Swartz Analytical Techniques in Combinatorial Chemistry

Methods NMR

Figure 1 Typical peptide 13 C Gel phase NMR spectrum in DMSO-d6. Product shown is obtained after deprotection reaction by BF3 .

79

80

Shapiro

Figure 2 19 F gel phase NMR spectrum for macrocyclization SNAr reaction for an oxytocin analog on Wang resin. Bottom spectrum is starting material and top spectrum is after a reaction time of 1 h. The broad 19 F signal arises from the probe components and can be used as an internal standard.

NMR Methods

81

obtained during the course of the reaction. The broad peak in the spectrum arises from fluorine-containing components of the Bruker quadranuclear probe and was conveniently used as an external standard. Using TentaGel resin linked compounds, 19F gel phase NMR data could be obtained in less than 10 min for the reactions shown in Fig. 3 (20). Since a wide variety of fluorine containing compounds are readily available, 19F gel phase NMR is expected to increase in utility.

Figure 3 375 MHz 19 F gel phase NMR spectrum for resin linked butyl amide. Conversion to product is monitored (a) on TentaGel resin and (b) in solution.

82

Shapiro

Figure 4 Horner-Wadsworth-Emmons reaction that was monitored by 31 P gel phase NMR.

As with 19F NMR gel phase, 31P NMR is useful because the resins currently used do not interfere with the signals produced. Spectra can be obtained in just 10 min with 50 mg of resin. The first use of 31P gel phase data was described by Geralt et al. in conjunction with oligonucleotide synthesis (21). An especially attractive application of 31P gel phase NMR was applied to the Horner-Wadsworth-Emmons olefination reaction depicted in Fig. 4 (22). The use of 31P NMR proved to be a highly sensitive method to follow the reaction on solid phase and the NMR spectra were used to rapidly identify a competing side reaction that would have been difficult to show using other analytical methodologies. Van Etten used gel phase 31P NMR to follow protection and deprotection of a phosphorylated benzyl group in peptide synthesis (23). Spectral resolution was sufficient to distinguish dibenzyl, monobenzyl, and nonbenzylated forms of a tyrosine phosphate group.

III. MAGIC ANGLE SPINNING NMR

A. 1H Magic Angle Spinning NMR

It would be of great utility if a better methodology could be provided to monitor polymer-bound reactions than gel phase heteronuclear NMR. However, as previously mentioned, the utility of gel phase proton NMR in solid phase synthesis monitoring is of little value, as the spectra obtained are generally quite broad.

NMR Methods

83

Spinning the sample at the magic angle (54.7° ), MAS overcomes the limitations of using 1H NMR for solvent-swollen resin samples. The broadening due to magnetic susceptibility and dipolar coupling are substantially removed. 1H MAS NMR have been applied to study properties of crosslinked polystyrene gels for some time (24). However, the application of MAS 1H NMR in combinatorial chemistry appeared only recently, where it has been demonstrated that ‘‘high-resolution’’ NMR data for resin-supported molecules can be obtained (25,26).

MAS NMR requires special equipment to obtain the data. High quality MAS NMR spectra can be obtained using specially designed probes that are now commercially available. While any standard MAS probe can also be used to collect NMR data, the resolution obtained will probably suffer due to mismatches in the magnetic susceptibility in the probes (27).

The quality of the NMR data is also dependent on the conditions by which the sample was prepared. All solvent/resin combinations do not yield high-quality 1H MAS NMR spectra (28,29). The most important factor in obtaining high-quality 1H MAS NMR data is the choice of resin, with TentaGel resins (TGT) generally giving rise to the best-quality data. The choice of swelling solvent can also play a critical role. For the most part, good solvents include CD2Cl2, CDCl3, C6D6, DMF-d7, and DMSO-d6. For example, spectra obtained from Fmoc-Asp(OtBu)-NovaSyn TGT gave good line widths, less than 6 Hz, for CD2Cl2, DMF-d7, and DMSO-d6 where as using benzene-d6 yielded 19.4 Hz. MAS data obtained on Fmoc-Asp(OtBu)-NovaSyn Wang yielded similar line widths for all the solvents with line widths ranging from 9 to 14 Hz. The compound attached to the resin also plays a role on the ‘‘high- resolution-like’’ appearance of the data (30).

