Supporting Information

© Wiley-VCH 2007

69451 Weinheim, Germany

Reassignment of the structure of the antibiotic A53868 reveals an unusual amino dehydrophosphonic acid

John T. Whitteck, Weijuan Ni, Benjamin M. Griffin, Andrew C. Eliot, Paul M. Thomas, Neil L. Kelleher, William W. Metcalf,

and Wilfred A. van der Donk∗

Tetrahydrofuran (Fischer, ACS Grade) was distilled over Na metal with benzophenone as an indicator under positive

argon pressure, dichloromethane (Fischer, ACS Grade) was distilled over CaH2 under positive argon pressure, N,N-dimethylformamide (Fischer, Sequence Grade) was kept over 4Å molecular sieves, and toluene (Sigma, ACS Grade). All water used was first filtered through a Barnsted E-pure system and had a resistance of at least 18.0 megaohms. Yields refer to chromatographically and spectroscopically pure compounds, except as noted. Starting materials were purchased from Sigma-Aldrich and used without further purification.

All reactions were monitored by thin layer chromatography (TLC) using 0.25" E. Merck precoated silica gel plates. Flash chromatography was performed with E. Merck silica gel 60 (particle size 0.040-0.063 mm). Solvents for chromatography were: hexanes (Fisher, ACS Grade), ethyl acetate (Fischer, ACS Grade), dichloromethane (Fischer, ACS Grade) and methanol (Fischer, ACS Grade) and were used without further purification.

1H, 13C, and 31P NMR spectra were recorded on a Varian Unity 400 or on a Varian Unity 500 spectrometer. Proton and carbon chemical shifts are reported in δ values relative to an external standard of 0.1% tetramethylsilane in CDCl3. Phosphorus shifts are reported in δ values relative to an external standard of 85% phosphoric acid. Data are presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet and/or multiple resonances), integration, assignment.

Mass spectrometry (except FTMS) was performed by the University of Illinois Mass Spectrometry Center. EI mass spectra were performed on a 70-VSE spectrometer. ESI mass spectra were performed on a Quattro spectrometer. FTMS was recorded on a Thermo Finnigan LTQ-FT mass spectrometer (San Jose, CA). Synthetic samples were dissolved in 49% methanol , 49% water, 2% formic acid for direct injection. For HRMS analyses, data were acquired in the FT cell at a nominal resolution of 50,000 and were summed over 1 minute. Collisionally induced dissociation (CID) spectra (50) were acquired in the ion trap at 35% normalized collision energy and 30 ms activation time. For the analysis of the naturally produced A53868, 25µL of an unconcentrated supernatant was injected onto a 2.0 x 75 mm Fusion C18 column (Phenomenex, Torrance, CA) and eluted into the mass spectrometer over a 40 min gradient from 95% water + 0.1% formic acid to 95% acetonitrile + 0.1% formic acid. During this time, both HRMS and CID scans were acquired. Infrared spectra (IR) were recorded on a Mattson Galaxy 5020 spectrophotometer in NaCl cells. Peaks are reported in cm-1.

Reverse phase HPLC purification and analyses were carried out on a Waters HPLC system (model code 6CE), equipped with UV absorbance detector (model 2487), at a wavelength of 220 nm. The products were analyzed and purified on preparative Vydac C18 columns (250 × 22 mm) at flow rate of 8 mL min-1. Production of A53868 by Streptomyces luridis

S. luridis (NRRL 15101) was obtained from the Agricultural Research Service Culture Collection (Peoria, IL). S. luridis was plated on ISP medium 4 (Difco, Sparks, MD) with the following composition (g/L): soluble starch (10), dipotassium phosphate (1.0), magnesium sulfate (1.0), sodium chloride (1.0), ammonium sulfate (2.0), calcium carbonate (2.0), agar (20), ferrous sulfate (0.001), manganous chloride (0.001), and zinc sulfate (0.001) at pH 7.2. After incubating at 30 °C for three days, the agar-solidified medium was liquefied by repeated freezing and subsequent thawing. The resulting supernatant was separated from the residual agar by filtration, before being concentrated ten-fold via rotary evaporation.

