Labeling of Peptides with Halocarbonyltungsten Complexes

Aug 13, 2009 - [Tp*WI(CO)(η2-Fmoc-Pgl-OH)] (2, Pgl = propargylglycine) was utilized to synthesize the side-chain-labeled enkephalin derivative ...
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Organometallics 2009, 28, 5090–5095 DOI: 10.1021/om9002297

Labeling of Peptides with Halocarbonyltungsten Complexes Containing Functional η2-Alkynyl Ligands Johannes Zagermann, Klaus Merz, and Nils Metzler-Nolte* Lehrstuhl f€ ur Anorganische Chemie I-Bioanorganische Chemie, Fakult€ at f€ ur Chemie und Biochemie, Ruhr-Universit€ at Bochum, Universit€ atsstrasse 150, 44801 Bochum, Germany Received March 25, 2009

Two different seven-coordinate iodocarbonyltungsten complexes incorporating tris(pyrazolyl)borate ligands and η2-coordinated functionalized alkynes have been synthesized and characterized. [Tp*WI(CO)(η2-HCC(CH2)2CO2H)] (1), which has been characterized in the solid state by X-ray diffraction, was readily coupled to the hexa- and pentapeptides pseudo-neurotensin(8-13) and enkephalin in solid phase peptide synthesis (SPPS) to provide the N-terminal-labeled bioconjugates [Tp*WI(CO)(η2HCC(CH2)2CO-NH-Lys-Lys-Pro-Tyr-Ile-Leu-OH)] (3) and [Tp*WI(CO)(η2-HCC(CH2)2CO-NH-TyrGly-Gly-Phe-Leu-OH)] (4), respectively. [Tp*WI(CO)(η2-Fmoc-Pgl-OH)] (2, Pgl = propargylglycine) was utilized to synthesize the side-chain-labeled enkephalin derivative H2N-Tyr-Gly-[Tp*W(I)(CO)(η2Pgl)]-Phe-Leu-OH (5). All new bioconjugates were comprehensively charaterized by HPLC, mass spectrometry, and multinuclear 1D and 2D spectroscopy. Their characteristic metal carbonyl IR band just over 1900 cm-1 and good stability against air and water make these Tp*W alkyne complexes valuable labels for biomolecules.

Introduction Recent years have seen heightened interest in the labeling of biologically active molecules (e.g., peptides, DNA, drugs) with metal-containing compounds to introduce properties usually not found in biomolecules.1-4 While classic coordination compounds are used for various diagnostic and therapeutic purposes,5 the use of organometallic molecules is limited by their stability under the conditions typically found in biological systems (aqueous medium, oxygen). Thus, there is a steady quest for compounds that are airand water-stable and provide “synthetic handles” for the facile synthesis of bioconjugates.6 One group of organometallic compounds frequently used in the modification of biomolecules are various carbonyl complexes of transition metals, mainly because of the spectroscopic window of their CO ligands in the 1800-2200 cm-1 region in IR spectroscopy. For example, acid-functionalized cymantrene (CpCO2H)Mn(CO)3 could recently be covalently attached to peptides,7 providing strong absorptions in the carbonyl region and making the resulting bioconjugates traceable by IR spectroscopy. Leong and co-workers *Corresponding author. Fax: þ49 (0) 234 3214378. Tel: þ49 (0) 234 3224153. E-mail: [email protected]. (1) Fish, R. H.; Jaouen, G. Organometallics 2003, 22, 2166–2177. (2) Metzler-Nolte, N. Angew. Chem., Int. Ed. 2001, 40, 1040–1043. (3) Severin, K.; Bergs, R.; Beck, W. Angew. Chem., Int. Ed. 1998, 37, 1634–1654. (4) Jaouen, G. Bioorganometallics; Wiley-VCH: Weinheim, 2005. (5) Alberto, R. J. Organomet. Chem. 2007, 692, 1179. (6) Metzler-Nolte, N. In Comprehensive Organometallic Chemistry III, 1st ed.; Parkin, G., Ed.; Elsevier: Amsterdam, 2006; Vol. 1. (7) N’Dongo, H. W. P.; Neundorf, I.; Merz, K.; Schatzschneider, U. J. Inorg. Biochem. 2008, 102, 2114–2119. pubs.acs.org/Organometallics

