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Inorganic Chemistry Communications 9 (2006) 1267–1270

Synthesis and characterization of novel N-para-ferrocenylbenzoyl dipeptide esters

David Savage a, Steven R. Alley a,b, Alok Goel a,b, Tara Hogan a, Yoshiteru Ida c,Paula N. Kelly a,b, Laurent Lehmann a, Peter T.M. Kenny a,b,*

a School of Chemical Sciences, Dublin City University, Dublin 9, Irelandb National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland

c School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Received 29 May 2006; accepted 10 June 2006Available online 27 June 2006

Abstract

N-para-ferrocenyl benzoyl dipeptide esters 2–5 were prepared by coupling para-ferrocenyl benzoic acid 1 to the dipeptide ethyl estersGlyGly(OEt) 2, GlyAla(OEt) 3, GlyLeu(OEt) 4 and GlyPhe(OEt) 5. The compounds were fully characterized by a range of NMR spec-troscopic techniques, mass spectrometry (ESI-MS, FABMS, MS/MS) and cyclic voltammetry (CV). The a1 and b1 ions appeared at onemass unit less than the expected mass in the tandem mass spectra. The CV curves of compounds 2–5 exhibited quasi-reversible redoxbehavior similar to the Fc/Fc+ redox couple and displayed oxidation potential values in the 50–55 mV range, against the Fc/Fc+ redoxcouple.� 2006 Elsevier B.V. All rights reserved.

Keywords: Ferrocene; Bioorganometallic chemistry; Tandem mass spectrometry; Dipeptides; CV

The organometallic compound ferrocene has severalnovel applications due to its stability, spectroscopic proper-ties, electrochemical properties and ease of use. Research inthe area of ferrocenyl derivatives has focused on the prep-aration of novel sensor compounds, peptide mimetic mod-els and unnatural drugs [1–7]. As amino acids and peptidesplay such diverse roles in biological systems, incorporationof a redox active group onto these compounds is of partic-ular interest. For example, the incorporation of a ferrocenegroup onto proteins has shown the mediation of electrontransfer between electrodes and the protein redox site [8].Also the synthesis and structural characterization ofN-ferrocenoyl and N-ferrocenyl amino acid and peptidederivatives has been reported [9–26]. In addition ferrocene

1387-7003/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.inoche.2006.06.013

* Corresponding author. Address: School of Chemical Sciences, DublinCity University, Dublin 9, Ireland. Tel.: +353 1 7005689; fax: +353 17005503.

E-mail address: peter.kenny@dcu.ie (P.T.M. Kenny).

has been incorporated in drugs such as antibiotics, aspirin,anti-malarials and anti-cancer drugs such as tamoxifen[27–30]. A review on the bioorganometallic chemistry offerrocene has been published [31]. Here, we now presentthe synthesis and structural characterization of a series ofnovel N-para-ferrocenyl benzoyl dipeptide ester deriva-tives. The aim of this research is to incorporate three keymoieties in the synthesis of unusual biological materials,namely, (i) an electroactive core, (ii) a conjugated linkerthat can act as a chromophore and (iii) an amino acid ordipeptide derivative that can interact with other moleculesvia hydrogen bonding. The ferrocene moiety is linked tothe dipeptide ester via a para-disubstituted benzoyl group.We recently reported the synthesis and structural charac-terization of a series of N-para, N-meta, and N-ortho-ferr-ocenyl benzoyl amino acid and N-meta-ferrocenylbenzoyl dipeptide ester derivatives. [32–36].

para-Ferrocenyl benzoic acid 1 was prepared as previ-ously reported [32]. Conventional peptide chemistry was

