Supporting Information

Mutasynthesis of Glycopeptide Antibiotics: Variations of Vancomycin’s AB-Ring Amino Acid 3,5-Dihydroxyphenylglycine

Stefan Weist, Claudia Kittel, Daniel Bischoff, Bojan Bister, Volker Pfeifer, Graeme J. Nicholson, Wolfgang Wohlleben and Roderich D. Süssmuth*

Department of Chemistry/Microbiology and Biotechnology, University of Tübingen, Auf der Morgenstelle,72076 Tübingen e-mail: [email protected]

General. NMR measurements were obtained using a Bruker Avance 400 MHz NMR spectrometer. Chemical shifts are given in parts per million (ppm). Positions of hydrogen or carbon atoms at the aromatic ring systems are numbered clockwise starting with the aromatic carbon atom attached to the α-carbon. LC-ESI-MS experiments were recorded on an Agilent 1100 HPLC system coupled to a Bruker Esquire 3000plus ESI-MS-ion trap spectrometer with the individually described parameters. MS spectra were recorded using a Finnigan TSQ-70 triple- stage quadrupole mass spectrometer in EI mode (70 eV). Preparative HPLC was performed using a Waters system (Eschborn, Germany) equipped with a Waters 600 pump, a Waters 481 UV detector and a Waters 740 data module with a Nucleosil C-18 column (250 x 20 mm, 5µm; Grom, Herrenberg, Germany) by isocratic elution employing 5% acetonitrile (0.1% TFA) in water (0.1% TFA) at a flow of 10 mL/min at a run time of 10 min. ESI-FTICR-MS analyses were performed on a Bruker APEX II FTICR-MS spectrometer (Bruker-Franzen, Bremen, Germany). Flash column chromatography was carried out with Merck Kieselgel 60 (230-400 mesh) and TLC was performed using Merck silica gel F254 precoated aluminium sheets (Merck, Darmstadt, Germany). Unless otherwise noted, materials were obtained from commercial sources and used without further purification. Dry solvents were purchased from Fluka (Neu-Ulm, Germany). 3,5- dihydroxymandelic acid (DMA), 3,5-Dihydroxyphenylglycine (DPg) and 3,5- dihydroxyphenylacetic acid (DPA) were purchased from Sigma (Neu-Ulm). 3- hydroxyphenylacetic acid (3-HPA) and 3-methoxyphenylacetic acid (3-MeOPA) were purchased from ABCR (Karlsruhe, Germany).

Mutants and bacterial strains. The mutant ∆dpgA was generated following a previously published procedure.[1] The mutant and the wild type strain of Amycolatopsis balhimycina (formerly A. mediterranei) were grown according to reported procedures.[1]

Supplementation Experiments. Amino acids and other biosynthesis precursors (mandelic and phenylacetic acids) were supplemented as previously reported1 in 10 mL cultures with final concentrations of 1 mg/mL. Feeding experiments were repeated three times. All substances used for supplementation were tested for antimicrobial activity against Bacillus subtilis ATCC6633 before supplementation and did not show any antibiotic activity. For the inhibition assay, 20 µL of the culture filtrate were brought on a filter plate which was placed on a culture of B. subtilis and incubated for 24 h at 37°C.

S1 Supporting Information JA0499389 Weist et al.

LCMS Analyses. LC-ESI-MS for the characterization of glycopeptide antibiotics in supplemented culture filtrates was performed after pre-purification with solid phase extraction using Varian BondElut cartridges (Varian, Darmstadt, Germany; size: 100 mg) eluting with water and water/MeOH solutions (1 mL each of 0%, 50% and 100% MeOH), and the fraction containing 50% MeOH was used for the LC-MS-experiment with an injection volume of 20 µL. LC was performed using a 125x4 mm LiChroCart Purospher RP-C18 column (VWR, Darmstadt, Germany; particle size: 5µm) with a flow of 1.5 mL/min using a gradient from 10 to 70% MeCN (0.1% TFA) in water (0.1% TFA) in 5 min.

