1. Conserved [3+2] Cycloaddition Reactions with Derivatives of Vitamin B6
Anna L. Pleto, David G. Hilmey*
Department of Chemistry, St. Bonaventure University, St. Bonaventure, New York
Vitamin B6 is a antioxidant cofactor in the human body. In the presence of light, singlet oxygen (1O2) is generated, causing lipid peroxidation resulting in a cell to lysis. A
connection was made between the proteins needed for biosynthesis of vitamin B6 and 1O2 generation in certain fungi. In order to investigate how Vitamin B6 is involved in this process, the
reaction between 1O2 and vitamin B6 was studied. In a previous study, observations and reactions determined Vitamin B6 quenches singlet oxygen (1O2) which led to our group determining the product(s)
yielded through subjecting aqueous, phosphate-buffered solutions of B6 to 1O2. Once determined, a low-temperature oxidation study suggested pyridoxine yielded a unique ring-contracted product, 3,4-Bis-
hydroxymethyl-5-hydroxy-5-(1-oxoethyl0-1,6-dihydropyrrole-2-one (Product 4), through a bicyclic endoperoxide intermediate. This suggests a [3+2] cycloaddition of 1O2 to the pyridine ring. Reactivity of Product
4 was tested with other reactive oxygen species, along with behavior in acidic and basic conditions. Other cycloaddition using pyridoxine have been recently been probed. During Thiamin pyrimidine
biosynthesis, PLP and His66 from the THI5 gene react in a cycloaddition fashion via a proposed mechanism with His66 being the alleged N=C-N donor. The possibility of a cycloaddition motivated us to simulate
the enzymatic environment and hopefully induce a the cycloaddition reaction between starting materials. Such starting materials were then modified to better mimic the active sites environment thereby
increasing its reactivity and simulating the His66 residue of the THI5 enzymatic environment.
Background
Layered 1H NMR time course of pyridoxine oxidation.
Time course is over 3 hours and proposed cycloaddition
mechanism.
Conclusion
Further Studies
• Singlet oxygen adds to the B6 vitamers by a
[3+2] cycloaddition mechanism
• Pyridoxine does not reaction with other
reactive oxygen species, such as hydrogen
peroxide and hypochlorite
• Unable to simulate enzymatic environment
of Thi5 to induce cycloaddition reaction
• Use heavy singlet oxygen (18O2) to further
support [3+2] cycloaddition of oxygen
• Perform photo-sensitized oxidation on
other vitamins, such as Vitamin D2 and
Vitamin E
• Continue to analyze pyridoxine and
Na2HPO4 reaction by time-dependent 1H
NMR scans
References
PLP and Active Site His66
• Hilmey, David G., David Samuel, and Kristen Norrell.
"Novel Ring Chemistry of Vitamin B6 with Singlet
Oxygen and an Activated ene: Isolated Products and
Identified Intermediates Suggesting an Operable [3+2]
Cycloaddition." Organic & Bimolecular Chemistry
(2012): n. pag. Print.
• Lai, R.-Y., Huang, S., Fenwick, M. K., & Hazra, A. (2012,
June). Thiamin pyrimidine biosynthesis in Candida
albicans: a remarkable reaction between histidine and
pyridoxal phosphate. National Institutes on Health.
• Ohta, B. K., & Foote, C. S. (2002, July). Characterization
of endoperoxide and hydroperoxide intermediates in
the reaction of pyridoxine with singlet oxgyen. JACS
Communication.
Pyridoxine Oxidation
Pyridoxine Reactivity Profile
Pyridoxine reactivity in various solutions
Proposed cycloaddition mechanism of PLP
and active site His66 to form HMP-P
Attempted Pyridoxine Reaction with
Reagents similar to enzymatic
environment
Active site of the C. albicans THI5p
showing PLP bound via an imine to Lys62
and His66 in close proximity to the PLP.
H3C___I
(25%)