Ferrocenyl-Labeled Sugar Amino Acids: Conformation and Properties

Article pubs.acs.org/Organometallics

Ferrocenyl-Labeled Sugar Amino Acids: Conformation and Properties Christoph Förster,† Monika Kovačević,‡ Lidija Barišić,*,‡ Vladimir Rapić,‡ and Katja Heinze*,† †

Institute of Inorganic and Analytical Chemistry, Johannes Gutenberg-University of Mainz, Duesbergweg 10-14, 55128 Mainz, Germany ‡ Department of Chemistry and Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia S Supporting Information *

ABSTRACT: Novel organometallic sugar amino acid conjugates 1−5 have been prepared by amide coupling of O-protected N-acetylmuramic acid and iso-muramic acid (2-[3-amino-2,5dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxypropanoic acid) with 1-aminoferrocene, 1-aminoferrocene-1′-carboxylic acid (H-Fca-OH), or 1,1′-diaminoferrocene, respectively. The influence of the ferrocenyl moiety and presence of additional remote potential hydrogen atom acceptors and donors at the ferrocenyl core on the conformation and lipophilicity is investigated by TLC, IR, NMR, and CD spectroscopic methods augmented by density functional calculations. Furthermore, the redox potential of the ferrocene/ferrocenium couple is tuned by the electron-withdrawing and -donating nature of the substituents at the ferrocenyl label.

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of at least one muramic acid and an α-amino acid. These muropeptides are considered to be promising immunomodulators and vaccine adjuvants.21 For example, Ellouz et al. reported N-acetylmuramyl-L-alanyl-D-isoglutamine (N-Ac-MurL-Ala-D-iGln, muramyldipeptide, MDP) (Chart 1, top) as a minimal adjuvant active subunit of bacterial cell walls that can potentiate immune responses.22 The conformation of muramyldipeptide features one 10-membered β-turn via an intramolecular hydrogen bond (IHB) involving the CONAcMur and the NHAla group (Chart 1, top).23 With the aim of boosting its immunostimulatory properties, the natural MDP has been chemically modified, giving several analogues, which should allow establishing structure−activity relationships.24,25 Recently, the first ferrocenyl-modified analogues of MDP (N-Ac-Mur-L/D-Ala-Fca-OMe)26 (A) have been reported (Chart 1, bottom left; H-Fca-OH = 1-aminoferrocene1′-carboxylic acid). In this organometallic derivative the Cterminal D-iGln moiety has been replaced by a ferrocene amino acid ester moiety (H-Fca-OMe27). Spectroscopic evidence suggested the presence of the expected 10-membered ring as found in the natural peptide MDP together with an additional intrachain IHB involving NHFca···OClac, giving a sevenmembered ring (Chart 1, bottom left). Apart from this example, only very few ferrocene-substituted sugar derivatives have been previously reported.12,13,28 Furthermore, ferrocene amino acid and its derivatives are robust yet electroactive building blocks for molecular peptidic wires and actuators,29 for molecular sensor devices,30−33 for

INTRODUCTION Bioorganometallic chemistry comprising pharmaceutical applications of organometallic compounds and conjugation of biomolecules with organometallics has become an important and vivid research area in the last decades.1 In this field the prototypical and extraordinarily stable organometallic complex ferrocene plays a pioneering role.2,3 The potential biologic activity of ferrocenyl-substituted derivatives of natural bioactive molecules is of great current interest, as exemplified by the famous organometallic analogoue of tamoxifen (ferrocifen)4 and the organometallic antimalaria drug ferroquine.5 Many conjugates of ferrocene with α- or β-amino acids6−11 and a few conjugates with carbohydrates12,13 have also been studied.2,3 Sugar amino acids (SAAs) are hybrids of carbohydrates and amino acids featuring both the sugar framework and the amino and acid functional groups.14 SAAs play a significant role in peptidomimetic studies.15 SAAs containing protected rigid furan and pyran rings are also suitable for incorporation into peptides, acting as nonpeptidic scaffolds in the resulting peptidomimetics. Several chiral centers at the carbohydrate moiety lead to immense structural and conformational diversity.16 Furthermore, the hydrophobic or hydrophilic nature can be tuned at the carbohydrate part via protection or deprotection of the sugar hydroxyl groups. An important example of a naturally occurring SAA is muramic acid (2-[3-amino-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxypropanoic acid), which is located in bacterial cell walls.17,18 Muramic acid is a constituent of glycan chains, which are cross-linked by oligopeptides to give peptidoglycans.19,20 The enzymatic degradation products of peptidoglycans in bacterial cell walls are the so-called muropeptides, which consist © 2012 American Chemical Society

Received: March 1, 2012 Published: April 27, 2012 3683

dx.doi.org/10.1021/om300174p | Organometallics 2012, 31, 3683−3694

Organometallics

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Chart 1. N-Ac-Mur-L-Ala-D-iGln, Muramyldipeptide (MDP, top) and Ferrocenyl-Labeled Muropeptides of Type A26 (bottom left) and B (bottom right)

tives. Two novel aspects are realized in these type B muropeptides, namely, a structural one and an electronic one: (i) The conformational space, i.e., the possible number and type of IHBs within the secondary structure, is modified by the absence (R = H) or presence of additional hydrogen atom acceptors (R = COOMe) or donors (R = NHAc) at the conformationally flexible ferrocene unit. This “conformational tuning” might have an impact on the potential biological activity of the organometallic muropeptides, for example by modification of the shape, the lipophilicity, and the metabolic stability of the labeled molecule. (ii) The redox potential of the ferrocene/ferrocenium redox couple is tuned by the electron-withdrawing or electrondonating nature of the substituents R. 37,38 This “electronic tuning” might be advantageous for potential applications as sensing probes for the electrochemical detection of carbohydrate−protein interactions.13 These tasks are tackled in this work by spectroscopic and analytical methods (IR, NMR, and CD spectroscopy, as well as cyclic voltammetry) and theoretical methods (density functional calculations).39

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RESULTS AND DISCUSSION Syntheses of Sugar Amino Acids with an Organometallic Label. The O-protected N-acetyl(iso)muramic acids III and IV were synthesized following a procedure reported in the literature (Scheme 1).40 The anomeric hydroxyl group of commercially available N-acetylglucosamine was protected by heating with benzyl alcohol in toluene solution to give the

34,35,13

redox-labeled biomolecules, and as constituents of redoxactivated cytotoxic pro-drugs.36 The aim of the present study focuses on the synthesis and characterization of novel ferrocenyl-modified muropeptides of type B (Chart 1, bottom right) with the complete C-terminal LAla-D-iGln dipeptide fragment replaced by ferrocenyl derivaScheme 1. Synthesis of N-Acetyl-Protected SAAs III and IV40 a

a

Reagents and conditions: (a) PhCH2OH, PTSA, toluene, reflux, 4 h, 79%; (b) PhCHO, (EtO)3CH, PTSA, DMF, dioxane, 24 h, 82%; (c) NaH, dioxane, 7 h, rac-CH3CHClCOOH, dioxane, 24 h; (d) MeI, DMF, rt, 24 h; (e) TLC separation; (f) 0.5 M NaOHaq, dioxane, 2 h, 98%. Atom numbering used for NMR and hydrogen bond numbering. 3684