Synthesis of Phenylglycines. General Procedure starting from aldehydes. A solution of the benzaldehyde derivative (10 mmol) in NH3/MeOH (7N, 50 mL) was cooled to 0°C. TMSCN (1.11 mL, 15 mmol, 1.5 eq) was added dropwise with stirring. After 10 min, the temperature was raised to 45°C and the mixture was stirred for 5 h. The solvent was removed in vacuo and the residue was purified by flash chromatography using chloroform/methanol (9/1) as the eluant to give the aminonitrile. Aminonitriles were hydrolyzed using 6N HCl (50 mL for a 10 mmol scale) by heating to reflux for 3-5 h until TLC (solvent: EtOAc/n-BuOH/H2O/HAc = 2/1/1/1) indicated completion of the reaction. Charcoal (100 mg) was added, the mixture refluxed for another 10 min and filtered. The solution was concentrated to 10 mL, and the amino acid started to precipitate. After Büchner funnel filtration, the amino acids were dried under vacuum.

3-Methoxyphenyl-α-aminoacetonitrile (1) yield: 82% 1HNMR (DMSO) δ = 7.30 (dd, J=7.8/8.1Hz, 1H, H-5), 7.08-7.04 (m, 2H, H-2, H-4), 6.91 (dd, J=8.3/2.3 Hz, 1H, H-6), 4.97 (s, 1H, H-α), 3.76 (s, 3H, OCH3).

13CNMR δ = 159.83, 140.34, 130.12, 122.55, 119.13, 114.04, 112.71, 55.54, 46.62.

LRMS (rel. Intens.) m/z: 162 (M+, 100%), 146 (18), 136 (38), 131 (84), 119 (14), 109 (24), 94 (12), 77 (16), 65 (10), 39 (7).

3-Methoxyphenylglycine hydrochloride (3-MeOPg) (2) yield: 76% from (1) 1 + HNMR (DMSO) δ = 8.76 (br s, 3H, NH3 ), 7.37 (t, J=8.0Hz, 1H, H-5), 7.11 (s, 1H, H-2), 7.05 (d, J=8.1 Hz, 1H, H-6), 6.99 (dd, J=8.1/2.0Hz, 1H, H-4), 5.03 (d, J=4.6Hz, 1H, H-α), 3.77 (s, 3H, OCH3).

13CNMR δ = 169.51, 159.65, 137.13, 129.99, 120.47, 115.28, 113.87, 57.27, 55.59.

HR-FTICR-MS: [M+H]+ m/z: calcd. 182.0811, found 182.0812.

S2 Supporting Information JA0499389 Weist et al.

3,5-Dimethoxyphenyl-α-aminoacetonitrile (3) yield: 65% 1HNMR (DMSO) δ = 6.68 (d, J=2.2 Hz, 2H, H-2, H-6). 6.48 (t, J=2.0Hz, 1H, H-4), 4.94 (s, 1H, H-α), 3.76 (s, 6H, OCH3).

13CNMR δ = 160.97, 141.11, 122.42, 105.14, 100.13, 55.65, 46.70.

3,5-Dimethoxyphenylglycine hydrochloride (DMeOPg) (4) yield: 60% 1 H-NMR (D2O) δ = 6.67 (d, J=2.2Hz, 2H, H-2, H-6), 6.62 (t, J=2.2Hz, 1H, H-4), 5.09 (s, 1H, H-α), 3.79 (s, 6H, OCH3).

13CNMR δ = 168.72, 159.29, 131.99, 104.79, 100.17, 102.96, 54.69, 53.97.

HR-FTICR-MS: [M+H]+ m/z: calcd. 212.0916, found 212.0904.

(3-Hydroxy-5-methoxy)phenyl-α-aminoacetonitrile (5) yield: 60% 1HNMR (DMSO) δ = 9.61 (br s, 1H, OH), 6.54 (t, J=2.1Hz, 2H, H-2, H-6), 6.29 (dd, J=2.2/2.3Hz, 1H, H-4), 4.89 (s, 1H, H-α), 3.71 (s, 3H, OCH3), 2.73 (br s, 2H, NH2).

