Picolyl alkyl amines as novel tyrosinase inhibitors: Influence of hydrophobicity and substitution

Article abstract of DOI:10.1021/jf902100k

Several novel picolyl alkyl amine derivatives (A-L) were synthesized, and the influence of hydrophobicity and substitution on the inhibition of mushroom tyrosinase toward both monophenolase and dlphenolase activities are described, α-, β-, and γ-picolyl amines are neither the substrates nor the inhibitors; however, the inhibition Is induced by the incorporation of an alkyl chain. The inhibition was strongly dependent on the substitution on a pyridine ring, and the inhibition follows the trend of a-picolyl alkyl amines (A, D, G) < β-picolyl alkyl amines (B, E, H) < γ -picolyl alkyl amines (C, F, I). The inhibition kinetics have been investigated, and γ-substituted derivatives were found to be a mixed type of inhibitor, whereas β-substituted derivatives were found to exhibit uncompetitive inhibition toward the oxidation of L-DOPA.

Full text of DOI:10.1021/jf902100k

9780 J. Agric. Food Chem. 2009, 57, 9780–9786
DOI:10.1021/jf902100k
Picolyl Alkyl Amines As Novel Tyrosinase Inhibitors:
Influence of Hydrophobicity and Substitution
P
UNAM
B
ANDYOPADHYAY,* SUJEETKUMAR
JHA
,
AND S. K. IMRAN
A
LI
Unilever Research India, 64 Main Road, Whitefield, Bangalore-560066, India
Several novel picolyl alkyl amine derivatives (A-L) were synthesized, and the influence of
hydrophobicity and substitution on the inhibition of mushroom tyrosinase toward both monopheno-
lase and diphenolase activities are described. R-, β-, and γ-picolyl amines are neither the substrates
nor the inhibitors; however, the inhibition is induced by the incorporation of an alkyl chain. The
inhibition was strongly dependent on the substitution on a pyridine ring, and the inhibition follows the
trend of R-picolyl alkyl amines (A, D, G) < β-picolyl alkyl amines (B, E, H) < γ -picolyl alkyl amines
(C, F, I). The inhibition kinetics have been investigated, and γ-substituted derivatives were found to
be a mixed type of inhibitor, whereas β-substituted derivatives were found to exhibit uncompetitive
inhibition toward the oxidation of L-DOPA.
KEYWORDS: Mushroom tyrosinase; picolyl amines; L-DOPA; enzyme-inhibition; mixed inhibitors;
uncompetitive inhibitors
INTRODUCTION
dopaquinone, and further reactions lead to melanin formation;
however, overproduction of melanin leads to hyperpigmentation.
It is well recognized that tyrosinase inhibitors are important for
their potential application in medical and cosmetic products that
may be used to prevent or treat hyperpigmentation. Tyrosinase
inhibitors may be clinically used for the treatment of some skin
disorders associated with melanin hyperpigmentation (9) and are
also cosmetically important for skin lightening effects (10, 11);
therefore, there is a need to identify the compounds that inhibit
mushroom tyrosinase activity. A number of tyrosinase inhibitors
from both synthetic (12) and natural sources that inhibit mono-
phenolase, diphenolase, or both of these activities have been
identified (13, 14).
It has been reported that gallic acid, which is a substrate of
mushroom tyrosinase, can become an inhibitor as an ester by the
incorporation of alkyl chains (7). There are reports where p-
substituted benzaldehyde derivatives (14), p-alkoxybenzoic
acid (15), alkylbenzoic acids (16), alkyl xanthates (17), and
synthetic n-alkyl dithiocarbamates with different alkyl chains (18)
have been investigated for inhibition toward mushroom tyrosi-
nase. In all these reports, the effect of hydrophobicity or alkyl
chain length has been studied toward inhibition of mushroom
tyrosinase activities. It is very important to understand the role of
hydrophobicity and substitution toward the inhibition of mush-
room tyrosinase, which can lead to the designing of new potent
inhibitors of mushroom tyrosinase.
Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase,
is a copper-containing multifunctional oxidase that catalyzes two
different reactions involving molecular oxygen, the hydroxylation
of monophenolic compounds to diphenolic compounds and the
oxidation of o-diphenols to o-quinones (1, 2). Tyrosinase widely
exists in plants and animals and is involved in the formation of
melanin pigments. In the food industry, tyrosinase is a very
important enzyme in controlling the quality and economics of
fruit and vegetable storage and processing including fruit pulp
manufacturing (3, 4). Tyrosinase catalyzes the oxidation of
phenolic compounds to the corresponding quinone and is
responsible for the enzymatic browning of fruits and vegetables.
