Journal ofElectroanalytical

Chemistry

Journal of Electroanalytical Chemistry 571 (2004) 283–287

www.elsevier.com/locate/jelechem

Activation of the electrochemical properties of thiamin andits phosphate esters in acidic solutions

Jeffrey Sutton, Masangu Shabangi *

Department of Chemistry, Southern Illinois University Edwardsville, Box 1652, Edwardsville, IL 62026, USA

Received 14 January 2004; received in revised form 6 April 2004; accepted 26 April 2004

Available online 2 July 2004

Abstract

An electrochemical investigation of free thiamin and its phosphate esters was performed in acidic solutions at an unmodified

platinum electrode by means of cyclic voltammetry. The results obtained provide information that reflects the redox properties of

thiamin itself rather than its usual interaction with mercury from polarographic analyses. In acidic media, the protonated thiamin

(A2þ) at the N10 position in the pyrimidine ring was found to be the electroactive species. A2þ was reduced in a one-electron transfer

mechanism to produce a very reactive radical cation, A�þ which underwent a fast dimerization [AA]2þ. In the reverse scan, the dimer

[AA]2þ was oxidized back to the starting material (A2þ) in a two-electron transfer process. Both pH and electrode materials played

an important role in the determination of the proposed mechanism.

� 2004 Elsevier B.V. All rights reserved.

Keywords: Thiamin; Electrochemistry; Acidic solutions

1. Introduction

Thiamin, commonly known as vitamin B1, is an im-

portant biological compound that is best known for the

coenzyme functions of thiamin pyrophosphate with a

class of enzymes that catalyze acyl group transfer reac-

tions [1–4]. Its deficiency in humans, which is caused bya poor diet or alcoholism, can result in an ultimately

fatal condition known as beriberi [5,6]. Of particular

interest is the fact that thiamin and its phosphate esters

undergo numerous structural changes when the pH is

altered [5,7–9]. In alkaline solutions, thiamin is con-

verted to monocyclic anion thiol, ylide, and thiochrome

forms via stable intermediates such as the pseudo-base

and the yellow form [8]. It is most stable in acidic media[5] and fully protonated with ionizable protons at the C2

(pKa � 19) and N10 (pKa � 5) positions of the thiazolium

and pyrimidine rings, respectively (see A2þ in Fig. 1)

[10–12]. Since the discovery by Breslow nearly five de-

* Corresponding author. Tel.: +1-618-650-2420; fax: +1-618-650-

3556.

E-mail address: mshaban@siue.edu (M. Shabangi).

0022-0728/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jelechem.2004.04.022

cades ago, that the ionization at the C2 position is key to

the reactivity of thiamin [13], most electrochemical

studies of thiamin conducted to date have attributed the

redox behavior observed to the thiazolium ring [14–18].

With the recent knowledge that the 40- aminopyrimidine

participates in catalysis (when protonated at the N10

position, A2þ) [11,12,19,20], this report is the first to il-lustrate the unusual electrochemical properties of thia-

min and its phosphate esters as activated by the

protonation at the N10 position in the pyrimidine ring.

While the electrochemistry of thiamin and its phos-

phate esters in alkaline solutions on modified and on

mercury electrodes has been known for a long time

[14,15,17,18], there has been very little effort devoted to

the study of the electrochemical behavior of thesecompounds in acidic media [16], the knowledge of which

may shed more light on the way thiamin dependent

enzymes catalyze reactions. To our knowledge, the po-

larographic study of thiamin in acidic media conducted

by Rao and co-workers two decades ago [16] provides

the only comprehensive report on the reduction of thi-

amin in acidic solutions that has come forth. Also, be-

cause of the tendency of mercury electrodes to bind to

N+

S

N1'

C4'

CH3

NH2

C2H4OR

CH3

C5'N3'

HC2

N+

S

N1'+

C4'

CH3

NH2

C2H4OR

CH3

C5'N3'

HC2

H

A

Acid

Base

B2+ +

Fig. 1. Thiamin structures in acidic (A2þ) and neutral (Bþ) solutionswhere R is H, H2PO4, and H3P2O7 for free thiamin, thiamin mono-

phosphate, and thiamin pyrophosphate, respectively.

