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REPORT ON THE STSM OF NATALYA SAPRYGINA
1.1.1.
Work plan of Natalya Saprygina for her STSM, from 13/05/2013 to 02/06/2013
Laser flash photolysis investigation of reactions of triplet excited TCBP with adenosine
monophosphate (AMP) and guanosine monophosphate (GMP) in the wide range (from 2 to 12) pH
of aqueous solutions, aimed at determining quenching rate constants of TCBP triplets. Knowledge
of quenching rate constants of TCBP triplets by GMP and AMP will allow us to interpret correctly
CIDNP data obtained in these reactions and will be compliment to our study of deprotonotion of
guanosyl cation radical [1]. These LFP data will help to understand in details the CIDNP formation
mechanism for these molecules and to discriminate between two factors that can contribute in the
geminate polarization magnitude: quenching factor and magnetic interactions (enhancement
coefficient). This will allow optimizing conditions for TR-CIDNP experiment and improving
hyperpolarization method for studying fast radical reactions of biologically relevant molecules.
Main Results and discussion
At the laboratory of Prof. Günter Grampp at the Institute of Physical and Theoretical
Chemistry, Graz University of Technology, Austria I continued to learn in details how to use a laser
flash photolysis apparatus working on the nanosecond-microsecond time scale. This set-up was
used for studying photoinduced radical reactions which lead to CIDNP of biologically relevant
molecules.
Using laser flash photolysis technique we investigated the reactions of triplet excited
3,3´,4,4´-benzophenone tetracarboxylic acid (TCBP) with guanosine-5´-monophosphate (GMP) as
well as adenosine-5´-monophosphate (AMP) in the wide range (from 2 to 12) pH of aqueous
solutions and determined rate constants of TCBP triplets quenching. This study continues the
project on pH-dependent photochemistry leading to hyperpolarization of biologically relevant
molecules. In the 1st STSM photoreactions of amino acids His and Tyr were studied, namely the
influence of amino group charge on the hyperpolarization magnitudes of these amino acids was
established.
Depending on the pH value of the aqueous solution reactants can exist in different
protonation states related to acid-base equilibrium. The 1st protonated state of phosphate group of
nucleotides with acidity constant pKa´<1 was not taken into account. The 2nd protonated state of
phosphate group of GMP and AMP is pKa´´~6. In the pH range from 2 to 12 protonated states and
pKa values of GMP are as follows: positively charged GH+, pKa1=2.4; neutral G, pKa2=9.4; and
negatively charged G(–H)–. AMP: positively charged AH+, pKa1=3.5; neutral A. For triplet TCBP,
pKa1*=2.1 and pKa2*=4.7.
1
The dye TCBP was chosen, as in the case of reaction with amino acids, because it is well
soluble in water, suitable for the laser initiation both on 308 nm and 355 nm, hyperfine coupling
constants of its radicals are well-known, that is important for correct interpretation of CIDNP data.
The decay of the triplet dye was measured at 550 nm (pH>pKa2*=4.7) and 590 nm (pH<pKa2*=4.7),
where the absorption of the triplet dye is much stronger than that of the radicals. In the presence of
the nucleotide the triplet decay becomes exponential with a pseudo-first-order rate constant k, which
is proportional to the concentration of quencher (GMP or AMP), c0 ; k=kobs×c0 , where kobs is the
quenching rate constant. In the presence of quencher lifetime of triplet TCBP is decreased and longlived radicals of TCBP and nucleotides appears. From Stern-Folmer plot we obtain quenching rate
constant for each pH value.
pH-dependences of quenching rate constants (Fig. 1) can be divided into several pH regions
with boundaries at pKa values of the initial compounds, which determine the nature of the reacting
species. Each pair of reactants, TCBP and quencher in the protonation states (i,j) is characterized by
quenching rate constant (kij), and the observed kobs can be treated as a sum of kij multiplied by the
fractions of the corresponding species (αi, αj) according to eqn. (1):
kobs   kij   i   j .
(1)
i, j
Intrinsic rate constants obtained from the best fit for each reactant pair are summarized in
Tab. 1.
