Native chemical ligation at glutamine

Article abstract of DOI:10.1021/ol300254y

The desulfurization reaction introduced by Yan and Dawson as a postnative chemical ligation step greatly expanded the scope of ligation chemistry beyond Xaa-Cys (Xaa is any amino acid) by making ligation at Xaa-Phe, Xaa-Val, Xaa-Lys, Xaa-Leu, Xaa-Thr, and

Full text of DOI:10.1021/ol300254y

ORGANIC
LETTERS
2012
Vol. 14, No. 6
1520–1523
Native Chemical Ligation at Glutamine
Peter Siman, Subramanian Vedhanarayanan Karthikeyan, and Ashraf Brik*
Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva, 84105,
Israel
abrik@bgu.ac.il
Received January 31, 2012
ABSTRACT
The desulfurization reaction introduced by Yan and Dawson as a postnative chemical ligation step greatly expanded the scope of ligation
chemistry beyond Xaa-Cys (Xaa is any amino acid) by making ligation at Xaa-Phe, Xaa-Val, Xaa-Lys, Xaa-Leu, Xaa-Thr, and Xaa-Pro junctions
accessible in the synthesis of functional proteins. A new ligation site based on Xaa-Gln utilizing γ-mercaptoglutamine is reported, and several
examples on the efficiency of ligation coupled with desulfurization are provided.
The combination of native chemical ligation (NCL)1
and global desulfurization2 has significantly increased our
ability to chemically synthesize proteins for biochemical
and structural analyses.3 To achieve this, an amino acid at
the desired ligation junction is modified at the β- or
γ-carbon with a thiol group to enable transthioesterification
with a peptide thioester and subsequent SÀN acyl transfer
via a favorable five- or six-membered ring to form the
backbone amide bond between the two peptides. The use
of Xaa-Ala (Xaa is any amino acid) ligation sites by
replacing Ala with Cys to enable NCL followed by a
selective reduction of the thiol group on Cys2 has inspired
the development of several other ligation junctions that
include Xaa-Phe,4 Xaa-Val,5 Xaa-Leu,6 Xaa-Thr,7 Xaa-
Pro,8 and Xaa-Lys.9 Several desulfurization conditions
such as nickel boride,2 Pd/Al2O3,10 and metal-free con-
ditions11 were found suitable to achieve efficient reduc-
tion. More recently, performing ligation and desulfuriza-
tion in situ was also reported.12 Moreover, carrying out
desulfurization in the presence of Cys residues is possible
thanks to the use of orthogonal protecting groups on the
Cys10,13 or by using selective deselenization in the presence
of unprotected Cys.14 Exploiting NCL principles, SÀN
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(10) Yang, Y. Y.; Ficht, S.; Brik, A.; Wong, C. H. J. Am. Chem. Soc.
2007, 129, 7690.
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Rohde, H.; Seitz, O. Biopolymers 2010, 94, 551. (c) Payne, R. J.; Wong,
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r
10.1021/ol300254y
Published on Web 02/24/2012
2012 American Chemical Society

