Mass spectrometry solid phase synthesis

Because the characterization of support-bound intermediates is difficult (see below), solid-phase reactions are most conveniently monitored by cleaving the intermediates from the support and analyzing them in solution. Depending on the loading, 5-20 mg of support will usually deliver sufficient material for analysis by HPLC, LC-MS, and NMR, and enable assessment of the outcome of a reaction. Analytical tools that are particularly well suited for the rapid analysis of small samples resulting from solid-phase synthesis include MALDI-TOF MS [3-5], ion-spray MS [6-8], and tandem MS [9]. MALDI-TOF MS can even be used to analyze the product cleaved from a single bead [5], and is therefore well suited to the identification of products synthesized by the mix-and-split method (Section 1.2). The analysis and quantification of small amounts of product can be further facilitated by using supports with two linkers, which enable either release of the desired product or release of the product covalently bound to a dye [10-13], to an isotopic label [11], or to a sensitizer for mass spectrometry [6,14,15] (e.g., product-linker-dye- analytical linker -Pol). 

Solid-phase synthesis practitioners should have elemental analysis, IR and NMR tools at hand for following reactions. Loadings of key intermediates should be measured and yields for new reactions reported. Mass spectrometry, HPLC and NMR will be needed for confirming structures after cleavage from the support. 

Some particular features of the analysis of products obtained by combinatorial methods have impaired the use of NMR spectroscopy in the initial phase of the development of this technique. Combinatorial chemistry produces large number of compounds in a very short period of time, in small quantities and instead of using traditional glassware for synthesis employs 96-well microtiter plates to store, transport and sometimes even to synthesize the compounds of interest. Another issue is the need to characterize solution and solid samples, since solid phase synthesis is extensively used in combichem. In this context, the need of an efficient and universal sample analysis remains a challenge. Actually, most combichem programs obtain mass spectrometry and UV (photodiode-array detection) data on their samples but clearly the use of NMR spectroscopy provides a structural characterization unparalleled by the aforementioned techniques. In the last years an increasing number of new NMR methods opened the possibility for the utilization of this analytical technique for monitoring combinatorial chemistry reactions. The first part of this chapter will focus on the recent developments introduced in NMR spectroscopy to overcome these difficulties. 

Fig. 20.10. Superimposed measurement in negative mode of a compound obtained by solid phase synthesis with ESl-quadrupole (A) and ESI-FT-ICR mass spectrometry (B). Starting points for elementary analysis [M-H] measured 452.148072 Da, charge -1, min./max. double bond equivalents (DBE) 0.5/30, tolerance 0.0005 Da. Elemetary analysis for Mr=452.148072 C26 03 N1 SO H21 F3 [M-H] calculated 452.1478911,15.5 DBE, deviation 0.4 ppm. The calculation of possible molecular formulae on the basis of the very precise ESI-FT-ICR measurement thus gave solely the correct composition.

Truncated sequences, core sequences, incompletely synthesized peptides. Imperfect conversion during acylation or deprotection of the temporary protecting group in solid-phase synthesis may lead to mismatch sequences that lack some amino acids and truncated sequences (core sequences). They occur, when the accessibility or reactivity of the peptide bound to the solid phase is insufficient difficult sequences). Truncated sequences may be classically identified and quantified by a modified Edman degradation, also called preview analysis. Alternatively, mass spectrometry provides efficient tools for the identification of truncated sequences. 

Polymeric supports of variable solubility have been investigated as an alternative to insoluble supports used in solid-phase synthesis. Reactions are performed in homogeneous media by choosing an appropriate solvent that solubilizes the polymer, and purification is performed by precipitation. This methodology benefits both solution-phase and solid-phase syntheses. Moreover compound characterization can be easily undertaken at any stage of the synthesis, since the support is soluble in standard spectroscopic solvents. A direct real-time control is possible, whereas a solid-phase protocol relies on a cleave and analyze strategy that consumes compound, imparts delay, and thus can only be accomplished at the end of synthesis. For these reasons soluble polymeric supports are preferred to conventional insoluble supports (resins, plastic pins), and they are compatible with analytical techniques such as NMR and mass spectrometry. 

Quality Control of Solid-Phase Synthesis by Mass Spectrometry... 

This literature review covers the applications of analytical techniques to solid phase organic chemistry and combinatorial chemistry published between June 96 and September 1997. Highlighted are mass spectrometry, NMR, IR and chromatographic analyses of solid phase synthesis reactions and combinatorial libraries. 

S. Beranova-Giorgianni and D. M. Desiderio. Fast Atom Bombardment Mass Spectrometry of Synthetic Peptides. In Methods in Enzymology Solid-Phase Peptide Synthesis, ed. G. B. Fields. Methods in Enzymology 289. Academic Press, San Diego, 1997, 478-499. 

Maux, D. Enjalbal, C. Martinez, J. Aubagnac, J.-L. Combarieu, R. Static Secondary Ion Mass Spectrometry to Monitor Solid-Phase Peptide Synthesis. J. Am. Soc. Mass Spectrom. 2001, 72, 1099-1105. 

A frequent complication in the use of an insoluble polymeric support lies in the on-bead characterization of intermediates. Although techniques such as MAS NMR, gel-phase NMR, and single bead IR have had a tremendous effect on the rapid characterization of solid-phase intermediates [27-30], the inherent heterogeneity of solid-phase systems precludes the use of many traditional analytical methods. Liquid-phase synthesis does not suffer from this drawback and permits product characterization on soluble polymer supports by routine analytical methods including UV/visible, IR, and NMR spectroscopies as well as high resolution mass spectrometry. Even traditional synthetic methods such as TLC may be used to monitor reactions without requiring preliminary cleavage from the polymer support [10, 18, 19]. Moreover, aliquots taken for characterization may be returned to the reaction flask upon recovery from these nondestructive... 

Monitoring reaction progress throughout a multistep synthesis is a relatively difficult task.22 Typical methods used for solution-phase synthesis, including thin-layer chromatography (TLC), GC, and most types of mass spectrometry (MS), are less informative for solid-phase methods. However, Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) are particularly useful in solid-phase strategies. 

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