A chemical kinetic isotope effect is a dependence of a rate of a unidirectional chemical process

an the isotopic content or structure of the reactant(s)

A B
A’ B’

The ‘kinetic isotope effect’ (KIE) on the reaction can be expressed as an a value
relating the abundance ratios of the substituted and unsubstituted reactants
([A’]/[A]) to the ratio of their rates of reaction (d[A’]/d[A])

(d[A’]/d[A])
‘KIE’ = a’reacted-reactant = [A’]
( /[A])

So, our job in understanding this phenomenon is to describe and predict how
concentration relates to rate, and how that relationship changes on isotopic substitution
 A couple of important examples

RuBisCO
• First steps of photosynthetic enzyme
carbon fixation
Ephemeral C6
CO2 (g) + Rubulose 1,5 Biphosphate 2xPGA
intermediate
+

• First steps of pyrolysis of buried organic matter

Kerogen

Gasses Condensates Oils

You’ve already seen an example of a process that is partly chemical-kinetic,
complexly mixed with equilibrium and physical-kinetic processes

Vapor

Interdiffusion with overlying

undersaturated air

Condensation Evaporation
of H2O of H2O

Liquid
 Placing the chemical kinetic effect in context

Classical Quantum
Physics Mechanics

Isotope Exchange Rxns

Reversible, • Heterogeneous
Gravitational
Steady State • Homogenous
• VPIE
A B

Diffusion
Unidirectional,
Chemical Kinetic
Time-Evolving
Thermal Escape
A B
 A primer on chemical kinetics
The characteristic patterns of time evolution for simple reactions

‘Rate’: d[product]
dt Reactant
d[i]
Product
dt

[i] or Mi Product

[i]

Reactant

‘Rate’: d[reactant] time

dt
ln(rate)

time
 A primer on chemical kinetics

Consider CO2 (g) uptake into aqueous solution:

CO2 (g) CO2 (aq)

(all [CO2] terms  d[CO2]/dt = -k•[CO2] where k is an empirically observed

refer to reactant gas) ‘rate constant’

‘k’ is a fundamental speed of the reaction, normalized for the strength of its driving force (i.e., activity
of the reactant or reactants)

d[CO2]/[CO2] = -k•dt

Integration from some initial time and concentration, t 0, [CO2]0,

up to some arbitrary time t and concentration [CO2]t yields:

=—

ln[CO2]t – ln[CO2]0 = -k∆t (∆t is t - t0)

ln([CO2]t/[CO2]0) = -k∆t
 
[CO2]t/[CO2]0 = exp-k∆t
 Measuring reaction rate

Rates of reactions are generally defined by change over time of amounts or concentrations
of reactants or products, multiplied by 1/c, where c is the stoichiometric coefficient

Consider carbonate precipitation: CO3=(aq) + Ca+2(aq) CaCO3 (s)

c: -1 -1 +1
Rate = —dMCO3=/dt = —dMCa /dt = dMcc/dt
+2

An abstract example with funny stoichiometry: 2 x A + 3 x B C

c: -2 -3 +1
Rate = —(dMA/dt)/2 = —(dMB/dt)/3 = dMC/dt

Thus the rate of reaction will be the same regardless of which reactant or product is
observed to accumulate or be consumed
 Quantifying integrated reaction progress

• The ‘reaction progress variable’ is a unit for reporting the integrated extent
of reaction; nomenclature is mixed, but it is sometimes given as V, (capital
‘Chi’),

V is generally calculated as total mols of accumulated product or lost reactant

(per unit volume, mass or total mols of system), divided by the stoichiometric
coefficient for that product or reactant

2xA+ 3 x B C

V= [B] – [B]0
-3

Where a critical reactant is being depleted following the Rayleigh distillation law, it is
also common to define ‘reaction progress’ as -ln(F), where F is the fraction of initial
reactant remaining; in this case, ‘reaction progress’ will be proportional to how much
time has passed as the reaction proceeds. For example above:
B
-ln(F) = -ln( )
B0
 More complex, or ‘higher order’ reactions
The preceding introduction focuses on on reactions with the simplest ‘rate laws’ — rate
depends linearly on the concentration of each reactant. This is just one of many possible
rate laws. A general formula is:

Rate = -k P [i]Xi
Where k is the experimentally determined rate constant, [i] is the concentration of any of
the reactants that participate in the reaction, Xi is an experimentally determined
exponent specific to that reactant, i, and the product summation is made over all
reactants
 More complex, or ‘higher order’ reactions