In addition to the undesirable color and flavor, the quinones
produced are highly reactive compounds and can polymerize
spontaneously to form high molecular weight compounds or
brown pigments, and in the browning reaction, they may irre-
versibly react with the amino and sulfhydryl groups of proteins
that enhance the brown color produced (5, 6). Browning can
cause deleterious changes in the appearance and organoleptic
properties of the food product, resulting into shorter shelf life,
loss of quality, and decreased commercial value. The quino-
ne-protein reaction results in decreased digestibility of the
protein and therefore low bioavailability of essential amino acids,
including lysine and cysteine. Tyrosinase inhibition may be a
potential approach to prevent and control the enzymatic brown-
ing reactions and improve the quality and nutritional value of
food products (7, 8).
In the literature, several copper chelators are known for
tyrosinase inhibition (19); although picolyl amines and deriva-
tives are known copper chelators (20-22), their tyrosinase
inhibition studies with structural modifications are scarce. In this
article, we have reported the tyrosinase inhibition efficiency of
picolyl amines, synthesized several picolyl alkyl amine deriva-
tives, and studied their inhibitory effect toward monophenolase
and diphenolase activities of mushroom tyrosinase. Furthermore,
Mammalian tyrosinase is the key enzyme in melanin biosynth-
esis, catalyzing the hydroxylation of
L-tyrosine to 3,4-dihydroxy-
L-phenylalanine (L-DOPA) and its subsequent oxidation to
*Corresponding author. E-mail: punam.bandyopadhyay@unilever.
com. Telephone: þ 91 080-39830993.
pubs.acs.org/JAFC
Published on Web 10/05/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 20, 2009 9781
Scheme 1. Synthesis of r-, β-, and γ-Picolyl Alkyl Amines
γ-Picolyl Propyl Amine (C). The molecular formula is C9H14N2,
and the calculated MW = 150. Mass spectra: Mþ peak observed at 149.6
along with Mþ þ 23 peak at 171.6 corresponding to [C9H13N2Na].
1H NMR in CDCl3 (300 MHz) δ ppm: 0.95 (t, 3H), 1.55 (m, 2H), 2.6
(t, 2H), 3.62 (s, 2H), 7.27 (d, 2H, J = 6.5 Hz), 8.53 (d, 2H, J = 6.5 Hz).
R-Picolyl Pentyl Amine (D). The molecular formula is C11H18N2,
and the calculated MW = 178. Mass spectra: Mþ peak observed at 177.7.
1H NMR in CDCl3 (300 MHz) δ ppm: 0.88 (t, 3H), 1.32 (m, 4H), 1.55
(m, 2H), 2.65 (t, 2H), 3.9 (s, 2H), 7.15 (dd, 1H, J = 8.1, 2.5 Hz), 7.3 (dd,
1H, J = 8.1, 2.5 Hz), 7.65 (ddd, 1H, J = 8.1, 8.1, 2.5 Hz), 8.55 (d, 1H, J =
9.0 Hz).
β-Picolyl Pentyl Amine (E). The molecular formula is C11H18N2,
and the calculated MW = 178. Mass spectra: Mþ peak observed at 177.7.
1H NMR in CDCl3 (300 MHz) δ ppm: 0.9 (t, 3H), 1.3 (m, 4H), 1.5 (m,
2H), 2.65 (t, 2H), 3.8 (s, 2H), 7.26 (m, 1H), 7.68 (d, 1H, J = 9 Hz), 8.49 (dd,
1H, J = 9, 2.1 Hz), 8.55 (d, 1H, J = 2.1 Hz).
γ-Picolyl Pentyl Amine (F). The molecular formula is C11H18N2,
and the calculated MW = 178. Mass spectra: Mþ peak observed at 177.7.