284 J. Sutton, M. Shabangi / Journal of Electroanalytical Chemistry 571 (2004) 283–287

electroactive species and participate in redox reactions

as catalysts [15,21], more definitive redox information

about thiamin obtained on inert or less reactive elec-

trode materials at a pH resembling that of biological

environments remains unavailable.

In the present study we discuss the electrochemical

reduction of thiamin and its phosphate esters in acidic

media on a platinum electrode to obtain informationthat would reflect the redox properties of thiamin itself

and not the mercury–thiamin interaction. The main

purpose of this paper is to show how the protonation at

the N10 position of the pyrimidine ring activates the

redox properties of thiamin and to elucidate the reaction

mechanism at the electrode surface using cyclic vol-

tammetry (CV).

2. Experimental

2.1. Chemicals

Free thiamin, thiamin monophosphate, and thiamin

pyrophosphate of analytical grade were obtained from

Sigma–Aldrich (St. Louis, MO) and used without anyfurther purification. Potassium chloride purchased from

EM Scientific, Inc. (Gibbstown, NJ) and hydrochloric

acid and sodium hydroxide purchased from Fisher Sci-

entific (Pittsburgh, PA) were of analytical grade. All

solutions were prepared with distilled water filtered

through a Millipore Milli-Q system (Bedford, MA).

2.2. Apparatus

The potentiostat used in this study was a BAS 100B

electrochemical analyzer (West Lafayette, IN). In the

CV analyses, the working electrodes examined were

platinum (MF-2013), gold (MF-2014), and glassy car-

bon (MF-2012) obtained from BAS and polished using

BAS polishing alumina (CF-1050). The reference elec-

trode, AgjAgClj3 M KCl, was obtained from BAS (RE-5B) and a 50 mm (length) x 1 mm (diameter) Pt wire

from Aldrich (Milwaukee, WI) was used as the auxiliary

electrode. Although all voltammograms presented

herein are not IR compensated, a few runs were per-

formed with IR compensation and the voltammograms

were not significantly altered.

The BAS bulk electrolysis (BE) cell (MF-1056) with a

platinum gauze working electrode was used in coulo-

metric studies. Experimental parameters set on the BAS100B potentiostat were as follows: reduction potential:

)0.7 V vs. AgjAgCl, sample interval: 1000 ms, end

current ratio: 10 per 1000, and sensitivity: auto. All BE

experiments were performed for 68 min with end current

ratios ranging from 1.6% to 2.4%. In all cases, the net

charge passed during BE experiments (total charge mi-

nus the background charge) was used in the determina-

tion of the number of electrons (n). The pH of solutions(2 mM ThPP in 1 M KCl and 1 M KCl used as a blank)

was adjusted with 0.1 MNaOH and 0.1 MHCl solutions

using a Fisher Scientific Accumet � model 15 pH meter.

3. Results and discussion

The electrochemical properties of free thiamin (Th),thiamin monophosphate (ThP) and thiamin pyrophos-

phate (ThPP) were studied in acidic solutions and found

to be similar with reduction/oxidation potentials (vs.

AgjAgCl at 100 mV/s) of )620/)470 mV, )672/)483mV, and )627/)526 mV, respectively. Due to the fact

that ThPP has received significant interest because of its

metabolic function as a coenzyme for a class of enzymes

that catalyze acyl group transfer reactions, the electro-chemical behavior of this class of compounds is dem-

onstrated here in greater detail using the ThPP form.