13CNMR δ = 160.91, 158.99, 140.85, 122.53, 106.56, 103.62, 101.08, 56.38, 55.39.

LRMS (rel. Intens.) m/z: 178 (M+, 8), 152 ([M-CN]+, 7), 84 (92), 66 (100%), 45 (40).

(3-Hydroxy-5-methoxyphenyl)glycine hydrochloride (HMeOPg) (6) yield: 65% 1 HNMR (D2O) δ =6.51 (s, 1H, H-6), 6.47 (s, 1H, H-4), 6.45 (s, 1H, H-2), 4.80 (s, 1H, H-α), 3.62 (s, 3H, OCH3).

13CNMR δ = 168.78, 158.80, 155.52, 132.15, 105.58, 103.65, 100.62, 54.45, 53.37.

HR-FTICR-MS: [M+H]+ m/z: calcd. 198.0761, found 198.0759.

S3 Supporting Information JA0499389 Weist et al.

3-Hydroxyphenylglycine hydrochloride (3-HPg) (7)

This compound was obtained from 3-Methoxyphenylglycine via demethylation using 57% aqueous HI according to literature[2] and purified using HPLC. yield: 50% 1 HNMR (D2O) δ = 7.27 (t, J=8.1Hz, 1H, H-5), 6.87 (m, 3H, H-2, H-4, H-6), 4.87 (s, 1H, H-α).

13CNMR δ = 170.90, 155.59, 133.31, 130.36, 119.23, 116.30, 114.13, 56.32.

HR-FTICR-MS: [M+H]+ (m/z): calcd. 168.0655, found 168.0653.

Synthesis of phenylacetic acids, General procedure To a solution of the benzaldehyde or benzoic acid (10 mmol) in dry THF (20 mL) under argon was added BH3·THF (2 eq; 20 mL of a 1N solution in THF) at 0°C. The mixture was stirred for 12 h before it was quenched by portionwise addition of water (50 mL). The layers were separated and the water phase was extracted with ethyl ether (2 x 20 mL). The solvent was removed to give the crude benzyl alcohol which was used for the next step without further purification. To a solution of the benzyl alcohol (5 mmol) in dry THF (10 mL), phosphorus tribromide (1 eq) was added dropwise under argon. The mixture was stirred for 2 hours before being quenched with water (5 mL). After separation of both layers, the aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined extracts were concentrated and purified by flash chromatography (hexanes:ethyl acetate 5:1) to yield the benzyl bromides. Benzyl bromides (5 mmol) were treated with dry sodium cyanide (1.5 eq) in dry N- methylpyrrolidone (5 mL). The mixture was stirred for 5 h before being diluted with brine. After extraction with ethyl ether and evaporation of the solvent, the crude nitriles (2 mmol) were hydrolyzed in conc. HCl (10 mL) at 70°C for 5 h. After dilution with water (20 mL) followed by extraction with ethyl ether (2 x 10 mL), the crude phenylacetic acids were purified by flash chromatography eluting with CHCl3:MeOH 90:5.

3,5-Dimethoxybenzyl alcohol (8) yield: 93% 1HNMR (DMSO) δ = 6.48 (d, J=2.3 Hz, 2H, H-2, H-6), 6.34 (t, J=2.0Hz, 1H, H-4), 5.19 (t, J= 5.8Hz, CH2OH), 4.42 (d, J= 5.8Hz, 2H, CH2OH), 3.72 (s, 6H, OCH3).

13CNMR δ = 160.73, 145.57, 104.37 , 98.89, 63.23, 55.44 .

LRMS (rel. Intens.) m/z: 168 (M+, 100%), 139 (55), 77 (8).

S4 Supporting Information JA0499389 Weist et al.

3,5-Dimethoxybenzyl chloride (9)

Methanesulfonyl chloride (1.2 eq) and triethylamine (1.1 eq) were used instead of PBr3 to give the chloride in 70% yield.

1 HNMR (CDCl3) δ = 6.46 (d, J=2.0Hz, 2H, H-2, H-6), 6.34 (t, J=2.0Hz, 1H, H-4), 4.44 (s, 2H, CH2), 3.72 (s, 6H, OCH3).

13CNMR δ = 161.33, 139.92, 106.86 , 100.85, 55.80, 46.76.

