Through Palladium-catalysed Cross-coupling

Metal-mediated carbon-carbon bond forming reactions involving triazine have been reported using an appropriately functionalised substituted triazine and unsaturated aliphatic compounds.^113,157 Carroll et al.¹⁵⁷ exploited methyl iodotriazine 133 in a metal-mediated Sonogashira-type reaction with phenylacetylene to afford phenylethynyltriazine 134 in 80%

yield (Scheme 3.6a). Carroll et al.¹⁵⁷ generated methyl iodotriazine 133 through diazotization and ensuing halogenation of methyl aminotriazine 118c, using isoamyl nitrite and diiodomethane, in a modest yield of 20%. Jackson and co-workers^158,159 have reported the synthesis of enantiomerically pure pyridylalanine amino acids through the palladium catalysed cross-coupling of serine-derived organozinc reagents with halopyridyl derivatives.

It was postulated that this Negishi-type cross-coupling could be extended to include other N-heterocycles such as iodotriazine 136 (Scheme 3.6). Therefore a synthetic strategy was devised based on the metal-mediated cross-coupling of 136 and protected iodoalanine 135 to generate (after removal of the methyl ester) Fmoc-triazinylalanine (TrzA) 137 (Scheme 3.6b). Triazinylalanine 137 would be suitable for use in the Fmoc-strategy for SPPS and hence be particularly appropriate for rapid functionalisation of peptide probe molecules.

Alternatively, it would be possible to use the acid functionality of 137 to derivatise onto small molecule probes.

Scheme 3.6: a) Reported transformation of methyl aminotriazine 118c into methyl iodotriazine 133 and subsequent carbonic addition to form alkynyl-triazine 134 using metal-mediated cross-coupling;¹⁵⁷ b) proposed synthetic route to Fmoc-triazinylalanine 137 via a Negishi-type

cross-coupling of iodoalanine 135 and iodotriazine 136, and subsequent methyl ester removal. 137 could be used in an Fmoc SPPS strategy to generate a fluorescent triazine-containing

fluorescent probe such as 138.

Iodotriazine 136 was formed in 30% yield through the diazotisation of aminotriazine 118a using isopentyl nitrite in diiodomethane at 55 °C (Scheme 3.7a). Although low, this yield is a 10% increase from the reported yield for formation of methyl iodotriazine 133 (Scheme 3.6).¹⁵⁷ It is possible that this difference is due to degradation of triazines 118c and 133 at the higher temperatures employed by Carrol et al.,¹⁵⁷ as observed previously when using aminotriazine 118a (Scheme 3.5b, Section 3.3.1).

Fmoc-iodoalanine methyl ester 135 was synthesised in three synthetic steps starting from serine methyl ester 139 (Scheme 3.7b).¹⁵⁸ L-Serine methyl ester 139 was sequentially reacted with Fmoc-OSu and p-toluenesulfonyl chloride to give Fmoc-Ser(OTs)-OMe 140 in an overall yield of 55%. SN2-substitution of the tosyl group of 140 with sodium iodide in a Finkelstein-type reaction generated iodoalanine methyl ester 135 in 52% yield. In this instance, the moderate yield was attributed to incomplete conversion of tosyloxyalanine methyl ester 140.¹⁶⁰

For zinc insertion of iodoalanine methyl ester 135 to form the corresponding organozinc reagant, the procedure reported by Jackson and co-workers was initially followed.¹⁵⁸ Commercial zinc dust and I2 (0.3 eq) were weighed into an oven dried flask which was evacuated and purged with nitrogen three times at 0 °C. Iodoalanine methyl ester 135 was dissolved in anhydrous DMF (freshly opened bottle), transferred to the reaction mixture, and stirred at 0 °C. After 2 hours, no formation of the activated organozinc reagent was observed by TLC. It was reasoned that a higher temperature may be needed for formation of the alkyl zincate of 135, and activation of zinc dust by catalytic amounts of iodine may be more effective in solution. Accordingly, formation of the organozinc reagent was attempted a second time. After evacuation and purging of an oven-dried flask containing zinc dust, anhydrous DMF (freshly opened bottle) and I2 (0.15 eq) were added in quick succession and stirred at room temperature. After 15 minutes, iodoalanine 135 was added followed by a further 0.15 equivalents of I2.ⁱ Formation of the corresponding organozinc reagent was observed after 2 hours; at which time iodotriazine 136, Pd(II) acetate and 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (SPhos) were added in quick succession. The reaction was stirred at 50 °C for 5 hours and after purification by column chromatography, triazinylalanine methyl ester 142 was recovered in 69% yield (Scheme 3.7b). In attempts to optimise Negishi-coupling, the palladium catalyst loading was altered and an alternative palladium catalyst (tris(dibenzylideneacetone)dipalladium(0)) was used;

