A new General Purpose Decontamination System for Chemical and Biological Warfare and Terrorism agents Sushil Khetan, Deboshri Banerjee, Arani Chanda, and Terry Collins Institute for Green Oxidation Chemistry Carnegie Mellon University, Pittsburgh, PA 15213
Joint Services Scientific Conference on Chemical & Biological Defense Research
November 20, 2003
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A new General Purpose Decontamination System for Chemical and Biological Warfare and Terrorism agents
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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18
Fe-TAML® Activator of Peroxide Y
n–
O
N
X
III
Fe
N X
N
N
O
R
O R
O X = H; R = CH 3 X = H; R = F X = Cl; R = F
‘Green’ Oxidizing System1 • • • •
FeB* FeBF2 FeDCBF 2
TAML Activators developed at Carnegie Mellon University
New Applications Rapid Inactivation of Bacterial Spores and degradation of organophosphorus triesters as surrogates of Biological and chemical Warfare Agents
Biomimetic System Non-toxic and non-corrosive Efficient user of peroxide High turnover in oxidative environment
Tested Applications1,2 • • • •
Effluent Treatment Bleaching in Pulp and Paper Desulfurization of Diesel Dye Transfer Inhibition Agent 1. Collins, T. J. Accounts of Chemical Research 2002, 35, 782-790 2. http:// www.cmu.edu/Greenchemistry
Activators of Hydrogen peroxide Relative Rates of Reactive Intermediates Formation • Bicarbonate Activated Peroxide* System (Aqueous Foam decon by Sandia National Laboratory)
HCO3- + H2O2
k
HCO4- + H2O
• Fe-TAML® activators of hydrogen peroxide k′ FeIII-TAML + H2O2 ‘Fe=O’ – Deep Oxidation capability – Non-toxic and non-corrosive
* Richardson, D. E. et al. J. Am. Chem. Soc. 2000, 122, 1729
k ≈ 10-3 M-1 s-1
k ′ ≈ 104 M-1 s-1
k′/k = 107
Biological Warfare Agents A microorganism or its by-product (toxin), which causes disease in man, plants or deterioration in material; used as weapons of warfare and/or terrorism Major Threats • Bacterial Diseases – Anthrax – Tularemia – Plague • Viral Diseases – Smallpox – Viral hemorrhagic fevers • Toxins – Botulinum Toxins – Ricin
Anthrax Spores
• • •
Dormant survival form of the vegetative bacterium Resistant to stress conditions, e.g. heat, UV radiations and chemical treatments Germinates on encountering favorable conditions
Bacterial Endospore Spore resistance is due to two protective shells that encase the organism
Spore Coat Multi-layered highly cross-linked polypeptide structure with numerous disulfide linkages
Spore Cortex Source: L. M. Prescott, Microbiology, McGraw-Hill, NY, 5th Ed., 2002
Thick layer of loosely crosslinked peptidoglycan structure with an overall negative charge
Spore Core • Normal cell structures with ribosomes and a nucleoid • Metabolically inactive and largely dehydrated
Bacterial Spore Deactivation Strategies and Mechanism Strategies •
•
•
•
Penetration of the spore coat with subsequent degradation of bacterial DNA Dissolution of spore peptidoglycan structure, exposing the vegetative cell elements Initiation of germination with weakening of spore wall followed by deactivation Inactivation of spore germination apparatus by destruction of germination-specific lytic enzymes
