Facile synthesis and altered ionization efficiency of diverse N ε-alkyllysine-containing peptides

Article abstract of DOI:10.1039/c1cc14711k

Protein lysine methylation is a biologically-important posttranslational modification. Given the increased use of alkyllysine-containing peptides to study protein methylation, we describe an efficient solid-phase synthesis to access various alkyllysine-containing peptides. As an exemplary application, we evaluated how ionization efficiency of the alkyllysine peptides affects mass spectrometric analysis.

Full text of DOI:10.1039/c1cc14711k

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COMMUNICATION
Facile synthesis and altered ionization efficiency of diverse
N -alkyllysine-containing peptideswz
Debjani Chakraborty, Kabirul Islam and Minkui Luo*
e
a
b
b
Received 31st July 2011, Accepted 19th September 2011
DOI: 10.1039/c1cc14711k
Protein lysine methylation is a biologically-important posttransla-
tional modification. Given the increased use of alkyllysine-containing
peptides to study protein methylation, we describe an efficient solid-
phase synthesis to access various alkyllysine-containing peptides. As
an exemplary application, we evaluated how ionization efficiency of
the alkyllysine peptides affects mass spectrometric analysis.
In contrast, the same events can be hijacked by cancer cells to
7
silence tumor suppressor genes.
Given the importance of protein methylation, multiple
approaches were described for site-specific incorporation of
8–10
methylated lysines into peptides or proteins.
Complementary
1
0
biochemical assays were also developed to trace these events.
e
Recently, the peptides that contain lysine-N -b unsaturated
substituents have emerged as valuable chemical entities to
e
Lysine-N amino groups of proteins undergo diverse posttransla-
11–15
study protein methylation and demethylation.
For example,
tional modifications such as ubiquitination, sumoylation, neddy-
1
lation, acetylation and methylation. Among the most known
several successful approaches have been reported to utilize
SAM derivatives, in combination with native or engineered
PKMTs, to transfer allyl derivatives to PKMT substrates
lysine posttranslational modifications is methylation, which is
featured by enzymatic installation of a methyl group from the
1
3–15
e
(
Fig. 1).
The resultant lysine-N derivatization enabled
1
biological methyl donor S-adenosyl-L-methionine (SAM)
2
Fig. 1). Despite its stable chemical nature, this modification
3,14
the PKMT substrates to be readily characterized. The
peptides carrying lysine-N -propargyl/allyl derivatives are potent
(
e
2
can be removed by demethylases (Fig. 1). The dynamic methyla-
1
1,12
inhibitors of the protein lysine demethylase LSD1 (Fig. 1).
tion and demethylation of targeted proteins play essential roles in
These applications thus triggered us to explore efficient
e
epigenetic regulation via modulating their localization, stability or
3
interacting partners. Their deregulation has been linked to many
chemical methods to assemble N -b-unsaturated moieties onto
1
1,12
3
diseases including cancer. For instance, histone H3 lysine 9
lysine side chain.
Among known synthetic strategies to access N -alkyllysines
e
(
H3K9) can be methylated by 7 human protein lysine methyl-
e
e
4
transferases (PKMTs). This modification is associated with gene
are base-promoted lysine-N alkylation, lysine-N reductive
alkylation, and leaving-group-facilitated S 2 C-amination of
N
silencing at euchromatin, facultative/constitutive heterochromatin,
11,12,16,17
6-hydroxynorleucine.
e
5,6
Base-promoted lysine-N -alkylation
or virus-integrating sites to maintain normal cellular functions.
is less selective for the degree of alkylation and often leads to
e
8,9
mono/di/tri-alkylated product mixtures. Lysine-N reductive
e
alkylation is facile to prepare N -monomethyllysine. The
monoalkylation selectivity was achieved by reductive benzylation
and then methylation through imine intermediates followed by
8
debenzylation. However, this approach is not suitable to
,9
e
install lysine-N -b-unsaturated substituents because of the
undesirable loss of the unsaturated moieties via 1,4-hydride
Fig. 1 Methylation, alkylation and demethylation on protein lysine.
PKMTs or their variants account for site-specific lysine modifications.
