AbstractA number of research studies on fatty acid derivatives of Ascorbic Acid (AA) have yielded interesting results, such as antimetastatic action of certain cancers, and inhibition of r-Phosphofructokinase (PFK-1), which helps regulate glycolysis and the metabolism of glucose. Other studies have concluded that cancer cells have a greater survival dependence on glucose than normal cells. Therefore, the inhibition of glycolysis should impact cancer cells at a greater rate than normal cells. Previous studies found that sixteen and eighteen carbon side chain derivatives, (ascorbic acid-6-palmitate (AAP) and ascorbic acid-6-stearate (AAS) decrease PFK-1 enzyme activity twelve-fold and ten-fold, respectively, relative to ascorbic acid (AA). Based on this observation, our hypothesis was that shortening the side chain and reducing the lipophilic characteristics should diminish hydrophobic interactions with r-PFK-1 and lower the inhibition.To test our assumption, we synthesized Ascorbic Acid-6-butyrate (AAB. Method used: Tanaka and Yamamoto) r-PFK-1 enzyme was purified from frozen rabbit muscle employing centrifugation and ion-exchange filtration. Inhibitions were carried out in buffered solutions of 95% ethanol. Our results show that AAB inhibited r-PFK-1 two-times greater than ascorbic acid (AA) but was only 0.16 as inhibitory as AAP, and only 0.20 as inhibitory as AAS.

(Under our experimental conditions, 95% ethanol, and methyl and ethyl butyrates, palmitates, and stearates showed no significant inhibitions of r-PFK-1. From the results, we conclude that the side chain length and lipophilic characteristics of the ascorbic fatty acid derivatives may be the major contributors in the observed inhibitions of r-PFK-1 activity.) IntroductionCancer is a leading cause of mortality. Therefore, development of effective drugs is of significant importance. Research studies have suggested a greater dependence on glycolysis for cancer proliferation. Previous research has shown that 6-ascorbic acid palmitate (AAP) and 6-ascorbic acid stearate (AAS) strongly inhibit r- PFK-1 enzymes, a mayor contributor in glycolysis. The aim of this research was to study the relationship of the length of ascorbic acid side chains in fatty-acid derivatives to enzyme inhibitions. AAS, AAP, and AAB were tested. Their inhibitory concentration (IC50) values were determined and compared. From the results, a mechanism by which ascorbic acid inhibits this enzymes was proposed.

Characterization of AAB. The IR spectrum in (Fig.3) yields distinctive peaks related to the hydroxyl groups (-OH) in the neighborhood of 3000 cm-1, and a carbonyl group (C=O) about 1700 cm-1 that are common to AA. The NMR spectrum in (Fig.4) shows six peaks which coincide with the number of protons and the relative chemical shifts of AAB. Analysis of AAB using IR and NMR confirmed the structure of AAB.

Comparative Inhibition. As expected, r-PFK-1 activity decreased due to presence of AAB (Fig.1). The half inhibitory concentration (IC50) was determined to be 0.130 mM. Comparing the IC50, AAB was two-times greater than AA. However, AAP has a six fold and AAS a five fold increase in strength when compared to AAB.

Results

DiscussionBy comparing the inhibitory strength of the 4 and 16 and 18 carbon chains of AAB, AAP, and AAS respectively, a possible correlation to rPFK1 inhibition can be based on the lipophilic character of the fatty derivatives. We propose that the increase in inhibitory strength is a result of a longer carbon chain.

ConclusionIn conclusion, further studies should be done to characterize ascorbic acid derivatives and their properties to treat cancer.

References Percy J. Russell, Anita Williams, Ami Abbot, et. al. “Characteristics of Rabbit Muscle adenylate Kinase inhibition by Ascorbate.” J. Enzyme Inhibition & Med. Chem. 2005: 1-7.

Jianwen Liu, Xiaodong Zhang, Fan Yang, et. al. “Antimetastatic effect of a lipophilic Ascorbic Acid Derivative with Antioxidation Through Inhibition of Tumor Invasion.” Cancer Chemother Pharmacol. 57 (2006): 584-90.