Published on Web 08/13/2009

have recently demonstrated intracellular IR imaging using the metal carbonyl vibration bands from Os carbonyl clusters.8 Jaouen et al. established carbonyl metallo immuno assays (CMIA),9 a method to label and detect drugs by coordinative attachment of metal carbonyl fragments such as alkyne-Co2(CO)6, cymantrenyl, and benchrotenyl, respectively. Introduction of alkyne-coordinated Co2(CO)6 fragments into biomolecules can also render them cytotoxic,10,11 a feature also found for modified peptides, making them potential antiproliferative drugs comparable to cis-platin.12 More recent investigations focus on the targeted release of CO, an endogenous signaling molecule,13 by metal carbonyl fragments to provide new medical applications.14,15 For example, (η5-C5H4R)Fe(CO)3 and Mn(CO)4X2 have been identified as possible CO-releasing molecules (CORMs) with rates of CO loss tunable by choice of R and X.15 Considering the rich chemistry of their carbonyl complexes in general, surprisingly little work is found concerning bioorganometallic compounds of the group 6 metals.16 Whereas carbonyl-containing (8) Kong, K. V.; Chew, W.; Lim, L. H. K.; Fan, W. Y.; Leong, W. K. Bioconjugate Chem. 2007, 18, 1370–1374. (9) Vessieres, A.; Salmain, M.; Brossier, P.; Jaouen, G. J. Pharm. Biomed. Anal. 1999, 21, 625–633. (10) Jung, M.; Kerr, D. E.; Senter, P. D. Arch. Pharm. 1997, 330, 173– 176. (11) Ott, I.; Schmidt, K.; Kircher, B.; Schumacher, P.; Wiglenda, T.; Gust, R. J. Med. Chem. 2005, 48, 622–629. (12) Neukamm, M. A.; Pinto, A.; Metzler-Nolte, N. Chem. Commun. 2008, 232–234. (13) Wu, L.; Wang, R. Pharmacol. Rev. 2005, 57, 585–630. (14) Johnson, T. R.; Mann, B. E.; Clark, J. E.; Foresti, R.; Green, C. J.; Motterlini, R. Angew. Chem., Int. Ed. 2003, 42, 3722–3729. (15) Mann, B. E.; Motterlini, R. Chem. Commun. 2007, 4197–4208. (16) Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.; Morgan, B. A.; Morris, H. R. Nature 1975, 258, 577–9. r 2009 American Chemical Society

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peptides following standard SPPS (solid phase peptide synthesis) protocols.17,28-41

Results

fragments of Mo and Cr, in particular fac-M(CO)3, are thoroughly investigated as metal-containing markers,17,18 similar use of tungsten organometallic compounds is limited to the (C5H5)W(CO)3 entity.19 Besides carbonyl ligands as a useful IR handle, the third-row element tungsten could serve as a high electron density tool in X-ray crystallography.20,21 Curran et al. proposed cyclic metallapeptides obtained by double alkyne coordination to W(dmtc)2 (dmtc = N,N-dimethyldithiocarbamate) fragments as covalent restraints for peptide conformation.22,23 As a promising entry to studies of tungsten bioorganometallic compounds, we were drawn to the rich chemistry of tungsten carbonyl complexes containing Tp* (Tp*=hydridotris(3,5-dimethylpyrazolyl)borate) ligands and their ability to form η2-alkyne complexes. The stability of complexes of the general formula Tp*W(CO)2(η2-alkyne) is found to be highly dependent on the alkyne ligand. For example, Seidel et al. reported Tp*W(CO)2(η2-bisbenzylthioacetylene)24,25 to be airstable, whereas Tp*W(CO)2(η2-diphenylethyne) is reported to be highly air-sensitive.26 Exchanging one carbonyl ligand for a halide gives chiral complexes of the type [Tp*W(CO)X(η2alkyne)] (X=Cl, Br, I), whose stability increases in the order Cl < Br < I.27 Therefore, we considered the [Tp*W(I)(CO)] moiety as a stable marker that is readily introduced by reaction of Tp*W(I)(CO)3 with alkynyl-functionalized biomolecules. Further, coordination of functional alkynes such as alkynoic acids should give building blocks that allow the subsequent labeling of peptides by established protocols. Herein, we report the synthesis and characterization of the two iodo-carbonyl-tungsten complexes [Tp*W(CO)(I)(HCC(CH2)2CO2H)] (1) and [Tp*W(CO)(I)(η2-Fmoc-Pgl)] (2) (Pgl = L-propargylglycine), shown in Figure 1. Additionally we demonstrate the use of these “building blocks” in the labeling of small

