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Figure 10 Schematic representation of nontilted 2D J-resolved experiment. Projection onto the chemical shift axis recovers the high resolution 1D NMR spectrum.

molecular structure to chemical shift information criteria only. Proton-proton spin coupling values can be obtained by performing a 2D J-resolved experiment (40,41). It was found that by using a nontilted projection from the 2D J-resolved experiment high quality as indicated in Figure 10, 1D proton NMR spectra of resin-bound molecules can be obtained. A comparison of the 1H MAS NMR spectrum, obtained under spin echo and by the projection of the nontilted 2-D J-resolved data, for the two methyl groups of DMF-swollen isoleucine on Wang resin is shown in Fig. 11. The methyl resonances can be assigned by observation of the coupling patterns. This increase in resolution is further exemplified for Alloc-Asp derivatized oxazolidinone attached via its side chain carboxyl to SCAL-linked aminomethylpolystyrene as seen in Fig. 12 (42).

In the J-resolved projection, the aromatic rings of the SCAL are clearly present where as the polymer resonances have ‘‘dropped out’’ of the spectrum. In addition, the proton coupling constants arising from alloc group are readily measured as seen in the expanded spectrum (Fig. 13). If a more detailed measure of the couplings is desired, then the full 2D J-resolved spectrum can be evaluated in the normal manner as exemplified in Fig. 14. A similar method to obtain accurate proton-proton coupling constants based on E.COSY spectra has also appeared recently (43).

It would be advantageous if we could retain the enhanced resolution afforded by the projection of the 2D J-resolved data and also obtain spin system connectivities. MAS spin-echo correlated spectroscopy (SECSY) (44) allows both spin connectivities and enhanced resolution to be obtained. The

Figure 11 Projection of nontilted 400 MHz MAS J-resolved NMR spectrum for methyl region for Ile on DMF-d6-swollen Wang resin.

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Figure 12 400 MHz MAS 1 H NMR spectrum for Alloc-Asp-derivatized oxazolidinone on SCAL-linked aminomethylpolystyrene. (a) Spin-echo 1 H NMR spectrum. (b) Nontilted projection from 2D J-resolved spectrum.

projection of the MAS SECSY data for isoleucine on Wang-1 resin shown in Fig. 15 illustrates the apparent increase in resolution. The apparent coupling constants observed in the 1D spectrum cannot be used (45).

The recent development of a gradient high-resolution MAS probe will extend the utility of 2D experiments by removing artifacts that generally accompany MAS 2D NMR data on resin samples (46). The lack of artifacts is illustrated by the high quality SECSY spectrum shown in Fig. 16. SECSY data contain the same information as a COSY spectrum, but the appearance of the spectrum is different. The diagonal lies along the F1 0 and the off-

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Figure 13 Partial NMR spectrum of projection of nontilted J-resolved NMR spectrum for Alloc-Asp-derivatized oxazolidinone attached via its side chain carboxyl to SCAL-linked aminomethyl polystyrene swollen with DMF-d7.

diagonal peaks occur in pairs along lines that make an angle of 135° with the diagonal (47). The ability to obtain structural data from a single resin bead represents the ultimate level of sensitivity for MAS NMR (48). Reasonable 1H NMR data can now be realized in about 1 h. This result, in principle, affords identification of the molecule on the resin directly without resorting to tagging methodologies (49). The incorporation of site specific 13C labels aids the ability to obtain heteronuclear NMR data.

Robotic sample changing and automated data collection is an increasingly important aspect in the efficient utilization of MAS NMR for combinato-

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Figure 14 Partial 2D J-resolved NMR spectrum for Alloc-Asp-derivatized oxazolidinone attached via its side chain carboxyl to SCAL-linked aminomethylpolystyrene swollen with DMF-d7.

rial chemistry. Sample changers for both MAS rotors and for nanoprobe samples are currently available from the instrument manufacturer.

V.MULTIPIN CROWNS

Combinatorial chemistry has developed using resin beads as a primary format for the synthesis of nonpeptide targets. An alternate format is the crown/pin system where resin is grafted onto a base polymer (50). FTIR, which is very useful for analysis of bead resin chemistry, as a general tool for analysis of crown chemistry is somewhat limited (51). The IR spectrum is hampered by interfering peaks that arise from the base plastic, the grafted resin, as well as from the linker molecule.

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(a)

(b)

(c)

Figure 15 (a) Spin echo (b) projection of the SECSY data MAS 1 H NMR spectrum for DMF-d7-swollen Fmoc-isoleucine on Wang resin 1 and (c) solution NMR spectrum for Fmoc-isoleucine methyl ester.

