Supporting Information
Ligand-induced expansion of the S1 site in the anthrax toxin lethal
factor
Kimberly M. Maize, Elbek K. Kurbanov, Rodney L. Johnson, Elizabeth Ambrose Amin,
and Barry C. Finzel*
308 Harvard St SE, 8-101 Weaver-Densford Hall, Minneapolis, MN, 55455, United
States
*To whom correspondence should be addressed. Phone: (612) 626 5979. Fax: (612) 626 3114.
Email: finze007@umn.edu
Representative Procedure for Synthesis of 10-12
(R)-tert-Butyl 2-(4-fluoro-3-methylphenylsulfonamido)-3-methylbutanoate (10)
D-Valine
tert-butyl ester hydrochloride (6, 0.6 g, 2.9 mmol) was added to a solution of K2CO3 (0.8 g, 6.0
mmol) in dioxane/water (6 mL, 1:1, v/v). To this, a solution of 4-fluoro-3-methylphenyl-sulfonylchloride (7,
0.5 g, 2.4 mmol) in dioxane (1 mL) was added immediately after with vigorous stirring. The mixture was
stirred at rt overnight. Upon consumption of the starting material as determined by TLC, the solvent was
reduced to one-third the reaction volume under reduced pressure. The aq. layer was extracted with
EtOAc (3 × 20 mL). The combined organic layers were washed with sat. aq. NH 4Cl (1 × 20 mL) and brine
(1 × 20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product
was purified over SiO2 using an eluent of EtOAc/hexane (1/5, v/v) to yield 10 as a white solid (0.8 g,
94%). 1H NMR (400 MHz, CDCl3) δ 7.77-7.72 (m, 2H), 7.10 (t, J = 8.4 Hz, 1H), 5.77 (d, J = 10.0 Hz, 1H,
NH), 3.66 (dd, J = 10.0 Hz, J = 4.4 Hz, 1H), 2.30 (s, 3H), 2.11-2.0 (m, 1H), 1.26 (s, 9H), 1.0 (d, J = 6.8 Hz,
3H), 0.88 (d, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.12, 163.46 (d, J = 251.2 Hz), 135.72 (d, J
= 3.8 Hz), 130.91 (d, J = 6.8 Hz), 127.26 (d, J = 9.9 Hz), 125.91 (d, J = 18.2 Hz), 115.43 (d, J = 23.5 Hz),
82.0, 61.31, 31.49, 27.46, 18.93, 17.02, 14.24 (d, J = 3.8 Hz).
(R)-tert-Butyl 2-(4-fluoro-3-methoxyphenylsulfonamido)-3-methylbutanoate (11)
White solid (202 mg, 29%). 1H NMR (400 MHz, CDCl3) δ 7.50-7.44 (m, 2H), 7.16 (t, J = 8.8 Hz, 1H), 5.57
(d, J = 10.4 Hz, 1H, NH), 3.11 (s, 3H), 3.64 (dd, J = 10.0 Hz, J = 4.4 Hz, 1H), 2.10-2.02 (m, 1H), 1.26 (s,
9H), 1.0 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 170.17, 154.73 (d, J =
252.8 Hz), 147.97 (d, J = 11.4 Hz), 136.04 (d, J = 3.8 Hz), 120.61 (d, J = 8.4 Hz), 116.14 (d, J = 19.8 Hz),
112.41 (d, J = 3.0 Hz), 82.28, 61.36, 56.37, 31.55, 27.58, 18.98, 17.02.
(R)-tert-Butyl 2-(3-(methoxymethyl)phenylsulfonamido)-3-methylbutanoate (12)
White solid (250 mg, 79%). 1H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.53 (d, J
= 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 5.54 (d, J = 10.0 Hz, 1H, NH), 4.49 (s, 2H), 3.65 (dd, J = 9.6 Hz, J
= 4.4 Hz, 1H), 3.39 (s, 3H), 2.08-2.01 (m, 1H), 1.22 (s, 9H), 0.99 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.4 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 170.12, 140.03, 139.77, 131.39, 128.99, 126.30, 126.02, 82.12, 73.47,
61.26, 58.22, 31.58, 27.57, 18.99, 17.05.
