A convenient preparation of N ε-methyl-l-lysine derivatives and its application to the synthesis of histone tail peptides

Article abstract of DOI:10.1007/s00726-014-1690-6

A convenient route is established for the preparation of N ^α-Fmoc-N ^ε-(Boc, methyl)-l-lysine and N ^α-Fmoc-N ^ε-dimethyl-l-lysine as building blocks to be used for the synthesis of methylated peptides. This methodology is based on the use of malonate derivatives and dibromobutane to produce key intermediates, l-2-Amino-6-bromohexanoic acid derivatives, which could be modified to the required group at the ε-position. Fmoc-protection is accessible, so these compounds can be used in solution as well as in solid-phase peptide synthesis. Also the peptides containing these methylated lysines have been proved to resist the action of trypsin and lysyl endopeptidase. Thus, this new method could be considered as an improvement of the synthesis of N ^ε-methyl-l-lysine derivatives.

Full text of DOI:10.1007/s00726-014-1690-6

Amino Acids
DOI 10.1007/s00726-014-1690-6
ORIGINAL ARTICLE
A convenient preparation of Ne-methyl-L-lysine derivatives
and its application to the synthesis of histone tail peptides
•
Hongfang Chi Md. Shahidul Islam
•
•
Tienabe K. Nsiama Tamaki Kato
•
Norikazu Nishino
Received: 29 October 2013 / Accepted: 31 January 2014
Ó Springer-Verlag Wien 2014
Abstract A convenient route is established for the
preparation of Na-Fmoc-Ne-(Boc, methyl)-L-lysine and Na-
Fmoc-Ne-dimethyl-L-lysine as building blocks to be used
for the synthesis of methylated peptides. This methodology
is based on the use of malonate derivatives and dibromo-
butane to produce key intermediates, L-2-amino-6-bromo-
hexanoic acid derivatives, which could be modified to the
required group at the e-position. Fmoc-protection is
accessible, so these compounds can be used in solution as
well as in solid-phase peptide synthesis. Also the peptides
containing these methylated lysines have been proved to
resist the action of trypsin and lysyl endopeptidase. Thus,
this new method could be considered as an improvement of
the synthesis of Ne-methyl-L-lysine derivatives.
DMAP
DMF
4-Dimethylaminopyridine
N,N-Dimethylformamide
9-Fluorenylmethyloxycarbonyl
Fmoc
Fmoc-OSu N-(9-
Fluorenylmethoxycarbonyloxy)succinimide
HBTU
O-(Benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate
HOBtꢀH2O 1-Hydroxybenzotriazole hydrate
HPLC
HRMS
(FAB)
LCMS
MCA
TFA
High-performance liquid chromatography
High-resolution fast atom bombardment
mass spectrometry
Liquid chromatography mass spectrometry
4-Methylcoumaryl-7-amide
Trifluoroacetic acid
TLC
Thin layer chromatography
Keywords L-2-Amino-6-bromohexanoic acid ꢀ Na-Fmoc-
Ne-(Boc, methyl)-L-lysine ꢀ Na-Fmoc-Ne- dimethyl-L-lysine
Abbreviations
Introduction
AcOH
AMC
Boc
Acetic acid
7-Amino-4-methylcoumarin
tert-Butyloxycarbonyl
Dicyclohexylcarbodiimide
Dichloromethane
The facile and selective synthesis of Ne-methyl-L-lysine
derivatives is important because of their biological and
medicinal application. Generally, lysine methylation,
which can be found in mono-, di-, or trimethylation state
in vivo, is one of the important post-translational modifi-
cations on the N-terminal peptide chain of the histone
proteins (Fig. 1).
DCC
DCM
DIEA
N,N-Diisopropylethylamine
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-014-1690-6) contains supplementary
material, which is available to authorized users.
