Chiral Ferrocene Amines Derived from Amino Acids and Peptides

Bioorganometallic Chemistry of Ferrocene. Dave R. van Staveren and Nils Metzler-Nolte. Chemical Reviews 2004 104 (12), 5931-5986. Abstract | Full Text...
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Inorg. Chem. 2000, 39, 5437-5443

5437

Chiral Ferrocene Amines Derived from Amino Acids and Peptides: Synthesis, Solution and X-ray Crystal Structures and Electrochemical Investigations Alexandra Hess, Jan Sehnert, Thomas Weyhermu1 ller, and Nils Metzler-Nolte* Max-Planck-Institut fu¨r Strahlenchemie, Stiftstrasse 34-36, D-45470 Mu¨lheim/Ruhr, Germany ReceiVed January 27, 2000

For the recognition of all but the simplest naturally occurring molecules, electrochemical sensors based on ferrocene will certainly require chiral centers. To advance the necessary chemistry, this work describes the synthesis and properties of ferrocene derivatives of enantiomerically pure amino acids, peptides, and other chiral amines. Ferrocene aldehyde is condensed with amino acid esters to yield the corresponding Schiff bases 2, which are reduced by NaBH4 in methanol to the ferrocene methyl amino acids 3. An X-ray single-crystal analysis was carried out on the phenylalanine derivative 3a (monoclinic space group P21, a ) 10.301(1) Å, b ) 9.647(1) Å, c ) 18.479(2) Å, β ) 102.98(2)°, Z ) 4). Further peptide chemistry at the C terminus proceeds smoothly as demonstrated by the synthesis of the ferrocene labeled dipeptide Fc-CH2-Phe-Gly-OCH3 5 (Fc ) ferrocenyl ((η-C5H4)Fe(η-C5H5))). We also report the synthesis of the C,N-bis-ferrocene labeled tripeptide Phe-Ala-Leu and its electrochemical characterization. Starting from the enantiomerically pure ferrocene derivative 9, which was synthesized from ferrocene aldehyde and L-1-amino-ethylbenzene, two diastereomers 10 were obtained by peptide coupling with N-Boc protected D- and L-alanine. There was, however, only very little diastereomeric induction if 0.5 equiv of a racemic mixture of alanine were used. This suggests that amino acid activation rather than coupling is the rate-determining step. A combination of NOESY (nuclear Overhauser effect spectroscopy) spectra and molecular modeling furnished detailed insights into the solution structures of 3, 9, and 10 and was used to rationalize their different reactivity.

Introduction Ferrocene serves as the redox active moiety in a large number of chemical sensors with applications ranging from use in simple anion sensors1 to the monitoring of glucose levels in the blood of diabetes mellitus patients2,3 and use in DNA sensors.4-7 For the binding of simple anions or cations, ferrocenes with covalently attached amines are most often employed. Through clever ligand design, remarkable selectivity between seemingly very similar targets is achievable; recently, Beer and co-workers reported the selective recognition of dihydrogenphosphate in the presence of hydrogensulfate.8 For sensors that are targeted to more complicated or diverse molecules of biological interest, chiral sensors will most likely be required to optimize sensitivity * To whom correspondence should be sent. New address: Institut fu¨r Pharmazeutische Chemie, Universita¨t Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg. Fax: +49-(0)6227-546430. E-mail: [email protected]. (1) Beer, P. D. Acc. Chem. Res. 1998, 31, 71-80. (2) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667-671. (3) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407-413. (4) Ihara, T.; Nakayama, M.; Murata, M.; Nakano, K.; Maeda, M. J. Chem. Soc., Chem. Commun. 1997, 1609-1610. (5) Bardea, A.; Dagan, A.; Ben-Dov, I.; Amit, B.; Willner, I. J. Chem. Soc., Chem. Commun. 1998, 839-840. (6) Bardea, A.; Dagan, A.; Willner, I. Anal. Chim. Acta 1999, 385, 3343. (7) Bardea, A.; Patolsky, F.; Dagan, A.; Willner, I. J. Chem. Soc., Chem. Commun. 1999, 21-22. (8) Beer, P. D.; Drew, M. G. B.; Smith, D. K. J. Organomet. Chem. 1997, 543, 259-261. (9) Bussmann, W.; Lehn, J. M.; Oesch, U.; Plumere, P.; Simon, W. HelV. Chim. Acta 1981, 64, 657-661.

and target discrimination. The recognition of chiral substrates with organic molecules has been actively persued by a number of groups.9-13 Organometallic compounds have successfully served as markers for the detection of biomolecules with a variety of techniques.14-21 We are interested in the synthesis and applications of bio-organometallics, e.g., for the selective recognition of nucleotides and DNA.22-24 In this project we have recently reported simple achiral ferrocene amines as redox labels (10) Behr, J. P.; Lehn, J. M.; Vierling, P. HelV. Chim. Acta 1982, 65, 18531867. (11) Russell, K. C.; Lehn, J. M.; Kyritsakas, N.; DeCian, A.; Fischer, J. New J. Chem. 1998, 22, 123-128. (12) Huskens, J.; Goddard, R.; Reetz, M. T. J. Am. Chem. Soc. 1998, 120, 6617-6618. (13) Hu, K.; Krakowiak, K. E.; Bradshaw, J. S.; Dalley, N. K.; Xue, G.; Izatt, R. M. J. Heterocycl. Chem. 1999, 36, 347-354. (14) Ryabov, A. D. Angew. Chem. 1991, 103, 945-955. (15) Ryabov, A. D.; Goral, V. N.; Gorton, L.; Cso¨regi, E. Chem.sEur. J. 1999, 5, 961-967. (16) Salmain, M.; Vessie`res, A.; Brossier, P.; Butler, I. S.; Jaouen, G. J. Immunol. Methods 1992, 148, 65-75. (17) Jaouen, G.; Vessie`res, A.; Butler, I. S. Acc. Chem. Res. 1993, 26, 361-369. (18) Di Gleria, K.; Nickerson, D.; Hill, H. A. O.; Wong, L.-L.; Fu¨lo¨p, V. J. Am. Chem. Soc. 1998, 120, 46-52. (19) Grotjahn, D. B.; Joubran, C.; Combs, D.; Brune, D. C. J. Am. Chem. Soc. 1998, 120, 11814-11815. (20) Severin, K.; Bergs, R.; Beck, W. Angew. Chem. 1998, 110, 17221743; Angew. Chem., Int. Ed. Engl. 1998, 37, 1634-1654. (21) Fish, R. H. Coord. Chem. ReV. 1999, 186, 569-584. (22) Hess, A.; Metzler-Nolte, N. J. Chem. Soc., Chem. Commun. 1999, 885-886. (23) Brosch, O.; Weyhermu¨ller, T.; Metzler-Nolte, N. Inorg. Chem. 1999, 5308, 8-5313. (24) Brosch, O.; Weyhermu¨ller, T.; Metzler-Nolte, N. Eur. J. Inorg. Chem. 2000, 323-330. (25) Hess, A.; Brosch, O.; Weyhermu¨ller, T.; Metzler-Nolte, N. J. Organomet. Chem. 1999, 589, 75-84.