LRMS (rel. Intens.) m/z: 186 (M+, 100%), 151 ([M-Cl]+, 94), 121 (37), 108 (17), 91 (36), 77 (28), 65 (18), 51 (17).

3,5-Dimethoxyphenylacetic acid (DMeOPA) (10) yield: 60% from chloride (9) 1HNMR (DMSO) δ = 12.31 (s, 1H, COOH), 6.42 (s, 2H, H-2, H-6), 6.38 (s, 1H, H-4), 3.72 (s, 6H, OCH3), 3.49 (s, 2H, CH2).

13CNMR δ = 172.83, 160.67, 137.46, 107.86, 55.49 , 41.27.

HR-FTICR-MS: [2M+Na-2H]- m/z: calcd. 413.1218, found 413.1217.

(3-tert-Butyldimethylsilyloxy-5-methoxy)benzyl alcohol

This compound was prepared by silylation of 3-hydroxy-5-methoxybenzaldehyde according to standard procedures[3] employing TBDMS-Cl and triethylamine in THF and subsequent reduction with borane-THF complex.

(3-tert-Butyldimethylsilyloxy-5-methoxy)benzaldehyde (11) yield: 84% 1 HNMR (CDCl3) δ = 9.88 (s, 1H, CHO), 7.02 (d, J=2.0Hz, 1H, H-6), 6.94 (d, J=2.0Hz, 1H, H-2), 6.65 (d, J=2.2Hz, 1H, H-4), 3.83 (s, 3H, OCH3), 0.99 (s, 9H, Si-tBu), 0.23 (s, 6H, Si-CH3).

13CNMR δ = 192.60, 161.84, 158.03, 115.24, 113.76, 107.22, 56.27, 26.26, 18.87, -3.76.

LRMS (rel. Intens.) m/z: 266 (M+, 32), 209 ([M-tBu]+, 100%), 181 (74), 166 (24), 151 (14), 75 (6), 57 (6).

S5 Supporting Information JA0499389 Weist et al.

(3-tert-Butyldimethylsilyloxy-5-methoxy)benzyl alcohol (12) yield: 74% 1HNMR (DMSO) δ = 6.51 (s, 1H, H-4), 6.41 (s, 1H, H-2), 6.23 (s, 1H, H-6), 5.17 (d, J=5.8Hz, 1H, OH), 4.41 (d, J=5.8Hz, 2H, CH2), 3.71 (s, 3H, OCH3), 0.95 (s, 9H, Si-tBu), 0.19 (s, 6H, Si-CH3).

13CNMR δ = 160.56, 156.36, 145.62, 110.37, 105.23, 104.40, 63.03, 55.36, 25.96, 18.29, -4.13.

+ + + LRMS (rel. Intens.) m/z 268 (M , 20), 211 ([M-tBu] , 26), 193 ([M-tBu-H2O] , 100 %), 181 (8), 135(10), 91 (9), 75(15), 43 (35).

(3-tert-Butyldimethylsilyloxy-5-methoxy)benzyl bromide (13) yield: 80% 1 HNMR (CDCl3) δ = 6.54 (d, J=1.5Hz, 1H, H-2), 6.48 (d, J=1.5Hz, 1H, H-6), 6.33 (d, J=2.3Hz, 1H, H-4), 4.39 (s, 2H, CH2), 3.78 (s, 3H, OCH3), 0.98 (s, 9H, Si-tBu), 0.21 (s, 6H, Si-CH3).

13CNMR δ = 160.68, 156.85, 139.54, 113.36, 107.50, 106.35, 55.32, 33.51, 25.63, 18.17, -4.44.

LRMS (rel. Intens.) m/z: 333 ([M(81Br)]+, 58), 331 ([M(79Br)]+, 60), 275 (58), 273 (60), 251 ([M-Br] +, 100%).