It has been studied that the transition between mono-, di-,
and trimethylation may control transcriptional regulation,
RNA maturation, DNA repair and genomic imprinting
(Grunstein 1998; Martin and Zhang 2005; Mai and Altucci
2009; Gavin and Sharma 2010). For the elucidation of the
specific biological function of different state of lysine
methylation, the preparation of site-specifically methylated
H. Chi ꢀ Md. S. Islam ꢀ T. K. Nsiama ꢀ T. Kato (&) ꢀ
N. Nishino
Graduate School of Life Science and Systems Engineering,
Kyushu Institute of Technology, Wakamatsu,
Kitakyushu 808-0196, Japan
e-mail: tmkato@life.kyutech.ac.jp
123

H. Chi et al.
Fig. 1 Ne-Methylation of lysine by histone methyltransferases (HMT)
model to mimic histone called for the synthesis of Ne-
methyl-L-lysine derivatives urgently. In recent years, the
developments of synthesis provided some methods to pre-
pare Na-methylated amino acids and their derivatives.
However, these methods are poor in the preparation of
methylated lysine at e-position, and achievement of selec-
tivity for mono-, di-, and trimethylated lysine (Aurelio et al.
2004; White and Konopelski 2005). But some impressive
progresses in the synthetic strategy of Ne-methyl-L-lysine
were also succeeded. Benoiton obtained Ne-methyl-L-lysine
by the formaldehyde–formic acid methylation of Na-carbo-
benzoxy-Ne-benzyl-L-lysine (Benoiton 1964). The direct
methylation of Na-Fmoc-lysine was also developed by
reductive methylation with formaldehyde and sodium cya-
noborohydride and quaternization with excess amount of
methyl iodide (Huang et al. 2006, 2007). The earlier methods
are costly and no large scale synthesis is shown. Herein, we
describe a different methodology from the past to prepare
Ne-methyl-L-lysine derivatives. In this strategy, protected
2-amino-6-bromohexanoic acid (Ab6, Fig. 2) has been
employed. Our group has reported before the use of deriva-
tives of 2-amino-n-bromo-alkanoic acid (Abn) as interme-
diates of functional amino acids (Watanabe et al. 2004a, b,
2005). By reacting mono- and dimethylamine to protected
Ab6, useful intermediates are available for Ne-methyl-lysine
derivatives such as Na-Fmoc-Ne-(Boc, methyl)-L-lysine and
Na-Fmoc-Ne-dimethyl-L-lysine. This method does not
require expensive reagents to allow the preparation of
designed methylated lysine in multi-grams. Thus, the con-
venient and economical preparation of useful Ne-methyl-L-
lysine derivatives has been achieved in large scale. Not only
the L-form but also the D-form of Ne-methyl-lysine deriva-
tives could be recovered if necessary. Since the methylation
of histone H3 protein is often observed at 4- and 9-positions,
H3 (1–12) peptides containing Ne-monomethyl-lysine has
been synthesized by standard solid-phase method in Fmoc-
chemistry. In addition, the fluorescent peptide (H3, 1–4)-
MCAs have also been synthesized to examine the suscepti-
bility to trypsin and lysyl endopeptidase. As a result, we
Fig. 2 Structure of 2-amino-6-
bromohexanoic acid (Ab6)
Br
CH
OH
H2N
C
O
observed that these peptides are hardly hydrolyzed at mono-
and dimethylated lysines.
Synthetic routes to Na-Fmoc-Ne-(Boc, methyl)-L-lysine
and Na-Fmoc-Ne-dimethyl-L-lysine
Since free Ab6 readily cyclizes to afford L-pipecolic acid
(Watanabe et al. 2005), we reacted methylamine to Ac-DL-
Ab6-OEt to prepare Na-Ac-Ne-methyl-DL-lysine ethyl ester
at the first step (Scheme 1). Subsequently, the side chain
was protected with Boc-group to Na-Ac-Ne-(Boc, methyl)-
DL-lysine ethyl ester (3). The racemic mixture of Na-Fmoc-
Ne-(Boc, methyl)-DL-lysine (4) was subjected to the action
of L-aminoacylase to obtain optically pure Ne-(Boc,
methyl)-L-lysine (5), whose a-amino group was finally
protected with Fmoc-group to give a useful derivative (6)
for peptide synthesis.
