Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally produced by oxidative stress and containing 3-nitrotyrosine

Article abstract of DOI:10.1007/s00726-015-2163-2

3-Nitrotyrosine (Nit) belongs to products of oxidative stress and could probably influence conformation of neurodegenerative proteins. Syntheses of peptides require availability of suitable synthon for introduction of Nit residue. Common phenolic protecti

Full text of DOI:10.1007/s00726-015-2163-2

Amino Acids (2016) 48:1087–1098
DOI 10.1007/s00726-015-2163-2
ORIGINAL ARTICLE
Rapid acidolysis of benzyl group as a suitable approach
for syntheses of peptides naturally produced by oxidative stress
and containing 3‑nitrotyrosine
Petr Niederhafner1,2 · Martin Šafarˇík2 · Eva Brichtová2 · Jaroslav Šebestík2
Received: 7 December 2015 / Accepted: 24 December 2015 / Published online: 14 January 2016
© Springer-Verlag Wien 2016
Abstract 3-Nitrotyrosine (Nit) belongs to products of
oxidative stress and could probably influence conforma-
tion of neurodegenerative proteins. Syntheses of peptides
require availability of suitable synthon for introduction of
Nit residue. Common phenolic protection groups are more
acid labile, when they are attached to Nit residue. We have
found that Fmoc–Nit(Bn)–OH is a good building block for
syntheses of Nit containing peptides by Fmoc/tBu strategy.
Interestingly, the peptides containing multiple Nit residues
can be available solely by use of Fmoc–Nit(Bn)–OH syn-
thon. Bn is removed rapidly with ca 80 % trifluoroacetic
acid in dark. The cleavage of Bn from Fmoc–Nit(Bn)–OH
proceeds via pseudo-first order mechanism with activation
barrier 32 kcal mol−1 and rate k = 15.3 s−1 at 20 °C. This
rate is more than 2,000,000 times faster than that for cleav-
age of benzyl from Tyr(Bn).
Keywords Nitrotyrosine · Peptide synthesis · Alpha-
synuclein · Reaction rate
Introduction
The aging population is progressively affected by neuro-
degenerative diseases such as Alzheimer’s disease (Butter-
field et al. 2011), Parkinson’s disease (Gurry et al. 2013)
and amyotrophic lateral sclerosis (Abe et al. 1995; Beal
et al. 1997; Strong et al. 1998). Moreover, transmissible
neurodegenerative diseases (Prusiner 1998) emerged as a
potential threat of entire society independently on the age.
In many of the above mentioned diseases, conformational
changes of proteins are considered to play the detrimental
role (Gurry et al. 2013; Prusiner 1998). Another detrimen-
tal factor for neurodegenerative diseases is an oxidative
stress (Butterfield et al. 2011; Radi 2013), which can affect
the system on molecular level. The attacked resources are
not only membrane lipids and proteins (Radi 2013; Bartes-
aghi et al. 2010) but also intracellular machinery respon-
sible for metabolic transformations. The oxidized proteins
can both gain or lose their functions and thus negatively
influence fragile equilibrium in life (Butterfield et al. 2011;
Radi 2013).
There are many free radical reactions responsible for
protein modifications (Radi 2013; Robinson and Evans
2012). Among them, the nitration of tyrosine (H–Tyr–OH)
residue is quite abundant (Radi 2004, 2013; Bartesaghi
et al. 2010; Robinson and Evans 2012). However, only
several structures containing 3-nitrotyrosine (H–Nit–OH)
were deposited in Protein Data Bank (Sept/1 2015). The
described structures mostly belong to family of oxidore-
ductases, i.e. proteins directly involved in redox reactions
and oxidative stress. Unfortunately, none of the deposited
Handling Editor: V. Soloshonok.
Standard abbreviations have been followed throughout this paper
(J Peptide Sci 12:1–12, 2006). Unless stated otherwise, amino
acids are of L-configuration.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-015-2163-2) contains supplementary
material, which is available to authorized users.
* Jaroslav Šebestík
jsebestik@seznam.cz; sebestik@uochb.cas.cz
1
Department of Chemistry of Natural Compounds,
Faculty of Food and Biochemical Technology, University
of Chemistry and Technology, Prague, Technická 5, 166
28 Prague 6, Czech Republic
2
Institute of Organic Chemistry and Biochemistry, Academy
of Sciences, Flemingovo námeˇstí 2, 16610 Prague 6, Czech
Republic
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P. Niederhafner et al.
structures belongs to the neurodegenerative proteins despite
many of them contain tyrosine residues (Fig. 1). In past, the
excessive nitration of proteins was observed during trans-
missible spongiform encephalopathies (Guentchev et al.
2000; Dear et al. 2007; Fernández et al. 2007), Alzheimer’s
disease (Butterfield et al. 2011; Lüth et al. 2002; Sacksteder
et al. 2006), Parkinson’s disease (Sacksteder et al. 2006;
Pennathur et al. 1999; Giasson et al. 2000), and amyo-
trophic lateral sclerosis (Abe et al. 1995; Beal et al. 1997;
Strong et al. 1998).
Nitration of proteins significantly alters their proper-
ties such as hydrophobicity and acidity (Radi 2013, 2004).
Moreover, it significantly changes the structure of proteins
(Butterfield et al. 2011). Thus, recognition of proteins by
cellular receptor can be affected. Prion proteins can play an
important role in transmembrane transport and signaling
(Laurén et al. 2009). It is hypothesized that neurotrophic
functions depend on protein–protein interactions, where the
prion protein is one of the interacting members (Martins
et al. 2010). Hence, the nitration of tyrosine in prion pro-
tein can change its role as the receptor.
Several nitro amino acids belong to family of tailor-
made amino acids (Sorochinsky et al. 2013a; Aceña et al.
2014), which can influence protein conformation and/or
lead to 3D-defined structures of proteins (Soloshonok et al.
1999; Sorochinsky et al. 2013a, b; Aceña et al. 2014).
Based on the previous findings, we can see that alpha-
synuclein and prion proteins are suitable targets for pro-
tein nitration. Nitration can affect both conformationally
ordered and unordered regions. The influence of nitration
on conformational changes of these proteins can have prac-
tical impact on the understanding of neurodegenerative
diseases.
To understand the influence of particular tyrosine nitra-
tion on neurodegenerative protein structure, the synthe-
sis of selectively nitrated peptides and proteins should be
available. For solid-phase peptide synthesis, a few pro-
tections of the nitrotyrosine have been employed so far:
Boc–Nit(Bn)–OH, Fmoc–Nit–OH and Fmoc–Nit(Trt)–OH
(Hanson and Law 1965; Mittoo et al. 2003; Song et al.
2006). These strategies have some limitations for syntheses
of neurodegenerative peptides and another protection of Nit
is required.
We have observed facile and fast cleavage of benzyl pro-
tection from nitrotyrosine in trifluoroacetic acid (Fig. 2).
This cleavage is ca 2,000,000 times faster than that from
Tyr(Bn) (Erickson and Merrifield 1973). Thus, Fmoc–
Nit(Bn)–OH (1) appears to be a good synthon for syntheses
of nitrotyrosine containing peptides by Fmoc/tBu strategy.
We suggest that the facile cleavage of benzyl from nitroty-
rosine containing peptides proceeds by neighboring group
assistance.
Experimental part
General methods
TLC was performed on silica gel-coated aluminium plates.
The compounds were visualized by exposure to UV light
Fig. 1 Tyrosine is quite abun-
dant in certain parts of “neu-
rodegenerative” proteins with
exception of model a, where
only one residue is presented.
