Synthesis of Biosynthetic Intermediates of Vibrioferrin and Enzyme Reactions Using Them as Substrates

Abstract

Biosynthetic intermediates of siderophore vibrioferrin (VF), O-citryl-L-serine, 2-aminoethyl citrate, and alanine-2-amidoethyl citrate were respectively synthesized as a mixture of stereoisomers. These compounds were used as substrates for enzyme reactions using recombinant PvsA, PvsB, and PvsE proteins as corresponding enzyme equivalents. The results of our study show that each enzyme reacts with a respective substrate and produces VF along the proposed biosynthetic pathway. Furthermore, the results of this study will contribute to the understanding of VF biosynthetic enzymes and may help in the development of antimicrobial drugs by inhibiting siderophore biosynthetic enzymes.

Introduction

Vibrioferrin (VF) is a siderophore produced and secreted by a Gram-negative marine bacterium, Vibrio parahaemolyticus, to uptake iron from the environment that the bacteria grow in.¹⁾ Iron is an essential element for living organisms as a cofactor for many enzymes catalyzing redox reactions. However, bacteria face problems in iron uptake because of the poor solubility of iron compounds under natural aerobic conditions and the existence of iron-binding proteins as iron ion sequestering agents in hosts. Thus, various microorganisms have evolved iron acquisition systems using low-molecular-weight iron chelators called siderophores.

The structure, including the absolute configurations of the VF, has been elucidated, as shown in Fig. 1, based on spectroscopic analysis followed by total synthesis.^2–4) VF is the epimeric mixture on the α-ketoglutarate (α-KG) N-hemiacetal moiety.

Fig. 1. The Structure of VF

The biosynthetic pathway of VF was elucidated through in vitro enzyme reactions using overexpressed and purified hexahistidine-tag (His) fused proteins His-PvsA, His-PvsB, PvsD-His, and His-PvsE as biosynthetic enzyme equivalents.⁵⁾ In the biosynthesis of VF, first, PvsD condenses L-serine (L-Ser) and citric acid (CA) to generate O-citryl-L-serine (L-Ser-CA), and second, PvsE decarboxylates L-Ser-CA to 2-aminoethyl citrate (AE-CA). Next, PvsA condenses L-alanine (L-Ala) and AE-CA to form L-alanine-2-amidoethyl citrate (L-Ala-AE-CA), and finally, PvsB condenses α-ketoglutaric acid (α-KG) and L-Ala-AE-CA to produce VF (Chart 1).

Chart 1. Biosynthetic Pathway of VF

To confirm the function of each enzyme, it is necessary to analyze the enzyme reactions using chemically synthesized biosynthetic intermediates of VF as corresponding substrates.

The biosynthetic intermediates of VF can be synthesized using the total synthesis method mentioned above. However, the synthetic route involves too many steps, including an optical resolution process. When confirming the function of biosynthetic enzymes of VF, their substrate specificity, including stereoselectivity, provides essential information. Consequently, the synthetic targets were set as the racemic AE-CAs 2a and 2b (2ab), and the diastereomeric mixtures L-Ser-CAs 1a and 1b (1ab), L-Ala-AE-CAs 3a and 3b (3ab), D-Ala-AE-CAs 4a and 4b (4ab) (Fig. 2).

Fig. 2. The Synthetic Substrates for Enzyme Reactions in This Study

In this paper, we present convenient syntheses of biosynthetic intermediates of VF and enzyme reactions using them as substrates for recombinant PvsA, PvsB, and PvsE proteins.

Results and Discussion

In our synthetic strategy for 1ab–4ab, cyclic anhydride 8 was used as a common intermediate.⁶⁾ Compound 8 was synthesized as follows (Chart 2): 1) Selective esterification of the outer two carboxy groups in CA to provide bis-methyl ester 5.⁷⁾ 2) Esterification on the remaining carboxy group to form tert-butyl ester 6.⁸⁾ 3) Hydrolysis of two methyl esters to form dicarboxylic acid 7. 4) Intramolecular dehydration of 7 was achieved using acetic anhydride.