Organometallics

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Scheme 2. Synthesis of Ferrocenyl-Labeled Muropeptides 1−5a

a

Reagents and conditions: (a) HCl (gaseous)/CH2Cl2; (b) NEt3/CH2Cl2; (c) 1. III/IV, HOBt/EDC, CH2Cl2, 2. TLC separation (CH2Cl2/EtOAc = 5:1). Atom numbering used for NMR and hydrogen bond numbering.

optically pure benzyl-protected α-anomer. This benzyl ether was stirred with benzaldehyde in the presence of triethyl orthoformate and p-toluenesulfonic acid (PTSA) to introduce the cyclic acetal protection at both C5 and C7 hydroxyl functions, giving the desired 2,5,7-O-protected sugar intermediate. The remaining hydroxyl group was treated with racemic 2-chloropropionic acid to give the crude mixture of diastereomeric lactate salts, which were converted to the mixture of esters N-Ac-Mur-OMe (I) and N-Ac-isoMur-OMe (II) by alkylation with methyl iodide. For NMR purposes, an appropriate portion of this mixture was separated by thin layer chromatography (CH2Cl2/EtOAc = 5:1) into I and II, respectively. According to TLC and NMR analysis, the diastereomers I and II were obtained in a 3:1 ratio. Alkaline hydrolysis of the I/II mixture in dioxane gave the N-protected SAAs III and IV as a distereomeric mixture. The N-Boc-protected ferrocenyl amines BocNH-Fn-R (V, R = H; VI, R = COOMe, R = NHAc, VII; Fn = 1,1′-ferrocenediyl; Boc = tert-butoxycarbonyl)the organometallic components necessary for conjugation with the above-described SAAs III/ IVwere prepared according to previously reported procedures.41,27a,10c Coupling of these N-deprotected organometallic amines with O-protected N-acetylmuramic (III) and N-acetyliso-muramic acid (IV) by using the standard HOBt/EDC peptide coupling protocol is depicted in Scheme 2 (HOBt = 1hydroxybenzotriazole, EDC = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)). Boc deprotection of the ferrocenyl amines was performed by action of gaseous HCl in CH2Cl2 to give the hydrochlorides HCl·H2N-Fn-R. Upon treatment with NEt3, the resulting amines were coupled to the HOBt-activated SAAs III and IV, respectively. The obtained novel yellow-colored diastereomeric pairs 1/2 and 3/4 were successfully separated by thin layer chromatography (CH2Cl2/EtOAc = 5:1) into 61% N-Ac-Mur-NH-Fc (1, Rf = 0.66), 62% N-Ac-isoMur-NH-Fc (2, Rf = 0.33), 71% N-Ac-Mur-NH-Fn-COOMe (3, Rf = 0.63), and 65% N-Ac-isoMur-NH-Fn-COOMe (4, Rf = 0.25). N-Ac-MurNH-Fn-NHAc (5, Rf = 0.33; 45%) was obtained as a pure diastereomer. The unambiguous assignment of the retention factors (Rf) to the respective diastereomers was achieved using small-scale syntheses of 1−5 starting from diastereomerically pure carbohydrate precursors I or II, respectively. As Rf values reflect the polarity or lipophilicity of the compounds, which is

of critical importance for potential pharmaceutical applications, we have a closer look at the retention factors. Indeed, 2 is much more polar than its diastereomeric counterpart 1. The introduction of a COOMe group at the ferrocenyl core increases the polaritiy only marginally (Rf = 0.66 (1) → 0.63 (3) and Rf = 0.33 (2) → 0.25 (4)). In contrast, 5 (Rf = 0.33), bearing an additional NHAc group at the ferrocenyl moiety, is significantly more polar than 1 (Rf = 0.66) or 3 (Rf = 0.63). Conformational Analysis of 1−5. To elucidate the conformational structures of 1−5 in solution, we begin with the conformational analysis of the starting sugar amino acid esters I and II. In the next step the effect of the ferrocenyl amide is investigated, and in the final increase of complexity a further substituent potent for hydrogen bonding at the distal cyclopentadienyl ring is considered. The comparison with esters I and II allows distinguishing substituent effects deriving from the sugar moiety from those deriving from the ferrocenyl label. The following study is conducted by the combination of IR, NMR, and CD spectroscopy supported by DFT calculations, a strategy that has been successfully applied in previous conformational analyses of simple and complex ferrocenyl amides, lactams, and ureas.10,39 In the IR spectra of the carbohydrate precursors I and II and of 1−5 the group frequencies of NH and CO are found in characteristic spectral regions (Table 1). Low-energy COester vibrations are observed for I and II around ν̃ ≈ 1730 cm−1, indicating hydrogen bonds to that group in solution and in the solid state for I but not in the solid state of II (ν̃ = 1749 cm−1). An inverse situation is encountered for ferrocenyl esters 3 and 4: according to the absorption energies of the COester groups, 3 and 4 appear to feature hydrogen bonds to the respective carbonyl in the solid state (ν̃ = 1715 cm−1) but only to a minor extent in solution (Table 1). Likewise, the solid-state IR spectra reveal that all NH groups are involved in hydrogen bonding with low-energy stretching vibrations below ν̃ = 3400 cm−1. In contrast, in CH2Cl2 solution both free and hydrogen-bonded NH groups are simultaneously present (Table 1, Supporting Information), as indicated by absorptions of NH stretching vibrations above and below ν̃ = 3400 cm−1, respectively. For ester I, with intense NH absorptions around 3356 cm−1 (and to a lesser extent for II, with weaker NH absorptions around 3283 cm−1), the combined IR data suggest IHBs of the type 3685


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Table 1. IR Spectroscopic Data (c = 10−2 M in CH2Cl2 and as CsI disk) of I, II, and 1−5 (ν̃/cm−1) NHfree CH2Cl2

I

3431 (w)

CsI CH2Cl2

II

3439 (m)

CsI CH2Cl2

1

3432 (m)

CsI CH2Cl2

2

3430 (m)

CsI CH2Cl2

3

3432 (m)

CsI CH2Cl2

4

3438 (m)

CsI CH2Cl2

5

CsI

3425 (m)

NHassoc

COester

3356 (m) 3300 (m) 3283 (w)

1732 (s)

1674 (s)

1737 (s)

1657 (s)

1733 (s)

1675 (s)

3296 (m) 3357 (m) 3300 (m) 3374 (mb) 3300 (m) 3355 (m) 3327 (m) 3367 (mb) 3327 (m) 3309 (mb) 3297 (m)

1749 (s)

1655 (s)