Representative Procedure for N-Alkylation (13-15)
(R)-tert-Butyl 2-(4-fluoro-N-isobutyl-3-methylphenylsulfonamido)-3-methylbutanoate (13)
To a solution of 10 (0.4 g, 1.0 mmol) and 1-iodo-2-methylpropane (0.3 mL, 3.0 mmol) in anhydrous DMF
(2.0 mL) was added K2CO3 (0.7 g, 5.0 mmol). The reaction mixture was stirred at room temperature. After
48 h, the solvent was removed under reduced pressure and the resulting residue was taken up in H 2O,
extracted with EtOAc (3 × 15 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified over SiO2 using an eluent of EtOAc/hexane (1/10, v/v) to yield 13 as a
colorless oil (240 mg, 59%). 1H NMR (400 MHz, CDCl3) δ 7.72-7.68 (m, 2H), 7.09 (t, J = 8.4 Hz, 1H), 4.0
(d, J = 10.4 Hz, 1H), 3.27CHA (dd, J = 14.8 Hz, J = 6.0 Hz, 1H), 3.05CHB (dd, J = 14.8 Hz, J = 8.8 Hz, 1H),
2.33 (s, 3H), 2.14-2.05 (m, 2H), 1.32 (s, 9H), 1.06 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.85 (t, J =
6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 169.27, 163.39 (d, J = 251.3 Hz), 136.58 (d, J = 3.8 Hz),
131.13 (d, J = 6.0 Hz), 127.46 (d, J = 9.1 Hz), 125.73 (d, J = 18.2 Hz), 115.38 (d, J = 23.5 Hz), 81.72,
67.15, 53.20, 29.1, 27.87, 27.74, 20.57, 20.26, 19.86, 19.40, 14.53 (d, J = 3.8 Hz).
(R)-tert-Butyl 2-(4-fluoro-N-isobutyl-3-methoxyphenylsulfonamido)-3-methylbutanoate (14)
Colorless oil (153 mg, 66%). 1H NMR (400 MHz, CDCl3) δ 7.49-7.43 (m, 2H), 7.16 (t, J = 8.4 Hz, 1H),
3.99-3.96 (m, 4H), 3.26 CHA (dd, J = 14.4 Hz, J = 6.0 Hz, 1H), 3.05 CHB (dd, J = 14.4 Hz, J = 8.8 Hz, 1H),
2.13-2.04 (m, 2H), 1.30 (s, 9H), 1.06 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 7.2 Hz, 6H).
13C NMR (100 MHz, CDCl ) δ 169.17, 154.64 (d, J = 252.8 Hz), 147.68 (d, J = 11.4 Hz), 136.92 (d, J = 3.8
3
Hz), 120.85 (d, J = 7.6 Hz), 116.14 (d, J = 19.0 Hz), 113.13 (d, J = 3.0 Hz), 81.9, 67.46, 56.53, 53.34,
29.16, 28.0, 27.76, 20.6, 20.37, 19.93, 19.43.
(R)-tert-Butyl 2-(N-isobutyl-3-(methoxymethyl)phenylsulfonamido)-3-methylbutanoate (15)
Colorless oil (163 mg, 56%). 1H NMR (400 MHz, CDCl3) δ 7.82-7.78 (m, 2H), 7.53 (d, J = 8.0 Hz, 1H),
7.47 (t, J = 7.6 Hz, 1H), 4.50 (s, 2H), 4.03 (d, J = 10.8 Hz, 1H), 3.39 (s, 3H), 3.26 CHA (dd, J = 14.8 Hz, J =
6.4 Hz, 1H), 3.07 CHB (dd, J = 14.0 Hz, J = 8.8 Hz, 1H), 2.14-2.04 (m, 2H), 1.29 (s, 9H), 1.04 (d, J = 6.4
Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 169.32, 141.08, 139.45, 131.16, 128.82, 126.66, 126.30, 81.70, 73.62, 67.12, 58.23, 53.14,
29.03, 27.79, 27.74, 20.59, 20.24, 19.84, 19.42.
Representative Procedure for tert-Butyl Deprotection (16-18)
To an ice-chilled solution of 13 (0.2 g, 0.6 mmol) in DCM (2.0 mL) was added TFA (1.0 mL) dropwise. The
reaction mixture was stirred for 6 hours at rt. Upon consumption of the starting material as determined by
TLC, the solvent was removed under reduced pressure, and the product was submitted to the next
reaction without further purification.