The quality of the spectra can be enhanced by using presaturation of the resin signal to reduce the intensity of the aromatic resonances from the polystyrene or polyethylene glycol resonances in TentaGel-like resins. The spin-echo experiment distinguishes between the narrow lines of the substrates and the much broader lines of the polymer and can be used to remove the peaks from the polymer as well (31–33).

B. 13C MAS NMR

In the late 1980s Frechet showed that high quality 13C NMR data of swollen gels could be readily obtained by MAS using a standard MAS probe (34–36). The first use of 13C MAS NMR data for combinatorial chemistry demonstrated that a potential reaction complication, the production of two similar compounds, could be monitored (37). The reaction of norbornane carboxylic acid

84

Shapiro

Figure 5 Utilization of MAS 13 C NMR for comparison of esterification reaction products for exo and endo norbornane-2-carboxylic acid mixture on Merrifield and Wang resins.

NMR Methods

85

on Merrifield and Wang resins to produce the ester were compared to evaluate the potential usability of the particular resin in a library synthesis (Fig. 5). It was found that the stereochemistry of addition was different for the two resins. In the case of the Wang resin a 60 : 40 ratio of exo/endo product was obtained while the Merrifield resin yielded an 80 : 20 ratio. The spectral quality of the 13C MAS data was virtually indistinguishable from that of the solution 13C NMR data. The MAS 13C NMR data can be collected in a much shorter time frame (20 min) than for gel phase 13C NMR data which generally take several hours.

C. Other Nuclei

MAS 19F NMR can also be used to follow appropriate reactions (19). The MAS data were seen to be an order of magnitude faster to collect than gel phase NMR for the same sample. While no reports of using MAS 31P NMR have appeared at this writing, there is no reason why this nucleus and others could not prove valuable in following solid phase chemical reactions.

Figure 6 Two-dimensional CH-correlated gel phase NMR spectrum for chloroformswollen Boc-Cys(Acm)-OCH2-Pab-copoly (styrene 1% divinylbenzene).

86

Shapiro

IV. TWO-DIMENSIONAL NMR

Two-dimensional NMR spectroscopy has proven of great value for product analysis in solution phase chemistry and by its nature should provide a platform by which enhanced data quality and ease of interpretation is obtained. Geralt demonstrated that the use of 2D CH correlated NMR spectra (Fig. 6) enhances the utility of gel phase NMR even though the proton NMR spectrum was broad (36). Since MAS NMR spectra of the swollen resins are of higher quality than gel phase NMR, it is possible to treat these samples as if they

Figure 7 Two-dimensional CH-correlated NMR spectrum for a mixture of endo (N) and exo (X) norbornane epimers. (Top) High-resolution solution data for the mixture of norbornane-2-carboxylic acid. (Bottom) MAS NMR on benzene-swollen Wang resin.

NMR Methods

87

were solutions and therefore use the same techniques that would be used in ‘‘high-resolution’’ NMR studies. The application of 2D NMR techniques to determine the structure on resin exemplifies this notion (26). The direct-ob- serve MAS CH-correlated data for the exo/endo norbornane mixture on resin shown in Fig. 7 illustrates the resolution obtainable. Assignments of both proton and carbon resonances in both major and minor isomers can be readily made. The combination of heteronuclear multiple quantum correlation (HMQC) and total correlation spectroscopy (TOCSY) data, as shown in Fig. 8 for Wang-Fmoc-lys-Boc, allows complete assignment of the proton and car- bon-13 spectra confirming the structure on resin (37). The step-by-step analysis of a multistep solid phase synthesis involving a Heck reaction was conveniently followed using MAS 2D NMR techniques (38). The use of 2D NMR

Figure 8 MAS HMQC and TOCSY NMR spectra for Fmoc-lys-tboc on benzeneswollen Wang resin.

88

Shapiro

TOCSY, HMQC, and nuclear Overhouser enhanced spectroscopy (NOESY)

(39)data as shown in Fig. 9 was instrumental in the evaluation of products. The main problem for NMR analysis of on-resin products, with the ex-

ception of TentaGel, is that the 1H NMR spectra are generally broad with featureless line widths around 10 Hz or more (28). The additional complication of large polymer resonances can be attenuated by using a spin-echo sequence but the residual peaks can still be problematic. Peak assignment can be difficult due to the loss of coupling information relegating the interpretation of the

Figure 9 MAS (A) TOCSY and (B) NOESY NMR spectra for Fmoc-lys-tboc on benzene-swollen Wang resin.