Demethylation is carried out by demethylases and can be inhibited by
reduction of the imine intermediatesy. So far, the reported
e
2
approach to chemically install lysine-N -b-sp/sp substituents
is to mesylate 6-hydroxynorleucine of resin-immobilized peptides,
e
N -propargyl/allyllysine-containing peptides.
followed by solution-phase S 2 displacement of the mesylate
N
b
with N -unsaturated amines (S
N
2-type C-amination), in which
11,12
a
Tri-Institutional Training Program in Chemical Biology,
Molecular Pharmacology and Chemistry Program,
multiple HPLC purifications are involved.
b
Here we describe a concise, efficient chemical approach to
e
Memorial Sloan-Kettering Cancer Center, New York, NY 10065,
USA. E-mail: luom@mskcc.org; Fax: +1 646-888-3166;
Tel: +1 646-888-3066
w This article is part of the ChemComm ‘Emerging Investigators 2012’
themed issue.
z Electronic supplementary information available: General materials
and methods, synthetic protocols and product characterization. See
DOI: 10.1039/c1cc14711k
install lysine-N -b-unsaturated substituents via solid-phase
Dess–Martin oxidation/reductive amination of hydroxy-
e
norleucine. Characterizing these lysine-N -derivatized peptides
e
also allowed us to compare ionization efficiency of diverse N -
alkyllysine-containing N-terminal H3 peptides on matrix-assisted
laser desorption/ionization mass spectrometry (MALDI-MS).
1
514 Chem. Commun., 2012, 48, 1514–1516
This journal is c The Royal Society of Chemistry 2012

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promising yields, the one-pot strategy served as a key starting
point to develop more efficient solid-phase synthesis (results below).
Encouraged by the success of solution-phase synthesis of
e
N -allyl/propargyl/2-butynyl lysine analogues 4a–c, we then
explored such chemical transformation of a peptide substrate
in solid phase (Scheme 2). The N-terminal H3 peptide (3–13 aa, 5)
with its H3K9 replaced by hydroxynorleucine was assembled
on Wang resin. In comparison with the prior solid-phase
peptide synthesis (SPPS) for hydroxynorleucine-containing
11,12
peptides,
the current approach is featured by incorporating
hydroxynorleucine and other amino acids without protecting
the hydroxynorleucine e-hydroxyl group. Similar to our
solution-phase strategy, the hydroxynorleucine moiety of
resin-immobilized peptide was efficiently converted to aldehyde
by Dess–Martin periodinane (from 5 to 6). Subsequent solid-
phase reductive amination of the aldehyde with 2 equiv. allyl
amine and then TFA-mediated global deprotection yielded the
desirable allyl peptide 8a with a remarkable yield of 30% for
23-step solid-phase reactions from Wang resin. The same
approach also allowed the synthesis of 7 other H3K9-alkylated
peptides (Me–, ethyl, CF CH –, propargyl, allyl, 2-butynyl,
e
Scheme 1 Synthesis of lysine analogues containing N -b-unsaturated
substituents via consecutive Dess–Martin oxidation/reductive amination
of hydroxynorleucine.
Although most H3K9-Ne alkyl substitutions (e.g. Me–,
CF CH –, propargyl, allyl, 2-butynyl and 2-butenyl) have no
3
2
significant effect on the ionization efficiency, ethylation and
benzylation alter the ionization efficiency by 2.5–5-fold. These
results therefore claimed the need to validate ionization efficiency
of native and alkyllysine peptides for direct quantification
through their MALDI-MS peak intensities.
3
2
2-butenyl and benzyl) from respective primary amines
(30–50% yields from 1 to 8a–h based on Wang resin,
Scheme 2, Fig. S2 and Section S2c in ESIz). The current
strategy is convenient because of its ability to access multiple
products from the commonly-shared hydroxynorleucine peptide
via simple solid-phase washing and a single HPLC purification.
In addition, Thr(tBut), Lys(Boc), Gln(Trt), Arg(Pbf),
Ser(tBut), Gly and Cys(Trt) are tolerant to Dess–Martin
oxidation/reductive amination conditions.z The robust and
general applicability of the solid-phase Dess–Martin oxidation/
reductive amination thus presents an efficient route to access
various alkyllysine-containing peptides. In comparison with
To develop strategies to access lysine derivatives containing
e
N -b-unsaturated substituents, we first focused on solution-
phase synthesis of N -allyllysine as a proof-of-concept target
e
(
Scheme 1). The reductive alkylation reaction of Fmoc-
protected lysine and acrolein underwent undesirable 1,4- and
e
then 1,2-imine reduction to give N -propyl rather than
e
N -allyllysine.y To avoid this 1,4-hydride reduction, we envi-
sioned a strategy through switching the positions of the
amine–aldehyde pair (reductive alkylation versus reductive
a
a
amination). Here N -Fmoc lysine was converted to N -Fmoc
1
1,12
hydroxynorleucine
1
via spontaneous hydrolysis of
1
a
the previous approaches,
the current method allows new
1,12,18
diazonium intermediate (Scheme 1).