acyl transfer assisted by proximity effect,15 a method
called sugar-assisted ligation for glycopeptide, was also
developed.16 In this strategy, the 2-acetamido group of the
glycan unit is modified with a thiol handle to promote
amide bond formation via a 14À15-membered ring inter-
mediate for SÀN acyl transfer. Our group has also devel-
oped a highly efficient method for peptide and protein
ubiquitination9a by modifying theδ-carbonof a lysine side
chain with a thiol group. This promotes a transthioester-
ification step with a ubiquitin thioester followed by iso-
peptide bond formation, which after a desulfurization step
furnishes the native structure.17
these advances, coupled with the need to have complete
flexibility in the retrosynthetic analysis of a protein target
by having the ability to perform ligation essentially at any
ligation junction, here we report NCL coupled with de-
sulfurization at Xaa-Gln ligation sites (Scheme 1). This
ligation is of particular interest due to the high abundance
of Gln in protein sequences19 relative to Cys (2%) and the
need to prepare native protein-containing polyQ repeats
that are involved in several inherited neurodegenerative
diseases for biochemical studies.20 For example, Hunting-
ton’s disease (HD) is caused by an expansion of a polyQ
repeat in the Huntingtin (Ht) gene.21 In future endeavors
of chemical synthesis of such proteins, the requirement for
Gln-Gln ligation site(s) is inevitable.
To enable ligation at Xaa-Gln sites, we sought to develop
a straightforward synthesis of thiol-modified Gln residue.
Scheme 1. Ligation at Glutamine Site Employing γ-Mercapto-
glutamine-NCL and Desulfurization
Scheme 2. Synthesis of γ -(R,S)-Mercapto-L-glutamine
The above-described strategies have contributed in sev-
eral ways to prepare proteins and posttranslationally mod-
ified analogues for a variety of studies.6a,12,18 Motivated by
Inspired by the previously reported work on the synthesis of
γ-fluorinated Gln,22 we reasoned that a similar approach
could be adopted to prepare the γ-mercaptoglutamine
using the Passerini three-component reaction (Scheme 2).
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Bavikar, S. N.; Spasser, L.; Moyal, T.; Ohayon, S.; Brik, A. Angew.
Chem., Int. Ed. 2011, 50, 6137. (c) Bavikar, S. N.; Spasser, L.;
Haj-Yahya, M.; Karthikeyan, S. V.; Moyal, T.; Kumar, K. S. A.; Brik,
A. Angew. Chem., Int. Ed. 2012, 51, 758.
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Physiol.: Cell Physiol. 1998, 274, C1101.
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Chellgren, V. M.; Byeon, I.-J. L.; Anjum, D. H.; Kodali, R.; Creamer,
T. P.; Conway, J. F.; Gronenborn, A. M.; Wetzel, R. Nat. Struct. Mol.
Biol. 2009, 16, 380.
(22) Qu, W.; Zha, Z.; Ploessl, K.; Lieberman, B. P.; Zhu, L.; Wise,
D. R.; Thompson, C. B.; Kung, H. F. J. Am. Chem. Soc. 2011, 133, 1122.
(18) (a) Bang, D.; Makhatadze, G. I.; Tereshko, V.; Kossiakoff,
A. A.; Kent, S. B. Angew. Chem., Int. Ed. 2005, 44, 3852. (b) McGinty,
R. K.; Kim, J.; Chatterjee, C.; Roeder, R. G.; Muir, T. W. Nature 2008,
453, 812. (c) Hejjaou, M.; Haj-Yahya, M.; Kumar, K. S. A.; Brik, A.;
Lashuel, H. A. Angew. Chem., Int. Ed. 2010, 50, 405. (d) Shang, S.; Tan,
Z.; Danishefsky, S. J. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5986.
Org. Lett., Vol. 14, No. 6, 2012
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The synthesis of modified glutamine started from com-
mercially available L-aspartic acid, which was converted
into aldehyde 1 in three steps according to a previously
reported procedure.23 Subsequently, the Passerini three-
component reaction was carried out between precursors 1,
2,4,6-trimethoxybenzyl isocyanide (Tmob-NC) 2, and bro-
moacetic acid. The 4-acyloxy substituted Gln derivative 3
was successfully obtained in 89% yield. Treatment of the
Passerini product3 with thiourea under basic conditions to
remove the bromoacetyl group gave Gln derivative 4 with
the free hydroxyl. Subsequently, mesylation was carried
out to give derivative 5, followed by nucleophilic attack
on the γ-position with thioacetic acid, which resulted in
γ-acylthio Gln 6. Hydrolysis of the acetyl group using
sodium hydroxide followed by trityl (Trt) protection yield-
ed the fully protected γ-mercaptoglutamine 7. Efforts to
directly introduce the Trt group by reacting the mesylated
derivative with Trt thiol failed to give the desired product.
Final basic hydrolysis of the methyl ester gave the γ-(R,S)-
mercapto-L-glutamine (mGln) 8, ready for solid phase
peptide synthesis (SPPS). At this stage, the presence of
two diastereomeric mixtures was not disturbing, as in the
and isolated in 20% yield (Scheme 3). To test the ligation
using mercaptoglutamine, four different model thioester
peptides 9À12 were prepared as previously reported
(Scheme 3)6a and subjected to ligation with peptide 26
(Figure 1 and the Supporting Information). Gratifyingly,
HPLC coupled with MS analysis showed that all ligation
reactions were successful and resulted in the ligation
products 13À16. The isolated yields with thioester peptides
9À12 were 70, 62, 56, and 50% respectively. The ligation
time for reaction completions, as measured by the disap-
pearance of the mGln-WW(23À40) 26 peak, in buffer con-
taining mercaptophenylacetic acid (MPAA) were as anti-
cipated,24 with Gly being the fastest (0.5 h), while Leu was the
slowest (1.5 h). In the case of the slow ligation, a side product
corresponding to dehydration of mGln-WW(23À40) was
observed (∼30%). A preliminary study indicated that this
side product is occurring because of the presence of mGln
residue, which might undergo nitrile formation of its side-
chain. We are currently further characterizing this side re-
action and providing solutions to minimize it.
Next, we examined the desulfurization step to generate
the native Gln residue. Interestingly, when using the metal-
free desulfurization conditions, no product was observed,
Scheme 3. Model Thioester Peptides Used in Our Liga-
tionÀDesulfurization Study with mGln-WW(23À40)
Figure 1. A representative example of ligation (top) and desul-
furization (bottom) of mGln-WW(23À40) 26 with LYRA-
thioester 10 after 1 h. Peak f corresponds to LYRA-MPAA
with the observed mass of 671.2 Da (calcd m/z 671.6 Da); peak g
corresponds to mGln-WW(23À40) 26 with the observed mass of
2478.6 Da (calcd m/z. 2478.7 Da); peak h corresponds to the
dehydration byproduct of mGln-WW(23À40) with the observed
mass of 2460.6 Da; peak i corresponds to the desired ligation
product 14 with the observed mass of 2982.1 Da (calcd m/z
2982.3 Da); peak j corresponds to the desired desulfurization
product 18 with the observed mass of 2950.0 Da (calcd m/z
2950.3 Da); peak * is thiol additive.
desulfurization step, this center will be converted to an
achiral one, which would furnish enantiomerically pure
peptide.
With the thiol modified glutamine derivative 8 in hand,
we incorporated it into a model peptide, mGln-WW-
(23À40) 26 derived from YAP65 WW domain polypep-
tide. This peptide was successfully prepared using SPPS
ꢀ
(23) Hernandez, J. N.; Ramırez, M. A.; Martın, V. S. J. Org. Chem.
´ ´
2002, 68, 743.
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Org. Lett., Vol. 14, No. 6, 2012