15N-labelled A53868 was produced in a similar manner using ISP medium 4 plates that contained 15N-ammonium sulfate (98+ atom% 15N, Isotec, Miamisburg, OH) as the sole nitrogen source. Double-labeled 15N13C A53868 was produced by S. luridis in broth culture using a modified ISP medium 4 that did not contain agar or soluble starch. The carbon source was 13C6-D-glucose (10 g/L, 99 atom% 13C, Isotec, Miamisburg, OH), and the nitrogen source was 15N-ammonium sulfate (2 g/L). The liquid cultures were incubated for three weeks at 30 °C for three days on a rotary shaker at 225 rpm. Biomass was removed by filtration through a 0.22 µM filter and the supernatant was concentrated ten-fold via rotary evaporation. Purification of Bacterial Cell Free Broth Supernatant was purified via reverse phase HPLC (C18) using the following conditions: Solvent A = 79.9% acetonitrile, 19.9% deionized H2O, 0.09% trifluoroacetic acid. Solvent B: 99.9% deinoized H2O, 0.1% trifluoroacetic acid. A linear gradient of 2:98 to 25:75 (A:B) over 45 min was used. The retention time (tR) for A53868 was 21 min. Acetonitrile and trifluoroacetic acid were removed under reduced pressure then the sample was flash frozen in liquid N2 and water was removed by lyophilization on a LABCONCO (model Freezone 4.5) at -49 oC. Unlabeled A53868 1H NMR (400 MHz, D2O) δ 0.73 (d, J = 6.2 Hz, 3H), 0.78 (d, J = 6.2 Hz, 3H), 1.46 (m, 3H), 3.34 (d, J = 11 Hz, 3H), 3.72 (s, 2H), 4.25 (m, 1H), 5.52 (d, J = 15 Hz, 1H), 6.01 (d, J = 36 Hz, 1H); 31P NMR (400 MHz, D2O) 10.5; HRMS (FTMS+) calcd

5

m/z (C11H22N3O5P+H+)+ 308.1365, found 308.1367 15N-Labeled A53868 1H NMR (400 MHz, D2O) 0.73 (d, J = 6.2 Hz, 3H), 0.78 (d, J = 6.2 Hz, 3H), 1.46 (m, 3H), 3.34 (d, J = 11 Hz, 3H), 3.72 (s, 2H), 4.25 (m, 1H), 5.52 (dd, J = 6, 16 Hz, 1H), 6.01 (dd, J = 2, 36 Hz, 1H); 31P NMR (400 MHz, D2O) 10.5 (d, J = 8.5 Hz) 15N,13C-Labeled A53868 13C NMR (400 MHz, D2O) 21.7 (d, J = 34 Hz), 22.9 (d, J = 34.9 Hz), 25.2 (m), 40.6 (m), 41.2 (m), 52.9 (d, J = 9 Hz), 54.2 (m), 117.1 (dd, J = 12, 73 Hz), 135.7 (ddd, J = 11, 73, 190 Hz), 167.9 ( dd, J = 17, 51 Hz), 174.2 (dd, J = 7.4, 14.4, 52.8 Hz) Synthesis of the previously reported structure of A53868