1268 D. Savage et al. / Inorganic Chemistry Communications 9 (2006) 1267–1270

employed in the preparation of the dipeptides. Thus equi-molar quantities of the N-Boc protected amino acids werereacted with the amino acid ethyl ester hydrochloride salts,1,3-dicyclohexylcarbodiimide (DCC), catalytic amounts of1-hydroxybenzotriazole (HOBt) and triethylamine in DCMat 0 �C. Deprotection of the amino terminal was achievedusing TFA. The deprotected dipeptides were coupled topara-ferrocenyl benzoic acid using equimolar amounts ofDCC and catalytic amounts of HOBt under basic condi-tions (Scheme 1). Purification by silica gel column chroma-tography furnished the pure products in yields of 52–67%and all gave analytical and spectroscopic data in accor-dance with the proposed structures [37]. The N-para-ferr-ocenyl benzoyl derivatives 2–5 were characterized by acombination of 1H NMR, 13C NMR, DEPT-135 and1HA13C COSY (HMQC) spectroscopy, ESI-MS, FABMS,MS/MS and CV. All the proton and carbon chemical shiftsfor compounds 2–5 were unambiguously assigned by acombination of DEPT-135 and 1H-13C-COSY (HMQC).The 1H and 13C NMR spectra for compounds 2–5 showedpeaks in the ferrocene region characteristic of a ferrocenylbenzoyl moiety [32–36]. The protons in the ortho positionof the (g5-C5H4) ring appear in the region d 4.61 to d4.64 whereas the protons in the meta position occur inthe range d 4.3 to d 4.32. The (g5-C5H5) ring appears inthe narrow region d 3.95 to d 3.98. The protons of thepara-disubstituted benzoyl group appear as two doubletsfrom d 7.43 to d 7.70. For example, in the case of N-{para-(ferrocenyl)benzoyl}-glycine-glycine ethyl ester 2,

the aromatic protons are present as two doublets at d7.45 and d 7.69, respectively. The (g5-C5H5) ring appearsas a singlet in the 1H NMR spectrum at d 3.96 whereas

+ H2N

OMe

O

Fe

O

NH

O

HN

R

O

OEt

2-5

Fe

Scheme 1. Synthesis of N-para-ferrocenyl benzoyl dipeptide esters 2–5; GlyGly5 �C, (ii) NaOH/MeOH, H2O, (iii) DCC, HOBt, Et3N, Dipeptide ethyl ester.

the meta and ortho protons on the (g5-C5H4) ring are pres-ent at d 4.31 and d 4.63, respectively. The 13C NMR spectraof compounds 2–5 show signals in the region d 67.2 to d83.9 indicative of a monosubstituted ferrocene subunit.The ipso carbon of the (g5-C5H4) ring appears in a verynarrow range of d 83.7 to d 83.9. This signal is absent inthe DEPT 135 spectra. The carbon atoms of the aromaticring are visible in the region d 126.2–144.5. The two quater-nary carbons of the aromatic ring and the methylenecarbon atoms of derivatives 2–5 were identified byDEPT-135. For example, in the 13C NMR spectrum ofN-{para-(ferrocenyl)benzoyl}-glycine-glycine ethyl ester 2,as is typical for a para disubstituted compound, there areonly four aromatic signals. The two phenyl ipso carbonsappear at d 144.5 and d 130.8 and the two other aromaticsignals appear at d 127.7 and d 126.3, respectively. The sig-nal at d 83.7 is absent in the DEPT 135 spectrum and there-fore it is assigned as the ipso ferrocenyl carbon. The (g5-C5H5) ring appears as an intense signal at d 70.2 and themeta carbons on the (g5-C5H4) ring appear at d 70.1. Theortho carbons on the (g5-C5H4) ring appear at d 67.2. Acomplete assignment of the 1H and 13C NMR spectra ofcompound 2 is presented in Table 1.

Since the introduction of soft ionization techniques suchas electrospray ionization (ESI) and fast atom bombard-ment mass spectrometry (FABMS) a wide range of thermo-labile and non-volatile compounds can be subjected tomass spectrometric analysis [38,39]. FAB and ESI wereemployed in the analysis of compounds 2–5 and confirmedthe correct relative molecular mass. Examination of themass spectra revealed the presence of intense radical-cat-ions, with cation adducts due to sodium and potassium

i

O

OMe

ii

O

OHiii

1

Fe

Fe

(OEt) 2, GlyAla(OEt) 3, GlyLeu(OEt) 4, GlyPhe(OEt) 5. (i) NaNO2, HCl,

Table 11H and 13C spectroscopic data for 2

Fe

O

NH

O

HN

O

O1

2

5

4

3

67

89

10

11

12

13 14

15

1617

1819

2021

22

23

Site 1H NMR 13C NMR HMQC

1 83.72, 5 4.63 67.23, 4 4.31 70.16–10 3.96 70.211 144.512, 13 7.45 126.314, 15 7.69 127.716 130.817 168.1a

18 4.12–15 4419 169.8a

20 4 41.821 170.122 4.12–15 62.123 1.21 14.5

a signals may be reversed.