3.1. Practical considerations of the electrode material

Both the pH of solutions and the electrode material

were found to play an important role in the electro-

chemical investigation of thiamin compounds. Mercuryelectrodes have been widely used in the voltammetric

determination of vitamin B1 in aqueous solutions [15–

18]. Their higher overpotential for hydrogen discharge

relative to other electrode materials such as platinum,

gold, and carbon, makes them the electrode material of

choice for the electrochemical study of many important

biological compounds [22]. Mercury, however, can bind

to thiamin [15] and influence its redox behavior. In thereport by Rao and co-workers [16] on the electrore-

duction of thiamin in neutral and acidic aqueous solu-

tions, multiple adsorption peaks attributed to pre- and

post-adsorption of thiamin and to the hydrogen dis-

charge were observed on the mercury electrode. This

behavior tends to make the elucidation of electrode re-

action mechanisms more complex and the accurate in-

terpretation of the actual redox properties of analytesmore difficult to achieve.

At a Pt electrode, the range of potentials to be used in

acidic solutions depends on the composition of a solu-

J. Sutton, M. Shabangi / Journal of Electroanalytical Chemistry 571 (2004) 283–287 285

tion and on its pH in particular [23,24]. In this investi-

gation, at a pH below 3.5, we observed two redox pro-

cesses, attributable to the reduction of Hþ and A2þ.Larger current peaks A and B in voltammograms I and

II, as shown in Fig. 2(a), that developed at )432 and)340 mV, respectively, from the CV analysis of thiamin

pyrophosphate (and a blank solution) in acidic media,

are typical for the reduction of protons (Eq. (1)) [24].

These peaks gradually disappeared as the pH increased

to 3.5 or higher (see voltammograms I and II, in

Fig. 2(b)). The current peaks observed at approximately

)627 and )526 mV (C and D in voltammograms I and I,

Fig. 2(a) and (b), respectively) were assigned to the re-dox process of thiamin pyrophosphate. The coulometric

analysis of a blank solution containing only the sup-

porting electrolyte (KCl) in acidic media (pH� 3.5) at a

Pt electrode also gave clear evidence of a two-electron

redox process (n value of 2.2 e� on the coulometric time

scale) for the hydrogen discharge (Eq. (1)). Other elec-

trode materials such as gold (Au) and glassy carbon

(GC) were examined in this investigation. A cathodicirreversible response was observed at )628 mV using the

Au electrode whereas the GC electrode displayed no

current response in acidic solutions.

Although these findings clearly demonstrated the

tendency of the background currents (hydrogen evolu-

tion) to mask the ThPP redox process, it is important to

note that the CV analysis of free thiamin and its phos-

phate esters can still be performed in acidic solutions at

Fig. 2. Cyclic voltammograms of 4.3 mM thiamin pyrophosphate in 1

M KCl solution (I) and a blank (II) at (a) pH 2.5 and (b) pH>3.5,

obtained at Pt vs. AgjAgCl reference electrodes with a scan rate of 100

mV/s. It should be noted that CVs are plotted with the cathodic cur-

rent positive upwards.

a pH much lower than 3.5 if the background current is

subtracted from the sample. However, working in a pH

above 3.5 where the instrument is less sensitive to the

decreased proton concentration is beneficial in avoiding

the proton redox feature.

2Hþ þ 2e� () H2 ð1Þ

3.2. The effect of pH on the electroreduction of thiamin

compounds

The effect of pH on the electroreduction of free thi-amin and its phosphate esters was studied by gradually

increasing the pH of a solution containing 4.3 mM of

thiamin compounds in 1 M KCl solution and recording

the resulting cyclic voltammograms. As shown in Fig. 3,

the current responses decreased with the increase in pH,

and diminished at about pH 6.5. The loss of redox ac-

tivities for ThPP was caused by the decrease of A2þ due

to the formation of Bþ in solutions. Despite the fact thatboth species exist in equilibrium with each other, the

decrease in current responses observed at a higher pH

where Bþ was present in a significant amount, demon-

strates that Bþ is a non-electroactive species under our

experimental conditions. While Bþ may have been ob-

served to be electroactive under other experimental

conditions (Hg electrodes and pH� 6.5) [16,18], in our

findings, we report with confidence that the protonatedspecies (A2þ) is the electroactive species observed at the

Pt electrode in acidic media.