Synthesis of Tungsten η2-Alkyne Complexes. Neutral seven-coordinate tungsten compounds 1 and 2 were prepared by alkyne substitution of two carbonyl groups in Tp*W(CO)3I in analogy with literature procedures.26 Reaction progress is easily monitored by a color change from red to dark green and a downfield shift of the signal for the terminal alkyne in 1H NMR spectra. Heating a mixture of Tp*W(CO)3I with a slight excess of 4-pentynoic acid in THF afforded 1 as green crystals in 53% yield. 1H NMR spectra indicated the existence of two isomers in 92:8 ratio (12.23, 13.13 ppm, singlet, 1H each), which differ in the orientation of the alkyne group relative to the M-CO vector. The X-ray single-crystal structure showed only one isomer, with the alkyne proton pointing away from the CO moiety, as depicted in Figure 2. In this compound, the W atom is a center of chirality. The molecule crystallizes in the space group P1 with Z = 2. Only one of the two molecules is shown in Figure 2. The other molecule in the unit cell is the enantiomer that is related to the shown enenatiomer by the crystallographic center of inversion. Assuming the alkyne moiety to occupy one coordination site, the coordination sphere of tungsten could be described as roughly octahedral with local C3v symmetry for the Tp* ligand. The W-Calkyne bond distances of 2.020(9) and 2.065(8) A˚ found in 1 are consistent with structural features seen in other d4 alkyne complexes of tungsten.26,42 Tungsten-coordinated propargylglycine 2 was synthesized in an analogous fashion by refluxing a mixture of Tp*W(CO)3I with a slight excess of Fmoc-protected propargylglycine in THF in 67% yield. Similar to 1, two isomers were detected in 90:10 ratio (12.35, 13.10 ppm, singlets, 1H each). Both compounds were found to be air-stable both as solids and in solution even after prolonged storage. Synthesis of the Tungsten-Functionalized Bioconjugates. The pentynoic acid-functionalized building block 1 was tested in SPPS by coupling to enkephalin (Enk = Tyr-Gly-Gly-PheLeu) and a pseudo-neurotensin fragment (pNT=Lys-Lys-ProTyr-Ile-Leu), two small peptides frequently used in our group as

(17) van Staveren, D. R.; Metzler-Nolte, N. Chem. Commun. 2002, 1406–7. (18) Lavastre, I.; Besanc-on, J.; Brossiert, P.; Moise, C. Appl. Organomet. Chem. 1991, 5, 143–149. (19) Gorfti, A.; Salmain, M.; Jaouen, G.; McGlinchey, M. J.; Bennouna, A.; Mousser, A. Organometallics 1996, 15, 142–151. (20) Salmain, M.; Caro, B.; Le Guen-Robin, F.; Blais, J. C.; Jaouen, G. ChemBioChem 2004, 5, 99–109. (21) Salmain, M. In Bioorganometallics; Jaouen, G., Ed.; Wiley-VCH: Weinheim, 2006. (22) Curran, T. P.; Grant, A. L.; Lucht, R. A.; Carter, J. C.; Affonso, J. Org. Lett. 2002, 4, 2917–2920. (23) Curran, T. P.; Yoon, R. S. H.; Volk, B. R. J. Organomet. Chem. 2004, 689, 4837–4847. (24) Seidel, W. W.; Ibarra Arias, M. D.; Schaffrath, M.; Bergander, K. Dalton Trans. 2004, 2053–2054. (25) Seidel, W. W.; Ibarra Arias, M. D.; Schaffrath, M.; Jahnke, M. C.; Hepp, A.; Pape, T. Inorg. Chem. 2006, 45, 4791–4800. (26) Feng, S. G.; Gamble, A. S.; Philipp, C. C.; White, P. S.; Templeton, J. L. Organometallics 1991, 10, 3504–12. (27) Bartlett, I. M.; Carlton, S.; Connelly, N. G.; Harding, D. J.; Hayward, O. D.; Orpen, A. G.; Ray, C. D.; Rieger, P. H. Chem. Commun. 1999, 2403–2404. (28) Kirin, S. I.; Noor, F.; Metzler-Nolte, N. J. Chem. Educ. 2006, 84, 108–111. (29) Metzler-Nolte, N. Chimia 2007, 61, 736–741.

(30) Kuchta, M. C.; Gross, A.; Pinto, A.; Metzler-Nolte, N. Inorg. Chem. 2007, 46, 9400–9404. (31) Kirin, S. I.; D€ ubon, P.; Weyherm€ uller, T.; Bill, E.; MetzlerNolte, N. Inorg. Chem. 2005, 44, 5405–5415. (32) Noor, F.; W€ ustholz, A.; Kinscherf, R.; Metzler-Nolte, N. Angew. Chem., Int. Ed. 2005, 44, 2429–2432. (33) Chantson, J.; Varga Falzacappa, M. V.; Crovella, S.; MetzlerNolte, N. ChemMedChem 2006, 1, 1268–1274. (34) Hoffmanns, U.; Metzler-Nolte, N. Bioconjugate Chem. 2006, 17, 204–213. (35) Caddy, J.; Hoffmanns, U.; Metzler-Nolte, N. Z. Naturforsch. B 2007, 62, 460–466. (36) Gross, A.; Metzler-Nolte, N. J. Organomet. Chem. 2008, 694, 1185–1188. (37) Kirin, S. I.; Ott, I.; Gust, R.; Mier, W.; Weyherm€ uller, T.; Metzler-Nolte, N. Angew. Chem., Int. Ed. 2008, 47, 955–959. (38) K€ oster, S. D.; Dittrich, J.; Gasser, G.; H€ usken, N.; Casta~ neda, I. C. H.; Jios, J. L.; Vedova, C. O. D.; Metzler-Nolte, N. Organometallics 2008, 27, 6326–6332. (39) Lemke, J.; Metzler-Nolte, N. Eur. J. Inorg. Chem. 2008, in press. olfl, S.; (40) Noor, F.; Kinscherf, R.; Bonaterra, G.; Walczak, S.; W€ Metzler-Nolte, N. ChemBioChem 2008, 10, 493–502. (41) Zagermann, J.; Kuchta, M. C.; Merz, K.; Metzler-Nolte, N. J. Organomet. Chem. 2008, 694, 862–867. (42) Davidson, J. L.; Green, M.; Sharp, D. W. A.; Stone, F. G. A.; Welch, A. J. J. Chem. Soc., Chem. Commun. 1974, 706–708.