Chart 2. Synthesis of Anhydride 8

In the esterification reaction of 8 using pyridine as a base, the reaction mixture turned red and the desired ester was not obtained. Thus, tri-n-butyl phosphine was used instead of an amine base in the esterification by 8.⁹⁾ Commercially available N-(tert-butoxycarbonyl (Boc))-L-serine benzyl ester was esterified by 8, and the resulting carboxy group was further reacted with benzyl chloride to give 9ab (Chart 3). Unfortunately, the diastereomers 9a and 9b could not be separated using silica gel column chromatography, so the subsequent reactions were carried out using the isomeric mixture. The hydrogenolysis of benzyl ester of 9ab and degradation of tert-butyl moiety by trifluoroacetic acid (TFA) provided the TFA salts of 1ab. In a subsequent examination, the diastereomers 1a and 1b were proved by HPLC to have separated. Apropos, it has been reported that O-citryl-L-serine (1) was rearranged to N-citryl-L-serine through an intramolecular acyl migration under neutral conditions.¹⁰⁾

Chart 3. Synthesis of the Diastereomeric Mixture 1ab TFA Salts

TFA salts of 2ab were prepared by N-Boc-2-aminoethanol and 8, followed by deprotection of the functional groups (Chart 4). Incidentally, when sodium bicarbonate was added to the hexadeuterodimethyl sulfoxide (DMSO-d₆) solution of the TFA salts of 2ab, an intramolecular rearrangement to form the amide product was deduced based on the one dimensional (1D) and 2D-NMR spectra.¹¹⁾

Chart 4. Synthesis of the Racemic Mixture 2ab TFA Salts

To synthesize 3ab and 4ab, amides 11 and 12 were first prepared by amidating N-Boc-Ala and 2-aminoethanol with DMT-MM as a condensing agent¹²⁾ (Chart 5). The TFA salts of 3ab and 4ab were then synthesized using 11 and 12, respectively, following the same procedure described above. The diastereomers 13ab and 14ab could not be separated by silica gel column chromatography.

Chart 5. Syntheses of the Diastereomeric Mixtures 3ab and 4ab TFA Salts

Enzymatic reactions were conducted on the synthesized substrates 1ab–4ab using recombinant PvsA, PvsB, and PvsE, and the reaction mixtures were analyzed by HPLC. The enzyme reaction between L-Ser and CA with PvsD to produce 1 was not evaluated, as a previous study had suggested that the intermediate L-Ser-CA would be unstable under neutral conditions in the enzyme reactions.⁵⁾ Thus, substrates 1ab–4ab were used in the enzyme reactions without neutralizing the TFA salts to prevent their decomposition.

The reaction of 1ab using PvsE followed by PvsA and PvsB produced VF (Fig. 3(B)). On a chromatogram, VF appeared as two peaks due to the epimers of the N-hemiacetal moiety. Even though HPLC did not provide clear evidence that 1a or 1b was transformed into 2, as the attributions of 1a, 1b, and 2 are unclear in Fig. 3(D), the appearance of VF in Fig. 3(B) shows that PvsE converted 1 into 2 via decarboxylation.

Fig. 3. HPLC Analysis of Enzyme Reaction Using 1ab

(A) 1ab in water; (B) 1ab, L-Ala, α-KG with PvsA/B/E; (C) L-Ser, CA, L-Ala, α-KG with PvsA/B/D/E; (D) 1ab with PvsE; (E) 2ab in water.

The reaction of 2ab using PvsA followed by PvsB produced VF (Fig. 4(B)). The enzyme reaction of 2ab with L-Ala using PvsA formed a single isomer 3a (Fig. 4(D)). Therefore, the absolute configuration of the CA moiety of 3a was deduced to be R according to that of VF. The result shows that PvsA recognizes the stereochemistry on the CA moiety of 2.

Fig. 4. HPLC Analysis of Enzyme Reaction Using 2ab

(A) 2ab in water; (B) 2ab, L-Ala, α-KG with PvsA/B; (C) L-Ser, CA, L-Ala, α-KG with PvsA/B/D/E; (D) 2ab with PvsA; (E) 3ab, α-KG without enzyme.

When 3ab and 4ab were subjected to the enzyme reaction using PvsB, VF was produced only from 3a (Fig. 5(B)). The results show the stereoselectivity of PvsB regarding the Ala and CA moiety.

