292; b) J. Liu, M. Zhao, C. Wang, S.Peng, Bioorg. Med. Chem. Lett. 2003, 13, 4065–4069; c) J.Zhang, M. Zhao, G. Cui, S. Peng, Bioorg. Med. Chem. 2008,16, 4019–4028; d) W. Bi, J. Cai, P. Xue, Y. Zhang, S. Liu, X.Gao, M. Li, Z. Wang, M. Baudy-Floc’h, S. A. Green, L. Bi,Bioorg. Med. Chem. Lett. 2008, 18, 1788–1794.

[7] a) A. Okamoto, T. Inasaki, I. Saito, Tetrahedron Lett. 2005,46, 791–795; b) V. Barone, M. Brustolon, P. Cimino, A. Poli-meno, M. Zerbetto, A. Zoleo, J. Am. Chem. Soc. 2006, 128,15865–15873; c) A. Collauto, M. Zerbetto, M. Brustolon, A.Polimeno, A. Caneschi, D. Gatteschi, Phys. Chem. Chem. Phys.2012, 14, 3200–3207; d) C. Rajadurai, V. Enkelmann, G. Zop-pellaro, M. Baumgarten, J. Phys. Chem. B 2007, 111, 4327–4334; e) H. Pramiti, M. A. Khaja, C. Rajadurai, Org. Biomol.Chem. 2012, 10, 2439–2446.

[8] a) M. Heise, F. H. Köhler, F. Mota, J. J. Novoa, J. Veciana, J.Am. Chem. Soc. 1999, 121, 9659–9667; b) A. Rajca, M. Pink,S. Mukherjee, S. Rajca, K. Das, Tetrahedron 2007, 63, 10731–10742; c) L. Talyatnyk, J. Vaara, Z. Rinkevicius, O. Vahtras, J.Phys. Chem. B 2004, 108, 1197–1206.

[9] a) H. Akpinor, J. A. Schlueter, P. M. Lathi, Chem. Commun.2013, 49, 3345–3347; b) A. Caneschi, D. Gatteschi, E.Rentschler, R. Sassoli, Gazz. Chim. Ital. 1995, 125, 283–286; c)Y. Pontillon, A. Caneschi, D. Gatteschi, A. Grand, E. Ressou-che, R. Sessoli, J. Schweizer, Chem. Eur. J. 1999, 5, 3616–3624;d) F. Lanfranc du Panthou, D. Luneau, J. Laugier, P. Rey, J.Am. Chem. Soc. 1993, 115, 9095–9100; e) G. Filippini, A. Gav-ezzotti, J. J. Novoa, Acta Crystallogr., Sect. B 1999, 55, 543–553; f) M. Deumal, J. Cirujeda, J. Veciana, M. Kinoshita, Y.Hosokoshi, J. J. Novoa, Chem. Phys. Lett. 1997, 265, 190–199;g) A. Zakrassov, V. Shteiman, Y. Sheynin, M. Botoshansky, M.Kapon, M. Kaftory, R. E. Del Sesto, J. S. Miller, Helv. Chim.Acta 2003, 86, 1234–1245.

[10] a) I. Ratera, J. Veciana, Chem. Soc. Rev. 2012, 41, 303–349;b) M. Minguet, D. Amabilino, J. Vidal-Gancedo, K. Wurst, J.Veciana, J. Mater. Chem. 2002, 12, 570–578.

K. Wright et al.FULL PAPER[11] a) K. Agawa, Y. Moruyama, Chem. Phys. Lett. 1989, 158, 556–

558; b) M. Tamura, Y. Nakazawa, D. Shiomi, K. Nozawa, Y.Hosokoshi, M. Ishikawa, M. Takahashi, M. Kinoshita, Chem.Phys. Lett. 1991, 186, 401–404.

[12] a) G. L. J. A. Rikken, E. Rampach, Nature 1997, 390, 493–494;b) G. L. J. A. Rikken, E. Rampach, Phys. Rev. E 1998, 58,5081–5084.

[13] a) A. Caneschi, D. Gatteschi, R. Sessoli, P. Rey, Acc. Chem.Res. 1989, 22, 392–398; b) D. Luneau, P. Rey, Coord. Chem.Rev. 2005, 249, 2591–2611.

[14] a) L. J. Berliner, J. Grunweld, H. O. Hankovszky, K. Hideg,Anal. Biochem. 1982, 119, 450–455; b) D. T. Warshaviak, L.Serbulea, K. N. Houk, W. L. Hubbell, J. Phys. Chem. B 2011,115, 397–405; c) D. T. Warshaviak, V. V. Kramtsov, D. Cascio,C. Altenbach, W. L. Hubbell, J. Magn. Reson. 2013, 232, 53–61.