On the other hand, since diethyl Boc-aminomalonate is
also commercially available, we prepared optically pure
Boc-L-Ab6-OH at first by the aid of subtilisin (Watanabe
et al. 2005) then fully protected it as tert-butyl ester
(Scheme 2, 10). Substitution reaction of 10 with dimeth-
ylamine gave 11, whose two tert-butyl groups were helpful
to extract 11 into ethyl acetate and in purification. Finally,
protecting groups of 11 were removed and reacted with
Fmoc-OSu to give 12. When we tried to synthesis of 12 by
the same procedure as described for 6, the water soluble
property of Na-Ac-Ne-dimethyl-DL-lysine reduced the yield
drastically. In addition, the isolation of the desired optically
pure product required tedious ion exchange procedure.
123

A convenient preparation of Ne-methyl-L-lysine derivatives
Scheme 1 Synthesis of Na-
Boc
CH 3
Br
Fmoc-Ne-(Boc, methyl)-L-lysine
N
(6). Reagents and conditions:
(a) (i) ethanol, sodium ethoxide,
reflux, 30 min; (ii) 1,
O
OEt
OEt
d, e
a, b, c
Ac
N
H
4-dibromobutane, reflux, 6 h;
o
(b) NaOH aq, 0 C, 4 h;
Ac
OEt
O
Ac
OEt
N
H
N
H
(c) toluene, reflux, 4 h; (d) KI,
methylamine/methanol, 30 min;
(e) dioxane, Boc2O, DIEA;
(f) NaOH aq., 2 h; (g) L-
O
O
1
2
3
aminoacylase, CoCl2ꢀ6H2O, pH
o
7–8, 38 C; (h) Na2CO3 aq.,
dioxane, Fmoc-OSu, pH 10
f
Boc
CH 3
Boc
CH 3
Boc
CH 3
N
N
N
g
h
Ac
OH
Fmoc
OH
OH
N
H
N
H
H2N
O
O
O
6
5
4
Scheme 2 Synthesis of Na-
Fmoc-Ne-dimethyl-L-lysine
(12). Reagents and conditions:
(a) (i) ethanol, sodium ethoxide,
reflux, 30 min; (ii) 1,4-
Br
Br
Et
O
O
a, b, c
d
Boc
Et
O
dibromobutane, reflux, 6 h;
o
(b) NaOH aq., 0 C, 4 h;
Boc
H
O
Boc
Et
O
N
H
N
H
N
H
O
(c) toluene, reflux, 6 h;
(d) subtilisin, DMF/H2O (3/1,
o
v/v), pH 7–8, 38 C, 3 h;
O
O
9
7
8
(e) tert-butyl alcohol, Boc2O,
DMAP; (f) DMF, KI,
dimethylamine hydrochloride;
(g) TFA; (h) Na2CO3 aq.,
dioxane, Fmoc-OSu, pH 10
e
Me
Me
Br
Me
Me
N
N
,
h
g
f
Boc
tBu
O
tBu
Fmoc
H
N
H
O
Boc
O
N
H
N
H
O
O
O
10
12
11
Materials and methods
were routinely checked by thin layer chromatography (TLC)
and/or high-performance liquid chromatography (HPLC).
TLC was performed on aluminum-backed silica gel plates
(Merck DC-Alufolien Kieselgel 60 F254) with spots visual-
ized by UV light or charring. Analytical HPLC was per-
formed on a Hitachi L-7100 instrument equipped with a
Unless otherwise noted, all solvents and reagents were
reagent grade obtained from WAKO, Japan. Flash chroma-
tography was performed using Merck silica gel 60 (230–400
mesh) eluting with solvents as indicated. All compounds
123

H. Chi et al.
chromolith performance RP-18e column (4.6 9 100 mm,
Merck). The mobile phases used were A: H2O with 0.1 %
TFA, B: CH3CN with 0.1 % TFA using a solvent gradient of
A–B over 15 min with a flow rate of 2 mL/min, with
detection at 220 nm. HRMS (FAB) measurements were
performed on a JEOL JMS-SX 102A instrument.
