Supplemental Section - Nature Research · Delta-Pak) using a linear gradient of 0-100%...
Transcript of Supplemental Section - Nature Research · Delta-Pak) using a linear gradient of 0-100%...
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