Anal. Chem. 1997, 69, 4731-4733
Correspondence
Mixture Analysis by NMR Spectroscopy Mengfen Lin and Michael J. Shapiro*
Department of Central Technology, Preclinical Research, Novartis Pharmaceuticals, 59 Route 10 East Hanover, New Jersey 07936
A strategy for NMR analysis of mixtures is described. The combination of HMBC with TOCSY allows for the complete identification of a complex mixture without resorting to physical separation of the components. The ability to perform mixture analysis by NMR spectroscopy without prior separation of the components has not been fully exploited. This is due to the paradigm that NMR spectra become too complex to analyze when there are many compounds present. However, if this paradigm were true, then the analysis of large peptides and small proteins would be an insurmountable problem. We know that this is not the case. An NMR methodology that could address the problem of mixtures without resorting to physical separation of components would be of great value in the analysis of small split-mix combinatorial mixtures. We have developed a strategy for performing mixture analysis from compounds generated from combinatorial split and mix synthesis.1 The split and mix synthesis by its nature generates mixtures of compounds. Typically these compounds contain a variety of substituents built around a common template. Let us consider the analysis of a 20 amino acid peptide analogy. This molecule would be the equivalent of a mixture of about seven compounds based on an average compound molecular weight of 200-300. For peptides, since almost all of the side chains form one contiguous spin system, the NMR assignments and hence the amino acid assignments are performed by a TOCSY experiment. The TOCSY experiment is powerful in this respect since it can identify networks of coupled protons. Recently it has also been shown that using TOCSY data each component in a mixture of inositols could be identified.2 In the sugar case, all the protons are connected by direct or long-range couplings. However, in the general case, this total spin connectivity cannot be accomplished due to nonreporting centers. This is illustrated by the examples shown below, where x and y can be chain extensions, heteroatoms, carbonyl group, etc.
X
X H3C
Y
CH3
H3C
Y
For most organic compounds, the spin systems are broken into several fragments, which must be connected to regain (1) (a) Geysen, H. M; Rodda, S. J.; Mason, T. J. Mol. Immunol. 1986, 23, 709-715. (b) Sebestven, F.; Dibo, G.; Kovacs, A.; Furka, A. Biomed. Chem. Lett. 1993, 3, 413-418. (c) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (2) Johnson, K: Barrientos, L. G.; Le, L.; Murthy, P. P. N. Anal. Chem. 1995, 231, 421. S0003-2700(97)00594-5 CCC: $14.00
© 1997 American Chemical Society
Figure 1. 500 MHz proton NMR spectrum for mixture of esters 1-6.
molecular identity. In such cases, additional NMR experiments will be required to complete the molecular linkage. As is the case with NMR assignments for peptides, we generally are not dealing with a de novo structure determination, but with structure verification, which makes the problem somewhat easier. We decided to see whether we could assign the structures of the various components from a mixture of six very similar esters 1-6. These molecules would serve to illustrate the potential O
O O 4
O 1 O
O O 2
O 5
O O O 3
O
O O 6
problems that might occur from mixtures generated by a small scale split-mix synthesis. The similarity of these structures can give rise to very complex NMR spectra, as shown in Figure 1. The strategy used for structure verification is similar to that employed to perform structure assignment in peptides. Initially, we obtained a TOCSY spectrum, which is used to establish the individual spin system connectivities in an analogous manner to the peptide example. For a peptide, the molecular linkage would next require a NOESY experiment where the amide protons of one amino acid have NOEs with R protons of the next amino acid. Analytical Chemistry, Vol. 69, No. 22, November 15, 1997 4731
The TOCSY spectrum for the mixture of six esters is shown in Figure 2. From the TOCSY spectra and consideration of chemical shift information, we can derive the following spin fragments: three acetyl groups, three propyl groups, one ethylene, and one methylene moiety next to carbon. There are also isobutyl, ethyl, propyl, isopropyl, and two butyl groups next to oxygen. In combination with the observed TOCSY correlations, the HMBC data shown in Figure 3 allow the connection of separate molecular groupings to identify molecules. We can step through a sequential connectivity as shown in the scheme below, to identify the structure by using the attachment to a common functional group as follows.