Fig. 5. HPLC Analysis of Enzyme Reactions Respectively Using 3ab and 4ab

(A) 3ab, α-KG without enzyme; (B) 3ab, α-KG with PvsB; (C) L-Ser, CA, L-Ala, α-KG with PvsA/B/D/E; (D) 4ab, α-KG with PvsB.

Conclusion

The enantiomeric or diastereomeric mixtures of the biosynthetic intermediates of VF were synthesized and used as substrates in the corresponding enzyme reactions. This study shows that the enzymes PvsA, PvsB, and PvsE react with their respective substrates and produce VF along the proposed biosynthetic pathway. PvsA and PvsB exhibited stereoselectivity in their enzyme reactions. However, it is uncertain whether stereoisomers of reactive substrates are completely unreactive. Therefore, further studies using stereoselectively synthesized substrates are required to clarify the substrate stereospecificity of these enzymes. The results of this study contribute to a better understanding of VF biosynthetic enzymes and may aid in the development of antimicrobial drugs by inhibiting siderophore biosynthetic enzymes.

Experimental

General

¹H- and ¹³C-NMR spectra were taken with a Bruker BioSpin AVANCE 500 spectrometer. Chemical shifts were given on a δ (ppm) scale with tetramethylsilane as an internal standard (s, singlet; d, doublet; t, triplet; m, multiplet; br, broad). The linear structures of the synthetic substrates used in the enzyme reactions were confirmed by H–H correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC) spectra, respectively. High-resolution electrospray ionization mass spectra (ESIMS) were obtained with a Bruker Daltonics micro TOF-Q spectrometer. HPLC analysis was performed on a Hitachi LaChrom Elite HPLC system (Tokyo, Japan).

Dimethyl 3-Carboxy-3-hydroxypentanedioate (5)

A solution of citric acid (50 g, 260 mmol) and boric acid (1.5 g, 24.3 mmol) in methanol (70 mL) and acetone (80 mL) was stirred at room temperature for three days. The newly formed crystals were collected by filtration under reduced pressure, followed by washing with acetone. The crystals were dried under reduced pressure to give 5 (48.4 g, 85% yield).; ¹H-NMR (500 MHz, DMSO-d₆) δ ppm (J in Hz): 2.75 (2H, d, J = 15.0), 2,84 (2H, d, J = 15.0), 3.57 (6H, s). ¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 42.6, 51.4, 72.5, 169.9, 174.2. ESI-TOFMS (neg.) m/z: 219.0510 [M − H]⁻ (Calcd for C₈H₁₁O₇: 219.0497).

Dimethyl 3-(tert-Butoxycarbonyl)-3-hydroxypentanedioate (6)

To a suspension of 5 (20.0 g, 90.8 mmol) in tert-butyl acetate (120 mL) under an argon atmosphere was added 60% perchloric acid (2.7 mL), followed by stirring at room temperature for three days. The reaction mixture was neutralized by carefully adding sodium bicarbonate (5.0 g, 60.0 mmol) and concentrating it under reduced pressure until the smell of the acetic acid disappeared. The concentrate was diluted with ethyl acetate and washed successively with water, saturated sodium bicarbonate aq., and brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give ester 6 (15.5 g, 63% yields) as a colorless oil.; ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.50 (9H, s), 2.77 (2H, d, J = 15.0), 2.87 (2H, d, J = 15.0), 3.69 (6H, s). ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 27.7, 43.3, 51.8, 73.0, 83.2, 170.1, 172.3. ESI-TOFMS (pos.) m/z: 299.1106 [M + Na]⁺ (Calcd for C₁₂H₂₀NaO₇: 299.1101).

