Supplemental Section

Supplemental protocols are provided below including synthetic details for each of the

compounds that are reported in the manuscript. Analytical characterization of the

compounds can be found following the methods section. In addition, two supplemental

figures and legends can be found at the end of this section.

General methods. Unless otherwise noted, all resin and reagents were purchased from

commercial suppliers and used without further purification. All solvents used were of

HPLC grade. All water-sensitive reactions were performed in anhydrous solvents and

under a positive pressure of argon. Reactions were analyzed by thin-layer

chromatography on Whatman 0.25 mm silica plates with fluorescent indicator. Flash

chromatography was carried out with EMD 230-400 mesh silica gel. Reverse-phase

HPLC was conducted on a C18 column using the ÄKTA explorer 100 (Amersham

Pharmacia Biotech). LCMS data were acquired using an API 150EX LC/MS system

(Applied Biosystems). High-resolution MS analyses were performed by Stanford

Proteomics and Integrative Research Facility using a Bruker Autoflex MALDI TOF/TOF

mass spectrometer.

General solid phase peptide synthesis method. The synthetic protocols for solid phase

synthesis of peptide AOMKs are a modification of the previously reported methods 1,2.

Halomethyl ketones (2a-f) and their solid support bound derivatives via carbazate linker

(5a-e) were synthesized with modification to the procedure as described below. Unless

otherwise noted, reactions were conducted in 12-mL polypropylene cartridges (Applied

Separations, Allentown, PA) with 3-way nylon stopcocks (BioRad Laboratories,

Hercules, CA). The cartridges were connected to a 20 port vacuum manifold (Waters,

Milford, MA) that was used to drain solvent and reagents from the cartridge. The resin

was gently shaken on a rotating shaker during solid-phase reactions.

General method for synthesis of halomethyl ketone derivatives of N-α-Fmoc-

protected amino acids (2a-f). A 0.2 M solution of the corresponding N-α-Fmoc amino

acid (1a-e, 5 mmol) in anhydrous THF was stirred in an ice/ acetone bath at -10° C. To

this solution, N-methylmorpholine (6.25 mmol, 1.25 equiv) and isobutylchloroformate

(5.75 mmol, 1.15 equiv) were sequentially added. Immediately after the addition of the

latter compound, a white precipitate formed. The reaction mixture was maintained at -

10° C for 25min. Diazomethane was generated in situ using the procedure described in

the Aldrich Technical Bulletin (AL-180). Ethereal diazomethane (16.6-21.4 mmol) was

transferred to the stirred solution of the mixed anhydride at 0° C. The reaction mixture

was warmed to room temperature over the course of 3 hours. To obtain the corresponding

chloromethyl ketones (2a-e), 15 mL of a 1:1 solution of concentrated hydrochloric acid

and glacial acetic acid was added dropwise to the reaction mixture at 0° C. Immediately

after the evolution of nitrogen gas stopped, the reaction mixture was diluted with ethyl

acetate and transferred to a separatory funnel. The reaction mixture was washed

sequentially with water, brine solution, and saturated aqueous NaHCO3. The organic

layer was dried over MgSO4. The solvent was removed under reduced pressure.

Alternatively, the bromethyl ketone (2f) was obtained by dropwise addition of 10 mL of a

1:2 solution of hydrogen bromide (30 wt. % solution in acetic acid) and water to the

reaction mixture at 0 °C. Workup was carried out as described for the chloromethyl

ketone synthesis. Chloromethyl ketones 2a (glycine), 2c (leucine), 2d (lysine), 2e

(aspartic acid) were obtained as a white solid (quantitative yield) and the bromomethyl

ketone 2f (aspartic acid) was obtained as a yellow oil (quantitative yield), and used

without any purification. Crude chloromethyl ketone 2b (arginine) was purified by

column chromatography (50-60% ethyl acetate in hexane) to obtain a white solid (3.13

mmol, 62 %).

Synthesis of carbazate linker on aminomethylpolystyrene resin.