Selected model peptides (red) in
the structures of alpha-synuclein
(a, b) and prion protein (c, d)
(protein databank access nos.
1XQ8 and 1QM2, respectively).
The tyrosine residues are
emphasized by all atom models.
a alpha-synuclein (25–53), b
alpha-synuclein (118–140), c
human prion protein (137–159)
and d human prion protein
(200–228) (color figure online)
1 3

Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally…
1089
shortly heated to 60 °C, then it was cooled to rt, diluted
with MeOH (80 mL) and made more alkaline by addi-
tion of 2 M NaOH (2.66 mL, 5.32 mmol). Then benzyl
bromide (2.1 mL, 17.7 mmol) was added and the mixture
was vigorously stirred at rt. After 1.5 h, second portion of
benzyl bromide (2.1 mL, 17.7 mmol) was added and the
mixture was stirred at rt for 1 h. The blue precipitate was
collected and washed with n-hexane (3 × 20 mL) and dried
60 min under vacuum. The complex [Cu(H–Nit(Bn)–O)2]
(3.5 g, 5.04 mmol) was dissolved in water (15 mL) with
Chelaton III (10.6 g, 28.5 mmol) and stirred on ice bath
for 10 min. Subsequently, Fmoc–OSu (3.52 g, 10.4 mmol)
in dioxane (15 mL) was added. The pH was maintained at
9.2 with 10 % aqueous Na2CO3 for 6 h at room tempera-
ture. The suspension was washed with ether (2 × 50 mL).
The aqueous layer was acidified with aqueous KHSO4
to pH 3. The aqueous solution was extracted with ethyl
acetate (3 × 50 mL). The combined organic layers were
collected, washed with brine and dried over anhydrous
Na2SO4. Solvent was removed in vacuo, the product was
obtained as a yellow solid (5.2 g, content of 1 according to
HPLC 220 nm was 90 %) which was recrystallized from
hot EtOAc/n-hexane to give 1 as a yellow crystal. Yield:
4.0 g, 42 %. Rf (CHCl3–CH3OH 9:1) 0.45; Rf (CH3OH–
toluene 3:10) 0.39; Rf (EtOAc–CH3OH 16:1) 0.24. HPLC
Rt (method B, 220 nm, 95 % purity) 12.7 min; UV–Vis:
λmaxNO2 = 338 nm. EA calcd. for C31H26N2O7 * 2.25 H2O:
C, 64.30; H, 5.31; N, 4.84. Found C, 64.87; H, 4.91; N,
4.76. ESI HRMS (m/z): for [M + Na]+ C31H26N2O723Na
calcd. 561.16322; found 561.16325 (0.05201 ppm).
Mp 103–105 °C. Specific rotation [α]2D0 16.9 (c 0.3,
acetone). IR (CHCl3, sat. sol., cm−1): ν(C=O) 1715 s,
Fig. 2 Cleavage of benzyl group from tyrosine or nitrotyrosine in
synthetic peptides
at 254 nm or by ninhydrin spraying. The products were
dried in a vacuum drying box at room temperature for 16 h.
During the syntheses, the molecular weights of the peptide
fragments were determined using a matrix assisted laser
desorption ionization and electrospray ionization mass
spectroscopies (MALDI–TOF and ESI, respectively). For
HPLC, an instrument with a quaternary pump, thermo-
stat, diode array detector and reverse-phase C18 columns
were used. The peptides were purified by semi-preparative
HPLC on the VYDAC 250 × 10 mm, 10 μm Vydac RP-18
column flow rate 3 mL/min using gradient 0–100 % ACN
(acetonitrile) in 0.05 % aqueous TFA. The analytical HPLC
was carried out on the Poroshell 120 SB-C18 2.7 mm,
3.0 × 50 mm column, flow rate 1 mL/min and diode array
detection using method A with gradient 10–10–100 % of
ACN in 0.05 % aqueous TFA within 0–1–10 min or method
B with gradient 5–5–100 % of ACN in 0.05 % aqueous
TFA within 0–1–21 min. Unless specified, the method A
was used.
O‑Benzyl‑N‑(9‑fluorenylmethoxycarbonyl)‑3‑nitro‑l‑
tyrosine 2.25 hydrate—Fmoc–Nit(Bn)–OH *2.25 H2O
(1) We have combined and modified the procedures for
syntheses of H–Tyr(Bn)–OH (Bodanszky and Bodanszky
1994) and H–Tyr[Bn(2,6-Cl2)]–OH (Erickson and Mer-
rifield 1973; Yamashiro and Li 1973). To commercially
available H–Nit–OH (4.0 g, 17.7 mmol) in 2 M NaOH
(17.7 mL, 35.4 mmol), a solution of CuSO4 5H20 (2.2 g
in 10 mL H2O, 8.84 mmol) was added. The mixture was
1
νas(NO2) 1533 vs, νs(NO2) 1352 m. H‑NMR (400 MHz,
DMSO-d6) δ 7.87 (d, J = 7.5 Hz, 2H, H4Fmoc), 7.84 (d,
J = 2.2 Hz, 1H, H2Nit), 7.78 (d, J = 8.6 Hz, 1H, NH), 7.62
(d, J = 7.5 Hz, 2H, H1Fmoc), 7.55 (dd, J = 8.7, 2.2 Hz,
1H, H6Nit), 7.46–7.24 (m, 10H, H2Fmoc + H2–4Bn + H3Fm
+ H5Nit), 5.24 (s, 2H, CH2,Bn), 4.18 (m, 4.2 Hz, 4H,
oc
CHαNit + CH2Fm + H9Fmoc), 3.12 (dd, J = 13.9, 4.4 Hz,
1H, CHβNit), 2.88 (dd, J = 13.9, 10.7 Hz, 1H, CHβNit). 13C‑
NMR (100 MHz, DMSO-d6) δ 173.44 (COOHNit), 156.44
(NHCOOFmoc), 150.12 (C4Nit), 144.18 (C9aFmoc), 141.15
(C4aFmoc), 139.61 (C3Nit), 136.5 (C1Bn), 135.60 (C6Nitt),
131.22 (C1Nit), 128.97 (C3Bn), 128.51 (C4Bn), 128.09
(C2Fmoc), 127.84 (C2Bn), 127.50 (C3Fmoc), 126.00 (C2Nit),
125.65 (C1Fmoc), 120.56 (C4Fmoc), 115.78 (C5Nit), 70.87
(CH2,Bn), 66.14 (CH2Fmoc), 55.59 (CHαNit), 47.01 (H9Fmoc),
35.43 (CHβNit).
N‑(9‑Fluorenylmethoxycarbonyl)‑3‑nitro‑l‑tyrosine–
Fmoc–Nit–OH (2) was prepared according to combination
of known procedures (Song et al. 2006; Fields and Noble
1990; Carpino and Han 1972). To a stirred solution of
1.0 g (44.2 mmol) of H–Nit–OH and 15 mL of water was
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P. Niederhafner et al.
added Fmoc–OSu (1.64 g, 48.6 mmol) in 15 mL of acetone
at 0 °C. The pH was maintained at 9.2 with 10 % aque-
ous Na2CO3 for 6 h at room temperature. The suspension
was washed with ether (2 × 50 mL). The aqueous layers
were acidified with aqueous KHSO4 to pH 3. The aque-
ous solution was extracted with ethyl acetate (3 × 50 mL).