neither of these changes resulted in a significant change in yield.

i After discussions with Christian Hedberg, catalytic I₂ was added once for zinc activation, and a second time for activation of the organozinc reagent.

Scheme 3.7: a) Diazotisation of aminotriazine 118a to give iodotriazine 136; b) successful synthesis of triazinylalanine 137 via Negishi-type cross coupling of zinc activated iodoalanine methyl ester 135 to 3-iodo-1,2,4-triazine 136 to generate 142 using palladium acetate and SPhos as a co-ligand. Methyl ester removal of 142 using trimethyltin hydroxide gave the desired product 137. Iodoalanine methyl ester 135 was synthesised in three steps from serine methyl ester 139.

In the optimised method for the metal-mediated cross-coupling of 135 and 136 to generate triazinylalanine methyl ester 142, the total 0.3 equivalents of iodine were added in two stages. This was to ensure consistent catalytic activation of zinc dust by iodine and subsequent formation of iodide. Iodide promotes in situ formation of dianionic zincate species RZnI3

2- (where R = alanine methyl ester). RZnI3

2- is formed from RZnI2

-, which in turn is generated from the organozinc reagent (RZnI). This doubly charged zincate species has been shown to be the active transmetallating agent in Negishi-type cross coupling reactions involving alkyl halides.¹⁶¹ Hence, iodine was exploited in this reaction in two ways: initially for activation of zinc towards nucleophilic addition, and secondly for the catalytic formation of a higher order zincate species. It should also be noted that Negishi coupling was only successful when using a freshly opened bottle of anhydrous DMF. This is due to the inherent sensitivity of organozinc reagents to oxygen and water.

Demethylation of Fmoc-TrzAla-OMe 142 to give the free acid 137 was achieved by stirring in DCE with trimethyltin hydroxide at 88 °C (Scheme 3.7b).¹⁶² Initial yields of 13% were reported, although alterations to the purification strategy of the crude product increased yields of 137 to 27%. In this case, the overall isolated yield of triazinylalanine 137 is limited by observable degradation of the triazinylalanines 137 and 142 at the elevated temperatures

required for deprotection; the methyl ester of 142 could not be removed at moderate temperatures of 55 °C. Base-catalysed deprotection of triazinylalanine methyl ester 142 was also attempted using LiOH, unfortunately, this led to concomitant Fmoc-group removal.

3.4 Conclusions

A novel strategy to generate an Fmoc-compatible triazine, triazinylalanine 137 has been developed (Scheme 3.7). The synthetic route towards 137 is robust and uses readily available and inexpensive starting materials. The route to 137 includes an optimised Negishi-type cross-coupling reaction between iodotriazine 136 and an organozinc reagent. It should be possible to couple iodotriazine 136 to a variety of alkyl- and aryl-organozinc reagents, which will ultimately increase the diversity of available (and synthetically accessible) triazine scaffolds. It should be possible to use triazinylalanine 137 in the Fmoc-strategy for SPPS to rapidly and efficiently functionalise a range of probes such as fluorescein isothiocyanate or carboxyfluorescein. The fully deprotected version of triazinylalanine 137 is similar in structure to a range of tyrosine-based scaffolds that have been genetically incorporated into proteins in response to an amber codon using evolved tyrosyl-tRNA synthetases.^145,163 Due to the promiscuity of these synthetases it is possible that fully deprotected triazinylalanine could be genetically incorporated into proteins.

Therefore, it should be possible to use triazinylalanine 137 and its derivatives as either counterpart of a bioorthogonal probe.