• When all Xi values are 1, rate is a simple function of the activity product of reactants:

A+B C
Rate = k x [A] x [B]

• Xi may be (but need not be) equal to the stoichiometric coefficient:

2xA + 3 x B C
Rate = k x [A]2 x [B]3

Thermal decomposition of acetaldehyde: CH3CHO CH4 + CO

Looks simple, but has a peculiar rate law: Rate = k x [CH3CHO]2

Thermal decomposition of N2O5 : 2N2O5 4 NO2 + O2

Complex stoichiometry but a simple rate law: Rate = k x [N2O5]

• If Xi is zero, [i] has no effect on reaction rate, despite the fact that it is part of the
reaction stoichiometry. This sounds weird, but we will see such ‘zero order’ kinetics
are an important part of our understanding of enzymatically catalyzed biochemistry
 Elementary reactions and their order

The preceding review presents the phenomenological description of the kinetics of net
reactions. More useful to us is the treatment of ‘elementary kinetics’.

Elementary kinetics describes the time evolution and concentration dependence of

individual atomistic steps, several of which might combine to yield a net reaction.

The general rule is that rate laws for elementary reactions have Xi values that exactly
equal the stoichiometry coefficient for that reactant, and that it is rare for an elementary
step to involve more than 1 or 2 molecules. Thus, these are the expected scenarios:
A B Rate = k[A]

2A B Rate = k[A]2

A+B C Rate = k[A][B]

We are more interested in elementary, ’atomistic’ kinetics because it will be possible for us
to call on chemical-physics principles to predict their isotope effects, whereas this generally
is not possible for phenomenological net reactions
Photosynthesis provides a good example of the difference between elementary
reactions and net phenomenological reactions

This is a pair of elementary reactions for which we can describe and predict KIE’s
RuBisCO
enzyme

Ephemeral C6
CO2 (g) + Rubulose 1,5 Biphosphate 2xPGA
intermediate
+

This is a net reaction that has nothing to do with atomistic reactions

This is fine; CO2 is a Not only is O2 not
reactant in C fixation directly produced at
CO2 + H 2O CH2O + O2 site of fixation, but it
actually competes with
CO2 for reaction with
RuBP is omitted Eventually C will RuBP
because it is in a Water is consumed, but
nowhere near site of CO2 end up in starches,
steady state of but several steps
consumption and fixation
intervene (p.s., this
regeneration Is the formula for
formaldehyde )
 Forward and back reactions

If a reaction can run in either direction, it is assigned a different rate constant for the
’forward’ (left to right) and ‘back’ (right to left) directions, each of which can have its
own KIE:
kf
A B
kb
kf’
A’ B’
kb ’
(d[A’]/d[A]) kf’
Forward KIE = a’A B = [A’] =
( /[A]) kf
(d[B’]/d[B]) kb’
Backward KIE = a’B A = [B’] =
( /[B]) kb
 Forward and back reactions

Note that the abundances of A, A’, B and B’ will evolve over time in a way that
reflects both the forward and back reactions:
kf
dA = -kf[A] + kb[B] dB = kf[A] - kb[B] A B
dA’ = -kf’[A’] + kb’[B’] dB’ = kf’[A’] - kb’[B’] kb
kf’
A’ B’
Initially, only rapid
forward reaction A steady state is reached kb’
is observed when forward and back
As B accumulates, reactions happen at the
back reaction begins B same rate
[i] or Mi A
A’
B’

time
 The relationship between kinetic and equilibrium fractionations

At steady state dA = dA’ = dB = dB’ = 0

kf[A] = kb[B] kf[A] = kb[B] kf

kf’[A’] = kb’[B’] kf’[A’] = kb’[B’] A B
kb
[A] kb [A’] kb’
= = kf’
[B] kf [B’] kf’
A’ B’
[A] x [B’] kb x kf’ kb’
=
[A’] x [B] kf x kb’

k f’
RB’ kf af
= = = aB-A at equilibrium
RA’ kb’ a b

kb

I.e., the equilibrium fractionation is simply the balance of forward and reverse KIE’s;
conversely, we can leverage the knowledge of equilibrium a’s to constrain relative
values of af and ab. Also, we might suspect KIE’s are related in some way to Q’/Q values
 The Arrhenius relationship and plot
Now let’s see how the energetics of k behaves, which will be our route to understanding KIE’s

