Native chemical ubiquitination using a genetically incorporated azidonorleucine

Article abstract of DOI:10.1039/c4cc03721a

A robust chemical ubiquitination method was developed. The method employed a genetically incorporated azidonorleucine as an orthogonal lysine precursor for the installation of a Gly residue bearing an Nα-auxiliary which mediated the ligation between ubiquitin(1-75)-thioester and the target protein. To demonstrate our methodology, a model protein, K48-linked diubiquitin, was synthesized with an overall yield of 35%. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03721a

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Native chemical ubiquitination using a genetically
incorporated azidonorleucine†
Cite this: Chem. Commun., 2014,
50, 7971
Renliang Yang,‡ Xiaobao Bi,‡ Fupeng Li, Yuan Cao and Chuan-Fa Liu*
Received 15th May 2014,
Accepted 4th June 2014
DOI: 10.1039/c4cc03721a
www.rsc.org/chemcomm
A robust chemical ubiquitination method was developed. The method to synthesize K6- and K29-linked diubiquitins.7 More recently,
employed a genetically incorporated azidonorleucine as an ortho- Cropp and Fushman modified the approach and applied it to the
gonal lysine precursor for the installation of a Gly residue bearing an synthesis of more complicated oligo-ubiquitins.8 Recently, Brik et al.
Na-auxiliary which mediated the ligation between ubiquitin(1–75)- also reported the expeditious synthesis of ubiquitinated peptides
thioester and the target protein. To demonstrate our methodology, employing orthogonal protection and chemical ligation.9 All these
a model protein, K48-linked diubiquitin, was synthesized with an methods generated ubiquitin conjugates with the native linkage.
overall yield of 35%.
Methods for preparing ubiquitin conjugates with non-native linkages
including a disulfide bond,10 oxime,11 triazole,12 thioether,13 as well as
Ubiquitination is one of the most important protein posttransla- an isopeptide bond with the C-terminal Gly76 mutated to Cys14 or
tional modifications in eukaryotic cells and is involved in almost Ala15 and an isopeptide bond with Ne-methylation14b,16 have also
all of the cellular processes, including protein degradation and been reported. While these non-native ubiquitin conjugates are
the regulation of gene expression.1 It refers to the linking of the useful for many studies, they do not reflect all the genius aspects
C-terminus of ubiquitin protein (76 amino acids) to the lysine side of the physiological properties of their native counterparts.
chain of the target protein through an isopeptide bond. To study
Among all these currently developed methods for synthesizing
the physiological roles of ubiquitination, it is crucial to generate native Ub-proteins, native chemical ligation17 as well as its
homogeneously ubiquitinated proteins. Biologically, ubiquitina- extended version – Na-auxiliary-mediated ligation18 – remain the
tion is achieved through the consecutive action of three enzymes, best choice due to their chemoselective and protection-free nature.
ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes Various ubiquitin conjugates have been created through chemo-
(E2) and ubiquitin ligases (E3).1 Due to the difficulties in identifying selective ligation between aubiquitin thioesterand thetarget protein
or isolating the substrate specific ligases, enzymatic ubiquitination of which the lysine side chain bears a ligatable functionality, such as
in vitro often has a limited practical value. Chemical ubiquitination a g- or a d-thiol group,3–6 or a Gly with an Na-auxiliary2 or Cys14,15 on
offers a better solution. Since the first report of Na-auxiliary the e-N. These existing ligation-based ubiquitination methods
mediated ubiquitination by Muir et al.,2 several different chemical shared a common feature that all these ligation functionalities are
ubiquitination approaches have been developed. Our group, pre-installedatthedesiredpositionduringtheproductionofpeptide
Brik’s group and Ovaa’s group reported the chemical synthesis or protein substrates through solid-phase peptide synthesis (SPPS)
of ubiquitinated peptides and proteins through g- or d-thiolysine or genetic incorporation. The pre-installation synthetic steps
mediated chemical ligations.3–5 Later, Chin et al. reported the are usually laborious and limit the overall yield. Therefore, we
genetic incorporation of d-thiolysine derivatives into recombi- have been actively seeking an alternative chemical ligation-based
nant proteins, which avoided the laborious work of installing ubiquitination method in which the ligatable functionality is
thiolysine into proteins through peptide synthesis and chemical post-synthetically or post-translationally installed through mani-
ligation.6 The same group also reported a genetically encoded pulating the full-length target protein.
