Selective synthesis of DOPA and DOPA peptides by native and immobilized tyrosinase in organic solvent

Article abstract of DOI:10.1002/cplu.201200300

3,4-Dihydroxyphenylalanine (DOPA)-containing peptides and proteins provide attractive design paradigms for pharmaceutical applications and engineering of synthetic polymers. An efficient and selective route to DOPA peptides by oxidation of ltyrosine deriv

Full text of DOI:10.1002/cplu.201200300

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DOI: 10.1002/cplu.201200300
Selective Synthesis of DOPA and DOPA Peptides by Native
and Immobilized Tyrosinase in Organic Solvent
Giorgia Botta,[a] Michela Delfino,[a] Melissa Guazzaroni,[a] Claudia Crestini,[b] Silvano Onofri,[a]
and Raffaele Saladino*[a]
3,4-Dihydroxyphenylalanine (DOPA)-containing peptides and
proteins provide attractive design paradigms for pharmaceuti-
cal applications and engineering of synthetic polymers. An effi-
cient and selective route to DOPA peptides by oxidation of l-
tyrosine derivatives with tyrosinase is reported. The efficiency
of the procedure was tested by using successively recycled ty-
rosinase immobilized on EupergitꢁC250L and coated with poly-
electrolytes by the layer-by-layer method.
Introduction
l-3,4-Dihydroxyphenylalanine
[2-amino-3-(3,4-dihydroxyphe-
tions.[12] An alternative to this strategy is the direct oxidation of
tyrosine residues. This procedure is usually performed with
toxic reagents and low selectivity owing to the formation of
cross-links involving reactive quinone intermediates.[13]
nyl)propanoic acid; l-DOPA] is a minor constituent of animal
and plant tissue derived from post-translational modification
of tyrosine.[1] l-DOPA is metabolized to catecholamine neuro-
transmitters, noradrenaline and adrenaline,[2] which are charac-
terized by different biological activities, including stimulation
of the central nervous system, control of the blood pressure,
and protection against neurodegenerative diseases.[3] Since the
1960s, l-DOPA has been the first drug of choice in the therapy
of Parkinson’s disease.[4] DOPA-containing peptides (DOPA-Pep)
also show important biological activities. For example, ana-
logues of the peptide alpha-factor with DOPA substituting for
tyrosine residue at position 13 acts by specific interactions (res-
idue-to-residue cross-link) with Ste2p, a Saccharomyces cerevi-
siae model G protein-coupled receptor.[5] DOPA-Pep inhibit the
oxidation of low-density lipoproteins in the atherosclerotic
plaque,[6] and are used both as delivering tool for improving
DOPA absorption in long-term treatment of Parkinson’s dis-
ease[7] and as skin moisturizer in cosmetic compositions.[8]
Moreover, DOPA-Pep provide attractive design tools for wet
adhesives and coatings.[9] To date, sequence-controlled DOPA-
Pep are prepared by multistep synthesis in solution or the
solid phase.[10,11] Although these reactions proceed with ac-
ceptable yields, the protection/deprotection sequences and ac-
tivation of DOPA residues require long and expensive proce-
dures, and limit the use of DOPA-Pep in industrial applica-
Recently, we reported the oxidation of tyrosine to DOPA
with 2-iodoxybenzoic acid.[14] Herein, we turn our attention to
tyrosinase (polyphenol oxidase, EC 1.14.18.1), an enzyme that
catalyzes the hydroxylation of phenols to catechols and that of
catechols to ortho-benzoquinones.[15] Tyrosinase works either in
purified or overexpressing mutant microorganisms.[16] Examples
of the application of tyrosinase in peptides and protein modifi-
cations in buffer have been reported,[17] with limitations owing