Aminomethylpolystyrene resin (1.1 mmol/g) was dried in vacuo overnight in a 12-mL

polypropylene cartridge. The resin was presolvated with DMF for 30 min and another 30

min with CH2Cl2. A 1 M solution of N, N’-Carbonyldiimidazole (6 equiv) in CH2Cl2 was

added to the resin, and the resin was shaken at room temperature for 3 h. The reagent

was drained and the resin was washed with CH2Cl2 followed by DMF. A 10 M solution

of hydrazine (60 equiv) in DMF was added to the resin, and the resin was shaken at room

temperature for 1 h. The resin was washed with DMF followed by CH2Cl2, dried in

vacuo, and stored at -4° C.

Loading of chloromethyl ketone derivatives and synthesis of 2,6-

dimethylbenzoyloxymethyl ketone derivatives (5a-e). A 0.5 M solution of the

chloromethyl ketone derivative of the corresponding N-a-Fmoc-L-amino acid (2a-e) in

DMF was added to the resin. The cartridge was tightly sealed and shaken at 50° C for

various time periods depending on the chloromethyl ketone . The glycine CMK (2a) was

incubated for 10 min; all others (2b-e) were incubated for 3 h. After the reaction the

solution was removed, and the resin was washed with DMF. Formation of the AOMK on

resin was performed using KF as reported for solution phase synthesis of AOMKs 3. This

method allowed the use of a reduced amount of the carboxylic acid. Specifically a 0.5 M

solution of 2,6-dimethylbenzoic acid (5 equiv) and potassium fluoride (10 equiv) in DMF

were added to the resin. The resin was shaken at room temperature overnight. After the

solution was removed, the resin was washed with DMF followed by CH2Cl2, and dried in

vacuo. The resin load was estimated by UV absorption of free Fmoc.

Synthesis of 2,6-dimethylbenzoyloxymethyl ketone derivative of N-a-Fmoc-L-

aspartic acid on Rink resin (5f). A 0.2 M solution of bromomethyl ketone derivative of

N-α-Fmoc-L-aspartic acid-β-tert-butyl ester (2f) in DMF was stirred at 0˚ C, and

potassium fluoride (3 equiv) was added as a solid. After 1min stirring at 0˚ C, 2,6-

dimethylbenzoic acid (1.2 equiv) was added as a solid, the reaction mixture was warmed

to room temperature. After overnight stirring, the reaction mixture was diluted with ethyl

acetate, and transferred to a separatory funnel. The reaction mixture was worked up

sequentially with water, brine solution, and saturated aqueous NaHCO3. The organic

layer was dried over MgSO4. The solvent was removed under reduced pressure. The

product was purified by flash chromatography (~17% ethyl acetate in hexane) yielding a

yellow oil (98% yield).

A 0.2 M solution of the product 2,6-Dimethylbenzoyloxymethyl ketone derivative

of N-α-Fmoc-L-Aspartic Acid-β-tert-butyl ester (3f) was dissolved in 25% v/v TFA/

CH2Cl2 and allowed to stand for 30 min with occasional shaking. The reaction mixture

was diluted with CH2Cl2. The cleavage solution was removed by coevaporation with

toluene. The product was further dried in vacuo.The crude product (4f; 96% yield) was

used without further purification.

Rink resin (0.75 mmol/ g) was presolvated by shaking in DMF for 1 h. The

Fmoc-protecting group on the resin was removed with 20% piperidine/ DMF for 15min.

The resin was washed with DMF followed by CH2Cl2. A 0.5 M solution of 2,6-

dimetylbenzoyloxymethyl ketone derivative of N-α-Fmoc-L-aspartic acid (4f, 1.25

equiv) and HOBT (1.25 equiv) was added to the resin followed by DIC (1.25 equiv).

After shaking for 2.5 h, the resin was washed with DMF, yielding the loaded resin (5f).

Resin load was determined by UV absorption of free Fmoc.

