The document discusses the identification of reaction mechanisms using intermediate trapping of reactive intermediates, which are short-lived and highly reactive molecules. It covers examples involving the trapping of oxocarbonium ions and single electron transfer intermediates, as well as their implications in enzyme-catalyzed reactions. Furthermore, the authors propose pathways and applications of intermediate trapping using various substrates and techniques to understand complex reaction mechanisms in chemistry.

1. Organic Pedagogical Electronic
Network
Deciphering Reaction Mechanism
with Intermediate Trapping
Shuangyu Ma & Yiling Bi
University of Utah

2. Identifying mechanism with intermediate trapping
Overview:
A reactive intermediate is a short-lived, high-energy, highly
reactive molecule. When generated in a chemical reaction, it
will quickly convert into a more stable molecule. When their
existence is indicated, reactive intermediates can help explain
how a chemical reaction takes place.
Wiki Page: http://en.wikipedia.org/wiki/Reactive_intermediate
Other References: Young, P. R.; Jencks, W. P. J. Am. Chem. Soc. 1977, 8238; Cordes, E.; Bull, H. Chem. Rev. 1974, 74,
581; Fife, T. Acc. Chem. Res. 1972, 78, 264.

3. Wiki Page: http://en.wikipedia.org/wiki/Reactive_intermediate
Other References: Young, P. R.; Jencks, W. P. J. Am. Chem. Soc. 1977, 8238; Cordes, E.; Bull, H. Chem. Rev. 1974, 74,
581; Fife, T. Acc. Chem. Res. 1972, 78, 264.
Early Examples: hydrolysis of acetals, ketals and ortho esters:
Identifying mechanism with intermediate trapping

4. Wiki Page: http://en.wikipedia.org/wiki/Reactive_intermediate
Other References: Young, P. R.; Jencks, W. P. J. Am. Chem. Soc. 1977, 8238; Cordes, E.; Bull, H. Chem. Rev. 1974, 74,
581; Fife, T. Acc. Chem. Res. 1972, 78, 264.
Trapping of the oxocarbonium ion intermediate by sulfite dianion:
Identifying mechanism with intermediate trapping
• Results: achieved up to 97% trapping; rate constants for
acid-catalyzed cleavage of ketals are independent of
[SO3
2-] (Table 1).
• Conclusion: [SO3
2-] is not present in the TS of the RLS.
Reaction proceeds through the oxocarbonium ion
pathway.
H2O
R'
OR
Oxocarbonium ion
OH
OR
R'
R'
O
SO3
2-
SO3
-
OR
R'
Disulfide buffer
OR
OR
R'
Monitor absorbance change at 262 nm

5. Identifying mechanism with intermediate trapping
References: Haberfield, P. J. Am. Chem. Soc. 1995, 3314; Bank, S.; Noyd, D. A. J. Am. Chem. Soc. 1973, 95,
8203.
Proposed two pathways for the SN2 reaction :
Nu X-X Nu +
Simultaneous transfer of elctron pairs
Single electron stransfer pathway (stepwise mechanism)
XNu +Nu X Nu+ X+ X-Nu +
Difficulty: the radical & radical anion intermediates
quickly continued to form the SN2 product,
concentration would be too low for trapping.
Early Examples: trapping of a single electron transfer intermediate.

6. Identifying mechanism with intermediate trapping
References: Haberfield, P. J. Am. Chem. Soc. 1995, 3314; Bank, S.; Noyd, D. A. J. Am. Chem. Soc. 1973, 95,
8203.
Early Examples: trapping of a single electron transfer intermediate.
Using identity reaction and very large concentration of scavenger (as solvent).
Br
H
O
Br
H
Br
H
O
Br
H
H
Hterbutylammonium bromide
cumene
100 °C, 66 h
H
Br
Also obatined other cumyl radical-derived products:
yield: 47%
cis & trans
Detectable
X+ X-X +X
X+X XX +
H
HX+ + X- +
H
H H ++