to the polymerization of quinone intermediates and inactiva-
tion of the enzyme. This drawback can be overcome by addi-
tion of ascorbic acid in buffer,[18] or by use of organic solvents
as opposed to conventional aqueous medium.[19] Nonaqueous
enzymology shows numerous advantages, such as increased
solubility of substrates, improved stability and selectivity, and
limited quinone polymerization. A detailed study on the char-
acterization of the behavior of mushroom tyrosinase towards
a representative series of phenolic and diphenolic substrates,
structurally related to tyrosine, in aqueous–organic solvent has
been reported.[20]
Results and Discussion
In this study we used a freshly purified tyrosinase from Agari-
cus bisporus obtained by modification of a previously reported
procedure.[21] Tyrosinase activity was assayed by the dopa-
chrome method with l-tyrosine (l-Tyr) as substrate.[22] All ex-
periments were performed in triplicate. In accordance with
data previously reported,[23] tyrosinase showed a specific activi-
[a] Dr. G. Botta, Dr. M. Delfino, Dr. M. Guazzaroni, Prof. Dr. S. Onofri,
Prof. Dr. R. Saladino
Department of Ecology and Biology
University of Tuscia, Largo dell’Universitꢀ
01100 Viterbo (Italy)
E-mail: saladino@unitus.it
, , and
ty value of 2568 UmgÀ1 Vmax 4ꢀ10À3 DAbs minÀ1 mgÀ1
[b] Dr. C. Crestini
Department of Chemical Science and Technology
University of Rome Tor Vergata
00133 Rome (Italy)
Km 257mm. Water plays a critical role in the solvation process
and enzyme activity,[24,25] so the amount of added buffer had
to be optimized for any specific catalytic system in organic sol-
vents.[26,27] For this reason, we evaluated the reaction rate
versus the concentration of buffer. In particular, Boc-Tyr-OMe 1
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201200300. It contains spectroscopic data
for the synthesized compounds.
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(Boc is the N-tert-butoxycarbonyl group) was selected as a rep-
resentative compound and the oxidation with tyrosinase
(57.8 IU) was performed with variation of the amount of Na
phosphate buffer (10–70 mL) in the presence of CH2Cl2 (2.5 mL).
The highest value of activity (2924 UmgÀ1) was obtained with
26.5 mL Na phosphate buffer. The kinetic parameters in CH2Cl2/
buffer were determined by measuring the tyrosinase activity at
different concentrations of compound 1 (range 330–1000 mm),
by using the amount of buffer previously optimized. The
Km value was 147 mm and Vmax was 3.4ꢀ10À3 DAbs minÀ1 mgÀ1
.
These values are of the same order of magnitude as those
measured for the enzyme in buffer alone.
Next, we analyzed the oxidation of 1 and peptide derivatives
3–11 containing one or more residues of tyrosine. The oxida-
tion of 1 (0.02 mmol) was performed with tyrosinase (400 IU)
in CH2Cl2 (2.5 mL) and traces of buffer (Na phosphate, 184 mL,
pH 7) at room temperature under an O2 atmosphere for 4 h.
After in situ reduction with sodium dithionite (Na2S2O4,
2.0 equiv in 2.5 mL added H2O), the Boc-DOPA-OMe 2 was ob-
tained with quantitative conversion of substrate and 95% yield
(Scheme 1).
Scheme 2. Oxidation of dipeptides bearing N-terminal tyrosine residues 3
and 4 with tyrosinase in CH2Cl2.
Scheme 1. Oxidation of compound 1 with tyrosinase (Tyro) in CH2Cl2.
After removal of the protective groups,[28] the correct stereo-
chemistry of 2 was verified by measurement of optical activity
and comparison with an authentic sample of l-DOPA ([a]2D7 =
À11.58, c=5 in 1.0m HCl). The optical rotation of 2 was in
good agreement with the reference data.