10.1021/ic000089+ CCC: $19.00 © 2000 American Chemical Society Published on Web 11/01/2000

5438 Inorganic Chemistry, Vol. 39, No. 24, 2000 for the C terminus of peptides.25 We now report the synthesis and characterization of ferrocene derivatives of chiral amines. From the natural chiral pool, amino acids are readily available, easy to handle but at the same time offer a variety of functionalities that can be exploited for further chemistry. Eckert et al. have already demonstrated that ferrocene derivatives of glycine (the simplest and achiral amino acid) can be very useful for the electrochemical detection of amino acids and peptides in liquid chromatography (ECD-HPLC).26,27 However, these authors were merely interested in reactivity studies and their products were never comprehensively or even structurally characterized.28 In a structural study, the complexation of Pd(II) ions by Schiff bases derived from ferrocene and amino acids was investigated by Beck and co-workers.29 In the following, we present a simple, high-yielding procedure for the synthesis of chiral ferrocene methylamines, their coupling to amino acids and peptides, along with spectroscopic (including electrochemical) and structural (including X-ray and molecular modeling) characterization. Experimental Section All reactions were carried out in ordinary glassware and solvents without further precautions except where indicated. Chemicals were purchased from Aldrich-Sigma GmbH and used as received. Enantiomerically pure L amino acids were used except where indicated. Elemental analyses were carried out by H. Kolbe, Analytisches Laboratorium, Mu¨lheim. IR spectra were recorded on a Perkin-Elmer system 2000 instrument as KBr disks, additionally in CH2Cl2 solution where indicated. Frequencies ν are given in cm-1. UV/vis spectra were recorded on a Perkin-Elmer Lambda 19 spectrometer; only the wavelengths of the lowest-energy ferrocene transition are given in nanometers,  (dm3 mol-1 cm-1) in brackets. Mass spectra were recorded by the mass spectrometry service group, Mu¨lheim, on a MAT 8200 (Finnigan GmbH, Bremen) instrument (EI, 70 eV) or on a MAT95 (Finnigan GmbH, Bremen) instrument (ESI, CH3OH solution, positive ion detection mode). Only characteristic fragments are given with intensities (%) and possible composition in brackets. Analytical HPLC on D/L amino acid derivatives were carried out on a Nucleosil-Chiral-2 column (ABIMED model 305 pump and Shimadzu SPD-M10 AV diode array detector) with cyclohexane and 0.5% propanol at a 0.8 mL/min flow rate. Samples were taken from the reaction mixtures, evaporated in vacuo, and redissolved in the HPLC solvent mixture. The ratio was determined at 215 nm, where the highest extinction coefficients were observed, and confirmed at 440 nm, where only bands from the ferrocene moiety are recorded. Cyclic voltammograms were obtained with a three-electrode cell and an EG&G Princeton Applied Research model 273A potentiostat. A Ag/AgNO3 (0.01 mol/L in AgNO3) reference electrode, a glass carbon disk working electrode of 2 mm diameter, and a Pt wire counter electrode were used. Square wave voltammograms were recorded with a step height of 1 mV, a 25 mV pulse amplitude, and a 40 Hz frequency. CH2Cl2 solutions (ca. 10-4 mol/L) contained 0.1 mol/L Bu4NPF6 as supporting electrolyte. As an internal standard, ferrocene was added in excess as a reference. NMR spectra were recorded in CDCl3 at room temperature on a Bruker ARX 250 (1H at 250.13 MHz and 13C), DRX 400 (1H at 400.13 MHz, 13C and 2D spectra), and DRX 500 (1H at 500.13 MHz, 13C, 15N, 2D). 1H and 13C spectra were referenced to TMS using the 13C signals or the residual protio signals of the deuterated solvents as internal standards (CDCl3 ≡ 7.24 (1H) and 77.0 (13C), DMSO ≡ 2.49 (1H) and 39.5 (13C)). Positive chemical shift values δ (in ppm) indicate a downfield shift from the standard, and only the absolute values of coupling constants (26) Eckert, H.; Koller, M. Z. Naturforsch., B: Chem. Sci. 1990, 45, 17091714. (27) Eckert, H.; Koller, M. J. Liq. Chromatogr. 1990, 13, 3399-3414. (28) Eckert, H.; Seidel, C. Angew. Chem. 1986, 98, 168-170; Angew. Chem., Int. Ed. Engl. 1986, 25, 159-161. (29) Freiesleben, D.; Polborn, K.; Robl, C.; Su¨nkel, K.; Beck, W. Can. J. Chem. 1995, 73, 1164-1174.