orthogonal protection and activated ligation (GOPAL) approach
The successful site-specific and post-synthetic installation of the
ligatable functionality relies on distinguishing the lysine involved in
ubiquitination from all the others which are not involved. We
realized that azidonorleucine (Anl) can serve this purpose. Recently,
azide has been recognized by peptide chemists as a lysine side
chain protection group for peptide condensation.19 However,
this application has not been performed on recombinant
Division of Structural Biology and Biochemistry, School of Biological Sciences,
Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
E-mail: cfliu@ntu.edu.sg; Fax: +65 67953021
† Electronic supplementary information (ESI) available: Detailed experimental
procedures; NMR, HPLC and MS data. See DOI: 10.1039/c4cc03721a
‡ These two authors contributed equally to this work.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 7971--7974 | 7971

View Article Online
Communication
ChemComm
Fig. 1 C18 analytical HPLC (A) and deconvoluted ESI-MS (B) of the key
intermediates during the installation of auxiliary to the side chain of
ubiquitin K48. (A): (a) purified ub 1; (b) crude ub 5 (peak ii: ub 5; peak i:
amine-form ub 1); (c) purified ub 5; (d) purified ub 6. Gradient: 0–80%
buffer B (90% acetonitrile, 10% H2O containing 0.045% TFA) in buffer A
(H2O containing 0.045% TFA) for 40 min. (B) (a–f): Deconvoluted ESI-MS
of ub 1–6. Average mass of 1, calculated 8617.70, observed 8618.0;
average mass of 2: Boc8, calculated 9418.62, found 9417.5, Boc7, calcu-
lated 9318.50, found 9317.8; average mass of 3: Boc8, calculated 9392.63,
found 9391.8, Boc7, calculated 9292.51, found 9292.4; average mass of 4:
calculated 9557.84; found 9557.2 (only Boc7 product was observed);
average mass of 5: calculated 8857.03; found 8856.9; average mass of
6: calculated 8845.02; found 8845.2.
Scheme 1 Synthesis of K48-linked diubiquitin through the combination of
genetic Anl incorporation and Na-auxiliary-mediated native chemical ligation.
Reagents and conditions: (i) Boc anhydride, DIEA, DMSO; (ii) 1 M TCEP in H2O,
DMSO; (iii) 9, DIEA, DMSO; (iv) TFA/TIS/H2O (95/2.5/2.5), 56% (four steps);
(v) 6 M GdnÁHCl, 0.2 M phosphate, 0.4 M MeONH2, pH 4.0, 85%; (vi) Ub(1–75)-
MES, 6 M GdnÁHCl, 0.2 M phosphate, 25 mM TCEP, 25 mM MPAA, pH 8.0,
85%; (vii) TFA/TIS/H2O (95/2.5/2.5), 86%.
protein expression within E. coli cells, two extra residues Ala and
Ser were introduced after the initiating residue. The expression of
ub 1 was done with MetRSL13A in the presence of 1 mM Anl. After
purification, homogeneous ub 1 was obtained with a yield of
proteins despite the successful genetic incorporation of Anl reported 10 mg LÀ1. Electrospray ionization mass spectrometry (ESI-MS)
long time ago.20 Previously, the genetically incorporated Anl was analysis of the protein confirmed that the initiating Anl had been
solely used as a source of azide functionality for protein labeling via completely removed (Fig. 1).
click chemistry.20 Here, we propose that the genetically incorporated
To install the ligation auxiliary at the K48 side chain of ub,
Anl can also serve as a temporary and orthogonal lysine precursor in all the free amines of ub 1 were first protected using (Boc)2O.
recombinant proteins for chemical ubiquitination (Scheme 1). To Under the conditions used (ESI†), the reaction was completed
do this, all the other amines in the substrate protein which are not in 1 h at room temperature and the crude ub 2 product was
involved in ubiquitination are first protected with Boc. A reduction obtained using diethyl ether precipitation. MS data (Fig. 1B
reaction then converts the Anl residue to Lys onto which a ligatable trace b) showed the presence of 7 or 8 Boc groups on the
functionality (9, Scheme 1) can then be installed. Subsequently, site- product. There are seven free amines in 1 and the eighth Boc
specific ubiquitination can be achieved through auxiliary-mediated group might have been added on the His residue in ub. It is
ligation (Scheme 1). To illustrate our methodology, a model protein, worth mentioning that, when an over-excessive amount of DIEA
K48-linked diubiquitin, was synthesized.