Next, we extended the procedure to a panel of selected di-
peptides and tripeptides, including Boc-Tyr-Phe-OMe 3, Boc-
Tyr-Leu-OMe 4, Boc-Gly-Tyr-OMe 5, Boc-Ala-Tyr-OMe 6, Boc-
Leu-Tyr-OMe 7, Boc-Val-Tyr-OMe 8, Boc-Gly-Leu-Tyr-OMe 9, and
Boc-Val-Tyr-Val-OMe 10, which bear one reactive tyrosine resi-
due at the N-terminal, C-terminal, or internal position. Boc-Tyr-
Tyr-Tyr-OMe 11, which bears three tyrosine residues, was also
studied as an example of a peptide reactive in all positions of
the sequence. Results are reported in Schemes 2–4.
Scheme 3. Oxidation of dipeptides bearing C-terminal tyrosine residues 5–8
with tyrosinase in CH2Cl2.
The oxidation of dipeptides bearing N-terminal tyrosine resi-
dues 3 and 4 afforded the corresponding DOPA derivatives
Boc-DOPA-Phe-OMe 12 and Boc-DOPA-Leu-OMe 13 in quantita-
tive yields (82 and 74% conversion of substrate, respectively),
besides unreacted substrates (Scheme 2). In a similar way, C-
terminal tyrosine dipeptides 5–8 afforded Boc-Gly-DOPA-
OMe 14, Boc-Ala-DOPA-OMe 15, Boc-Leu-DOPA-OMe 16, and
Boc-Val-DOPA-OMe 17 in quantitative yield (conversion of sub-
strate in the range of 74–90%), besides unreacted substrates
(Scheme 3). The yield of products is calculated on converted
substrates. This finding indicates that dipeptides containing C-
terminal tyrosine residues show a reactivity similar to that of
N-terminal ones. As a general trend, in the case of C-terminal
tyrosine dipeptides, the conversion decreases gradually with
increasing steric hindrance of the substituent in the side chain,
and reaches the minimum value with leucine.
Notably, no dimers or quinone derivatives were detected,
thus confirming the benign role of the organic solvent in the
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As selected examples, the stereochemical integrity of pep-
tides 14–17 and 20 was confirmed by comparison of the opti-
cal rotatory power with authentic samples after the deprotec-
tion procedure described above:[28] Gly-l-DOPA: found [a]D = +
46, reported [a]D = +46.07 and l-DOPA-l-DOPA-l-DOPA: found
[a]D = +13.7, reported [a]D = +13.76 (1.0m HCl, c 1.0, T=
258C);[29] Ala-l-DOPA: found [a]D = +25.0, reported [a]D = +
24.91, Leu-l-DOPA: found [a]D = +40, reported [a]D = +39.99,
and Val-l-DOPA: found [a]D = +27, reported [a]D = +26.93
(1.0m HCl, c 0.8, T=258C).[30] Compound 1 was also analyzed,
as previously reported, by chiral HPLC (CHIROBIOTIC T, Aldrich)
with commercial l-DOPA as reference.[12,14] The presence of the
peak attributable to l-DOPA and the absence of d-DOPA in the
HPLC profile of the sample prepared according to our proce-
dure further confirmed the selectivity of the oxidation.
With the aim to further improve the procedure, the oxida-
tion of Boc-Tyr-OMe 1 and the selected dipeptide Boc-Gly-Tyr-
OMe 5 was repeated by using immobilized tyrosinase on Eu-
pergit C250L (Tyro/E) and the latter catalyst after coating by
the layer-by-layer (LbL) technique (Tyro/E-LbL).[23] Figure 1
shows the structure of the heterogeneous catalysts.
Briefly, tyrosinase (5.0 mg, 12840 IU) was suspended in Na
phosphate buffer (0.1m, pH 7) in the presence of Euper-
gitꢁC250L (1.0 g) for 24 h at room temperature, followed by
treatment with glycine to block residual epoxy groups, to yield
Tyro/E. Under these experimental conditions Tyro/E retained
about 37% of the native activity (4750 UgÀ1). Then the LbL
technique was applied by coating the particles through se-
quential deposition of alternately charged polyelectrolytes.