(9H-Fluoren-9-yl)methyl 2-((benzyloxy)phosphono)allylcarbamate (5) Prior to use, all solvents for this reaction were degassed by bubbling Ar through the solvent for 1 h. To a mixture of diphenyl phosphinic acid (12.3 mg, 4 mol%) and Ni(cod)2 (8 mg, 2 mol%) was added a solution of dibenzyl phosphite (350 mg, 1.34 mmol, 1 eq), dimethyl phenyl phosphine (13.8 mg, 8 mol%) and 2 (350 mg, 1.34 mmol, 1 eq) in THF (5 mL). The resulting suspension was heated at 65 oC on an oil bath for 5 h, allowed to cool to room temperature, concentrated to 2 mL under reduced pressure, and purified by silica gel flash chromatography (1:1 EtOAc:Hexanes – 100% EtOAc) to yield 5 (318 mg, 0.59 mmol, 44%) as an oil and the E-isomer 6 (166 mg, 0.31 mmol, 23%) as an oil. Compound 5: 1H NMR (400 MHz, CDCl3) δ 3.92 (m, 2H, N-CH2), 4.21 (m, 1H, CH-CH2, Fmoc), 4.37 (m, 2H, CH-CH2, Fmoc), 5.03 (m, 4H, CH2-Ph), 5.90 (d, 47.2 Hz, 1H, C=CH), 6.04 (d, J=21 Hz, 1H, C=CH), 7.39-7.22 (m, 14H, Ar), 7.77 (d, J=7.2 Hz, 2H, Ar); 13C NMR (400 MHz, CDCl3) δ 156.0, 143.7, 141.2, 135.7, 130.8, 128.4, 127.9, 127.6, 126.9, 124.9, 119.8, 67.5, 66.6, 47.0, 42.2; 31P NMR (400 MHz, CDCl3) δ 19.0; IR (thin film) 3291, 1721, 1521, 1450, 1246, 995; HRMS (ESI+) calcd m/z for (C32H31NO5P+Na+)+ 540.1948, found: 540.1940.

HN

PO

OBnOBn

Fmoc

Compound 6. 1H NMR (400 MHz, CDCl3) δ 3.89 (bs, 2H, N-CH2), 4.2 (t, J = 5.6 Hz, 1H, CH-CH2), 4.44 (d, J = 6.8 Hz, 2H, CH-CH2), 4.70 (bs, 1H), 5.03-5.00 (m, 4H, CH2-Ph), 5.70-5.65 (m, 1H, C=CH), 6.78-6.62 (m, 1H, C=CH), 7.41-7.27 (m, 14H, Ar), 7.77-7.41 (m, 4 H, Ar); 13C NMR (400 MHz, CDCl3) δ 148.9, 148.8, 143.7, 141.3, 128.6, 128.5, 128.4, 128.0, 127.7, 127.6, 124.9, 120.0, 116.3, 67.4, 67.3, 60.4, 47.1, 42.8; 31P NMR (400 MHz, CDCl3) δ 19.0; IR (thin film) 3413, 1718, 1534, 1450, 1246, 996; HRMS (ESI+) calcd m/z for (C32H31NO5P+Na+)+ 540.1948, found: 540.1940.

-Aminomethyl-vinyl)-phosphonic acid dibenzyl ester (7). To a solution of 5 (312 mg, 0.58 mmol, 1 eq) in 3 mL CH2Cl2

-methyl}-vinyl)-phosphonic acid dibenzyl ter (8). A mixture of 7 (24 mg, 0.076 mmol, 1 eq), HOBt (12 mg, 0.083 mmol, 1.1 eq) and Z-Gly-Leu-OH (23 mg, 0.076

H2NPO

OBnOBn

(1was added 1,8-diazabicyclo[5.4.0]undec-7-ene (159 µL, 1.04 mmol, 1.8 eq). The solution was stirred at room temperature for 30 min and then purified by silica gel flash chromatography (1:10 CH3OH:CH2Cl2, Rf = 0.3) to yield 7 (13 mg, 0.38 mmol, 67%) as an oil. 1H NMR (400 MHz, CDCl3) δ 3.45 (d, J = 9.6 Hz, 2H, N-CH2), 5.02 (d, J = 8.4 Hz, 4H, CH2-Ph), 5.95 (d, J = 48.8 Hz, 1H, C=CH), 6.11 (d, J = 22.8 Hz, 1H, C=CH), 7.32 (s, 10H, Ph); 13C NMR (400 MHz, CDCl3) δ 137.5, 136.0, 130.3, 130.2, 128.6, 128.5, 128.3, 128.2, 128.0, 127.9, 127.7, 67.3, 67.2, 49.2 (d, J = 12.5 Hz); 31P NMR (400 MHz, CDCl3) δ 20.0; HRMS (ESI+): calcd m/z for (C17H21NO3P+H+)+ 318.1259, found 318.1252.