Representative Procedure for the Synthesis of 19-21
(2R)-2-(4-Fluoro-N-isobutyl-3-methylphenylsulfonamido)-3-methyl-N-((tetrahydro-2H-pyran-2yl)oxy)butanamide (19)
To a solution of 16 (130 mg, 0.38 mmol) in DMF (2 mL), HOBt (62 mg, 0.46 mmol), NMM (125 μL, 1.14
mmol), THPONH2 (137 mg, 1.17 mmol) and EDC (102 mg, 0.53 mmol) were added. The mixture was
stirred at rt overnight. Upon consumption of the starting material as determined by TLC, H 2O (10 mL) was
added. The aq. layer was extracted with EtOAc (3 × 20 mL).The combined organic layers were washed
with saturated solution of NaHCO3 (1 × 20 mL), brine (1 × 20 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified over SiO 2 using an eluent of
EtOAc/hexane (1/4, v/v) to yield a colorless oil (50 mg, 30%). 1H NMR (400 MHz, CDCl3) δ 9.43 (s, 1H),
7.72-7.68 (m, 2H), 7.09 (t, J = 8.4 Hz, 1H), 4.95 (s, 1H), 4.0-3.90 (m, 1H), 3.69-3.54 (m, 2H), 3.30-3.21
(m, 1H), 3.01-2.96 (m, 1H), 2.33 (s, 3H), 2.25-2.05 (m, 2H), 1.92-1.6 (m, 6H), 0.93-0.81 (m, 9H), 0.5-0.39
(m, 3H). 13C NMR (100 MHz, CDCl3) δ 167.10, 163.39 (d, J = 251.3 Hz), 136.58 (d, J = 3.8 Hz), 131.13 (d,
J = 6.0 Hz), 127.46 (d, J = 9.1 Hz), 125.73 (d, J = 18.2 Hz), 115.38 (d, J = 23.5 Hz), 102.1, 63.67, 62.13,
52.74, 27.83, 27.35, 26.36, 24.99, 20.38, 20.15, 19.93, 19.25, 18.28, 14.53 (d, J = 3.8 Hz).
(2R)-2-(4-Fluoro-N-isobutyl-3-methoxyphenylsulfonamido)-3-methyl-N-((tetrahydro-2H-pyran-2yl)oxy)butanamide (20)
Colorless oil (160 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 9.43 (s, 1H), 7.72-7.68 (m, 2H), 7.09 (t, J = 8.4
Hz, 1H), 4.95 (s, 1H), 4.01-3.94 (m, 4H), 3.72-3.62 (m, 2H), 3.33-3.27 (m, 1H), 3.03-2.98 (m, 1H), 2.242.05 (m, 2H), 1.92-1.6 (m, 6H), 0.93-0.81 (m, 9H), 0.62-0.52 (m, 3H). 13C NMR (100 MHz, CDCl3) δ
167.10, 163.39 (d, J = 251.3 Hz), 136.58 (d, J = 3.8 Hz), 131.13 (d, J = 6.0 Hz), 127.46 (d, J = 9.1 Hz),
125.73 (d, J = 18.2 Hz), 115.38 (d, J = 23.5 Hz), 102.1, 63.67, 62.13, 56.54, 52.74, 27.83, 27.35, 26.36,
24.99, 20.38, 20.15, 19.93, 19.25, 18.28.
((2R)-2-(N-Isobutyl-3-(methoxymethyl)phenylsulfonamido)-3-methyl-N-((tetrahydro-2H-pyran-2yl)oxy)butanamide (21)
Colorless oil (173 mg, 97%). 1H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 7.43-7.37 (m, 2H), 7.16-7.11 (m,
2H), 4.57 (s, 1H), 4.12 (s, 2H), 3.71-3.52 (m, 1H), 3.35-3.23 (m, 2H), 3.02 (s, 3H), 2.93-2.89 (m, 1H),
2.65-2.50 (m, 1H), 1.84-1.67 (m, 2H), 1.44-1.21 (m, 6H), 0.55-0.42 (m, 9H), 0.13-0.01 (3, H). 13C NMR
(100 MHz, CDCl3) δ 167.06, 140.45, 140.02, 131.63, 129.11, 126.33, 125.96, 101.98, 63.56, 62.0, 60.29,
58.24, 52.70, 27.83, 27.35, 26.36, 24.99, 20.38, 20.15, 19.93, 19.25, 18.28.
Representative Procedure for THP Deprotection (3-5)
(R)-2-(4-Fluoro-N-isobutyl-3-methylphenylsulfonamido)-N-hydroxy-3-methylbutanamide (3)
To an ice-chilled solution of 19 (0.05 g, 0.11 mmol) in DCM (2.0 mL) was added TFA (1.0 mL) dropwise.
