Mass spectrometry thioesters

The N-linked pentasaccharide core Man3(GlcNAc)2 glycopeptide bearing the extracellular MMP inducer sequence 517 (emmprin 34—58) has been successfully linked to G(l) PAMAM aminodendrimer by thioester activation (Fig. 63). The resulting octameric 30 kDa construct was obtained in low yield, but was purified by preparative electrophoresis and fully characterized by MALDI-TOF mass spectrometry. The multivalent architecture was built to evaluate the requirement of emmprin multimerization for inducing MMP expression.384... 

The first extender we constructed is shown in Fig. 9.7. Mass spectrometry showed we could modify caspase-3 cleanly and quantitatively with this molecule, even though the large subunit of the enzyme contains four other cysteine residues. We could also fully deprotect the thioester to reveal a free thiol. Screens... 

Scheme 18 illustrates the proposed stages in 6-MSA biosynthesis in which the first and second condensation steps proceed with inversion to give the triketide (63). Ketoreduction gives the alcohol (64) and then elimination followed by a final malonyl condensation generates the tetraketide (65) which cyclises via an intramolecular condensation and enolises to give the aromatic nucleus of (66). In the first set of experiments (J )- and (S)-[l- C, H]nialonales were incubated separately with 6-MSA synthase purified from Penicillium patulum [56]. Isotope incorporations were determined by mass spectrometry. All the possible isotope patterns for retention or loss of the pro-J or pro-S hydrogens from C-3 and C-5 were permutated. Comparison with the actual spectra obtained demonstrated that opposite prochiral hydrogens were eliminated. The absolute stereochemistry was established in an analogous experiment [57] where the chiral malonates were incubated with acetoacetyl CoA rather than acetyl CoA. Subsequent mass spectral analysis showed that it is the Hr proton that is retained at C-3 of 6-MSA and so it can be deduced that the hydrogen at C-5 must be derived from the opposite prochiral hydrogen, Hg. The overall result is summarised in Scheme 18. In a recent collaborative study we have synthesised the triketide alcohol (64) as its NAC thioester and shown that it is indeed a precursor as, on incubation with 6-MSA synthase and malonyl CoA, 6-MSA production is observed [unpublished results]. Current work is aimed at synthesis of both enantiomers of (64) to study the overall stereochemistry of the ketoreduction and elimination reactions.

Subsequently, Walsh et al. identified a kutzneride hologenase for piperazinate (KthP). This halogenase, which uses O2 as co-substrate, acts on the piperazyl ring tethered by a thioester linkage to the holo forms of the thiolation domains. MS (mass spectrometry) analysis of the protein-bound product confirmed the chlorination of the piperazate framework from the (35)- but not the (3/f)-piperazyl-5-pantetheinyl thiolation protein [215]. 

J Kopka, JB Ohlrogge, JG Jaworski. Analysis of in vivo levels of acyl-thioesters with gas chromatography/mass spectrometry of the butylamide derivative. Anal Chem 224 51-60, 1995. 

Alternatively, the ethanol can be removed by evaporation and if required an IR spectrum of the residue can be obtained as described in section 2.2.2.2.S. This would be the case if the cold-ethanol treatment had removed a significant amount of material, such as a thioester thereby giving a more intense spectrum of the other components. If confirmation of the IR examination is required the residue can be examined by mass spectrometry. 

The substrate selectivity of PsyA KSl has been previously examined using SNAC-thioesters, with acetyl-SNAC being shown as the preferred substrate, despite a rather promiscuous substrate profile (see Sect. 3.2.3). It was postulated that the use of the corresponding acyl-ACPs would represent a more realistic approximation of the native system. Moreover, de-acylation of the small ACP domain could be monitored by mass spectrometry. The assay consisted of incubation of PsyA... 

These relatively new techniques viz laser desorption/ionization Fourier transform mass spectrometry and fast atom bombardment and laser desorption Fourier transform ion cyclotron resonance mass spectrometry have been applied to the determination of non volatile polymer additives (thioester, phosphite, phosphonate and hindered amine antioxidant types) and antioxidants, ultraviolet absorbers and amide waxes. 

Analyze samples by 15 % SDS-PAGE to check loading and cleavage efficiency. Combine protein thioester containing fractions and verify identity with mass spectrometry. Include always a sample of beads, because protein thioester might precipitate that will in turn reduce yield, if not realized. 

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