(1-{[2-(3-Imino-4-oxo-4-phenyl-butyrylamino)-4-methyl-pentanoylamino]

NH O

HN

O

NH

PO

OBnOBnO

O

esmmol 1 eq) was dissolved in 1 mL of DMF. While stirring, diisopropylcarbodiimide (11 mg, 0.083 mmol, 1.1 eq) was added and the yellow solution was stirred at room temperature for 6 h. This was diluted with EtOAc (50 mL), washed with 5% aqueous citric acid (25 mL), saturated NaHCO3 (25 mL), and brine (30 mL). The organic layer was dried over MgSO4, filtered, and concentrated via rotary evaporation. Purification by silica gel flash chromatography (100% EtOAc - 1:30 CH3OH:CH2Cl2) yielded 8 (37 mg, 0.61 mmol, 81%) as an oil. 1H NMR (500 MHz, CDCl3) δ 0.88 (m, 6H, CH3), 1.75-1.50 (m, 3H, CH2 + CH), 3.79-3.78 (m, 2H, N-CH2), 3.97-3.93 (m, 2H, CH2-C=C), 4.43 (m, 1H, N-CH), 4.99 (m, 4H, CH2-Ph), 5.10 (s, 2H, O-CH2-Ph), 5.83 (s, 1H), 5.85 (d, J = 47.0 Hz, 1H, C=CH), 6.00 (d, J = 22 Hz, 1H, C=CH), 6.36 (d, J = 8.5 Hz, 1H, NH), 6.82 (s, 1H, NH), 7.36-7.28 (m, 15H, Ar); 13C NMR (500 MHz, CDCl3) δ 171.7, 169.4, 156.7, 136.0, 135.7, 135.6, 133.9, 130.8, 128.7, 128.6, 128.4, 128.2, 128.0, 67.8 (J = 5.6 Hz), 67.1, 51.7, 44.9, 41.2, 40.5, 24.7, 24.6, 22.9, 21.7; 31P NMR (CDCl3) δ 19.0; IR (thin film) 3290, 2956, 1715, 1538, 1455, 1239, 995; HRMS (ESI+) calcd m/z for (C33H41N3O7P+H+)+ 622.2682, found 622.2678.

6

.1 mol, 1 eq) was dissolved in 1 mL of dry toluene. The solution was cooled to 0 C on an ice-water bath and a 1 M BBr3 lution in dichloromethane (0.3 mL, 0.3 mmol, 3 eq) was added while stirring. The solution was heated at 70 oC on an oil bath r 5 h and then allowed to cool to room temperature. To this solution was added 1 mL of MeOH and the reaction was stirred

Figure S1. X-ray structure of Synthesis of A53868

N-Carboxybenzyl-glycinyl-leIn a 500 mL round bottom flask L-leucine methyl ester hydrochloride (10.4 g, 50 mmol, 1 eq.), N-carboxybenzyl-glycin d O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (20.8 g, 55 mm mL of DMF. While stirring, N-methylmorpholine (12

n was stirred overnight at room temperature. The solution was poured

H2NO

HN

O

NH

PO

OHOH

(1-{[2-(2-Amino-acetylamino)-4-methyl-pentanoylamino]-methyl}-vinyl)-phosphonic acid (2). Compound 7 (60mg, 0

omsofofor 10 min, and dried under reduced pressure. The remaining residue was dissolved in water (20 mL) and washed with EtOAc (2x20 mL). The aqueous layer was lyophilized and purified by preparative reverse phase HPLC (C18) via isocratic elution with 5:95 CH3CN:H2O (with 0.1% TFA). 1H NMR (500 MHz, CDCl3) δ 0.88 (m, 6H, CH2), 1.75-1.50 (m, 3H, CH2+CH), 3.70 (s, 2H, N-CH2), 3.97-3.84 (d, J = 8.5 Hz, 2H, CH2-C=C), 5.54 (d, 43.5 Hz, 1H, C=CH), 5.69 (d, J = 21.5 Hz, 1H, C=CH); 13C NMR (500 MHz, CDCl3) δ 175.7, 168.1, 140.1 (J = 168 Hz), 125.5 (J = 7.3 Hz), 53.8, 41.7 (J = 19.3 Hz), 41.3, 40.9, 40.9, 25.3, 23.0, 22.7, 21.8, 21.5; 31P NMR (500 MHz, CDCl3) δ 14.0; HRMS (ESI+) calcd m/z for (C11H23N3O5P+H+)+ 308.1371, found 308.1378.

synthetic peptide 2.