Hess et al. are given in hertz. 15N spectra were referenced to the absolute frequency of 50.696 991 0 MHz, which was the resonance frequency of neat nitromethane under the same experimental conditions. All resonances were assigned by 2D NMR (H-H-COSY and 1H-13C-HMQC for 1J and long-range couplings). Where unambiguous or proven through spectroscopy, the following conventions are used: δ/δ′ denotes pairs of signals originating from s-cis/s-trans isomers, integration nH/2 indicates one signal of one rotational isomer only, “δ and δ′” denotes pairs of diastereotopic signals. 15N chemical shifts and coupling constants were taken from the F1 projection of indirect detection 1H15 N correlated 2D spectra with 1024/256 data points in F1/F2, processed after applying a matched cosine function and zero filling in both dimensions. Mo¨ssbauer data were recorded on a spectrometer with alternating constant accelaration and a 57Co source in a 6 µm Rh matrix. The minimum experimental line width was 0.24 mm s-1 full width at half-maximum. The sample temperature was maintained constant in an Oxford Instruments VARIOX cryostat. Isomer shifts are quoted relative to iron metal at 300 K. Molecular modeling was carried out with the SPARTAN program, version 5, on a Silicon Graphics Indigo2 workstation (Wavefunction Inc., 18401 Von Karman Avenue, Suite 370, Irvine, CA). X-ray Structure Determination of 3a. A transparent orange single crystal of 0.70 × 0.32 × 0.14 mm3 was sealed in a glass capillary and mounted on a Siemens SMART CCD-detector diffractometer system at ambient temperature. Cell constants were obtained from a leastsquares fit of 5541 reflections. A total of 17 847 intensities (7590 independent with Rint ) 0.0364) were collected by a hemisphere run taking frames at 0.30° in ω and corrected for Lorentz and polarization effects. The program SADABS (G. Sheldrick, University of Go¨ttingen, Germany, 1994) was used for absorption correction. The Siemens ShelXTL software package (Siemens Analytical X-ray Instruments, Inc.) was used for solution and refinement of the structure. Neutral atom scattering factors were taken from the usual sources.60 All non-hydrogen atoms were refined anisotropically. H atoms were placed at calculated positions and refined as riding atoms with isotropic displacement (30) Osman, A. M.; El-Maghraby, M. A.; Hassan, K. M. Bull. Chem. Soc. Jpn. 1975, 48, 2226. (31) Cano, J.; Benito, A.; Martı´nez-Ma´n˜ez, R.; Soto, J.; Paya´, J.; Lloret, F.; Julve, M.; Marcos, M. D.; Sinn, E. Inorg. Chim. Acta 1995, 231, 45-56. (32) Beer, P. D.; Chen, Z.; Drew, M. G. B.; Johnson, A. O. M.; Smith, D. K.; Spencer, P. Inorg. Chim. Acta 1996, 246, 143-150. (33) Eckert, H.; Forster, B.; Seidel, C. Z. Naturforsch., B: Chem. Sci. 1991, 46, 339-352. (34) Kraatz, H.-B.; Lusztyk, J.; Enright, G. D. Inorg. Chem. 1997, 36, 2400-2405. (35) Lin, L.; Berces, A.; Kraatz, H.-B. J. Organomet. Chem. 1998, 556, 11-20. (36) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265322. (37) Therien, M. J.; Chang, J.; Raphael, A. L.; Bowler, B. E.; Gray, H. B. Struct. Bonding (Berlin) 1991, 75, 109-129. (38) Winkler, J. R.; Gray, H. B. Chem. ReV. 1992, 92, 369-379. (39) Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B. Annu. ReV. Biophys. Biomol. Struct. 1992, 21, 349-377. (40) Gray, H. B.; Winkler, J. R. Annu. ReV. Biochem. 1996, 65, 537-561. (41) Langen, R.; Colo´n, J. L.; Casimiro, D. R.; Karpishin, T. B.; Winkler, J. R.; Gray, H. B. J. Biol. Inorg. Chem. 1996, 1, 221-225. (42) Fernando, S. R. L.; Kozlov, G. V.; Ogawa, M. Y. Inorg. Chem. 1998, 37, 1900-1905. (43) Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.; Bossmann, S. H.; Turro, N. J.; Barton, J. K. Science 1993, 262, 1025-1029. (44) Holmlin, R. E.; Dandliker, P. J.; Barton, J. K. Angew. Chem. 1997, 109, 2830-2848; Angew. Chem., Int. Ed. Engl. 1997, 36, 2714-2730. (45) Meggers, E.; Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 12950-12955. (46) Giese, B.; Wessely, S.; Spormann, M.; Lindemann, U.; Meggers, E.; Michel-Beyerle, M. E. Angew. Chem. 1999, 111, 1050-1052; Angew. Chem., Int. Ed. 1999, 38, 996-998. (47) Meggers, E.; Giese, B. Nucleosides Nucleotides 1999, 18, 1317-1318. (48) Diederichsen, U. Angew. Chem. 1997, 109, 2411-2413; Angew. Chem., Int. Ed. Engl. 1997, 36, 2317-2319. (49) Dong, T.-Y.; Chang, C.-K.; Cheng, C.-H. J. Organomet. Chem. 1999, 587, 46-48. (50) Arduengo, A. J., III; Harlow, R. L.; Kline, M. J. Am. Chem. Soc. 1991, 113, 361-363.