1H NMR in CDCl3 (300 MHz) δ ppm: 0.9 (t, 3H), 1.35 (m, 4H), 1.52
(m, 2H), 2.65 (t, 2H), 3.82 (s, 2H), 7.26 (d, 2H, J = 6.5 Hz), 8.53 (d, 2H,
J = 6.5 Hz).
the aim was also to carry out a kinetic study of the inhibition of
the diphenolase and monophenolase activity of tyrosinase by
these derivatives to understand the inhibition mechanism as a
function of substitution and to evaluate the kinetic parameters
and inhibition constants.
R-Picolyl Heptyl Amine (G). The molecular formula is C13H22N2,
and the calculated MW = 206. Mass spectra: Mþ þ 23 peak observed at
227.8 corresponding to [C13H21N2Na].
1H NMR in CDCl3 (300 MHz) δ ppm: 0.88 (t, 3H), 1.3 (m, 8H), 1.55 (m,
2H), 2.65 (t, 2H), 3.9 (s, 2H), 7.15 (dd, 1H, J = 8.1, 2.5 Hz), 7.3 (dd, 1H, J =
8.1, 2.5 Hz), 7.65 (ddd, 1H, J = 8.1, 8.1, 2.5 Hz), 8.55 (d, 1H, J = 9.0 Hz).
β-Picolyl Heptyl Amine (H). The molecular formula is C13H22N2,
and the calculated MW = 206. Mass spectra: Mþ þ 23 peak observed at
227.8 corresponding to [C13H21N2Na].
MATERIALS AND METHODS
Reagents. R-Pyridine carboxaldehyde, β-pyridine carboxaldehyde,
γ-pyridine carboxaldehyde, R-picolyl amine, β-picolyl amine, γ-picolyl
amine, n-propyl amine, n-pentyl amine, n-heptyl amine, n-nonyl amine,
L
-tyrosine, and 3,4-dihydroxy-L-phenyl alanine (L-DOPA) were pur-
1H NMR in CDCl3 (300 MHz) δ ppm: 0.85 (t, 3H), 1.25 (m, 8H), 1.5
(m, 2H), 2.65 (t, 2H), 3.8 (s, 2H), 7.25 (m, 1H), 7.68 (d, 1H, J = 9.0 Hz),
8.49 (dd, 1H, J = 9, 2.1 Hz), 8.55 (d, 1H, J = 2.1 Hz).
chased from Aldrich and CuSO4 5H2O from SD Fine Chemicals,
3
India. Methanol and dimethyl sulfoxide (DMSO) were of high perfor-
mance liquid chromatography (HPLC) grade obtained from Merck,
India.
γ-Picolyl Heptyl Amine (I). The molecular formula is C13H22N2,
and the calculated MW = 206. Mass spectra: Mþ þ 23 peak observed at
227.8 corresponding to [C13H21N2Na].
Mushroom tyrosinase (polyphenol oxidase (EC 1.14.18.1)) was pur-
chased from Worthington Biochemical Corporation, New Jersey, USA.
Stock solutions: 0.1 M phosphate buffer (KH2PO4, pH 6.5), 3.5 mM
1H NMR in CDCl3 (300 MHz) δ ppm: 0.88 (t, 3H), 1.28 (m, 8H), 1.52
(m, 2H), 2.65 (t, 2H), 3.82 (s, 2H), 7.26 (d, 2H, J = 6.5 Hz), 8.53 (d, 2H,
J = 6.5 Hz).
L-tyrosine in phosphate buffer, 3.5 mM L-DOPA in phosphate buffer,
1 mg/mL mushroom tyrosinase enzyme (100 kU) in phosphate buffer, and
10 mM CuSO4 in water. Also, 1000 mM inhibitor solutions were prepared
in DMSO and further diluted in phosphate buffer to give
10 mM and 20 mM inhibitor stock solutions; thus, the amount of DMSO
in test assays were almost negligible.
R-Picolyl Nonyl Amine (J). The molecular formula is C15H26N2,
and the calculated MW = 234. Mass spectra: Mþ peak observed at 233.9.
1H NMR in CDCl3 (300 MHz) δ ppm: 0.87 (t, 3H), 1.24 (m, 12H), 1.52
(m, 2H), 2.65 (t, 2H), 3.92 (s, 2H), 7.15 (dd, 1H, J= 8.1, 2.5 Hz), 7.28 (dd, 1H,
J= 8.1, 2.5 Hz), 7.65 (ddd, 1H, J=8.1, 8.1, 2.5Hz), 8.55(d, 1H,J=9.0Hz).