3-(tert-Butoxycarbonyl)-3-hydroxypentanedioic Acid (7)

To a cold (0 °C) solution of ester 6 (5.2 g, 18.8 mmol) in methanol (20 mL) was added 2.0 mol/L sodium hydroxide aq. (20 mL), followed by stirring while gradually warming to room temperature, and stirred for six hours at this temperature. To the reaction mixture was added a 1.0 mol/L potassium bisulfate aq. (20 mL) and it was concentrated under reduced pressure. The concentrate was diluted with ethyl acetate and washed with a 1.0 mol/L potassium bisulfate aq. The aqueous layer was extracted with ethyl acetate and the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure to give dicarboxylic acid 7 (3.6 g, 76% yields) as a colorless solid.; ¹H-NMR (500 MHz, DMSO-d₆) δ ppm (J in Hz): 1.39 (9H, s), 2.60 (2H, d, J = 15.0), 2.74 (2H, d, J = 15.0). ¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 27.4, 42.9, 72.9, 80.6, 171.2, 171.7. ESI-TOFMS (pos.) m/z: 271.0816 [M + Na]⁺ (Calcd for C₁₀H₁₆NaO₇: 271.0788).

3-(tert-Butoxycarbonyl)-3-hydroxypentanedioic Anhydride (8)

A solution of dicarboxylic acid 7 (50 g, 201 mmol) in acetic anhydride (110 mL) was stirred for two hours at 60 °C. The reaction mixture was concentrated under reduced pressure. The residue was recrystallized from ethyl acetate–hexane (1 : 9) to give anhydride 8 (38.5 g, 83% yields) as a colorless needles.; ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.51 (9H, s), 2.94 (2H, d, J = 17.0), 3.04 (2H, d, J = 17.0). ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 27.7, 40.4, 69.7, 86.5, 163.6, 170.9. ESI-TOFMS (pos.) m/z: 253.0691 [M + Na]⁺ (Calcd for C₁₀H₁₄NaO₆: 253.0683).

N-Boc-O-benzyl-L-serinyl Benzyl 3-(tert-Butoxycarbonyl)-3-hydroxypentanedioate (9ab)

To a solution of N-Boc-L-serine benzyl ester (2.95 g, 10.0 mmol) in dichloromethane (20 mL) under an argon atmosphere was added 8 (2.3 g, 10.0 mmol) and tri-n-butylphosphine (2.5 mL, 10.0 mmol), followed by stirring at room temperature for three days. The reaction mixture was diluted with ethyl acetate and washed successively with 10% potassium bisulfate aq. and brine. The water layer was extracted with ethyl acetate, and the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (10 mL) and sodium bicarbonate (1.1 g, 13.0 mmol), benzyl chloride (1.4 mL, 12.0 mmol) and potassium iodide (116 mg, 1.0 mmol) were added and then stirred at 120 °C for two hours. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with hexane–ethyl acetate (2 : 1) to give the diastereomeric mixture 9ab (3.3 g, 54% yield in two steps) as a colorless viscous oil.; ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.40–1.49 (18H, m), 2.65–2.89 (4H, m), 4.00 (0.5H, s), 4.03 (0.5H, s), 4.27 (0.5H, dd, J = 4.1, 12.0), 4.41 (0.5H, dd, J = 3.3, 11.2), 4.47 (0.5H, dd, J = 3.4, 11.2), 4.59 (1H, m), 5.10–5.20 (4H, m), 5.43 (0.5H, d, J = 8.3), 5.60 (1H, d, J = 8.2), 7.34 (10H, m). ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 27.7, 27.7, 28.2, 43.1, 43.2, 43.5, 53.0, 64.6, 66.6, 67.6, 72.8, 72.8, 83.4, 83.5, 128.3, 128.4, 128.5, 128.5, 128.6, 135.1, 135.4, 169.1, 169.3, 172.1. ESI-TOFMS (pos.) m/z: 638.2596 [M + Na]⁺ (Calcd for C₃₂H₄₁NNaO₁₁: 638.2572).