The combined organic layers were collected and dried over
anhydrous Na2SO4. Solvent was removed in a vacuum, and
the product was obtained as a yellow solid. Yield: 1.8 g,
90 %. Rf (CHCl3–CH3OH, 9:1) 0.53; Rf (EtOAc–CH3OH,
16:1) 0.43. HPLC Rt (gradient B, 220 nm, 95 %) 12.1 min.;
UV–Vis: λmaxNO2 358 nm. EA calcd. for C24H20N2O7: C,
64.28; H, 4.50; N, 6.25. Found C, 64.57; H, 4.56; N, 5.86.
ESI HRMS (m/z): for [M + Na]+ C24H20N2O7Na calcd.
471.11627; found 471.11627 (0.09827 ppm). Mp 143–
144 °C. Specific rotation [α]2D0 −3.7 (c 0.3, acetone). IR
(CHCl3, sat. sol., cm−1): ν(C=O) 1721 vs, νas(NO2) 1541 s,
its life time was very short. E.g. 0.05 % TFA caused com-
plete conversion of Nit(Trt) compounds to corresponding
Nit within 20 min, thus re-running of purified peak pro-
vided mixture of Fmoc–Nit–OH and Trt–OH. To analyze
each compound from the reaction mixture, we used HPLC
separation with collecting of peaks in 0.01 M NaOH and
subsequent MS. However, during prolonged storage, com-
pounds spontaneously decompose to Fmoc–Nit–OH and
Trt–OH. The UV–Vis λmaxNO2 can serve as a proof of
phenolic group protection (see section UV–Vis spectra of
Nit derivatives in Supplementary Information). Fmoc–
Nit(Trt)–OH (3): HPLC Rt (gradient B) 16.9 min; UV–
Vis: λmaxNO2 328 nm. ESI HRMS (m/z): for [M + H]+
C43H34N2O7Na calcd. 713.22582, found 713.22611
(0.40786 ppm). Fmoc–Nit–O–Trt (4): HPLC Rt (gradi-
ent B) 17.9 min; UV–Vis: λmaxNO2 358 nm. ESI HRMS
(m/z): for [M + H]+ C43H34N2O7Na calcd. 713.22582,
found 713.22599 (0.23193 ppm). Fmoc–Nit(Trt)–O–Trt
(5): HPLC Rt (gradient B) 21.3 min; UV–Vis: λmaxNO2
328 nm. ESI HRMS (m/z): for [M + H]+ C62H48N2O7Na
calcd. 955.33537, found 955.33583 (0.48096 ppm).
Hydrochloride of methyl 3‑nitro‑l‑tyrosinate—
HCl.H–Nit–OMe (6) was prepared according to published
method (Bodanszky and Bodanszky 1994). To a stirred sus-
pension of H–Nit–OH (1.0 g, 4.42 mmol) and 10 mL of dry
methanol was added thionyl chloride (1 mL, 13.3 mmol)
dropwise at −10 °C. Subsequently, 20 mL of methanol
was added. The mixture was then refluxed 6 h and stirred
overnight at room temperature. The solution was concen-
trated in a vacuum. The product 6 (1.18 g, 4.27 mmol)
was obtained as a yellow–brown oil. Yield: 1.18 g,
96 %. Rf (CHCl3–CH3OH, 9:1) 0.55. HPLC Rt (gradi-
ent B, 220 nm, 95 %) 4.24 min. ESI HRMS (m/z): for
[M + H]+ C10H12N2O5 calcd. 241.08190; found 241.08194
(0.17764 ppm). Mp 183–184 °C, (195–197 °C)0.20 1H‑
NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 2.3 Hz, 1H,
H2Nit), 7.11 (dd, J = 8.6, 2.4 Hz, 1H, H6Nit), 6.77 (d,
J = 8.6 Hz, 1H, H5Nit), 3.58 (s, 3H, OCH3), 3.49 (m, 1H,
CHαNit), 2.73 (dd, J = 13.6, 6.0 Hz, 1H, CHβNit), 2.65
(dd, J = 13.5, 7.0 Hz, 1H, CHβNit). 13C‑NMR (100 MHz,
DMSO) δ 175.39 (COOHNit), 158.64 (C4Nit), 135.89
(C6Nit + C3Nit), 125.67 (C2Nit), 123.37 (C5Nit), 122.59
(C1Nit), 55.64 (CαNit), 51.39 (OCH3), (CβNit, hidden by sol-
vent, identified by HSQC).
1
νs(NO2) 1324 s. H‑NMR (400 MHz, DMSO-d6) δ 10.81
(s, 1H, OH), 7.87 (d, J = 7.5 Hz, 2H, H4Fmoc), 7.84 (d,
J = 2.2 Hz, 1H, H2Nit), 7.76 (d, J = 8.6 Hz, 1H, NHNit),
7.61 (dd, J = 7.5, 3.8 Hz, 2H, H1Fmoc), 7.46 (dd, J = 8.6,
2.2 Hz, 1H, H6Nit), 7.40 (t, J = 7.5 Hz, 2H, H3Fmoc), 7.28
(dt, J = 8.7, 7.5, 1.1 Hz, 2H, H2Fmoc), 7.04 (d, J = 8.6 Hz,
1H, H5Nit), 4.21–4.13 (m, 4H, CHαNit + CH2,Fmoc + CHF-
moc), 3.07 (dd, J = 13.9, 4.4 Hz, 1H, CHβNit), 2.84 (dd,
J = 13.9, 10.7 Hz, 1H, CHβNit). 13C‑NMR (100 MHz,
DMSO) δ 173.02 (COOHNit), 155.96 (NHCOOFmoc),
150.85 (C4Nit), 143.67 (C9aFmoc), 140.68 (C4aFmoc), 136.22
(C6Nit + C3Nit), 129.17 (C1Nit), 127.63 (C3Fmoc), 127.03
(C2Fmoc), 125.47 (C2Nit), 125.21 (C1Fmoc), 120.12 (C4Fmoc),
118.98 (C5Nit), 65.65 (CH2,Fmoc), 55.23 (CαNit), 46.55
(CHFmoc), 35.01 (CβNit).
Attempt
to
prepare
O‑trityl‑N‑(9‑
fluorenylmethoxycarbonyl)‑3‑nitro‑l‑tyrosine—Fmoc–
Nit(Trt)–OH (3), Fmoc–Nit–O–Trt (4) and Fmoc–
Nit(Trt)–O–Trt (5) Compound Fmoc–Nit–OH (2, 1 g,
2.5 mmol) and trityl chloride (700 mg, 2.5 mmol) were
suspended in 50 mL of anhydrous acetonitrile, and redis-
tilled triethylamine (0.7 mL, 5 mmol) was added dropwise
(Song et al. 2006). The mixture was stirred at room tem-
perature for 5 h, then the solvent was evaporated and the
crude product was stirred with 50 mL water and ice. The
yellow precipitate was filtered and dried in a vacuum for
3 h (Figure SI 1). In our hands, the attempt to prepare com-
pound 3 was not successful (Song et al. 2006), we obtained
mixture of Fmoc–Nit–OH, Fmoc–Nit(Trt)–OH, Fmoc–
Nit–OTrt, Fmoc–Nit(Trt)–OTrt, and Trt–OH as confirmed
by HPLC (Figure SI 1), UV–Vis and MS. Any attempts
to separate this mixture lead to removal of Trt protection
from the phenolic group. Even silica pretreated with vari-
ous bases, e.g. pyridine or triethylamine leads to decompo-
sition of reaction mixture to starting material and Trt–OH.