Optimization of base deprotection of peptide AOMKs. Before solid phase peptide

synthesis could be carried out for extended peptides a survey of optimal bases for

deprotection of the Fmoc group was performed to identify conditions that allowed Fmoc

removal without displacement of the AOMK group. Eighteen aliquots of N-α-Fmoc-L-

leucine 2,6-dimethylbenzoyloxymethyl ketone loaded resin (5c, ~1 mg, ~3.7 x 10-4

mmol) were solvated with DMF for 30 min. DMF solutions of each of the bases were

added to each well, and the reactions were shaken for 20 min. The resins were washed

with DMF followed by CH2Cl2. Acetic anhydride (10 equiv) and DIEA (15 equiv) in 250

µL DMF were added to each well to acylate the deprotected free amine. The reactions

were shaken for 15 min and the resin washed with DMF followed by CH2Cl2. The

reaction block was placed under vacuum for ~15 min. 200 µL of cleavage cocktail (95%

TFA, 5% H2O) was added to the resin. After 1 h the cleavage mixture were collected,

diluted in methanol, and analyzed by direct infusion ion-spray mass spectrometry.

Solid phase peptide synthesis on aminomethylpolystyrene. N-Fmoc-protected 2,6-

dimethylbenzoyloxymethyl ketone derivatives linked to aminomethylpolystyrene or Rink

resin (5a-f) were presolvated in DMF for 30 min. N-terminal Fmoc group was removed

by treatment with a 5% diethylamine solution in DMF for 15min followed by another 15

min treatment with fresh solution. The resin was washed with DMF followed by CH2Cl2.

A 0.2 M solution of N-Fmoc-protected amino acid (3 equiv) (Z-protected amino acid for

8, 9 a-c), HOBT (3 equiv) in DMF and DIC (3 equiv) were sequentially added to the

resin. The resin was shaken at room temperature for 2 h, and washed with DMF followed

by CH2Cl2. For each subsequent step of the solid phase peptide synthesis, the same

deprotection and coupling reactions were followed. Deprotection and coupling reactions

were monitored by the ninhydrin test for primary amine. Capping of the N-terminal

amine for compound (7a-f, 10a-b) was achieved by shaking the resin with a 0.5 M

solution of acetic anhydride (10 equiv) and DIEA (15 equiv) in DMF. After shaking at

room temperature for 15min, the resin was washed with DMF followed by CH2Cl2, and

dried in vacuo.

General method of cleavage from aminomethylpolystyrene resin. The 3-way nylon

stopcocks were replaced with TFA-resistant polypropylene needle valve (Waters). A

solution of 95% TFA/ 5% H2O was added to the resin. After standing at room

temperature for 1.5 h, the cleavage mixture was collected, and the resin was washed with

fresh cleavage solution. The combined mixture was precipitated in cold ether at -20° C

for 2 h. The precipitated peptide was collected by centrifugation at 3,000 rpm at -10° C

for 15 min. The pellet was dried by positive flow of argon, dissolved in a minimal

amount of DMSO. The product was purified on a C18 reverse phase HPLC (Waters,

Delta-Pak) using a linear gradient of 0-100% water-acetonitrile. Fractions containing

product were pooled, then lyophilized to dryness. The identity of the product was

confirmed by mass spectrometry.

The general method of cleavage from Rink resin. The same procedure as for

aminomethylpolystyrene resin was followed except that a solution of 20% TFA/ 2.5%

triisopropylsilane in CH2Cl2 was added to the resin, and the reaction time was shortened

to 15 min.

Characterization of compounds. All final compounds used for biological studies were

purified by HPLC and characterized by high-resolution mass spectrometry (HRMS) using

a Bruker Autoflex TOF/TOF mass spectrometer. Data files for these studies can be found

in the Supplementary Data section. Overall yields for the complete synthesis and HRMS

data are listed below.

Compound 7a. 5.0% yield. [MNa]+ calcd for C45H63N7NaO10S, 915.4307; found,

915.42999. Compound 7b. 2.1% yield. HRMS (m/z): [MH]+ calcd for C49H73N10O10S,

993.5232; found, 993.5428. Compound 7c. 11% yield. HRMS (m/z): [MNa]+ calcd for

C49H71N7NaO10S, 971.4983; found, 971.4899. Compound 7d. 1.5% yield. HRMS (m/z):

[MH]+ calcd for C49H73N8O10S, 965.5170; found 965.5004. Compound 7e. 8.4% yield.