The reaction mixture was stirred overnight at rt. Upon consumption of the starting material as determined
by TLC, the solvent was removed under reduced pressure and the resulting residue was purified by
preparatory TLC using DCM/MeOH (19/1, v/v) to yield a white solid (0.02 g, 50%). The HPLC retention
time is 18.7 min. Compound purity is >99% as determined by two-wavelength HPLC analysis (254 nm
and 215 nm). 1H NMR (400 MHz, CD3OD) δ 7.77-7.68 (m, 2H), 7.20 (t, J = 8.8 Hz, 1H), 3.74 (d, J = 11.2
Hz, 1H), 3.45 CHA (dd, J = 14.4 Hz, J = 6.8 Hz, 1H), 3.0 CHB (dd, J = 14.8 Hz, J = 8.0 Hz, 1H), 2.34 (s, 3H),
2.16-2.08 (m, 2H), 0.89-0.82 (m, 12H). 13C NMR (100 MHz, CD3OD) δ 167.06, 163.56 (d, J = 250.4 Hz),
136.1 (d, J = 3.8 Hz), 130.79 (d, J = 6.8 Hz), 127.24 (d, J = 9.9 Hz), 126.07 (d, J = 18.9 Hz), 115.18 (d, J
= 23.5 Hz), 64.10, 52.70, 27.96, 27.67, 19.49, 19.2, 18.83, 18.30, 12.99 (d, J = 3.8 Hz). MS (ESI) 361.20
[M + H]+.
(R)-2-(4-Fluoro-N-isobutyl-3-methoxyphenylsulfonamido)-N-hydroxy-3-methylbutanamide (4)
White solid (40 mg, 31%). The HPLC retention time is 18.1 min. Compound purity is 94% by 254 nm
and 97% by 215 nm as determined by HPLC analysis. 1H NMR (400 MHz, CD3OD) δ 7.50-7.43 (m, 2H),
7.25 (t, J = 8.4 Hz, 1H), 3.95 (s, 3H), 3.8 (d, J = 10.8 Hz, 1H), 3.46 CHA (dd, J = 14.8 Hz, J = 6.8 Hz, 1H),
3.0 CHB (dd, J = 14.4 Hz, J = 8.0 Hz, 1H), 2.18-2.1 (m, 2H), 0.91-0.80 (m, 12H). 13C NMR (100 MHz,
CD3OD) δ 167.16, 154.73 (d, J = 252.7 Hz), 148.13 (d, J = 10.6 Hz), 136.68 (d, J = 3.8 Hz), 120.71 (d, J =
8.3 Hz), 115.78 (d, J = 19.8 Hz), 112.14 (d, J = 3.1 Hz), 64.19, 55.68, 52.75, 27.91, 27.59, 19.54, 19.23,
18.83, 18.32. MS (ESI) 377.10 [M + H]+.
(R)-N-Hydroxy-2-(N-isobutyl-3-(methoxymethyl)phenylsulfonamido)-3-methylbutanamide (5)
White solid (80 mg, 57%). The HPLC retention time is 15.5 min. Compound purity is 97% by 254 nm and
86% by 215 nm as determined by HPLC analysis. 1H NMR (400 MHz, CD3OD) δ 7.83 (s, 1H), 7.78 (d, J =
7.2 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 4.53 (s, 2H), 3.78 (d, J = 10.4 Hz, 1H), 3.473.38 (m, 4H), 3.02 CHB (dd, J = 14.4 Hz, J = 7.6 Hz, 1H), 2.16-2.10 (m, 2H), 0.91-0.80 (m, 12H). 13C NMR
(100 MHz, CD3OD) δ 167.14, 140.51, 139.79, 131.50, 128.81, 126.29, 125.99, 73.21, 64.06, 57.28,
52.69, 27.88, 27.61, 19.59, 19.27, 18.85, 18.39. MS (ESI) 373.10 [M + H]+.
Figure S1. Ligand omit map (mFo-DFc) for structure 4XM6 (complex with compound 3). Contoured at
3 (dark blue) and 2 (light blue).
Figure S2. Ligand omit map (mFo-DFc) for structure 4XM7 (complex with compound 4). Contoured at
3 (dark blue) and 2 (light blue).
Figure S3. Ligand omit map (mFo-DFc) for structure 4XM8 (complex with compound 5). Contoured at
3 (dark blue) and 2 (light blue).