Large-scale production of N,N′-diBoc-dityrosine and dityrosine by HRP-catalyzed N-Boc-l-tyrosine oxidation and one-step chromatographic purification

Article abstract of DOI:10.1016/j.procbio.2010.07.031

Dityrosine (DY) can be used as a biomarker to detect oxidative protein damage and selective proteolysis. It is generally prepared by horseradish peroxidase (HRP)-catalyzed oxidation of l-tyrosine (Y) followed by multistep chromatographic separations. In this study, we present an alternative method for the preparation of DY by HRP-catalyzed synthesis of N,N′-diBoc-dityrosine (DBDY) from N-Boc-l-tyrosine (BY). The presence of the tert-butoxycarbonyl (Boc) group ensured that the fraction of further oxidized by-products (e.g., trimers and pulcherosine) was quite low. The yield of DBDY (37.5%) was comparable to that reported for DY (> 26%). DBDY could be purified by a simple one-step silica column chromatography procedure that resulted in a purity of 89.7%. DBDY is considered to be better than DY for subsequent chemical reactions (for binding to polymers, amino acids, drugs, antibodies, etc.) because such reactions can be selectively performed by using the carboxylic acid and amine groups in the following sequence: first, the carboxylic acid groups are used; next, the Boc groups are removed; and finally, the amino groups are used. To prepare DY, the Boc groups in DBDY were simply and completely removed by treatment with trifluoroacetic acid.

Full text of DOI:10.1016/j.procbio.2010.07.031

Process Biochemistry 46 (2011) 142–147
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Large-scale production of N,Nꢀ-diBoc-dityrosine and dityrosine by HRP-catalyzed
N-Boc-l-tyrosine oxidation and one-step chromatographic purification
Dong-Ik Lee, Jee-Yun Choi, Chan-Jin Kim, Ik-Sung Ahn∗
Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Dityrosine (DY) can be used as a biomarker to detect oxidative protein damage and selective proteolysis.
It is generally prepared by horseradish peroxidase (HRP)-catalyzed oxidation of l-tyrosine (Y) followed
by multistep chromatographic separations. In this study, we present an alternative method for the prepa-
ration of DY by HRP-catalyzed synthesis of N,Nꢀ-diBoc-dityrosine (DBDY) from N-Boc-l-tyrosine (BY). The
presence of the tert-butoxycarbonyl (Boc) group ensured that the fraction of further oxidized by-products
(e.g., trimers and pulcherosine) was quite low. The yield of DBDY (37.5%) was comparable to that reported
for DY (> 26%). DBDY could be purified by a simple one-step silica column chromatography procedure that
resulted in a purity of 89.7%. DBDY is considered to be better than DY for subsequent chemical reactions
(for binding to polymers, amino acids, drugs, antibodies, etc.) because such reactions can be selectively
performed by using the carboxylic acid and amine groups in the following sequence: first, the carboxylic
acid groups are used; next, the Boc groups are removed; and finally, the amino groups are used. To pre-
pare DY, the Boc groups in DBDY were simply and completely removed by treatment with trifluoroacetic
acid.
Received 31 March 2010
Received in revised form 27 July 2010
Accepted 28 July 2010
Keywords:
N,Nꢀ-diBoc-dityrosine
Dityrosine
Horseradish peroxidase
Large-scale production
Chromatography
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Several methods have been reported for the preparative-scale
synthesis of DY. Nonenzymatic preparation methods involving the
Dityrosine (DY) is formed when proteins are exposed to reac-
tive oxidizing agents (e.g., hydrogen peroxide, hydroxyl radical,
and superoxide radical), ionizing radiation, or peroxidase, and it
is associated with Alzheimer’s and Parkinson’s diseases [1–3], cys-
tic fibrosis [4], and atherosclerosis [5]. Yoburn et al. [6] reported
that DY formation in ␥b-crystallin, calmodulin, ribonuclease A,
and bovine serum albumin resulted in protein denaturation and
enhanced precipitation.