Experimental observations indicate a characteristic dependence of k’s on Temperature:

-Ea -Ea
k= A x eR x T or k= A x e kB x T

A: The pre-exponential factor

ln(k) R: The gas constant
kB: The Boltzmann constant
Ea: ‘Activation energy’, in molar or atom
units (for R or kB, respectively)
T: Temperature in K
1
(K-1)
T

• ‘A’ can be interpreted as a frequency of events (e.g., collisions) where reactant(s)

take on an orientation that permits reaction

• Ea can be thought of as an energy barrier that must be overcome for appropriately

oriented reactant(s) to react
KIE’s are generally temperature dependent

12
C2H5D vs. 12C2H6

13 12
C CH6 vs. 12C2H6
This temperature dependence implies that A and Ea are themselves isotope dependent

-Ea
A k= A x e kB x T
A’ k -Ea’
Slope = Ea/R or Ea/kB
k’ = A’ x e kB x T
ln(k) k’ -∆Ea
k’ A’
KIE = = x e kB x T
k A
Slope = Ea’/R or Ea’/kB Where ∆Ea = Ea’ - Ea

1
(K-1)
T

This observation, combined with the insight that KIE’s have a connection to
equilibria, and thus Q’/Q values, gives us some attractive ‘leads’ for constructing
a predictive atomistic model
Transition state theory and the Eyring-Evans-Polanyi equation

The reactant and transition state

interconvert, establishing a sort
of local equilibrium

But conversion of the transition

state to product is irreversible (for
one direction of an elementary rxn)

Unimolecular rxn: A [‘activated’ A] Product

Bimolecular rxn: A+B [A—B] Product

 Transition state theory and the Eyring-Evans-Polanyi equation

This is the Arrhenian Ea

-∆G
k x kB x T kB x T
k= e
h
All this is the Arrhenian A
k: Rate of reaction
k: ‘transmission’ coefficient, which is the probability a TS molecule goes to product
∆G: Gibbs free energy difference between reactant and TS
kB: Boltzmann constant
h: Plank constant
T: Temperature in Kelvin
So, question is how to evaluate ∆G and k

Note: you will sometimes see ∆G deconvolved into to terms, with ∆S between
reactant and TS pulled out and used as part of the Arrhenian ‘A’, with ∆H
taking the role of ‘Ea’
Urey-Meyer-Bigeleisen theory provides us a way of evaluating free energy differences
between reactant and transition state

H
A + B A + B
H

A H B

∆E for transition state A—H—B complex

AH exists in exchange TS irreversibly
equilibrium with TS turns into product

B A
A H
B
H

∆E for reactant AH ∆E for product BH

Reaction coordinate
Urey-Meyer-Bigeleisen theory provides us a way of evaluating free energy differences
between reactant and transition state

Reactant-TS equilibrium constant

-∆G
k x kB x T kB x T
k= e
h

-∆∆G
k’ k’ kB x T
KIE = = xe
k k
Where ∆∆G = ∆G’ - ∆G

-∆∆G
keq for TS-reactant kB x T (QTS’/QTS) bTS
isotope exchange equilibrium = e = =
(QReactant’/QReactant) breactant
(under constant P condition, where we can relate Q to ∆G)
 Urey-Meyer-Bigeleisen theory provides us a way of evaluating free energy differences
between reactant and transition state

H
A + B A + B
H

A H B

b for transition state A—H—B complex

k’ k’ x bTS
AH exists in exchange TS irreversibly KIE = =
equilibrium with TS turns into product k k breactant
B A
A H
H
B b for product BH
b for reactant AH

Two tasks:
Reaction coordinate
• How do I evaluate bTS?
• What do I do with this k’/k nonsense?
The potential energy landscape of possible inter-atomic geometries

Energy

Motion 2

Motion 1
 Interatomic motions (including, but not limited to bond vibrations) can be understood
as a consequence of the shape of the energy landscape
d2E
= k — the spring constant for motion along x coordinate
dx2
k positive; a harmonic ‘real’ frequency

Recall n for a motion in the

dimension of x is given by the
harmonic oscillator eqn.:
1/2
1 k m1m2
n= µ=
2π µ m1+m2

k negative; this ‘frequency’ is an imaginary number; a motion can occur, but does not
harmonically repeat – it is an irreversible translation, like a spring-loaded trap that fires
 Interatomic motions (including, but not limited to bond vibrations) can be understood
as a consequence of the shape of the energy landscape