In the pH range of this investigation (3.5–6.5), the

N10 nitrogen of the 40-aminopyrimidine ring is known to

undergo protonation (pKa � 5) [11,12], which activates

the redox properties of thiamin compounds at a Pt

electrode. Other groups [16 and references within]

have attributed the redox behavior of thiamin to the

Fig. 3. Cyclic voltammograms of 4.3 mM thiamin pyrophosphate in a

1 M KCl solution at various pHs (3.5, 4.0, 5.0, 5.5, 6.0, and 6.5) with a

scan rate of 100 mV/s at Pt vs. AgjAgCl reference electrodes.

Fig. 4. Cyclic voltammograms (two complete cycles) of 4.3 mM thia-

min pyrophosphate in 1 M KCl solution (pH 3.5) with various scan

rates (I: 0.1 V/s, II: 1 V/s, III: 10 V/s) at Pt vs. AgjAgCl reference

electrodes.

286 J. Sutton, M. Shabangi / Journal of Electroanalytical Chemistry 571 (2004) 283–287

protonation of the –NH2 group at the C4 position as

well as those at both the pyrimidine nitrogen positions

(N10 and N30). In recent years, through the work of

Jordan et al. [11,12] and others [25] it has been dem-

onstrated that the N10 nitrogen is the most basic, andonce protonated, the 40 amino group becomes a weak

acid which interacts with the N30 nitrogen through hy-

drogen bonding. In this investigation it is assumed that

the N10 nitrogen is the only atom on the pyrimidine

moiety that undergoes protonation in acidic media.

At pH 7, no current response was observed because

A2þ existed in trace amounts and was undetectable un-

der our experimental conditions. The coulometricanalysis for number of electrons (n) transferred in the

reduction of ThPP at pH� 7 gave the value of

0.33� 0.03, attributable to the redox activity of trace

amounts of A2þ in solution or regenerated from Bþ due

to the time scale of coulometric measurements (68 min).

3.3. Proposed redox mechanism

The first step in elucidating the redox mechanism for

the electroreduction of thiamin compounds was the es-

tablishment of the redox site between the two aromatic

rings (thiazolium and pyrimidine). Most electrochemical

properties of thiamin reported to date have been at-

tributed to the thiazolium ring without any apparent

rational basis [14–18]. In this investigation, based on the

pH profile as discussed above, it is now evident that A2þ

is the electroactive species at the Pt electrode. Also, with

the pKa falling between 17 and 19 for the ionization of

thiamin compounds at the C2 position [10], it is im-

portant to note that the possibility of ionizing this po-

sition (C2) to form ylide intermediates is undoubtedly

ruled out and dismissed under the pH range of our in-

vestigation. The fact that no structural change occurred

at the thiazolium ring as the pH was increased from 3.5to 6.5 provides the clearest evidence that the electro-

chemical analysis of thiamin compounds (Th, ThP, and

ThPP) in acidic media at the Pt electrode is activated by

the protonation of the N10 nitrogen of the 40-amino-

pyrimidine ring. This effect makes the pyrimidine ring a

much more electron-withdrawing group which is sus-

ceptible to an electrochemical reduction.

In acidic solutions and at a Pt electrode, A2þ is re-duced in a one-electron transfer reaction to produce a

very reactive radical cation, A�þ (most likely at the C40

position of the pyrimidine ring [26–28]) which undergoes

a fast dimerization (Eqs. (2) and (3)) [26–28]. In a proton

rich environment such as acidic media, the dimerization

process might be expected to compete with the proton

abstraction pathway, which may lead to a dihydro-thi-

amin system upon further reduction [28]. However, theliterature data for rate constants for the dimerization

(8� 5� 105 mol�1 s�1) and the proton abstraction (7

mol�1 s�1) of a simple pyrimidine anion radical mea-

sured in acetonitrile with residual water [26,27] suggest

that the dimerization of A�þ, which is much faster than

its proton abstraction, will be the most dominant

pathway.