DY can be used as a biomarker to detect oxidative protein dam-
age and selective proteolysis [7–9]. DY occurs naturally in fungal
cell-wall proteins, insect eggs, and sea-urchin envelopes as well
as in extensible proteins such as elastin, resilin, and calmodulin
[10–14]. In these proteins, DY plays the role of a crosslinking agent
and can help in increasing the mechanical strength. To adequately
measure DY formation in biological samples, an efficient method
is required to produce this compound in sufficient quantities [15].
There has been an attempt to produce novel polymeric materials in
which DY functions not only as the crosslinking agent but also as
the fluorescent core [16]. The above discussion indicates that there
is a growing need for the large-scale production of DY.
use of oxidizing agents such as potassium ferricyanide, sodium
persulfate, and ferrichloride have been reported. However, the
yield was less than 5% in these cases [17,18], and large amounts
of higher oxidation products such as trityrosine and isotrityro-
sine were inevitably formed [18]. Several elegant but complicated
schemes have been suggested for producing DY in high yields.
For example, Nishiyama et al. [19] successfully obtained a yield of
28% by protection of the amino and carboxylic groups of tyrosine,
iodination at both the ortho positions of the phenol group, oxida-
tive electrolysis, zinc reduction, catalytic hydrogenation, and acidic
hydrolysis. Hutton and Skaff [20] succeeded in obtaining a yield
of 39% by employing the following series of reactions: protection
of the amino and carboxylic groups of tyrosine, iodination at the
ortho position of a phenol group, Miyaura borylation, and Suzuki
coupling. However, high-level skills and experience of organic syn-
thesis are required to attain this level of productivity. We have
earlier reported a simple one-step synthesis reaction for DY prepa-
ration using Mn(III) as the oxidizing agent [21]. However, a common
feature of these chemical preparation methods described above is
that several purification steps such as precipitation, filtration, and
chromatographic separation are necessary because various impu-
rities are present in addition to DY.
Enzymatic preparation of DY generally employs a peroxidase
to catalyze the oxidative coupling of Y, and a yield of more than
∗
Corresponding author. Tel.: +82 2 2123 2752; fax: +82 2 312 6401.
E-mail address: iahn@yonsei.ac.kr (I.-S. Ahn).
1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.07.031

D.-I. Lee et al. / Process Biochemistry 46 (2011) 142–147
143
26% has been achieved with this method [22]. However, due to the
zwitterionic property of DY and the presence of by-products such
as isodityrosine, trityrosine, and pulcherosine, at least two chro-
matographic separation steps (e.g., gel filtration chromatography,
ion exchange chromatography, and thin-layer chromatography)
are required in this process [18,22].
The purpose of this study was to develop an alternative method
for the enzymatic preparation of DY. Similar to other enzymatic
methods, horseradish peroxidase (HRP) was chosen as the model
peroxidase. N-Boc-l-tyrosine (BY) was used as the substrate, and
N,Nꢀ-diBoc-dityrosine (DBDY) was the expected product. As DBDY
does not have a free amino group, it is not a zwitterion.
Similar to our study, Amado et al. [18] used N-acetyl-l-tyrosine
(AY) as the substrate of HRP and synthesized N,Nꢀ-diacetyl-
dityrosine (DADY). Deblocking (i.e., removal of acetyl groups) was
performed by hydrolysis in 6 M HCl solution under N2 reflux
for 20 h. Hydrolysis under such harsh conditions resulted in sev-
eral impurities and affected the subsequent reactions of DY (e.g.,
amide bond formation between DY and other materials) [23].
Consequently, three chromatographic separation steps had to be
employed in this process, which was similar to the process in
which Y was used as the HRP substrate [18]. The Boc group used to
protect the amino group is generally removed by hydrolysis with
trifluoroacetic acid (TFA) in methylene chloride for 30 min at room
temperature and 1 atm [24–27]. The resulting Boctrifluoroacetate
and residual TFA are easily removed without additional chromatog-
raphy steps.