from the oily starting material. To the solution of the ester
in ethanol (100 mL), 2 M NaOH (36 mL) was added. After
2 h, reaction mixture was acidified with AcOH (5.72 mL,
100 mmol) and concentrated to give an oil. The acid was
extracted into ethyl acetate at pH 3–4 using solid citric
acid. The organic layer was washed with brine, dried over
MgSO4, and evaporated to get compound 4 (14.5 g,
48 mmol, 100 %). LCMS m/z found: 303.1, calcd. for
[M ? H]? C14H27N2O5: 303.1.
Preparation of Na-Fmoc-Ne-(Boc, methyl)-L-lysine
Ac-DL-Ab6-OEt (2 in Scheme 1)
Ne-(Boc, methyl)-L-lysine (5)
Metallic sodium (2.23 g, 95 mmol) was dissolved in
dehydrated ethanol (100 mL) and diethyl acetamidomalo-
nate 1 (21.7 g, 100 mmol) was added to the solution. The
mixture was refluxed for 30 min for the complete disso-
lution and then 1,4-dibromobutane (64.2 g, 300 mmol) was
added. After 3 h reflux, the reaction mixture was cooled on
an ice bath and hydrolysis was carried out by the addition
of 1 M NaOH (50 mL). After selective hydrolysis, ethanol
was evaporated and the unreacted 1,4-dibromobutane was
removed by extraction with diethyl ether under alkaline
condition. Then the desired half ester was extracted with
ethyl acetate (pH about 3 with solid citric acid) and washed
with brine, dried over MgSO4 and evaporated to obtain
white solid of monoester monoacid (19.6 g, 61 mmol). The
solution of the half ester in toluene (100 mL) was refluxed
for 5 h. The removal of solvent gave an oil, which was
purified by silica gel column chromatography using hex-
ane/ethyl acetate (2/1, v/v) to give an oily 2 (15.5 g,
55 mmol, 55 %). LCMS m/z found: 280, calcd. for
[M ? H]? C10H19BrNO3: 280.
The racemic acid 4 (14.5 g, 48 mmol) was dissolved in water
(240 mL) and pH of the solution was adjusted to 7 by the
addition of 1 M NaOH at 38 °C. To this neutral solution,
CoCl296H2O (57 mg) and Aspergillus genus L-aminoacy-
lase (1.20 g, TCI) were added and incubated overnight. The
pH of the solution was maintained neutral by the addition of
1 M NaOH. The reaction mixture was acidified with dilute
hydrochloric acid and unreacted Na-Ac-Ne-(Boc, methyl)-D-
lysine was extracted into ethyl acetate.
Na-Fmoc-Ne-(Boc, methyl)-L-lysine (6)
To the aqueous solution containing 5, 10 % Na2CO3
(15.3 g, 144 mmol) and dioxane (200 mL) were added and
allowed to react with Fmoc-OSu at pH around 10 at room
temperature overnight. After evaporation of dioxane, the
reaction mixture was shaken with ether (100 mL) and
acidified with 1 M hydrochloric acid to pH 2–3. The
desired product 6 was extracted into ethyl acetate and
purified with a silica gel column using chloroform/metha-
nol (49/1, v/v). The yield of 6 was 9.3 g (19.2 mmol,
80 %). HRMS (FAB) m/z found: 483. 2530, calcd. for
[M ? H]? C27H35N2O6; 483.2495.