HRMS (m/z): [MH]+ calcd for C47H66N7O12S, 952.4490; found, 952.4402. Compound

7f. 8% yield. HRMS (m/z): [MH]+ calcd for C47H67N8O11S, 951.4650; found, 951.6500.

Compound 8. 13.4% yield. HRMS (m/z): [MNa]+ calcd for C28H28N2NaO7, 527.1794;

found, 526.8497 Compound 9a. 10.8% yield. HRMS (m/z): [MH]+ calcd for

C29H31N2O6, 503.2182; found, 503.2118. Compound 9b. 3.6% yield. HRMS (m/z):

[MH]+ calcd for C26H33N2O6, 469.2339; found, 469.2374. Compound 9c. 14.6% yield.

HRMS (m/z): [MH]+ calcd for C33H40N5O6, 602.2979; found, 602.2874. Compound 10a.

13.2% yield. HRMS (m/z): [MH]+ calcd for C32H36N3O7, 574.2553, found, 574.2504.

Compound 10b. 12.5% yield. HRMS (m/z): [MH]+ calcd for C36H45N6O7, 673.3350;

found, 673.3274. Coupound 11. 4.7% yield. HRMS (m/z): [MH]+ calcd for

C57H82N9O16S, 1180.5600, found, 1180.5564.

Radiolabeling of inhibitors. A 1.5 mL microcentrifuge tube was coated with 100 mg of

IODO-GEN® (Pierce, Rockford, IL). Probe (62.5 µl of a 0.2 mM solution in phosphate

buffer pH 7.4) was added to the tube. Na125I (1 mCi, 10 ml) was added to the tube and

incubation continued for 20 min. Labeled inhibitor was purified by application to a Sep-

Pak® (Waters, Milford, MA) column containing a C18 stationary phase. After sample

application, the column was washed with 25 mL of phosphate buffer pH 7.5. Labeled

inhibitor was eluted using 100% acetonitrile. Fractions of 1mL were collected and

samples with the largest number of counts were pooled and used without further

purification.

Small scale radiolabeling of probes. A 1.5mL microcentrifuge tube was coated with 50

µg of IODO-GEN®. Probe (6.5 µl of a 0.2 mM solution in phosphate buffer pH 7.4) was

added to the tube. Na125I (0.1 mCi, 1 µl) was added to the tube and incubation continued

for 20 min. Labeled inhibitor was transferred to a fresh tube containing 100 µL of 100%

acetonitrile and used without further purification.

Labeling of gingipains in crude cell lysates. Pelleted cells from P. gingivalis grown in

batch culture for 24 h (early stationary phase) were washed three times with 20 mM Bis-

Tris, 150 mM NaCl, 0.02% NaN3, pH 6.8 containing 1.5 mM 4,4’-dithiopyridine

disulfide, followed by sonication at 1,500 Hz for 10 min. Unbroken cells and large debris

were removed by centrifugation (27,000 x g, 60 min, 4oC) and the resulting supernatant

diluted into reaction buffer (50mM Tris, 10mM DTT, 5mm MgCl2, pH 7.6) to a final

concentration of 1 mg/ml. Total crude lysates (10 µg) were labeled with 125I-labeled P1

AOMK probes (1X106 total counts per minute) for 30 min at RT and labeled proteases

visualized by SDS-PAGE followed by autoradiography.

Supplemental References

1. Wood, W. J., Huang, L. & Ellman, J. A. Synthesis of a diverse library of

mechanism-based cysteine protease inhibitors. J Comb Chem 5, 869-80 (2003).

2. Lee, A., Huang, L. & Ellman, J. A. General solid-phase method for the

preparation of mechanism-based cysteine protease inhibitors. Journal of the

American Chemical Society 121, 9907-9914 (1999).

