In the Laboratory
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Linkage Isomerization by Two-Dimensional 31P Nuclear Magnetic Resonance Spectroscopy An Undergraduate Inorganic Laboratory Experiment Rathindra N. Bose* Department of Chemistry, Kent State University, Kent, OH 44240; *
[email protected] Ahmad M. Al-Ajlouni Department of Chemistry, Jordan University of Science & Technology, Irbid, Jordan Erika Volckova Department of Chemistry, Kent State University, Kent, OH 44240
Nuclear magnetic resonance spectroscopy is perhaps the most widely used technique for structural characterization and dynamics studies of compounds ranging from small to macromolecules. During the last 20 years, the development of highfield instruments and their applications in solving chemical and biological problems has grown exponentially. These applications include elucidation of three-dimensional structures of molecules (especially proteins), reaction dynamics, and NMR imaging (1–3). Incorporation of NMR spectroscopy into the undergraduate curriculum has recently been emphasized by the ACS Committee on Professional Training for securing the certification by the American Chemical Society (4 ). In fact, the requirement of an NMR spectrometer has been spelled out in the guidelines under the instrumentation requirements for an ACS approved B.S. degree in chemistry (4 ). Almost all major universities and colleges with ACS-approved programs are equipped with NMR instruments that are used for undergraduate education. Since almost all upper-class undergraduate chemistry textbooks from analytical to physical chemistry started covering the application of 2-D NMR, an introduction of some two-dimensional NMR experiments in the undergraduate laboratory curriculum seems timely. A modern NMR instrument is equipped with at least two frequency channels and adequate software to perform simple two-dimensional experiments. Here we report a simple 31 P COSY experiment to characterize two linkage isomers of the [Co(NH3)4(H2P3O10)] complex. These isomers are formed via two alternative chelation modes of the triphosphate moiety: the metal center is coordinated either through two adjacent phosphate groups (β,γ-chelate) or through two terminal phosphate groups (α,γ-chelate). This experiment is ideal for an integrated senior undergraduate laboratory course, which could be part of the core inorganic curriculum for an ACS-certified B.S. degree. 31 P NMR spectroscopy has been widely used to characterize phosphato complexes of diamagnetic metal ions and is an excellent tool for determining the binding modes of phosphate ligands including nucleoside di- and triphosphates (5–13). In the experiment described here, students prepare linkage isomers of tetraammine(triphosphato)cobalt(III) complexes, make unequivocal assignments of 31P resonances for the isomers by using 2-D NMR, and determine their abundance in solution. Furthermore, during the formation or isomerization of these complexes, metal-promoted phosphate hydrolysis takes place,
generating orthophosphate and pyrophosphate ions. The triphosphate hydrolysis can be used as a model for the roles of metal ions in ATP and other phosphate hydrolyses by a variety of enzymes (7, 11, 12). Overview of the Experiment The experiment has two parts. In the first part, students prepare linkage isomers, [Co(NH3)4(β,γ-H2P3O10] (1) and [Co(NH3)4(α,γ-H2P3O10] (2) in situ (see structures). In the second part, 1-D and 2-D (COSY) 31P NMR spectra are recorded and assignments are made for the isomers and hydrolyzed products. + NH3 Co NH3
P3O105 −
O
NH3
O
C O
H2O/H +
NH3
O
OH P
NH3 NH3
NH3 OH O O P Co O O P OH NH3 O
1
ONH3
+
NH3 Co NH3
NH3
O OH O P O
OP
O
P
O
O
O
OH
2
Experimental Procedure
Reagents The preparation of tetraamine(carbonato)cobalt(III) nitrate, [Co(NH3)4CO3]NO3, was according to published methods (14, 15). It is recommended that students prepare this complex in a preliminary experiment. Preparation of α,γ and β,γ Linkage Isomers (6) The aqua complex tetraaminediaquacobalt(III) was generated by dissolving [Co(NH3)4CO3]NO3 (50 mg, 0.20 mmol) in 10 mL of deionized water, adding 1.0 M HCl in 10% molar
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excess over the cobalt complex. Ten milliliters of a solution of sodium triphosphate, Na5P3O10⭈5H2O (74 mg, 0.20 mmol), was prepared in another 25-mL flask and adjusted to pH 5 by 1.0 M HCl. The triphosphate solution was mixed with the aquacobalt(III) complex. The solution was brought to pH 3 with 1.0 M HCl and heated on a water bath at 80 °C for 3–10 min. The resulting violet-pink solution was kept on an ice bath until the NMR measurements. The pH of the solution was readjusted to 7.5 to 8.0 by adding 1.0 M NaOH solution.