Chiral Ferrocene Amines parameters. The absolute structure was determined reliably (Flack parameter -0.01(2)) in accordance with the known stereochemistry (L) of the amino acid. General Synthesis of Ferrocene-Methylamine Derivatives 3 and 9. The amino acid hydrochloride, triethylamine, and ferrocene aldehyde (1 equiv each, typical scale 2 mmol) were refluxed in dry CHCl3 for 3 h. After evaporation of the solvent on a rotary evaporator, the imine was obtained quantitatively as a yellow solid, the purity of which was checked by 1H NMR (see text and data below). The imine was dissolved in dry CH3OH, and 4 equiv of solid NaBH4 were added in small portions at 0 °C. After the mixture was stirred for 30 min, 20 mL of 1 mol/L aqueous NaOH was added and the organic phase extracted with CHCl3 (3 × 100 mL). The combined organic phases were dried and evaporated to dryness to afford the pure ferrocene methyl amino acid in 90-100% yield. 3a. 1H NMR: δ 7.30-7.17 (5H, mult, HPh), 4.13 (1H, HCp,o), 4.11 (1H, HCp,o), 4.06 (2H, HCp,m), 4.03 (5H, s, Cp), 3.65 (3H, s, OCH3), 3.61 (1H, t, J ) 6.8 Hz; CRH), 3.46 (1H, d, J ) 12.8 Hz, Cp-CH2), 3.34 (1H, d, J ) 12.8 Hz, Cp-CH2), 2.98 (2H, mult, CβH2), 2.0 (1H, br, NH). 13C NMR: δ 174.9 (CO2), 137.2 (Ci), 129.2, 128.4, 126.8 (CPh,p), 86.6 (Cpi), 68.3 (5C, Cp), 68.2, 68.1, 67.7, 67.6 (all Cp), 62.4 (CR), 51.6 (OCH3), 47.1 (N-CH2), 39.7 (Cβ). IR (KBr): 1728vs, 1719vs cm-1. MS (m/z): 377 (50, M+•), 312 (2), 218 (5), 199 (100). CV: +2 mV. Anal. Calcd for C21H23FeNO2 (377.27 g/mol): C, 66.9; H, 6.1; N, 3.7. Found: C, 66.8; H, 6.2; N, 3.5. 2a. 1H NMR: δ 7.88 (1H, s, CHdN), 4.58 (1H, HCp,o), 4.54 (1H, HCp,o), 4.30 (2H, HCp,m), 4.07 (1H, dd, J ) 9.3 Hz, J ) 4.8 Hz, CRH), 3.96 (5H, s, Cp), 3.69 (3H, s, OCH3), 3.29 (1H, mult, CβH), 3.10 (1H, mult, CβH). 3c. 1H NMR: δ 4.18 (1H, HCp,o), 4.12 (1H, HCp,o), 4.11 (5H, s, Cp), 4.07 (2H, HCp,m), 3.70 (3H, s, OCH3), 3.44 (1H, d, J ) 12.6 Hz, CpCH2), 3.33 (1H, d, J ) 12.6 Hz, Cp-CH2), 3.31 (1H, app t, J ) 7.2 Hz, CRH), 1.68 (1H, mult, CγH), 1.45 (2H, t, J ) 7.2 Hz, CβH2), 0.90 (3H, d, J ) 6.6 Hz, CH3), 0.85 (3H, d, J ) 6.6 Hz, CH3). 13C NMR: δ 176.5 (CO2), 86.6 (Cpi), 68.4 (5C, Cp), 67.9, 67.7 (2C), 67.6 (all Cp), 59.5 (CR), 51.6 (OCH3), 47.2 (NCH2), 42.9 (Cβ), 25.0 (Cγ), 22.7, 22.4 (both CH3). IR (KBr): 1735vs cm-1. UV (nm (, dm3 mol-1 cm-1)): 437 (109). MS (m/z): 343 (71, M+•), 278 (5), 199 (100). CV: +2 mV. Anal. Calcd for C18H25FeNO2 (343.25 g/mol): C, 63.5; H, 7.3; N, 4.1. Found: C, 62.5; H, 6.9; N, 4.2. 2c. 1H NMR: δ 8.11 (1H, s, CHdN), 4.65 (2H, HCp,o), 4.35 (2H, HCp,m), 4.15 (5H, s, Cp), 3.89 (1H, dd, J ) 5 Hz, J ) 9.3 Hz, CRH), 3.69 (3H, s, OCH3), 1.80 (1H, mult, CβH), 1.70 (1H, mult, CβH), 1.56 (1H, mult, CγH), 0.90 (3H, d, J ) 6.7 Hz, CH3), 0.86 (3H, d, J ) 6.4 Hz, CH3). 3d. 1H NMR: δ 4.18 (1H, HCp,o), 4.12 (1H, HCp,o), 4.11 (5H, s, Cp), 4.06 (2H, HCp,m), 3.71 (3H, s, OCH3), 3.46 (1H, d, J ) 12.6 Hz, CpCH2), 3.41 (1H, dd, CRH), 3.34 (1H, d, J ) 12.6 Hz, Cp-CH2), 2.57 (2H, mult, CγH2), 2.07 (3H, s, S-CH3), 1.92 (1H, mult, CβH), 1.83 (1H, mult, CβH). 13C NMR: δ 175.5 (CO2), 86.6 (Cpi), 68.4 (5C, Cp), 68.3, 67.8, 67.7, 67.6 (all Cp), 59.7 (CR), 51.8 (OCH3), 47.2 (NCH2), 32.9 (Cβ), 30.5 (Cγ), 15.4 (SCH3). IR (KBr): 1735vs cm-1. UV (nm (51) Arduengo, A. J., III; Dias, H. V. R.; Dixon, D. A.; Harlow, R. L.; Klooster, W. T.; Koetzle, T. F. J. Am. Chem. Soc. 1994, 116, 68126822. (52) Denk, M.; Green, J. C.; Metzler, N.; Wagner, M. J. Chem. Soc., Dalton Trans. 1994, 2405-2410. (53) Roberts, G. C. K. NMR of Macromolecules - A Practical Approach; Oxford University Press: Oxford, 1993; Vol. 134. (54) Meissner, A.; Haehnel, W.; Vahrenkamp, H. Chem.sEur. J. 1997, 3, 261-267. (55) Knipp, B.; Mu¨ller, M.; Metzler-Nolte, N.; Balaban, T. S.; Braslavsky, S. E.; Schaffner, K. HelV. Chim. Acta 1998, 81, 881-888. (56) Turner, D. L. Prog. Nucl. Magn. Reson. Spectrosc. 1985, 17, 281358. (57) Perrin, C. L.; Dwyer, T. J. Chem. ReV. 1990, 90, 935-967. (58) Neuhaus, D.; Williamson, M. The Nuclear OVerhauser Effect in Structural and Conformational Analysis; VCH Publishers: New York, 1989. (59) Tropp, J. J. Chem. Phys. 1980, 72, 6035-6043. (60) International Tables for Crystallography; Kynoch Press: Birmingham, 1974; Vol. 4.