Figure 1. Seven-coordinate tungsten building blocks 1 and 2.

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Figure 2. ORTEP drawing of [Tp*W(CO)(I)(HCC(CH2)2CO2H)] (1) (thermal ellipsoids at 30%, hydrogen atoms omitted for clarity). Only one molecule in the unit cell (Z = 2) is shown, the other being the enantiomer. Selected bond lengths (A˚): W(1)-C(16) 1.954(9); W(1)-C(17) 2.020(9); W(1)-C(18) 2.065(8); W(1)-N(6) 2.152(7); W(1)-N(4) 2.241(7); W(1)-N(1) 2.265(7); W(1)-I(1) 2.7917(12).

Figure 3. Tp*WI(CO)-alkyne pseudo-neurotensin derivative 3.

model peptides.12,17,28,30,31,34,35,38,39,41,43 Enkephalin is a neuropeptide that serves as a natural ligand to opioid receptors, acting inter alia as a natural analgesic. Neurotensin is a 13 amino acid peptide with central as well peripheral functions. Moreover, it acts as a growth factor for cells, and the neurotensin receptor is frequently overexpressed on tumor cells. The six amino acid Nteminal sequence of neurotensin (NT(8-13)) used in this work has been identified as the minimal binding motif for the neurotensin receptor. The N-terminally functionalized bionconjugates [Tp*WI(CO)(HCC(CH2)2CO-pNT-OH)] (3) (Figure 3) and [Tp*WI(CO)(HCC(CH2)2CO-Enk-OH)] (4) (Scheme 1) were obtained by coupling 1 in the last step of standard Fmoc-SPPS after activation with HOBt, TBTU, and triethylamine as outlined exemplarily for 4 in Scheme 1. The side-chainfunctionalized enkephalin derivative H-Tyr-Gly-[Tp*W(I)(CO)(η2-Pgl)]-Phe-Leu-OH (5) was obtained by replacement of one glycine with the tungsten-functionalized amino acid 2 and continuation of the peptide synthesis as depicted in Scheme 1. All functionalized peptides were cleaved from the resin by treatment with trifluoroacetic acid (20% v/v in DMF) and purified by RP-HPLC (chromatograms and accompanying ESI mass spectra are given in the Supporting (43) Dirscherl, G.; K€ onig, B. Eur. J. Org. Chem. 2008, 597–634.

Information). The pale blue solids 3-5 were unambiguously characterized by mass spectrometry, 1H NMR, and IR spectroscopy. For 3, electrospray ionization MS (ESI-MS, positive mode) shows two signals, one centered around m/z= 1477.30 corresponding to the molecular ion and a second around m/z=675.33 corresponding to the dication with loss of the iodide ligand, both matching the calculated isotope patterns. All 1H NMR signals could be assigned and indicate, in accordance with the isomeric distribution found for the starting material 1, two isomers in 92:8 ratio. A likewise distribution was found in the characterization of 4. ESI-MS (negative mode) of 4 displays two signals, one for the molecular ion around m/z=1270.27 and an additional signal for the doubly charged molecular ion around m/z=634.37. 1 H NMR spectra and assignment of the signals for compound 5 indicate two isomers in 90:10 ratio. ESI-MS (positive mode) of 5 gives one signal for the molecular ion around m/z=1230.26 ([M þ H]þ). A second signal around m/ z = 594.41 missing the characteristic W isotope pattern indicates loss of the tungsten-containing [Tp*W(CO(I)] moiety. Solid state IR spectra of the bioconjugates show one single sharp band for the carbonyl ligand located at 1904 cm-1 for 3 and 4 and 1914 cm-1 for 5, respectively, indicating little change to the coordination sphere of tungsten by the bioconjugate coupling.