O NH

O

O

HN

O

OMe

ucine methyl ester (12) with a magnetic stir bar, a mixture of

e (9 g, 50 mmol, 1 eq.) anol, 1.1 eq.) was dissolved in 250

mL, 110 mmol, 2.2 eq.) was added. The yellow solutiointo a 1 L separatory funnel, diluted with 500 mL of EtOAc, washed with aqueous 5% citric acid (1x200 mL), saturated aqueous NaHCO3 (1x300 mL), and brine (1x200 mL). The organic layer was saved and dried over Na2SO4, filtered, and concentrated via rotary evaporation. Purification by flash silica gel chromatography (1:1 EtOAc:hexanes) afforded (12) (15.2 g, 45.2 mmol, 90%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 0.91 (d, J = 4.9, 6H, CH3), 1.61 (m, 3H, CH, CH2), 3.71 (s, 3H, OCH3), 3.91 (m, 2H, N-CH2), 4.63 (m, 1H, N-CH), 5.12 (s, 2H, Ph-CH2), 5.54 (bs, 1H, NH), 6.53 (bs, 1H, NH), 7.34 (m, 5H, Ph); 13C NMR (400 MHz, CDCl3) δ 22.0, 23.0, 25.0, 41.6, 44.6, 50.9, 52.6, 67.4, 128.3, 128.4, 128.7, 136.3, 156.8, 169.0, 173.5; HRMS (ESI+) calcd m/z (C17H24N2O5+H+)+ 337.1763, found: 337.1755

7

O NH

O

O

HN

O

NH2

N-Carboxybenzyl-glycinyl-leucinamide (13) In a 500 mL round bottom flask with a magnetic stir bar (12) (11 g, 33 mmol) was dissolved in 100 mL of methanol. To this colorless solution was then added 100 mL of NH4OH (28-30% ammonia) and the reaction mixture was stirred for 2 d at room temperature. The yellow solution was poured into a 1 L separatory funnel and diluted with 200 mL of brine. The product was extracted with EtOAc (3x300 mL). The organic layers were combined and washed with 100 mL brine, dried over Na2SO4, filtered and concentrated to dryness under reduced pressure to afford 8.45 g of (13) (26.2 mmol, 80%) as a white foam. 1H NMR (400 MHz CD3CN) δ 0.89 (d, J = 6.2 Hz, 3H, CH3), 0.92 (d, J = 6.3 Hz, 3H, CH3), 1.583 (m, 3H, CH + CH2), 3.75 (d, J = 6.0 Hz, 2H, N-CH2), 4.32 (m, 1H, N-CH), 5.10 (d, J = 1.5, 2H, Ph-CH2), 6.01 (bs, 1H, CO-NH), 6.20 (t, J = 5.8 Hz, 1H, NH-CH2), 6.36 (bs, 1H, CO-NH), 7.04 (d, J = 7.5, 1H, NH-CH) 7.39 (m, 5H, Ph); 13C NMR (400 MHz, CD3CN) δ 21.0, 22.7, 40.8, 44.3, 51.7, 66.6, 128.1, 128.2, 128.7, 137.2, 157.2, 169.8, 175.0 HRMS (ESI+); calcd m/z (C16H23N3O4+H+) 322.1767, found 322.1763

Acetylphosphonate dimethyl ester (14)