Inorganic Chemistry, Vol. 39, No. 24, 2000 5439 (, dm3 mol-1 cm-1)): 437 (118). MS (m/z): 361 (92, M+•), 296 (3), 280 (6), 199 (100). CV: -3 mV. Anal. Calcd for C17H23FeNO2 (361.29 g/mol): C, 56.5; H, 6.4; N, 3.9. Found: C, 56.4; H, 6.4; N, 4.1. 2d. 1H NMR: δ 8.18 (1H, s, CHdN), 4.71 (1H, HCp,o), 4.65 (1H, HCp,o), 4.39 (2H, HCp,m), 4.19 (5H, s, Cp), 4.04 (1H, dd, CRH), 3.73 (3H, s, OCH3), 2.54 (1H, mult, CβH), 2.42 (1H, mult, CβH), 2.21 (2H, mult, CγH2), 2.07 (3H, s, S-CH3). 4. Compound 3a (0.38 g, 1 mmol) was suspended in aqueous 2 N NaOH solution. After 1 h of refluxing followed by filtration, the clear solution was cooled to 0 °C and concentrated HCl was added slowly to it. The orange precipitate was filtered off and dried to afford the pure acid 4 (0.34 g, 94%). 1H NMR (DMSO): δ 7.30-7.22 (5H, mult, HPh), 4.41 (1H, HCp,o), 4.38 (1H, HCp,o), 4.23 (2H, HCp,m), 4.17 (5H, s, Cp), 4.16 (1H, mult, CRH), 3.94 (2H, br, Cp-CH2), 3.33 (1H, dd, J ) 14.0 Hz, J ) 4.7 Hz, CβH), 3.05 (1H, dd, J ) 14.0 Hz, J ) 8.7 Hz, CβH). 13C NMR (DMSO): δ 169.5 (CO2), 135.1 (Ci), 129.6, 128.8, 127.5 (CPh,p), 76.2 (Cpi), 71.0, 70.9, 69.2 (all Cp), 69.0 (5H, Cp), 59.4 (CR), 45.2 (N-CH2), 35.2 (Cβ). IR (KBr): 1623sh, 1602vs cm-1. MS (m/z): 363 (100, M+•), 251 (4), 199 (93). Mp: 330 °C (dec). 5. To a solution of 4, glycine methyl ester and NEt3 (1 equiv each, 1 mmol) in CH3CN was added 1 equiv of HBTU (O-(1H-benzotriazol1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate). After the mixture was stirred for 15 min, brine was added. The solution was diluted with ethyl acetate, and the organic phase was washed with aqueous 2 N HCl, water, aqueous NaHCO3, and water. The organic phase was dried and evaporated to dryness to afford 5 (0.37 g, 85%) as a yellow solid. 1H NMR: δ 7.81 (1H, br, t, J ) 5.1 Hz, NHGly), 7.30 (3H, mult, HPh), 7.23 (2H, mult, HPh), 4.10 (1H, HCp,o), 4.06 (2H, d, J ) 5.1 Hz, CR,GlyH), 4.03 (2H, HCp,m), 3.95 (1H, HCp,o), 3.87 (5H, s, Cp), 3.73 (3H, s, OCH3), 3.46 (1H, dd, J ) 10.6 Hz, J ) 3.8 Hz, CR,PheH), 3.44 (1H, d, J ) 13.2 Hz, Cp-CH2), 3.27 (1H, dd, J ) 13.9 Hz, J ) 3.8 Hz, CβH2), 3.20 (1H, d, J ) 13.2 Hz, Cp-CH2), 2.68 (1H, dd, J ) 13.9 Hz, J ) 10.6 Hz, CβH2). 1H NMR (DMSO): δ 8.33 (1H, br, NHGly), 7.27 (4H, mult, HPh), 7.21 (1H, mult, HPh), 4.15 (1H, HCp,o), 4.08 (1H, HCp,o), 4.04 (2H, HCp,m), 3.99 (5H, s, Cp), 3.88 (1H, d, J ) 6.0 Hz, CR,GlyH), 3.64 (3H, s, OCH3), 4.02 (1H, mult, CR, PheH), 3.41 (1H, d, J ) 12.4 Hz, Cp-CH2), 3.18 (1H, d, J ) 12.4 Hz, CpCH2), 2.96 (1H, dd, J ) 13.8 Hz, J ) 8.3 Hz, CβH2), 2.73 (1H, dd, J ) 13.8 Hz, J ) 5.3 Hz, CβH2). 13C NMR (CDCl3): δ 174.2, 170.3 (both CO), 137.5 (Ci), 129.0, 128.9, 127.1 (CPh,p), 86.3 (Cpi), 68.3 (5H, Cp), 67.8, 67.7, 67.5, 67.3 (all Cp), 63.8 (CR, Phe), 52.3 (OCH3), 47.7 (N-CH2), 40.8 (CR, Gly) 39.4 (Cβ). 15N NMR: δ -281 (NGly). IR (KBr): 3360br, 1752vs, 1674vs cm-1. MS (m/z): 434 (42, M+•), 390 (1), 369 (2), 291 (4), 214 (18), 199 (100). Anal. Calcd for C23H26FeN2O3 (434.32 g/mol): C, 63.6; H, 6.0; N, 6.5. Found: C, 63.8; H, 6.1; N, 6.4. 6. Details of the preparation and properties of 6 will be given elsewhere. Only preliminary data are given for comparison. 1H NMR: δ 7.76/7.66 (1H, br, NHLeu), 7.13 (2H, mult, HPh), 7.06 (2H, mult, HPh), 5.25/4.89 (1H, mult, CR,LeuH), 4.85 (1H/2, d, CH2), 4.59 (1H/2, d, CH2), 4.46 (1H, d), 4.44 (1H/2, s), 4.38 (1H/2, d), 4.17 (1H/2, s, Cp), 4.15 (1H/2, s, Cp), 4.12 (1H, s, Cp), 4.01 (1H/2), 4.08 (1H), 4.10/4.08 (5H, s, Cp), 4.01 (1H/2, d), 4.00 (1H, d), 3.51/3.42 (1H, quart, J ) 7.0 Hz, CR,AlaH), 2.33/2.32 (3H, s, Ar-CH3), 1.68, 1.56, 1.43 (3H, several mult, Cβ,LeuH2, Cγ, LeuH), 1.35/1.29 (3H, d, J ) 7.0 Hz, CH3,Ala), 1.08/0.96 (3H, d, J ) 6.5 Hz, CH3,Leu), 0.79, 0.75 (6H, d, J ) 6.0 Hz, CH3,Leu). C28H37FeN3O2 (503.47 g/mol). MS (m/z): 503 (100), 438 (66), 360 (61), 199 (77). 7. 7 was prepared analogously to 5 (70% yield). 1H NMR: δ 7.84/ 7.76 (1H, d, J ) 7.9 Hz, NHAla), 6.82/6.73 (1H, d, J ) 8.6 Hz, NHLeu), 7.32-7.03 (10H, several mult, HPh), 5.22/4.89 (1H, mult, CR,LeuH), 4.83 and 4.06, 4.60 and 4.02, 4.37 and 4.03 (1H each, all pairs of diastereotopic N-CH2), 4.53 (1H, s, N-CH2), 4.45 (1H, CR,AlaH), 4.40 (1H, Cp), 4.15-3.94 (7H, Cp), 4.09/4.08 (5H, Cp), 3.91/3.89 (5H, Cp), 3.46 (1H, mult, CR,PheH), 3.44/3.20 (2H, Phe-CH2-Cp), 2.33 (s, ArCH3), 1.67/1.52 (1H, mult, Cγ,LeuH), 1.63 and 1.45/1.50 and 1.30 (2H, mult, Cβ,LeuH2), 1.36/1.31 (3H, d, J ) 7.0 Hz, Cβ,AlaH), 1.03, 0.91, 0.76, 0.71 (3H/2 each, d, J ) 6.4 Hz, 6.5 Hz, 6.4 Hz, 6.2 Hz, Cδ,LeuH3). 13C NMR: δ 173.6/173.5 (CO Phe), 172.2/171.8 (COLeu), 171.7/171.4 (COAla), 137.4 (Ci,Phe), 137.3/137.0, 133.9/133.1 (Cquart,Ar), 129.5, 129.3, 129.0, 128.8, 127.9, 127.0, 126.8 (aromatic C), 86.2/86.1 (Phe-CH2Cpi), 82.6/82.0 (Cpi), 68.8/68.6, 68.1/68.2 (10H, Cp), 69.6-67.6 (all