Na-Ac-Ne-(Boc, methyl)-DL-lysine ethyl ester (3)
To a solution of 2 (14.1 g, 50 mmol) in DMF (50 mL), KI
(8.3 g, 50 mmol) was dissolved and then added methyl-
amine in methanol (9 M, 55 mL, 500 mmol) and the
mixture was allowed to stand for 30 min at room temper-
ature. Solvents and excess methylamine were removed by
evaporation to give a syrup, which was dissolved in diox-
ane (100 mL). To this solution, DIEA (13.1 mL, 75 mmol)
and di-tert-butyl dicarbonate (Boc2O) (13.1 g, 60 mmol)
were added at room temperature. After overnight reaction
the desired compound 3 was isolated and purified with a
silica gel column using hexane/ethyl acetate (1/4, v/v).
Colorless oil was obtained as 3 (15.8 g, 48 mmol, 96 %).
LCMS m/z found: 331.2, calcd. for [M ? H]?
C16H31N2O5: 331.2.
Preparation of Na-Fmoc-Ne-dimethyl-L-lysine
Boc-DL-Ab6-OEt (8)
Compound 8 was prepared by the same procedure as
described for compound 2 except that diethyl Boc-amino-
malonate 7 (21.1 g, 100 mmol) was employed as starting
material. Compound 8 could be obtained in 40–60 % yield.
LCMS m/z found: 338.1, calcd. for [M ? H]? C13H25-
BrNO4: 338.1.
Boc-L-Ab6-OH (9)
Na-Ac-Ne-(Boc, methyl)-DL-lysine (4)
Compound 8 (4.39 g, 13 mmol) was suspended on a mixed
solvent of DMF (39 mL) and water (13 mL) (3/1, v/v) at
38 °C using a mechanical stirrer. The pH was adjusted at
7–8 by adding 1 M ammonia. Then subtilisin Carlsberg
The ethyl ester 3 (15.8 g, 48 mmol) was dissolved in eth-
anol (50 mL) and flashed once to remove ethyl acetate
123

A convenient preparation of Ne-methyl-L-lysine derivatives
from Bacillus licheniformis (13 mg, 1 mg enzyme per
mmol of substrate, SIGMA) was added. The reaction was
completed within 3 h and then the mixture was concen-
trated. The unreacted Boc-D-Ab6-OEt was extracted with
diethyl ether under alkaline condition. The desired com-
pound 4 was extracted with ethyl acetate at pH 3–4 by
acidification with solid citric acid. The organic layer was
washed with brine, dried over MgSO4, and concentrated to
dryness. The yield of 9 was 1.95 g (6.30 mmol, 48 %).
LCMS m/z found: 310, calcd. for [M ? H]? C11H21-
BrNO4: 310.
Table 1 Mass spectrum data and HPLC purities for all synthetic
peptides
Histone tail peptides
MS [M ? H]?
Purity
(%)
ARTK(Me)QTARK(Me)STG
(H3 1–12)
1,332.8 (calcd. 1,332.8)
688.4 (calcd. 688.4)
98
Ac-ARTK(Me)-MCA (H3 1–4)
100
100
Ac-ARTK(Me)2-MCA (H3 1–4) 702.4 (calcd. 702.4)
Peptides synthesis
Boc-L-Ab6-OtBu (10)
Automated solid-phase peptide synthesis
Compound 9 (1.12 g, 3.62 mmol) was dissolved in 10 mL
of t-butyl alcohol. To this solution, Boc2O (1.11 g,
5.10 mmol) and DMAP (0.14 g, 1.10 mmol) were added.
After 1 h reaction at room temperature, the solution was
concentrated and the residues were passed through a silica
gel column using ethyl acetate/hexane (1/6, v/v). Colorless
oil was obtained as 10 (1.1 g, 3.0 mmol, 66 %). LCMS m/z
found: 366.1, calcd. for [M ? H]? C15H29BrNO4: 366.1.