Weakening of spore coat through oxidation of the disulfide bonds
Protein Fragment
Cysteic acid
Model compound: Dialkyldisulfide (e.g. Cystine)
Modeling Studies Oxidation of Di-alkyl Disulfides Dissociation of disulfide bonds also observed in di-tert-butyl disulfide
Cysteic acid formation confirmed by HPLC and UV-Visible Spectroscopy
Result obtained from ESI-MS studies
Deactivation Studies with Bacillus spores
Bacillus atrophaeus (formerly B. globigii)
Spore-forming harmless soil bacterium B. atrophaeus (ATCC 9372) was tested as surrogate for Bacillus anthracis in spore deactivation studies
Optimization of Reaction Conditions Variation of Fe-TAML® concentrations • Studies conducted at two H2O2 concentrations 0.05M
• Exponential relationship between spore deactivation and Fe-TAML® concentration 0.5M
• Optimized Fe-TAML® concentration: 10 µM N0 = Initial number of spores N = Number of surviving spores
• Reactions carried out for 1 hour at 30°C • Spore Population of 5×107 CFU/ml • Na-carbonate/bicarbonate (0.1 M) buffer, pH 10.0
Optimization of Reaction Conditions Variation of H2O2 concentrations • Linear relationship between spore deactivation and concentration of H2O2 • Optimized H2O2 concentration: 0.5M
N0 = Initial number of spores N = Number of surviving spores
• Reactions carried out for 1 hour at 30°C • Spore Population of 5×107 CFU/ml • Na-carbonate/bicarbonate (0.1 M) buffer, pH 10.0
Use of Cationic Surfactant Cationic Surfactants • Enhance penetrability of Fe-TAML® activators across the spore coat • Increase dispersion of hydrophobic spores in aqueous phase • Can cause collapse of spore peptidoglycan structure through ionic interactions Optimized concentration: 0.03% (close to cmc value) N0 = Initial number of spores N = Number of surviving spores
Time Dependence of Spore Kill Time Dependence of Spore Kill
10,000×
10,000×
Control
95% mortality with hydrogen peroxide
N0 = Initial number of spores N = Number of surviving spores 10,000×
99.98% mortality with Fe-TAML® activator, and hydrogen peroxide
• • • •
99.98% (4-log) kill of spores Treatment time: 2 hours Fe-TAML®: 10 µM; H2O2: 0.5 M Spore population: 1×108 cfu/ml
Enhanced Spore Mortality
N0 = Initial number of spores N = Number of surviving spores
75% with tBuOOH
99.99999% with Fe-TAML® + tBuOOH
• • • •
99.99999% (7-log) kill of spores Treatment time: 1 hour Fe-TAML®: 5 µM; tBuOOH: 0.3 M Spore population: 1×108 cfu/ml
Oxidative Detoxification of Organophosphorus Triesters and Dialkyl sulfides
TAML®-activated H2O2 Treatment of Fenitrothion S
H 3C
P O2N
O
OMe OMe
Fenitrothion
UV-Visible spectroscopic study
TAML®-activated H2O2 Treatment of Fenitrothion UV-Vis Kinetic Studies
HPLC of the Reaction Mixture Fenitrothion
With TAML/H2O2 With H2O2 only
0.3
0.2
0.1
0.0 0
1000
2000
3000
4000
5000
Time (sec)
Kinetics of decomposition of fenitrothion is followed through absorption at 396 nm In UV-Vis. Rapid hydrolysis is seen followed by degradation of p-nitrocresol.
Absorbance (265 nm)
Absorbance (396 nm)
0.4
p-nitrocresol Fenitrooxon
H2O 2
1
2
3
4
5
6
7
8
Retention time (min)
Time-lapsed analysis of the reaction mixture by HPLC shows initial formation of p-nitrocresol and fenitrooxon. In subsequent stage, most of p-nitrocresol is degraded.
