Temperature and pH Dependence of the Cyclization of Creatine:
A Study via Mass Spectrometry
William J. Meadows, Brian J. Diamond and William D. Price
Marshall University, Huntington, WV
Introduction
NH
HN
ln(k)
0
6
8
time (min)
3
10
12x10
Creatinine:Creatine Ionization Efficiency
pH = 3
0
1000
2000
3000 4000
time (min)
5000
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
6000
7000
0.0
30 °C
40 °C
50 °C
60 °C
70 °C
80 °C
90 °C
99 °C
a)
pH = 5
2000
3000 4000
time (min)
5000
6000
Figure 3. Kinetic plots for the cyclization of creatine at a) pH 1, b) pH 3
and c) pH 5 for temperatures ranging from 30 - 99 oC. Note the rates of
cyclization are fastest at pH 3 and slowest at pH 1. The reaction is
reversible and an equilibrium can be established. For Arrhenius plots,
the initial rates are used (data points with lines through them).
0.8
1.0
2
2
4
6
Creatinine:Creatine (actual)
8
10
Figure 2. Relative ionization efficiencies for creatinine:creatine mixtures for
concentration ratios ranging from 10:1 to 1:10. The slope of 1.52 (R = 0.999) indicates
that, under the conditions of these experiments, creatinine is preferentially protonated
over creatine. All data is corrected for this bias.
-1
100
time (min)
150
k =0.020 min
-1
-1
-2
pH 3
-3
k =0.035 min
-1
-4
0
20
40
60
time (min)
80
100
0
c)
ln(k)
-6
k =0.0028 min
-2
k =0.079 min
pH 5
Creatine
Creatine Ethyl Ester
0
-8
pH 1 Ea = 83.1 kJ/mol logA = 9.6 R = 0.9997
pH 3 Ea = 93.6 kJ/mol logA = 11.8 R = 0.9991
pH 5 Ea = 79.1 kJ/mol logA = 9.0 R = 0.9997
2.8
2.9
3.0
-1
-1
T (K )
-3
3.1
3.2x10
Creatine Microspecies
H2N
+
N
HO
Protonated
O
NH
H2N
N
HO
Neutral
O
20
40
time (min)
60
80
oC
Figure 4. Arrhenius plots for the cyclization of creatine at pH 1, pH 3 and
pH 5 for temperatures ranging from 30 - 99 oC. Note the activation
energy is highest for the pH 3 solution even though the reaction rates
are the fastest at this pH. These data indicate that the reaction is
entropically favored at pH 3, compare log A values.
NH2
-1
-1
-4
-6
1.2
4
b)
-4
2.7
0.6
k =0.0095 min
50
0
Arrhenius Plots for Creatine Cyclization
-10
0.4
pH 1
0
0.0
0.2
-1
-0.8
7000
ln (rate)
1000
0.5
0.0
k =0.0044 min
-0.4
-1.2
0
1.5
6
Figure 6 a) Relative concentration of microspecies of creatine as a function of pH
based on equilibrium data from Wang and Yin3 at 20°C. b) Expanded plot with an
estimated ΔGrxn = 16.6 kJ. Proton concentration as a function of pH has been
added to show where acid concentration would become a limiting factor.
Cyclization rate appears to track neutral species concentration.
ln (rate)
ln(k)
c)
14
1.0
-1.0
-2.0
Experimental
Creatinine:Creatine (experimental)
4
Creatinine
Solution-phase kinetics for the cyclization of creatine were obtained for buffered creatine
solutions at pH 1, 3 and 5 with temperatures ranging from 303 K to 372 K and for reaction
times as long as nine days. Reactions were quenched by placing in an ice bath and
bringing the pH to 7.5. Samples were analyzed with ESI-MS on a ThermoFinnigan LCQ at
a flow rate of 3 uL/min, spray voltage of 4.1 kV, and a capillary voltage of 25 V. Prior to
analysis, the pH of quenched reaction solutions was adjusted to pH 1.5 for optimum signal.
Relative ionization efficiencies of creatine and creatinine were obtained from standards.
Similar kinetic studies for the cyclization of ethyl ester of creatine were conducted. Ethyl
ester was prepared by acid catalyzed esterification of creatine. Final concentrations and
ionization efficiencies of creatine and ethyl ester were determined by the method of
standard addition.
Data
8
Neutral
Protonated
Zwitterion
Proton
x
0.0
ln(k)
Creatine
the rate of cyclization of creatine using different pH at constant temperature. Her data
showed that the rate of cyclization peaks at pH 3.4 and is diminished on either side of
the peak, instead of being directly proportional to the concentration of hydrogen protons.
Jencks2 has proposed a two step mechanism which describes a shift in the rate limiting
steps caused by changes in pH. For this experiment, mass spectrometry was used to
determine the rate of creatine cyclization as a function of temperature and pH. From
these measurements the activation energies and entropies are obtained. Additionally, to
further understand the mechanisms responsible for the unusual pH dependence of
creatine cyclization, the kinetics for creatine ethyl ester cyclization was also syudied.
The esterification will eliminate the formation of the zwitterion ion which is the dominant
species found in the acidic solution conditions used in these experiments.
10
2
N
b)
Neutral
Protonated
Zwitterion
Proton
pH = 1
-4
Figure 1. Scheme for cyclization of creatine to creatinine
12
-2
-3
O
O
x
+ H2 O
N
b)
-1
ln (rate)
HO
a)
0
a)
NH
H 2N
Creatine Microspecies Concentration
Kinetics of Creatine Cyclization
The creatine to creatinine cyclization is of significant biological importance,
providing energy for muscle cells and indicating proper renal function. From a chemical
perspective, the rate at which creatine cyclizes to form creatinine has interested
researchers since the late 1920s, but the cyclization is poorly understood both
energetically and mechanistically. In 1985, Witkowska1 conducted experiments to
explore
H2N
+
NH2
N
O
Zwitterion
O
Figure 5 Microspecies of creatine in solution determined by Wang and Yin.3
Figure 7. 90
kinetic plots for cyclization of creatine (red) and the ethyl
ester of creatine (blue) at a) pH 1, b) pH 3 & c) pH 5. Compare
monotonically increasing rates for creatine ethyl ester as pH increases
with the more complex creatine pH-rate relation.
Conclusions
• ESI-MS can be used to monitor the rates and Arrhenius parameters for a solutionphase cyclization reaction if the relative ionization efficiencies of the reactants and
products can be obtained.
• Creatine cyclization rate has a maximum at pH 3.
• Reaction is entropically driven at pH 3.
• Cyclization of creatine ethyl ester increases monotonically with increasing pH.
• Rate of cyclization of creatine depends on the solution concentration of the neutral
microspecies.
References
1.  Witkowska, A. Acta Alimentaria Polononica. 1985: 9 (2): 263-269.
2.  Jencks, W. JACS. 1959; 81: 475-481.
3. Wang, X. Yin, Q. J. Chem. Eng. of Chinese Univ. 2003; 17 (5): 569-574