In the Laboratory
Carbon–Carbon and Carbon–Nitrogen Spin–Spin Coupling in NMR Spectroscopy
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Simple Examples Based on Isotope-Labeled Glycines Leif Grehn, Ulf Ragnarsson, and Christopher J. Welch University of Uppsala, Biomedical Center, S-751 23 Uppsala, Sweden Whereas 13C NMR spectroscopy is nowadays a routine analytical technique whose elements are taught to most chemistry undergraduates, 15N NMR is not so well established. This is mainly owing to the nearly one-order-ofmagnitude lower sensitivity and to the natural abundance of the nuclei being 1.1% for 13C and and 0.37% for 15N. Therefore, without isotopic enrichment, acquisition of 15N spectra generally requires more concentrated sample solutions and long recording times. The increasing availability of high-field spectrometers, with their inherent enhancement in sensitivity due to greater population differences and improved detection techniques, also makes 15N NMR spectroscopy more accessible for routine measurements. A well-known consequence of the low natural abundance of the 13C nucleus is the absence of spin–spin coupling in such spectra, all signals appearing as singlets assuming broad band decoupling. This may sometimes have pedagogical and other advantages, but the price paid for this apparent simplicity is considerable. Carbon–carbon spin–spin coupling constants, like proton–proton ones, provide valuable information about the geometry of the immediate surroundings of the nuclei. It goes without saying that C–N spin– spin coupling at natural abundance is exploited even less. In this paper, we demonstrate the existence of both C–C and C–N spin–spin coupling in some recently prepared simple isotope-labeled Boc-glycines (Boc = tert-butoxycarbonyl) (1, 2). Although their synthesis is rather simple, the present cost of the labeled precursors, especially those containing 13C, places restrictions on this type of work outside an expert laboratory. However, with the cost for [ 15N]ammonium chloride being less than U.S. $5/mmol, we believe that the occasional use of this isotope for small-scale experiments by students is justified.
13C and 15 N NMR Spectra Illustrating Spin–Spin Coupling
As a first example on 13C–13C spin–spin coupling, Figure 1 shows the 13C spectrum of the double-labeled bromoacetate used as precursor (y/z = 13/13). The carbons show up as doublets at 26 and 167 ppm (1 JCC = 65 Hz). Bromine causes upfield shifts of 5 ppm (13CH2) and 4 ppm (13CO) in comparison with those of ethyl acetate. The 15N spectrum of Boc-[15N]glycine (x/y/z = 15/12/12) is shown in Figure 2, upper part. The appearance of two signals is due to the presence of two E/Z conformers, E |
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Figure 1. 100.4 MHz proton noise decoupled of Br13 CH213CO–OEt in CDCl 3.
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Synthetic Summary We recently prepared the complete set of 13 C/15 N labeled Boc-glycines according to the following scheme: Boc2xNK + Br yCH2zCO–OEt → Boc 2xNyCH2zCO–OEt → Boc–xNHyCH2zCO–OEt → Boc–xNHyCH2zCOOH (x = 14/15; y and z = 12/13) Boc2 NH is easily prepared and is a useful alternative to phthalimide in the Gabriel reaction (3). The three Br-yCH2 z CO–OEt isotopomers needed are commercially available at prices not less than U.S. $250/g. The yield in the alkylation as well as in each subsequent partial deprotection step is 95% or better (2). Glycine isotopomers have been used as precursors in asymmetric synthesis of other backbone-labeled amino acids (4) needed in the synthesis of peptides (5). 82
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Supplementary materials for this article are available on JCE Online at http://jchemed.chem.wisc.edu/Journal/Issues/1997/Dec/ index.html .
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Figure 2. 40.4 MHz proton noise decoupled NMR spectra of Boc-15NHCH 2COOH (upper) and Boc-15NH 13CH2COOH (lower) in CDCl3 .
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In the Laboratory
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Figure 3. 100.4 MHz proton noise decoupled 13 C NMR spectra of Boc-15NH13CH2COOH (upper) and Boc-15NH13CH213COOH (lower) in CDCl3.
(major) at 75.5 ppm and Z (minor) at 79.5 ppm (6). On introduction of 13C next to nitrogen (x/y/z = 15/13/12) (Fig. 2, lower part), 13C–15N spin–spin coupling takes place, which results in the appearance of doublets for both the nitrogen signals (1 JNC(E) = 14 and 1 JNC(Z) = 12 Hz). The existence of conformers is evident also from the corresponding 13C spectrum (Fig. 3, upper part). It allows both the latter coupling constants to be measured as well. A further split of the CH2 E/Z signals at 42.2/43.2 ppm due to additional 13C–13C spin–spin coupling, giving rise to doublets of doublets, results on labeling of the carbonyl also. This is shown in Figure 3, lower part (1JCC(E) = 1JCC(Z) = 59 Hz, 1J 1 13 CN(E) = 14 Hz, JCN(Z) = 12 Hz). The E/Z CO signals also appear as doublets. On the other hand, the 15N spectrum of this compound does not exhibit any new features in comparison with those of Figure 2 (not shown), since no 2-bond 15N– 13C spin–spin coupling takes place between the carboxyl carbon and nitrogen in an amino acid. The presence of 15N–13C spin–spin coupling can be demonstrated already in the 13C NMR spectrum of Boc215NH, the potassium salt of which was used above (Fig. 4). In addition, it can be seen in Boc2 15NCH2 CO–OEt, the first intermediate in our previous synthetic sequence, and therefore we have elaborated a fast and simple small-scale synthetic experiment to provide enough material for a 13C-NMR spectrum only. This new experiment is based on alkylation of Boc215NH using phase-transfer catalysis instead of nucleophilic alkylation of the corresponding potassium salt. The spectrum of the crude product is shown in Figure 5. Both the carbamate and the methylene carbons couple with the 15N nucleus, giving different coupling constants (23 and 13 Hz, respectively). Phase-transfer catalysis is an efficient methodology for a wide range of organic conversions in cases when the substrate and the reagent are not mutually soluble in a suitable solvent to a sufficient extent. One way to overcome this obstacle is to use a two-phase system—one organic, the other usually aqueous or solid. To facilitate the reaction at the phase interface, certain catalysts are added. Thus, our novel procedure allows the direct alkylation of Boc215NH in excellent yield, using ethyl bromoacetate in toluene containing powdered KOH in the presence of small amounts of tetrabutylammonium hydrogen sulfate (7). 15N- and, especially, 13C-labeling significantly affect absorption of IR radiation. Details on the IR spectra of isotopomers of Boc-glycine are given in our original paper (2). In this context it should be mentioned that isotope labeling induces changes in the pKa, which can be determined by NMR techniques as was demonstrated for [15N]glycine (8). Experimental Procedure Boc215NH can be prepared in accordance with our previously described detailed procedure (9).