Information). A further different pattern is displayed by the trisamide 5 (Supporting Information). This already suggests that the COOMe substituent in 3 and 4 is less influential than the NHAc substituent in 5 concerning the secondary structure of these ferrocenyl muropeptides in solution. As a powerful tool to evaluate individual hydrogen-bonding situations and the strength of individual hydrogen bonds 1H NMR spectroscopy is used. Indeed, all resonances of the amide protons could be assigned in the 1H NMR spectra by their coupling pattern (NHa, doublet) and by two-dimensional NMR techniques. Even a distinction between the resonances for NHb and NHc in trisamide 5 with very similar chemical environments for the NHCO units was possible in two solvents, CDCl3 and [D6]-DMSO (for atom numbering see Schemes 1 and 2). In CDCl3 the NHb resonance of 5 is assigned on the basis of its correlations to the cyclopentadienyl carbon atoms C14 and C18 as well as to the carbonyl atom C13Ob, which itself correlates to the methyl protons H12 of the lactate unit (Supporting Information). In [D6]-DMSO an unambigous assignment of the NHb resonance is achieved in an anlogous fashion, and the resonance of NHc is identified by its correlation to C24Oc, which itself correlates to the methyl protons H25 (Supporting Information). Having assigned all proton resonances, especially the indicative NH resonances, we focus on their respective chemical shifts in different solvents (Table 2, CDCl3, [D6]DMSO). Generally, chemical shifts of amide protons above δ = 7 ppm in CDCl3 as a non-hydrogen-bonding solvent are indicative of a significant involvement in hydrogen bonding. The NHa doublet resonance of the N-acetylated sugar moiety of I is found at δ = 7.49 ppm, indicating a hydrogen bond (likely to the lactate ester carbonyl COb, vide supra). The chemical shift varies only slightly, by Δδ = 0.34 ppm, when changing the solvent to the strong hydrogen acceptor [D6]DMSO, suggesting a very strong IHB. Furthermore, the vicinal coupling constant to H3 of 3JHH = 4.3 Hz points to an ordered conformation with a HaNC3H3 torsion angle θ ≈ 126°.42 For diastereomer II the NHa resonance is found at δ = 5.65 ppm in CDCl3 and with a large shift, Δδ = 2.44 ppm, to lower field in [D6]-DMSO and a vicinal coupling constant to H3 of 3JHH = 8.8 Hz, pointing to a torsion angle around θ = 0°/180°. These data suggest that II features mainly non-hydrogen-bonded forms in CDCl3, in agreement with the IR data. These interpretations

amide I

1732 (sh),1679 (s) 1677 (s), 1658 (s) 1732 (sh), 1678 (s) 1677 (s), 1658 (s) 1744 (sh) 1715 (s) 1733 (sh) 1715 (s)

1708 (s), 1679 (s) 1677 (s), 1659 (s) 1708 (s), 1680 (s) 1677 (s), 1659 (s) 1733 (sh), 1716 (sh), 1671 (s) 1677 (s), 1669 (s), 1655 (s)

NHa···ObC (Scheme 1) in equilibrium with open forms in solution. For the bis- and trisamides 1−5 the situation is less clear-cut. Upon dilution from c = 10−2 M to 10−3 M the intensity ratio and position of the IR bands for hydrogenbonded and free NH groups remain unchanged. This indicates essentially intramolecular hydrogen-bonding patterns rather than intermolecular hydrogen bonds within this concentration range. An assignment of absorption bands to individual NH stretching and amide vibrations for 1−5 featuring several amide groups is intrinsically impossible without site-specific isotopic labeling. However, we note a striking difference within the diasteromeric pairs 1/2 and 3/4, respectively, but a similarity between like diastereomers with/without the ester substituent at the ferrocenyl moiety 1/3 and 2/4, respectively (Supporting

Table 2. 1H NMR Data (δ/ppm) and Chemical Shift Variations (Δδ/ppm) of the NH Protons 1−5 (for atom numbering see Schemes 1 and 2) I II 1 2 3 4 5

CDCl3 [D6]-DMSO CDCl3 [D6]-DMSO CDCl3 [D6]-DMSO CDCl3 [D6]-DMSO CDCl3 [D6]-DMSO CDCl3 [D6]-DMSO CDCl3 [D6]-DMSO

δ(NHa)

Δδ(NHa)

7.49 7.83 5.65 8.09 5.95 8.30 5.80 8.19 6.42 8.30 6.05 8.18 6.35 8.28

0.34

δ(NHb)

Δδ(NHb)

7.74 8.87 8.05 8.40 7.75 8.87 8.07 8.37 8.05 8.60

1.13

δ(NHb)

Δδ(NHc)

8.13 9.14

1.01

2.44 2.35 2.39 1.88 2.13 1.93

3686

0.35 1.12 0.30 0.55


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NHb) < δ(2, NHb), δ(3, NHb) < δ(4, NHb) and Δδ(1, NHb) > Δδ(2, NHb), Δδ(3, NHb) > Δδ(4, NHb). These observations suggest that NHb···Ox IHBs are more dominant/stronger for the iso-muramic conjugates 2 and 4 as compared to the muramic acid derivatives 1 and 3. Thus, the conformational ensemble of all conjugates 1−4 features strong NHb···Ox IHBs with a small contribution of NHa···Ox IHBs for 1 and 3. By DFT/B3LYP calculations four local energetic minimum geometries have been identified for the simplest ferrocenyl derivatives 1 and 2, respectively (Figure 2). The lowest energy conformations of 1 and 2 (1-10,5-ring and 2-10,5-ring) feature bifurcated IHBs of the type NHb···OaC and NHb···Oe with H···O distances between 2.0 and 2.2 Å, resulting in 10membered and five-membered rings, in accordance with the NMR analysis. An analogous hydrogen-bonding pattern has been suggested for the natural muropeptide MDP.23 The DFTcalculated torsional angles of 1-10,5-ring and 2-10,5-ring amount to θ = −160° and θ = −158°, respectively, in very good agreement with the experimental estimation (vide supra).43 The conformers analogous to esters I-8a-ring and II-8a-ring featuring eight-membered rings and NHa···ObC IHBs are slightly destabilized by 8.2 kJ mol−1 (1-8a-ring) and significantly by 32.4 kJ mol−1 (2-8a-ring), again in line with the NMR interpretation of a slight contribution of NHa···Ox for 1. The torsional angles θ = 121°/122° of I-8a-ring/II-8a-ring would be in contradiction with the experimentally observed angles of the main conformers. In a third low-energy conformation, one of the protected OH groups (Of) also acts as a hydrogen acceptor for NHb by formation of an eightmembered ring with a NHb···Of hydrogen bond (1-8b-ring: 1.99 Å, 17.7 kJ mol−1; 2-8b-ring: 2.19 Å, 12.7 kJ mol−1). This is also compatible with the NMR data of 1 and 2, suggesting a larger NHb···Ox fraction in 2 as compared to 1. Conformers of even higher energy feature a hydrogen bond between NHb···Oe (1-5-ring: 2.04 Å, 37.6 kJ mol−1; 2-5-ring: 2.00 Å, 47.4 kJ mol−1), but they are considered to be less important. Thus, the calculations suggest that NHb can bind to several acceptors, namely, to COa, Oe, and Of, while for NHa only COb is available for hydrogen bonding. For 1 the latter situation of 1-8a-ring is competitive to some extent (8.2 kJ mol−1), while it is less accessible for 2 (2-8a-ring: 32.4 kJ mol−1). This underlines the favorable hydrogen bonding of NHb in 2, as already suggested by the NMR data (vide supra). Essentially the same arguments as compiled for 1 and 2 hold for 3 and 4, bearing an additional COOMe group as potential hydrogen acceptor at the distal cyclopentadienyl ring. A slight impact is noticed on the chemical shift and shift variation of NHa as compared to their unsubstituted counterparts 1 and 2. The resonance δ(NHa) appears at lower field, and Δδ(NHa) is somewhat smaller. This suggests that NHb of 3 and 4 behaves essentially identically to NHb of 1 and 2, while NHa of 3 and 4 engages in a slightly stronger hydrogen-bonding activity as compared to NHa of 1 and 2. Similar to 1 and 2, DFT/B3LYP calculations for 3 and 4 (Figure 3) reveal the dominance of the conformer 3-10,5-ring (NHb···OaC: 2.23 Å; NHb···Oe: 2.11 Å; θ = −160°). For 3 this conformation is followed by 3-8a-ring (NHa···ObC: 1.89 Å; θ = 121°; 6.5 kJ mol−1) and 3-8b-ring (NHb···Of: 1.97 Å; θ = −178°; 16.9 kJ mol−1). An additional conformer, 3-8a,6-ring-P, with a cooperative hydrogen bond, is found at low energy, featuring both NHa and NHb hydrogen bonds (NHa···ObC: 1.83 Å; NHb···OdC: 2.03 Å; θ = 120°; 9.3 kJ mol−1). For 4 only

