Studies on the Biosynthesis of Paraherquamide

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The remaining oil was distilled under reduced pressure to afford 22 as an ... to 150˚C and 2-[13C2H3]-bromoethane was distilled through a short path distillation.

Studies on the Biosynthesis of Paraherquamide: Concerning the Mechanism of the Oxidative Cyclization of L-Isoleucine to ß- Me-Proline Emily M. Stockinga, Rodolfo A. Martinez,b Louis A. Silks,b Juan F. Sanz-Cerverac, and Robert M. Williamsa* a

b

Department of Chemistry, Colorado State University Fort Collins, Colorado 80523

NIH Stable Isotope Resource, Los Alamos National Laboratory Los Alamos, New Mexico 87545 c

Departamento de Química Orgánica, Facultad de Química, Universidad de Valencia, Calle Dr. Moliner, 50. E-46100 Burjassot, Spain

Supporting information: (1) Synthesis of L-[ 5-13C,5-2H3]isoleucine (14): General considerations. Commercial reagents were used unless otherwise specified. 1H NMR spectra and 13C NMR spectra were obtained on a 300 or 400 MHz Varian NMR spectrometer at the Chemistry Department at Colorado State University. NMR spectra were taken in CDCl3 (1H, 7.24 ppm;

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C 77.0 ppm) and D2 O (1 H, 4.8 ppm) obtained from Cambridge Isotope Labs.

Electrospray mass spectra were obtained using a FisonsVG AutoSpec and exact masses were obtained using a VG Quattro-SQ at the Chemistry Department at Colorado State University.

SCH3

2.

21

13

2

Me

S

C ( H)3 I (65%)

13

N O 24

S O2

(2H)313CCH2MgBr

2. Mg , ether

C ( H) 3

23

22

Me

Me

O

13

1. ( H)3 CCH2MgBr Me

0

2

Me 2

Br , 150 ˚C

1.

1. n-BuLi

2. O=N Cl 3. 1N HCl ( 13%, 5 steps)

Me H313 C

2

NHOH N O 25

S O2

1. Zn, 1N HCl, HOAc 2. LiOH 3. DOWEX ion exch.

Me

OH H NH2 13 2 C H3

(77%, 3 steps) 26

2-[13C2H3]-ethylphenylsulfide (22): To a stirring solution of thioanisole (21, 4.2 mL, 36.1 mmol) in THF at 0˚C, 19.18 mL of a 2.5 M solution on n-BuLi was added dropwise. The resultant mixture was slowly brought to room temperature and stirred for 12h at which time the mixture was again cooled to 0ºC and 13

C2H3-methyl iodidei (4.59 g, 39.7 mmol) was added dropwise to the reaction. The reaction

mixture was slowly brought to room temperature and stirred for 4h. A saturated solution of NH4Cl

(aq)

was added and the mixture was extracted three times with EtOAc. The combined

organic extracts were washed with saturated NH4Cl

(aq)

, dried over anhydrous Na2SO4 and

concentrated in vacuo. The remaining oil was distilled under reduced pressure to afford 22 as an oil. Yield: 3.28 g, 23.5 mmol, 65%. 1

H NMR (400 MHz, CDCl3): ∂ 2.91 (2H, s), 7.13-7.32 (5H, m).

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C NMR (100 MHz, CDCl3): ∂ 13.5 (septet, JC-2H = 20.0 Hz), 27.3 (d, Jc-c= 35.3 Hz), 125.7,