Tyro/E was suspended in positively charged polyallylamine hy-
drochloride (PAH) (2 mgmLÀ1 in 0.5m NaCl), filtered, and then
treated with negatively charged polystyrene sulfonate (PSS;
2 mgmLÀ1 in 0.5m NaCl) for three runs. The immobilized LbL
enzymes retained approximately 87% of the Tyro/E activity
(4133 UgÀ1). The structure and morphology of particles of
Tyro/E and Tyro/E-LbL in CH2Cl2 was similar to that in Na phos-
phate buffer.[23]
Scheme 4. Oxidation of dipeptides bearing one or three tyrosine residues 9–
11 with tyrosinase in CH2Cl2.
inhibition of undesired side reactions. Finally, the oxidation of
tripeptides 9–11 bearing one or three reactive tyrosine resi-
dues was studied. As reported in Scheme 4, the oxidation of 9
and 10 afforded Boc-Gly-Leu-DOPA-OMe 18 and Boc-Val-DOPA-
Val-OMe 19 in high yield (95 and 97%, respectively) and con-
version (90 and 93%, respectively), besides unreacted sub-
strate.
Again, the efficiency of the reaction was not influenced by
the position of the tyrosine residue in the peptide (that is, C-
terminal versus internal position). This finding was further con-
firmed by oxidation of 11 in which case all three tyrosine resi-
dues were oxidized with the formation of Boc-DOPA-DOPA-
DOPA-OMe 20 as the only recovered product (95% yield and
85% conversion).
Figure 2 shows scanning electron microscopy (SEM) images
of the section of Tyro/E (panel A) and Tyro/E-LbL particles
(panel B). The surface of Tyro/E appears granular and heteroge-
neous, whereas in the second case the presence of the poly-
electrolytes makes the surface uniform. Reactions were per-
formed with 0.02 mmol of the appropriate substrate, with
Figure 1. Preparation of Tyro/E and Tyro/E-LbL.
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products. In accordance with the usual selectivity of enzymatic
transformations, the reaction proceeds with complete reten-
tion of the stereochemistry, as shown in the oxidation of 1,
14–17, and 20 as selected representative examples. The oxida-
tion of tyrosine is efficient irrespective of its position in the
peptide (that is, C-terminal versus central or N-terminal posi-
tions). On the other hand, the conversion of the substrate
slightly decreased with increasing steric hindrance of the vici-
nal a-amino acid residue to tyrosine (see, for example, the oxi-
dation of peptide 14 versus 16, Scheme 3). The reusability of
immobilized Tyro/E and Tyro/E-LbL was determined in the oxi-
dation of 1 under previously reported experimental conditions.
As shown in Table 1, Tyro/E and Tyro/E-LbL remained active
after three runs. Moreover, the presence of the polyelectrolyte
coating further stabilized the enzyme, thereby ensuring
a higher value of conversion of the substrate. This behavior
suggests that microcapsules create an internal microenviron-
ment able to protect the enzyme from denaturing agents. Fur-
ther studies are in progress to evaluate the oxidation of large
peptides and small proteins with the aim of synthesizing new
drugs for the treatment of Parkinson’s disease.
Figure 2. SEM images of the section of Tyro/E (A) and Tyro/E-LbL parti-
cles (B).
Tyro/E and Tyro/E-LbL (400 IU) and Na phosphate buffer
(184 mL; pH 7) at room temperature under an O2 atmosphere
for 4 h, followed by in situ reduction with sodium dithionite
(Na2S2O4, 2.0 equiv in 2.5 mL of added H2O).
As reported in Table 1, both heterogeneous systems catalyze
the oxidation of selected compounds to give the correspond-
ing DOPA derivatives 2 (Table 1, entries 1 and 2) and 14
(Table 1, entries 3 and 4) in yields similar to that of the native
enzyme. The experiments were repeated in triplicate.
Table 1. Oxidation of 1 and 5 under heterogeneous conditions.