are confirmed by DFT/B3LYP calculations with a polarized continuum model (Figure 1, CHCl3 as solvent). In both cases I

Figure 1. DFT-optimized low-energy conformations of I and II along with their torsion angles θ and relative energies (Erel in kJ mol−1) in parentheses (solvent model CHCl3).43

and II the minimum structures feature eight-membered rings with IHBs (NHa···ObC: 1.94 Å (I-8a-ring), 1.83 Å (II-8a-ring)) (Figure 1).43 The open conformers are destabilized by 68.1 kJ mol−1 (I-open) and by only 22.8 kJ mol−1 (II-open). This remarkable destabilization of II-8a-ring relative to II-open might be a result of an unfavorable orientation of the methyl group C12 toward the benzylidene protection group in the case of II-8a-ring (Figure 1). The HaNC3H3 torsion angles derived from DFT calculations are around θ = 122° for the ring structure and θ = −16° for the non-hydrogen-bonded conformer, in good agreement with the experiment. According to the 1H NMR chemical shifts δ(NHa) < 6.4 ppm and the solvent-induced shift variation Δδ(NHa) > 1.8 ppm, the NHa group of the bis- and trisamides 1−5 is only marginally involved in hydrogen bonding, suggesting the dominance of a fundamentally different hydrogen bond as compared to esters I and II. Small differences are noted within the pairs 1/2 and 3/ 4, with δ(1, NHa) > δ(2, NHa), δ(3, NHa) > δ(4, NHa) and Δδ(1, NHa) < Δδ(2, NHa), Δδ(3, NHa) < Δδ(4, NHa), suggesting a slightly higher presence of NHa···Ox IHBs in the ensemble mixtures of 1 and 3 as compared to 2 and 4. The NHa−H3 coupling constants of 3JHH = 9.2−9.9 Hz suggest a torsion angle θ = −159 ± 3° for all derivatives 1−5, also significantly distinct from the torsion of I and II. In all cases the proton NHb resonates at δ(NHb) > 7.7 ppm, suggesting hydrogen bonds involving this group. Diluting CDCl3 solutions of 1−5 from concentrations of 50 mM to 1 mM reveals that δ(NH b ) is practically concentration independent (Δδ(NHb) < 0.1 ppm; see Supporting Information). This finding substantiates the IR-based interpretation that NH groups are involved in intramolecular hydrogen bonds. Again, differences for NHb are noted within the 1/2 and 3/4 pairs with an inverse behavior as compared to proton NHa: δ(1, 3687


Organometallics

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Figure 2. DFT-optimized low-energy conformations of 1 and 2 along with their torsion angles θ and relative energies (Erel in kJ mol−1) in parentheses (solvent model CHCl3).43

conformer 4-8b-ring is of low energy (NHb···Of: 2.25 Å; θ = −168°; 11.4 kJ mol−1), while 4-8a-ring (NHa···ObC: 1.82 Å; θ = 120°; 34.0 kJ mol−1) and 4-8a,6-ring-P (NHa···ObC: 1.79 Å; NHb···OdC: 1.95 Å; θ = 118°; 29.7 kJ mol−1) are higher in energy, leaving less available conformations for 4 as compared to 3, similar to the 2/1 case. The presence of low-energy

conformers with IHBs to the ester carbonyl COd explains the slight remote influence of the COOMe group on the hydrogen bonding of NHa. Less relevant conformers of 3 and 4 with higher energy are given in the Supporting Information. The trisamide 5 features three NH groups, which are all engaged in IHBs in the ensemble mixture of 5, however to a 3688