[15] a) R. Marchetto, S. Schreier, C. R. Nakaie, J. Am. Chem. Soc.1993, 115, 11042–11043; b) C. Toniolo, M. Crisma, F.Formaggio, Biopolymers (Pept. Sci.) 1998, 47, 153–158.

[16] a) C. Toniolo, M. Crisma, F. Formaggio, C. Peggion, Biopoly-mers (Pept. Sci.) 2001, 60, 396–419; b) R. Bardi, A. M. Piaz-zesi, C. Toniolo, M. Sukumar, P. Balaram, Biopolymers 1986,25, 1635–1644; c) A. Santini, V. Barone, A. Bavoso, E. Bened-etti, B. Di Blasio, F. Fraternali, F. Lelj, V. Pavone, C. Pedone,M. Crisma, G. M. Bonora, C. Toniolo, Int. J. Biol. Macromol.1988, 10, 292–299; d) M. Crisma, G. M. Bonora, C. Toniolo,E. Benedetti, A. Bavoso, B. Di Blasio, V. Pavone, C. Pedone,Int. J. Biol. Macromol. 1988, 10, 300–304.

[17] a) C. M. Venkatachalam, Biopolymers 1968, 6, 1425–1436; b)G. D. Rose, L. M. Gierasch, J. A. Smith, Adv. Protein Chem.1985, 37, 1–109; c) C. Toniolo, CRC Crit. Rev. Biochem. 1980,9, 1–44.

[18] a) C. Toniolo, E. Benedetti, Trends Biochem. Sci. 1991, 16, 350–353; b) K. A. Bolin, G. L. Millhauser, Acc. Chem. Res. 1999,32, 1027–1033; c) I. L. Karle, P. Balaram, Biochemistry 1990,29, 6747–6756; d) E. Benedetti, B. Di Blasio, V. Pavone, C. Pe-done, C. Toniolo, M. Crisma, Biopolymers 1992, 32, 453–456;e) M. Crisma, F. Formaggio, A. Moretto, C. Toniolo, Biopoly-mers (Pept. Sci.) 2006, 84, 3–12.

[19] a) W. Kowalczyk, D. Sobolewski, A. Prahl, I. Derdowska, L.Borovicková, J. Slaninová, B. Lammek, J. Med. Chem. 2007,50, 2926–2929; b) P. W. Schiller, G. Weltrowska, T. M. Nguyen,C. Lemieux, N. N. Chung, B. J. Marsden, B. C. Wilkes, J. Med.Chem. 1991, 34, 3125–3132; c) A. Lukaszuk, H. Demaegt, I.Van den Eynde, P. Vanderheyden, G. Vauquelin, D. Tourwé, J.Pept. Sci. 2011, 17, 545–553.

[20] a) J. F. Lohier, K. Wright, C. Peggion, C. Toniolo, M. Waksel-man, J.-P. Mazaleyrat, Tetrahedron 2006, 62, 6203–6213; b) K.Wright, A. Moretto, M. Crisma, M. Wakselman, J.-P. Mazaley-rat, F. Formaggio, C. Toniolo, Org. Biomol. Chem. 2010, 8,3281–3286; c) K. Wright, A. Blanco-Alvarez, M. Crisma, A.Toffoletti, F. Formaggio, C. Toniolo, Helv. Chim. Acta 2012,95, 2446–2459.

[21] a) Z. Getahun, C.-Y. Huang, T. Wang, B. De Léon, W. De-Grado, F. Gai, J. Am. Chem. Soc. 2003, 125, 405–411; b) K.Aprilakis, H. Taskent, D. Raleigh, Biochemistry 2007, 46,12308–12313; c) C. Fang, J. D. Bauman, K. Das, A. Remorino,E. Arnold, R. W. Hochstrasser, Proc. Natl. Acad. Sci. USA2008, 105, 1472–1477; d) K. C. Schultz, L. Supekova, Y. Riu,J. Xie, R. Perera, P. G. Schultz, J. Am. Chem. Soc. 2006, 128,13984–13985; e) B. A. Lindquist, K. E. Fursc, S. A. Corcelli,

www.eurjoc.org © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2014, 1741–17521752

Phys. Chem. Chem. Phys. 2009, 11, 8119–8132; f) J. Zimmer-mann, M. C. Thielges, Y. S. Seo, P. E. Dawson, F. E. Romes-berg, Angew. Chem. 2011, 123, 8483; Angew. Chem. Int. Ed.2011, 50, 8333–8337; g) C. G. Bazewicz, J. S. Lipkin, E. E.Smith, M. T. Liskov, S. H. Brewer, J. Phys. Chem. B 2012, 116,10824–10831; h) R. F. Wissner, S. Batjargal, C. M. Fadzen, E. J.Petersson, J. Am. Chem. Soc. 2013, 135, 6529–6540; i) H. Kim,M. Cho, Chem. Rev. 2013, 113, 5817–5847; j) A. T. Fafarman,S. G. Boxer, J. Phys. Chem. B 2010, 114, 13536–13544.