We can trap the Fmoc–Nit(Trt)–OH by HPLC; however,
Methyl
N‑(9‑fluorenylmethoxycarbonyl)‑3‑nitro‑
l‑tyrosinate—Fmoc–Nit–OMe (7) synthesis was adapted
according to published procedure (Sever and Wilker 2001).
Fmoc–OSu in 40 mL of dioxane (9.35 g, 27.7 mmol)
was added dropwise to emulsion of H–Nit–OMe (5 g,
20.8 mmol) in a mixture of 10 % Na2CO3 (42.5 mL) and
dioxan (20 mL) at 0 °C. After 20 h of stirring with grad-
ual warming to rt, the solution was poured into ice/water
(500 mL) and extracted with EtOAc (3 × 70 mL). The
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Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally…
1091
extract was dried (Na2SO4), and evaporated to an oil,
which crystallized from EtOAc/hexane or from metha-
nol as yellow crystals. Yield: 6.52 g, 68 %. Rf (EtOAc–
hexan, 1:3) 0.20; Rf (EtOAc–hexan, 1:1) 0.61; Rf
(CH3OH–toluene, 2:5) 0.77; Rf (CH3OH–toluene, 2:4)
0.75; Rf (CHCl3–CH3OH, 9:1) 0.92. HPLC Rt (gradient
B, 220 nm, 95 %) 12.32 min.; UV–Vis: λmaxNO2 358 nm.
EA calcd. for C25H22N2O7: C, 64.96; H, 4.80; N, 6.06.
Found C, 64.95; H, 4.54; N, 5.95. ESI HRMS (m/z): for
[M + H]+ C25H23N2O7 calcd. 463.14975; found 463.14998
(−0.49160 ppm); for [M + H]+ C25H22N2O7 23Na calcd.
485.13180, found 485.13192 (−0.25306 ppm). Mp 133–
134 °C. Specific rotation [α]2D0 −14.6 (c 0.3, acetone).
IR (KBr, cm−1): ν(C=O) 1724 s, 1691 vs, νas(NO2) 1536
DMSO-d6) δ 7.77 (d, J = 2.0 Hz, 1H, NHNit), 7.63 (d,
J = 8.2 Hz, 1H, H2Nit), 7.43 (dd, J = 8.5, 2.2 Hz, 1H,
H6Nit), 7.04 (d, J = 8.5 Hz, 1H, H5Nit), 4.27–4.17 (m, 1H,
CHαNit), 3.97–3.87 (m, 2H, CH2,Et), 3.62 (s, 3H, OCH3),
3.01 (dd, J = 13.9, 4.8 Hz, 1H, CHβNit), 2.81 (dd, J = 13.8,
10.4 Hz, 1H, CHβNit), 1.09 (t, J = 7.1 Hz, 3H, CH3, Et). 13C‑
NMR (101 MHz, DMSO-d6) δ 172.43 (COONit), 156.38
(NHCOOEtoc), 151.15 (C4Nit), 136.49 (C3Nit + C6Nit),
128.92 (C1Nit), 125.71 (C2Nit), 119.28 (C5Nit), 60.29
(CH2,Et), 55.41 (CHαNit), 52.24 (OCH3, Nit), 35.29 (CHβNit),
14.75 (CH3, Et).
Attempts for tert.‑butylation of Fmoc–Nit–OMe (7)
and Etoc–Nit–OMe (8)
1
vs, νs(NO2) 1335 vs. H‑NMR (400 MHz, DMSO-d6) δ
10.83 (s, 1H, OH), 7.90 (d, J = 8.4 Hz, 1H, NHNit), 7.87 (d,
J = 7.6 Hz, 2H, H4Fmoc), 7.83 (d, J = 2.2 Hz, 1H, H2Nit),
7.61 (dd, J = 7.6, 2.4 Hz, 2H, H1Fmoc), 7.47–7.36 (m, 3H,
H6Nit + H3Fmoc), 7.29 (q, J = 7.9 Hz, 2H, H2Fmoc), 7.05 (d,
J = 8.5 Hz, 1H, H5Nit), 4.30–4.20 (m, 3H, CH2,Fmoc + CHF-
moc), 4.17 (m, 1H, CHαNit), 3.64 (s, 3H, OCH3), 3.05 (dd,
J = 13.9, 4.9 Hz, 1H, CHβNit), 2.86 (dd, J = 13.9, 10.4 Hz,
1H, CHβNit). 13C‑NMR (100 MHz, DMSO-d6) δ 172.31
(COONit), 156.18 (NHCOOFmoc), 151.18 (C4Nit), 143.89
(C9aFmoc), 140.94 (C4aFmoc), 136.45 (C3Nit + C6Nit),
128.94 (C1Nit), 127.91 (C3Fmoc), 127.28 (C2Fmoc), 125.77
(C2Nit), 125.38 (C1Fmoc), 120.37 (C4Fmoc), 119.29 (C5Nit),
65.93 (CH2,Fmoc), 55.43 (CαNit), 52.31 (OCH3), 46.82
(CHFmoc), 35.20 (CβNit).
Fmoc–Nit(tBu)–OMe, method A
Isobutene (70 mL, 733.5 mmol) was condensed to the
thick-walled, well-stoppered Champagne bottle with mix-
ture of Fmoc–Nit–OMe (7, 1.0 g, 2.16 mmol), DCM
(30 mL) and H2SO4 (60 μL, 96 %, 1.08 mmol). The mix-
ture was shaken for 4 days at rt (Beyerman and Bonte-
koe 1962). The bottle was carefully and slowly cooled in
CO2 (s)/ethanol bath, the cooled bottle was opened, subse-
quently NaHCO3 (0.188 g, 2.16 mmol) was added and the
reaction mixture was gradually warmed to rt. Following
careful degassing, the rest of isobutene gas and DCM was
evaporated, and the residue was dissolved in 20 mL MeOH.
The precipitate was filtered off and dried in a vacuum for
3 h. The filtrate was concentrated and dried in a vacuum.
Only the starting material was recovered as a yellow solid
compound (0.8 g) as confirmed by HPLC, TLC and NMR.
Yield 0 %.
Methyl
N‑(9‑ethoxycarbonyl)‑3‑nitro‑l‑tyrosi‑
nate—Etoc–Nit–OMe (8) synthesis was based on pub-
lished method (Song et al. 2006). A stirred solution of
0.8 g (2.9 mmol) of H–Nit–OMe.HCl and 10 mL of water
was neutralized by NaOH to pH 7. Subsequently, 0.45 g
of NaHCO3 (5.35 mmol) was added. The reaction mix-
ture was cooled and stirred in an ice bath and solution of
ethyl chloroformate (0.44 mL, 4.62 mmol) in chloroform
(10 mL) was slowly added. The mixture was kept at rt for
4 h. The organic layer was washed with Na2SO4, and dried
over anhydrous Na2SO4 and solvent was removed in a
vacuum. The product was obtained as a yellow solid. Yield
(0.51 g, 56.2 %). Rf (CHCl3–CH3OH, 9:1) 0.82. HPLC Rt
(gradient B, 220 nm—95 %) 8.3 min; UV–Vis: λmaxNO2
358 nm. EA calcd. for C13H16N2O7: C, 50.00; H, 5.16;
N, 8.97. Found C, 50.13; H, 5.13; N, 8.77. ESI HRMS
(m/z): for [M + H]+ C13H17N2O7 calcd. 313.10307, found
313.10303 (0.12106 ppm), [M + H]+ C13H16N2O7Na
calcd. 335.08507, found 335.08497 (0.28521 ppm). Mp
75–77 °C. Specific rotation [α]2D0 +3.6 (c 0.3, CH3OH)
lit. (Song et al. 2006) [α]2D0 +10.1 (c 0.6, CHCl3). IR
(KBr, cm−1): ν(C=O) 1745 vs (ester), 1721 vs, 1621 vs,
Fmoc–Nit(tBu)–OMe, method B
Attempt to prepare Fmoc–Nit(tBu)–OMe was inspired by
the method described (Pícha et al. 2013; Mathias 1979).