(3-Hydroxy-5-methoxy)benzyl cyanide (14)

Prepared from above by substitution with sodium cyanide in NMP as described. yield: 56% 1 HNMR (CDCl3) δ = 6.36 (s, 1H, H-2), 6.34 (s, 1H, H-6), 6.28 (s, 1H, H-4), 3.90 (s, 2H, CH2), 3.70 (s, 3H, OCH3).

13CNMR δ = 161.10, 159.26, 133.46, 119.54, 107.84, 105.08, 100.57, 55.38, 22.74.

LRMS (rel. Intens.) m/z: 163 (M+, 45), 99 (18), 75 (100%).

(3-Hydroxy-5-methoxy)phenylacetic acid (HMeOPA) (15) yield: 69% 1HNMR (DMSO) δ = 12.23 (br s, 1H, COOH), 9.37 (s, 1H, OH), 6.27 (s, 2H, H-2, H-6), 6.21 (s, 1H, H-4), 3.67 (s, 3H, OCH3), 3.40 (s, 2H, CH2).

13CNMR δ = 173.03, 160.65, 158.69, 137.36, 109.24, 106.36, 99.81, 55.24, 41.49.

HR-EI-MS: [M-H]- m/z: calcd. 181.0579, found 181.0584.

S6 Supporting Information JA0499389 Weist et al.

Synthesis of mandelic acids

A) 3-Hydroxymandelic acid[4] (3-HMA)

3-Hydroxymandelonitrile (16)

A suspension of 3-hydroxybenzaldehyde (35.3 g, 0.25 mol) in 1N sodium bisulfite solution (300 mL) was heated to 50°C for 1h. After cooling to –5°C, a solution of sodium cyanide (13.5 g, 0.275 mol) in water (50 mL) was added dropwise. The mixture was stirred for 12h and extracted with ethyl ether (2x100 mL). Evaporation of the solvent gave 65% of the title compound as a colorless oil.

1HNMR (DMSO) δ =9.64 (s, 1H, Ar-OH), 7.23 (t, J=7.6Hz, 1H, H-5), 6.96 (d, J=6.6 Hz, 1H, H-6), 6.89 (m, 2H, H-2, H-4), 6.79 (s, 1H, α-OH), 5.65 (d, J= 6.3Hz, 1H, H-α).

13CNMR δ = 158.02, 138.95, 130.27, 120.94, 117.21, 116.18, 113.51, 62.02.

LRMS (rel. Intens.) m/z: 122 ([M-HCN]+, 100%), 93 (52), 65 (28), 39 (18).

3-Hydroxymandelic imino ethyl ester hydrochloride (17)

Dry HCl gas was bubbled into a solution of 3-hydroxymandelonitrile (16) (20 g) in dry Et2O (150 mL) and EtOH (13 mL) until no more exothermic reaction was observed. The solution is stored at 5°C for 5 h. During this time, the imino ester separated as a clear gum. After decantation, the gum was desiccated over NaOH in vacuo to give 19 g (69%) of a white powder.

1 + HNMR (DMSO) δ =11.6 (br s, 2H, NH2 ), 9.76 (s, 1H, ArOH), 7.26 (s, 1H, H-6), 7.19 (t, J=7.8 Hz, 1H, H-5), 6.85 (m, 2H, H-2, H-4), 6.79 (s, 1H, α-OH), 5.42 (s, 1H, H-α), 4.45 (q, J=7Hz, 2H, CH2CH3), 1.25 (t, J=7.1 Hz, 3H, CH2CH3).

13CNMR δ = 179.82, 158.11, 139.22, 130.15, 117.81, 116.37, 114.11, 71.22, 70.24, 13.71.

3-Hydroxymandelic acid ethyl ester (18)

The above crude imino ethyl ester (17) (19 g) was dissolved in 0.2N HCl (200 mL) and ethyl ether (100 mL) was added. The solution was stirred for 2h. The layers were separated, the organic layer was dried and concentrated. Filtration through a plug of silica gel eluting with chloroform and evaporation of the solvent yielded the title compound ((19); 14.6 g, 88%).