β-Picolyl Nonyl Amine (K). The molecular formula is C15H26N2,
and the calculated MW = 234. Mass spectra: Mþ peak observed at 233.8.
1H NMR in CDCl3 (300 MHz) δ ppm: 0.86 (t, 3H), 1.25 (m, 12H), 1.5
(m, 2H), 2.65 (t, 2H), 3.8 (s, 2H), 7.26 (m, 1H), 7.68 (d, 1H, J = 9.0 Hz),
8.49 (dd, 1H, J = 9, 2.1 Hz), 8.56 (d, 1H, J = 2.1 Hz).
Dopachrome formation was studied by reading the absorbance at
450 nm using a TECAN spectrophotometer.
Synthesis of r-, β-, and γ-Picolyl Alkyl Amine. n-Alkyl amine
(1.0 mmol) was taken in dry methanol (10mL) and to this stirring solution,
(R/β/γ-pyridine carboxaldehyde (1.1 mmol) was added over a period of 1 h
(Scheme 1). The solution was left stirring for 3 h under N2 atmosphere.
Progress of reaction was monitored by thin layer chromatography (TLC).
After the completion of the reaction (as noted by TLC), the reduction of
corresponding imine was done by adding sodium borohydride (1.5 mmol).
After stirring for 2 h, the methanol was evaporated under vacuum, and the
solid was washed with saturated brine solution (5 ꢀ 10 mL) and the residue
extracted in chloroform.
γ-Picolyl Nonyl Amine (L). The molecular formula is C15H26N2,
and the calculated MW = 234. Mass spectra: Mþ peak observed at 233.9.
1H NMR in CDCl3 (300 MHz) δ ppm: 0.88 (t, 3H), 1.28 (m, 12H), 1.5
(m, 2H), 2.62 (t, 2H), 3.82 (s,2H), 7.26 (d, 2H, J = 6.5 Hz), 8.54 (d, 2H,
J = 6.5 Hz).
Enzyme Activity Assay. o-Monophenolase and o-diphenolase activ-
ities of mushroom tyrosinase were determined with
L-tyrosine and
The residue was purified by column chromatography to obtain pure
picolyl alkyl amine derivatives as a yellowish liquid (Table 1). All of the
derivatives were characterized by mass and NMR spectroscopy.
R-Picolyl Propyl Amine (A). The molecular formula is C9H14N2,
and the calculated MW = 150. Mass spectra: Mþ peak observed at 149.6
along with Mþ þ 23 peak at 171.6 corresponding to [C9H13N2Na].
1H NMR in CDCl3 (300 MHz) δ ppm: 0.95 (t, 3H), 1.55 (m, 2H), 2.65
(t, 2H), 3.9 (s, 2H), 7.16 (dd, 1H, J = 8.1, 2.5 Hz), 7.3 (dd, 1H, J = 8.1,
2.5 Hz), 7.65 (ddd, 1H, J = 8.1, 8.1, 2.5 Hz), 8.55 (dd, 1H, J = 9 Hz).
β-Picolyl Propyl Amine (B). The molecular formula is C9H14N2,
and the calculated MW = 150. Mass spectra: Mþ peak observed at 149.6
along with Mþ þ 23 peak at 171.6 corresponding to [C9H13N2Na].
1H NMR in CDCl3 (300 MHz) δ ppm: 0.94 (t, 3H), 1.51 (m, 2H), 2.52
(t, 2H), 3.56 (s, 2H), 7.27 (m, 1H), 7.68 (d, 1H, J = 9.0 Hz), 8.48 (dd, 1H,
J = 9, 2.1 Hz), 8.55 (d, 1H, J = 2.1 Hz).
L-DOPA, respectively as the substrate by measuring the rate of dopa-
chrome formation at 450 nm (ε = 3700 M-1 cm -1) (14).
Sixty microliters of 10 mM inhibitor solution was taken to have the
resultant inhibitor concentration of 3 mM, 8 μL of mushroom tyrosinase
enzyme, and 3.5 mM -tyrosine (57 μL, 1 mM) for monophenolase
L
activity, and 3.5 mM L-DOPA (57 μL, 1 mM) for diphenolase activity.
The reaction volume was adjusted (q.s.) to 200 μL with KH2PO4 buffer,
and the progress of dopachrome formation was observed by measuring the
absorbance at 450 nm. Assays were performed in a flat bottom 96 well
microlitre plate.
