Fmoc-S-acetamidomethyl-D-cysteine 4-alkoxybenzyl alcohol resin

Two functionalised supports for the solid-phase synthesis of peptides under mild reaction conditions were prepared: 4-chloromethylphenoxyacetamidomethyl-copoly (styrene-1%-divinylbenzene) and 4-chloromethylphenoxyacetyl-norleucyl-poly (dimethylacrylamide). They were devised in order to avoid the danger of racemization which exists during base-catalyzed esterification of the first protected amino acid to the 4-alkoxybenzyl alcohol resins formerly employed in combination with N alpha-9-fluorenylmethoxycarbonyl and tert.-butyl side-chain protecting groups. Esterification of N alpha-protected amino acids to the new resins can be achieved easily and without significant levels of racemization by means of their caesium salts, while cleavage from the supports is possible by treatment with trifluoroacetic acid. The 4-chloromethylphenoxyacetyl polystyrene resin was tested by the synthesis of Leu-enkephalin which was cleaved, at the end of the synthesis, from the solid support in 91% yield by 60% trifluoroacetic acid in methylene chloride, and was shown to be more than 99% pure by ion-exchange chromatography and reverse phase high pressure liquid chromatography.

Esterification of glycosylated serine and cysteine derivatives with a 4-alkoxybenzyl alcohol (Wang) resin is described. The classical methods of ester bond formation (symmetrical anhydride, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate [TBTU]/4-dimethylaminopyridine [DMAP] with or without 1-hydroxybenzotriazole [HOBT], pentafluorophenyl [Pfp] esters gave high percentages of racemization of the glycosylated serine or cysteine residues. To reduce the D-amino acid content, we found that the best results were obtained with the highly efficient MSNT reagent (2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide), which gave a high yield of substitution of the resin and the lowest percentage of racemization. A difference in behavior was observed between the two amino acids. The glycosylated cysteine derivative always gave lower racemization than the analogous glycosylated serine.

The quality of preloaded Wang resins is very important for the success of solid-phase peptide syntheses (SPPS). A critical factor is the capping of remaining hydroxyl groups after loading with the first amino acid, since these free alcohols lead to truncated sequences during the following SPPS steps. Because the detection of hydroxyl groups by color tests is difficult and unreliable, the capping efficiency is often controlled by time-consuming peptide test syntheses. Here, we describe a two-dimensional, high resolution magic angle spinning NMR method for the quantitative determination of remaining 4-alkoxybenzyl alcohols in Fmoc-Xaa-Wang resins with a detection limit of 1 mol-%. The NMR method was validated with samples of known ratios between Fmoc-Ala-Wang and 4-alkoxybenzylalcohol resin. Application to a set of preloaded Fmoc-Ala- and Fmoc-Thr(tBu)-Wang test resins demonstrated that the full range of essential amino acids can be quantified without further spectrometer calibration. Compared to established test synthesis protocols, the NMR method represents not only advantages in terms of time and cost savings but also eliminates all inaccuracies due to further sample treatment like SPPS and cleavage from the resin.