1. Abstract
Introduction
Characterization of creatine salts by
NMR References
Acknowledgements
Barry University, Miami Shores, Florida 33161
Megan Henneberry, Anton S Wallner, PhD
Characterization and the effect of pH and temperature on
the degradation of creatine ascorbate, di-creatine
ascorbate and creatine di-ascorbate
The purpose of this research is to use proton NMR to characterize three newly
synthesized creatine salts; creatine ascorbate, di-creatine ascorbate, and creatine
di-ascorbate. Additionally, data from the stability study to determine the kinetic
degradation and shelf life of the three compounds will be reported. The effect of
different temperatures, solvents and pH will be observed for the three creatine salts
over a 45 day time range to explore the effect on kinetic degradation. Comparison
of these results to previous studies on the degradation of other creatine salts
performed within our research group will be presented.
Creatine is an amino acid that plays a crucial role in the energy metabolism of cells.
Creatine aids in the transportation of ATP through a process of phosphorylation by
creatine kinase1. The creatine-creatine phosphate system helps to delay the
depletion of ATP when there is a shortage of oxygen and/or blood supply
necessary for cell metabolism. Only a percent of creatine undergoes
phosphorylation which prevents the migration across the plasma membrane, as a
result the administration and benefits of creatine are limited1. Thus, the synthesis of
a more soluble creatine salt derivative is highly desirable, but also difficult to
achieve due to the intrinsic character of creatine2. The zwitterionic property of
creatine monohydrate has the potential to form salt derivatives with acids that are
more acidic than the carboxylic acid functional group in creatine (pKa 2.79 and
12.1)2. Here we have used ascorbic acid (pKa 4.17 and 11.57)3 which has two
reactive positions at C2 and C3 that are open to derivatization towards producing a
salt with desirable chemical and physical properties.
[1] Ellis A. et al., CNS drugs, 2004, 18(14), 967-980.
[2] Gufford B. et al., Journal of DietarySupplements, 2010, Vol. 7(3), 241.
[3] Olabisi, A. O., The chemistry of L-ascorbic acid derivatives in the asymmetric synthesis
of c2-and c3- substituted aldono-y-lactones. Ph.D. Dissertation, Wichita State University,
Wichita, KS, 2005.
[4] Wallner, T. PhD, Personal communication, 2015.
[5] Kuhl, B., Wallner, A.S. 243rd ACS meeting, San Diego, CA, 2012.
[6] Llaneras, J, 249th ACS meeting, Dallas, TX, 2014.
Purpose
The purpose of this research project is to synthesize creatine salt derivatives;
creatine ascorbate, di-creatine ascorbate and creatine di-ascorbate, and
characterize the compounds through proton NMR analysis and to determine the
kinetic degradation of each compound through a stability study.
Proposed structures:
Discussion/Conclusion
Figure 3: NMR of creatine ascorbate
Compound CH2 Peak (ppm)
from creatine
CH3 Peak (ppm)
from creatine
CH on C4 peak (ppm)
from ascorbic acid
CH on C5 Peak
(ppm) from ascorbic
acid
CH2 on C6 Peak
(ppm) from ascorbic
acid
D2O Peak (ppm)
Creatine monohydrate 3.75 2.85 ------- -------- -------- 4.65
L-ascorbic acid ------ ------ 4.77 3.90 3.59 4.65
Creatine ascorbate 4.06 2.93 4.52 3.86 3.57 4.65
Di-creatine ascorbate 3.97 2.90 4.38 3.84 3.56 4.65
Creatine di-ascorbate 4.08 2.93 4.59 3.88 3.56 4.65
Table 1: Peak Shifts
Future Directions
•Re-run kinetic study on creatine ascorbate and creatine diascorbate
•Further purification through recrystallization
•Elemental Analysis of all products for comparison to NMR results
Michael Wise for NMR support
Dr. Karen Callaghan, Dean of CAS for undergraduate travel support
Figure 2: NMR of ascorbic acidFigure 1: NMR of creatine
monohydrate
Table 2: Peak integrations
Compound Number of protons for
CH2 peak (ppm) from
creatine
Number of protons for
CH3 peak (ppm)