The extent of inhibition by the compounds is expressed as the inhibitor
concentration leading to 50% decrease in enzyme activity (IC50).
Mode of Enzyme Inhibition Assay. In order to determine the mode
of enzyme inhibition, detailed kinetic studies were done. The assay was

9782 J. Agric. Food Chem., Vol. 57, No. 20, 2009
Bandyopadhyay et al.
Table 1. Chemical Yields, log Po/w, IC50, Inhibition Type, Inhibition Constants for Diphenolase
iinhibition constants (mM)
substitution on
pyridine ring
log Po/w
(predicted)
inhibition type
(monophenolase)
inhibition type
(diphenolase)
compound
R-
yield (%)
IC50 (mM)
KI
KIS
Aa
B
nC3H7
nC3H7
nC3H7
R
β
γ
R
β
γ
R
β
γ
R
β
γ
1.09
1.09
70
77
74
75
80
76
79
81
80
77
74
80
>12
12
uncompetitive
uncompetitive
uncompetitive
mixed
31
37
C
1.09
5.8
>12
12
23
20
18
Da
E
nC5H11
nC5H11
nC5H11
nC7H15
nC7H15
nC7H15
nC9H19
nC9H19
nC9H19
2.152
2.152
2.152
3.215
3.215
3.215
4.278
4.278
4.278
uncompetitive
uncompetitive
uncompetitive
mixed
29
42
F
4.2
>12
9.5
6.8
Ga
H
uncompetitive
uncompetitive
uncompetitive
mixed
24
32
I
Jb
Kb
Lb
a Inhibition type could not determined as the inhibition was very low. b Inhibitory effects of J, K, and L could not be tested because of the low solubility in the assay media.
Chart 1. Inhibition Efficacy of Different Picolyl Alkyl Amines toward the
Monophenolase and Diphenolase Activity of Mushroom Tyrosinase at
3 mM Picolyl Alkyl Amines
Figure 1. Chemical structures of R-picolyl amine, β-picolyl amine, and
γ-picolyl amine.
performed by varying the enzyme concentration at different inhibitor
concentrations at the same substrate (L-DOPA) concentration. To the
KH2PO4 buffer (q.s. to 200 μL) in a flat bottom 96 well plate was added 20,
30, 40, and 50 μL of 20 mM inhibitor solutions to obtain desired inhibitor
concentrations of 2, 3, 4, and 5 mM in assay medium. Then to each set of
inhibitor concentration, 2, 4, 6, 8, and 10 μL of mushroom tyrosinase
enzyme was added, which ultimately resulted in the enzyme units 2, 4, 6, 8,
and 10, respectively. Finally, 3.5 mM L-DOPA (57 μL, 1 mM) was added
to the resultant solution, and dopachrome formation was observed by
measuring the absorbance at 450 nm. The mode of inhibition was
determined from the nature of the graph when the enzyme concentration
was plotted against the reaction velocity (absorbance/min).
were tested for their effect toward monophenolase and dipheno-
lase activities of mushroom tyrosinase.
R-, β-, and γ-picolyl amines (Figure 1) are known copper
chelators, when tested for their action toward monophenolase
and diphenolase activities of mushroom tyrosinase, they did not
inhibit any of the two activities. However, introduction of alkyl
chain (C3, C5, and C7) into the picolyl amines influenced the
inhibitory action toward both monophenolase and diphenolase
activities of mushroom tyrosinase. C9 derivatives could not be
studied because of the low solubility in assay media.
The inhibition was not greatly induced with R-substitution,
and the inhibition toward monophenolase and diphenolase
activities were estimated to be in the range of 4-15% for C3,
C5, and C7 picolyl alkyl amines.
Alkyl chain introduction at the β-position influenced the
inhibition activity to a greater extent as compared to that at the
R-position. The inhibition for C3, C5, and C7 was estimated to be
12%, 12%, and 24% for monophenolase activity and 27%, 28%,
and 36% for diphenolase activity.
It was further observed that γ-picolyl alkyl amine led to the
maximum inhibition toward both the monophenolase and diphe-
nolase activities of the mushroom tyrosinase. The inhibitions for
C3, C5, and C7 were estimated to be in the range of 40-55%
toward both monophenolase and diphenolase activities. Com-
parison of the percentage of inhibition of monophenolase and
diphenolase activities of mushroom tyrosinase by these deriva-
tives at 3 mM are shown in Chart 1.