1HNMR (DMSO) δ = 9.37 (s, 1H, ArOH), 7.13 (t, J=7.8 Hz, 1H, H-5), 6.82 (m, 2H, H-2, H-6), 6.69 (m, 1H, H-4), 5.89 (s, 1H, α-OH), 5.01 (s, 1H, α-H), 4.08 (q, J=7.1Hz, 2H, CH2CH3), 1.14 (t, J=7.1 Hz, 3H, CH2CH3).

13CNMR δ = 172.95, 157.64, 141.30, 129.51, 117.62, 115.14, 113.83, 72.81, 60.67, 14.31.

S7 Supporting Information JA0499389 Weist et al.

LRMS (rel. Intens.) m/z: 196 (M+, 30), 123 (100%), 121 (12), 95 (68), 77 (24), 65 (7).

3-Hydroxymandelic acid (3-HMA) (19)

The above 3-hydroxymandelic acid ethyl ester ((18); 14.6 g) was dissolved in THF (300 mL) and 1N LiOH (300 mL) was added. The reaction mixture was stirred for 12h. After evaporation of the organic layer, the aqueous phase was acidified and extracted 10 times with EtOAc (50 mL). The combined extracts were dried over sodium sulfate to give 3-hydroxymandelic acid ((19); 60%).

1HNMR (DMSO) δ = 12.53 (s, 1H, COOH), 9.37 (s, 1H, ArOH), 7.12 (t, J=7.6 Hz, 1H, H-5), 6.83 (m, 2H, H-2, H-6), 6.67 (d, J=7.6 Hz, 1H, H-4), 5.75 (br s, 1H, α-OH), 4.93 (s, 1H, H-α).

13CNMR δ = 174.52, 157.61, 142.03, 129.52, 117.85, 115.02, 113.94, 72.82.

HR-FTICR-MS: [2M-H]- m/z: calcd. 335.0772, found 335.0771.

B) 3-Hydroxy-5-methoxymandelic acid (HMeOMA)

(3-Benzyloxy-5-methoxy)mandelonitrile (20)

To 3-benzyloxy-5-methoxybenzaldehyde[5] (5 mmol) was added sodium bisulfite solution (1N, 50 mL) and the mixture was heated to 50°C for 30 min. The solution was cooled to 0°C. Ethyl ether (5 mL) was added followed by dropwise addition of a solution of sodium cyanide (5.5 mmol) in water (1 ml). The mixture was stirred for 8 h. Both layers were separated and the organic layer was dried and evaporated to give crude 3-benzyloxy-5-methoxymandelonitrile (21) in 50% yield.

1 HNMR (DMSO) δ = 7.54-7.32 (m, 5H, OCH2Ph), 7.05 (d, J=6.6Hz, 1H, α−ΟH), 6.73 (s, 1H, H-4), 6.65 (s, 1H, H-6), 6.61 (s, 1H, H-2), 5.66 (d, J=6.6Hz, 1H, H-α), 5.10 (s, 2H, OCH2Ph), 3.75 (s, 3H, OCH3).

13CNMR δ = 161.02, 160.08, 139.89, 137.15, 128.81, 128.27, 128.12, 120.74, 105.53, 104.81, 101.58, 69.76, 61.94, 55.72.

LRMS (rel. Intens.) m/z: 242 ([M-HCN]+, 36), 224 (6), 91 (Bn, 100%), 65 (9), 45 (8).

(3-Hydroxy-5-methoxy)mandelic acid (HMeOMA) (21)

A suspension of the above mandelonitrile ((20); 3 mmol) was hydrolyzed using conc. HCl (10 mL) by heating for 2h at 70°C. After having cooled to room temperature, the solution was poured into water (50 ml) and extracted with ethyl acetate. The solvent was evaporated to give crude 3- hydroxy-5-methoxymandelic acid, which was purified by preparative HPLC. yield: 50% starting from (20) 1HNMR (DMSO) δ = 9.41 (br s, 1H, ArOH), 6.44 (s, 2H, H-2, H-6), 6.22 (s, 1H, H-4), 4.88 (s, 1H, H-α), 3.68 (s, 3H, OCH3).

S8 Supporting Information JA0499389 Weist et al.

13CNMR δ = 174.32, 160.48, 106.71, 103.64, 100.58, 73.09, 55.30.