L-Ser-CA (1) TFA Salt

To a solution of 9ab (1.55 g, 2.52 mmol) in ethyl acetate (13 mL) under an argon atmosphere was added 10% Pd on charcoal (25 mg), followed by vigorous stirring at room temperature overnight under a hydrogen atmosphere. The reaction mixture was filtered through celite and concentrated under reduced pressure. Next, TFA (8.0 mL) was added to the residue and stirred for two hours at room temperature. The reaction mixture was concentrated under reduced pressure to give the TFA salt of 1ab (1.04 g, quantitative yield in two steps) as a highly hygroscopic colorless amorphous solid.; ¹H-NMR (500 MHz, DMSO-d₆) δ ppm (J in Hz): 2.68 (1H, d, J = 15.0), 2.78–2.90 (3H, m), 4.28–4.46 (2H, m), 4.31 (1H, m), 8.47 (3H, br s). ¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 14.1, 27.1, 42.1, 42.3, 42.7, 51.3, 51.4, 59.8, 61.6, 61.7, 72.4, 73.4, 115.2, 117.6, 158.2, 158.5, 168.3, 168.4, 169.0, 169.1, 171.1, 171.2, 174.4, 174.5. ESI-TOFMS (neg.) m/z: 280.0678 [M − H]⁻ (Calcd for C₉H₁₄NO₉: 280.0663).

N-Boc-2-aminoethyl Benzyl 3-(tert-Butoxycarbonyl)-3-hydroxypentanedioate (10ab)

To a solution of 8 (1.00 g, 4.34 mmol) in dichloromethane (10 mL) under an argon atmosphere was added N-Boc-2-aminoethanol (670 µL, 4.34 mmol) and tri-n-butylphosphine (1.1 mL, 4.34 mmol), followed by stirring at room temperature for three days. The reaction mixture was diluted with ethyl acetate and washed successively with 10% potassium bisulfate aq. and brine. The water layer was extracted with ethyl acetate and the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (4.5 mL) and sodium bicarbonate (450 mg, 5.35 mmol), benzyl chloride (600 µL, 5.21 mmol), and potassium iodide (66 mg, 0.4 mmol) were added and then stirred at 120 °C for two hours. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with hexane–ethyl acetate (3 : 2) to give the racemic ester 10ab (1.66 g, 79% yield in 2 steps) as a colorless viscous oil.; ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.44 (18H, s), 2.78 (1H, d, J = 15.1), 2.82 (1H, d, J = 15.6), 2.84 (1H, d, J = 15.1), 2.90 (1H, d, J = 15.6), 3.36 (2H, m), 4.09 (2H, m), 4.22 (1H, m), 5.10 (1H, br s), 5.11 (1H, d, J = 15.0), 5.14 (1H, d, J = 15.0), 7.33 (5H, m). ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 27.7, 28.3, 39.5, 43.5, 43.6, 64.2, 66.7, 73.0, 83.5, 128.3, 128.5, 135.4, 169.2, 169.5, 172.4. ESI-TOFMS (pos.) m/z: 504.2217 [M + Na]⁺ (Calcd for C₂₄H₃₅NNaO₉: 504.2204).

AE-CA (2ab) TFA Salt

To a solution of 10ab (1.66 g, 3.45 mmol) in ethyl acetate (17 mL) under an argon atmosphere was added 10% Pd on charcoal (30 mg), followed by vigorous stirring at room temperature overnight under a hydrogen atmosphere. The reaction mixture was filtered through celite and concentrated under reduced pressure. Next, TFA (10 mL) was added to the residue and stirred for two hours at room temperature. The reaction mixture was concentrated under reduced pressure to give the TFA salt of 2ab (1.40 g, quantitative yield in two steps) a highly hygroscopic colorless amorphous solid.; ¹H-NMR (500 MHz, DMSO-d₆) δ ppm (J in Hz): 2.68 (1H, d, J = 15.6), 2.80 (1H, d, J = 15.6), 2.81 (1H, d, J = 15.5), 2.90 (1H, d, J = 15.5), 3.08 (2H, m), 4.16 (1H, dt, J = 5.5, 12.2), 4.22 (1H, dt, J = 5.5, 12.2), 7.99 (3H, br s). ¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 42.8, 42.9, 60.6, 72.5, 115.2, 117.5, 158.2, 158.4, 158.6, 169.3, 171.1, 174.6. ESI-TOFMS (neg.) m/z: 236.0790 [M − H]⁻ (Calcd for C₇H₁₄NO₇: 236.0765).

