Dual native chemical ligation at lysine

Article abstract of DOI:10.1021/ja905491p

(Chemical Presented) A thiol group introduced on the γ-carbon of lysine mediates robust native chemical ligation at both the α- and ε-amines in two consecutive steps. Desulfurization then affords the final product, in which the lysine residue at the ligat

Full text of DOI:10.1021/ja905491p

Published on Web 09/03/2009
Dual Native Chemical Ligation at Lysine
Renliang Yang,† Kalyan Kumar Pasunooti,‡ Fupeng Li,† Xue-Wei Liu,*,‡ and Chuan-Fa Liu*,†
DiVision of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological UniVersity,
60 Nanyang DriVe, Singapore 637551, and DiVision of Chemistry and Biological Chemistry, School of Physical and
Mathematical Sciences, Nanyang Technological UniVersity, 1 Nanyang Walk, Singapore 637616
Received July 3, 2009; E-mail: xuewei@ntu.edu.sg; cfliu@ntu.edu.sg
Protein chemical synthesis through chemoselective peptide ligation
is becoming an increasingly important enabling technology in the study
of proteins.1 A particularly useful ligation method is native chemical
ligation (NCL), which involves the reaction of a thioester peptide with
another cysteinyl peptide to form an Xaa-Cys bond at the ligation
junction.2,3
Starting from (S)-Boc-Asp-OtBu 1, the O-silyl-protected 1,3-diol 2 was
prepared in enantiopure form through a key δ-lactam intermediate using
known literature procedures.10 The terminal alcohol in 2 was mesylated
and further converted to an azide.10 The azide was then reduced by
catalytic hydrogenation to an amine, which was protected using CbzCl
to afford 3. After deprotection of the silyl ether in 3 with TBAF,
mesylation of the secondary alcohol followed by nucleophilic substitu-
tion of thioacetate afforded 4 in good yields. Following saponification,
the thiol was protected in disulfide form using MMTS to furnish 5
according to literature methods.8,9b Acidolytic deprotection with TFA
and further protection with Boc anhydride yielded 6, the protected form
of 4-mercaptolysine ready for use in peptide synthesis.
Since cysteine is an uncommon amino acid in naturally occurring
proteins, several strategies have been developed to expand the appli-
cation scope of NCL. One strategy uses the appending of a removable
thiol-containing auxiliary group on the N-terminal amine to mediate
ligation.4 Recently, this strategy was employed to introduce a ubiquitin
moiety through ligation at a Gly-Gly junction on the side chain of
Lys.5 A drawback of this approach is that the ligation reaction exhibits
very slow kinetics because of steric hindrance imposed by the resultant
secondary amine; thus, its practical utility is limited only to unhindered
ligation junctions. Ligation at Met is made possible through the use
of homocysteine, which is converted to methionine postligationally
by S-methylation.6 Another strategy employs desulfurization to convert
the post-NCL cysteine to alanine.7 Similarly, other amino acids contain-
ing a surrogate thiol have been used in NCL followed by desulfur-
ization, making phenylalanine8 and valine9 accessible as ligation sites.
Herein, we report a dual native chemical ligation strategy through
the use of a 4-HS-lysine residue (Scheme 1). Lysine is a frequently
occurring amino acid in proteins. In addition, the ε-amino group of
lysine provides a platform for post-translational protein modifications,
such as methylation, acetylation, and ubiquitination or sumoylation.
The side-chain amine of lysine is also often used as an anchor point
for labeling proteins with a biophysical or biochemical tag. We realized
that a single thiol group introduced on the γ-carbon of lysine would
mediate ligation at both the R- and ε-amines, in each case via a six-
membered-ring reaction intermediate, to form native and isopeptide
bonds, respectively (Scheme 1). Therefore, by using a proper protecting
group, one can perform two consecutive ligation steps via the same
thiol to synthesize proteins containing specifically modified (e.g.,
ubiquitinated or biotinylated) lysine residues.
Scheme 2. Synthesis of 6a
a Reagents and conditions: (a) ref 10; (b) MsCl, DIPEA, 0 °C; (c) NaN3,
DMF, 80 °C, 83% yield over two steps; (d) H2, Pd/C, ethyl acetate, rt,
quantitative yield; (e) CbzCl, NaHCO3, 2:1 dioxane/water, 0 °C, 81%; (f) TBAF,
THF, 0 °C, 77%; (g) MsCl, DIPEA, 0 °C; (h) CH3COSK, DMF, 40 °C, 70%
over two steps; (i) NaOH, MeOH, rt; (j) (S)-methyl methanethiosulfonate
(MMTS), triethylamine, CH2Cl2, rt, 50% over two steps; (k) 95% TFA, H2O,
rt; (l) Boc2O/TEA, MeOH, rt, 78% over two steps.
The 4-mercaptolysine derivative was introduced as the N-terminal
residue of the small peptide 7 with Cbz remaining on its ε-amine.
Peptide 7 was directly used in ligation reactions, as the free-thiol form
8 could be readily generated in situ under the reducing conditions of
the ligation reaction (Scheme 3).
Scheme 1. General Scheme for Dual Chemical Ligation: (a) First
Ligation with P1-COSR; (b) Deprotection; (c) Second Ligation with
P3-COSR; (d) Desulfurization
Robust ligation was observed when 7 was subjected to reaction with
the thioester peptide H-LSTEA-COSR at pH 8. After reaction for 1 h
at 37 °C, the 4-mercaptolysyl peptide limiting reactant was completely
consumed, and the ligation product 9a was obtained in ∼90% yield
based on HPLC analysis. Ligation with another thioester peptide with
a larger C-terminal Leu residue, H-LSTEL-COSR, also proceeded