Discussion This work introduces the Tp*W(I)(CO) moiety as a new group for the labeling of biomolecules. This fragment reacts readily with terminal alkynes to yield very stable W-η2alkyne complexes. Owing to the excellent stability of the Tp*W(I)(CO)-alkyne fragment, the use of either alkynefunctionalized acids or amino acids permits the introduction of this metal fragment at any stage during solid phase synthesis. N-Terminal labeling has been achieved by several

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Scheme 1. Synthesis of the Bioconjugates 4 and 5 by SPPS

different metal carboxylates before,6,7,17,28-33,36,37,39-41,44,45 but sometimes still poses a problem when functionalized amino acids require special and/or rather harsh conditions for side-chain deprotection. In comparison, internal peptide modification with organometallics is very limited.12,38,46,47 Internal peptide modification has been made possible via the amino acid propargyl-glycine in this work. It is potentially more versatile with respect to biochemical studies, but only possible when the metal complex withstands the conditions of all subsequent steps in solid phase peptide synthesis. On the other hand, metal modification at internal positions is particularly important if a free N-terminal amino group is required for receptor binding. This is indeed the case for enkephalin. Moreover, previous structure-activity studies on this peptide have shown that there should be sufficient space to accommodate a bulky substituent on the third amino acid (Gly3 position).48-51 Use of complexes such as the W(CO)-alkyne in this work mark the road ahead for the use of heavy atom peptide derivatives with additional possibilities for IR detection in biochemical and receptor binding studies. Interestingly, propargyl-glycine has been identified in naturally occurring proteins of some bacteria. It may thus be possible to design a specific tag for this unusual amino acid by employing metal fragments such as the one described in this work. (44) Neundorf, I.; Hoyer, J.; Splith, K.; Rennert, R.; Peindy N’Dongo, H. W.; Schatzschneider, U. Chem. Commun. 2008, 5604– 5606. (45) Peindy N’Dongo, H. W.; Ott, I.; Gust, R.; Schatzschneider, U. J. Organomet. Chem. 2009, 694, 823–827. (46) Gasser, G.; H€ usken, N.; K€ oster, S. D.; Metzler-Nolte, N. Chem. Commun. 2008, 3675–3677. (47) Gasser, G.; Neukamm, M. A.; Ewers, A.; Brosch, O.; Weyherm€ uller, T.; Metzler-Nolte, N. Inorg. Chem. 2009, 48, 3157–3166. (48) Eberle, A.; Leukart, O.; Schiller, P.; Fauchere, J.-L.; Schwyzer, R. FEBS Lett. 1977, 82, 325–328. (49) Morley, J. S. Annu. Rev. Pharmacol. Toxicol. 1980, 20, 81–110. (50) So os, J.; Berzetei, I.; Bajusz, S.; R onai, A. Z. Life Sci. 1980, 27, 129–133. (51) Fournie-Zaluski, M.-C.; Gacel, G.; Maigret, B.; Premilat, S.; Roques, B. P. Mol. Pharmacol. 1981, 20, 484–491.

Experimental Part General Remarks. Unless noted otherwise, all preparations except the SPPS were carried out under an inert gas atmosphere of Ar or N2 using standard Schlenk techniques and a M-Braun glovebox. All reagents and anhydrous solvents were purchased from commercial sources and used as received. [Tp*WI(CO)3] was prepared following literature methods.26 Fmoc-Propargylglycine, HOBt, TBTU, and DIPEA were purchased from Iris Biotech (Germany). NMR spectra were recorded at ambient temperature on Bruker DPX 250 and DRX 600 spectrometers. The chemical shifts (δ) are reported in ppm relative to the residual proton chemical shifts of the deuterated solvent set relative to external TMS. Absolute values of the coupling constants (J) are given in Hertz. 13C{1H} assignments were obtained from standard attached proton test (APT) and heteronuclear single quantum coherence (HSQC) experiments. IR spectra were recorded on a Bruker Tensor 27 spectrometer equipped with a Pike MIRacle Micro ATR accessory as solid samples. Electrospray ionization mass spectra (ESI-MS) were recorded on a Bruker Esquire 6000 spectrometer. The analytical and preparative HPLC were carried out on a customized Varian Prostar instrument using RP Varian Dynamax columns (C18 microsorb 60 A˚, diameter 4.5 mm, length 250 mm). Eluents were water and acetonitrile both containing 0.1% v/v TFA using a linear gradient of 15-100% acetonitrile for 30 min at flow rates of 1 mL/min (analytical) and 3 mL/min (preparative). Elemental analyses were carried out at the RUBiospek Biospectroscopy Department, Ruhr-Universit€ at Bochum. Products from solid phase synthesis likely contain at least traces of excess solvents, TFA, and salts, as well as remaining water from lyophilization. Therefore, no meaningful elemental analyses can be obtained for peptidic compounds (3, 4, and 5 in this work). [Tp*WI(CO)(η2-HCC(CH2)2CO2H)] (1). 4-Pentynoic acid (88 mg, 0.89 mmol) and [Tp*WI(CO)3] (560 mg, 0.81 mmol) were dissolved in THF (15 mL), and the dark red solution was heated to reflux for 12 h open to a bubbler. Removal of the solvent yielded a green solid, which was dissolved in diethyl ether (10 mL). Addition of cold pentane (0 °C, 10 mL) gave a pale green solid, which was washed with pentane (2  5 mL) and dried under lowered pressure (315 mg, 0.43 mmol, 53%). An analytical sample was obtained by slow evaporation of a dichloromethane solution as the adduct 1*CH2Cl2. Anal. Calcd