2. Materials and methods
2.1. Chemicals
N-Boc-l-tyrosine (98%), l-tyrosine (99%), HRP, boric acid (99%), sodium tetrab-
orate decahydrate (99.5%), TFA (99%), N,N-dimethylformamide (DMF) (99.8%),
dimethyl sulfoxide-d6 (DMSO-d6) (99.9%), D2O, and 2-mercaptoethanol (98%) were
purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Acetonitrile and
water (HPLC grade) were purchased from Baker Chemical Co. (Phillipsburg, NJ, USA).
Silica gel 60 (70–230 mesh) was purchased from Merck Chemical. Co. (Darmstadt,
Germany). Ammonia water (25–30%), n-propanol (99%), H2O2 (28%), and methylene
chloride (99%) were purchased from Duksan Pure Chemical Co. (Ansan, GyonggiDo,
Korea). A Whatman GF/C glass microfiber filter membrane (Whatman, UK) with a
pore size of 1.2 ␮m and an Advantec PTFE filter membrane (Advantec, Japan) with
a pore size of 0.2 ␮m were used for filtration.
Fig. 1. The apparatus used for silica column chromatography. Dry silica gel 60 was
packed into a stainless steel column (150 cm × 5 cm i.d.) that was plugged with a
stainless steel net and sea sand (20–30 mesh; Junsei, Japan) at both ends. Deionized
water was first passed through the silica column to remove any trapped air. After
equilibration with an eluent composed of n-propanol and ammonia:water (90:10,
v/v), the column was connected to an injector, which consisted of a glass column
with two screw caps (30 cm × 2 cm i.d.).
2.2. Preparation of DBDY
For the large-scale synthesis of DBDY, 1 L of 5 mM BY solution was pre-
pared in 0.1 M borate buffer (pH 9.1). HRP was added to an activity of
through the column. Separation was performed at a flow rate of 60 ml/min and at a
temperature below 15 ◦C to minimize the volatilization of ammonia. HPLC analysis
of the elution fractions revealed that DBDY had a retention time of approximately
90 min. The fractions containing DBDY were collected and concentrated by vacuum
evaporation at 45 ◦C.
2260 purpurogallin units/L. Oxidation was initiated by adding H2O2 to
a con-
centration of 2.5 mM. After 20 min, the reaction was terminated by adding
2-mercaptoethanol to a concentration of 5 mM. For comparison, DY was directly pre-
pared from Y using HRP under the same conditions. All the reactions were performed
at 39 ◦C [22].
To minimize the loading volume in silica column chromatography, DBDY was
concentrated 100 times by evaporating under vacuum at 45 ◦C. Evaporation at a
temperature higher than 50 ◦C was found to result in the decomposition of the Boc
group (data not shown). Precipitated buffer components were removed by filtration
through a Whatman GF/C glass microfiber filter membrane. The concentrated solu-
tion was stored at 4 ◦C prior to purification. Additional precipitates formed during
storage were also removed by filtration.
2.4. Removal of the Boc groups in DBDY
To remove the Boc groups, 0.5 g of purified DBDY was dissolved in 20 ml methy-
lene chloride. The deblocking reaction was initiated by adding 20 ml TFA [24–27].
After 30 min, the solution was evaporated under vacuum at 45 ◦C. The residue was
then dissolved in deionized water, and undissolved materials were removed by fil-
tration through an 0.2 ␮m PTFE filter membrane. Deionized water was removed by
evaporation under vacuum at 45 ◦C. The residue was dissolved in DMF and finally
reprecipitated in an excess of methylene chloride. The precipitate (DY) was collected
by filtration through a PTFE membrane of pore size 0.2 ␮m.