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accordance with the NMR data concerning the hydrogen bonding of NHb and NHc as well as the estimated torsion angles around θ = −163° (5-13c,5-ring-P/M: NHb···Oe, NHc···OaC; 5-10,5,7c-ring-P/M: NHb···Oe, NHc···ObC). The seven-membered rings involving the ferrocene backbone observed for the diamido ferrocene derivative 5 in 5-10,5,7cring-P/M and 5-8a,7b-ring-P/M have been previously found in simpler derivatives.10c Further high-energy conformations of 5 have also been located on the energy landscape but are not considered further due to their high energy (see Supporting Information). Thus, conformers 5-8a,7b-ring-P/M account for the presence of NHa hydrogen bonds, while conformers 513c,5-ring-P/M and 5-10,5,7c-ring-P/M account for the dominance of NHb and NHc hydrogen bonds. Interestingly, the NHb···Oe IHB resulting in the fivemembered ring seems to be common to most low-energy conformations of muramic amides 1−5 (1-10,5-ring, 2-10,5ring, 3-10,5-ring, 4-10,5-ring, 5-13c,5-ring-P/M, 5-10,5,7c-ringP/M, 5-10,5-ring; Figures 2−4). In several cases this IHB is augmented in a bifurcated IHB, giving the preferred 10membered ring (1-10,5-ring, 2-10,5-ring, 3-10,5-ring, 4-10,5ring, 5-10,5,7c-ring-P/M, 5-10,5-ring; Figures 2−4). Cooperative hydrogen bonds featuring six-membered or sevenmembered rings also seem to be important in the remotely substituted derivatives, e.g., in 3-8a,6-ring-P, 4-8a,6-ring-P (Figure 3), 5-10,5,7c-ring-P/M, and 5-8a-7b-ring-P/M (Figure 4). Similar cooperative hydrogen-bonding patterns featuring eight-membered rings have been previously observed in oligoferrocene amides PG-NH-Fn-[CONH-Fn]n-X (n = 1−4; PG = Ac, Boc, Fmoc; X = H, COOMe).29a,b The sign of the Cotton effect observed for 1−4 at the ferrocene absorption band around 460 nm is largely determined by the stereochemistry of the nearest stereogenic center, namely, the configuration of C11 of the lactate unit (Supporting Information, Table 3). An R-configured C11 (1, 5) gives a positive Cotton effect, and an S-configured C11 (2, 4) results in a negative Cotton effect. The intensity of the CD signal is higher for the unsubstituted 1 and 2 as compared to esters 3 and 4. The absolute intensity of the CD effect of the isomuramic acid derivatives 2 and 4 is higher than the intensity of the corresponding muramic acid derivatives 1 and 3, respectively. This can be accounted for by the larger number of available conformations for 1 and 3 as compared to 2 and 4 (vide supra). For the diaminoferrocene alanine conjugate AcNH-FnNHCO-CαH(Me)-NHBoc a positive Cotton effect has been reported for the S-configured Cα of alanine and a negative one for the R-configured enantiomer.10b However, an inverse situation is found for the diaminoferrocene lactate conjugate 5, with a positive Cotton effect for 5 with an R-configured C11 atom (Table 3). This apparent contradiction is a result of the different preferred conformations of the alaninyl derivative and the sugar derivative 5 due to different IHB options to further donors and acceptors.10b Cyclic Voltammetry of 1−5. Each of the cyclic voltammograms of 1−5 features a one-electron oxidation for the ferrocene/ferrocenium redox couple (Figure 5). Introducing an electron-withdrawing COOMe substituent at the 1′-position of the ferrocenyl derivatives 1/2 (E1/2 = −125/−100 mV vs FcH/FcH+) increases the half-wave potential significantly, by +245 to +250 mV (3: E1/2 = 120/130 mV vs FcH/FcH+). A similar shift of +200 mV has been previously observed for the related pair FcNHAc and AcNH-Fn-COOH.27b The redox

Figure 3. DFT-optimized low-energy conformations of 3 and 4 along with their torsion angles θ and relative energies (Erel in kJ mol−1) in parentheses (solvent model CHCl3).43

different extent (Table 2). According to chemical shifts δ and shift variations Δδ, the NHa group of 5 forms hydrogen bonds to a small degree similar to NHa of 3, while NHc and NHb form hydrogen bonds to a much higher extent than NHa. Conformational searches on the trisamide 5 by DFT/B3LYP methods yield the expected three conformations analogous to 1 and 3, namely, 5-10,5-ring, 5-8a-ring, and 5-8b-ring (Figure 4). Similar to ester 3, the carbonyl group of the remote substituent can act as a hydrogen acceptor, resulting in the low-energy conformation 5-8a,7b-ring-P/M, with two cooperative hydrogen bonds (5-8a,7b-ring-P: NHa···ObC: 1.81 Å; NHb···OcC: 1.83 Å; θ = 120°; 11.0 kJ mol−1; 5-8a,7b-ring-M: NHa···ObC: 1.83 Å; NHb···OcC: 1.79 Å; θ = 120°; 12.2 kJ mol−1; Figure 4). No significant differences in energy between corresponding Pand M-helical ferrocene conformers have been observed. As the remote NHAc substituent also features the hydrogen donor group NHc, two further opportunities arise that indeed have the lowest energy calculated for 5 (5-13c,5-ring-P/M and 5-10,5,7cring-P/M, Figure 4). Both low-energy conformers are in full 3689


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Figure 4. DFT-optimized low-energy conformations of 5 along with their torsion angles θ and relative energies (Erel in kJ mol−1) in parentheses (solvent model CHCl3).43 3690


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Table 3. UV/Vis Spectroscopic and Chiroptical Data of 1−5 in CH2Cl2 λmax/nm (ε/M−1 cm−1) 1 2 3 4 5

457 454 487 454 447

(240) (220) (235) (340) (235)

Scheme 3. Schematic View of Relevant Conformations with Hydrogen-Bonded Rings

λmax/nm (θ/deg cm2 dmol−1) 464 465 465 466 456

(+290) (−1020) (∼0) (−475) (375)

Figure 5. Cyclic voltammograms of 1, 3, and 5 in CH2Cl2/n-Bu4NPF6.

waves of the diastereomeric pairs 1/2 and 3/4 are very similar, although 2 and 4 appear slightly more difficult to oxidize than their diastereomeric counterparts 1 and 3. Whether this observation is reflecting the preferred conformations of 2 and 4 with the stronger NHb···O hydrogen bonds is an open point, as redox potentials are based on the energtic difference between the redox partners. As the preferred secondary structures of the cations 1+−4+ are unkown, this is an unsolved issue. A slight shift of −40 mV in the opposite direction is found for derivative 5, bearing a further electron-donating NHAc group in the 1′-position of the ferrocene unit (E1/2 = −165 mV vs FcH/FcH+). These shifts excellently agree with the empirically derived substituent effects reported by Lever et al. (+250 mV for an additional COOMe substituent and −40 mV for an additional NHAc substituent).37

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CONCLUSION Five novel ferrocenyl-labeled derivatives of the sugar amino acids N-acetylmuramic acid and N-acetyl-iso-muramic acid bearing remote substituents at the distal cyclopentadienyl ring (R = H (1, 2), COOMe (3, 4), NHAc (5)) have been prepared in diastereomerically pure form. Their preferred conformation in solution has been elucidated using IR, NMR, and CD spectroscopy together with DFT molecular modeling.39 The preferred conformation of 1−4 encompasses an intramolecular hydrogen bond from NHb to COa (10membered ring, β-turn) assisted by an NHb···Oe hydrogen bond (five-membered ring), giving the 10,5-ring conformation (Scheme 3, A). For the N-acetylmuramic acid derivatives 1 and 3 a further distinct conformation with a hydrogen bond from NHa to COb (eight-membered ring) is energetically accessible (8a-ring conformation, Scheme 3, B). For the N-acetyl-isomuramic acid derivatives 2 and 4 this conformation B is strongly destabilized. The presence of a remote ester group in 3 and 4 allows for a stabilization of this eight-membered ring by a cooperative hydrogen bond forming an additional hydrogen bond of NHb to COd (six-membered ring, Scheme 3, B). A NHb···Of hydrogen bond giving an eight-membered ring is energetically feasible for 2 and 4 (Scheme 3, C). All these

factors together generate a higher diversity of conformations for 1, 3, and 4 as compared to 2. Even more energetically relevant conformations are possible by installing a remote NHAc group at the distal cyclopentadienyl ring (5). A cooperative hydrogen bond NHb to COc forming a seven-membered ring stabilizes the eightmembered ring (5-8a,7b-ring, similar to 3-8a,6-ring and 4-8a,6ring, Scheme 3, D), and a cooperative hydrogen bond NHc to COb (seven-membered ring) stabilizes the original 10membered ring (5-10,5,7c-ring, Scheme 3, A). However, the most favorable conformation of 5 features a 13-membered ring including the sugar and the remote substituent with a NHc to COa hydrogen bond and the NHb to Oe hydrogen bond pattern (five-membered ring) (Scheme 3, E). The remote substituents R not only influence the conformational freedom of these novel organometallic sugar amino acids but also tune the redox potential of the ferrocene/ferrocenium redox couple by up to 285 mV. 3691


