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 ChemistryCarnegie Mellon University, Pittsburgh, PA 15213

Joint Services Scientific Conference on Chemical & Biological Defense Research

November 20, 2003

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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18

Fe-TAML® Activator of Peroxide

‘Green’ Oxidizing System1

• 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

NO

NO

R

R

ON

O

NFe

Y n–

III

X

X

X = H; R = CH 3 FeB*X = H; R = F FeBF2X = Cl; R = F FeDCBF 2

Rapid Inactivation of Bacterial Spores and degradation of organophosphorus triesters as surrogates of Biological and chemical Warfare Agents

New Applications

TAML Activators developed at Carnegie Mellon University

Activators of Hydrogen peroxideRelative Rates of Reactive Intermediates Formation

* Richardson, D. E. et al. J. Am. Chem. Soc. 2000, 122, 1729

FeIII-TAML + H2O2 ‘Fe=O’

k′/k = 107– Deep Oxidation capability– Non-toxic and non-corrosive

HCO3- + H2O2 HCO4

- + H2O

• Bicarbonate Activated Peroxide* System(Aqueous Foam decon by Sandia National Laboratory)

k k ≈ 10-3 M-1 s-1

• Fe-TAML® activators of hydrogen peroxidek ′ k ′ ≈ 104 M-1 s-1

Biological Warfare Agents

Major Threats• Bacterial Diseases

– Anthrax– Tularemia– Plague

• Viral Diseases– Smallpox– Viral hemorrhagic

fevers• Toxins

– Botulinum Toxins– Ricin

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

• Dormant survival form of the vegetative bacterium

• Resistant to stress conditions, e.g. heat, UV radiations and chemical treatments

• Germinates on encountering favorable conditions

Anthrax Spores

Bacterial Endospore

Source: L. M. Prescott, Microbiology, McGraw-Hill, NY, 5th Ed., 2002

Spore Core

• Normal cell structures with ribosomes and a nucleoid

• Metabolically inactive and largely dehydrated

Thick layer of loosely cross-linked peptidoglycan structure with an overall negative charge

Spore Cortex

Spore CoatMulti-layered highly cross-linked polypeptide structure with numerous disulfide linkages

Spore resistance is due to two protective shells that encase the organism

Bacterial Spore DeactivationStrategies and Mechanism

• 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

StrategiesWeakening of spore coat through oxidation of the disulfide bonds

ProteinFragment

Cysteic acid

Model compound: Dialkyldisulfide (e.g. Cystine)

Modeling StudiesOxidation of Di-alkyl Disulfides

Dissociation of disulfide bonds also observed in

di-tert-butyl disulfide

Result obtained from ESI-MS studies

Cysteic acid formation confirmed by HPLC and UV-Visible Spectroscopy

Deactivation Studies with Bacillus spores

Spore-forming harmless soil bacterium B. atrophaeus(ATCC 9372) was tested as surrogate for Bacillus anthracisin spore deactivation studies

Bacillus atrophaeus(formerly B. globigii)

Optimization of Reaction ConditionsVariation of Fe-TAML® concentrations

• 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

• Studies conducted at two H2O2 concentrations

• Exponential relationship between spore deactivation and Fe-TAML® concentration

• Optimized Fe-TAML®

concentration: 10 µMN0 = Initial number of sporesN = Number of surviving spores

0.05M

0.5M

N0 = Initial number of sporesN = Number of surviving spores

Optimization of Reaction ConditionsVariation of H2O2 concentrations

• 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

• Linear relationship between spore deactivation and concentration of H2O2

• Optimized H2O2

concentration: 0.5M

Use of Cationic Surfactant

Optimized concentration: 0.03%(close to cmc value)