7. Application of intermediate trapping in understanding enzyme catalyzed
complex reaction mechanism
References: Mehta, A. P.; Abdelwahed, S. H.; Xu, H.; Begley, T. P. J. Am. Chem. Soc. 2014, 136, 10609;
Mehta, A. P.; Abdelwahed, S. H.; Begley, T. P. J. Am. Chem. Soc. 2013, 135, 10883.
MoaA/MoaC catalyzed rearrangement reaction of GTP to cPMP:
MoaA/MoaC
44
33
22
11O N
N66
N NH
NH2
O
HO OH
55
4-O9P3O
N
NH
NH2
O
H
N
22
11
N
H
66
33
44
O
O
O
P
O
55
O
O
S-adenosyl methionine
(SAM)
B
Proposed mechanism:
44
33
22
11O N
N
66
N NH
NH2
O
HO OH
55
PPPO
44
33
22
11O N
N
66
N NH
NH2
O
HO OH
55
PPPO
H
P = phosphate group
radical SAM
5'-deoxyadenosine
44
33
22
11O N
N66
N
NH
NH2
O
HO OH
55
PPPO
44
33
22
11O N
H
N66
N
NH
NH2
O
HO OH
55
PPPO
44
33
22
11O H
N
H2N
N
NH
NH2
O
O OH
55
PPPO
Intermediate II
66
O
H
44
33 22
11
O
H
N
H2N
N
NH
NH2
O
O OH
55
PPPO
66
OH
H+
44
33 22
11
OH N
H2N
N
NH
NH2
O
O OH
55
PPPO
66
OH
H
B
H+
44
33 22
11
OH
H
N
H2N
N
NH
NH2
O
O OH
55
PPPO
66
OH
H
B
H+
44
33 22
11
OH
H
N
H2N
N
NH
NH2
O
O OH
55
PPPO
66
OH
H+
Intermediate I

8. Application of intermediate trapping in understanding enzyme catalyzed
complex reaction mechanism
References: Mehta, A. P.; Abdelwahed, S. H.; Xu, H.; Begley, T. P. J. Am. Chem. Soc. 2014, 136, 10609;
Mehta, A. P.; Abdelwahed, S. H.; Begley, T. P. J. Am. Chem. Soc. 2013, 135, 10883.
44
33
22
11
OH
NH
HN
N
NH
NH2
O
HO
HO
55
PPPO
66
HO
Intermediate III
44
33
22
11
OH
NH
HN
N
NH
NH2
O
HO
H2O
55
PPPO
66
O
44
33 22
11
OH N
H
N
N
NH
NH2
O
HO
55
PPPO
66
O
44
33 22
11
OH N
H
N
N
NH
NH2
O
HO
55
PPPO
66
O
H+
44
33 22
11
O N
H
H
N
N
NH
NH2
O
HO
55
PPPO
66
O
44
33 22
11
O N
H
H
N
N
NH
NH2
O
O
55
O
66
O
P
O
O
Trapping of the key intermediates using substrate analogues:
Trapping of radical intermediate I by 2'-chloroGTP
44
33
22
11O N
N
66
N NH
NH2
O
HO Cl
55
PPPO H
44
33 22
11O N
N
66
N NH
NH2
O
HO Cl
55
PPPO
radical SAM
5'-deoxyadenosine
MoaA
44
33 22
11
O N
N
66
N NH
NH2
O
O
55
PPPO
H
-Cl
B
H+
N
H
N
N
NH
NH2
O
O
PPPO
O
Observed by LC-MS
O
O
Observed by LC-MS
HOOC
S
O
O
Trap with 3-mercaptobenzoic acid