[22] K. Wright, E. Scola, A. Toffoletti, F. Formaggio, C. Toniolo,in: Peptides 2012: Proceedings of the 32nd European PeptideSymposium (Eds.: G. Kokotos, V. Constantinou-Kokotou, J.Matsoukas), University of Athens, Athens, Greece, 2012, p.622–623.

[23] S. Kotha, E. Brahmachary, J. Org. Chem. 2000, 65, 1359–1365.[24] a) B. Drouillat, K. Wright, J. Marrot, F. Couty, Tetrahedron:

Asymmetry 2012, 23, 690–696; b) G. Helmchen, D. Nill, D.Flockerzi, M. S. K. Joussef, Angew. Chem. 1979, 91, 65; Angew.Chem. Int. Ed. Engl. 1979, 18, 63–65.

[25] A. El-Faham, F. Albericio, Chem. Rev. 2011, 111, 6557–6602.[26] O. Backerberg, B. Staskun, J. Chem. Soc. 1962, 3961–3963.[27] C. Hirel, K. Vostrikova, J. Pecaut, V. Ovcharenko, P. Rey,

Chem. Eur. J. 2001, 7, 2007–2014.[28] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397–4398.[29] C. Peggion, A. Moretto, F. Formaggio, M. Crisma, C. Toniolo,

Biopolymers (Pept. Sci.) 2013, 100, 621–636.[30] C. H. Görbitz, Acta Crystallogr., Sect. B 1989, 45, 390–395.[31] a) J. K. Fawcett, N. Camerman, A. Camerman, Acta Crys-

tallogr., Sect. B 1975, 31, 658–665; b) R. J. Fletterick, C. Tsai,R. E. Hughes, J. Phys. Chem. 1971, 75, 918–922; c) Y. Tokuma,T. Ashida, M. Kakudo, Acta Crystallogr., Sect. B 1969, 25,1367–1373.

[32] a) M. Palumbo, S. Da Rin, G. M. Bonora, C. Toniolo, Makro-mol. Chem. 1976, 177, 1477–1492; b) M. H. Baron, C. De Lozé,C. Toniolo, G. D. Fasman, Biopolymers 1978, 17, 2225–2239;c) C. Toniolo, G. M. Bonora, J. Polym. Sci. Polym. Chem. Ed.1976, 14, 515–518; d) C. Toniolo, G. M. Bonora, Makromol.Chem. 1975, 176, 2547–2558.

[33] a) T. Ashida, M. Kakudo, Bull. Chem. Soc. Jpn. 1974, 47,1129–1133; b) D. Cremer, J. A. Pople, J. Am. Chem. Soc. 1975,97, 1354–1358.

[34] a) C. Peggion, M. Jost, W. M. De Borggraeve, M. Crisma, F.Formaggio, C. Toniolo, Chem. Biodiversity 2007, 4, 1256–1268;b) R. A. Jose, M. De Zotti, C. Peggion, F. Formaggio, C. Toni-olo, W. M. De Borggraeve, J. Pept. Sci. 2011, 17, 377–382.

[35] S. Stoll, A. Schweiger, J. Magn. Reson. 2006, 178, 42–55.[36] a) G. Loidl, H.-J. Musiol, N. Budisa, R. Huber, S. Poirot, D.

Fourmy, L. Moroder, J. Pept. Sci. 2000, 6, 139–144; b) E. Sar-tori, A. Toffoletti, C. Corvaja, L. Moroder, F. Formaggio, C.Toniolo, Chem. Phys. Lett. 2004, 385, 362–367.

[37] M. C. Burla, M. Camalli, B. Carrozzini, G. L. Cascarano, C.Giacovazzo, G. Polidori, R. Spagna, J. Appl. Crystallogr. 2003,36, 1103.

[38] G. M. Sheldrick, Acta Crystallogr., Sect. A 2008, 64, 112–122.[39] A. L. Spek, Acta Crystallogr., Sect. D 2009, 65, 148–155.[40] a) A. Bax, D. G. Davis, J. Magn. Reson. 1985, 65, 355–360; b)

C. Grisinger, G. Otting, K. Wüthrich, R. R. Ernst, J. Am.Chem. Soc. 1988, 110, 7870–7872.

[41] O. Felix, M. W. Hosseini, A. De Cian, J. Fischer, L. Catala, P.Turek, Tetrahedron Lett. 1999, 40, 2943–2946.

Received: November 25, 2013Published Online: January 14, 2014