NMR Measurements The 31P NMR measurements were carried out on either 300-MHz (GN 300) or 500-MHz (Varian unity plus) spectrometers. All measurements were performed at ambient temperature (T ≈ 22 °C). The chemical shifts are reported with respect to 85% phosphoric acid. In a typical experiment, 0.30 mL of D2O and 0.20 mL of 0.20 M Na2EDTA were added to a 2.5-mL solution of the reaction mixture of triphosphato complexes prepared above. The addition of this sequestering agent helps to narrow the line-width presumably by binding to paramagnetic impurities in the reaction mixture. The pH was adjusted to 7.5–8.0 with 2 M NaOH. A 1.5-mL aliquot of this mixture was transferred to a 5-mm NMR tube and subjected to NMR measurements One-dimensional 31P NMR spectra were recorded with a 90° pulse. Typical parameters were 5000 Hz sweep-width, 11 µs pulse width, 2 s pulse delay, and 16K data points. Usually 8 transients were sufficient to observe a well-resolved spectrum with signal-to-noise ratio >50. A line-broadening
factor of 2 Hz was introduced during the Fourier transformation. The spin–lattice relaxation time, T1 was measured by the conventional inversion recovery method. The measured T1 values for all phosphorus atoms in the complex and free triphosphate lie in the range 0.8 to 1.2 s. An absolute mode COSY experiment (listed as COSY in the Varian Manual [16 ], Fig. 1) was performed with both 90° p1 and p2 pulses of 11 µ s. Usually, identical data points were selected in both frequency domains with a sweep-width of 3000 Hz. A sinebell multiplication function was used during the 2-D data processing. When both frequency domains contained identical data points, the spectra were symmetrized to remove artificial cross peaks that might exist. The double quantum filtered COSY (DQF-COSY) experiment was performed using the same parameters as in the COSY experiment except that p2 was selected to be 8 µs and a third 90° pulse (p3 = p1) was applied. The spectra were not symmetrized. Hazards Cobalt (II) nitrate hexahydrate is a strong oxidizer; therefore it should be handled with care to avoid contact with the skin and eyes. It can also cause allergic respiratory and skin reactions. Use of gloves and safety goggles is recommended. In proximity to the NMR instrument strong magnetic and radiofrequency fields are present that could cause serious injury or death to persons with implanted or attached medical devices such as pacemakers and prosthetic parts.
Figure 1. 200-MHz 31P COSY spectra of the reaction mixture repor ted in Figure 2b. The COSY connectivities for the free ligand and β,γ- and α,γtriphosphato chelates are indicated by i, ii, and iii.
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Results and Discussion Usually, linkage isomerism is introduced in coordination chemistry to explain the mode of coordination to transition metal ions by ligands with alternative binding sites. One of the most common examples is the N- and S-bonded thiocyanato complexes of ruthenium(III), Ru(NH3)5(SCN)2+ (17). In general, one major linkage isomer is formed in greater abundance over the others. In some cases, the minor isomers are either not observed or difficult to isolate. Instability and interconversion of these isomers often lead to unsuccessful separations. The experiment described herein should aid students in grasping the concept of alternative sites of coordination. The reaction of [Co(NH3)4CO3]NO3 with triphosphate moiety in equimolar concentrations affords both linkage isomers of tetraammine(triphosphato)cobalt(III).W In the β,γ isomer, the triphosphate ligand exhibits a 6-membered chelate ring, whereas an 8-membered chelate ring is formed in the α,γ isomer. The 31P NMR spectrum of the free ligand exhibits a doublet at ᎑5.4 ppm ( 2J = 20.4 Hz) and a triplet at ᎑19.9 ppm with the same coupling constant. The 31P NMR spectrum of a reaction mixture containing equimolar concentrations of the carbonato–cobalt(III) complex and tripolyphosphate
Figure 2. 200 MHz 31P NMR spectra of (a) Na5P3O10 at pH 8.0; (b) a reaction mixture containing equimolar concentration (0.01 M) of Na5 P3O10 and [Co(NH3)4CO 3]NO3 recorded at pH 8 after heating for 3 min at 80 °C at pH 3.0; (c) the same reaction mixture in b after heating for 10 min.