2-Hydroxyethyl N-Boc-L-alanine Amide (11)

To a solution of N-Boc-L-alanine (1.9 g, 10.0 mmol) in methanol (20 mL) was added 2-aminoethanol (660 µL, 11.0 mmol) and DMT-MM (3.6 g, 13.0 mmol), followed by stirring at room temperature for overnight. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The water layer was extracted with ethyl acetate and the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with chloroform–methanol (9 : 1) to give the amide 11 (2.3 g, 99% yield) as a colorless solid.; ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.36 (3H, d, J = 7.5), 1.44 (9H, s), 3.38 (1H, m), 3.45 (1H, m), 3.58 (1H, m), 3.69 (2H, m), 4.18 (1H, br s), 5.42 (1H, br s), 7.02 (1H, br s). ¹³C-NMR (125 MH, CDCl₃) δ ppm: 18.5, 28.3, 42.2, 50.3, 61.6, 80.2, 155.7, 173.9. ESI-TOFMS (pos.) m/z: 255.1327 [M + Na]⁺ (Calcd for C₁₀H₂₀N₂NaO₄: 255.1315).

2-Hydroxyethyl N-Boc-D-alanine Amide (12)

To a solution of N-Boc-D-alanine (1.9 g, 10.0 mmol) in methanol (20 mL) was added 2-aminoethanol (660 µL, 11.0 mmol) and DMT-MM (3.6 g, 13.0 mmol), followed by stirring at room temperature for overnight. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The water layer was extracted with ethyl acetate and the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with chloroform–methanol (9 : 1) to give the amide 12 (2.4 g, quantitative yield) as a colorless solid.; The ¹H, ¹³C-NMR spectra and the results of accurate mass measurement were identical with 11.

N-Boc-L-alanine-2-amidoethyl Benzyl 3-(tert-Butoxycarbonyl)-3-hydroxypentanedioate (13ab)

To a solution of 11 (2.30 g, 10.0 mmol) in dichloromethane (20 mL) under an argon atmosphere was added 8 (2.3 g, 10.0 mmol) and tri-n-butylphosphine (2.5 mL, 10.0 mmol), followed by stirring at room temperature for three days. The reaction mixture was diluted with ethyl acetate and washed successively with 10% potassium bisulfate aq. and brine. The water layer was extracted with ethyl acetate and the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (10 mL) and sodium bicarbonate (1.1 g, 13.0 mmol), benzyl chloride (1.4 mL, 12.0 mmol) and potassium iodide (116 mg, 1.0 mmol) were added and then stirred at 120 °C for two hours. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with hexane–ethyl acetate (1 : 3) to give the diastereomeric mixture 13ab (3.5 g, 63% yield in two steps) as a colorless viscous oil.; ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.35 (1.5H, d, J = 7.0), 1.37 (1.5H, d, J = 7.0), 1.40–1.46 (18H, m), 2.75–2.93 (4H, m), 3.34 (0.5H, m), 3.45 (0.5H, m), 3.53 (0.5H, m), 3.65 (0.5H, m), 4.03–4.30 (3H, m), 5.12 (1H, d, J = 12.2), 5.15 (1H, d, J = 12.2), 7.34 (5H, m). ¹³C-NMR (125 MH, CDCl₃) δ ppm: 18.4, 38.2, 43.8, 44.0, 63.8, 66.7, 73.2, 83.5, 83.7, 128.4, 128.6, 135.4, 169.0, 169.4, 173.0. ESI-TOFMS (pos.) m/z: 575.2602 [M + Na]⁺ (Calcd for C₂₇H₄₀N₂NaO₁₀: 575.2575).

N-Boc-D-alanine-2-amidoethyl Benzyl 3-(tert-Butoxycarbonyl)-3-hydroxypentanedioate (14ab)

To a solution of 12 (700 mg, 3.01 mmol) in dichloromethane (6 mL) under an argon atmosphere was added 8 (695 mg, 3.01 mmol) and tri-n-butylphosphine (750 µL, 3.01 mmol), followed by stirring at room temperature for three days. The reaction mixture was diluted with ethyl acetate and washed successively with 10% potassium bisulfate aq. and brine. The water layer was extracted with ethyl acetate and the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was dissolved in N,N-dimethylformamide (3 mL) and sodium bicarbonate (330 mg, 3.91 mmol), benzyl chloride (420 µL, 3.61 mmol), and potassium iodide (50 mg, 0.3 mmol) were added, and then stirred at 120 °C for one hour. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with hexane–ethyl acetate (1 : 3) to give the diastereomeric mixture 14ab (1.1 g, 67% yield in 2 steps) as a colorless viscous oil.; The ¹H, ¹³C-NMR spectra, and the results of accurate mass measurement were identical with 13ab.