Experimental Section
Entry
Substrate
Catalyst
Product
Yield [%] (conv. [%])
Mushroom tyrosinase was extracted from A. bisporus. Eupergit
C250L, poly(sodium 4-styrenesulfonate) (PSS, MW 70000), poly(al-
lylamine hydrochloride) (PAH, MW 56000), l-tyrosine (l-Tyr), 2,2’-
azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), bovine
serum albumin (BSA), ammonium sulfate [(NH4)2SO4], dichlorome-
thane (CH2Cl2), ethyl acetate (EtOAc), anhydrous sodium sulfate
(Na2SO4), trimethylchlorosilane (TMCS), peptides, and Boc2O were
purchased from Sigma–Aldrich. Water used during extraction was
degassed and stored at 48C until use. All spectrophotometric
measurements were made with a Varian Cary 50 UV/Vis spectro-
photometer equipped with a single cell Peltier device. Dichlorome-
thane was dried on anhydrous Na2SO4 prior to use. NMR spectra
were recorded on a Varian Mercury 400 (1H, 400 MHz) spectrome-
1
2
3
4
5
6
7
8
9
10
1
1
5
5
1
1
1
1
1
1
Tyro/E
Tyro/E-LbL
Tyro/E
Tyro/E-LbL
Tyro/E
Tyro/E
2
2
14
14
2
2
2
2
2
90Æ2 (81Æ2)
96Æ1 (91Æ1)
92Æ2 (>98)
90Æ1 (90Æ1)
88Æ2 (76Æ2)
89Æ1 (70Æ1)
89Æ2 (65Æ2)
91Æ1 (89Æ2)
93Æ1 (88Æ1)
92Æ1 (85Æ2)
Tyro/E
Tyro/E-LbL
Tyro/E-LbL
Tyro/E-LbL
2
To evaluate the reusability of heterogeneous catalysts, the
oxidation of 1 was repeated for three runs after recovering the
catalyst from the reaction mixture. Tyro/E showed a decrease
of its conversion efficiency (Table 1, entries 5–7). A different
pattern was observed for Tyro/E-LbL (Table 1, entries 8–10), in
which case the yield and conversion were similar to those of
the first run, thus confirming the benign role of the LbL coat-
ing in the stability of the system.
1
ter. For H and 13C NMR data, d values of chemical shifts are report-
ed in ppm. Mass spectra were obtained on a GC/LC–MS Varian
spectrometer. Chromatographic purifications were performed on
columns packed with silica gel, 230–400 mesh, for a flash tech-
nique.
Extraction of tyrosinase
Tyrosinase was obtained from commercial A. bisporus.[21a,b] In a typi-
cal procedure, the sporocarps were cleaned to remove earthy resi-
dues and then washed with 20 mm ascorbic acid maintained at
48C. After drying, the sporocarps were sliced and frozen at À208C
at least 1 day before extraction. Frozen sporocarps (1 kg) were ho-
mogenized twice in acetone (1.2 L) at À208C in a blender for
1 min. The obtained solid pulp was filtered through a Buchner
funnel and homogenized with 30% v/v acetone (1.0 L) in water for
2–3 min. The mixture was centrifuged at 9000 g for 20 min. Vol-
umes of acetone (1.5 mL) at À208C were added dropwise to the
supernatant under vigorous stirring. The mixture was allowed to
settle at 48C for 2–3 h; most of the supernatant fluid was decanted
and discarded, the remainder was centrifuged and the precipitate
was dissolved in water and subjected to precipitation with calcium
acetate (1% saturation). The turbid mixture was frozen at À208C.