2.3. Purification of DBDY by one-step silica column chromatography
The apparatus for silica column chromatography is shown in Fig. 1. Dry sil-
ica gel 60 was packed into a stainless steel column (150 cm × 5 cm i.d.) that was
plugged with a stainless steel net and sea sand (20–30 mesh; Junsei, Japan) at both
ends. Deionized water was first passed through the silica column to remove any
trapped air. After equilibration with an eluent composed of n-propanol and ammo-
nia:water (90:10, v/v), the column was connected to an injector, which consisted of
a glass column with two screw caps (30 cm × 2 cm i.d.). Concentrated reaction solu-
tion (5–10 ml) was added to the injector. As soon as the sample was loaded onto the
silica column, the injector was filled with the eluent. Passage of vaporized ammo-
nia caused a crack in the packed silica gel; therefore, a diaphragm pump (model
no. 07090-62; Masterflex AG, Gelsenkirchen, Germany) was used to pass the eluent
2.5. Analysis
The products of the HRP-catalyzed oxidation reaction and fractions from the
silica chromatography column were analyzed by using a HPLC system (Waters 510,
Waters Corp., USA) equipped with a UV absorbance detector (Waters 486, Waters
Corp., USA) [2] and a mass spectrometer (Triple Quadrupole Tandem Mass Spectrom-
eter, Micromass and Waters, USA). Separation was carried out on a C-18 column
˚
(5 ␮m particle size, 250 mm × 4.6 mm, 80 A pore size, Phenomenex, USA) using a
mixture of 0.1% (v/v) trifluoroacetic acid solution and acetonitrile as the eluent. For

144
D.-I. Lee et al. / Process Biochemistry 46 (2011) 142–147
Fig. 3. Mass spectrum of DBDY obtained using
a mass spectrometer (Triple
Quadrupole Tandem Mass Spectrometer, Micromass and Waters, USA).
better separation of the oxidation products, elution was carried out with a linearly
increasing gradient of acetonitrile (from 0% to 60%) in 30 min. The flow rate of the
eluent was maintained at 1 ml/min.
The structures of DBDY and DY were identified based on their 1H NMR spec-
tra that were recorded on a Bruker Advance 500-MHz nuclear magnetic resonance
spectrometer (Bruker BioSpin Corp., Germany). Prior to analysis, DBDY and DY were
dissolved in D2O. The 1H NMR spectrum of DBDY was compared with that of DMF
to determine the purity of purified DBDY. DBDY and DMF were dissolved in DMSO
for this analysis. The DBDY purity was calculated using the ratio of the peak area of
the aromatic ring protons in DBDY to that of methyl protons in DMF.
3. Results and discussion
3.1. Characterization of the oxidation products
HRP-catalyzed oxidation of BY was monitored by HPLC, and the
result is shown in Fig. 2. The compounds present in the six major
peaks had retention times (RT) of 21.7, 25.7, 30.6, 31.6, 33.0, and
Fig. 4. The HPLC chromatograms of DBDY (—) and DY (- - -). DBDY was purified
from the products of BY oxidation by one-step silica column chromatography. DY
was then prepared by the removal of Boc groups in DBDY. The apparatus used for
silica column chromatography is shown in Fig. 1. The purification of DBDY was ini-
tiated by adding the concentrated reaction solution (5–10 ml) to the injector. As
soon as the sample was loaded onto the silica column, the injector was filled with
the eluent. To avoid the passage of vaporized ammonia, a diaphragm pump (model
no. 07090-62; Masterflex AG, Gelsenkirchen, Germany) was used to pass the eluent
through the column. Separation was performed at a flow rate of 60 ml/min and at a
temperature below 15 ◦C to minimize the volatilization of ammonia. HPLC analysis
of the elution fractions revealed that DBDY had a retention time of approximately
90 min. The fractions containing DBDY were collected and concentrated by vacuum
evaporation at 45 ◦C. The condition of the HPLC separation is described in the leg-
end for Fig. 2. For the removal of Boc groups, purified DBDY was trated with TFA
for 30 min. After vacuum evaporation at 45 ◦C, the residue was dissolved in deion-
ized water, and undissolved materials were removed by filtration through an 0.2 ␮m
PTFE filter membrane. Deionized water was removed by evaporation under vacuum
at 45 ◦C. The residue was dissolved in DMF and finally reprecipitated in an excess of
methylene chloride. The precipitate (DY) was collected by filtration through a PTFE
membrane of pore size 0.2 ␮m.