efficiently (Figure 1), yielding the ligation product 9b in 92% yield
by HPLC analysis after reaction for 1 h at 37 °C, indicating that the
ligation reaction was not affected by the bulkiness of Leu in either
reaction rate or yield. The Cbz group in 9 was then removed using a
cocktail containing TFMSA in order to expose the ε-NH2 of 4-mer-
captolysine for the second ligation. First, the purified peptide 10a was
reacted with a small peptide thioester, H-LSTEG-COSR. Similar to
the first ligation at the R-amine, the second ligation proceeded very
Scheme 2 shows the synthesis of the protected γ-thiol-substituted
lysine analogue 6. Essentially, the method of Guichard and co-
workers10 was adopted to prepare a 4-hydroxylysine derivative of
which the 4-hydroxyl group was subsequently converted to a thiol.
† Division of Chemical Biology and Biotechnology.
‡ Division of Chemistry and Biological Chemistry.
9
13592 J. AM. CHEM. SOC. 2009, 131, 13592–13593
10.1021/ja905491p CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Scheme 3. Demonstration of Peptide Dual Ligationa
Figure 2. HPLC monitoring of ligation between peptide 10b and ubi(1-76)-
MES at (bottom) 3 and (top) 45 min. Gradient: 0-36% buffer B in 18 min,
36-45% in 18 min. Peak a, peptide 10b; peak b, ubi(1-76)-MES; peak c,
ligation product 11b; peak *, unidentified. Ligation conditions: 6.6 mM 10b,
1.2 mM ubi(1-76)-MES, 6 M Gdn·HCl, 0.2 M phosphate, 60 mM TCEP,
pH 8.0, 37 °C.
a See the Supporting Information for experimental details and charac-
terization data.
11c in 90% yield by HPLC analysis. Free-radical desulfurization afforded
the final biotinylated peptide 12c in 80% yield based on HPLC analysis.
The above results show that in a unique one-stone-two-birds fashion,
a γ-SH group on an N-terminal lysine mediates facile chemical ligation
at both its R- and ε-amines. The unhindered nature of the two primary
amines likely accounts for the robustness of the ligation reactions. If used
without the second ligation step, our method would allow conventional
linear NCL at Lys, a notable expansion of the application scope of NCL
in view of the abundance of lysine in proteins. Through the use of the
dual ligation scheme, it is possible to synthesize complex protein molecules
that are acylated on specific lysine side chains. We believe that our method
will be particularly useful for the chemical synthesis of lysine-rich and
lysine-modified proteins.
efficiently, reaching completion in 1.5 h to give the product 11a in
92% yield by HPLC analysis. A control experiment performed with
thethioesterpeptideH-LSTEA-COSRandlysylpeptideH-KGAKAFA-
NH2 for 2 h showed no detectable amount (<2%) of direct aminolysis
at either the R- or ε-amine. This indicated the vital role of the γ-SH
group in mediating ligation at both the R- and ε-amino groups of the
4-mercoptolysine residue. Next, to verify whether our method can be
used for site-specific peptide ubiquitination, peptide 10b was reacted
with a large (76 amino acids) ubiquitin thioester, ubi(1-76)-CO-
-
SCH2CH2SO3 [ubi(1-76)-MES], generated by thiolysis of a
ubiquitin-intein fusion protein.11 As shown in Figure 2, ligation with
the ubiquitin thioester was complete within only 45 min, giving a very
clean product in >90% yield based on HPLC analysis. The efficiency
of the reaction in our dual NCL scheme makes it a particularly viable
method for the synthesis of complex protein conjugates such as
ubiquitinated proteins for the functional elucidation of such post-
translational modifications on lysine.
Acknowledgment. We thank A*Star of Singapore (BMRC 08/
1/22/19/588 to C.-F.L.) and Nanyang Technological University for
financial support.
Supporting Information Available: Experimental procedures and
compound characterization data. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
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Figure 1. HPLC monitoring of ligation between H-LSTEL-COSR and peptide
8 at (bottom) 10 min and (top) 1 h. Gradient: 0-60% buffer B in 30 min.
Peak a, H-LSTEL-COSR; peak b, peptide 8; peak c, ligation product peptide
9b; peak d, H-LSTEL-COSBz; peak *, H-LSTEL-OH. Ligation conditions:
28 mM H-LSTEL-COSR, 20 mM 8, 6 M Gdn·HCl, 0.2 M phosphate, 60
mM TCEP, 1% benzyl mercaptan, pH 8.0, 37 °C.
To generate native Lys at the ligation junction, we first tried the
Raney nickel-mediated desulfurization method.7 Desulfurization of 11a
gave only a moderate yield of 44% (based on quantitative HPLC
analysis) after 8 h of reaction. Moreover, this metal-based method did
not work for 11b, which is much larger and more complex than 11a.
We then tried the recently developed free-radical desulfurization
approach.12 Desulfurization of 11b using VA-044 reached completion
within 5 h to give the final ubiquitinated peptide 12b, and the
conversion was near quantitative based on MS analysis (see the
Supporting Information).
(6) (a) Tam, J. P.; Yu, Q. Biopolymers 1998, 46, 319. (b) Saporito, A.; Marasco,
D.; Chambery, A.; Botti, P.; Monti, S. M.; Pedone, C.; Ruvo, M.
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To further test whether our method can be used for specific biotinylation
on the lysine side chain, peptide 10b was reacted with a biotin thioester.
The reaction was complete in 3 h and gave the biotinyl ligation product
(12) Wan, Q.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2007, 46, 9248.
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