is shown in Figure 2b. The assignments for product signals can be made on the basis of chemical shift data, coupling patterns, signal intensities, and J values. These assignment strategies have been employed for a variety of cobalt(III), Pt(II), and Rh(III)–triphosphato and –ATP complexes (5–13). For example, the coordinated phosphate groups in the β,γ isomer show appreciable coordination chemical shifts (3 to 10 ppm), whereas the uncoordinated αphosphate group exhibits very little change in δ compared to the free ligand. Furthermore, the β-phosphate group shows a doublets of doublet feature due to coupling with two nonequivalent phosphorus atoms. However, owing to a small difference in coupling constants resulting from β–γ and β–α spin couplings, the two central peaks may merge and these doublets of doublet may appear as a triplet, especially when the line-width is comparable to ∆ J, the difference in coupling constants. Likewise, for the α,γ isomer, significant changes in chemical shift for the bound α- and γ-phosphate groups are expected with very little change in the chemical shift of the unbound βphosphate group. In our case, at pH 7.5, the β,γ isomer exhibits a doublet at 4.4 and a doublets of doublet at ᎑ 9.1 ppm for the coordinated γ- and β-phosphate groups. The signal for the uncoordinated α-phosphate group is masked under the doublet of the free ligand, which can be revealed from the COSY spectra discussed below. The α,γ isomer exhibits two sets of signals, a doublet at 0.8 ppm for the two magnetically equivalent α- and γ-phosphate groups and a triplet at ᎑21.7 ppm for the uncomplexed β-phosphate group. Note that the chemical shifts depend upon pH values due to the successive loss of protons from the bound triphosphate ion in the complex. Assignments of 1-D NMR signals based on chemical shifts and coupling arguments may not be unambiguous, especially when a number of products are present in a reaction mixture. Furthermore, since some signals for complexes may be masked under the resonances of the free ligand, unequivocal assignment would be difficult. Two-dimensional COSY spectra, by virtue of direct correlation among the coupled nuclei, help us to make an unambiguous assignment. Figure 1 shows COSY spectra of the products formed in the reaction between the cobalt(III) carbonato complex and tripolyphosphate as well as the free ligand. As can be seen, a direct correlation exists between β (᎑19.9 ppm) and two equivalent α,γ phosphate groups (᎑5.4 ppm) of the free ligand. Two additional sets of correlations are also observed. In one set, the triplet at ᎑9.1 ppm is connected to two doublets at 4.4 and ᎑5.4 ppm. The doublet at ᎑5.4 ppm must be a composite signal of α phosphate of free triphosphate and the β,γ isomer, since these signals show correlation with those from both the free ligand and the complex. A second set can be recognized from the connectivity between the doublet at 0.8 ppm and the triplet at ᎑21.7 ppm. By using the COSY connectivity, chemical shift, and coupling data , it can be concluded that the first set of signals belongs to the major product, β,γ isomer, whereas the second set is for the minor α,γ isomer. The product yields and ratio depend on pH, temperature, and heating time during the reaction. For example, after 3 min of heating at 80 °C, the yield of the major product was ca. 50%, and a small amount, ca. 7% of the minor product was formed. After the solution is heated for 10 min, ca. 20% of the triphosphate remained uncomplexed, as was evident from the appearance of the NMR signals of the free ligand.