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in dry CH2Cl2 (pH ≈ 8) and added to the mixture of activated III/IV. After the total consumption of free amine (as monitored by TLC using EtOAc as eluent), a second portion of ferrocenyl amine hydrochloride salt/NEt3 (0.25 mmol) was added. After stirring at room temperature for 48 h, the mixture was washed twice with a saturated solution of NaHCO3, then with a 10% aqueous solution of citric acid and brine and dried over Na2SO4. The solution was evaporated to dryness in vacuo. The residue was purified by TLC on silica gel (CH2Cl2/EtOAc, 5:1). After scraping the adsorbent off the plate the orange-colored products 1−5 were extracted from the adsorbent by methanol and diethyl ether. N-Ac-Mur-NH-Fc (1). Yield: 0.15 g, 0.22 mmol, 61%. Rf = 0.66. Mp: 110−114 °C. IR (CsI): ν̃ 3300 (m, NHassoc), 1677 (s), 1658 (s, amide I), 1552 (s, amide II) cm−1. IR (CH2Cl2): ν̃ 3432 (m, NHfree), 3357 (m, NHassoc), 1732 (sh), 1679 (s, amide I), 1543 (s, amide II) cm−1. 1H NMR (CDCl3, 400 MHz): δ 1.44 (d, 3H, 3JHH = 6.6 Hz, H12), 1.80 (s, 3H, H9), 3.64−3.67 (m, 2H, H4, H5), 3.76 (pt, 1H, 2JHH = 10.1 Hz, H7a), 3.83−3.87 (m, 1H, H6), 3.93 (s, 1H, H17), 3.96 (s, 1H, H16), 4.04 (q, 1H, 3JHH = 6.8 Hz, H11), 4.10 (s, 5H, H19−H23), 4.23 (dd, 1H, 3JHH = 4.5 Hz, 2JHH = 10.0 Hz, H7b), 4.32−4.39 (m, 1H, H3), 4.46 (s, 1H, H18), 4.47 (d, 1H, 2JHH = 11.6 Hz, H1a), 4.70 (d, 1H, 2 JHH = 11.8 Hz, H1b), 4.88 (d, 1H, 3JHH = 3.8 Hz, H2), 4.92 (s, 1H, H15), 5.58 (s, 1H, H8), 5.95 (d, 1H, 3JHH = 9.2 Hz, NHa), 7.28−7.46 (m, 10H, HPh), 7.74 (s, 1H, NHb) ppm. 13C{1H} NMR (CDCl3, 100 MHz): δ 20.0 (C12), 23.4 (C9), 52.5 (C3), 60.9 (C18), 61.5 (C15), 63.0 (C6), 63.9 (C17), 64.8 (C16), 68.8 (C7), 69.1 (C19−C23), 70.0 (C1), 79.6 (C4), 79.6 (C11), 81.7 (C5), 94.3 (C14), 97.4 (C2), 101.4 (C8), 125.9−129.1 (CHPh), 136.5, 136.9 (CPh), 170.9 (C10), 171.2 (C13) ppm. MS(FD): m/z (%) 654.6 (100) [M]•+. Anal. Calcd for C35H38N2O7Fe·MeOH: C, 62.98; H, 6.18; N, 4.07. Found: C, 62.85; H, 6.11; N, 4.05. CV (CH2Cl2, n-Bu4NPF6, vs FcH/FcH+): E1/2 = −125 mV. N-Ac-iso-Mur-NH-Fc (2). Yield: 0.05 g, 0.08 mmol, 62%. Rf = 0.33. Mp: 117−119 °C. IR (CsI): ν̃ 3300 (m, NHassoc), 1677 (s), 1658 (s, amide I), 1552 (s, amide II) cm−1. IR (CH2Cl2): ν̃ 3430 (m, NHfree), 3374 (m, NHassoc), 1732 (sh), 1678 (s, amide I), 1540 (s, amide II) cm−1. 1H NMR (CDCl3, 400 MHz): δ 1.34 (d, 3H, 3JHH = 6.8 Hz, H12), 1.98 (s, 3H, H9), 3.72−3.78 (m, 4H, H4, H5, H16, H17), 3.80 (d, 1H, 2JHH = 9.2 Hz, H7a), 3.87 (dd, 1H, 3JHH = 4.7 Hz, 3JHH = 9.3 Hz, H6), 4.02 (s, 6H, H15, H19−H23), 4.22 (s, H18), 4.24 (m, 1H, 3JHH = 5.6 Hz, H7b), 4.34 (q, 1H,3JHH = 6.8 Hz, H11), 4.44 (ptd, 1H, 3JHH = 3.7 Hz, 3JHH = 9.5 Hz, H3), 4.48 (d, 1H, 2JHH = 11.8 Hz, H1a), 4.72 (d, 1H, 2 JHH = 11.8 Hz, H1b), 4.81 (d, 1H, 3JHH = 3.7 Hz, H2), 5.59 (s, 1H, H8), 5.80 (d, 1H, 3JHH = 9.9 Hz, NHa), 7.30−7.50 (m, 10H, HPh), 8.05 (s, 1H, NHb) ppm. 13C{1H} NMR (CDCl3, 100 MHz): δ 19.0 (C12), 23.5 (C9), 53.2 (C3), 60.8 (C18), 61.0 (C15), 63.1 (C6), 64.1 (C17), 64.3 (C16), 68.9 (C7), 69.0 (C19−C23), 70.2 (C1), 75.2 (C4), 76.9 (C11), 81.5 (C5), 94.1 (C14), 97.6 (C2), 102.3 (C8), 126.5−129.5 (CHPh), 136.4, 136.7 (CPh), 170.1 (C10), 171.1 (C13) ppm. MS(FD): m/z (%) 654.6 (100) [M]•+. Anal. Calcd for C35H38N2O7Fe ·OEt2: C, 64.29; H, 6.64; N, 3.84. Found: C, 64.22; H, 6.72; N, 3.85. CV (CH2Cl2, n-Bu4NPF6, vs FcH/FcH+): E1/2 = −100 mV. N-Ac-Mur-NH-Fn-COOMe (3). Yield: 0.19 g, 0.26 mmol, 71%. Rf = 0.63. Mp: 115−118 °C. IR (CsI): ν̃ 3327 (m, NHassoc), 1715 (COester), 1677 (s), 1659 (s, amide I), 1548 (s, amide II) cm−1. IR (CH2Cl2): ν̃ 3432 (m, NHfree), 3355 (m, NHassoc), 1744 (sh, COester), 1708 (s), 1679 (s, amide I), 1539 (s, amide II) cm−1. 1H NMR (CDCl3, 400 MHz): δ 1.44 (d, 3H, 3JHH = 6.7 Hz, H12), 1.93 (s, 3H, H9), 3.68 (pt, 1H,3JHH = 9.2 Hz, H5), 3.72 (s, 3H, H25), 3.74−3.80 (m, 2H, H4, H7a), 3.88 (dd, 1H, 3JHH = 4.6 Hz, 3JHH = 9.6 Hz, H6), 3.99 (m, 2H, H16, H17), 4.10 (q, 1H,3JHH = 6.7 Hz, H11), 4.25 (dd, 1H, 3JHH = 4.6 Hz, 2JHH = 10.1 Hz, H7b), 4.32−4.37 (m, 3H, H3, H21, H22), 4.49 (d, 1H, 2JHH = 11.8 Hz, H1a), 4.69−4.74 (m, 4H, H1b, H15, H18, H20, H23), 4.91 (d, 1H, 3JHH = 3.7 Hz, H2), 5.58 (s, 1H, H8), 6.42 (d, 1H, 3 JHH = 9.2 Hz, NHa), 7.28−7.47 (m, 10H, HPh), 7.75 (s, 1H, NHb) ppm. 13C{1H} NMR (CDCl3, 100 MHz): δ 19.9 (C12), 23.5 (C9), 51.6 (C25), 51.7 (C3), 62.0, 62.3 (C15, C18), 63.0 (C6), 66.0 (C16, C17), 68.8 (C7), 70.0 (C1),71.0, 71.1 (C20, C23),72.2 (C21/C22), 72.3 (C19, C21/ C22), 78.8 (C4), 79.1 (C11), 82.0 (C5), 95.7 (C14), 97.4 (C2), 101.5 (C8), 125.9−129.1 (CHPh), 136.6, 136.9 (CPh), 171.0 (C10), 171.5

