Synthesis of Bacillithiol and the Catalytic Selectivity of FosB-type Fosfomycin Resistance Proteins

Abstract

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, D-glucosamine hydrochloride, in 11 steps and 8-9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive microorganisms associated with the deactivation of the antibiotic fosfomycin. The in vitro results indicate that the preferred thiol substrate and metal ion for the FosB from Staphylococcus aureus are BSH and Ni(II), respectively. However, the metal ion selectivity is less distinct with FosB from Bacillus subtilis, Bacillus anthracis or Bacillus cereus.

Low-molecular weight thiols are critical to living systems for maintaining a reducing environment in the cytosol and preventing oxidation of cysteine residues in proteins. Eukaroyotes typically utilize glutathione for these purposes while most Gram-positive bacteria and Archae lack glutathione and instead utilize novel cysteine derived thiols.¹ For example, the N-acetyl cysteine derivative mycothiol (MSH, Figure 1) is found in most actinomycetes including mycobacteria and streptomycetes.² In 2009 a structurally related thiol, bacillithiol (BSH, Figure 1), was detected in Gram-positive bacteria, including Bacilli and Staphylococcus aureus and then isolated from Deinoccus radiodurans and characterized as its S-bimane derivative (BSmB, Figure 1).^3,4 Subsequently, a biosynthetic pathway for BSH was proposed and the first chemical synthesis was reported.^5,6

Figure 1.

Mycothiol, bacillithiol, S-bimane bacillithiol and bacillithiol disulfide.

Although the functions of BSH in biology are not fully understood, recent reports suggest that it may be the preferred substrate for the FosB-type fosfomycin resistance proteins.^5,6 FosB is a metallo-enzyme, found in Gram-positive microorganisms, that catalyzes the general reaction illustrated in Scheme 1 and confers resistance to the antibiotic, fosfomycin.⁷ Previous work in our laboratory suggested that the prefered thiol substrate and divalent metal ion might be L-Cys and Mg²⁺, respectively.⁷ The discovery of BSH and the availability in our lab of FosB enzymes from several Gram-positive microorganisms prompted us to initiate an investigation of their substrate and metal ion selectivity. In this report we describe a complete chemical synthesis of BSH from inexpensive starting materials, a new continuous ³¹P NMR assay of FosB enzymes and its use to further elucidate the substrate and metal ion selectivity of FosB enzymes from four Gram-positive microorganisms.

Scheme 1.

Our synthetic strategy (Scheme 2) targeted the disulfide BSSB and the use of D-glucosamine as a starting point. While our synthetic studies were underway Hamilton and coworkers⁶ reported the chemical synthesis of bacillithiol starting trichloroacetimidate 1 prepared by the oxidative azidation (CAN, NaN₃)⁸ of D-glucal. We also started from trichloroacetimidate, 1, but in our case derived 1 from D-glucosamine by way of a known⁹ diazo transfer-acetylation reaction (TfN₃ then Ac₂O) followed by selective removal of the C1 acetate (NH₂NH₂, MeOH)¹⁰ and trichloroacetimidate formation (Cl₃CCN, NaH).¹¹ Coupling of trichloroacetimidate 1 with di-tert-butylmalate 2¹² under TMSOTf activation afforded 3 in yields ranging from 61 to 91% (3:1 α/β), with highest yields observed on a 10 gram reaction scale. Alpha (3a) and beta (3b) glycosides are separable by chromatography leading to an isolated yield of 52% for the desired 3a. Reduction of azide 3a was first examined using polymer-supported triphenylphosphine followed by treatment of the crude product with 4N HCl-dioxane leading to isolation of the corresponding hydrochloride salt (4•HCl) albeit in low yield (ca. 30%). It was subsequently determined that hydrogenation (45 psi) of azide 3b over palladium-carbon in methanol afforded crude amine (4) of sufficient purity to be condensed directly with commercially available Boc protected cysteine disulfide (5) to afford disulfide 6 in 80% yield (two-steps). Bacillithiol disulfide was derived from 6 by a two-step deprotection sequence starting with removal of acetyl groups (NaOMe, MeOH, −40 to −20 °C) to provide disulfide 7 in 92% yield. Next, treatment of 7 with a 50% TFA dichloromethane solution served to remove remaining acid labile protecting groups and following elution thru a strong cation exchange column with 5% acetic acid-methanol yielded BSSB disulfide as its ammonium acetate salt (BSSB•2HOAc).