5440 Inorganic Chemistry, Vol. 39, No. 24, 2000

Hess et al.

Scheme 1

Cp), 63.6/63.5 (CR,Phe), 48.4/48.3 (CR,Ala), 47.8/47.6 (CR,Leu), 47.7/47.6 (Phe-CH2-Cp), 49.0, 46.5, 45.4, 44.0 (all remaining CH2), 43.5/42.2 (Cβ,Leu), 39.4 (Cβ,Phe), 24.8/24.6 (Cγ,Leu), 23.6, 23.4, 21.6, 21.6 (Cδ,Leu), 21.1/21.0 (Ar-CH3), 18.4/18.0 (Cβ,Ala). 15N NMR: δ -262, -263. IR (KBr): 3092w, 3026vw, 2956m, 2927m, 2868w, 1638vs,br cm-1. MS (m/z): 848 (19, M+•), 783 (10), 705 (17), 199 (100). CV: +60 mV. Mp: 72-73 °C. Anal. Calcd for C48H56Fe2N4O3 (848.69 g/mol): C, 67.9; H, 6.7; N, 6.6. Found: C, 67.7; H, 6.7; N, 6.7. 9. Brown oil. 1H NMR: δ 7.33 (4H, mult, HPh), 7.25 (1H, mult, HPh), 4.13 (1H, mult, HCp,o), 4.12 (1H, mult, HCp,o), 4.07 (2H, HCp,m), 4.05 (5H, s, Cp), 3.80 (1H, quart, J ) 6.6 Hz, CH), 3.55 (1H, d, J ) 13.0 Hz, CH2), 3.25 (1H, d, J ) 13.0 Hz, CH2), 1.32 (1H, d, J ) 6.6 Hz, CH3). 13C NMR: δ 145.6 (Ci), 128.4, 126.8 (CPh,p), 126.6, 87.2 (Cpi), 68.4, 68.3 (5C), 68.1, 67.7, 67.6 (all Cp), 57.5 (CH), 46.6 (NCH2), 24.6 (CΗ3). IR (KBr): 3084m, 3024, 2962m, 2924, 1492m, 1450m cm-1. MS (m/z): 390 (100, M+•), 253 (18), 200 (25), 199 (32). CV: -6 mV. General Synthesis of Ferrocene Amino Acids 10. The amino acid (1.15 equiv), DhbtOH (1.15 equiv), and 9 (1 equiv, typical scale 1 mmol) were dissolved in 25 mL of DMF. After addition of 25 mL of CH2Cl2 and cooling to 0 °C, DCC (1.25 equiv) was added, and stirring continued for 2 h and an additional 12 h at room temperature. A white precipitate of dicyclohexylurea was removed by filtration and washed twice with CH2Cl2. The combined organic phases were washed with saturated NaHCO3 (3 × 50 mL), brine (50 mL), KHSO4 (3 × 50 mL), and brine (50 mL). The organic phase was dried over MgSO4 and the solvent removed on a rotary evaporator. The oily residue was chromatographed over a silica column with ether/pentane (5:1). D-10b. 1H NMR: δ 7.40-7.15 (5H, mult, HPh), 5.79/5.12 (1H, quart, J ) 6.8 Hz, C*H-Ph), 5.44/5.26 (1H, d, J ) 8.0 Hz, NHBoc), 4.66 (1H, mult, CRH), 4.44/4.05 (1H, d, J ) 6.8 Hz, CH2), 4.40/3.81 (1H, d, J ) 8.8 Hz, CH2), 4.18 (1H/2 see text, HCp), 4.11 (1H/2, HCp), 4.05/ 4.02 (5H, s, Cp), 3.96 (2H/2, HCp), 3.92 (2H/2, HCp), 3.85 (2H/2, HCp), 3.81 (1H/2, HCp), 1.55/1.53 (3H, d, J ) 6.8 Hz, C*H-CH3), 1.43/1.41 (9H, s, C(CH3)3)), 1.28/1.12 (3H, d, J ) 6.8 Hz, CβH3). 13C NMR: δ 174.3/173.1, (COAmide), 155.1/155.0 (COBoc), 140.6/139.9 (Ci), 128.7, 128.4, 127.7, 127.3, 127.2, 127.1 (CPh, p), 84.8/84.4 (Cpi), 79.5 (C(CH3)3), 68.8/68.5 (5H, Cp), 70.2, 69.9, 69.5, 69.1, 68.2, 67.8, 67.7, 67.2 (all Cp), 55.1/52.0 (C*H), 46.7/46.6 (CR), 44.0/42.1 (N-CH2), 19.9/18.9, 19.3/17.2 (both Cβ). 15N NMR: δ -288/-287. IR (KBr): 3340br, 1706vs, 1639vs cm-1. UV (nm (, dm3 mol-1 cm-1)): 436 (113). MS (m/z): 490 (100, M+•), 434 (52), 416 (28), 369 (41), 199 (66). CV: +20 mV. Anal. Calcd for C27H34FeN2O3 (490.43 g/mol): C, 66.1; H, 7.0; N, 5.7. Found: C, 66.0; H, 7.1; N, 5.6. L-10b. 1H NMR: δ 7.39-7.21 (5H, mult, HPh), 5.82/5.11 (1H, quart, J ) 7.0 Hz, C*H-Ph), 5.47/5.38 (1H, d, J ) 7.6 Hz, NHBoc), 4.73 (1H, mult, CRH), 4.54/and 3.70 (1H, d, J ) 14.4 Hz, CH2), 4.31/4.04 (1H, d, J ) 16.0 Hz, CH2), 4.14 (2H/2 see text, HCp), 4.09 (2H/2, HCp), 4.04/4.00 (5H, s, Cp), 3.98 (2H/2, HCp), 3.62 (2H/2, HCp), 1.57/ 1.40 (3H, d, J ) 7.0 Hz, C*H-CH3), 1.44/1.40 (9H, s, C(CH3)3)), 1.19/1.12 (3H, d, J ) 6.4 Hz, CβH3). 13C NMR: δ 173.8/172.9, (COAmide), 155.1/155.0 (COBoc), 140.6/139.9 (Ci), 128.6, 128.5, 127.9, 127.7, 127.6/127.0 (CPh, p), 84.8/84.4 (Cpi), 79.4 (C(CH3)3), 68.8/68.5 (5H, Cp), 70.0, 69.8, 69.7, 68.8, 68.6, 68.0, 67.3, 67.2 (all Cp), 52.1

Table 1. Summary of Crystallographic Details for 3a chemical formula fw space group (No.) a, Å b, Å c, Å β, deg V, Å3 Z D (calcd), g/cm-3 temp (K) λ, Å abs coeff (mm-1) R1a (I > 2σ(I)) wR2a (all data) data/restraints/parameters GOF on F2

C21H23FeNO2 377.25 P21 10.301(1) 9.647(1) 18.479(2) 102.98(2)° 1789.4(3) 4 1.40 298 0.71073 0.856 0.0599 0.1648 7287/3/457 1.062

a R1 ) (∑F | - |F |/∑F |); wR2 ) [∑(F 2 - F 2)2/∑F 4]1/2; GOF ) o c o o c o [∑(Fo2 - Fc2)2/(no. of reflns - no. of params)]1/2.

(C*H), 47.0/46.6 (CR), 43.2/42.4 (N-CH2), 30.3/29.7 (C*H-CH3), 28.4/28.3 (CBoc), 19.6/18.4, 19.3/16.7 (Cβ). 15N NMR: δ -287.5. IR (KBr): 1709vs, 1638vs cm-1. UV: 445 (115). MS (m/z): 490 (98, M+•), 434 (44), 416 (81), 369 (36), 199 (61), 105 (100). CV: +15 mV. Anal. Calcd for C27H34FeN2O3 (490.43 g/mol): C, 66.1; H, 7.0; N, 5.7. Found: C, 66.2; H, 7.0; N, 5.7. 10c. 1H NMR: δ 7.40-7.23 (5H, mult, HPh), 5.78/5.22 (1H, quart, J ) 7.0 Hz, C*H-Ph), 5.2 (1H, br, NHBoc), 4.87 (1H, mult, CRH), 4.58 and 3.65 (1H, d, J ) 14.4 and 14.6 Hz, N-CH2), 4.35 and 3.88 (1H, d, J ) 16.0 and 16.3 Hz, N-CH2), 4.11-3.91 (4H, HCp), 4.06/ 4.01 (5H, s, Cp), 1.58/1.36 (3H, d, C*H-CH3), 1.45/1.37 (9H, s, C(CH3)3)), 1.52/1.18 (2H, mult, CβH2), 1.70 (1H, mult, CγH), 0.96/ 0.89 (3H, d, J ) 6.4 Hz/6.7 Hz, CδH3), 0.85/0.82 (3H, d, J ) 6.8 Hz, CδH3). 13C NMR: δ n. o. (COAmide), n. o. (COBoc), 140.6 (Ci), 128.6, 128.5, 127.9, 127.6, 127.5/127.2 (CPh,p), 84.9/84.6 (Cpi), 79.5 (C(CH3)3), 68.8/68.6 (5H, Cp), 70.1, 70.0, 69.9, 69.8, 69.7, 68.9, 68.8, 68.1 (all Cp), 54.7/52.3 (C*H), 49.7/48.9 (CR), 43.4/42.5 (N-CH2), 42.9/42.8 (Cβ), 25.0 (Cγ), 28.4/28.3 (CBoc), 23.6, 23.4, 21.9, 21.8 (all Cδ), 18.6/ 16.7 (C*H-CH3). IR (KBr): 1708 vs, 1636 vs cm-1. MS (m/z): 532 (100, M+•), 476 (41), 458 (52), 411 (33), 199 (67). Mp: 50 °C. Anal. Calcd for C30H40FeN2O3 (532.51 g/mol): C, 67.7; H, 7.6; N, 5.3. Found: C, 67.5; H, 7.5; N, 5.1.