N0 = Initial number of sporesN = Number of surviving spores

• 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

Cationic Surfactants

Time Dependence of Spore Kill

N0 = Initial number of sporesN = Number of surviving spores

Control 95% mortalitywith hydrogen peroxide

99.98% mortalitywith Fe-TAML® activator,and hydrogen peroxide

10,000× 10,000×

10,000ו 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

Time Dependence of Spore Kill

N0 = Initial number of sporesN = Number of surviving spores

Enhanced Spore Mortality

99.99999% with Fe-TAML®

+ tBuOOH

75% with 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

O

H3C

O2N

Fenitrothion

P

S

OMe

OMe

UV-Visible spectroscopic study

Time (sec)

0 1000 2000 3000 4000 5000

Abs

orba

nce

(396

nm

)

0.0

0.1

0.2

0.3

0.4

With TAML/H2O2With H2O2 only

UV-Vis Kinetic Studies

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.

Retention time (min)1 2 3 4 5 6 7 8

Fenitrothion

Abs

orba

nce

(265

nm

)

Fenitrooxon

p-nitrocresol

H2O2

HPLC of the Reaction Mixture

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 H2O2 Treatment of Fenitrothion

TAML-activated peroxide decomposition of Fenitrothion

PO

Fenitrooxon

NO2

CH3

OCH3

OCH3

p-Nitrocresol

OHNO2

CH3

OP

OH3CO

H3CO H

Dimethyl phosphateFenitrothion

PS

NO2

CH3

OCH3

OCH3

TAML/H2O2

TAML/H2O2 HO2-

HydrolysisTAML/H2O2

+

Oxidative degradation

Oxidative hydrolysis

Desulfuration

Fenitrothion and Fenitrooxon Degradation - pH Dependence

Initial rate measurements following p-nitrocresol formation (395 nm)

pH

7 8 9 10 11 12Rat

e of

Fen

itrot

hion

Deg

rada

tion

(M/s

)

0.0

2.0e-6

4.0e-6

6.0e-6

8.0e-6

1.0e-5

1.2e-5

1.4e-5

1.6e-5

TAML/peroxideperoxide

TAML/H2O2 mediatedFenitrothion degradation

TAML®: Fenitrothion: peroxide (1: 25: 50,000) in phosphate buffer (0.1M)

pH7 8 9 10 11R

ate

of F

enitr

ooxo

n D

egra

datio

n (M

/s)

0.0

5.0e-8

1.0e-7

1.5e-7

2.0e-7

2.5e-7

Peroxide assistedFenitrooxon degradation

Fenitrooxon: peroxide (1:2000) in phosphate buffer (0.1M)

p-Nitrocresol Degradation – pH dependenceOptimization of Reaction Conditions

At higher pH, the reaction rate increases, but catalyst gets inactivated faster

Optimum pH range 9.5-10.0

p H

7 8 9 10 11

Rat

e of

p-n

itroc

reso

l Deg

rada

tion

0.0

4.0e-7

8.0e-7

1.2e-6

Initial rate measurements

Time (s)

0 10 20 30 40 50 60

Deg

rada

tion

of p

-nitr

ocre

sol

0.0

0.2

0.4

0.6

0.8

1.0 pH 8.0pH 9.0

pH 10.0

pH 10.5

Kinetics of oxidative degradation

pH 9.5-10.0, phosphate buffer (0.1 M), 25°CTAML®: Fenitrothion: Peroxide 1 : 25 : 50,000

Optimal reaction conditions for Total degradation of fenitrothion

Summary of Fenitrothion degradation Study

Fenitrothion – Oxidative hydrolysis

p-Nitrocresol – Oxidative degradation

Fenitrooxon – Peroxy anion assisted hydrolysis

9.0 10.0 11.0

>9.5

<10.5

9.0-11.0

Limiting condition

Optimal pH

Methyl maleic acid

Fe

Oxalic Acid Maleic acid

CO2 + H2O + SO4

2- + NO3- + NO2

--+

Oxidative hydrolysis

Minor Pathway

Peroxide assisted Hydrolysis

Oxidation

Fe-TAML®

H2O2Major pathway

+

Formic AcidHO2C CO2HO O

OHHO

HO2C CO2HH-CO2H +

O

H3C

O2N

Fenitrothion

P

S

OMe

OMe

Fenitrooxon

O

H3C

O2NP

O

OMe

OMe

OH

H3C

O2N

p-nitrocresol

Mineralization

P

O

H3CO

H3COOH

Total Degradation of Fenitrothion

Fenitrothion DegradationAquatic Toxicity

Fenitrothion (99%)

MicroToxEC50 (15 min.)