was used, a significant side product with 17–18 Da less than the
The synthesis of K48-linked diubiquitin starts with the expected product was observed (data not shown). This was
expression of the base ubiquitin with K48 replaced by Anl (ub 1). possibly due to deamination or dehydration under the over-
Anl has been successfully incorporated into proteins through alkaline conditions. After Boc protection, the azide group of Anl
an engineered methioninyl-tRNA synthetase (MetRS) in Met- in ub 2 was reduced to amine by tris(2-carboxyethyl)phosphine
auxotrophic E. coli cells.20 It has been reported that MetRS with hydrochloride (TCEP) in DMSO. The reduction reaction was
a single mutation (L13G) is sufficient for the efficient Anl finished in 3 h at room temperature as confirmed by ESI-MS
incorporation. In our study, we found that MetRS with L13A (Fig. 1B trace c). The reduced product ub 3 was ether-precipitated
mutation (MetRSL13A) could also catalyze the incorporation of and used directly for introducing the ligation auxiliary using
Anl efficiently. There are no other Met residues except the compound 9 (Scheme 1). After 45 min, the crude ub 4 product
initiator Met present in the ubiquitin sequence. To express was obtained by ether precipitation (Fig. 1B trace d). Finally,
ub 1 (Scheme 1), the codon coding for K48 was mutated to that global Boc deprotection was done on the crude ub 4 using TFA
for Met. To ensure that the initiating Anl can be removed after for 20 min. After ether precipitation, the crude deprotection
7972 | Chem. Commun., 2014, 50, 7971--7974
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
product was analyzed by C18 analytical HPLC (Fig. 1A, trace b)
which showed an excellent purity profile of the desired product
5 with only a small amount of the amine form of ub 1 as a
noticeable side product. After HPLC purification, 4.9 mg of 5
was obtained. The overall yield for the first four steps was 56%.
This good yield is mostly due to that only one HPLC purifica-
tion step was employed in the 4-step process of synthesizing 5.
The analytical HPLC and deconvoluted ESI-MS of 5 are shown
in Fig. 1 (panel A, trace c and panel B, trace e).
After the installation of the auxiliary group, the thiazolidine
protection group of 5 was removed by methoxylamine. So
2.9 mg of 5 was dissolved in 750 mL of the reaction solution
(6 M GdnÁHCl, 0.2 M phosphate, 0.4 M MeONH2, pH 4). After
5 h at 37 1C, 2.5 mg of deprotected product 6 was obtained after
HPLC purification (yield 85%) (Fig. 1A trace d and B, trace f).
To form diubiquitin 7, ub 6 was ligated with the 75-aa ubiquitin
thioester ub(1–75)-MES through auxiliary-mediated ligation. The
thioester was generated through the thiolysis of a ubiquitin–intein
fusion protein21 with sodium mercaptoethanesulfonate (MESNa).
For the auxiliary-mediated ligation, initially we chose MESNa as
the thiol additive. 2.0 mg of ub(1–75)-MES and 1.4 mg of ub 6 were
dissolved in 200 mL of ligation buffer (6 M GdnÁHCl, 0.2 M
Fig. 3 (A) SDS-PAGE with coomassie blue staining (lane 1 and 2) and
western blot (lane 3 and 4) of the synthesized K48-linked diubiquitin 8.
Lanes 1 and 3: ub1; lanes 2 and 4: diub8. (B) The raw and deconvoluted
ESI-MS of 8. Average mass calculated 17 138.43; found 17 137.1.
(C) Deubiquitinase assays of 8 performed with IsoT and A20CD monitored
by C8 analytical HPLC. Peak a: 8; peak b: ub 1; peak c: wild type ub.
phosphate, 0.1 M MESNa, 20 mM TCEP, pH 8). At room tempera- the presence of MPAA at such a concentration. After 7 h at room
ture, the ligation reaction was extremely sluggish. After 24 h, less temperature, most ub 6 was consumed to form the ligation
than 20% of the ligation product was formed. The majority of the product. After another 5 h, almost all of the 6 was consumed
ubiquitin thioester were hydrolyzed. After incubation for another (Fig. 2A). After HPLC purification, 2.9 mg ligation product 7 was
24 h, there was no significant improvement in ligation yield (ESI†). obtained with an isolated yield of 85%. Based on these observa-
Based on these observations, we realized that MESNa was not a tions, we concluded that our auxiliary-mediated ligation was very
good thiol additive for our auxiliary-mediated ligation. The use efficient with MPAA as the thiol additive even though the ligation
of thiol additives in NCL has long been practiced22 and more was at a sterically hindered secondary amine.