As an example of the use of Na-Fmoc-Ne-(Boc, methyl)-L-
lysine in the automated solid-phase peptide synthesis, we
synthesized H3 (1–12) peptide with monomethylation at
4- and 9-positions. The protected peptide fragments were
assembled on Wang resin (substitution 0.7 mmol/g) by
peptide synthesizer (Applied Biosystems 433A) using
Fmoc/piperidine strategy in FastMoc Chemistry at
0.25 mmol scale. HBTU and HOBtꢀH2O were used as
activating agents. The cleavage and removal of protecting
groups were carried out by the treatment with TFA con-
taining phenol (5 %), thioanisole (5 %), ethanedithiol
(2.5 %) and water (5 %). The peptides were precipitated by
tert-butyl methyl ether. The methylated peptide ARTK(-
Me)QTARK(Me)STG was purified by Sephadex G-25 with
10 % AcOH and HPLC, analyzed by LCMS (Table 1).
HPLC profiles and LCMS data are provided in Supple-
mentary data.
Boc-L-Lys(Me)2-OtBu (11)
Compound 10 (1.1 g, 3.0 mmol) was dissolved in 5 mL of
DMF. To this solution, KI (0.48 g, 2.40 mmol), dimeth-
ylamine hydrochloride [(CH3)2NH9HCl] (0.5 g, 6 mmol)
and triethylamine (0.3 mL, 6 mmol) were added and stir-
red overnight. After completion of the reaction, DMF was
evaporated and the residue was dissolved in ethyl acetate.
The solution was washed with saturated sodium carbonate
and then dried over anhydrous Na2CO3, filtered and
evaporated to get the oily 11 (0.8 g, 2.4 mmol, 80 %).
LCMS m/z found: 331.2, calcd. for [M ? H]?
C17H35N2O4: 331.2.
Fluorescent peptide-MCAs
To a chilled solution of Na-Fmoc-Ne-(Boc, methyl)-L-
lysine (1.9 g, 4 mmol) in DCM (10 mL), DCC (0.42 g,
2 mmol) was added. After 1 h on ice bath, AMC (0.36 g,
2 mmol) was added to the mixture. The suspension was
stirred at room temperature overnight, then the product was
filtered and washed with methanol. Na-Fmoc-Ne-(Boc,
methyl)-L-lysine-MCA was obtained as crystalline solid
(2.3 g, 90 %). Na-Fmoc-Ne-dimethyl-L-lysine-MCA was
synthesized as described in the same manner above. Yield,
2.0 g, 92 %.
Na-Fmoc-Ne-(Boc, methyl)-L-lysine-MCA (160 mg,
0.25 mmol) was dissolved in 20 % piperidine/DMF (2 mL)
at room temperature. After 30 min, the reagent and solvent
were removed by evaporation. The residues were extracted
into ethyl acetate, the solution being washed with sat.
Na2CO3 and dried over anhydrous Na2CO3. The filtrate
was concentrated to yield Ne-(Boc, methyl)-L-lysine-MCA
as free amine (104 mg, 0.25 mmol, 100 %). Na-Fmoc-Ne-
Na-Fmoc-Ne-dimethyl-L-lysine (12)
Compound 11 (0.8 g, 2.4 mmol) was dissolved in TFA
(2.4 mL) at 0 °C and kept for 3 h at room temperature.
After evaporation of TFA, the residue was crystallized with
the mixture of ether/petroleum ether (1/4) as TFA salt. It
was then dissolved in the 10 % Na2CO3 (1.21 g,
11.4 mmol) and dioxane (14 mL) and allowed to react with
Fmoc-OSu at pH around 10 with stirring at room temper-
ature overnight. After evaporation of dioxane, the reaction
mixture was washed with ether (10 mL) and acidified with
1 M hydrochloric acid to adjust pH about 3. The purifica-
tion with a silica gel column using chloroform/methanol
(19/1, v/v) as eluent, gave compound 12 (0.75 g,
1.15 mmol, 60 %). HRMS (FAB) m/z found: 397.2049,
calcd. for [M ? H]? C23H28N2O4: 397.2127.
123

H. Chi et al.
dimethyl-L-lysine-MCA was treated with 20 % piperidine
in DMF as described above. 82 mg (0.25 mmol, 100 %) of
free amine Ne-dimethyl-L-lysine-MCA was obtained.