P

O

O

OMe

OMe

In a dry three-necked round bottom flask with a magnetic stir bar under positive N2 pressure, acetyl chloride (3.5 mL, 50 mmol, 1 eq) was cooled to 0 oC in an ice-water bath. Then, trimethyl phosphite (5.9 mL, 50 mmol, 1 eq) was slowly added over a period of 1 h, which resulted in evolution of MeCl. After full addition of trimethyl phosphite, the colorless solution was allowed to warm to room temperature and stirred until evolution of gas ceased (about 1 h). Unreacted materials were removed under vacuum to afford (14) (7.3 g, 48 mmol, 96%) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 2.47 (d, J = 5.28 Hz, 3H, CH3), 3.85 (d, J = 10.71, 6H, OCH3); 13C NMR (400 MHz, CDCl3) 30.90 (d, J = 59.5 Hz), 54.07 (d, J = 7.00 Hz), 208.40 (d, J = 170.4 Hz); 31P NMR (500 MHz, CDCl3) δ 0.02 HRMS (EI) calcd m/z (C4H9O4P) 152.0328, found 152.0326

O NH

O

O

HN

O

NH

PO

OMe

OMe

N-Carboxybenzyl-glycinyl-leucinyl-1-amidoethylene-1-phosphonate dimethyl ester (15). In a 250 mL round bottom flask equipped with a Dean-Stark apparatus, Ar was bubbled through 75 mL of toluene for 1 h. 4-Hydroxyanisole (253 mg, 2.04 mmol, 20 mol%) was added with stirring. After all 4-hydroxyanisole was dissolved, 13 (3.28 g, 10.2 mmol, 1 eq) and 14 (1.55 g, 10.2 mmol, 1 eq) and p-toluenesulfonic acid monohydrate (194 mg, 1.02 mmol, 10% mol) were added and the suspension was heated under reflux overnight resulting in a yellow solution. The reaction mixture was allowed to cool to room temperature and toluene was removed via rotary evaporation. The resulting red foam was taken up in 500 mL of EtOAc, washed with saturated aqueous NaHCO3 (1x200 mL), and brine (1x150 mL). The organic layer was dried over Na2SO4, filtered, and concentrated via rotary evaporation. Purification by silica gel flash chromatography (30:1 DCM:MeOH) yielded 15 (519 mg, 1.14 mmol, 11.2%) as a white foam. 1H NMR (500 MHz, CDCl3) δ 0.86 (d, J = 5.5 Hz, 3H, CH2), 0.89 (d, J = 5.4 Hz, 3H, CH3), 1.55 (m, 3H, CH + CH2), 3.69 (d, J = 11.2 Hz , 6H, OCH3), 3.89 (d, J = 5.38 Hz, 2H, N-CH2), 4.62 (m, 1H, N-CH), 5.08 (s, 2H, Ph-CH2), 5.58 (d, J = 19.1 Hz, 1H, C=CH), 5.95 (m, 1H, NH), 6.61 (d, J = 41.7 Hz, 1H, C=CH), 7.05 (d, J = 8.7 Hz, 1H, NH), 7.30 (m, 5H, Ph), 8.29 (d, J = 7.8 Hz, 1H, NH); 13C NMR (500 MHz, CDCl3) 22.09, 23.08, 24.92, 40.90, 44.50, 52.63, 53.58 (d, J = 5.5 Hz), 67.35, 116.29 (d, J = 10.0 Hz), 128.30, 128.45, 128.76, 130.22 (d, J = 201 Hz), 136.37, 156.97, 170.00, 172.07 (d, J = 10.0 Hz); 31P NMR (400 MHz, CDCl3) 15.7; HRMS (ESI+) calcd m/z (C20H30N3O7P+H+)+ 456.1900 found 456.1901

H2NO

HN

O

NH

PO

OMe

OH

Glycinyl-leucinyl-1-amidoethylene-1-phosphonate monomethyl ester (3) In a clean 25 mL round bottom flask equipped with a magnetic stir bar, 15 (433 mg, 0.95 mmol, 1 eq) was dissolved in 5 mL of dry DCM. Then, triethylsilane (303 µL, 1.9 mmol, 2 eq), palladium(II) chloride (16 mg, 0.09 mmol, 10% mol), and triethylamine (50 µL, 0.66 mmol, 70%) were added while stirring. The black suspension was stirred at room temperature for 2 h, filtered over a column of Celite 545®, and concentrated to dryness under reduced pressure to yield a yellow oil. This oil was