Kim Han Jip, et al. "The Mitochondrial Warburg Effect: A Cancer Enigma." Interdisciplinary Bio Central 1 (2009): 1-6. Academic Search Complete. Web. 5 Oct. 2012.

Hiroyoshi Tanaka, Ryuichi Yamamoto., Shionogi & Co., Ltd.

Comparison of r-Phosphofructokinase (r-PFK-1) Enzyme Inhibition by Ascorbic-6-Butyrate (AAB), Ascorbic Acid-6-Palmitate (AAP) and Ascorbic Acid-6-Stearate (AAS)

Materials and Methodsr-PFK-1 purification: 50g of frozen rabbit muscle was homogenized in EDTA/DTT/NaF, heat treated, centrifuged and washed at every step. r-PFK-1 enzyme was collected using ion-exchange filtration with solutions of 40TP8/DTT and 600TP8/DTT. Finally, the enzyme was condensed by ammonium sulfate forming a pellet for later use. AAB synthesis: Esterification of Vitamin C and butyric acid was attained utilizing sulfuric acid as a catalyst. Chloroform and ethyl acetate were used to extract organic material, including AAB. Dehydration agent, vacuumed filtration, and rotary evaporator completed the synthesis. Verification of AAB: was conducted using Eft-60 spectrometer and NMR by Anastasi Instruments, Inc. Inhibition studies: r-PFK-1 pellet was re-suspended and incubated for two hours in several concentrations of AAB with Tris pH 8. The enzyme activity was tested using assay and Beckman DU800 spectrophotometer at 340.0 nm wavelength.

Relative r-PFK-1 Activity vs. AAB mM Con.

0

0.2

0.4

0.6

0.8

1

1.2

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35Concentration AAB mM

Rela

tive

Activ

ity rP

FK1

Relative to sample 1

Sample Concentration

AAB mM

r-PFK-1 Activity(Eu/ml)

RelativeActivity

1 0 0.0117 12 0.02 0.0076 0.65

3 0.06 0.0070 0.60

4 0.10 0.0062 0.53

5 0.14 0.0053 0.45

6 0.32 0.0042 0.35

Table 1. r-PFK-1 activity after two hours of incubation

Inhibitor Name IC50 (mM)0.250/IC50 Inhibitor

1 AA 0.250 1.0R=CH3(CH2)2 AAB 0.130 1.9

R=CH3(CH2)14 AAP 0.021 11.9

R=CH3(CH2)16 AAS 0.026 9.6

Table 2. Comparison of half inhibitory concentration (IC50) of ascorbate on PFK-1 activity.

1 AA

AA & fatty acid derivatives

Fig. 1 Plot of table 1. r-PFK-1 inhibition by AAB

Fig. 2 Cellular respiration diagram. The r-PFK-1 is an early enzyme in the glycolysis processes illustrated by the downward arrows.

H2O+C4H8O6+CH3(CH2)2COOH−−H2

SO4→AAB+C4H8O2+H2O

1. Chemical reaction with excess butyric acid.2 . Extraction of aqueous solution with chloroform.3. Extraction of aqueous solution with ethyl acetate.4. Dry with Na2SO4. Followed by vacuum filtration.5. Solvent evaporation and recrystallization. 6. Structure confirmed by mp, IR and NMR.

Synthesis of AAB

Fig. 3 IR of synthesized AAB Fig. 4 NMR of synthesized AAB.

AcknowledgmentsNIH-NIGMS GRANT 5R25GM073590-7, Bridges to the Baccalaureate

San Diego Mesa College: E. Alexander Ph.D., J. Corliss., and E. Erickson. A. Hussein., M. Graves., P. Duyen-Anh.

University of California, San Diego: G.Sadler Ph.D., L. Alfred Ph.D., and P. Russell Ph.D., Gratitude to A. Williams, A. Abbott, and E. Betancourt.

(r-PFK-1)

AA

Alberto Palacios, Abdirisak Hussein, Mary Graves, Mohamed Musse, Edward Alexander, Ph.D., San Diego Mesa College, Department of Chemistry,

Bridges to the Baccalaureate Program