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for C22H30BCl2IN6O3W: C, 32.26; H, 3.69; N, 10.26. Found: C, 32.22; H, 2.93 ; N, 10.24. 1H NMR (CDCl3): major isomer (92%) δ 12.23 (s, 1H), 6.10, 5.81, 5.67 (s, 1:1:1, Tp*CH), 4.12 (m, 1H), 3.85 (m, 1H), 3.12 (m, 1H), 2.98 (m, 1H), 2.80, 2.62, 2.53, 2.40, 2.32, 1.59 (s, 3:3:3:3:3:3, Tp*CH3); minor isomer (8%) δ 13.13 (s, 1H), 6.11, 5.78, 5.66 (s, 1:1:1, Tp*CH), 4.16 (m, 1H), 3.80 (m, 1H), 3.17 (m, 1H), 2.93 (m, 1H), 2.83, 2.62, 2.55, 2.41, 2.29, 1.66 (s, 3:3:3:3:3:3, Tp*CH3). 13C NMR (CDCl3): major isomer δ 233.9 (CO), 206.89 (HCCR), 204.39 (HCCR), 177.38 (CO2H), 155.17-143.38 (Tp*CCH3), 108.46-107.24 (Tp*CH), 34.43 (CH2CO2H), 32.87 (CH2CH2CO2H), 18.43-12.56 (Tp*CCH3) ppm. IR (solid): 1907, 1714 cm-1. X-ray Structure Determination of 1. A crystal of 1 (green needle), obtained by slow evaporation of the pentane washing solution, was placed on a glass capillary in perfluorinated oil and measured in a cold gas flow. The intensity data were measured with a Bruker axs area detector (Mo KR radiation 0.71073 A˚, ω scan) at -60 °C. C21H28BIN6O3W, M = 734.04, triclinic, a = 8.236(4) A˚, b=10.387(5) A˚, c=17.389(7) A˚, R=82.035(9)°, β= 84.714(10)°, γ=78.758(9)°, V=1441.7(11) A˚3, space group P1, Z = 2, 7975 reflections collected, 4952 unique (Rint = 0.0459), wR2(F2)=0.1495 (all data). Bruker-axs-SMART 1000 CCD was used. Structure solution was with direct methods52 and data were refined against F2 with all measured reflections.52,53 CCDC 722677 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam. ac.uk/data_request/cif. [Tp*W(I)(CO)(η2-Fmoc-Pgl)] (2). Tp*WI(CO)3 (340 mg, 0.49 mmol)) and Fmoc-propargylglycine (180 mg, 0.54 mmol) were dissolved in THF (15 mL). The red solution was heated to reflux for 12 h open to a bubbler. After cooling to room temperature, the green solution was filtered and the solvent removed under reduced pressure. The resulting green solid was washed with pentane (2  5 mL) and recrystallized from diethyl ether/hexane to give dark green crystals (321 mg, 0.33 mmol, 67%). Analytical samples were found to contain one mole of diethyl ether per mole of 2 even after prolonged drying and heating. Anal. Calcd for C40H49BIN7O6W: C, 45.96; H, 4.72; N, 9.38. Found: C, 46.15; H, 4.27; N, 9.61. 1H NMR (CDCl3): major isomer (90%) δ 12.34 (s, 1H), 7.74-7.31 (m, 8H, Fmoc), 6.08, 5.81, 5.66 (s, 1:1:1, Tp*CH), 5.14 (m, 1H, R-H), 4.55-4.18 (m, 5H, Fmoc CH and CH2, β-CH2), 2.80, 2.61, 2.53, 2.41, 2.32, 1.55 (s, 3:3:3:3:3:3, Tp*CH3). 13C NMR (CDCl3): δ 232.8 (CO), 208.1 (HCCR), 203.8 (HCCR) 175.7 (CO2H), 155.2 (NHCO2), 155.0-141.4 (Tp*CCH3 and Cq of Fmoc), 127.8-125.4 (CH), 108.8-107.6 (Tp*CH), 67.7 (Fmoc-CH2), 47.3 (Fmoc-CH), 38.8 (HCCCH2), 18.7-12.8 (Tp*CH3) ppm. IR (solid): 1902, 1716 cm-1. [Tp*WI(CO)(η2-HCC(CH2)2CO-NH-Lys-Lys-Pro-Tyr-IleLeu-OH)] (3). Resin-bound pseudo-neurotensin Lys(IvDde)-Lys(IvDde)-Pro-Tyr(2Cl-Trt)-Ile-Leu-(2Cl-Trt-resin) was obtained by standard SPPS starting from Fmoc-Leu loaded 2-Cl-Trt resin (116 mg, load 0.86 mmol/g).28 After Fmoc deprotection of the second lysine, the tungsten complex 1 was coupled to the peptide as detailed here: [Tp*WI(CO)(η2-HCC(CH2)2CO2H)] (1) (173 mg, 0.21 mmol), HOBt (54 mg, 0.40 mmol), TBTU (125 mg, 0.39 mmol), and DIPEA (101 μL, 0.60 mmol) were mixed in DMF (3 mL), and the solution was stirred for 5 min. The homogeneous green solution was then added to the resin-bound peptide, and the mixture was shaken for ca. 20 h. After filtering the reaction mixture, the resin-bound product was washed with DMF (5  2 mL) and dichloromethane (5  2 mL) and dried under reduced pressure. The IvDdE protecting groups of lysine were removed by shaking the resin with 3% v/v hydrazine in DMF (4 mL) for 15 min followed by washing with DMF (5  2 mL). The product was (52) Sheldrick, G. M. University of G€ ottingen, Germany, 1997. (53) Van der Sluis, P.; Spek, A. L. Acta Crystallogr. 1990, Sect. A46, 194-201.