HR-FTICR-MS: [2M-H]- m/z: calcd. 395.0984, found 395.0982.

C) 3,5-Dimethoxymandelic acid (DMeOMA)

To a solution of 3,5-dimethoxybenzaldehyde (2 g, 12 mmol) in MeCN (40 mL) was added TMSCN (1.8 mL; 18.1 mmol; 1.5 eq). The solution was heated to 45°C for 4h and evaporated to dryness after cooling to room temperature. The resulting oil was purified by flash chromatography using CHCl3 as the eluant. The mandelonitrile was hydrolyzed using 6N HCl (50 mL) heating to 70°C for 6h. Charcoal (100 mg) was added, the solution cooled, poured into 100 ml of water and filtered. After evaporation to dryness, the residue was purified using reverse phase flash chromatography eluting with water.

O-TMS-(3,5-dimethoxy)mandelonitrile (22) yield: 89% 1HNMR (DMSO) δ = 6.43 (d, J=2Hz, 2H, H-2, H-6), 6.36 (t, J=2.3Hz, 1H, H-4), 5.75 (s, 1H, H-α), 3.77 (s, 6H, OCH3), 0.18 (s, 9H, Si(CH3)3).

13CNMR δ = 161.20 , 139.34, 120.18, 104.65, 100.95, 62.99, 55.78, 0.03.

3,5-Dimethoxymandelic acid (DMeOMA) (23) yield: 59% from (22) 1HNMR (DMSO) δ = 6.64 (d, J=2.2Hz, 2H, H-2, H-6), 6.48 (t, J=2.2Hz, 1H, H-4), 5.85 (br s, 1H, α-OH), 4.94 (s, 1H, H-α), 3.73 (s, 6H, OCH3).

13CNMR δ = 173.90, 160.22, 142.49, 104.63 , 99.34, 72.36, 55.16.

LRMS (rel. Intens.) m/z: 212 (M+, 85), 167 (80), 139 (100%), 124 (41), 108 (16), 77 (14).

HR-FTICR-MS: [2M-H]- m/z: calcd. 423.1297, found 423.1302.

D) 3-Methoxymandelic acid (3-MeOMA)

Prepared analogously to (C).

O-TMS-(3-methoxy)mandelonitrile (24) yield: 76% 1 HNMR (CDCl3) δ = 7.33 (t, J=7.8Hz, 1H, H-5), 7.05-7.01 (m, 2H, H-2, H-6), 6.92 (dd, J=8.3/1.5 Hz, 1H, H-4), 5.47 (s, 1H, H-α), 3.84 (s, 3H, OCH3), 0.24 (s, 9H, Si(CH3)3).

13CNMR δ = 160.00, 137.69, 130.01, 119.11, 118.50, 114.89, 111.82, 63.52, 55.33, -0.24.

S9 Supporting Information JA0499389 Weist et al.

LRMS (rel. Intens.) m/z: 235 (M+, 46), 220 (100%), 205 (8), 165(8), 146 (30), 135 (19), 89 (10), 84 (16), 75 (8).

3-Methoxymandelic acid (3-MeOMA) (25) yield: 55% 1HNMR (DMSO) δ = 12.53 (br s, 1H, COOH), 7.26 (t, J=8.1Hz, 1H, H-5), 6.99-6.96 (m, 2H, H-2, H-6), 6.85 (dd, J=7.8/2.0Hz, 1H, H-4), 5.85 (br s, 1H, OH), 4.99 (s, 1H, H-α), 3.74 (s, 3H, OCH3).

13CNMR δ = 174.52, 159.62, 142.26, 129.71, 119.41, 113.58, 112.70, 72.80, 55.53.

HR-FTICR-MS: [2M-H]- m/z: calcd. 363.1085, found 363.1083.

S10 Supporting Information JA0499389 Weist et al.