8

suspended in 3 mL of an aqueous solution of 10% NaOH and stirred vigorously overnight at room temperature. The resulting yellow solution was diluted to 10 mL with H2O, filtered through a 0.22µm filter unit, and purified by HPLC (C18) using the same method to isolate A53868 from cell free broth to yield 175 mg of (3) (0.57 mmol, 60%) as a white solid. 1H NMR (400 MHz, D2O) δ 0.68 (d, J = 5.5 Hz, 3H, CH3), 0.72 (d, J = 5.7 Hz, 3H, CH3), 1.45 (m, 3H, CH2, CH), 3.28 (d, J = 11 Hz. 3H, OCH3), 3.65 (s, 2H, N-CH2), 4.21 (m, 1H, N-CH), 5.53 (d, J = 16 Hz, 1H, C=CH), 5.95 (d, J = 36 Hz, 1H, C=CH); 13C NMR (400 MHz, D2O) 20.69, 22.14, 24.4, 39.7, 40.3 52.1 (d, J = 5.2), 53.4, 117.6 (d, J = 11.7), 134.5 (d, J = 190.4), 167.2, 173.8; 31P NMR (400 MHz, D2O) 10.5; HRMS (FTMS+) calcd m/z (C11H22N3O5P+H+)+ 308.1365, found 308.1367

9

NMR Spectra 1H NMR of Isolated Unlabeled A53868

A53868

31P NMR of Isolated Unlabeled A53868

A53868

10

HRMS of Unlabeled A53868

A53868

1H NMR of Isolated 15N Labeled A53868

15N Labeled A53868

11

31P NMR of Isolated 15N Labeled A53868

15N Labeled A53868

13C NMR of Isolated 15N13C Labeled A53868

15N13C Labeled A53868

12

1H NMR of (1-{[2-(2-Amino-acetylamino)-4-methyl-pentanoylamino]-methyl}-vinyl)-phosphonic acid (2)

H2NO

HN

O

NH

PO

OHOH

13

13C NMR of (1-{[2-(2-Amino-acetylamino)-4-methyl-pentanoylamino]-methyl}-vinyl)-phosphonic acid (2)

H2NO

HN

O

NH

PO

OHOH

14

1H NMR of N-Carboxybenzyl-glycinyl-leucine methyl ester

O NH

O

O

HN

O

OMe

15

13C NMR of N-Carboxybenzyl-glycinyl-leucine methyl ester

O NH

O

O

HN

O

OMe

HRMS of N-Carboxybenzyl-glycinyl-leucine methyl ester

O NH

O

O

HN

O

OMe

16

1H NMR of N-Carboxybenzyl-glycinyl-leucamide

O NH

O

O

HN

O

NH2

13C NMR of N-Carboxybenzyl-glycinyl-leucamide

O NH

O

O

HN

O

NH2

17

HRMS of N-Carboxybenzyl-glycinyl-leucinamide

O NH

O

O

HN

O

NH2

1H NMR of Acetylphosphonate dimethyl ester

P

O

O

OMe

OMe

18

13C NMR of Acetylphosphonate dimethyl ester

P

O

O

OMe

OMe

31P NMR of Acetylphosphonate dimethyl ester

P

O

O

OMe

OMe

19

1H NMR of Glycinyl-leucinyl-1-amidoethylene-1-phosphonate monomethyl ester (A53868)

H2NO

HN

O

NH

PO

OMe

OH

20

13C NMR of Glycinyl-leucinyl-1-amidoethylene-1-phosphonate monomethyl ester (A53868).

H2NO

HN

O

NH

PO

OMe

OH

31P NMR of Glycinyl-leucinyl-1-amidoethylene-1-phosphonate monomethyl ester

H2NO

HN

O

NH

PO

OMe

OH

21

HRMS of Glycinyl-leucinyl-1-amidoethylene-1-phosphonate monomethyl ester

H2NO

HN

O

NH

PO

OMe

OH

22