Zagermann et al. cleaved from the resin by treatment with 20% v/v TFA in DMF (4 mL) for 2 h. The resulting green solution was filtered, and the resin washed with methanol (2  2 mL). The solutions were combined, and TFA and methanol were removed under lowered pressure. The product was obtained as a pale green-blue solid by addition of cold diethyl ether. It was isolated by filtration, dried under lowered pressure, and purified by HPLC (57 mg, 0.04 mmol, 56% based on the resin load). 1H NMR (CD3OD) [note: assignments are based, in part, on comparison with literature data of parent neurotensin54]: major isomer (92%) δ 12.36 (s, 1H), 6.98 (d, J=8.0, 2H, Tyr), 6.62 (d, J=8.0, 2H, Tyr), 6.08, 5.82, 5.69 (s, 1:1:1, Tp*CH), 4.44-4.01 (m, 8H, R-CH and CH2), 3.72 (m, 2H, CH2), 3.24 (m, 2H, β-CH2), 3.04 (m, 4H, Lys ε-CH2), 2.88, 2.85, 2.68, 2.52, 2.50 (s, 3:3:3:3:3, Tp*CH3), 1.90 (m, 4H, Lys β-CH2), 1.79-1.73 (m, 5H, Lys γ-CH2 and Ile β-CH), 1.68 (m, 4H, Lys NH2), 1.60 (s, 1H), 1.62-1.59 (m, 6H, Leu β-CH2, γ-CH and Tp*CH3), 1.16 (m, 2H, Ile γ-CH2), 0.92-0.78 (m, 12H, CH3 of Ile and Leu) ppm. ESI-MS (pos.): 1477.30 ([M þ H]þ), 739.25 ([M þ 2H]2þ), 675.33 ([M - I þ H]2þ). Exact mass for C59H90BIN14O10W = 1476.56. IR (solid): 1904, 1643 cm-1. [Tp*WI(CO)(η 2 -HCC(CH 2 )2 CO-NH-Tyr-Gly-Gly-PheLeu-OH)] (4). Resin-bound enkephalin Tyr(2-Cl-Trt)-Gly-GlyPhe-Leu(2Cl-Trt-resin) was obtained by standard SPPS starting from 2-Cl-Trt resin (82 mg, load 0.86 mmol/g) preloaded with leucine.28 After Fmoc deprotection of tyrosine, the metal complex was coupled to the peptide as detailed here: [Tp*WI(CO)(η2-HCC(CH2)2CO2H)] (1) (78 mg, 0.11 mmol), HOBt (44 mg, 0.28 mmol), TBTU (89 mg, 0.28 mmol), and DIPEA (72 μL, 0.43 mmol) were mixed in DMF (3 mL) and stirred for 5 min. The homogeneous green solution was then added to the resin-bound peptide, and the mixture was shaken for ca. 20 h. After filtering the reaction mixture, the resin-bound product was washed with DMF (5  2 mL) and dichloromethane (5  2 mL) and dried under reduced pressure overnight. The product was cleaved from the resin by treatment with 20% v/v TFA in DMF (4 mL) for 2 h. The resulting green solution was filtered, and the resin was washed with methanol (2  2 mL). The solutions were combined, and TFA and methanol were removed under lowered pressure. The product precipitated from the solution as a pale green-blue solid upon addition of cold diethyl ether. It was isolated by filtration, dried under lowered pressure, and purified by HPLC (44 mg, 0.034 mmol, 29% based on the resin load). 1H NMR (CD3OD) [note: assignments are based, in part, on comparison with literature data30,55]: major isomer (92%) δ 12.37 (s, 1H), 7.27-7.19 (m, 5H, C6H5 of Phe), 6.63 (d, J=8.0, 2H, Tyr), 6.01 (d, J=8.0, 2H, Tyr), 6.14, 5.87, 5.73 (s, 1:1:1, Tp*CH), 4.67 (dd, J=9.0, 5.5, 1H, R-CH), 4.50-4.31 (m, 3H, R-CH and CH2), 3.86-3.62 (m, 6H, CH2 and Gly R-CH2), 3.22 (m, 2H, β-CH2), 2.98 (m, 3H, β- CH2 and Tp*CH3), 2.85, 2.75, 2.69, 2.57 (s, 3:3:3:3, Tp*CH3), 1.67-1.59 (m, 3H, R-CH, β-CH2), 1.56 (s, 1H, Tp*CH3), 0.88 (d, J= 6.3, 3H, δ-CH3 of Leu), 0.84 (d, J=6.3, 3H, δ-CH3 of Leu) ppm. ESI-MS (neg.): 1270.27 ([M - H]-), 634.37 ([M - 2H]2-). Exact mass for C49H63BIN11O9W=1271.35. IR (solid): 1904, 1634 cm -1. H2N-Tyr-Gly-[Tp*W(I)(CO)(η2-Pgl)]-Phe-Leu-OH (5). Resin-bound enkephalin functionalized with a tungsten-propargylglycine moiety was obtained by standard SPPS starting from Fmoc-Leu loaded 2-ClTrt resin (123 mg, load 0.86 mmol/g).28 For the third coupling, [Tp*W(I)(CO)(η2-Fmoc-Pgl)] (2) (154 mg, 0.16 mmol), HOBt (65 mg, 0.42 mmol), TBTU (133 mg, 0.41 mmol), and DIPEA (107 μL, 0.64 mmol) were dissolved in DMF (3 mL). After stirring the solution for 5 min, it was added to the resin and the mixture was shaken for 20 h. After filtration and washing with DMF (5  2 mL), the Fmoc protection group was removed and the peptide sequence was (54) Coutant, J.; Curmi, P. A.; Toma, F.; Monti, J. Biochemistry 2007, 46, 5656–5663. (55) Picone, D.; D’Ursi, A.; Motta, A.; Tancredi, T.; Temussi, P. A. Eur. J. Biochem. 1990, 192, 433–439.