EXPERIMENTAL SECTION

General Procedures. All reactions were performed under an inert atmosphere (Schlenk techniques, glovebox). CH2Cl2 used for synthesis and FT-IR spectroscopy was dried (P2O5), distilled over CaH2, and stored over molecular sieves (4 Å). All solvents were dried according to general procedures for purification of solvents, unless indicated otherwise. All other reagents were used as received from commercial suppliers (Acros, Sigma-Aldrich). Products were purified by preparative thin layer chromatography on silica gel (Merck, Kieselgel 60 HF254), using CH2Cl2/EtOAc mixtures. Analytical TLC was performed on Fluka silica gel (with fluorescent indicator 254 nm) plates (0.2 mm), and pure sugar (ferrocene-unlabeled) compounds were visualized with ultraviolet light and stained with 10% sulfuric acid. Melting points were determined with a Büchi apparatus. IR spectra were recorded as CH2Cl2 solutions or as CsI disks by using a Bomem MB 100 mid FT-IR spectrometer or a BioRad Excalibur FTS 3100 spectrometer. NMR spectra were recorded on a Varian EM 360 or Varian Gemini 300 spectrometer in [D6]-DMSO solutions at 300 MHz (1H), 75 MHz (13C{1H}) or on a Bruker Advance DRX 400 spectrometer in CDCl3 at 400.31 MHz (1H), 100.66 MHz (13C{1H}). All resonances are reported in ppm versus the solvent signal as internal standard [[D6]-DMSO (1H: δ = 2.50; 13C: δ = 39.4 ppm), CDCl3 (1H: δ = 7.24; 13C: δ = 77.0 ppm)]. Double resonance experiments (COSY, NOESY, TOCSY, HMBC, and HSQC) were performed in order to assist in signal assignment. (s) = singlet, (d) = doublet, (t) = triplet, (q) = quartet, (m) = muliplet, (dd) = doublet of doublets, (ddd) = doublet of doublet of doublets, (td) triplet of doublets, (od) = with neighboring signals overlapping doublet, (om) = with neighboring signals overlapping multiplet, (pt) = pseudotriplet (unresolved doublet of doublets), (ptd) = pseudotriplet of doublets (unresolved ddd). Electrochemical experiments were carried out on a BioLogic SP-50 voltammetric analyzer using platinum wires as counter and working electrodes and a 0.01 M Ag/AgNO3 electrode as reference electrode. The cyclic voltammetry measurements were carried out at a scan rate of 50−100 mV s−1 using 0.1 M n-Bu4NPF6 as supporting electrolyte in CH2Cl2. Potentials are referenced to the ferrocene/ferrocenium couple (E1/2 = 270 ± 5 mV under the experimental conditions). CD spectra were measured as CH 2 Cl 2 solutions with a Jasco-810 CD spectrophotometer. FD mass spectra were recorded on a FD Finnigan MAT90 spectrometer. Elemental analyses were performed by the microanalytical laboratory of the chemical institutes of the University of Mainz. The sugar amino acids N-Ac-Mur-OH III and N-Ac-isoMurOMe IV were prepared in improved yields by modification of literature procedures starting from commercially available Nacetylglucosamine.40 Ferrocenyl amine precursors V, VI, and VII were synthesized in the form of N-Boc-protected derivatives (Fc-NHBoc,41 Boc-NH-Fn-COOMe,27a Boc-NH-Fn-NH-Ac10c) by Curtius rearrangement of the corresponding azide precursors in tert-butyl alcohol. The spectroscopic data of prepared carbohydrates and ferrocenyl precursors match the respective literature data. Computational Method. Density functional calculations were carried out with the Gaussian09/DFT series of programs.44 The B3LYP formulation of density functional theory was used employing the LANL2DZ basis set.44 No symmetry constraints were imposed on the molecules. All points were characterized as minima (Nimag = 0) by frequency analysis. Solvent modeling was done employing the integral equation formalism polarizable continuum model (IEFPCM, chloroform). General Procedure for Coupling of O-Protected Muramic and iso-Muramic Acid to Ferrocenyl Amines. A solution of NBoc-protected ferrocenyl amine (BocHN-Fn-R, R = H (V), R = COOMe (VI), R = NHAc (VII)) (0.5 mmol) in ethylacetate was cooled to 0 °C and treated with gaseous HCl for 0.5 h. After strirring at room temperature for 0.5 h, the solvent was evaporated in vacuo to give respective yellow amines HCl·H2N-Fn-R as hydrochloride salts. A diastereomeric mixture of N-acetylmuramic acid III and N-acetyl-isomuramic acid IV was activated by stirring with EDC (1.5 mmol) and HOBt (1.5 mmol) in CH2Cl2 for 2 h at 0 °C. The ferrocenyl amine hydrochloride salt (0.25 mmol, 0.5 equivalent) was treated with Et3N 3692