A subsequent direct
functionalities such as ethyl, CF CH –, 2-butynyl, 2-butenyl
3
2
oxidation of hydroxynorleucine’s primary alcohol to aldehyde
proved to be challenging. All the attempts for such oxidation,
concomitant with the loss of 1, gave a mixture of products,
which either showed complex profiles (e.g. with tetra-n-propyl
ammonium perruthenate, pyridinium chlorochromate, or
chromium(VI) oxide/pyridine as oxidants) or decomposed in
the course of purification (e.g. oxalyl chloride/DMSO for
Swern oxidation or periodinane for Dess–Martin oxidation).
Given that the product profile under the Dess–Martin condition
is simpler than others, we reasoned that the failure of Dess–Martin
periodinane to yield the desirable aldehyde could be due to the
challenge of product purification. This problem was gratifyingly
circumvented using a step-wise one-pot reaction (Scheme 1). Here
and benzyl groups (8c and 8e–h) to be installed in lysine side
chains of peptides. The peptides carrying such functionalities
can be further examined as potential mechanism-based inhibitors
of protein lysine methyltransferases or demethylases.
a
N -Fmoc hydroxynorleucine was first oxidized to the corres-
ponding aldehyde 2 with Dess–Martin periodinane. After simple
concentration, the aldehyde product was reacted with allyl amine
(
2 equiv. to suppress multi-alkylation), followed by cyanoboro-
e
hydride-mediated reduction, to yield N -allyllysine 3. Without
e
further purification, N -allyllysine was masked by Boc-carbamate
a
with a final yield of 15% from N -Fmoc hydroxynorleucine 1 to
4
a. The generality of this synthesis was also confirmed by its
e
a
application to prepare N -Fmoc-N -(Boc, propargyl)-lysine 4b
a
Scheme 2 Solid-phase synthesis of alkyllysine-containing H3
N-terminal peptides via Dess–Martin oxidation/reductive amination.
NL = norleucine.
e
and N -Fmoc-N -(Boc, 2-butynyl)-lysine 4c in comparable
5–20% yields (Scheme 1, Section S2b in ESIz). Despite less
1
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1514–1516 1515

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peptides also underscores the importance of validating the
MS parameter for applying MALDI-MS to quantify alkylated
e
peptides. Here N -alkyllysine-containing peptides can be readily
prepared as the MS standards for such validation through the
described synthesis.
We thank Drs Ouerfelli, Sukenick, Liu and Fang for helpful
discussion and compound analysis; the supports from NIH
(
R01GM096056, NIH Director’s New Innovator Award DP2-
OD007335), March of Dimes Foundation (Basil O’connor
Starter Scholar Award), The Starr Cancer Consortium, Alfred
W. Bressler Scholars Endowment Fund, Mr William H.
Goodwin and Mrs Alice Goodwin the Commonwealth
Foundation and The Experimental Therapeutics Center of
MSKCC. D.C. is supported by the fellowship of Tri-Institutional
Training Program in Chemical Biology.
Notes and references
a
Fig. 2 Comparison of MALDI-MS ionization efficiency of H3K9
with representative H3K9-alkyllysine derivatives. Equal molar native
and alkyllysine peptides were co-injected into MS. Ionization efficiency
of 8e (ethyl) and 8h (benzyl) was altered by 2.5–5 fold.
y Upon reacting N -Fmoc-lysine with acrolein, NaCNBH
3
and then
a
e
2
Boc O, N -Fmoc-N -(Boc, propyl)lysine was isolated as the only
major product, which arises from 1,4-imine reduction.
z Thr(tBut), Lys(Boc), Gln(Trityl), Arg(Pbf), Ser(tBut) and Gly were
installed in the course of synthesizing the 3–13 aa H3 peptides. The
success of the peptide synthesis therefore indicated that these amino-
acid building blocks are tolerant to Dess–Martin oxidation/reductive
amination. The tolerance of Cys(Trt) was confirmed independently by
synthesizing C-terminal-Cys(Trt)-appended 3–13 H3 peptide (data not
shown). Other amino acids (Met, Tyr and Trp) remain to be examined.