TAML-activated peroxide decomposition of Fenitrothion CH3
CH3 NO2
S P
Fenitrothion
OCH3 OCH3
TAML/H2O2 Oxidative hydrolysis
TAML/H2O2 Desulfuration
NO2
H3 CO OH
+ H3 CO
p-Nitrocresol
HO2Hydrolysis
O NO2
P
Fenitrooxon
OCH3 OCH3
Oxidative degradation
P OH
Dimethyl phosphate
TAML/H2O2 CH3
O
Fenitrothion and Fenitrooxon Degradation - pH Dependence Initial rate measurements following p-nitrocresol formation (395 nm) Peroxide assisted Fenitrooxon degradation
1.6e-5
TAML/peroxide peroxide
1.4e-5 1.2e-5 1.0e-5 8.0e-6 6.0e-6 4.0e-6 2.0e-6 0.0 7
8
9
10
11
12
pH
TAML®: Fenitrothion: peroxide (1: 25: 50,000) in phosphate buffer (0.1M)
Rate of Fenitrooxon Degradation (M/s)
Rate of Fenitrothion Degradation (M/s)
TAML/H2O2 mediated Fenitrothion degradation 2.5e-7 2.0e-7 1.5e-7 1.0e-7 5.0e-8 0.0 7
8
9
10
pH
Fenitrooxon: peroxide (1:2000) in phosphate buffer (0.1M)
11
p-Nitrocresol Degradation – pH dependence Optimization of Reaction Conditions Kinetics of oxidative degradation
Degradation of p-nitrocresol
Rate of p-nitrocresol Degradation
Initial rate measurements
1.2e-6
8.0e-7
4.0e-7
0.0
7
8
9
10
11
pH 8.0 pH 9.0 pH 10.0
1.0 0.8 0.6 0.4
pH 10.5 0.2 0.0 0
10
pH
20
30
40
50
60
Time (s)
At higher pH, the reaction rate increases, but catalyst gets inactivated faster Optimum pH range 9.5-10.0
Summary of Fenitrothion degradation Study Optimal pH Fenitrothion – Oxidative hydrolysis
Limiting condition 9.0-11.0
p-Nitrocresol – Oxidative degradation
<10.5
Fenitrooxon – Peroxy anion assisted hydrolysis
>9.5 9.0
10.0
11.0
Optimal reaction conditions for Total degradation of fenitrothion pH 9.5-10.0, phosphate buffer (0.1 M), 25°C TAML®: Fenitrothion: Peroxide 1 : 25 : 50,000
Total Degradation of Fenitrothion Fe
O
H 3C
P O
O2N
OMe OMe
Minor Pathway
S
H 3C
P O
O2N
OMe
Fe-TAML® H2O2
OMe
Fenitrooxon
Major pathway Oxidative hydrolysis
Fenitrothion
Peroxide assisted Hydrolysis
H 3C
O
O2N
OH
P OH + H3CO H3CO
Oxidation
HO
OH CO2 + H2O +
H-CO2H + Formic Acid
p-nitrocresol
O O Oxalic Acid
HO 2C
CO2H
Maleic acid
HO 2C
CO2H
Methyl maleic acid
+ SO 2- + NO - + NO -4 3 2
Mineralization
Fenitrothion Degradation Aquatic Toxicity
MicroTox Vibrio fischeri
Dafnia Magna
Fenitrothion (99%)
EC50 (15 min.) Mg/L
2.33
D. Magna EC50 Mg/L 14.1
TAML catalyst (FeBF2)
58.00
NA
Reaction mixture (pH 10, quenched with catalase)
57.25
>530
Reduction in toxicity
25-fold
>38-fold
Reaction of Dialkyl sulfides with TAML/peroxide TAML/H2O 2 1: 2000
S
Sulfoxide
O
S
<5 mts.
Dibutyl sulfide
O O
TAML/ (CH3)3COOH in MI
S
S O
Dibutyl sulfide TAML/H2O 2 1: 10,000 HO S
OH
Bis (2-hydroxyethyl) sulfide
5 mts.
