Total Synthesis of (±)-3-Thiaglutamate

Abstract

The total synthesis of (±)-3-thiaglutamate is reported. Central to our strategy is an thiol addition to an imine to form the thioaminal of the natural product. The resulting thioaminal product is then subjected to triflic acid global deprotection to produce 3-thiaglutamate as a triflate salt. This work constitutes the first total synthesis of 3-thiaglutamate and demonstrates that the hemithioaminal group in 3-thiaglutamate can be stabilized under acidic conditions.

Graphical Abstract

1. Introduction

Many microorganisms produce natural products that mimic essential compounds found in primary metabolism.¹ For example, the plant pathogen Pseudomonas syringae is known to produce antimetabolites that mimic jasmonic acids found in plants.² More recently, P. syringae was reported to produce 3-thiaglutamate (1), a natural product identified by genome mining.³ Structurally, 3-thiaglutamate resembles glutamic acid with a carbon-to-sulfur atom substitution at the β position (Figure 1a).^3-5 Moreover, 3-thiaglutamate is the class defining natural product of pearlins with unusual biosynthetic origins.⁶ The natural product is biosynthesized from L-cysteine which is attached to a ribosomal peptide scaffold and maturated through post-translational modifications to become a 3-thiaglutamate residue at the C-terminus of the peptide.^6,7 Proteolytic cleavage liberates the natural product and regenerates the peptide scaffold. The stereocenter of 1 was confirmed to be in the L stereochemical configuration by microcrystalline electron diffraction (MicroED)⁸ studies of an intermediate with the amino acid bound to a peptide.³ 3-Thiahomoleucine (2) is another 3-thiaamino acid natural product,⁹ which along with the ammosamides (ammosamide A shown, 3),^10,11 compromise all pearlin natural products known to-date.

Figure 1.

(a) The pearlin class of amino acid-derived natural products. (b) Retrosynthesis of 3-thiaglutamate.

3-Thiaglutamate and 3-thiahomoleucine contain a hemithioaminal functional group and are expected to be extremely unstable. Hemithioaminals have seen use as self-immolative linkers in prodrugs and are known to undergo spontaneous fragmentation of the carbon-sulfur bond to form an imine and a thiol.¹² Thus, it is a highly unusual functional group in natural products. Unsurprisingly, the 3-thiaamino acids have not been previously synthesized or isolated in pure form. Recently, natural products that are unstable to isolation have recently received much interest from the organic synthesis and biosynthesis communities.^13,14 Chemical synthesis represents an alternative method for structural elucidation of natural products that have yet to be isolated from their natural sources.¹⁵ While there are previous syntheses of protected thioaminals and the coupling of these amino acids to peptides have been reported,^3,16-20 none of these compounds possess a free hemithioaminal. Based on the reactivity of hemithioaminals, it is not known if 3-thiaglutamate is stable to fragmentation. To validate the natural product’s unusual structure and evaluate its reactivity, we set out to prepare 3-thiaglutamate by total synthesis.

Due to the presumed instability of 3-thiaglutamate, protonation of the amino group by strong acid was envisioned to stabilize the natural product enabling its purification. As such, protecting groups amenable to acid-mediated removal were strategically selected for late-stage global deprotection. An addition of a thiol to an imine was envisioned to form thioaminal 4 (Figure 1b). The imine can be assembled by elimination of a bromoglycine derivative. Radical bromination would then allow for functionalization of a protected glycine by formation of the captodative radical and bromination of the α carbon.

2. Results and Discussion

The synthesis of racemic 3-thiaglutamate commenced with commercially available N-Boc-glycine tert-butyl ester (5, Scheme 1). In the first step, 5 was subjected to radical bromination conditions with N-bromosuccinimide and irradiation with 254 nm ultraviolet light affording α-bromoglycine 6 in 98% yield.²¹ Using conditions reported by Kobayashi and co-workers, 6 was treated with piperidinemethyl polystyrene resin in toluene for 30 minutes at room temperature.²² This process formed imine 7 which can be easily separated from the solid resin by decantation. 2-Mercaptoacetic acid benzyl ester 8²³ was added to imine 7 to produce thioaminal 9 in 74% yield over two steps. At this point, global deprotection necessitated the removal of three different protecting groups. Notably, when compound 9 was treated with triflic acid, thioanisole, ethanedithiol, and trifluoroacetic acid this resulted in efficient deprotection of the amino acid. The addition of thioanisole and ethanedithiol scavengers were necessary to sequester the highly reactive cations formed in the reaction.²⁴

Scheme 1.