completed within 1 h and afforded the desired products
17À20 in high purity and ∼60% yield (Figure 1 and the
Supporting Information).
With the lessons gained from our study on the model
peptides, wethenturnedoureffortstothe synthesisofWW
domain (Scheme 3). The N-terminal fragment, i.e., WW-
(1À21) thioester 21, was prepared using N-methylcysteine-
mediated thioester formation25 in 30% yield (Supporting
Information). With the two fragments in hand, we then
employed our optimized conditions for Gln-NCL to con-
struct the full length WW domain (1À40) 22, which upon a
desulfurization step furnished the native WW domain 23
with an overall yield of 25%.
To verify that the chiral integrity of the R-carbon in
γ-(R,S)-mercaptoglutamine was not affected during any
step of the synthesis, we carried out the following study. We
synthesized the WW domain (1À40) using SPPS, where
either L-Gln or D-Gln was introduced at position 23 to yield
the peptides 24 and 25, respectively. The two isomers were
separated by HPLC, and to our delight, the desulfurization
product 23 overlapped with the L-Gln containing WW
domain (1À40) prepared by SPPS (Figure 2B,C and the
Supporting Information). Moreover, the circular dichroism
(CD) measurement of the ligationÀdesulfurization product
23 and the prepared WW domain (1À40) 24 using SPPS
exhibited similar spectra (Supporting Information). These
spectra were also very similar to the previously reported
one.26 These results further support the integrity of the Gln-
mediated ligation.
In summary, we have presented a straightforward and
high-yielding synthesis of γ-mercaptoglutamine. Using
this γ-mercaptoglutamine, we were able to perform several
Xaa-Gln ligation sites and the synthesis of WW domain. In
these cases, the desulfurization using nickel boride was
efficient, contrary to metal-free desulfurization that led
mainly to unidentified byproduct. We believe that ligation
at Gln junctions will be useful in the synthesis of several
other proteins, in particular Gln rich proteins such as
proteins containing polyQ repeat. We are currently ex-
ploiting the applicability of this method.
Figure 2. (A) Ligation of WW(1À21)-thioester 21 and mGln-
WW(23À40) 26 after 80 min. (B) Desulfurization of mGln-
WW(1À40) 22. (C) WW(1À40) prepared by SPPS with L-Gln at
position 23. Peak a corresponds to mGln-WW(23À40) 26 with
the observed mass of 2478.6 Da (calcd m/z 2478.7 Da); peak b
corresponds to WW(1À21) thioester 21 with the observed mass
of 2317.4 Da (calcd m/z 2317.4 Da); peak c corresponds to
WW(1À21)-MPAA with the observed mass of 2379.0 Da (calcd
m/z 2379.4 Da); peak d corresponds to the dehydration bypro-
duct of mGln-WW(23À40) with the observed mass of 2460.6
Da; peak e corresponds to the desired ligation product WW-
(1À40) 22 with the observed mass of 4689.5 Da (calcd m/z 4690.1
Da); peak f corresponds to the desired desulfurization product,
WW domain 23 with the observed mass of 4657.1 Da (calcd m/z
4658.1 Da); peak g corresponds to WW domain prepared by
SPPS with L-Gln at position 23 with the observed mass of 4657.1
Da (calcd m/z 4658.1 Da); peak * is thiol additive.
and the purified ligation product was abolished to give
products with unidentified masses. This could arise from
undesired reactivity of the formed radical on the γ-position
during the desulfurization step. Consequently, we turned
our attention to the nickel boride desulfurization
conditions,2 which do not involve radical intermediates.
To our delight, under these conditions the reaction was
Acknowledgment. We gratefully acknowledge the
Israel Ministry of Science and Technology for their financial
support (A.B. and P.S.). We also wish to thank Prof. Hilal
A. Lashuel and Dr. Loay Awad from the Swiss Federal
Institute of Technology-Lausanne (EPFL) for their valu-
able discussions.
Supporting Information Available. Experimental pro-
cedures and spectroscopic data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Sci. U. S. A. 1999, 96, 10068.
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Haj-Yahya, M.; Dawson, P. E.; Brik, A. Org. Biomol. Chem. 2010, 8, 2392.
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The authors declare no competing financial interest.
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