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Although more β,γ isomer was produced upon prolonged heating, most of the α,γ isomer suffered decomposition leading to hydrolyzed products as discussed below. Note that for the quantitative analysis based on signal integrations, the delay time between pulses must be ≥5T1 to ensure reestablishment of equilibrium magnetization. Three singlets at 3.2, 4.9, and ᎑6.0 ppm were detected, which exhibit no correlations with other signals in the spectra. Lack of correlation with the other resonances indicates that these signals are not part of the triphosphato complexes. In fact, these singlets can be eliminated or substantially reduced by performing a double quantum filtered COSY (DQFCOSY) experiment, which, in principle, should only reveal signals for the coupled nuclei. Figure 3 shows DQF-COSY spectra indicating substantially reduced singlets compared to Figure 1. The singlet at ᎑6.0 ppm is due to pyrophosphate impurity present in the starting triphosphate sample, which can be verified independently by recording the spectrum of the triphosphate moiety. The singlet at 4.9 ppm is for a pyrophosphatocobalt(III) complex, [Co(NH3)4(HnP2O7)]3–(4–n) (n = 0 to 4), 3 (6 ). The strong signal at 3.2 ppm was assigned to an orthophosphato complex, [Co(NH3)4(HnPO4)]3–(4–n) (n = 0 to 3), 4 (6 ). The chelate mode of the pyrophosphate ligand is apparent from its singlet appearance. These products are formed through the hydrolysis of the coordinated triophosphate moiety. Certainly, COSY spectra alone cannot be used to make specific assignment for the orthophosphate and pyrophosphate signals. Chemical shift data from independent experiments or analysis of coordination chemical shift would
help to make correct assignments. O OH O P Co O O P OH NH3 O NH3
NH3 NH3 NH3 NH3 Co NH3
NH3
O OH O P O
OP
O O
P
O
PO43-
3 H2O
O
OH NH3 NH3 Co NH3
NH3
O
O P
O
+
P2O74-
OH
4
This experiment demonstrates the unique application of 1-D and 2-D 31P NMR spectroscopy to study the linkage isomerism of cobalt(III)-triphosphato complexes. Students have the opportunity to learn 1-D and 2-D NMR spectroscopy in general and 31P NMR in particular, and how to use an NMR instrument. Aspects of 2-D NMR including the meaning of preparation, evolution and mixing times, spin-lattice relaxation times, and “nuts and bolts” of setting and processing 2-D spectra can be addressed through this experiment. The relative sensitivity of 31P and 1H NMR can be addressed. Mechanistic implications of metal-assisted triphosphate hydrolysis to ATP and other biological phosphates can be discussed.
Figure 3. 200-MHz 31P DQF-COSY spectra of the reaction mixture reported in Figure 2b. Note that the three singlets in Figure 1 are substantially reduced in their intensities.
86
+
Journal of Chemical Education • Vol. 78 No. 1 January 2001 • JChemEd.chem.wisc.edu
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This experiment has been successfully implemented in the Senior Inorganic Laboratory course (Chem 40364) at Kent State University and is well received by students. Acknowledgments We thank Mahinda Gangoda for technical assistance with the NMR experiments and reviewers of this manuscript for their incisive comments. W
Supplemental Material
Detailed laboratory documentation is available in this issue of JCE Online. Literature Cited 1. Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions; Clarendon: Oxford, 1987. 2. Derome, A. Modern NMR Techniques for Chemistry Research; Pergamon: Oxford, 1987. 3. Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York, 1986. 4. Undergraduate Professional Education in Chemistry: Guidelines and Evaluation Procedures; American Chemical Society: Washington, DC, 1999.
5. Merritt, E. A.; Sundralingam, M.; Cornelius, R. D. J. Am. Chem. Soc. 1980, 102, 6151. 6. Cornelius, R. D. Inorg. Chem. 1980, 19, 1286. Reibenspies, J.; Cornelius, R. D. Inorg. Chem. 1984, 23, 1563. 7. Cornelius, R. D.; Hart, P. A.; Cleland, W. W. Inorg. Chem. 1977, 16, 2799. 8. Bose, R. N.; Viola, R. E.; Cornelius, R. D. J. Am Chem. Soc. 1984, 106, 3336. 9. Bose, R. N.; Cornelius, R. D.; Viola, R. E. J. Am. Chem. Soc. 1986, 108, 4403. 10. Bose, R. N.; Slavin, L. L.; Cameron, J. W.; Luellen, D.; Viola, R. E. Inorg. Chem. 1993, 32, 1795. 11. Lue, Z.; Shorter, A. L.; Lin, I.; Dunaway-Mariano, D. In Mechanisms of Enzymatic Reactions: Stereochemistry; Frey, P. A., Ed; Elsevier: New York, 1985; pp 141, 149. 12. Hendry, P.; Sargeson, A. M. In Progress in Inorganic Chemistry: Bioinorganic Chemistry; Lippard, S. J., Ed.; Wiley: New York, 1990; p 201. 13. Tafesse, F.; Massoud, S. S.; Milburn, R. M. Inorg. Chem. 1985, 24, 2591. 14. Angelici, R. J. Synthesis and Technique in Inorganic Chemistry; Saunders: Philadelphia, 1977; p 13. 15. Schlessinger, G. Inorg. Synth. 1960, 6, 173. 16. Guide to the NMR Experiments; Pub. No. 87-190140-03; Varian Associates, USA, 1996. 17. Lee, R. A.; Earley, J. E. Inorg. Chem. 1981, 20, 1739.
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