Conclusion
Usually, DOPA peptides are prepared by solid-phase synthesis,
which requires complex and long reaction conditions.[12] An al-
ternative to this procedure is the direct oxidation of l-tyrosine
and l-tyrosine derivatives into DOPA and DOPA peptides with
tyrosinase, an enzyme that catalyzes the oxidation of phenols
to catechols.[15] To develop novel procedures of industrial inter-
est, the tyrosinase was immobilized on the epoxy resin Euper-
gitꢁC250L and coated by the layer-by-layer technique to afford
Tyro/E and Tyro/E-LbL catalysts. The reactions performed in or-
ganic solvent at 258C were selective to produce the corre-
sponding DOPA peptides in high yield as the only recovered
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Samples obtained from several days were stored frozen at this
stage. They were then thawed, mixed, and centrifuged. Powdered
(NH4)2SO4 was added to the collected supernatant to make a 35%
saturated solution. The resulting solution was allowed to stand for
30 min at 48C and centrifuged at 9000 g for 20 min. (NH4)2SO4 was
added to the supernatant to make a 70% saturated solution. The
solution was allowed to stand for 2 h at 48C and centrifuged. The
precipitate was dissolved in a minimal volume of cold water and
then dialyzed against water and concentrated by means of a Viva-
flow 50 system equipped with a polyethersulfone (PES) membrane
(10000 molecular weight cutoff). The resulting enzyme solution
was lyophilized and stored at À208C.
Figure 3. Lineweaver–Burk plot for Boc-Tyr-OMe 1.
Determination of protein concentration
Protein concentration was determined spectrophotometrically at
595 nm according to the Bradford method with BSA as a standar-
d.[31a,b]
sured at 475 nm as described above. Reactions in aqueous systems
were performed under similar experimental conditions.[33]
Tyrosinase immobilization on EupergitꢀC250L
Activity assay
The enzyme immobilization was performed by a modification of lit-
erature procedures.[35a,b] Dry EupergitꢁC250L (1.0 g) was added to
Na phosphate buffer (8 mL, 0.1m, pH 7) containing tyrosinase
(5 mg, 12840 IU). The mixture was incubated for 24 h at room tem-
perature with orbital shaking. At the end of the coupling period,
the beads were isolated by filtration and washed (5ꢀ8 mL) with
Na phosphate buffer (0.1m, pH 7) until no activity was detected in
the washing liquid. The obtained beads were incubated with gly-
cine (3m) for 2 h to block residual epoxy groups,[36] then washed
with buffer and finally air-dried and stored at 48C to give Tyro/E.
The amount in milligrams and the units of coupled tyrosinase were
calculated by the difference between the amount/units loaded and
that recovered in the washings by the conventional Bradford test.
Tyrosinase assay was performed by the dopachrome method as
previously described.[32] Briefly, l-Tyr solution (1 mL, 2.5 mm) was
mixed with Na phosphate buffer (1.9 mL, 0.1m, pH 7) and incubat-
ed at 258C for 10 min in a series of vials. Then, an appropriate
amount of free or immobilized enzyme in Na phosphate buffer
(100 mL) was added to the mixture and the vials were agitated on
a horizontal shaker at 150 rpm. Each vial was removed at a prede-
termined time and the initial rate was measured immediately as
a linear increase in optical density at 475 nm, as a result of dopa-
chrome production. In the case of immobilized enzyme the catalyst
was removed quickly by centrifugation. One unit of enzyme activi-
ty was defined as an increase in absorbance of 0.001 minÀ1 at pH 7
and 258C, in a 3 mL reaction mixture containing 0.83 mm l-Tyr and
67 mm Na phosphate buffer of pH 7.
Tyrosinase immobilization by the layer-by-layer procedure
Tyro/E was coated with the layer-by-layer (LbL) method according
to literature procedures.[37] Briefly, PAH and PSS solutions
(2.0 mgmLÀ1 in 0.5m NaCl) were alternately added to the Tyro/E
system: each polyelectrolyte layer was adsorbed for 20 min at
room temperature with orbital shaking and then washed with
0.5m NaCl to remove excess polyelectrolytes. The deposition of
polyelectrolytes started with PAH and was repeated to obtain
three layers (PAH-PSS-PAH). Immobilized tyrosinase (Tyro/E-LbL)
was air-dried and stored at 48C.