Figure 2. 2D TOCSY NMR spectrum, region from δ ) 0.5 to 2.7, for the mixture of esters 1-6.
Figure 3. 2D HMBC NMR spectrum for mixture of esters 1-6.
However, in the case of organic molecules, this relationship is generally missing and often NOEs are too small to be observed readily. Further connectivities can be made by HMBC.3 The HMBC experiment is a particularly useful experiment in that it connects protons to carbons via two or three bond couplings. This allows the connection of protonated carbons to nonprotonated centers, thereby allowing the spin systems derived from the TOCSY experiment to be linked as shown in the diagram.
This union is similar to an alternate method used in peptides to make main chain assignments and connectivities.4 4732
Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
The methylene protons on the butyl group at δ ) 4.06 are connected to the ester moiety at δ ) 172.9 via a three-bond proton-carbon coupling as indicated in Figure 4B. This methylene group has TOCSY correlations to resonances at δ ) 1.46, 1.26, and 0.83 designating the spin system as a butyl function, as shown in Figure 4A. The ester carbonyl group is also connected to an ethylene spin system at δ ) 2.46 and 2.25. This methylene moiety has an HMBC correlation with a carbonyl resonance at δ ) 204.9 that is attached to an acetyl group at δ ) 1.70. These connectivities then uniquely identify molecule 3. The same process was used to identify all of the other molecules in the mixture, for example, as indicated in Figure 4B for ester 5. A possible drawback to this approach may occur when the carbon-13 chemical shifts for the common connecting groups are insufficiently resolved to allow assignment. In this case, diffusion encoded spectroscopy methods can be used to aid the structure assignments.5 However, this method can be more time consuming than the TOCSY/HMBC combination and is more difficult to obtain on a routine basis. The strategy for NMR using the combination of HMBC with TOCSY allows for the complete identification of a complex mixture without resorting to physical separation of the components. EXPERIMENTAL SECTION All compounds were obtained from Aldrich Chemicals and used without further purification. The mixture was made as an approximately eqimolar ratio of components except for 5, which was present at ∼0.2 equiv of the other compounds. All NMR data were collected on a Bruker DMX-500 spectrometer system. The TOCSY data were collected with a sweep width of 8992.8 Hz. The spin-locking field of 10 KHz was generated with a 25 µs pulse at a power of 19.2 dB. A matrix of 4K × 512 was obtained with a mixing time of 70 ms using a m/ev17 sequence and the data were (3) Bax, A.; Summers, M. J. Am. Chem. Soc. 1986, 108, 2093. (4) (a) Bermel, W.; Griesenger, C.; Kessler, H.; Wagner, K. Magn. Reson. Chem. 1987, 25, 325. (b) Bradley, E. K. J. Magn. Reson., Ser. B 1996, 110, 195. (5) Lin, M.; Shapiro, M. J. J. Org. Chem. 1996, 61, 7617.
Figure 4. (A) Portion of the 2D TOCSY spectrum. The correlations from proton at δ ) 4.06 for ester 3 are indicated. (B) Portion of the HMBC NMR spectrum shown in Figure 3. Correlations for esters 3 and 5 are indicated by the lines. Assignment for all of the ester carbonyls is also shown.
zero-filled to 4K × 1024. Eight scans were obtained per increment. TPPI phase cycling was used to generate phase-sensitive data. The data were processed in both dimensions with a sine bell squared function shifted by 90°. The HMBC data were collected with a sweep width of 6009 Hz in F1 and 26 411 Hz in F2 dimensions. The data matrix size was 4K × 512 and was linear predicted to 1K and zero-filled to 2K words. Sixteen scans per increment were collected. The 1JCH filter was set to 3.57 ms, and the delay for the evolution of the long-range coupling was set to
50 ms. A 3-sine gradient pulse program was employed for the duration of 1 ms each and amplitudes of 50%, 30%, and 40%.
Received for review June 9, 1997. Accepted August 17, 1997.X AC970594X X
Abstract published in Advance ACS Abstracts, October 1, 1997.
Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
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