The transition state is the lowest saddle point on the energy surface between the
reactant and some other stable (or metastable) product

The reaction coordinate: the

direction of motion out of the
deep, confined ‘well’ of the
reactant through the transition
state

As this irreversible motion occurs, the transition state has stable, bound vibrations in
other directions. Thus, one of the reactant molecules degrees of free motions (its
fundamental modes) has been turned into a free translation, while the others (or
things like them…) continue to harmonically oscillate in the normal way
 A concrete example: interconversion of hydrogen cyanide and hydrogen isocyanide

Linear tri-atomic Non-linear tri-atomic Linear tri-atomic

3x3-5= 4 fundamental, 3x3-6= 3 modes 3x3-5= 4 fundamental,
modes; all are ’real’ 2 are real modes; all are real
• CN stretch • CN stretch • CN stretch
• CH stretch • Another I don’t know • NH stretch
• CH bend • NH bend
(x2 degenerate) 1 is ‘imaginary’ (x2 degenerate)
• H migration
 Reduced partition function ratio
of the transition state, considering
only its harmonically oscillating
A + B A +
H
B ’real’ modes
H

A H B

b for transition state A—H—B complex

k’ k’ x bTS
AH exists in exchange TS irreversibly KIE = =
equilibrium with TS turns into product k k breactant
B A
A H
H
B b for product BH
Reduced partition
b for reactant AH
function ratio of the
reactant, considering
Reaction coordinate all of its fundamental
modes
Relative rates of motion associated with
the ‘imaginary’ frequency
 Bigeleisen, 1949 ’semiclassical’ theory for KIE’s

The b value of the transition state, ignoring

motions along the reaction coordinate
(3N-6 for linear transition state)

The ratio of imaginary frequencies for The b value of the reactant

motion across the saddle point; this
(3N-5 for linear reactant)
term should be T-independent and
equal to (µ2/µ1)1/2
 Hopefully you found that coherent, but unfortunately you will find alternate treatments
of k’/ k almost everywhere you look
Reduced partition function ratio
of the transition state, considering
only its harmonically oscillating
’real’ modes

k’ k’ x bTS
KIE = =
k k breactant

• Ratio of imaginary frequencies Reduced partition

• Assumed ~1
• Retrieve from isotope effect on
entropies of reactant and TS,
(∆S), computed by first-principles
models; in this case the b is replace
with simply the ∆ZPE
function ratio of the
reactant, considering
all of its fundamental
modes
A primary isotope effect arises when a substitution in a site
that gains or lose a bond during the reaction slows or speeds
up the reaction; these are usually faster for light species than A H
heavy because the atom is relatively free to move in the TS.
This is the origin of the expectation that KIE’s ’normally’ favor b for transition state A—H complex
light isotopes A
H
Disproprtionation
KIE ~ breactant
A
(a bit lower if TS has some sort H
of harmonic interaction between H b for reactant AH
and A)
Reaction coordinate
A H B

b for transition state A—H—B complex

Atom transfer
KIE ~ breactant
B A
A H
H
B b for product BH
b for reactant AH (a bit lower if TS has some sort
of harmonic interaction between
Reaction coordinate H and A or B)
A secondary isotope effect arises when a substitution in a site that doesn’t gain or lose a
bond during the reaction slows or speeds up the reaction; generally, this is because of the
reactant and TS differ in bonding environment for that atom
Here the C-H bond
Here the C-H bond is in a trigonal
is in a tetragonal planar geometry
geometry
with a strong C-Br
bond Deuteration in this site will likely
lead to a secondary isotope effect
because the modes of motion
involving the C-H bond
will have different k’s and µ’s

Secondary KIE’s are generally an order of magnitude lower than primary KIEs
because differences in bonding environments are subtle
An inverse isotope effect arises when the heavy species reacts faster than the
light; this is usually because of a secondary isotope effect where the TS has a
tighter bonding environment than the reactant for the atom in question

Ea for light > Ea for heavy b for transition state complex

b for reactant
b for product

Reaction coordinate

(note in this sort of cartoon, the energy well for the TS is an abstract depiction of
all the motions orthogonal to the reaction coordinate)
The ‘sp2-sp3’ transition is the iconic example of a secondary isotope effect
that is often ‘inverse’

(kH/kD)
 Mapping out primary and secondary isotope effects is a good way to develop an
atomistic understanding of a reaction