Fig. 2. HPLC chromatograms of oxidation products sampled at different reaction
times. The time of sampling is denoted by “ts.”. For the synthesis of DBDY, 5 mM
BY solution was prepared in 0.1 M borate buffer (pH 9.1). HRP was added to an
activity of 2260 purpurogallin units/L. Oxidation was initiated by adding H2O2 to
a concentration of 2.5 mM. After 20 min, the reaction was terminated by adding
2-mercaptoethanol to a concentration of 5 mM. The reaction temperature was main-
tained at 39 ◦C. The HPLC separation was carried out on a C-18 column (5 ␮m particle
˚
size, 250 mm × 4.6 mm, 80 A pore size, Phenomenex, USA) using a mixture of 0.1%
(v/v) trifluoroacetic acid solution and acetonitrile as the eluent. Elution was carried
out with a linearly increasing gradient of acetonitrile (from 0% to 60%) in 30 min.
The flow rate of the eluent was maintained at 1 ml/min.

D.-I. Lee et al. / Process Biochemistry 46 (2011) 142–147
145
Fig. 5. 1H NMR spectra of DBDY (A) and DY (C) obtained after the removal of Boc groups in DBDY. These spectra were recorded on a Bruker Advance 500-MHz nuclear
magnetic resonance spectrometer (Bruker BioSpin Corp., Germany). Both compounds were dissolved in D2O. The chemical structures of DBDY and DY are shown in (B) and
(D), respectively.
34.0 min, respectively. These were then analyzed by mass spec-
troscopy (positive mode detection). The compound with an RT of
31.4 min disappeared during the concentration step, and its mass
spectrum could not be obtained. The compound in the tiny peak
with an RT of 21.7 min, which was labeled as I in Fig. 2, showed a
mass value (m/z) of 462.2, indicating a molecular mass of 461 Da.
The data indicated that this compound is N-Boc-dityrosine, and it
is believed to be formed by the accidental loss of one Boc group
from DBDY. The compound with an RT of 25.7 min showed a mass
value (m/z) of 283.2, indicating that it is residual BY of molecular
weight 282 Da. A fluorescent compound in the largest peak with RT
of 30.6 min showed a mass value (m/z) of 562 (see Fig. 3), indicating
that it is DBDY. The compound with an RT of 31.6 min, which was
labeled as II in Fig. 2, also showed a mass value (m/z) of 562. Its
mass spectrum (data not shown) indicated that it is another dimer
of BY, i.e., N,Nꢀ-diBoc-isodityrosine. Compounds with RT values of
33.0 and 34.0 min showed a mass value (m/z) of 841, indicating
that they are trimers of BY such as N,Nꢀ,Nꢀꢀ-triBoc-trityrosine and

146
D.-I. Lee et al. / Process Biochemistry 46 (2011) 142–147
Table 1
Peak areas of BY, DBDY, Y, and DY in the HPLC chromatograms of samples that were taken during HRP-catalyzed oxidation of BY and Y. Detailed conditions of the reaction
and the HPLC separation are described in the legend for Fig. 2.
Reaction time (min)
BY
DBDY
0.0
6433.2
12595.3
14206.2
Reaction time (min)
l-Tyrosine
DY
0.0
5.0
10.0
20.0
13259.8
6808.7
4617.3
2148.0
0.0
40
80
9421.7
4005.5
3802.9
2770.0
0.0
3100.8
4975.1
4985.4
120
N,Nꢀ,Nꢀꢀ-triBoc-pulcherosine (see the peaks labeled as III and IV in
Fig. 2).