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(C13), 171.6 (C24) ppm. MS(FD): m/z (%) 712.6 (100) [M]•+. Anal. Calcd for C37H40N2O9Fe: C, 62.37; H, 5.66; N, 3.93. Found: C, 62.30; H, 5.78; N, 3.71. CV (CH2Cl2, n-Bu4NPF6, vs FcH/FcH+): E1/2 = 120 mV. N-Ac-iso-Mur-NH-Fn-COOMe (4). Yield: 0.06 g, 0.08 mmol, 65%. Rf = 0.25. Mp: 121−123 °C. IR (CsI): ν̃ 3327 (m, NHassoc), 1715 (COester), 1677 (s), 1659 (s, amide I), 1548 (s, amide II) cm−1. IR (CH2Cl2): ν̃ 3439 (m, NHfree), 3367 (m, NHassoc), 1733 (sh, COester), 1708 (s), 1680 (s, amide I), 1539 (s, amide II) cm−1. 1H NMR (CDCl3, 400 MHz): δ 1.38 (d, 3H, 3JHH = 6.6 Hz, H12), 1.99 (s, 3H, H9), 3.73 (s, 1H, H17), 3.75 (s, 3H, H25), 3.77−3.79 (m, 3H, H5, H7a, H16), 3.92 (dd, 1H, 3JHH = 4.5 Hz, 3JHH = 9.6 Hz, H6), 3.98 (m, 1H, 3 JHH = 9.7 Hz, H4), 4.05 (s, 1H, H18), 4.18 (s, 1H, H15), 4.23−4.28 (m, 3H, H7b, H21, H22), 4.39 (m, 1H, 3JHH = 6.3 Hz, H11), 4.44 (dd, 1H, 3 JHH = 2.8 Hz, 3JHH = 9.8 Hz, H3), 4.49 (d, 1H, 2JHH = 11.9 Hz, H1a), 4.65, 4.66 (s, 2H, H20, H23), 4.73 (d, 1H, 2JHH = 11.9 Hz, H1b), 4.83 (d, 1H, 3JHH = 2.8 Hz, H2), 5.62 (s, 1H, H8), 6.05 (d, 1H, 3JHH = 9.6 Hz, NHa), 7.29−7.50 (m, 10H, HPh), 8.07 (s, 1H, NHb) ppm. 13C{1H} NMR (CDCl3, 100 MHz): δ 19.0 (C12), 23.4 (C9), 51.6 (C25), 54.4 (C3), 62.0, 62.3 (C15, C18), 62.9 (C6), 65.6 (C17), 66.0 (C16), 69.0 (C7), 70.1 (C1),70.8, 71.0 (C20, C23),72.7 (C19), 72.4 (C21, C22), 74.9 (C4), 77.2 (C11), 81.4 (C5), 95.6 (C14), 97.6 (C2), 102.3 (C8), 126.5− 129.5 (CHPh), 136.6, 136.8 (CPh), 170.2 (C10), 171.5, 171.6 (C13, C24) ppm. MS(FD): m/z (%) 712.6 (100) [M]•+. Anal. Calcd for C37H40N2O9Fe: C, 62.37; H, 5.66; N, 3.93. Found: C, 62.35; H, 5.43; N, 3.80. CV (CH2Cl2, n-Bu4NPF6, vs FcH/FcH+): E1/2 = 130 mV. N-Ac-Mur-NH-Fn-NHAc (5). Yield: 0.12 g, 0.16 mmol, 45%. Rf = 0.33. Mp: 119−121 °C. IR (CsI): ν̃ 3297 (m, NHassoc), 1677 (s), 1659 (s), 1655 (s, amide I), 1562 (s, amide II) cm−1. IR (CH2Cl2): ν̃ 3425 (m, NHfree), 3309 (mb, NHassoc), 1733 (sh), 1716 (sh), 1671 (s, amide I), 1540 (s, amide II) cm−1. 1H NMR (CDCl3, 400 MHz): δ 1.45 (d, 3H, 3JHH = 6.7 Hz, H12), 1.90 (s, 3H, H9), 2.01 (s, 3H, H25), 3.66−3.71 (m, 2H, H4, H5), 3.78 (pt, 1H, 2JHH = 10.2 Hz, H7a), 3.87 (m, 1H, 3JHH = 4.6 Hz, 3JHH = 9.5 Hz, H6), 3.94 (pq, 1H, 3JHH = 1.0 Hz, H22), 3.97 (pq, 1H, 3JHH = 1.2 Hz, H16), 4.00 (pq, 1H, 3JHH = 1.2 Hz, H21), 4.05 (m, 2H, 3JHH = 1.0 Hz, H17, H20), 4.11 (q, 1H, 3JHH = 6.8 Hz, H11), 4.24 (dd, 1H, 3JHH = 4.7 Hz, 2JHH = 10.2 Hz, H7b), 4.34 (ptd, 1H, 3JHH = 3.9 Hz, 3JHH = 9.4 Hz, H3), 4.39 (pt, 1H, 3JHH = 1.0 Hz, H18), 4.44 (pt, 1H, 3JHH = 1.0 Hz, H15), 4.49 (d, 1H, 2JHH = 11.8 Hz, H1a), 4.58 (pt, 3JHH = 1.0 Hz, H23), 4.72 (d, 1H, 2JHH = 11.8 Hz, H1b), 4.90 (d, 1H, 3JHH = 3.9 Hz, H2), 5.58 (s, H8), 6.35 (d, 1H, 3JHH = 9.4 Hz, NHa), 7.31−7.48 (m, 10H, HPh), 8.05 (s, 1H, NHb), 8.13 (s, 1H, NHc) ppm. 13C{1H} NMR (CDCl3, 100 MHz): δ 19.8 (C12), 23.4 (C9), 23.9 (C25), 52.7 (C3), 62,9 (C15), 63.0 (C6), 64.1 (C18), 64.9 (C16/C17/ C20/C21/C22), 65.2 (C23), 65.3, 65.4, 65.6 (C16/C17/C20/C21/C22), 68.8 (C7), 70.1 (C1), 79.1 (C4), 79.1 (C11), 81.7 (C5), 93.8 (C14), 94.1 (C19), 97.3 (C2), 101.5 (C8), 125.9−129.1 (CHPh), 136.4, 136.9 (CPh), 169.3 (C24), 171.1 (C10), 172.3 (C13) ppm. MS(FD): m/z (%) 711.6 (100) [M]•+. Anal. Calcd for C37H41N3O8Fe·CH2Cl2: C, 57.3; H, 5.44; N, 5.28. Found: C, 57.16; H, 5.87; N, 5.05. CV (CH2Cl2, nBu4NPF6, vs Fc/Fc+): E1/2 = −165 mV.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (grant number 0581191344-3122). We thank the Katholischer Akademischer Ausländer-Dienst KAAD for a fellowship to M.K., and the National Foundation for Science, Higher Education and Technological Development of the Republic of Croatia for financial support to M.K.

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ASSOCIATED CONTENT

S Supporting Information *

Graphical representation of IR and CD spectra; NMR data of 1−5 in tabulated form; 13C,1H-HMBC NMR spectra of 5 in CDCl3 and [D6]DMSO; concentration-dependent 1H NMR chemical shifts; figures of all DFT-optimized conformers of I, II, and 1−5 and their Cartesian coordinates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(K.H.) Fax: int + 49-6131-3927277; e-mail: [email protected]. (L.B.) Fax: int + 385-4836-082; e-mail: [email protected]. 3693


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