128.8, 129.0. HRMS (EI+): Calcd for C713C1H72H3S2: 142.0725. Found 147.0720 (M+). 2-[13C2H3]-bromoethane: A mixture of 22 (810 mg, 5.69 mmol) and benzyl bromide (1.35 mL, 11.26 mmol) was heated to 150˚C and 2-[13C2H3]-bromoethane was distilled through a short path distillation apparatus over a period of 9 hours.. The crude material was used immediately in the next reaction without further purification. Yield: 557 mg, 4.9 mmol, 88%. (2S, 2’S, 3’S)-N-[2’-(Hydroxyamino)-3’methyl-5’13C2H3-pentanoyl] borane-10,2-sultam (24)ii: An ethereal solution (2 mL) of dibromoethane (61µL, 0.71 mmol) was added dropwise to Mg0 turnings (105 mg, 4.32 mmol in a two neck flask fitted with a reflux condenser. When the bubbling subsided, an ethereal solution (1.2 mL) of 2-[13C 2H3]-bromoethane (370 mg, 3.27 mmol) was added dropwise. The mixture was allowed to react until bubbling subsided and most of the magnesium was consumed (~30 minutes). The Grignard reagent, 23, was directly added via cannula to a 0.07 M solution of 24iii in THF at -78˚C. The reaction was stirred at -78˚C for a total of 36 h at which time a 1M THF solution of 1-chloro,1-nitrosocyclohexaneiv was added dropwise. After stirring an additional 1.5 h, 1 M HCl

2

(aq)

was added to the reaction, the cooling

bath was removed and stirring was continued for 30 minutes before removing the THF in vacuo. An additional 20 mL of 1M HCl

(aq)

was added and the mixture was extracted three times with

hexanes. The aqueous layer was made basic (pH=10) by the addition of solid NaHCO3 and extracted five times with dichloromethane. After drying over anhydrous Na2SO4 and evaporating in vacuo, the crude product (25) was purified by flash column chromatography using a 1:10 mixture of diethylether and dichloromethane. Yield: 124 mg, 0.355 mmol, 15%. 1

H NMR (300 MHz, CDCl3): ∂ 0.92 (3 H, d, J = 7.0 Hz), 0.93 (3H, s), 1.10 (1H, m), 1.15 (3H, s),

1.36 (2 H, m), 1.62 (1H, d, J = 13.2 Hz), 1.90-1.76 (4 H, m), 2.06 (1H, dd, J = 7.7, 13.9 Hz), 2.25 (1 H, m), 3.41 (1H, d, J = 13.9 Hz), 3.50 (1H, d, J = 13.6 Hz), 3.91 (1H, dd, J = 4.7, 7.7 Hz), 4.00 ( 1H, d, J = 7.7 Hz), 5.77 (2H, bs). 13

C NMR (75 MHz, CDCl3): ∂ 10.2 (septet, JC-2H = 18.8 Hz), 16.2, 19.6, 20.6, 24.2 (d, JC-C = 34.9

Hz), 26.4, 32.8, 34.2, 38.3, 44.6, 47.8, 48.6, 53.1, 65.4, 69.0 (d, JC-C =4.0 Hz). HRMS (FAB+): Calcd for C1513C1H262H3N2O4S1: 349.2054. Found 349.2056 (M+H). L-[ 5-13C,5-2H3]isoleucine (14): A mixture of 25 (119 mg, 0.34 mmol) and activated Zn0 powder (887 mg) was stirred in a 2:1 solution of HCl: AcOH for 2 days at 0˚C. The mixture then was filtered through glass wool, the solvent removed in vacuo and the residue made basic by the addition of aqueous NaHCO3. The aqueous solution was extracted three times with dichloromethane. Drying over anhydrous Na2SO4 and concentration in vacuo afforded (2S, 2’S, 3’S)-N-[2’-amino-3’methyl-5’13C2H3-pentanoyl] borane-10,2-sultam as a solid ( 102 mg, 0.31 mmol, 90%). Without further purification, (2S, 2’S, 3’S)-N-[2’-amino-3’methyl-5’13C2H3-pentanoyl] borane-10,2-sultam (82 mg, 0.247 mmol) was saponified by stirring in a 1:4 mixture of 1N LiOH in THF (2.5 mL) at room temperature over 24 hours. The THF was removed under reduced pressure and the residue was partitioned between dichloromethane and water. The aqueous layer was brought to pH=7, evaporated to dryness and re-suspended in de-ionized water before loading onto a DOWEX (50W X2-100) column. The column was rinsed with several portions of water (until the elutant remained clear upon the addition of AgNO3) and then was eluted with 2% NH4OH (aq). Removal of most of the NH4OH (aq) in vacuo followed by lyophilization provided 14 as a colorless white solid.v Yield: 28.5mg, 0.21 mmol, 85%.