Diisopropylcarbodiimide (3.15 mL, 20 mmol) and CuCl
(20 mg, 0.21 mmol) were added to tert.-butyl alcohol
(1.9 mL, 20 mmol), and the mixture was stirred for 12 h at
room temperature to allow the formation of O-tert-butyl-
N,N-diisopropylurea. A solution of Fmoc–Nit–OMe (7,
85 mg, 0.18 mmol) in DCM (5 mL) was slowly added to the
isourea upon cooling to 0 °C, and the reaction mixture was
refluxed overnight. After the reaction mixture cooled, the
precipitated urea was filtered off, the filtrate was evaporated
in a vacuum, and then the filtrate was redissolved in toluene
(20 mL). The second crop of urea was filtered off, and the
solvent was evaporated in a vacuum again. The crude mate-
rial (brown–yellow oil) contained only starting compound
as was confirmed by HPLC, TLC and NMR. Yield 0 %.
1
νas(NO2) 1539 vs, νs(NO2) 1333 vs. H NMR (401 MHz,
1 3

1092
P. Niederhafner et al.
Etoc–Nit(tBu)–OMe
Calculations
Isobutene (34 mL, 356.3 mmol) was condensed to the
thick-walled, well-stoppered Champagne bottle with mix-
ture of Etoc–Nit–OMe (8, 3.4 g, 11 mmol), DCM (26 mL)
and H2SO4 (0.5 mL, 96 %, 0.09 mmol). The mixture was
shaken for 4 days at rt (Beyerman and Bontekoe 1962).
The bottle was carefully and slowly cooled in CO2(s)/
ethanol bath, the cooled bottle was opened, and the reac-
tion mixture was gradually warmed to rt. Following careful
degassing, the rest of isobutene gas and DCM was evapo-
rated, and the residue was dissolved in 50 mL EtOAc. The
solution was washed with 1 M NaHCO3 (3 × 50 mL). The
organic layer was washed with sat. Na2SO4 (3 × 30 mL),
water (3 × 30 mL) and dried over Na2SO4. The solvent was
removed in a vacuum. To the resultant yellow oil, hexane
was added and the precipitate was filtered and dried in a
vacuum for 3 h. The starting material was recovered as a
yellow solid (2 g) as confirmed by HPLC, TLC and NMR.
Yield 0 %.
In the calculations of heat of formation of cations (Fig. 4,
compounds 9a–c and their cations 10a–c), tyrosine was
simplified to the p-cresol (11) and 3-nitrotyrosine as
2-nitro-p-cresol (12). Geometries were optimized and ener-
gies were calculated using Gaussian09 program (Frisch
et al. 2009). The energies were obtained using B3LYP
functional (Becke 1993; Vosko et al. 1980; Lee et al. 1988;
Miehlich et al. 1989), 6-31 + G** basis set, and CPCM
(Barone and Cossi 1998; Cossi et al. 2003; Tomasi et al.
2005) solvent model (which is the Gaussian implementa-
tion of the COSMO model (Klamt and Schürmann 1993))
with parameters of acetic acid as a solvent.
Peptide synthesis
The peptides were synthesized by the Fmoc/tBu method
(Fields and Noble 1990) by automatic solid-phase synthe-
sizer ABI 433A (Applied Biosystems) using the FastMoc
0.1 mmol program (SynthAssist™ version 3.1) with a sin-
gle coupling; 10 eq of an excess of protected amino acids
and HBTU coupling reagent and 20 eq of an excess of
DIPEA were used. Nitrotyrosine containing peptides were
prepared using either Fmoc–Nit–OH or Fmoc–Nit(Bn)–OH
for compounds 14a, 16a–22a and 14b, 16b–22b, respec-
tively. The peptides were cleaved from the resin by mix-
ture of TFA (4.5 mL), H2O (150 μL), EDT (150 μL), thio-
anisole (150 μL) and TIS (50 μL) for 4 h. If benzylated
products were detected, the cleavage was repeated with the
same mixture for another 4 h (20b). All the peptides were
prepared in more than 95 % purity. Retention times are
summarized in Table 1.
Alpha‑synuclein(25–53)—H–Gly–Val–Ala–Glu–
Ala–Ala–Gly–Lys–Thr–Lys–Glu–Gly–Val–Leu–Tyr–
Val–Gly–Ser–Lys–Thr–Lys–Glu–Gly–Val–Val–His–
Gly–Val–Ala–OH (13)—For C125H209N35O40 (2840.54)
found MALDI-MS, m/z: 2841.6 ([M + H]+); 2864.6
([M + Na + H]+); 2881.6 ([M + K + H]+). Amino acid
analysis: Ala 4.11 (4), Glu 3.50 (3), Gly 6.00 (6), Tyr 1.20
(1), Thr 1.88 (2), Ser 0.94 (1), His 1.73 (1), Lys 4.26 (4),
Leu 1.12 (1), Val 5.27 (6).
Reactivity study
The reaction kinetics of benzyl cleavage from Fmoc–
Nit(Bn)–OH (1) were investigated according to adapted
procedure for acridine reactions (Zawada et al. 2011).
Briefly, Fmoc–Nit(Bn)–OH (ca. 1 mg) was dissolved
in acetonitrile (200 μL). The solution was stirred for
10 min to ensure temperature equilibration. In another
working vial, trifluoroacetic acid (180 μL) and anisole
(20 μL) were stirred for 10 min to ensure temperature
equilibration. After addition of 20 μL of Fmoc–Nit(Bn)–
OH (1) solution to TFA system, the mixture was vigor-
ously stirred for 20 s and then 20 μL of reaction mix-
ture was injected into the HPLC apparatus in desired
time. The reaction mixture was analyzed by HPLC at
301 nm with the following solvent gradient: 20–20–70–
100–100 % ACN within 0–1–7–10–12 min. The purity
of the Hamilton syringe was crucial; after each injec-
tion it was rinsed ten times with ACN (full volume of the
syringe), the syringe was dried with dry air for around
20 s, and the piston was wiped dry. The temperature of
the syringe and piston was allowed to partially equili-
brate with that of the reaction mixture. Before injec-
tion, the Hamilton syringe was rinsed three times with
the reaction mixture. The time (τ) dependence of the
amount of reactant (n) was fitted by a first-order kinetic
model, dn/dτ = A × exp(−kτ). By using the Arrhenius
relation [k = P × exp(−E*/RT)], the activation energy
E* was calculated from the rate constants (k) obtained at
five different temperatures (T) within the interval 273–
298 K; R is the universal gas constant and P is a constant
independent of temperature.