9. Application of intermediate trapping in understanding enzyme catalyzed
complex reaction mechanism
References: Mehta, A. P.; Abdelwahed, S. H.; Xu, H.; Begley, T. P. J. Am. Chem. Soc. 2014, 136, 10609;
Mehta, A. P.; Abdelwahed, S. H.; Begley, T. P. J. Am. Chem. Soc. 2013, 135, 10883.
44
33
22
11
OH
NH
HN
N
NH
NH2
O
HO
H
55
PPPO
66
O
44
33
22
11
OH N
H
N
N
NH
NH2
O
HO
55
PPPO
66
HO
44
33
22
11
OH
NH
HN
N
NH
NH2
O
HO
H
55
PPPO
66
HO
Trapping purine ribose adduct intermediate II by 2',3'-deoxyGTP
44
33
22
11O N
H
N66
N
NH
NH2
O
H H
55
PPPO
44
33 22
11O N
N
66
N NH
NH2
O
H H
55
PPPO
O2
44
33
22
11O N
N66
N
NH
NH2
O
H H
55
PPPO
phosphatase
hydrolysis
44
33
22
11O N
N66
N
NH
NH2
O
H H
55
HO
H+/H2O
44
33
2211
O
N
H
N
66
N
NH
NH2
O
55
HO
HO
Observed by LC-MS
Trapping intermediate III by 2'-deoxyGTP
44
33 22
11O N
N66
N NH
NH2
O
HO H
55
PPPO
44
33 22
11
OH N
H
H
N
N
NH
NH2
O
HO
55
PPPO
66
OH2
H+
44
33
22
11
OH N
H
N
N
NH
NH2
O
HO
55
PPPO
66
H+
44
33 22
11
OH N
N
N
NH
NH2
O
HO
55
PPPO
66
Observed by LC-MS

10. Problems:
1. The scheme shown below is the
proposed intramolecular anodic coupling
amides and olefins. A second single-
electron oxidation followed by solvent
(MeOH) trapping of the
subsequent cation would afford the final
product 4.
Based on the information, draw two
possible pathways for the trapping of
cation by the alcohol. (J. Org. Chem.,
2014, 79, 379−391)

11. Problems:
2. Thymidylate synthases (TSases)
catalyze the last step in the de novo
biosynthesis of the DNA nucleotide
dUMP to dTMP, using CH2H4 folate as a
methylene donor. (J. Am. Chem. Soc.,
2012, 134, 4442−4448)
Figure 1 shows the total ion counts
measured at various reaction times for
substrate dUMP and product dTMP along
with their sum (which represents the total
amount of material due to these species).
Please explain why intermediate
accumulates according to data shown in
Figure 1 and draw suggesting
intermediate accumulation curve.
Figure 1. Single-turnover FDTS reaction.
Total ion counts for dUMP and dTMP. dUMP
(purple◆) and dTMP (blue■). The sum of the
counts for dUMP and dTMP (black▲) was
not conserved during the reaction

12. Problems:
(3) The figure shows (a) 14C-labeled
dUMP or (b)14C-labeled CH2H4 folate.
The labeled carbon is shown in red. In
both cases, the 14C-containing trapped
intermediate eluted at∼11 min,
representing the same trapped species.
According to the data shown below,
please determine whether the trapped
species already contains the methylene
from the cofactor CH2H4 folate and justify
your answer.

13. Solutions
1. The key part of the question is to come up with the equilibrium in the amidyl radical
reactions. The radical products trapped by the alcohol are 3 and 4.

14. Solutions
2. In the figure, during 0.5 to 10 s, the sum of the ion counts for dUMP and dTMP
was substantially less than those at the beginning and the end of the reaction. This
observation suggests that a reaction intermediate accumulated during this time
period. The suggesting intermediate accumulation curve is shown in red line.

15. Solutions
3. [11-14C] CH2H4 folate radiolabeled, a new radioactive peak had developed that
had the same retention time as the peak observed when starting with [2-14C]
dUMP. It means it is the same reactive intermediate. So it shows that the
intermediate nucleotide being chemically trapped during the acid quenching
had already undergone the condensation and that the carbon−carbon bond
between the C5 of dUMP and the methylene had been formed prior to the formation
of that intermediate.

16. This work is licensed under a
Creative Commons Attribution-
ShareAlike 4.0 International
License.
Contributed by:
Shuangyu Ma & Yiling Bi (Undergraduate Students)
University of Utah
2014