3. Mujica, T. M. & Jung, G. A novel approach to the solid phase synthesis of

(acyloxy)methyl ketones. Synlett 12, 1933-5 (1999).

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

0

10

20

30

40

50

60

70

80

90

99 1.62 5.80

12.32

5.27

Compound 7a

Ch

ann

el 1

, mA

u

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6894.4

133.2

498.4 762.5744.3560.3366.3397.2

149.3 876.5335.5 916.8111.4 944.6 1007.6781.6185.1 241.1 611.4 726.6532.0466.6 674.7 812.4 1089.8 1146.6

Inte

nsi

ty, C

PSHN

NH

O

HN O

O O

HN

NH

O

O

O

O

SHN

NH

O

OH

H

H

1.73 4.97 12.48

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 7b

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 10001050110011501200

m/z, amu

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e6993.5497.5

597.5397.1

310.5 434.4 639.3 861.7133.1

560.4880.5796.7

447.3

Inte

nsi

ty, C

PSHN

NH

O

HN O

O O

HN

NH

O

O

O

O

SHN

NH

O

NH

NH2HN

OH

H

H

1.69 6.55 12.48

6.27

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 7c

950.7

133.1

818.6554.5

977.8422.4

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

Inte

nsi

ty, C

PSHN

NH

O

HN O

O O

HN

NH

O

O

O

O

SHN

NH

O

OH

H

H

1.73 4.9012.48

9.37

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 7d

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

965.6

133.1

833.6

483.6

474.4

397.3815.4179.1

852.5

102.1

Inte

nsi

ty, C

PSHN

NH

O

HN

O O

HN

NH

O

O

O

O

OH

O

SHN

NH

O

NH2

H

H

12.50

1.64

5.73

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 7e

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

1.00e5

2.00e5

3.00e5

4.00e5

5.00e5

6.00e5

7.00e5

8.00e5

9.00e5

1.00e6

1.10e6952.6

133.1

153.1 820.6424.1

560.2

spc ,ytisne tn IHN

NH

O

HN

O O

HN

NH

O

O

O

O

OH

O

OH

O

SHN

NH

O

H

H

12.31

1.55

5.50

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 7f

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

spc ,ytisne tn I

951.7

133.1

819.5

933.6423.1

973.7560.1 783.5

HN

O

NH

OHN

O

HN O

SHN

NH

O

NH

O

O

O

O

NH2

O

OH

H

H

1.72 7.7212.43

7.05

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

spc ,ytisne tn I

505.1

461.3

1010.0

353.4

314.3

Compound 8

O

O

O

OHN

NH

O

O

O

1.51 21.36

32.21

19.61

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 9a

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

spc ,ytisne tn I

503.0132.9

459.4

525.4223.3

1005.6

O

O

OHN

NH

O

O

O

1.71 12.49

6.06

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 9b

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

spc ,ytisn etnI

601.8

750.5

O

O

OHN

NH

O

O

O

NH

NH2HN

21.261.52

32.22

20.98

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 9c

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

spc ,ytisne tn I

132.9469.2

491.3425.3223.2

319.3

O

O

OHN

NH

O

O

O

1.63 6.77

12.39

6.14

5.551.85

Compound 10a

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

spc ,ytisne tnI

574.2

369.11147.7

353.3

280.0

133.1

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

uO

O

OHN

NH

O

HN

O

O

OH

12.40

5.361.64

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

10

20

30

40

50

60

70

80

90

99

Ch

ann

el 1

, mA

u

Compound 10b

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

spc ,ytisne tn I

673.5

510.5

O

O

OHN

NH

O

HN

O

O

OH NH

H2N NH

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time, min

0

10

20

30

40

50

60

70

80

90

99

Compound 11C

han

nel

1, m

Au

12.29

5.63

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

m/z, amu

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

spc ,ytisnetn I

1180.7

133.1

1048.8

HN

O

NH

HN

O

O

O

NH

OH

HN

O

S

NH

HNO

HN

O

O

OO

OH

NH

O

O

OHO

H

H