Sulfone
S
TAML/H2O 2 1:5,000
HO
S O
OH
Sulfoxide
Sulfoxide 100%
TAML:substrate = 1:1,000; pH 8; Phosphate buffer, 25°C
Conclusions TAML®-peroxide technology: ! Effectively deactivate bacterial spores, the toughest of all microorganisms, in aqueous solution achieving 99.99999% (7-log) of spore destruction ! Rapidly detoxify organo-phosphorus triesters, followed by the deep oxidation of hydrolysates ! Selectively oxidize dialkyl sulfides to less toxic sufoxide ! Promises an environmentally friendlier superior technology for destruction of all chemical-biological warfare agents
New Decon System Features " " " " " " " "
Catalytic – Requires very low catalyst and low peroxide concentration Designed to be Non-toxic – No toxic elements or functionality Aqueous based – Compatible with wide variety of surfaces and technologies; can be used on sensitive equipment Broad-spectrum activity – Detoxify and degrade largerange of chemicals and inactivate bacterial spores Performance previously unavailable – Truly biomimetic with deep oxidation capability (leaves no toxic biproducts) Robust system – Stable and functional over wide range of pH Rapid acting and safe - for people and environment Easy to use – Used at ambient conditions, offers a practical approach
Acknowledgements Anindya Ghosh Dr. Peter Berget Dr. Edwin Minkley NSF DURIP
Nucleophile assisted Hydrolytic Detoxification of Chemical Warfare Agents Peroxy Anion (OOH-) RO
O
O
O
P F CH3
OOH- HF
RO
H2O2
P OO-
-O2, H2O
-O3S O
RO
P O-
CH3
CH3 R = CH(CH3)2 R = CH(CH3)[C(CH3)3]
Sarin Soman
RO
NR'2
OOH-
P S
NR'2
CH3 VX
R = CH2CH3
R' = CH(CH3)2
The rate of nucleophile aided hydrolysis of esters is increased by cationic micelles (e.g. -OOH/CTABr). Wagner and Yang, 2002. Ind. Eng. Chem. Res., 41(8), 1925-1928
Fe-TAML peroxide oxidant system mimics Cytochrome 450 O
S MeO
CH 3
S O
MeO
P
"Fe IV =O"
P NO 2
CH 3 O
H 3 CO
NO 2
H 3 CO
Fenitrothion
S
O
S
CH 3
O
CH 3
S
P
P H 3 CO
O
NO 2
NO 2
H3 CO
H 3 CO
H 3 CO
O
NO2
H3 CO
Three-membered 'phospho-oxythiarane' intermediate
Minor pathway
CH3
P O
H 3 CO
O
O H
H
Major pathway
SO 4 2 - +
H 3 CO H 3 CO
O P O
Fenitrooxon
S
CH 3 NO 2
SO 4 2 -
H 3 CO H 3 CO
OH
Dimethyl phosphate
+
P
O P
H3 CO H3 CO
CH3
O O H
HO
NO2
H
p-Nitrocresol
TAML-activated peroxide treatment of fenitrothion possibly results in a common 3-membered ring intermediate formation leading to fenitrooxon and p-nitrocresol
TAML/H2O2 Degradation of Organophosphorus Triesters A Versatile and Robust Process R1 O R1 O
S P
Fe-TAML
ROH +
H 2O 2
OR
R1 O
Minor pathway
R1 O R1 O
O
P
OH
+ SO4 2-
HOO Small alephatic acids + CO2 + H 2O
OR
CH3 R =
O
∆
O P
R1 O
NO2
Cl
N
-O
Cl N
R1 = -CH3 , −C2H 5
Chlorpyriphos
N
-O
-O N
Cl
Fenitrothion
CH3
Quinalphos
N H 3C
CH3
Diazinon
Catalysis of Phosphate Triester Hydrolysis by Cationic Micelles •
Nucleophile (such as peroxide anion) aided hydrolysis is the most preferred reaction to detoxify phosphorus esters.
•
The rate of nucleophile aided hydrolysis of esters is increased by cationic micelles (e.g. -OOH/CTABr).1,2
•
CTABr has significantly enhanced hydrolytic rate of phosphorus esters, (depending on substrate, 20-300 fold enhancement) with hypochlorite.1
•
Aqueous cationic micelles accelerate spontaneous hydrolysis of dinitrophenyl phosphate and acyl phosphate dianions, with an extensive P-O bond cleavage in the transition state.3
______________________
1. Dubey, Gupta et al., Langmuir, 2002, 18, 10489-10492 2. Couderc and Toullec, Lanmuir, 2001, 17, 3819-3828. 3. Brinchi, profio et al., Langmuir, 2000, 16, 10101-10105
Bacterial Endospore Spore Cortex
• Loosely cross-linked peptidoglycan composed of Nacetyl glucosamine and N-acetylmuramic acid with short peptide side-chains • Maintains spore dormancy and heat resistance; hydrolyzes during germination • An overall negative charge — from the phosphate backbone of teichoic acid (20-40% of dry weight of cortex)
Teichoic Acid