Evidence of a one-electron transfer reduction of thi-amin and other related pyrimidine systems in aqueous

solutions has appeared in the literature [17,26,27]. In

this study, the coulometric analysis of ThPP was per-

formed to determine the number of electrons (n) trans-ferred in the reduction of A2þ at various pHs. Values for

n determined in the reduction of 2 mM ThPP in 1 M

KCl solutions at average pHs of 3.5, 5.0, and 7.0 were

1.72� 0.19, 1.16� 0.21, and 0.33� 0.03, respectively. AtpH 3.5, the ThPP and Hþ (mole ratio of ThPP deter-

mined based on its formal concentration to that of Hþ:1.0–0.2) underwent a one- and two-electron transfer

reduction, respectively, resulting in an average n value of1.72� 0.19. This observation was confirmed in the

coulometric analysis of a blank solution (1 M KCl)

adjusted to a pH of approximately 3.5, yielding an nvalue of 2.1� 0.33. At pH 5 (mole ratio: 1.0–5.0� 10�3), the reduction of Hþ had less effect on the nvalue of ThPP and provided clear evidence of a one-

electron transfer in the electro-reduction of ThPP. As

the pH was increased to 7 (mole ratio: 1.0–5.0� 10�5)

where ThPP (A2þ) significantly diminished in solution

due to its conversion to Bþ, no substantial charge

transfer attributable to the reduction of A2þ or Hþ was

observed. These results suggest that A2þ undergoes aone-electron transfer reduction at the Pt electrode in

acidic media, followed by a fast dimerization (the exact

structure of [AA]2þ was not characterized in this study).

In the reverse scan, the resulting dimer ([AA]2þ) wasoxidized back to the starting material (A2þ). Depending

on the scan rate, this either took place by a single two-

electron process or via two successive one-electron

processes (Eqs. ()()()(4)–(6)). The reduction process ob-served at )627 mV from voltammograms obtained in

multiple scans (Fig. 4) suggests that the starting material

is entirely regenerated at the electrode surface in the

J. Sutton, M. Shabangi / Journal of Electroanalytical Chemistry 571 (2004) 283–287 287

oxidation process. However, proving that the fate of

[AA]2þ is controlled by reactions displayed in Eqs. (4)–

(6) rather than the reverse of Eq. (2) is the issue at

hand.

At a lower scan rate (100 mV/s), voltammograms(Fig. 2, CV II and Fig. 3) exhibited nearly identical

single cathodic and anodic waves, which may suggest

the reverse of Eq. (2) during the oxidation scan. How-

ever, because of the electron stoichiometry and the

knowledge of a one-electron reduction process estab-

lished above, we attributed the characteristics of the

anodic wave described here to a simultaneous two-

electron transfer oxidation reaction of [AA]2þ (Eq. (6)).When the scan rate was increased to 10 V/s, the vol-

tammograms (Fig. 4) displayed two poorly resolved

anodic waves suggesting two consecutive one-electron

oxidation reactions of the dimer (Eqs. (4) and (5)). The

fact that the anodic wave shape changed significantly at

a faster scan rate confirms the electro-irreversibility of

A�þ and the existence of two possible oxidation paths

with respect to the scan rate.

A2þ þ e� ! A�þ ð2Þ

2A�þ ! ½AA�2þ ðfast dimerizationÞ ð3Þ

½AA�2þ ! ½AA��3þ þ e� ! A2þ þ A�þ ð4Þ

A�þ ! A2þ þ e� ð5Þ

½AA�2þ ! ½AA�4þ þ 2e� ! 2A2þ ð6Þ

4. Conclusion

Both the pH of thiamin solutions and the electrode

material played a crucial role in the electrochemical in-vestigation of thiamin and its phosphate esters. In acidic

solutions, the redox properties of these types of com-

pounds are activated by the protonation of the N10 ni-trogen of the pyrimidine ring of thiamin compounds. At

a Pt electrode, thiamin and its phosphate esters are re-

duced in a one-electron transfer reaction to produce

very reactive radical cations, which undergo fast di-

merization.

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