Enzyme Kinetic Studies. To the KH2PO4 buffer (q.s. to 200 μL) in a
flat bottom 96 well plate was added 20-120 μL of 20 mM inhibitor
solution in order to have the resultant inhibitor concentration in the range
of 2-12 mM. Then 8 μL of enzyme was added to each well. Finally, 29, 40,
57, and 100 μL of 3.5 mM L-DOPA was added to each set to yield 0.5, 0.7,
1.0, and 1.75 mM final concentration of L- DOPA, respectively, and
dopachrome formation was observed for 10 min by measuring the
absorbance at 450 nm. The inhibition type was assessed by Linewea-
ver-Burke plots using initial velocities for the dopachrome formation,
and the inhibition constants were determined by the secondary plots of the
kinetic parameters versus the inhibitor concentrations (23).
Effect on Lag Phase during Monophenolase Activity. The mono-
phenolase activity with L-tyrosine as the substrate was performed in way
similar to that described for the enzyme activity assay and the progress of
dopachrome formation was immediately monitored (23).
UV Absorption Studies. Copper chelation efficiency of picolyl alkyl
amines was studied by UV absorption spectroscopy (220-350 nm) using
CuSO4 (in molar ratio, 1:2). To get more insight into the mechanism of
inhibition, mushroom tyrosinase enzyme (10 U) was incubated with the
inhibitor solutions (10 mM) for 30 min at room temperature, the solutions
were diluted to 1 mL with 0.1 M phosphate buffer (KH2PO4, pH 6.5) to
study the absorbance spectra (220-350 nm) (24).
Enzyme activity was found to decrease with increasing con-
centration of inhibitors. The plot of remaining enzyme activity
versus the concentration of enzyme at various concentrations of
H and I gave a family of straight lines which passed through the
origin, as shown in Figures 2 and 3, respectively. It was found that
RESULTS AND DISCUSSION
Series of R-, β-, and γ-picolyl alkyl amines (C3, C5, C7, and C9)
were synthesized using Schiff-base condensation reaction between
pyridine carboxaldehyde and n-alkyl amines. These derivatives

Article
J. Agric. Food Chem., Vol. 57, No. 20, 2009 9783
Figure 2. Relationship of enzyme activity and the enzyme concentration
at different concentrationsofH where()) 0mM, (0) 3 mM, (4) 5 mM, (ꢀ)
7 mM, and (/) 10 mM.
Figure 4. Ability of picolyl alkyl amine derivatives to inhibit the enzymatic
activity of mushroom tyrosinase was measured using L-DOPA as sub-
strate. (a) IC50 for H and (b) IC50 for I.
Figure 3. Relationship of enzyme activity to the enzyme concentration at
the different concentrations of inhibitor I where ()) 0 mM, (0) 2 mM, (4)
3 mM, (ꢀ) 4 mM, and (/) 5 mM.
increasing the inhibitor concentration resulted in a descending
slope of the line, which is indicative of the reversible effect of
inhibitor on the enzyme (16). At higher concentrations of enzyme,
a plateau was obtained, indicating that the reaction may be
limited by the substrate concentration (L-DOPA). All of the
derivatives of β-picolyl alkyl amines and γ-picolyl alkyl amines
were found to be the reversible inhibitors of mushroom tyrosinase
for the oxidation of L-DOPA.
In order to compare the inhibitory potencies, the IC50 values
(the concentration leading to 50% activity lost) of all of the tested
inhibitors were determined under the same experimental condi-
tions. The IC50 values of picolyl alkyl amine derivatives ranged
from 4 mM to 10 mM comparable to that of arbutin (6 mM), a
well-known tyrosinase inhibitor (11) not as potent as kojic acid
(0.07 mM) another well-known tyrosinase inhibitor (25). Plots to
obtain the IC50 values for derivatives H and I are shown in
Figure 4a and b, respectively, and Table 1 summarizes the IC50 of
tested compounds for comparison.
To obtain further information on the mechanism of action of
these derivatives on mushroom tyrosinase, kinetics of the reaction
at various concentrations of L-DOPA and inhibitors was studied.