recently it has been shown that aromatic thiols are more effective
The final step for the synthesis of K48-linked diubiquitin 8 is
additives than alkyl thiols.23 So next, we tested the aromatic thiol to remove the auxiliary. To do this, 2.9 mg of 7 was treated with
4-mercaptophenylacetic acid (MPAA) which is a very efficient thiol a cocktail containing TFA/TIS/H2O (95/2.5/2.5) for 20 min on
additive.23 For a typical ligation reaction, 2.8 mg of ub(1–75)-MES ice. After purification, 2.5 mg of 8 was obtained with an isolated
and 1.7 mg of ub 6 were dissolved in 200 mL of ligation buffer yield of 86%. The SDS-PAGE, ESI-MS and C8 analytical HPLC
(6 M GdnÁHCl, 0.2 M phosphate, 25 mM MPAA, 25 mM TCEP, analyses of 8 were shown in Fig. 3A (lane 2), B and C (trace a).
pH 8). Here we used a minimum concentration of MPAA to avoid The overall yield for our seven-step diubiquitin synthesis was
the possible overlap of the MPAA peak and ubiquitin peaks on about 35%, which makes our approach one of most efficient
HPLC profiles. To our delight, the ligation underwent efficiently in methods for chemical diubiquitin synthesis.
To generate native-folded K48-linked diubiquitin, the chemically
synthesized diubiquitin 8 was refolded through dialysis (ESI†).
To test whether the refolded diubiquitin was biologically active,
the diubiquitin was analyzed by western blot using ubiquitin
monoclonal antibody P4D1. A single band corresponding to the
diubiquitin was detected in western blot (Fig. 3A). Deubiquitinase
assays with two ubiquitin deconjugating enzymes, isopeptidase T
(IsoT) and A20 catalytic domain (A20CD), were also performed
(Fig. 3C; ESI†). It was observed that with a 350 : 1 substrate-to-
enzyme ratio, IsoT could effectively hydrolyze almost all the K48
diubiquitin in 2 h at 37 1C. Compared to IsoT, A20CD was found to
be less efficient in hydrolyzing K48 diubiquitin. With a substrate
enzyme ratio of 24 : 1, only about 60% of the diubiquitin was
hydrolyzed by A20CD after 2 h at 37 1C. After incubation for another
Fig. 2 (A) C8 analytical HPLC monitored ligation reaction between ub 6
and ub(1–75)-MES with MPAA as the thiol additive at 0 h, 7 h and 12 h. Peak
a: at 0 h and 7 h: mixture of 6 and ub(1–75)-MES; at 12 h, only ub(1–75)-
MES remaining; peak b: ub(1–75)-OH; peak c: ligation product 7. (B) The
raw and deconvoluted ESI-MS of 7. Average mass calculated 17 334.7;
found 17 333.6.
2 h, about 85% of the diubiquitin was hydrolyzed by A20CD
.
In conclusion, we have developed a novel and efficient native
chemical ligation-based method for protein ubiquitination.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 7971--7974 | 7973

View Article Online
Communication
ChemComm
T. Van Den Bergh, E. Snip, M. Lutz, F. El Oualid and H. Ovaa, Chem.
Sci., 2013, 4, 4494–4498.
6 S. Virdee, P. B. Kapadnis, T. Elliott, K. Lang, J. Madrzak,
D. P. Nguyen, L. Riechmann and J. W. Chin, J. Am. Chem. Soc.,
2011, 133, 10708–10711.
7 S. Virdee, Y. Ye, D. P. Nguyen, D. Komander and J. W. Chin, Nat.
Chem. Biol., 2010, 6, 750–757.
Different from all the previously reported methods, ubiquitin
ligation is facilitated by an auxiliary which is installed on a
recombinant protein containing a genetically incorporated Anl.
The method combines the advantageous features of Muir’s
Na-auxiliary-mediated site-specific ubiquitination approach and
Chin’s GOPAL approach. The key ubiquitination step in our
approach is based on the highly efficient and selective NCL
which allows the use of completely unprotected reaction partners
for ligation in aqueous buffer. The only step that requires
protection in our scheme is the installation of the auxiliary group
on the substrate protein. This is different from the GOPAL
approach in which the key ubiquitination reaction step involves
Ag+-mediated activation of a C-terminal thioester24 in the acyl
ubiquitin for condensation with the amine partner in DMSO and
requires protection of all the uninvolved amines in both reaction
partners, and the large size of the reaction components also
limits the efficiency of condensation. Our work shows an inter-
esting new application of the Anl genetic incorporation method.