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Figure 16 Gradient 2D SECSY NMR spectrum for Alloc-Asp-derivatized oxazolidinone on SCAL-linked aminomethylpolystyrene swollen with DMF-d7 obtained with a 4-mm high-resolution gradient MAS probe.

One dimensional MAS NMR is not affected to the same extent as is IR. Reasonable NMR data were obtained using a 7-mm high-resolution MAS probe utilizing the spin-echo pulse sequence, allowing the removal of the broader resonances due to the plastic as well as the signals from the slower moving components of the resin. Use of MAS NMR allows the same crown to be sequentially monitored and returned to the reaction conditions until the desired transformation is complete (Scheme 1). The ability to follow the transformation of alcohol to aldehyde and then elaboration to olefin on the same crown exemplifies this technique (Fig. 17). The stereochemistry of the olefin was found to be trans as determined from the measured coupling constant of 16 Hz. Confirmation of the olefinic peaks was obtained by COSY (52). MAS NMR, following cleavage, on the same crown, showed complete absence of product peaks.

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Scheme 1

Figure 17 (A) 400 MHz spin-echo MAS 1 H NMR spectrum of a crown with aldehyde product attached to the grafted polymer and (B) with olefin attached, swelled in DMF-d7.

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VI. SOLUTION TECHNIQUES

A. HPLC/NMR

While the majority of attention in combinatorial syntheses has been on solid phase analysis, the use of traditional solution phase organic chemistry to form compound collections should not be disregarded. MS techniques are widely used for evaluation of mixtures produced by combinatorial chemistry (4). However, a potential problem with the MS methodology involves a situation when isomolecular weight compounds are present. The compounds can be stereoisomers, positional isomers or by chance identical molecular weight materials. In these cases, the identity of the substance as determined by MS can be ambiguous.

The recent redevelopment of HPLC-NMR allows for complete identification of individual compounds in complex mixtures (53). While the majority of reports using HPLC-NMR have been in the drug metabolism area, the utility for organic compounds and for peptides has been recently demonstrated (54). It is presently possible to obtain routine high-quality NMR data using this technique with as little as 5 mg of compound in the chromatogram peak, with the detection limit using this technology presently being on the order of 100 ng (55).

The utility of HPLC-NMR for analysis of isomolecular weight compounds is illustrated by the analysis of a mixture of dimethoxybenzoylglycines prepared by split-mix synthesis having aromatic ring positional isomers. The results from the dimethoxybenzoylglycines was analyzed by stop-flow HPLCNMR where individual components are evaluated as each peak enters the NMR probe while the separation is paused. The assignment of structure from the separated components is straightforward by consideration of the shifts and the coupling patterns for the region of interest shown in the stacked plot NMR spectrum shown in Fig. 18.

On-flow HPLC-NMR analysis can also be performed when sufficient material is available. It involves collecting the NMR data continuously as the sample passes through the probe. This is the most efficient method for structure evaluation by HPLC-NMR. The NMR data are represented in a 2-D plot where the x direction contains chemical shift information and the y direction is representative of the LC retention time. The individual spectra can be extracted from the 1D slices along the x axis if so desired. The resolutions in the individual spectra are of somewhat lower quality than in the stop-flow method; however, the introduction of the second dimension allows for easy structure assignment even for overlapping peaks in the LC separation. As seen in Fig. 19, the on-flow HPLC-NMR characterization shows four distinct sets of resonances.

The HPLC/NMR for peptides is illustrated by the data obtained for pen-

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Figure 18 500 MHz 1 H HPLC/NMR stack plot of chemical shift vs. retention time for a mixture of isomolecular weight dimethoxybenzoylglycines.

tapeptide mixtures FNXEF-OH where X D, Q, I, K, or T. In each of these five component mixtures there are two compounds having identical molecular weights; therefore, it would be difficult to unambiguously assign structures by MS without resorting to special techniques. Stop-flow HPLC-NMR allowed all of the compounds to be unambiguously assigned utilizing 2D TOCSY data as exemplified in Fig. 20 for the compound having a retention time of 10.8 min. The TOCSY spectrum showed resonances at δ 1.3, 1.6, 2.9, and 4.1, indicative of a lysine residue. The additional resonances are consistent with the amino acids; phenylalanine δ 4.2 and 3.1, and 4.6, 3.18, and 2.98; asparagine δ 4.7, 2.7, 2.55; and glutamate δ 4.27, 2.34–2.05. This clearly identifies the peptide as FNKEF-OH. The isomolecular weight complication of lysine vs. glutamine was readily resolved. In another example, using standard HPLC conditions, assignment of a majority of compounds from a library of 27 tripeptides (AYM) was achieved (56). This task, which was accomplished in a single pass using on-flow HPLC-NMR, would have been difficult if not impossible by any other technique.