Under the conditions employed in the present investigation
with L-DOPA as the substrate to study the diphenolase activity of
mushroom tyrosinase in the presence of synthesized derivatives
(I), the oxidation reaction followed the Michaelis-Menten equa-
tion by the Lineweaver-Burke plot (Figure 5). The plot resulted
in a family of straight lines with different slopes and intercepts,
but they intersect one another in the second quadrant. This
indicates that I exhibits a mixed type of inhibition, which means
that it can bind to the free enzyme as well as the enzyme-sub-
strate complex; likewise, other γ-substituted derivatives also
showed a mixed type of inhibition. The inhibition constants for
inhibitor (I) binding with the free enzyme, KI, was determined
Figure 5. Lineweaver-Burke Plots for the inhibition of mushroom tyrosi-
naseinthe presenceof I where()) 0 mM, (4) 1 mM, (ꢀ) 2 mM, (/) 3 mM,
(O) 4 mM, and (þ) 5 mM.
from a plot of the slopes (Km/Vm) versus the inhibitor concentra-
tion, and for the enzyme-substrate complex, KIS was determined
from a plot of the intercept on the Y-axis versus the inhibitor
concentration, as shown in the inset of Figure 5. KI and KIS for I
are found to be 18 mM and 30 mM, respectively.
In the presence of H, the Lineweaver-Burke plot (Figure 6)
gave a family ofparallel straight lines withthe same slopes. Values
of both Km and Vmax were enhanced, but the ratios of Km/Vmax
remained constant with increasing concentrations of H. The
slopes were found to be independent of the concentration of H,
which indicates that it is an uncompetitive type of inhibition with
respect to L-DOPA oxidation. This means that the inhibitor
binds at a site distinct from the substrate and combines with the
enzyme-substrate complex and not with the free enzyme. The
inhibition constant, KIS, for binding with the enzyme-substrate
complex was obtained from a plot of the vertical intercept (1/Vm)
versus the inhibitor concentration, which is linear and is shown in
the inset of Figure 6. The KIS value was estimated to be 24 mM.
The inhibitory activities of the synthesized picolyl alkyl amines on
mushroom tyrosinase, the type of inhibition obtained from the
Lineweaver-Burke plot, and the inhibitor constants are listed
in Table 1. The mode of inhibition could not be studied with
R-substituted derivatives because of low inhibition.

9784 J. Agric. Food Chem., Vol. 57, No. 20, 2009
Bandyopadhyay et al.
Figure 6. Lineweaver-Burke Plots for the inhibition of mushroom tyrosi-
nase in the presence of H where ()) 0 mM, (0) 2 mM, (/) 6 mM, (O)
7 mM, and (þ) 9 mM.
Figure 9. UV Spectrum of I (1) without and (2) with tyrosinase.
of F, extended to 150 s in the presence of 3 mM of F, and to 200 s
at 6 mM of F (Figure 7). Hence, lag time extended up to 50% with
3 mM and to 100% in the presence of 6 mM of F.
At various concentrations of
reaction was lengthened by the γ-substituted derivatives to different
extents. Figure 8 shows that with increase in -tyrosine concentra-
L-tyrosine, the lag period of the
L
tion, the lag time increases at a particular concentration of F.
It is well known that tyrosinase exists in three different forms,
met-, oxy-, and deoxy-tyrosinase. During the monophenolase
reaction, the oxy-form of the enzyme participates in the hydro-
xylation of L-tyrosine, whereas during the diphenolase reaction,
both the oxy- and met- forms participate in L-DOPA oxidation.
No effect on lag period during the monophenolase reaction in the
presence of R- and β-picolyl alkyl amines indicates that they may
preferentially be interacting with met-tyrosinase, whereas with
γ-picolyl alkyl amines, the substantial enhancement in lag time is
indicative of an interaction with both the oxy- and met-forms of
tyrosinase (29).
Figure 7. Inhibition by F on monophenolase activity of mushroom tyrosi-
nase showing that the lag time had been lengthened by F where 1, 2, and
3 represent 0, 3, and 6 mM F, respectively.
The copper chelation efficiency of these inhibitors was studied
through change in their UV absorption in presence of CuSO4.
R-Picolyl alkyl amines and γ-picolyl alkyl amines were found to
form complexes with copper, whereas β-picolyl alkyl amines did
not form complexes with copper. The length of the alkyl chain
length did not affect the copper chelation efficiency of these
derivatives.