from creatine
Number of protons for
C4 of ascorbic acid
Number of protons for
C5 of ascorbic acid
Number of protons for
C6 of ascorbic acid
Creatine monohydrate 2 protons 3 protons ------ ------ ------
L-ascorbic acid ------ ------ 1 proton 2 protons 3 protons
Creatine ascorbate 1.00 1.50 0.51 0.75 1.35
Di-creatine ascorbate 4.17 6.18 1.00 1.06 2.14
Creatine di-ascorbate 3.70 5.39 1.00 3.39 7.07
Ascorbic AcidCreatine monohydrate
Department of Physical Sciences
Barry University
11300 NE 2nd Ave.
Miami Shores, FL 33161
Phone: (305) 801-7972
megan.henneberry@mymail.barry.edu
Figure 4: NMR of di-creatine ascorbate Figure 5: NMR of creatine di-ascorbate
Di-creatine Ascorbate Creatine Di-ascorbate
Creatine Ascorbate
Kinetic Study
pH 9.0 Rate
constant
(day-1)
Fraction
degraded in 63
days
R2
Creatine4
4 Co 0.0005 0.0315 0.9788
Room 0.0005 0.0315 0.9831
37 Co 0.0065 0.4095 0.9668
Di-
Creatine
Ascorbat
e
4 Co 0.0059 0.3717 0.9409
Room 0.0067 0.4221 0.9831
37 Co 0.0095 0.5985 0.9565
pH 2.0 Rate
constant
(day-1)
Fraction
degraded in 63
days
R2
Creatine4
4 Co
0.0005 0.0315 0.9788
Room 0.0005 0.0315 0.9831
37 Co
0.0065 0.4095 0.9668
Di-
Creatine
Ascorbate
4 Co
0.0035 0.2205 0.9662
Room 0.0044 0.2772 0.9721
37 Co
0.0075 0.4725 0.9978
pH 7.4 Rate
constant
(day-1)
Fraction
degraded in 63
days
R2
Creatine4
4 Co 0.0005 0.0315 0.9788
Room 0.0005 0.0315 0.9831
37 Co 0.0065 0.4095 0.9668
Di-
Creatine
Ascorbate
4 Co 0.0067 0.4221 0.9730
Room 0.0079 0.4977 0.9949
37 Co 0.0084 0.5292 0.9383
Table 3: Kinetic comparison of creatine to creatine ascorbate
salts
Table 4: Kinetic comparison of creatine to creatine ascorbate
salts
Table 5: Kinetic comparison of creatine to creatine
ascorbate salts
Figure 8: First order plots at different pH and
temp
Figure 6: First order plots at different pH and temp
Figure 7: First order plots at different pH and temp
The OH of ascorbic acid at C2 and C3 have pKa values of 11.793 and 4.253, respectively,
based on that information we proposed that C3 is the site that will undergo de-protonation
to form creatine ascorbate, because it is more acidic. For di-creatine ascorbate, the OH of
ascorbic acid at C2 and C3, again, have pKa values of 11.793 and 4.253, respectively,
based on that information we proposed that C2 and C3 are the two sites that will undergo
de-protonation as they are most acidic. For creatine di-ascorbate we have proposed that
the first site of deprotonation will be at the C3 on ascorbic acid and another at the O- on
creatine. The hydrogen atoms nearest to the site of de-protonation should exhibit the
largest ppm shift, and likewise the hydrogen atoms further from the site of deprotonation
should exhibit the smallest ppm shift. NMR analysis revealed that a shift in all peaks
occurred (Table 1) giving reason to suggest there was a change in the chemical
environment. The C4 peak of ascorbic acid appeared to experience the largest shift in ppm
for all three compounds, which is in agreement with our proposed site of de-protonation at
the C3 of ascorbic acid; the proton on the neighboring carbon (C4) should experience the
largest shift as it is closest to the site of de-protonation. Our proposed structure is also in
agreement with NMR because as you can see in table 1, the further away the carbon atoms
of ascorbic acid are from the site of de-protonation, a lower shift in ppm occurs (less
impacted). The resonances are coupled or split by interactions they have with neighboring
protons, or neighboring NMR active signals. More often than not these interactions exist
through bonds, however, interactions can occur through space interaction when the
molecules geometry brings the proton nearby causing through space interaction instead of
through bonds. The relative areas of various absorptions in the NMR spectrum obtained
suggest that the creatine ascorbate, di-creatine ascorbate and creatine di-ascorbate salts
are of 1:1, 2:1 and 1:2 molar ratios, respectively, as summarized in table 2. The stability
comparisons of creatine monohydrate to di-creatine ascorbate are shown in figures 6-8 and
table 3-6. Creatine monohydrate was found to be more stable then di-creatine ascorbate at
each pH and temperature used. Although the solubility of the salt is increased compared to
creatine monohydrate, its stability is decreased according to our results. We have made a
more bioavailable salt but a slightly less stable one in solution.