Diels-Alder reaction

Isoprene Maleic anhydride Cyclohexene products

Assumed 0 ‰
-10 ‰
1‰ Fractionation factors
0‰
-32 ‰ dD d13C
(e values; negative is ‘inverse’)
-44 ‰ 17 ‰
22 ‰ -62 ‰
-92 ‰
Thus, the reaction is ‘concerted’ (involves both ends of the isoprene boomarang),
and the hydrogens on those carbons undergo conversion from sp2 to sp3 electronic structure
Singleton and Thomas, 1995
 When you are not sure what to think about the transition state structure, reaction
energetics provides a guide

TS is ‘ergo neutral’ – reacting

atoms relatively free of bonds TS is reactant-like TS is product-like

If you ever get serious about a problem like this, use a Density Functional Theory
software packages to predict transition state structure (they will even give you KIE’s
without any extra effort on your part)
 The energy surface and KIE associated with photosynthetic carbon fixation’s first steps

K13CO2/K12CO2 ~ 0.97

(unfilled are 13C KIE’s;

filled are 18O)

The KIE for each version of the RuBisCO

enzyme varies with how selective it is for
CO2 and against O2 (S). A good guess is
that the enzyme is manipulating the
transition state structure to select for
modes of motion that involve carbon
(e.g., bending and asymmetric stretch)
Tcherkez et al., 2006
 KIE’s are generally strongly position specific

in vitro pyruvate decarboxylation

Pyruvate 20
er ve d
Obs
10

d13C (C2) — d13C (C1) (‰)

0

KIE ~25 ‰
-10

-20
Low d13C
C1 -30 rium
Low d13C Equilib
C2
High d13C CO2
-40
Acetyl group 10 20 30 40
T (˚C)

DeNiro and Epstein, 1977

 Position specificity is often ’passed’ from precursor to product, meaning isotopic
structures of organics are a record of several steps in their biosynthetic pathways
Lipid synthesis inherits the acetyl group ‘fingerprint’

CO2

KIE ~25 ‰
Low d13C ‘Elongation’
Pyruvate
Acetyl
High d C
13 group

“C=O” C=O C=O C=O C=O C=O C=O C=O

“CH3” CH3 CH3 CH3 CH3 CH3 CH3 CH3

d13C

DeNiro and Epstein, 1977; Monson and Hayes, 1982

 Mass laws of kinetic isotope effects

k’ k’ x bTS
KIE = =
k k breactant
This term generally acts like
This term generally acts like a the equilibrium mass law; i.e.:
diffusive mass law for
1 1
interdiffusion, where the -
ln(a2i-j) = m1 m2
controlling variable is the l2/3 =
reduced mass; i.e.: ln(a3i-j) 1 1
-
m1 m3

At the high-temperature limit,

li/j = but generally ’flatter’ slopes at
lower temperatures

Independent of temperature,
but very dependent on reduced
mass of reaction coordinate motion
An example of a mass law for a net metabolic reaction: sulfate reduction to H 2S
 Clumped isotope effects can arise through both distinctive b values of reactants
and transition states and differences in reduced mass of motion on reaction coordinate

Additionally, mixing/inheritance effects arise, and can be surprisingly strong

Pyrolysis of Octadecane to yield methane:

CH4 inherits 3 hydrogens from methyl precursor, and one

Very low D/H hydrogen from another source; once they
are combined they are in symmetrically equivalent sites,
yet the probability of two D’s in the same molecule has
been suppressed

Model prediction

Work of Guannan Dong

 Some closing comments about how this works in practice

• Most KIE’s are constrained by experimental observations of net (not elementary)

reactions, or system/organismal level net processes, at a single temperature.

• The most common modern approach to estimating KIE’s of elementary reactions is

density functional theory modeling, which can tell you about potential energy
surfaces, transition state structures and KIE’s with minimal effort. Just don’t get
comfortable – it always looks cool but is often wrong.

• Linking models of elementary reactions to systems scale processes has to happen

through multi-step models of intermediate fluxes and reservoirs; we’ll discuss
examples on Wednesday.

• The end result is that even well constrained KIE’s have real errors of 10’s of %,
relative when you apply them to new experimental or natural contexts.

• The material you learned in this lecture is a kind of road map to understanding what
these data sources are trying to tell you, and developing your own intuitions about
how KIE’s are likely to behave in new contexts. Good luck!