[Nit39]Alpha‑synuclein(25–53)—H–Gly–Val–Ala–
Glu–Ala–Ala–Gly–Lys–Thr–Lys–Glu–Gly–Val–Leu–
Nit–Val–Gly–Ser–Lys–Thr–Lys–Glu–Gly–Val–Val–His–
Gly–Val–Ala–OH (14a)—For C125H208N36O42 (2885.52)
found MALDI-MS, m/z: 2887.6 ([M + 2H]+); 2909.6
([M + Na + H]+); 2925.6 ([M + K]+). Amino acid analy-
sis: Ala 4.02 (4), Glu 3.50 (3), Gly 6.00 (6), Thr 1.94 (2),
Ser 0.91 (1), His 1.10 (1), Lys 4.03 (4), Leu 1.12 (1), Val
5.21 (6), Nit 0.84 (1).
[Nit39]Alpha‑synuclein(25–53)—H–Gly–Val–Ala–
Glu–Ala–Ala–Gly–Lys–Thr–Lys–Glu–Gly–Val–Leu–
1 3

Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally…
1093
Table 1 Yields and retention
times of synthesized peptides
Peptide
Yield (%)
HPLC RT (min)
Alpha-synuclein (25–53) (13)
[Nit39]Alpha-synuclein (25–53) (14a, 14b)
4a
3b, 3c
2a
4b, 2c
1b, 3c
3b, 6c
5b, 4c
4b, 5c,d
4b, 6c
0b,e, 7c
3.2
4.5
3.3
4.0
3.3
3.5
4.2
4.2
4.2
4.3
Alpha-synuclein (118–140) (15)
[Nit125]Alpha-synuclein (118–140) (16a, 16b)
[Nit133]Alpha-synuclein (118–140) (17a, 17b)
[Nit136]Alpha-synuclein (118–140) (18a, 18b)
[Nit125, Nit133]Alpha-synuclein (118–140) (19a, 19b)
[Nit125, Nit136]Alpha-synuclein (118–140) (20a, 20b)
[Nit133, Nit136]Alpha-synuclein (118–140) (21a, 21b)
[Nit125, Nit133, Nit136]Alpha-synuclein (118–140) (22a, 22b)
a
Synthesis with Fmoc–Tyr(tBu)–OH
b
Synthesis with Fmoc–Nit–OH
c
Synthesis with Fmoc–Nit(Bn)–OH
d
After 4 h cleavage with TFA, only mono- and bis-benzylated products were isolated with overall yields 2
and 3 %, respectively. In another batch, the desired peptide was obtained using 2 × 4 h cleavage
e
The deletion peptide lacking one Nit residue was isolated with overall yield 3 %
Nit–Val–Gly–Ser–Lys–Thr–Lys–Glu–Gly–Val–Val–His–
Gly–Nit–Gln–Asp–Tyr–Glu–Pro–Glu–Ala–OH (17a)—For
C114H156N26O50S (2721.02) found MALDI-MS, m/z:
2744.9 ([M + H + Na]+); 2760.9 ([M + K]+). Amino acid
analysis: Ala 1.90 (2), Asp + Asn 4.07 (4), Glu + Gln 7.29
(7), Gly 0.89 (1), Pro 3.07 (3), Tyr 2.00 (2), Ser 0.77 (1),
Met 0.69 (1), Val 1.00 (1), Nit 0.80 (1).
Gly–Val–Ala–OH (14b)—For C125H208N36O42 (2885.52)
found MALDI-MS, m/z: 2887.4 ([M + 2H]+); 2909.4
([M + Na + H]+); 2925.4 ([M + K]+). Amino acid analy-
sis: Ala 4.02 (4), Glu 3.50 (3), Gly 6.00 (6), Thr 1.94 (2),
Ser 0.90 (1), His 1.10 (1), Lys 4.03 (4), Leu 1.12 (1), Val
5.21 (6), Nit 0.84 (1).
[Nit133]Alpha‑synuclein(118–140)—H–Val–Asp–Pro–
Asp–Asn–Glu–Ala–Tyr–Glu–Met–Pro–Ser–Glu–Glu–
Alpha‑synuclein(118–140)—H–Val–Asp–Pro–Asp–
Asn–Glu–Ala–Tyr–Glu–Met–Pro–Ser–Glu–Glu–Gly–
Gly–Nit–Gln–Asp–Tyr–Glu–Pro–Glu–Ala–OH
(17b)—
Tyr–Gln–Asp–Tyr–Glu–Pro–Glu–Ala–OH
(15)—For
For C114H156N26O50S (2721.02) found MALDI-MS, m/z:
2745.1 ([M + H + Na]+); 2761.1 ([M + K]+). Amino acid
analysis: Ala 1.88 (2), Asp + Asn 3.68 (4), Glu + Gln 7.24
(7), Gly 1.00 (1), Pro 2.85 (3), Tyr 2.01 (2), Ser 0.78 (1),
Met 0.78 (1), Val 0.82 (1), Nit 0.94 (1).
C114H157N25O48S (2676.03) found MALDI-MS, m/z:
2699.0 ([M + Na]+); 2716.0 ([M + K]+). Amino acid anal-
ysis: Ala 2.00 (2), Asp + Asn 3.62 (4), Glu + Gln 7.34 (7),
Gly 0.98 (1), Pro 3.22 (3), Tyr 3.14 (3), Ser 0.78 (1), Met
0.82 (1), Val 0.87 (1).
[Nit136]Alpha‑synuclein(118–140)—H–Val–Asp–Pro–
Asp–Asn–Glu–Ala–Tyr–Glu–Met–Pro–Ser–Glu–Glu–
[Nit125]Alpha‑synuclein(118–140)—H–Val–Asp–Pro–
Asp–Asn–Glu–Ala–Nit–Glu–Met–Pro–Ser–Glu–Glu–
Gly–Tyr–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH
(18a)—
Gly–Tyr–Gln–Asp–Tyr–Glu–Pro–Glu–Ala–OH
(16a)—
For C114H156N26O50S (2721.02) found MALDI-MS, m/z:
2745.0 ([M + H + Na]+); 2761.0 ([M + K]+). Amino acid
analysis: Ala 1.85 (2), Asp 4.05 (4), Glu 7.22 (7), Gly 0.87
(1), Pro 3.00 (3), Tyr 2.04 (2), Ser 0.80 (1), Met 0.75 (1),
Val 1.06 (1), Nit 0.70 (1).
For C114H156N26O50S (2721.02) found MALDI-MS, m/z:
2745.1 ([M + H + Na]+), 2761.1 ([M + K]+). Amino acid
analysis: Ala 1.94 (2), Asp + Asn 3.89 (4), Glu + Gln 7.44
(7), Gly 0.96 (1), Pro 3.00 (3), Tyr 1.95 (2), Ser 0.82 (1),
Met 0.81 (1), Val 1.00 (1), Nit 1.04 (1).
[Nit136]Alpha‑synuclein(118–140)—H–Val–Asp–Pro–
Asp–Asn–Glu–Ala–Tyr–Glu–Met–Pro–Ser–Glu–Glu–
[Nit125]Alpha‑synuclein(118–140)—H–Val–Asp–Pro–
Asp–Asn–Glu–Ala–Nit–Glu–Met–Pro–Ser–Glu–Glu–
Gly–Tyr–Gln–Asp–Tyr–Glu–Pro–Glu–Ala–OH (16b)—
For C114H156N26O50S (2721.02) found MALDI-MS, m/z:
2744.9 ([M + H + Na]+), 2760.9 ([M + K]+). Amino acid
analysis: Ala 1.88 (2), Asp + Asn 3.74 (4), Glu + Gln 7.32
(7), Gly 1.00 (1), Pro 2.67 (3), Tyr 2.04 (2), Ser 0.80 (1),
Met 0.76 (1), Val 0.90 (1), Nit 0.91 (1).