Results and Discussion Synthesis. Ferrocene aldehyde 1 reacts readily with amino acids to form Schiff bases 2.29,30 These imines were subsequently reduced by NaBH4 in methanol to yield ferrocene methyl derivatives 3 of the corresponding amino acids in good yield (phenylalanine 3a, alanine 3b, leucine 3c, methionine 3d, Scheme 1).29,31,32 In fact, formation of the Schiff base and subsequent reduction may be carried out in a one-pot reaction in methanol without isolation of the Schiff base. To our surprise,

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Scheme 2

Scheme 3

aldehyde and enantiomerically pure L-1-amino-ethylbenzene (Scheme 3). Compound 9 reacted smoothly with amino acids such as alanine and leucine and in an equally facile manner with both enantiomers of alanine. The ferrocene-labeled amino acids 10 were obtained by our standard protocol in good yield (Scheme 3). Spectroscopic Characterization and Discussion

this reaction did not yield the corresponding glycine derivative 3e despite various efforts. This corresponds to an observation by Eckert et al., who obtained 3e by a slightly different route.26,33 Also, the alanine derivative 3b could not be obtained analytically pure, although the corresponding Schiff base 2b formed in quantitative yield. The NaBH4 reduction appears to work better for the larger amino acids such as leucine and phenylalanine, but we were unable to isolate even one defined reaction product from the glycine or alanine reductions. Compounds 3 are orangeyellow oils or solids, and crystalline material for a single-crystal X-ray analysis was obtained for the phenylalanine derivative 3a (Table 1). For further derivatization, the methyl ester in 3a was hydrolyzed by aqueous NaOH to yield the free acid 4. Coupling of glycine methyl ester to 4 proceeds smoothly via an HBTU mediated coupling scheme to yield the dipeptide FcCH2-Phe-Gly-OMe 5 (Fc ) ((η-C5H4)Fe(η-C5H5))) (Scheme 1). By the same method, the bis-ferrocene tripeptide 7 can be prepared from 4 and the dipeptide H-Ala-Leu-N(CH2pCH3-C6H4)-CH2-Fc 6 in good yield (Scheme 2). In an earlier study, we prepared a number of N-terminally functionalized amino acids and peptides by reaction of activated amino acids with the secondary ferrocene amine Fc-CH2-NH-CH2pCH3-C6H4 (8).25 To our surprise, we were unable to couple activated amino acids to the secondary amino group in 3a. To test whether steric congestion at the amino center might be the reason for this unexpected result, we prepared the slightly smaller methylamino-ferrocene derivative 9 from ferrocene Scheme 4

The reaction of 1 with amines proceeds smoothly in refluxing CHCl3 or CH3OH with quantitative yield. The purity of the Schiff bases was established by the disappearance of the 1H NMR signal of the aldehyde proton at 10 ppm and the appearance of a new signal at 7.9 ppm for the Fc-CHdN-R imino proton. After reduction with NaBH4, this signal disappears again, while a broad signal at 2.0 ppm (in CDCl3 at 300 K) for the amino proton and a signal for the newly formed methylene group are observed. As a consequence of the chiral CR atom of the attached amino acid, the two protons of this CH2 group are diastereotopic and give rise to an AB signal. For the same reason, all protons and carbon atoms of the substituted Cp ring become nonequivalent and four methine 13C NMR signals are observed from this ring. Even at 500 MHz, not all signals in the 1H NMR spectra are equally well resolved and usually only three broad signals with unresolved couplings and intensities 1:1:2 are observed. Hydrolysis of the methyl ester proceeds cleanly in very good yield, as does the coupling of the free acid 4 to glycine methyl ester and dipeptide 6. The resulting tripeptide 7 was purified by HPLC and its identity established by electron-spray ionization mass spectrometry (ESI-MS). A chiral Schiff base was also prepared by stirring a mixture of ferrocene aldehyde 1 and L-1-amino-ethylbenzene in CHCl3 over solid NaHCO3. This Schiff base was readily reduced to the ferrocene-methyl-N-(1-amino-ethylbenzene) (9) by NaBH4. No racemization was observed in this reduction or the formation of 3a-d. We have previously reported a simple coupling reaction of secondary ferrocene amines 8 to the C terminus of amino acids and peptides (Scheme 4). The same reaction could be applied to 9 to yield amides 10. It should be noted that a diastereomeric pair is formed after reaction with D- and L-alanine. In addition, the amides exist as two isomers due to rotation about the tertiary amide bond as indicated in Scheme 3. Both facts are nicely illustrated by the 1H NMR spectra of D-10b and L-10b in Figure 1. In previous work we have

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Hess et al.

Figure 2. ORTEP plot of one molecule of 3a with numbering scheme, selected bond lengths and angles: Fe-CCp (av) 2.047 Å; Fe-Cp(centroid) 1.653 and 1.652 Å (unsubstituted Cp); C(10)-C(11) 1.514(7) Å; Cp(centroid)-Fe-Cp(centroid) 178.9 Å.

Figure 1. 1H NMR spectra of D-10b (top) and L-10b (bottom) between 3.5 and 6.0 ppm (see text).

unsuccessfully tried to induce a preference for one rotational isomer by hydrogen bonding or steric bulk.25 It is noteworthy that in diastereomers 10b, one rotational isomer is in slight excess as indicated by integration of the 1H NMR signals in Figure 1. We have used molecular modeling to gain further insight into the geometry of both isomers (named s-cis and s-trans conformers, vide infra). For diastereomeric compounds such as 10b, two different values for the barrier of rotation about the central amide bond are expected. In D6-DMSO, coalescence for the two methyl groups in both diastereomers of 10b was easily observed between 20 and 60 °C, giving activation energies of 70.5 kJ/mol (D-10b) and 70.7 kJ/mol (L-10b). These values are equal within the accuracy of determination ((1 kJ/mol) and match the value previously determined for the achiral N-Bocglycine amide 12e (70.5 ( 0.5 kJ/mol, Scheme 4).25 All new compounds show reversible electrochemical waves in the cyclic voltammograms around 0 mV vs ferrocene (see Experimental Section) which are attributed to oxidation of the iron center. For compounds 3, the shift potential difference does not exceed 5 mV and is thus unsuitable for electrochemical differentiation of the attached amino acid.34,35 Interestingly, a small difference of 5 mV is also observed for the two diastereomers of 10b. Comparable to the previously investigated pair of 8 and 12e, the electrochemical potential of the ferrocene moiety shifts more than 20 mV between amine 9 and amides 10. This shift difference is large enough to be analytically useful in monitoring the process of the peptide coupling reaction. The electronic interaction between metal centers at a defined distance36 has raised considerable interest among chemists, e.g., in the question of electron transfer through peptides37-42 or the stacked bases of helical DNA.43-48 The square wave voltammogram of 7 is not significantly different from the sum of the square wave voltammograms of 3a and 6, with its peak potential comparable to that of 3a and 6 and a width at a half-potential w1/2 of 230 mV (compared to a w1/2 of 118 and 119 mV for 3a and 6, respectively). This result does not support a direct electronic interaction between the ferrocene centers.49 Indeed, molecular modeling suggests a minimum distance between the metal centers of about 10 Å even in a helical structure which places the metal centers in proximity. Chiral Induction The most significant difference between ferrocene amines 8