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1

H NMR (400 MHz, D2O): ∂ 1.00 (3H, d, J= 7.0 Hz), 1.23 (1H, m), 1.45 (1H, m), 1.93 (1H, bs),

3.56 (1H, bs). 13

C NMR (400 MHz, D2O): ∂ 10.4 (septet, JC-2H = 18.5 Hz), 15.0, 24.2 (d, JC-C = 35 Hz), 36.5,

60.0, 176.1. [α]D25= +11.82˚ (c=0.11, H2O).

(2) Calculation of Percentage Incorporation from Mass Spectral Data: Because the

13

C peaks of interest were partial buried under neighboring signals, nmr

could not be used to determine percentage of incorporation. Instead, results were calculated using electrospray mass spectroscopy. The base peak was the M+1 peak and was set to 100%. Percentage of isotopic enrichment in paraherquamide A from a doubly labeled biosynthetic precursor was determined by subtracting the percentage of base peak intensity (BPI) of the M+3 peak in the labeled paraherquamide A from the percentage of BPI of the M+3 peak in the control paraherquamide A. The percentage of precursor incorporated was determined by multiplying the percentage of isotopic enrichment by the mmol of paraherquamide A produced, dividing this number by the mmol of precursor multiplied by the percentage of isotopic enrichment of the precursor and multiplying the answer by 100. For the feeding experiment with 5-[13C 2H3]-Lisoleucine, the percentage BPI for M+3 was 8.78. The percentage BPI of M+3 for the control was 6.29. Thus the percentage of isotopic enrichment from 5-[13C2H3]-L-isoleucine was 2.49%. 8.2mg, 0.0166 mmol, of Paraherquamide A was isolated and 16.2 mg, 0.1198 mmol, of 100% enriched 5-[13C2H3]-L-isoleucine was used in this feeding experiment. Thus the percentage of 5[13C2H3]-L-isoleucine incorporated was calculated by multiplying 2.49 by 0.0166, dividing this by the product of 0.1198 and 100 and multiplying the answer by 100. We determined in this manner that 0.34% of [13C]-L-isoleucine was incorporated into the paraherquamide A.

4

(3) HSQC spectrum of paraherquamide A from the feeding experiment with 5-[13C2H3]-L-ile. C-16, CH 2

Solvent: CDCl3 Temp. 25.0 C / 298.1 K File: ems_091400_hsqc INOVA-500 "rwindigo2" PULSE SEQUENCE: HSQC Relax. delay 1.000 sec Acq. time 0.160 sec Width 3870.0 Hz 2D Width 16096.6 Hz 16 repetitions 2 x 256 increments OBSERVE H1, 400.1063232 MHz DECOUPLE C13, 100.6148376 MHz Power 43 dB on during acquisition off during delay GARP-1 modulated DATA PROCESSING Gauss apodization 0.074 sec F1 DATA PROCESSING Gauss apodization 0.014 sec FT size 2048 x 2048 Total time 0 min, -1 sec

F2 (ppm)

C-16,

13

2

1

CHH

2.0

H- 16b

2.2

2.4

2.6

2.8

C-20 H- 20

3.0

H- 16a

3.2

3.4

53.5

53.0

52.5

52.0

51.5

51.0

50.5

50.0

49.5

F1 (ppm)

The 13C signal from the deuterium coupled C-16 shows connectivity with the proton at 2.22 ppm, H-16b, but not with the proton at 3.21 ppm, H-16a. This experiment proves H-16a is the deuteron and H-16b is the proton.

i

Synthesis to be disclosed in due course. Also commercially available from Aldrich Chemical Co. Prepared according to the procedure outlined in (a) Oppolzer, W.; Tamura, O., Tetrahedron Lett. 1990, 31, 991-94, (b) Oppolzer, W.; Tamura, O.; Deerberg, J., Helv. Chim. Acta 1992, 75, 1965-1973. iii Prepared according to the procedure outlined in Oppolzer, W.; Barras, J.-P., Helv. Chim Acta 1987, 70, 1666. iv Prepared according to the procedure outlined in Diekmann, H.; Luttke, W., Angew. Chem. Int. Ed., 1968, 7, 387388. v A small amount of allo-L-isoleucine was detected by 1H nmr. ii

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