Gly–Tyr–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH
(18b)—
For C114H156N26O50S (2721.02) found MALDI-MS, m/z:
2745.0 ([M + H + Na]+), 2760.9 ([M + K]+). Amino acid
analysis: Ala 2.03 (2), Asp + Asn 3.78 (4), Glu + Gln 7.26
(7), Gly 1.07 (1), Pro 3.11 (3), Tyr 2.18 (2), Ser 0.86 (1),
Met 0.85 (1), Val 0.88 (1), Nit 1.00 (1).
[Nit125
,
Nit133]Alpha‑synuclein(118–140)—H–Val–
[Nit133]Alpha‑synuclein(118–140)—H–Val–Asp–Pro–
Asp–Asn–Glu–Ala–Tyr–Glu–Met–Pro–Ser–Glu–Glu–
Asp–Pro–Asp–Asn–Glu–Ala–Nit–Glu–Met–Pro–Ser–
Glu–Glu–Gly–Nit–Gln–Asp–Tyr–Glu–Pro–Glu–Ala–OH
1 3

1094
P. Niederhafner et al.
(19a)—For C114H155N27O52S (2766.00) found MALDI-
MS, m/z: 2790.1 ([M + H + Na]+); 2806.1 ([M + K]+).
Amino acid analysis: Ala 2.00 (2), Asp + Asn 3.77 (4),
Glu + Gln 7.67 (7), Gly 0.96 (1), Pro 2.87 (3), Tyr 1.02 (1),
Ser 0.79 (1), Met 0.61 (1), Val 0.96 (1), Nit 1.61 (2).
[Nit125, Nit133]Alpha‑synuclein(118–140)—H–Val–Asp–
Pro–Asp–Asn–Glu–Ala–Nit–Glu–Met–Pro–Ser–Glu–Glu–
Gly–Nit–Gln–Asp–Tyr–Glu–Pro–Glu–Ala–OH (19b)—For
C114H155N27O52S (2766.00) found MALDI-MS, m/z: 2790.1
([M + H + Na]+); 2806.1 ([M + K]+). Amino acid analysis:
Ala 1.91 (2), Asp 3.79 (4), Glu 7.54 (7), Gly 0.94 (1), Pro
3.01 (3), Tyr 1.00 (1), Ser 0.79 (1), Met 0.84 (1), Val 0.85 (1),
Nit 1.62 (2).
([M + H + Na]+); 2806.1 ([M + K]+). Amino acid analysis:
Ala 1.90 (2), Asp + Asn 3.72 (4), Glu + Gln 7.38 (7), Gly
0.91 (1), Pro 2.86 (3), Tyr 1.00 (1), Ser 0.76 (1), Met 0.76 (1),
Val 0.90 (1), Nit 1.54 (2).
[Nit125, Nit136]Alpha‑synuclein(118–140)—H–Val–Asp–
Pro–Asp–Asn–Glu–Ala–Nit–Glu–Met–Pro–Ser–Glu–Glu–
Gly–Tyr–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH (20b)—For
C114H155N27O52S (2766.00) found MALDI-MS, m/z: 2790.1
([M + H + Na]+); 2806.1 ([M + K]+). Amino acid analysis:
Ala 2.00 (2), Asp 4.02 (4), Glu 7.86 (7), Gly 1.01 (1), Pro
3.62 (3), Tyr 1.13 (1), Ser 0.89 (1), Met 0.72 (1), Val 0.94 (1),
Nit 1.61 (2).
[Nit133
,
Nit136]Alpha‑synuclein(118–140)—H–Val–
[Nit125, Nit136]Alpha‑synuclein(118–140)—H–Val–Asp–
Pro–Asp–Asn–Glu–Ala–Nit–Glu–Met–Pro–Ser–Glu–Glu–
Gly–Tyr–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH (20a)—For
C114H155N27O52S (2766.00) found MALDI-MS, m/z: 2790.1
Asp–Pro–Asp–Asn–Glu–Ala–Tyr–Glu–Met–Pro–Ser–
Glu–Glu–Gly–Nit–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH
(21a)—For C114H155N27O52S (2766.00) found MALDI-
MS, m/z: 2790.1 ([M + H + Na]+); 2806.1 ([M + K]+).
Amino acid analysis: Ala 2.00 (2), Asp + Asn 3.84 (4),
Glu + Gln 7.72 (7), Gly 0.97 (1), Pro 3.06 (3), Tyr 1.00 (1),
Ser 0.83 (1), Met 0.82 (1), Val 0.83 (1), Nit 1.72 (2).
[Nit133
,
Nit136]Alpha‑synuclein(118–140)—H–Val–
Asp–Pro–Asp–Asn–Glu–Ala–Tyr–Glu–Met–Pro–Ser–
Glu–Glu–Gly–Nit–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH
(21b)—For C114H155N27O52S (2766.00) found MALDI-
MS, m/z: 2790.1 ([M + H + Na]+); 2806.0 ([M + K]+).
Amino acid analysis: Ala 1.96 (2), Asp + Asn 3.73 (4),
Glu + Gln 7.23 (7), Gly 1.01 (1), Pro 3.10 (3), Tyr 1.03 (1),
Ser 0.80 (1), Met 0.79 (1), Val 1.00 (1), Nit 1.79 (2).
Trial for synthesis of [Nit125, Nit133, Nit136]alpha‑
synuclein(118–140)—H–Val–Asp–Pro–Asp–Asn–Glu–
Ala–Nit–Glu–Met–Pro–Ser–Glu–Glu–Gly–Nit–Gln–Asp–
Nit–Glu–Pro–Glu–Ala–OH (22a)—The desired peptide
was not obtained. Instead, a peptide without one Nit residue
was obtained as confirmed by MS and amino acid analysis:
H–Val–Asp–Pro–Asp–Asn–Glu–Ala–Glu–Met–Pro–Ser–
Glu–Glu–Gly–Nit–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH
For C105H146N26O50S (2602.94) found MALDI-MS, m/z:
2627.1 ([M + H + Na]+); 2643.1 ([M + K]+). Amino acid
analysis: Ala 1.69 (2), Asp + Asn 3.84 (4), Glu + Gln 7.59
Fig. 3 Kinetic of benzyl removal from Fmoc–Nit(Bn)–OH with
ca 80 % TFA at 25 °C (left). The amount of Fmoc–Nit(Bn)–OH (1)
and Fmoc–Nit–OH (2) is shown in red and black color, respectively.
The data were fitted with first-order kinetic providing k 38.8 s−1
.
The dependence of rate constant on temperature (right) was fit-
ted by Arrhenius equation. The activation energy from the fit is
32 kcal mol−1
Table 2 Reaction rates of
Reactant
k [s−1 b
]
ΔE [kcal mol−1 a
]
protection group cleavage
and energies of model cation
formation
Protonation
TFA
Cleavage
TFA
HCl
HBr
HCl
HBr
Tyr(Bn)
Nit(Bn)
Tyr(tBu)
6.36 × 10−6 (c)
15.3
3.12 (d)
42
33
36
33
25
27
30
22
24
7
8
8
7
8
8
7
8
8
a
Reaction: reactant + acid → protonated cation + anion → product + anion + cleaved cation
b
c
d
In TFA
Erickson and Merrifield (1973) and Tam et al. (1983)
Lundt et al. (1978)
1 3

Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally…
1095
(7), Gly 1.07 (1), Pro 3.00 (3), Ser 0.92 (1), Met 0.91 (1),
Val 0.93 (1), Nit 1.82 (2).
