gsh reactivity: an in chemico method and overview of its database

T. W. Schultz

Presented at the Logan Workshop

March 23-24, 2010

Background Method & Data Base Reactions & Coverage What We are Currently Doing

No consensus in number > 25 < 50 (40)Vary in preferred target moiety

Typically 45% -SH and 45% -NH2

Vary in structural domain Simple: isothiocyanate RN=C=S Complicated: Michael addition Complex: SNAr addition

In order of increasing hardness include:

Thiol-group of cysteine S-atom of methionine Primary amino-group of lysine Secondary amino-group of histidine

CH2=CH- k(thiol) K(amine) ______________________________________________________

C(=O)OMe 0.011 0.00076

C#N 0.0027 0.00020

C(=O)NH2 0.00046 0.000026

Different nucleophiles can differ in their absolute reactivity towards a given electrophile, but relative

reactivity is well correlated over a range of nucleophiles within the same mechanism.

Use in a similar context to in vitro or in silicoQuantitative, rapid, inexpensive

experiments with model nucleophilesVerify reaction-based rules of reactivityDefine the chemical space of a reactionProvides a measure of relative potencyUseful in refining categories and modeling

Full Kinetics- measured at several time intervals with several initial concentrations of electrophile (100 chemicals)

Partial Kinetics- measured at several time intervals with one initial concentrations of electrophile (O’Brien Assay)

Concentration giving 50% reaction in a fixed time- measured at one time with several initial concentrations of electrophile (1000 chemicals)

Extent of reaction after a fixed time- measured at one time with one initial concentrations of electrophile (Gerberick Assay)

Quantitative with kinetics-linked endpointSimple, rapid, repeatable, and inexpensiveCysteine-based thiol targetDepletion-based (% free thiol) Analyses by

Concentration-Response (RC50)

Full kinetic

Readily availableConcentration can be analyzed by simple

methodsOdorless, non-hazardousWater soluble but NOT readily soluble in

organic solventsDoes not lead itself to HPLC

Initial concentration of electrophile that gives a half-life of 120 minutes

Good if electrophile is in excess Adequate if concentrations of

electrophile and GSH are similar Poor if GSH is in excess; RC50 values are

then extrapolated

> 2,600 individual assays≈ 1,000 separate structure> 25 different organic reactions (mechanisms)> 300 not reactive because of structure< 100 not reactive because of solubility< 50 not reactive because of color interference

Highly relevant with multiple domains1) Michael Addition2) Nucleophilic substitution (N-sub) of

haloaliphatics3) N-sub of haloaromatics (SNAr)

1) Michael Addition > 250 compounds (cpds)

2) Pre-Michael Addition > 50 cpds

3) N-sub of Haloaliphatics > 150 cpds

4) N-sub of Haloaromatics > 125 cpds

Highly relevant with simple domains 1) disulfide exchange 2) O-heterocyclic ring opening 3) N-sub of alkyl sulfates & sulfonates 4) nitroso- & N-oxides 5) disulfide formation

1) disulfide exchange, >10 cpds 2) O-heterocyclic ring opening, 20 cpds 3) N-sub of alkyl sulfates, 5 cpds 4) N-sub of alkyl sulfonates, >10 cpds 5) nitroso-compounds, >5 cpds 6) N-oxides, 10 cpds All demonstrate GSH reactivity &

are related to sensitization

Less relevant 1) arenesulfinic acid substitution 2) azomethyne addition 3) thiocyanate addition 4) mercury thiolate formation 5) others

Other chemical classes 1) unsaturated alcohols 2) secondary amines 3) dialkyl acetals 4) lactates 5) anhydrides (hydrolysis) 6) aldehydes (Schiff-base formers) 7) diones (cycloaddition to diamines)

only ,-unsaturates are reactive

Compounds RC50 (mM) _______________________________________________ C=CC(O)Cn 25 - 50 C#CC(O)Cn 1.0 – 3.0 OCC=CCn 5.0 - 10.0 OCC#CCn 3.0 - 5.0

1-pentyn-3-ol 1.3, 1.7 4-CH3-1-pentyn-3-ol 16, 13 3,4-CH3-1-pentyn-3-ol NR at 50mM

Base Structure and Special Features

R1C(X)YR2

Y = C6H5 > C#C > C=C, etc

X = I > Br > Cl > F

R1 = H > CnH(2n +1)

R2 no effect

Base Structure and Special Features

XC6H3Y2

Y = NO2, > in-ring-N > CHO > CN

X = F > Cl > Br > IPosition of leaving group in relationship to activity groups effects potency

Thank you