A limitation of our approach lies with that Anl would substitute
for all Met residues in the target protein. Nevertheless, Met is a
rare amino acid and can often be replaced by other amino acids
such as Leu without affecting protein function.25 The synthesis of
K48-linked diubiquitin with native isopeptide linkage at an over-
all yield of 35% demonstrates the robustness of our approach.
This, coupled with the relative easiness to express Anl-containing
proteins, makes our method an excellent addition to the list of
chemical ubiquitination methods which together have made the
difficult-to-make ubiquitinated proteins more accessible for the
concerned research community.
˜
8 C. Castaneda, J. Liu, A. Chaturvedi, U. Nowicka, T. A. Cropp and
D. Fushman, J. Am. Chem. Soc., 2011, 133, 17855–17868.
9 K. S. Ajish Kumar, L. Spasser, S. Ohayon, L. A. Erlich and A. Brik,
Bioconjugate Chem., 2011, 22, 137–143.
10 (a) C. Chatterjee, R. K. McGinty, B. Fierz and T. W. Muir, Nat. Chem. Biol.,
2010, 6, 267–269; (b) J. Chen, Y. Ai, J. Wang, L. Haracska and Z. Zhuang,
Nat. Chem. Biol., 2010, 6, 270–272; (c) F. Meier, T. Abeywardana, A. Dhall,
N. P. Marotta, J. Varkey, R. Langen, C. Chatterjee and M. R. Pratt, J. Am.
Chem. Soc., 2012, 134, 5468–5471; (d) B. Fierz, C. Chatterjee,
R. K. McGinty, M. Bar-Dagan, D. P. Raleigh and T. W. Muir, Nat. Chem.
Biol., 2011, 7, 113–119.
11 A. Shanmugham, A. Fish, M. P. A. Luna-Vargas, A. C. Faesen, F. El Oualid,
T. K. Sixma and H. Ovaa, J. Am. Chem. Soc., 2010, 132, 8834–8835.
12 (a) S. Eger, M. Scheffner, A. Marx and M. Rubini, J. Am. Chem. Soc.,
2010, 132, 16337–16339; (b) N. D. Weikart and H. D. Mootz, Chem-
BioChem, 2010, 11, 774–777; (c) S. Sommer, N. D. Weikart,
A. Brockmeyer, P. Janning and H. D. Mootz, Angew. Chem., Int.
Ed., 2011, 50, 9888–9892.
13 (a) E. M. Valkevich, R. G. Guenette, N. A. Sanchez, Y.-C. Chen, Y. Ge
and E. R. Strieter, J. Am. Chem. Soc., 2012, 134, 6916–6919;
(b) V. H. Trang, E. M. Valkevich, S. Minami, Y.-C. Chen, Y. Ge and
E. R. Strieter, Angew. Chem., Int. Ed., 2012, 51, 13085–13088.
14 (a) X. Li, T. Fekner, J. J. Ottesen and M. K. Chan, Angew. Chem., Int.
Ed., 2009, 48, 9184–9187; (b) N. Haj-Yahya, M. Haj-Yahya,
˜
C. A. Castaneda, L. Spasser, H. P. Hemantha, M. Jbara, M. Penner,
A. Ciechanover, D. Fushman and A. Brik, Angew. Chem., Int. Ed.,
2013, 52, 11149–11153.
¨
15 (a) R. K. McGinty, M. Kohn, C. Chatterjee, K. P. Chiang, M. R. Pratt
and T. W. Muir, ACS Chem. Biol., 2009, 4, 958–968; (b) B. Fierz,
S. Kilic, A. R. Hieb, K. Luger and T. W. Muir, J. Am. Chem. Soc., 2012,
134, 19548–19551.
16 M. Haj-Yahya, N. Eltarteer, S. Ohayon, E. Shema, E. Kotler, M. Oren
and A. Brik, Angew. Chem., Int. Ed., 2012, 51, 11535–11539.
17 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. H. Kent, Science,
1994, 266, 776–779.
18 (a) P. Botti, M. R. Carrasco and S. B. H. Kent, Tetrahedron Lett., 2001,
42, 1831–1833; (b) D. W. Low, M. G. Hill, M. R. Carrasco, S. B. H. Kent
and P. Botti, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 6554–6559;
(c) J. Offer, C. N. C. Boddy and P. E. Dawson, J. Am. Chem. Soc., 2002,
124, 4642–4646; (d) J. Offer, Biopolymers, 2010, 94, 530–541.