(2), Fmoc–Nit(Trt)–OH (3), Fmoc–Nit–OTrt (4), Fmoc–
Nit(Trt)–OTrt (5) and Trt–OH (see Figure SI 1 in support-
ing information). The tritylation did not lead to completion;
attempts of chromatographic purification using mobile
phase with various bases led to Trt cleavage. It appears that
Nit(Trt) is even more acid labile than Tyr(Trt) (Barlos et al.
1991). Since Fmoc–Nit–OH can be used for syntheses of
peptides on solid phase (Mittoo et al. 2003) and the Fmoc–
Nit(Trt)–OH (3) is very labile compound, we suppose that
the previously prepared peptides (Song et al. 2006) were
obtained using mixture of Fmoc–Nit–OH (2) and Fmoc–
Nit(Trt)–OH (3).
[Nit125, Nit133, Nit136]Alpha‑synuclein(118–140)—H–
Val–Asp–Pro–Asp–Asn–Glu–Ala–Nit–Glu–Met–Pro–Ser–
Glu–Glu–Gly–Nit–Gln–Asp–Nit–Glu–Pro–Glu–Ala–OH
(22b)—For C114H154N28O54S (2810.99) found MALDI-
MS, m/z: 2835.1 ([M + H + Na]+). Amino acid analysis:
Ala 1.91 (2), Asp 3.78 (4), Glu 7.46 (7), Gly 1.00 (1), Pro
3.03 (3), Ser 0.82 (1), Met 0.77 (1), Val 0.82 (1), Nit 2.73
(3).
Results and discussions
We have also tried to protect phenolic group of Fmoc–
Nit–OMe (7) and Etoc–Nit–OMe (8) (Song et al. 2006;
Goeshen et al. 2011) using analogous procedure for prepa-
ration of Fmoc–Tyr(tBu)–OMe with isobutene (Beyerman
and Bontekoe 1962; Adamson et al. 1991); however, we
have just recovered the starting materials.
Since the O-protected Nit is more vulnerable to acids
than analogous O-protected Tyr, we have used more
acid stable benzyl group commonly cleavable by strong
For introduction of 3-nitrotyrosine into synthetic pep-
tides, the use of Boc–Nit(Bn)–OH, Fmoc–Nit–OH (2) and
Fmoc–Nit(Trt)–OH (3) was described in literature (Hanson
and Law 1965; Mittoo et al. 2003; Song et al. 2006). We
have found that the described synthesis (Song et al. 2006)
of Fmoc–Nit(Trt)–OH (3) does not provide pure Fmoc–
Nit(Trt)–OH in our hands but a mixture of Fmoc–Nit–OH
Fig. 4 Formation of cation
from protected peptides. Cal-
culated geometries of parent
peptide models (considered
only red part) and their cations.
a Tyr(Bn), b Nit(Bn), and c
Tyr(tBu) (color figure online)
1 3

1096
P. Niederhafner et al.
acids such as liquid HF, TFMSA or HBr/AcOH. We have
observed rapid cleavage of benzyl protection with mild tri-
fluoroacetic acid. At 25 °C, 50 % of the benzyl group is
removed within 1.1 min with ca 80 % TFA (Fig. 3).
Our observation of rapid cleavage of Nit(Bn) by tri-
fluoroacetic acid confirmed that Nit(Bn) is not compat-
ible with Boc/Bn strategy. This instability of benzyl at
Boc–Nit(Bn)–OH during mild acid treatment was already
observed (Hanson and Law 1965). Generally, the benzyl
can be cleaved with TFA from OBn protected phenols with
activating group (Marsh and Goodman 1965; Fletcher and
Gunning 2008).
formation of internal hydrogen bond between nitro group
and hydroxyl group of Nit prevented the interaction with
solvent, whereas the Tyr hydroxyl can freely interact with
water in mobile phase.
We have determined the activation energy for cleavage
of benzyl from Fmoc–Nit(Bn)–OH (1) to be 32 kcal mol−1
.
The reaction fitted to first-order kinetic equation, which
corresponds to the same order as for the acidolytic cleav-
age of benzyl group from Tyr(Bn) (Erickson and Merrifield
1973). For the acidolytic cleavage of Tyr(Bn), the rate con-
stant k was 6.36 × 10−6 s−1 at 20 °C (Erickson and Mer-
rifield 1973). In the case of Nit(Bn), we have determined
k as 15.3 s−1 at 20 °C. Thus, the cleavage of benzyl from
Nit(Bn) proceeds more than 2,000,000 times faster than
that from Tyr(Bn). Although we compare the cleavage in
50 % (Erickson and Merrifield 1973; Tam et al. 1983) and
80 % TFA, the concentration should not influence the rate
because the pseudo-first order kinetic is achieved in both
cases. Interestingly, since cleavage of tert.-butyl from
Z–Tyr(tBu)–OH proceeded with k 3.12 s−1 (Lundt et al.
1978), the cleavage of benzyl from Nit(Bn) is ca five times
faster. This clearly confirmed that the nitro group made the
O-benzyl or O-tert. butyl protection extremely vulnerable
towards acidolysis.
Fig. 5 Stabilization of transition state by formation of hydrogen bond
Using simple model systems, where the peptide chain
was substituted with hydrogen atoms, energies of cation
formation before the benzyl group deprotection (cleavage)
were calculated using density functional theory (Table 2).
The energy correlates with reaction rate of various tyrosine
deprotection reactions (Figure SI 2). Based on the inspec-
tion of neutral and protonated states (Fig. 4), the nitro
group stabilizes the cation by its protonation (Exner and
Böhm 2005) i.e. the nitro group is protonated instead of
phenolic oxygen in the cation. However, in transition state,
the phenolic oxygen became protonated just before bond
cleavage (Fig. 5, SI movies 1 and 2). Transition state is sta-
bilized by neighboring nitro group via hydrogen bonding.
The experimental evidence for the formation of internal
hydrogen bond between HOH and ONO2 (Fig. 6) can be seen
from HPLC data (Table 1). Normally, the introduction of
nitro group to an alkane led to increase of permittivity and
polarity e.g. n-hexane and nitromethane have permittivities
1.8819 and 36.562, respectively (Frisch et al. 2009). How-
ever, the retention times of Nit peptides at reverse phase
are higher than those of Tyr analogues (Table 1). Thus, the
Fig. 6 Internal hydrogen bond is probably responsible for higher
hydrophobicity of nitrotyrosine containing peptides at low pH
1 3

Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally…
1097
We have compared the syntheses of neurodegenerative
peptides derived from alpha-synuclein using Fmoc–Nit(Bn)–
OH (1) and Fmoc–Nit–OH (2). For syntheses of alpha-
synuclein(25–53) containing one Tyr residue, the yields
of the peptides prepared with both Nit derivatives were the
same (Table 1, 14a and 14b). The situation was different for
derivatives of alpha-synuclein(118–140). At the N-terminus,
the unprotected phenolic group can cause less side reac-
tions. Thus, only peptides 16a and 19a with 125Nit provided
higher yields without benzyl protection. For the combina-
tion of 125Nit and 136Nit, the desired peptide resisted to com-
mon 4 h deprotection procedure. Complete benzyl removal
was achieved after 8 h cleavage (20b). For alpha-synu-
clein(118–140) with all three tyrosines nitrated, the desired
peptide 22b was obtained only with Fmoc–Nit(Bn)–OH. In
the absence of benzyl protecting group, the synthesis pro-
vided only deletion peptides lacking one Nit residue (22a).
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