Mg/L

D. MagnaEC50

Mg/L

2.33 14.1

Reaction mixture(pH 10, quenched with catalase)

57.25 >530

Reduction in toxicity 25-fold >38-fold

TAML catalyst (FeBF2) 58.00 NA

Dafnia MagnaVibrio fischeri

STAML/ (CH3)3COOH in MI

S

OSulfoxide

Dibutyl sulfide

Bis (2-hydroxyethyl) sulfide

TAML:substrate = 1:1,000; pH 8; Phosphate buffer, 25°C

Reaction of Dialkyl sulfides with TAML/peroxide

S S OHHOO

TAML/H2O21: 10,000

5 mts.HO OHSulfoxide

100%

S <5 mts.

TAML/H2O21: 2000

TAML/H2O21:5,000

S

S

O

O O

Sulfoxide

Sulfone

Dibutyl sulfide

! 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

Conclusions

TAML®-peroxide technology:

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 large-range 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 GhoshDr. Peter Berget

Dr. Edwin MinkleyNSF

DURIP

Wagner and Yang, 2002. Ind. Eng. Chem. Res., 41(8), 1925-1928

Nucleophile assisted Hydrolytic Detoxification of Chemical Warfare Agents

ROP

O

FCH3

ROP

O

OO-RO

P

O

O-

ROP

O

SNR'2

OOH-

OOH--O2, H2O

H2O2

CH3

-O3SNR'2

CH3

CH3

R = CH(CH3)2R = CH(CH3)[C(CH3)3]

SarinSoman

VX R = CH2CH3 R' = CH(CH3)2

- HF

Peroxy Anion (OOH-)

The rate of nucleophile aided hydrolysis of esters is increased by cationic micelles (e.g. -OOH/CTABr).

O NO2

CH3

PSMeO

MeO"Fe IV =O" O NO2

CH3

PH3CO

H3CO

SO

O NO2

CH3

PH3CO

H3CO

S O

O NO2

CH3

PH3CO

H3CO

S O

O NO2

CH3

POH3CO

H3COSO4

2-

Fenitrothion

Three-membered 'phospho-oxythiarane' intermediate

Fenitrooxon

Minor pathway

+

OP

OH3CO

H3CO H

Dimethyl phosphate

SO42-

O NO2

CH3

PH3CO

H3CO

S O

OH H

HO NO2

CH3

PH3CO

H3CO

S O

O H

H p-Nitrocresol

+

Major pathway

Fe-TAML peroxide oxidant system mimics Cytochrome 450

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

O

S

R

R1O

R1OP ROH +

O

O

H

R1O

R1OP + SO4

2-Fe-TAML

O

O

R

R1O

R1OP Small alephatic acids

+ CO2 + H2O

H2O2

HOO-

∆

R = -ON

Cl

Cl

Cl

N

N-O

NN

CH3H3C

CH3

-O

R1 = -CH3 −C2H5,

Minorpathway

O NO2

CH3

Fenitrothion Chlorpyriphos Quinalphos Diazinon

Catalysis of Phosphate Triester Hydrolysis by Cationic Micelles

1. Dubey, Gupta et al., Langmuir, 2002, 18, 10489-104922. Couderc and Toullec, Lanmuir, 2001, 17, 3819-3828.3. Brinchi, profio et al., Langmuir, 2000, 16, 10101-10105

• 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

______________________

• Loosely cross-linked peptidoglycan composed of N-acetyl 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)

Bacterial EndosporeSpore Cortex

Teichoic Acid