This work is supported by the Agency for Science, Technol-
ogy and Research (A*STAR) of Singapore (grant No. SERC 112
120 2017) and Nanyang Technological University.
Notes and references
1 (a) M. Hochstrasser, Nat. Cell Biol., 2000, 2, E153–E157; (b) C. M. Pickart,
Annu. Rev. Biochem., 2001, 70, 503–533; (c) O. Kerscher, R. Felberbaum 19 (a) H. Katayama, H. Hojo, T. Ohira and Y. Nakahara, Tetrahedron Lett.,
and M. Hochstrasser, Annu. Rev. Cell Dev. Biol., 2006, 22, 159–180;
(d) M. Hochstrasser, Nature, 2009, 458, 422–429; (e) Z. J. Chen and
2008, 49, 5492–5494; (b) H. Katayama, H. Hojo, I. Shimizu, Y. Nakahara
and Y. Nakahara, Org. Biomol. Chem., 2010, 8, 1966–1972.
L. J. Sun, Mol. Cell, 2009, 33, 275–286; ( f ) F. Ikeda, N. Crosetto and 20 (a) A. J. Link, M. K. S. Vink and D. A. Tirrell, J. Am. Chem. Soc., 2004,
I. Dikic, Cell, 2010, 143, 677–681.
126, 10598–10602; (b) A. J. Link, M. K. S. Vink, N. J. Agard,
J. A. Prescher, C. R. Bertozzi and D. A. Tirrell, Proc. Natl. Acad. Sci.
U. S. A., 2006, 103, 10180–10185; (c) D. M. Abdeljabbar, T. J. Klein,
S. Zhang and A. J. Link, J. Am. Chem. Soc., 2009, 131, 17078–17079;
(d) J. T. Ngo, J. A. Champion, A. Mahdavi, I. C. Tanrikulu,
K. E. Beatty, R. E. Connor, T. H. Yoo, D. C. Dieterich,
E. M. Schuman and D. A. Tirrell, Nat. Chem. Biol., 2009, 5, 715–717.
2 (a) C. Chatterjee, R. K. McGinty, J.-P. Pellois and T. W. Muir, Angew.
Chem., Int. Ed., 2007, 46, 2814–2818; (b) R. K. McGinty, J. Kim,
C. Chatterjee, R. G. Roeder and T. W. Muir, Nature, 2008, 453,
812–816.
3 (a) R. Yang, K. K. Pasunooti, F. Li, X.-W. Liu and C.-F. Liu, J. Am.
Chem. Soc., 2009, 131, 13592–13593; (b) R. Yang, K. K. Pasunooti,
F. Li, X.-W. Liu and C.-F. Liu, Chem. Commun., 2010, 46, 7199–7201. 21 T. W. Muir, D. Sondhi and P. A. Cole, Proc. Natl. Acad. Sci. U. S. A.,
4 (a) K. S. A. Kumar, M. Haj-Yahya, D. Olschewski, H. A. Lashuel and 1998, 95, 6705–6710.
A. Brik, Angew. Chem., Int. Ed., 2009, 48, 8090–8094; (b) K. S. A. Kumar, 22 J. P. Tam, Y.-A. Lu, C.-F. Liu and J. Shao, Proc. Natl. Acad. Sci. U. S. A.,
L. Spasser, L. A. Erlich, S. N. Bavikar and A. Brik, Angew. Chem., Int. Ed., 1995, 92, 12485–12489.
2010, 49, 9126–9131; (c) K. S. A. Kumar, S. N. Bavikar, L. Spasser, T. Moyal, 23 E. C. B. Johnson and S. B. H. Kent, J. Am. Chem. Soc., 2006, 128,
S. Ohayon and A. Brik, Angew. Chem., Int. Ed., 2011, 50, 6137–6141. 6640–6646.
5 (a) F. El Oualid, R. Merkx, R. Ekkebus, D. S. Hameed, J. J. Smit, A. De 24 S. Aimoto, Biopolymers, 1999, 51, 247–265.
Jong, H. Hilkmann, T. K. Sixma and H. Ovaa, Angew. Chem., Int. Ed., 25 H. Yang, L. Liua, M. Wang, J. Li, N. S. Wang, G. Du and J. Chen,
2010, 49, 10149–10153; (b) R. Merkx, G. De Bruin, A. Kruithof,
Appl. Environ. Microbiol., 2012, 78, 7519–7526.
7974 | Chem. Commun., 2014, 50, 7971--7974
This journal is ©The Royal Society of Chemistry 2014