D. Savage et al. / Inorganic Chemistry Communications 9 (2006) 1267–1270 1269

also present. Fragment ions were not observed or were ofvery low intensity. Therefore tandem mass spectrometrywas employed to confirm the integrity of the structures.As the radical-cation species were of greater intensity thanthe [M + H]+ species these ions were selected as the precur-sor ions. Sequence specific ions were observer in all theMS/MS spectra for compounds 2–5 confirming that theglycine residue was linked to the benzoyl spacer group.Important product ions were present at m/z 261, m/z 289,m/z 317 and m/z 345 (Fig. 1). The ions at m/z 261 andm/z 289 are due to the ferrocenylphenyl and ferro-cenylbenzoyl subunits respectively. However, the expecteda1 and b1 product ions at m/z 318 and m/z 346 were notobserved, instead a1-1 and b1-1 product ions were observedat m/z 317 and m/z 345 respectively. Accurate mass mea-surement of the MS/MS spectrum for compound 4 gavean elemental composition of C18H15N1O1Fe1 and a molec-ular mass of m/z 317.0497 for the ion at nominal mass m/z317. This corresponds to a ppm error of 0.1420. Obviouslya hydrogen atom has also been lost during the fragmenta-tion process. The hydrogen atom is either one of the glycinemethylene hydrogen atoms or the hydrogen on the glycine

Fe

O

NH

HN

O

O

O

261 289 -H317 -H345

R

Fig. 1. Product ions observed in the MS/MS spectra of compounds 2–5.

amide nitrogen. This is unusual as these a1 and b1 fragmentions are usually produced without loss of a hydrogen atom[40].

The CV curves of compounds 2–5 exhibit quasi-revers-ible redox behavior, similar to the Fc/Fc+ redox couple,and display oxidation potential values ca. 51–55 mV higherthan ferrocene. For example, the Eo0 value for compound 2

is calculated to be 55 mV with respect to ferrocene. Theslight increase in the oxidation potential with respect tounsubstituted ferrocene is explicable in terms of substituenteffects. Electron withdrawing amide groups on phenyl ringmake the ferrocene moiety electron deficient and more dif-ficult to oxidize.

In conclusion, the novel N-para-ferrocenyl benzoyldipeptide esters 2–5 were prepared in good yields usingorganic peptide synthetic protocols. The compounds werecharacterized by NMR spectroscopic technique, tandemmass spectrometry and CV. The tandem mass spectrarevealed the loss of a hydrogen atom in the productionof the a1 and b1 type product ions. In addition the com-pounds are electroactive, with oxidation potentials compa-rable to ferrocene and have the ability to interact withother molecules via hydrogen bonding, which make thesecompounds suitable as potential receptors for sensordevices with electrochemical detection.

Acknowledgements

D.S. thank the Irish American Partnership and DublinCity University for the funding of a studentship award1999–2002. This research was partly supported by the Na-tional Institute for Cellular Biotechnology under the Pro-gramme for Research in Third Level Institutions (PRTLI,round 3, 2001–2006).

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m.p. 165–167 �C, Eo0 ¼ 55 mV (vs Fc/Fc+).Analysis: found: C, 61.46; H, 5.56; N, 6.36.C23H24N2O4Fe requires: C, 61.62; H, 5.40; N, 6.25.Mass spectrum: found: [M]+ 448C23H24N2O4Fe requires: 448I.R. mmax (KBr): 3321, 2924, 1742, 1622, 1574, 1501, 1311 cm�1.UV-Vis kmax EtOH; 352 (e 2140), 447 (e 620) nm.1H NMR (400 MHz) d (CDCl3): 7.69 (2H, d, J = 8 Hz, ArH), 7.45(2H, d, J = 8 Hz, ArH), 7.09 (1H, br.s, ACONHA), 6.85 (1H, br.s,ACONHA), 4.63 {2H, s, ortho on (g5-C5H4)}, 4.31 {2H, s, meta on(g5-C5H4)}, 4.12–4.15 (4H, m, AOCH2CH3, ANHCH2CO), 4.00(2H, d, J = 5.2 Hz, ANHCH2COA), 3.96 {5H, s, (g5-C5H5)}, 1.21(3H, t, J = 7.6 Hz, -OCH2CH3). 13C NMR (100 MHz) d (CDCl3):170.1, 169.8, 168.1, 144.5, 130.8, 127.7, 126.3, 83.7, 70.2, 70.1, 67.2,62.1 (-ve DEPT), 44.0 (-ve DEPT), 41.8 (-ve DEPT), 14.5.

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