Article completed by coupling of glycine and tyrosine. The product was cleaved from the resin by shaking it with 20% v/v TFA in DMF (4 mL) for 2 h. The resulting green solution was filtered, and the resin was washed with methanol (2  2 mL). The solutions were combined, and TFA and methanol were removed under lowered pressure. The product precipitated from the solution as a pale turquoise solid upon slow addition of cold (0 °C) diethyl ether. It was isolated by filtration, dried under lowered pressure, and purified by HPLC (25 mg, 0.02 mmol, 24% based on resin load). 1 H NMR (CD3OD) [note: assignments are based, in part, on comparison with literature data56]: major isomer 90%, δ 12.35 (s, 1H), 7.29-7.16 (m, 5H, C6H5 of Phe), 7.13 (d, J=8.0, 2H, Tyr), 6.78 (d, J=8.0, 2H, Tyr), 6.14, 5.88, 5.74 (s, 1:1:1, Tp*CH), 4.72 (dd, J = 9.0, 5.5, 1H, R-CH), 4.41 (dd, J = 9.0, 5.5, 1H, R-CH), 4.31 (dd, J=9.0, 5.5, 1H, R-CH), 3.89 (m, 2H, R-CH2 of Gly), 3.21 (m, 3H, β-CH2), 2.97 (m, 2H, β-CH2), 2.74, 2.58, 2.55, 2.41, 2.36 (s, 3:3:3:3:3 Tp*CH3), 1.61-1.55 (m, 6H, β-CH2 γ-CH of Leu, Tp*CH3), 0.88 (d, J=6.3, 3H, δ-CH3 of Leu), 0.83 (d, (56) Willisch, H.; Hiller, W.; Hemmasi, B.; Bayer, E. Tetrahedron 1991, 47, 3947–3958.

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J = 6.3, 3H, δ-CH3 of Leu) ppm. ESI-MS (pos.): m/z 1230.26 ([M þ H]þ), 594.41 ([M - (Tp*WICO) þ H]þ). Exact mass for C47H61BIN11O8W=1229.34. IR (solid): 1914, 1637 cm-1.

Acknowledgment. The authors are grateful for an insightful discussion with Dr. W. Seidel (then in M€ unster), which inspired this work. Helpful advice in Tp chemistry from Dr. Matthew Kuchta is also gratefully acknowledged. J.Z. is a fellow of the Ruhr-University Research School. This work was supported by the DFG through the Research Unit “Biological Function of Organometallic Compounds” (FOR 630, www.rub.de/for630). Supporting Information Available: HPLC and ESI-MS data for peptidic compounds 3, 4, and 5. This material is available free of charge via the Internet at http://pubs.acs.org. X-ray crystal data for compound 1 can be obtained from The Cambridge Crystallographic Data Centre, see Experimental Section for details.