L-Ala-AE-CA (3ab) TFA Salt

To a solution of 13ab (1.18 g, 2.14 mmol) in ethyl acetate (10 mL) under an argon atmosphere was added 10% Pd on charcoal (20 mg), followed by vigorous stirring at room temperature overnight under a hydrogen atmosphere. The reaction mixture was filtered through celite and concentrated under reduced pressure. Next, TFA (6 mL) was added to the residue and stirred for two hours at room temperature. The reaction mixture was concentrated under reduced pressure to give the TFA salt of 3ab (1.06 g, quantitative yield in two steps) as a highly hygroscopic colorless amorphous solid.; ¹H-NMR (500 MHz, DMSO-d₆) δ ppm (J in Hz): 1.34 (3H, d, J = 7.5), 2.68 (1H, d, J = 15.5), 2.77 (0.5H, d, J = 15.0), 2.78 (0.5H, d, J = 15.0), 2.80 (1H, d, J = 15.5), 2.85 (0.5H, d, J = 15.0), 2.86 (0.5H, d, J = 15.0), 3.33–3.42 (2H, m), 3.80 (1H, m), 3.97–4.12 (2H, m), 8.10 (3H, br s), 8.42 (1H, br s). ¹³C-NMR (125 MH, DMSO-d₆) δ ppm: 17.1, 27.1, 37.8, 42.7, 42.9, 48.2, 62.2, 62.3, 72.5, 169.4, 169.7, 171.1, 174.5. ESI-TOFMS (neg.) m/z: 307.1145 [M − H]⁻ (Calcd for C₁₁H₁₉N₂O₈: 307.1136).

D-Ala-AE-CA (4ab) TFA Salt

To a solution of 14ab (1.11 g, 2.01 mmol) in ethyl acetate (10 mL) under an argon atmosphere was added 10% Pd on charcoal (20 mg), followed by vigorous stirring at room temperature overnight under a hydrogen atmosphere. The reaction mixture was filtered through celite and concentrated under reduced pressure. Next, TFA (6 mL) was added to the residue and stirred for two hours at room temperature. The reaction mixture was concentrated under reduced pressure to give the TFA salts of 4ab (1.07 g, quantitative yield in two steps) as a highly hygroscopic colorless amorphous solid.; The ¹H, ¹³C-NMR spectra, and the results of accurate mass measurement were identical with 3ab.

Enzyme Reaction

Enzyme reactions were performed using 1 µM of His-PvsA, His-PvsB and His-PvsE as the VF biosynthetic enzyme equivalents in a VF biosynthesis buffer [15 mM magnesium sulfate, 100 mM Tris–hydrochloric acid buffer (pH 8.0), 100 mM sodium chloride, and 15 mM ATP]. As needed, each 3 mM of the corresponding synthesized substrates 1ab–4ab, L-Ser, CA, L-Ala, and α-KG were added and incubated at 37 °C for two hours. In particular, in the enzyme reaction using His-PvsE, 100 µM of pyridoxal phosphate and 1ab were added immediately before incubation.

HPLC Analysis of Enzyme Reaction Mixtures

Each enzyme reaction was stopped by heat denaturation at 70 °C for 15 min and centrifuged at 16000 × g for two minutes to separate the supernatants. The supernatant was then filtered through a 0.22 µM cellulose-acetate filter and then the filtrates were directly injected into the HPLC with a C₈ reverse phase column (4.6 × 150 mm, GL Sciences, Tokyo, Japan). The HPLC mobile phase consisted of 0.1% TFA aq. (solvent A) and 30% acetonitrile aq. containing 0.1% TFA (solvent B). The HPLC separation consisted of an isocratic step for 5 min at 5% solvent B, a linear increase from 5% solvent B to 75.8% solvent B over 25 min, and a 10 min isocratic step at 100% solvent B with a 0.5 mL/min flow rate. The detection absorbance was 220 nm.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (C) (23K06555) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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