The protected peptide Ac-AR(Pmc)T(tBu)-OH was
assembled on Barlos resin (substitution 0.6 mmol/g) by
manual solid-phase synthesis using Fmoc/piperidine strat-
egy. HBTU and HOBtꢀH2O were used for condensation.
After capping the N-terminal with acetic anhydride, the
protected peptide was cleaved from the resin in DCM
containing TFE and AcOH for 2 h. The protected peptide
(177 mg, 0.25 mmol) was condensed with Ne-(Boc,
methyl)-L-lysine-MCA (104 mg, 0.25 mmol) and Ne-
dimethyl-L-lysine-MCA (82 mg, 0.25 mmol) by the aid of
HBTU/HOBt method, respectively. The protected peptide-
MCAs were treated with deprotection cocktail by the same
procedure as described in automated synthesis. The HPLC
purified peptide-MCAs (Table 1) were analyzed by HRMS
(FAB). HPLC profiles and HRMS (FAB) data are provided
in Supplementary data.
groups and increased in hydrophobicity. Actually this
compound was easily extracted into ethyl acetate for iso-
lation. Since Boc-group cannot be cleaved by L-amino-
acylase, the enzymatic resolution was carried out by the
enantio-specific hydrolysis of ester bond by subtilisin.
Two peptide-MCAs corresponding to the H3 (1–4)
sequence were synthesized. Upon the action of trypsin and
lysyl endopeptidase, neither Ac-ARTK(Me)-MCA nor Ac-
ARTK(Me)2-MCA was hydrolyzed. This fact suggests the
drastic change in susceptibility by methylation toward
these enzymes.
Conclusion
In summary, the convenient and economical routes have
been developed for the synthesis of Na-Fmoc-Ne-(Boc,
methyl)-L-lysine and Na-Fmoc-Ne-dimethyl-L-lysine to be
used as building blocks for the synthesis of methylated
peptides. Ne-Methyllysine residues have been efficiently
incorporated to the H3 (1–12) peptide by automated solid-
phase peptide synthesis. Na-Fmoc-Ne-(Boc, methyl)-L-
lysine and Na-Fmoc-Ne-dimethyl-L-lysine are condensed
with 7-amino-4-methylcoumarin to prepare fluorescent
peptide-MCAs, which are used to examine the suscepti-
bilities to trypsin and lysyl endopeptidase. They showed no
activity to mono- and dimethylated lysine peptides.
Results and discussion
In the present synthetic routes, the key intermediates are
Ab6 derivatives, which are conveniently prepared from
diethyl acetamido- or Boc-aminomalonate and dibromo-
butane by a few steps. Since the Ne-modification with Boc-
group is provided enough hydrophobicity for the extraction
of the intermediate such as Na-Ac-Ne-(Boc, methyl)-DL-
lysine (4), acetamide malonate was better choice. However,
dimethyl modification afforded hydrophilic property to the
intermediate, therefore, resulted in difficulty in isolation.
To clean this serious problem, we employed diethyl Boc-
aminomalonate (7). In both routes (Schemes 1, 2), the
L-amino acid derivatives were obtained in high optical purity
by enzymatic resolution with L-aminoacylase and subtilisin,
respectively, for well-established in high optical purity.
For the monomethylation, the synthesis started with
commercially available diethyl acetamidomalonate 1, the
reaction of 2 with methyl amine in DMF in the presence of
KI was followed by Boc-protection without further purifi-
cation. After saponification of 3, racemic 4 was subjected
to the resolution by Aspergillus genus L-aminoacylase
(TCI). The Na-Ac-Ne-(Boc, methyl)-D-lysine could be
recovered by extraction into ethyl acetate. The free amino
acid Ne-(Boc, methyl)-L-lysine in the aqueous layer was
reacted with Fmoc-OSu in the presence of Na2CO3. The
desired compound Na-Fmoc-Ne-(Boc, methyl)-L-lysine was
obtained in good yield.
Conflict of interest The authors declare that they have no conflict
of interest.
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