Table S1: Supplementation of mutant ∆dpgA with amino acids, phenylacetic acids and mandelic acids

Y COOH

R2 R1 compound abbreviation R1 R2 Y Antibiotic LC-MS activity of detection of culture glycopeptide broth*) derivatives 3,5-dihydroxyphenylglycine DPg OH OH NH2 + + 3,5-dihydroxymandelic acid DMS OH OH OH + + 3,5-dihydroxyphenylacetic DPA OH OH H + + acid 3-hydroxyphenylglycine 3-HPg OH H NH2 + + 3-hydroxymandelic acid 3-HMA OH H OH + + 3-hydroxyphenylacetic acid 3-HPA OH H H - - 3-methoxyphenylglycine 3-MeOPg OMe H NH2 + + 3-methoxymandelic acid 3-MeOMA OMe H OH - - 3-methoxyphenylacetic acid 3-MeOPA OMe H H - - 3-hydroxy-5-methoxy- HMeOPg OMe OH NH2 + + phenylglycine 3-hydroxy-5- HMeOMA OMe OH OH - - methoxymandelic acid 3-hydroxy-5- HMeOPA OMe OH H - - methoxyphenylacetic acid 3,5-dimethoxyphenylglycine DMeOPg OMe OMe NH2 + + 3,5-dimethoxymandelic acid DMeOMA OMe OMe OH - - 3,5-dimethoxyphenylacetic DMeOPA OMe OMe H - - acid *) assay: Bacillus subtilis (cf. page S1)

S11 Supporting Information JA0499389 Weist et al.

Table S2: HR-FTICR Mass Spectrometry of metabolites from supplementation with 3- methoxyphenylglycine

OH OH R4O

CH2OH OO Cl O O

3 Cl R O OH O H O H H N N N R5 O N NH H H H H O H O NH+ O O 6 H B H R NH -OOC 2 A R1 R2 OH

Mass R1 R2 R3 R4 R5 R6 ABa Mol. Formula found calcd. ∆(ppm) [M+H]+ [M+H]+ c d 1627 OMe H urvcn ovcn Me H + C75H88Cl2N11O26 1628.52959 1628.52735 1.37 c 1486 OMe H urvcn H Me H + C68H77Cl2N10O24 1487.44821 1487.44838 0.11 d 1429 OMe H ovcn H Me H + C66H73Cl2N9O23 1430.42657 1430.42691 0.24 1288 OMe H H H Me H + C59H62Cl2N8O21 1289.34885 1289.34793 0.71 1304 OMe H H H Me H - C60H66Cl2N8O21 1305.37786 1305.37923 0.21 1290 OMe H H H H H - C59H64Cl2N8O21 1291.36331 1291.36358 1.05 c 1128 OMe H H H H H - C53H54Cl2N8O16 1129.31151 1129.31076 0.66 aindicates whether the AB Ring (AA 5→7) is closed (+) or open (-).bnon-glucosylated (Aglycon). curvcn: ureido-vancosamine. dovcn: 4-oxovancosamine.

S12 Supporting Information JA0499389 Weist et al.

Table S3: Mutasynthetic Generation of balhimycin derivatives via supplementation of different 3,5-Dihydroxyphenylglycine analogs

[M+H]+ R1 R2 R3 R4 R5 R6 AB Supplemented m/z ringb mutasynthon 1291.3¶ OH H H H Me H - 1305.3¶ OH H H H Me Me - 3-hydroxyphenylglycine 1129.2¶,c OH H Hc H Me H - (3-HPg) ¶,c c or 1143.2 OH H H H Me Me - 3-hydroxymandelic acid 1430.3 OH H ovcnd H Me H + (3-HMS) 1303.2 OH H H H Me Me + 1628.5 OMe H urvcne ovcnd Me H + 1487.4 OMe H urvcne H Me H + 1430.2 OMe H ovcnd H H H + 3-methoxyphenylglycine 1289.1 OMe H H H H H + (3-MeOPg) 1303.1 OMe H H H Me H + 1291.2¶ OMe H H H H H - 1305.2¶ OMe H H H Me H - 1305.1 OH OMe H H H H + 3-hydroxy-5-methoxy- 1319.1 OH OMe H H Me H + phenylglycine 1319.2 OMe OMe H H H H + 3,5-dimethoxyphenylglycine

References

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