At 2<pH<4.7 (for GMP) and 2<pH<6.0 (for AMP) for reactant pairs TH4 and GH+, TH4 and
G, TH22– and G as well as TH4 and AH+(HPO4–), TH22– and AH+(HPO4–), TH22– and A(HPO4–), T4–
and A(HPO4–) quenching rate constants are relatively high (≥109 M-1s-1) and it is impossible to the
mechanism of the quenching from LFP data. E-transfer and PCET could be both proposed. With the
deprotonation of dye triplets (pKa2*=4.7) the rate constant of the reaction between TCBP and GMP
decreases by 4 times (reactant pair T4– and G) and we can assume two reaction mechanisms in this
case H-atom transfer and PCET. At pH higher than 9.4, the quenching reaction rate of fully
deprotonated TTCBP* by G(–H)– molecule was very slow (kq<3×107M-1s-1), since G(–H)– has no
abstractable H atoms and e-transfer is restricted in this case due to Coulombic repulsion between
negatively charged T4– and G(–H)–. It is interesting that for AMP the influence of protonated state
of phosphate residue was observed: with the 2nd deprotonation of phosphate group the quenching
reaction rate decreased in about 10 times. We cannot explain this effect right now and additional
TR-CIDNP measurements are required.
Obtained LFP results are in a good agreement with CIDNP data obtained earlier in the
photoreactions of TCBP with GMP: maximum of polarization was achieved at acidic and neutral
pH while at basic solution no significant CIDNP of reaction products was observed. Complimentary
2
TR-CIDNP data for the photoreaction of TCBP with AMP will be obtained soon in ITC
(Novosibirsk). In general two factors can influence the magnitude of geminate CIDNP:
concentration of radicals (in other words, how effective dye quenching by electron- or H-transfer
proceeds) and magnetic interactions in the radical pair. If concentration of quencher is high enough
quenching factor does not affect the geminate CIDNP intensity. Using obtained LFP results we can
optimize TR-CIDNP experiment conditions, under which we will be able to study only magnetoresonance properties of short-lived radicals. On the other hand we will use these LFP results to
optimize conditions for obtaining 13C hyperpolarized signals of nucleotides to investigate structure
of their radicals. Also obtained data will be used to study “chemical reparation” of chosen
nucleotides radicals by amino acids and proteins.
Main results and conclusions
Using laser flash photolysis we investigated the reactions of triplet excited TCBP with with
guanosine-5´-monophosphate (GMP) as well as adenosine-5´-monophosphate (AMP) in the wide
range (from 2 to 12) pH of aqueous solutions. pH dependence of quenching rate constants of TCBP
triplets by GMP and AMP was revealed and explained in terms of pKa of reactants.
Obtained LFP data will help to understand in details the CIDNP formation mechanism for
these molecules, to optimize conditions for TR-CIDNP experiment and to improve
hyperpolarization method for studying fast radical reactions of biologically relevant molecules.
[1] O. B. Morozova, N. N. Saprygina, O. S. Fedorova and A. V. Yurkovskaya, Deprotonation
of Transient Guanosyl Cation Radical Catalyzed by Buffer in Aqueous Solution: TR-CIDNP Study,
Appl. Magn. Reson., 2011, 41, 239-250.
Fig. 1. pH dependencies of the quenching rate constants of TCBP triplets by AMP (black dots)
3
and GMP (red dots). Solid lines – the best fits obtained according eqn. 1.
Table 1. Quenching rate constants of TCBP triplet by GMP and AMP
Quencher
pH region
Reactant pair
kq (M-1s-1)
Guanosinemonophosphate
(GMP)
pH<2.4
TH4 and GH+
1.2×109
Adenosinemonophosphate
(AMP)
2.4<pH<2.8 TH4 and G
1.2×109
2.8<pH<4.7 TH22– and G
1.4×109
4.7<pH<9.4 T4– and G
3.2×108
pH>9.4
T4– and G(–H)–
<3×107
pH<2.8
TH4 and AH+(HPO4–)
1.0×109
2.8<pH<3.5 TH22– and AH+(HPO4–) 1.1×109
3.5<pH<4.7 TH22– and A(HPO4–)
9.5×108
4.7<pH<6.0 T4– and A(HPO4–)
1.1×109
pH>6.0
T4– and A(PO42–)
1.5×108
4
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