Optimization of water requirement for the oxidation in or-
ganic solvent
The optimum quantity of buffer necessary for hydration of tyrosi-
nase was determined by applying our previously reported proce-
dure.[23] Briefly, Boc-Tyr-OMe 1, selected as a representative sub-
strate[33] (0.02 mmol), tyrosinase (57.8 IU), and CH2Cl2 (2.5 mL) were
placed in vials at 258C under an O2 atmosphere, to which Na phos-
phate buffer (0.1 m, pH 7, 10–70 mL) was added. The reaction mix-
ture was stirred vigorously for 30 min. Every 15 min, aliquots were
removed and their absorbance at 389 nm, owing to DOPA produc-
tion, was measured; the aliquots were returned to the vials as rap-
idly as possible. One unit of enzyme activity (IU) was defined as an
increase in absorbance of 0.001 minÀ1 at 389 nm, 258C, Na phos-
phate buffer (0.1 m, pH 7).
Procedure for the preparation of protected amino acids
Amino acid and peptides (1 mmol) were dissolved in MeOH (5 mL)
and stirred at room temperature. Freshly distilled TMCS (2.0 mmol)
was added and the resulting solution or suspension was stirred at
room temperature for 48 h. After completion of the reaction (moni-
tored by TLC), the mixture was concentrated on a rotary evapora-
tor to give the product amino acid or peptide ester hydrochlo-
ride.[38] A solution of the above tetramethyl ester salt (1.0 mmol),
NaHCO3 (4 mmol), and Boc2O (2.4 mmol) in THF/H2O (2.4:1) was
stirred at room temperature. After 24 h, the pH was adjusted to 3.0
with HCl and the solution was extracted with EtOAc. The combined
extracts were concentrated in vacuo and the crude products were
purified by flash chromatography to give the corresponding Boc-
protected products.[39]
Kinetic assay
Kinetic parameters (Km and Vmax) were determined by measuring
enzyme activity at different concentrations of substrate and plot-
ting data to a double reciprocal plot (Lineweaver–Burk plot,
Figure 3).[23,34] For studying the catalytic properties of enzymes in
the organic solvent media, reactions were performed with different
concentrations of Boc-Tyr-OMe 1 (in the range of 330–1000 mm)
and optimum amounts of aqueous buffer. Absorbance was mea-
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[12] Z. Liu, B. H. Hu, P. B. Messersmith, Tetrahedron Lett. 2008, 49, 5519–
5521.
Procedure for the oxidation of 1 and peptides 3–11
The reactions were performed under both homogeneous and het-
erogeneous conditions in CH2Cl2/buffer. As a general procedure
the appropriate substrate (0.02 mmol), tyrosinase (400 IU), and the
optimal amount of Na phosphate buffer (184 mL, 0.1m, pH 7) were
dissolved in CH2Cl2 (2.5 mL) at 258C in an O2 atmosphere under
vigorous stirring. The reaction was monitored by thin-layer chro-
matography (TLC). After the disappearance of the substrate, the
catalyst was recovered by filtration (immobilized enzymes) and the
organic layer was treated with an equal volume of sodium dithion-
ite solution (1%, w/w) to reduce benzoquinones to catechols. The
mixture was stirred for 5 min and the phases were separated. The
aqueous phase was acidified with HCl (1.0 n) and extracted twice
with EtOAc. The organic extracts were treated with a saturated so-
lution of NaCl and dried over anhydrous Na2SO4, then filtered and
concentrated under vacuum to yield a colored crude product. The
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[17] M. A. Rosei, L. Antonilli, R. Coccia, C. Foppoli, Biochem. Int. 1989, 19,
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Acknowledgements
The project Prin MIUR 2008 is acknowledged.
Keywords: biocatalysis
·
DOPA peptides
·
enzymes
·
immobilization · synthetic methods
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Received: November 26, 2012
Revised: January 21, 2013
Published online on February 21, 2013
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