To examine the activities of protein methyltransferases, a
common strategy is to quantify enzymatic modification with
MALDI-MS. Some prior experiments relied on direct
comparison of ion intensities of modified peptides (products)
1
9
versus unmodified peptides (starting materials). However, given
1
B. C. Smith and J. M. Denu, Biochim. Biophys. Acta, 2009, 1789,
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P. A. Cole, Nat. Chem. Biol., 2008, 4, 590–597.
the uncertain ionization efficiency of the alkylated peptides,
1
4
3,15,20
caution should be made for such direct comparison.
For
2
example, prior to MALDI-based MS quantification, comparable
ionization efficiency on MALDI-MS was confirmed for
N-terminal H3 peptide and its (E)-hex-2-en-5-ynyl
3 S. R. Bhaumik, E. Smith and A. Shilatifard, Nat. Struct. Mol.
Biol., 2007, 14, 1008–1016.
4
5
T. Kouzarides, Cell, 2007, 128, 802–803.
D. C. Leung, K. B. Dong, I. A. Maksakova, P. Goyal,
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U. S. A., 2011, 108, 5718–5723.
1
3
derivative.
Given our newly-gained ability to access various alkyllysine
peptides, their ionization efficiency on MALDI-MS was eval-
uated in a systematic manner. Upon co-injecting equal-molar
N-terminal H3 3–13 aa peptide and its H3K9-alkylated deri-
6
X. Cheng and R. M. Blumenthal, Biochemistry, 2010, 49,
2
999–3008.
7 A. Spannhoff, W. Sippl and M. Jung, Int. J. Biochem. Cell Biol.,
009, 41, 4–11.
2
vatives (Me–, Et–, CF
-butenyl and benzyl) on MALDI-MS, their ion intensities
were compared (Fig. 2, Fig. S2 in ESIz). Although the H3K9
peptides containing Me–, CF CH –, propargyl, allyl, 2-butynyl
3 2
CH –, propargyl, allyl, 2-butynyl,
8
Z. P. Huang, J. T. Du, X. Y. Su, Y. X. Chen, Y. F. Zhao and
Y. M. Li, Amino Acids, 2007, 33, 85–89.
2
9
Z. P. Huang, X. Y. Su, J. T. Du, Y. F. Zhao and Y. M. Li,
Tetrahedron Lett., 2006, 47, 5997–5999.
0 Y.-S. Wang, B. Wu, Z. Wang, Y. Huang, W. Wan, W. K. Russell,
3
2
1
and 2-butenyl modifications showed MALDI-MS ionization
efficiency comparable to that of the native peptide, ethylation
reduced and benzylation enhanced the ionization efficiency by
P.-J. Pai, Y. N. Moe, D. H. Russell and W. R. Liu, Mol. BioSyst.,
2
010, 6, 1575–1578.
1
1
1
1
1
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´
4 R. Wang, G. Ibanez, K. Islam, W. Zheng, G. Blum, C. Sengelaub
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2.5- and 5-fold, respectively (Fig. 2). This finding thus argues
that, although H3K9 methylation, allylation and propargylation
have negligible effect on the peptide’s MALDI-MS ionization
efficiency, other H3K9 alkylations (e.g. ethylation or benzylation)
may alter such a parameter dramatically.
In conclusion, a convenient synthesis has been developed
through consecutive solid-phase Dess–Martin oxidation and
reductive amination. The unprecedented approach enables the
ready access to alkyllysine-containing peptides via SPPS, in
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e
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1
8 P. R. Thompson, H. Kurooka, Y. Nakatani and P. A. Cole,
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Given that similar peptide derivatives have been explored as
intermediates for labeling PKMT substrates and as inhibitors
of histone-modifying enzymes, these applications can be benefited
by diversifying the peptide entities with the delineated synthesis.
Analyzing ionization efficiency of alkyllysine-containing
19 R. E. Collins, M. Tachibana, H. Tamaru, K. M. Smith, D. Jia,
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2
0 A. Patel, V. Dharmarajan, V. E. Vought and M. S. Cosgrove,
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1
516 Chem. Commun., 2012, 48, 1514–1516
This journal is c The Royal Society of Chemistry 2012