Scheme 2.

Synthesis of bacillithiol.

Access to BSH allowed us to screen several FosB enzymes from Staphylococcus aureus (FosB^SA), Bacillus cereus (FosB^BC), Bacillus anthracis (FosB^BA) and Bacillus subtilis (FosB^BS). Time courses for the reactions were followed by integrated peak-intensities in a continuous ³¹P-NMR assay, which is quite convenient with diamagnetic metals. The assay results for the FosB^SA and FosB^BC enzymes with combinations of Ni(II), Mg²⁺, L-Cys and BSH are illustrated in Figure 2. The results indicate that there is a very clear preference of the FosB^SA enzyme for BSH and Ni(II). We detected no activity with L-Cys in the presence of Mg²⁺. The enzymes from bacilli also show a substantial preference for BSH but exhibit far less discrimination between the two metals (Figure 2B) and Figure S2 (Supporting Information). In other experiments we have found Zn(II) is an excellent inhibitor of the FosB enzymes (data not shown).

Figure 2.

Time course of the FosB catalyzed-addition of BSH or L-Cys to fosfomycin in the presence of Mg²⁺ or Ni(II). Results for FosB^SA and FosB^BS are shown in panels A and B, respectively. Reactions were run at 25° C in 20 mM HEPES (pH 7.0) with 4 mM fosfomycin and 0.5 μM enzyme in the presence of ( ) 2 mM BSH and 10 μM Ni(II), ( ) 2 mM BSH and 1 mM Mg²⁺, (▼) 2 mM L-Cys and 10 μM Ni(II) or ( ) 2 mM L-Cys and 1 mM Mg²⁺.

Although the continuous ³¹P-NMR assay is quick and convenient, it is not suitable with paramagnetic metals nor for performing detailed steady-state kinetics. Nevertheless, approximate minimum turnover numbers (k_cat) can be estimated from the initial rates of the reactions. The apparent k_cat for FosB^SA with BSH and Ni(II) is ∼30 s⁻¹ but with Mg²⁺ drops to ∼1 s⁻¹. The FosB^SA activity in the presence of Ni(II) also drops to ∼0.9 s⁻¹ when L-Cys is the substrate. The results with Mg²⁺ are consistent with previous observations.⁶ The apparent k_cat values for FosB^BS with BSH and Ni(II) or Mg²⁺ are ∼6 s⁻¹ and 3 s⁻¹, respectively. With L-Cys, the k_cat values are much lower, on-the-order-of 0.05 to 0.2 s⁻¹.

In summary, BSH is clearly the preferred in vitro thiol substrate for FosB enzymes from several Gram-positive microorganisms. However, the metal-ion preferences of the enzymes are not so clear and bear further investigation particularly with respect to what the preferred metal is in vivo. It is interesting to note that the preferred metal in the reaction catalyzed by the anthrax enzyme (FosB^BA) is Mg²⁺ (Figure S2A) Supporting Information. The availability of synthetic BSH opens the door to more detailed kinetic and metal-ion preference studies of the enzymes as well as structural investigation of the FosB•BSH and FosB•product complexes.

By way of the described 10-step reaction sequence we have prepared over one gram of BSSB•2HOAc.Reduction of BSSB disulfide to afford BSH is easily accomplished by stirring with [tris(2-carboxyethyl)phosphine] (TCEP) reducing gel in water. The bromobimane derivative (BSmB) was prepared and its properties compared favorably by LC-MS and NMR analysis to an authentic sample of BSmB.³