To get more insight into the interaction of picolyl alkyl amines
with tyrosinase, the UV absorption spectrum (220-350 nm) of
these derivatives were studied (24). It was found that when I was
incubated with tyrosinase there was increase in the absorption
(λ max = 260 nm) of the I (Figure 9), indicating a different local
environment (may be copper chelation), whereas incubation of
tyrosinase with β-substituted derivatives (H) did not result in any
change in its absorption behavior, indicating no interaction with
the free enzyme. Although R-substituted derivatives were found
to be copper chelators, they did not show any change in the
absorption pattern, indicating inaccessibility to the enzyme.
Inhibition of tyrosinase activity by β-substituted derivatives
indicates that hydrophobicity plays an important role in the
inhibition of tyrosinase activity.
Figure 8. Effect of monophenol concentration on lengthening the lag time
of mushroom tyrosinase by F where ()) 0.5 mM, (0) 0.75 mM, and (4)
1 mM of L-tyrosine.
All the derivatives of β- and γ-picolyl alkyl amines showed
uncompetitive inhibition toward the monophenolase activity of
tyrosinase (Table 1).
These derivatives were also tested for their effect on lag period
during monophenolase activity. When the enzymatic reaction
was started by the action of tyrosinase on L-tyrosine, a marked lag
period, characteristic of monophenolase activity was observed,
and the system reached a steady state after the lag period, which
was obtained by the extrapolation of the linear portion of the
curve (26). The lag period is known to be dependent on both
enzyme and substrate concentrations in the reaction medium, and
many monophenolase inhibitors are known for extending the lag
time such as tropolone (27) or cumic acid (13). There are some
inhibitors such as kaempferol (24) or R,β-unsaturated alde-
hydes (28), which do not lengthen the lag phase.
R- and β-picolyl alkyl amines did not show any effect on lag
time; however, steady state was influenced, whereas γ-substituted
derivatives were found to influence both the lag time and the
steady state. The lag time was estimated to be 100 s in the absence
The γ-substituted derivatives have been found to be the most
potent inhibitors as compared to R- and β-substituted derivatives,
which indicates that γ-substituted derivatives can be well posi-
tioned and could be easily incorporated in the hydrophobic
pocketof the enzyme (16). The other reason for the ineffectiveness
of R-substituted derivatives and the low inhibition activities of
β-substitutedderivativescouldbe stericcrowding, which can cause
spatial blocks, making the inhibitors unable to interact with the
free enzyme whereas, the γ-substituted derivatives which are least
sterically crowded showed considerable inhibition. The marginal
increase in inhibition with increase in alkyl chain length indicates
that the longer the chain length, the higher the hydrophobic
interaction with the hydrophobic pocket of the enzyme, although

Article
J. Agric. Food Chem., Vol. 57, No. 20, 2009 9785
derivatives with higher chain length were synthesized but could
not be tested because of low solubility.
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ꢀ
ꢀ
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, L. Polyphenol oxidase and peroxidase in fruits and
(6) Vamos-Vigyazo
Since the structure of mushroom tyrosinase is largely un-
known, the precise explanation for how these derivatives are
interacting with the enzyme on a molecular basis is still unknown,
but possibly, the hydrophobicity enhances the van der Waal’s
interaction with the protein residue, and the tertiary structures of
the enzyme can also get disrupted because of intermolecular
hydrogen bonding. Low activities of R-picolyl alkyl amine could
also be attributed to the fact that in R-substituted derivatives the
chances of intramolecular hydrogen bonding are higher, thus
reducing the chances of interaction with the enzyme via inter-
molecular hydrogen bonding interactions (30).
Because of the planar structure of γ-picolyl alkyl amines, the
chances of intermolecular bonding is higher, and the known low
conformational stabilities of native proteins make them easily
susceptible to denaturation by altering the balance of the weak
nonbonding forces that maintain the native conformation. There-
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alkyl amine. In addition, we have also studied the preincubation
of the enzyme with inhibitors in the absence of the substrate
L-DOPA, where the enzyme activity was found to remain un-
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inhibitors rather than inactivators of enzymes (31).
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ACKNOWLEDGMENT
We thank all of the reviewers for their valuable suggestions and
criticism for improving the quality of this work.
Supporting Information Available: Spectroscopic data for
the picolyl alkyl amines. This material is available free of charge
via the Internet at http://pubs.acs.org.
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