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Figure 4. 100.4 MHz proton noise decoupled 13C NMR spectra (selected parts) of Boc2NH (upper) and Boc215NH (lower) in CDCl 3.
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Small-Scale Preparation of Boc215 NCH2CO–OEt In a small tube equipped with a stopper, a mixture of Boc215NH (50 mg, 0.23 mmol), Bu 4N+HSO4 { (50 mg, 0.15 mmol) and finely ground KOH (50 mg, 0.91 mmol) was treated with a solution of ethyl bromoacetate (50 mg, 0.30 mmol) in toluene (0.5 mL) with thorough mixing. After vigorous stirring for 1 h, the resulting pinkish slurry was partitioned between diethyl ether (10 mL) and 1 M aq KHSO 4 (5 mL). The colorless ether extract was washed sequentially with KHSO4, 1 M aq NaHCO3, and brine (5 mL each) and dried (MgSO4 ). Removal of the solvent on a rotatory evaporator left a colorless oil, weighing 70 mg (ca. 100%) after drying in vacuo with an oil pump. Thin-layer chromatography on a silica plate (light petroleum/diethyl ether 2:1) gave
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Figure 5. 100.4 MHz proton noise-decoupled NMR spectrum of Boc2 15N-CH2CO-OEt (crude product) in CDCl 3.
one spot after brief heating and spraying with 0.1% ninhydrin in n-BuOH/HOAc 97:3. 1 H NMR indicated < 2% Boc215NH. The 13C NMR spectrum is shown in Figure 5. C AUTION: Ethyl bromoacetate is a strong lachrymator. Therefore this experiment should be conducted in a fume hood. All 13C and 15N proton noise decoupled NMR spectra were recorded in 0.05 M CDCl3 solutions (Fig. 1: 0.1 M), corresponding to 5–6 mg of labeled material, at 100.4 and 40.4 MHz, respectively, on a JEOL JMN EX 400 spectrometer at 25 °C. The acquisition of the spectra was carried out during 2–3 h to obtain high-quality illustrations. In most cases, however, a satisfactory spectrum showing the heteronuclear coupling pattern could be recorded in 15 min. Conclusions Various homonuclear and heteronuclear coupling constants provide valuable information for the elucidation of miscellaneous structural features, such as bond angles and charge-density parameters. The above spectra of glycine de-
rivatives, selectively labeled with the stable nuclei 13C and 15N, demonstrate that 13C–13 C and 13C–15N spin–spin coupling constants could be easily measured from conventional onedimensional NMR spectra under standard conditions. Although there are a number of modern techniques for further enhancement of sensitivity that also make it possible to obtain such information from natural-abundance 13C- and 15N NMR spectroscopy, the time and sensitivity gained justify isotopic labeling. For teaching purposes the original data (in the form of FID or as processed spectra) can be made available free of charge. Please contact the authors for details. This material is also available on JCE Online at http://jchemed. chem.wisc.edu/Journal/Issues/1997/Dec/index.html. Acknowledgments Generous economic support from the Swedish Natural Science Research Council (NFR, including the NMR spectrometer) and the Swedish National Board for Technical Development (NUTEK) is gratefully acknowledged. Literature Cited 1. Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991; p 327. 2. Grehn, L.; Pehk, T.; Ragnarsson, U. Acta Chem. Scand. 1993, 47, 1107. 3. Ragnarsson, U.; Grehn, L. Acc. Chem. Res. 1991, 24, 285. 4. Lankiewicz, L.; Nyasse, B.; Fransson, B.; Grehn, L.; Ragnarsson, U. J. Chem. Soc., Perkin Trans. 1 1994, 2503. 5. Nyasse, B.; Grehn, L.; Ragnarsson, U. J. Chem. Soc., Chem. Commun. 1994, 2005. 6. Branik, M.; Kessler, H. Tetrahedron 1974, 17, 781. 7. McIntosh, J. M. J. Chem. Educ. 1978, 55, 235. 8. Rabenstein, D. L.; Mariappan, S. V. S. J. Org. Chem. 1993, 58, 4487. 9. Degerbeck, F.; Grehn, L.; Ragnarsson, U. Acta Chem. Scand. 1993, 47, 896.
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