[29,30]. Therefore, 1,2-ethanedithiol was added to inhibit such an
alkylation reaction, if any. However, the alkylation reaction did not
occur, and all DBDY was converted to DY (see Fig. 4), irrespective
of whether or not 1,2-ethanedithiol was added.
3.2. Accumulation of DBDY during HRP-catalyzed oxidation of BY
The 1H NMR spectrum of DY, which was obtained by deblocking
of DBDY, is shown in Fig. 5C. A singlet for methyl protons (1.36 ppm)
designated as Hf (see Fig. 5A and B) is observed in Fig. 5A but is
not seen in Fig. 5C, which proves that the Boc groups were com-
pletely removed (compare Fig. 5B and D). Moreover, the spectrum
shown in Fig. 5C is identical to the 1H NMR spectrum of DY reported
elsewhere [19–21].
The peak areas of BY, DBDY, Y, and DY in the HPLC chro-
matograms (see Fig. 2) are summarized in Table 1. Approximately
5 min was required for 50% reaction of BY, but approximately
40 min was needed for Y. In the last two samples, the increase in the
peak area of DBDY was coupled with a decrease in the peak area of
BY. However, the peak area of DY did not change even though the
peak area of Y clearly decreased. This indicates that the conversion
of Y to DY and the subsequent oxidation of DY occurred simulta-
neously during this time period. Further oxidation such as this was
much lower in the case of DBDY, leading to a continuous increase
in its peak area. Amado et al. [18] reported similar results with AY.
Michon et al. [28] recently reported the kinetics of the HRP-
catalyzed oxidation of Y, AY, N-acetyltyrosine amide (AYA), and
tyrosine-containing peptides. They found that the dimerization of
AY and AYA (i.e., the conversion of AY and AYA to DADY and N,Nꢀ-
diacetyl-dityrosine amide) was about 20 times faster than that of Y
(i.e., the conversion of Y to DY). They suggested that the dimeriza-
tion rate might depend on the positive ionization of the ␣-amino
group because AY and AYA were devoid of a free amino group. They
also found that the polymerization of AY and tyrosine-containing
peptides (i.e., the subsequent oxidation of their dimers) was less
than that of Y. Steric hindrance was suggested to be a reason for this
result [28]. Their study of HRP kinetics also demonstrated that the
polymerization was hindered due to the enzyme inhibition by the
dimers of AY and tyrosine-containing peptides. Hence, the faster
conversion of BY to DBDY than Y to DY can be explained by the
absence of a free amino group in BY. The subsequent oxidation of
DBDY is believed to have been hindered due to steric hindrance and
the inhibition of HRP by DBDY, which resulted in the accumulation
of DBDY.
4. Conclusions
In this study, HRP-catalyzed oxidation of N-Boc-l-tyrosine fol-
lowed by one-step silica column chromatography was presented as
a method for the simple and large-scale preparation of dityrosine
(DY). The presence of the tert-butoxycarbonyl (Boc) group allowed
faster production of N,Nꢀ-diBoc-dityrosine (DBDY), the target prod-
uct of the HRP-catalyzed oxidation reaction, and a higher yield. The
yield and purity of DBDY were up to 37.5% and 89.7%, respectively.
Reaction with trifluoroacetic acid permitted the easy removal of
Boc groups in DBDY, and all of the purified DBDY was completely
converted to DY. DBDY is considered to be better than DY for sub-
sequent chemical reactions (for binding to a polymer, amino acid,
drug, antibody, etc.) because these reactions can be selectively
performed by using the carboxylic acid and amine groups in the
following sequence: first, the carboxylic acid groups are used; next,
the Boc groups are removed; and finally, the amino groups are used.
Acknowledgement
This research was supported by the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education, Science
and Technology (2009-0084190).
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