Total synthesis of (±)-3-thiaglutamate (1•HOTf). Reagents and conditions: a) 5 (1.0 equiv), NBS (1.0 equiv), CCl₄, hν (254 nm), 1.5 h, 98%; b) 6 (1.0 equiv), piperidinemethyl polystyrene (3.0 mmol/g, 1.1 equiv), PhMe, RT, 0.5 h; c) 7 (1.0 equiv), 8 (1.1 equiv), PhMe, RT, 1 h, 74% over two steps; d) 9 (1.0 equiv), TfOH/thioanisole/EDT (2:2:1, v/v/v), TFA, 0 °C to RT, 2 h, 65%. NBS = N-bromosuccinimide, EDT = ethanedithiol, tBu = tert-butyl, Bn = benzyl, TFA = trifluoroacetic acid.

Given the instability and polarity of the amino acid natural product, conventional purification methods failed to deliver the natural product. After extensive optimization of purification conditions, a suitable procedure was developed that involved quenching the reaction with water and directly subjecting the reaction solution to reverse-phase column chromatography to afford 3-thiaglutamate (1) as a triflate salt. The structure of the natural product was unambiguously determined by X-ray crystallography.²⁵ In the crystal structure, the amine was protonated and resulting positively charged amino group was interacting with the triflate counterion. The closest proton of the ammonium salt was 2.2 Å away from the triflate showing an ion pair interaction. When protonated the natural product is stabilized presumably by lone pair occupation of the amine preventing n to σ*_C-S delocalization and fragmentation of the hemithioaminal. This is supported by the normal bond length of the carbon-sulfur bond (1.8 Å) in the crystal structure.²⁵

To interrogate the stability of the hemithioaminal, 1•HOTf was subjected to aqueous conditions at neutral pH (Scheme 2). Upon treatment of the triflic acid salt of 3-thiaglutamate with sodium phosphate buffer at pH 7, 1•HOTf converted to hemithioacetal 10 in 30% yield and the hemiacetal of glyoxylic acid monohydrate (11) in 15% yield. These results illustrate that 3-thiaamino acids are unstable at physiological pH and readily deaminate under physiological conditions.

Scheme 2.

Deamination of 1•HOTf at pH 7. Reagents and conditions: 1•HOTf (1.0 equiv), 50 mM Na₂HPO₄, H₂O, pH 7, RT, 1 h, 30% yield of 10 and 15% yield of 11. ^aNMR yield

With these experimental results, we propose a mechanism for the hydrolysis of 3-thiaglutamate. The mechanism likely proceeds by deprotonation of the positively charged ammonium to generate the free hemithioaminal. The amino group then fragments to the thiol and iminium. The iminium can then be hydrolyzed to liberate ammonia and form glyoxylic acid, which then reacts with the in situ generated thiol to give hemithioacetal 10 (See supporting information). The formation of 11 also indicates that the carbon-sulfur bond is labile which supports the proposed mechanism. Given the stability of the compound in acid, the mechanism is unlikely to involve direct loss of ammonia from 3-thiaglutamate. If this were the case, the compound would be more unstable under acidic condition, which is not observed.

3. Conclusion

In conclusion, we have reported a 4-step total synthesis of racemic 3-thiaglutamate. This work constitutes the first total synthesis of a 3-thiaamino acid natural product and illustrates a general strategy towards this class of compounds. By acid-mediated stabilization of 3-thiaglutamate, this work also validates the structure of 3-thiaglutamate, a compound that has not been previously isolated in pure form. Unstable secondary metabolites have untapped potential and represent a formidable challenge and opportunity for chemical synthesis. Given that thioaminals and thioaminoketals functional groups are commonly found in other natural product classes such as epithiodiketopiperazines (ETPs)²⁶ and peptides,^27,28 this work may find broader application in the synthesis of other thioaminal natural products. This work is currently ongoing in our lab and will be reported in due course.

Data Availability Statement

The data underlying this study are available in the Supporting Information.