and 9 is the chiral center in 9. In fact, 9 was used to investigate whether a chiral induction would occur in the coupling reaction of 9 to alanine as a model amino acid. To this end, the pure diastereomers D-10b and L-10b were prepared independently and characterized spectroscopically and by HPLC. Then, a racemic mixture of alanine was activated with DhbtOH and DCC and 0.5 equiv of 9 were added. Two parallel experiments were carried out at 0 and 22 °C in DMF, and samples from both runs were investigated by HPLC after 15 min and after 6 h. At both temperatures, there was only a slight excess of the diastereomer originating from the D enantiomer (58% at 22 °C and 59% at 0 °C), which did not change with time. The HPLC findings could be confirmed by 1H NMR by careful integration of characteristic signals of both diastereomers (see Figure 1) in the reaction mixtures. This result is consistent with previous electrochemical experiments in which activation of the amino acid was found to be the rate-determining step in DhbtOH/DCC mediated coupling reactions. DMF is the preferred solvent for peptide couplings. However, the above findings were reproduced in THF, suggesting little if any solvent-dependence of the ratedetermining step. X-ray Analysis of 3a and Structural Investigations by Molecular Modeling An X-ray single-crystal analysis was carried out on 3a at 100 K. The result shows two independent molecules per asymmetric unit. A graphical representation of molecule 1 is shown in Figure 2. The main difference from molecule 2 is a clockwise rotation about the C(15)-C(16) bond by 36°. In the solid state, 3a forms discrete molecules with the amino acid pointing away from the ferrocene moiety and the phenyl ring being almost perpendicular to the cyclopentadienyl rings. Both Cp rings are almost eclipsed as concluded from an average dihedral angle H-C-C-H of 0.3°. All bond lengths and angles are well within the range usually observed for simpler achiral ferrocene amines.25,31,32 Eckert et al. reported the synthesis of some related ferrocene amino acid derivatives.28 However, these compounds were never structurally characterized. The crystal structure of 3a presents unambiguous proof for the proposed constitution of the chiral ferrocene derivatives 3. To rationalize their reactivity, a more detailed insight into their conformation in solution structures is required. We were surprised to find that coupling of activated amino acids to ferrocene amines was possible for 9 but not for 3a. Unfortunately, X-ray quality crystals of 9 could not be obtained. We have therefore used molecular modeling (SYBYL force field; see Experimental Section) to compare the steric congestion

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Inorganic Chemistry, Vol. 39, No. 24, 2000 5443

Figure 4. Molecular modeling structures of the s-cis and s-trans isomers of L-10b (see text for details).

Figure 3. Comparison of the molecular structure of 3a derived from an X-ray single-crystal structure (A, left) and molecular modeling (B, middle). On the right, the molecular modeling structure of 9 is also shown (C). See text for discussion of the structures and details on the molecular modeling.

around the secondary nitrogen center in 9 and 3a (Figure 3). Figure 3 compares the X-ray solid-state structure of 3a (left, A) with the calculated minimum solution structure of 3a (middle, B). Given the limitations of the force field approach, especially when dealing with transition metal compounds, the overall similarity of the two structures is quite convincing. The calculated solution structure of 9 is shown on the right (C). A comparison between B and C reveals that the ester group in B points in the same direction as the lone pair of the nitrogen atom in B and in C. This offers a compelling explanation for the different reactivity of 3a and 9 toward activated amino acids in that the carbonyl oxygen atom O(1) shields the lone pair on the nitrogen from attack of the carbon electrophile. This electronic shielding is not present in the simpler ferrocene methylamine 9, and thus, amide bonds are as readily formed, as in the sterically less demanding achiral ferrocene methylamine 8. Among others, a related reasoning has been used to explain the stability of Arduengo-type carbenes.50-52 We have used a combined NMR and molecular modeling approach to elucidate the structures of the s-cis and s-trans conformers of 10b (Scheme 3).53 While structural analysis of this kind is very common for biopolymers such as proteins and oligonucleotides, the analysis of small molecules such as 10 is complicated by their high degree of flexibility.54,55 To search the conformational space, 72 starting structures were generated by stepwise rotation around the N-CH bond and around the C-Ph bond and subsequently optimized without constraints. Figure 4 shows the minimum energy structures for the two families of molecules. Both structures exhibit a similar orientation of the phenylethylamine substituent relative to the ferrocene group and a voluminous Boc group pointing far away from the rest of the molecule. The NMR situation is further complicated by chemical exchange between the related protons sites of the two rotational isomers. This gives rise to cross-peaks of opposite sign relative to NOE (nuclear Overhauser effect) cross-peaks between proton sites in spatial proximity.56,57 To avoid extensive secondary NOE as a consequence of interconversion between the two rotational isomers, a mixing time of 300 ms at 300 K sample temperature was chosen. Under these conditions, many

NOE cross-peaks58 are observed and confirm the overall molecular structure of the molecule but are of little diagnostic value with regard to the s-cis/s-trans problem. However, a few diagnostic NOE cross-peaks confirm the molecular modeling structures for both rotational isomers qualitatively; for the s-trans isomer, a medium NOE is observed between the alanine methyl group and one of the diastereotopic protons of the CH2 group. From molecular modeling, the distance Cβ,Ala (a good mean value for the three hydrogen atoms on a freely rotating methyl group)53,59 to the CH2 atoms is 2.9 and 4.3 Å. These distances are 4.9 and 5.8 Å in the s-cis isomer, corresponding to a very small NOE cross-peak only. On the other hand, the alanine methyl group gets closer to the hydrogen center at the chiral carbon atom of the phenylethylamine moiety in the s-cis isomer (4.2 Å vs 5.1 Å), and indeed, a weak NOE cross-peak between these groups is detectable for the s-cis isomer only. In the s-cis isomer, the protons at both chiral centers are only 2.2 Å apart (4.4 Å in the s-trans isomer), and indeed, a strong NOE in the s-cis isomer is observed. These data demonstrate that a combination of NMR spectroscopy and molecular modeling may be successfully used to elucidate the solution structures even of relatively flexible bioorganometallics.53 For a comprehensive treatment, a molecular dynamics simulation would certainly be appropriate. However, the agreement between crystallographic data and the minimum structure for 3a in solution is convincing. A qualitative interpretation of NOE data further supports the proposed structure, and this is used in turn to rationalize the fundamentally different reactivity of the two superficially very similar molecules 3a and 9 toward activated amino acids. This work shows a simple and high-yielding route to ferrocene derivatives of highly functionalized biomolecules with well-defined and predictable conformation. The development of biosensors based on ferrocene peptide conjugates presented in this work is in progress in our group. Acknowledgment. The technical assistance of Agnetha Schmidt is gratefully appreciated. The authors are also grateful to Kerstin Sand and Jo¨rg Bitter for running numerous NMR spectra and to Manuela Trinoga for expert HPLC services. N.M.N. thanks Prof. K. Wieghardt for his support of our work. Supporting Information Available: An X-ray crystallographic file in CIF format for the structure determination of 3a. This material is available free of charge via the Internet at http://pubs.acs.org. IC000089+