Chemical synthesis of N-peptidyl 2-pyrrolidinemethanethiol for peptide ligation

Article abstract of DOI:10.1016/j.tetlet.2013.05.013

Peptides carrying a C-terminal 2-pyrrolidinemethanethiol (PMT) unit were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS), and were shown to ligate efficiently with cysteinyl-peptides. This novel PMT-mediated ligation tolerated many different C-terminal residues and was successfully applied to a one-pot N-to-C sequential ligation reaction and the semi-synthesis of lysine 16 acetylated histone H4, demonstrating the utility of the method in peptide and protein synthesis.

Full text of DOI:10.1016/j.tetlet.2013.05.013

Tetrahedron Letters 54 (2013) 3777–3780
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Chemical synthesis of N-peptidyl 2-pyrrolidinemethanethiol for peptide ligation
⇑
Renliang Yang, Le Qi, Yanling Liu, Yingjie Ding, Milton Sheng Yi Kwek, Chuan-Fa Liu
Structural Biology and Biochemistry Division, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptides carrying a C-terminal 2-pyrrolidinemethanethiol (PMT) unit were synthesized using 9-fluore-
nylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS), and were shown to ligate efficiently
with cysteinyl-peptides. This novel PMT-mediated ligation tolerated many different C-terminal residues
and was successfully applied to a one-pot N-to-C sequential ligation reaction and the semi-synthesis of
lysine 16 acetylated histone H4, demonstrating the utility of the method in peptide and protein synthesis.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 10 March 2013
Revised 24 April 2013
Accepted 3 May 2013
Available online 13 May 2013
Keywords:
2-Pyrrolidinemethanethiol
Peptide ligation
N?S acyl transfer
Protein synthesis
Thioester
a
Peptide C -thioesters are the key building blocks for many con-
vergent peptide synthesis strategies, including the well-known na-
tive chemical ligation1 (NCL), traceless Staudinger ligation,2 and
Ag+-mediated thioester ligation.3 Due to the importance of such
molecules, the chemical synthesis of thioesters has become a sig-
nificant research topic in peptide chemistry.4 Typically, peptide
thioesters are prepared using tert-butyloxycarbonyl-(Boc) based
solid-phase peptide synthesis (SPPS) whereby the peptide chain
is assembled directly on a thiol linker.5 Despite the effectiveness
of this strategy, peptide thioesters bearing acid-sensitive modifica-
tions such as glycosylations and phosphorylations are difficult to
access using Boc-chemistry due to the repeated use of TFA during
deprotection, and the use of HF during the final cleavage step. In
addition, the hazardous HF represents a deterring element to many
research laboratories. Alternatively, many strategies based on 9-
fluorenylmethoxycarbonyl (Fmoc) chemistry have been developed
for the synthesis of peptide thioesters.4,6–11 The direct synthesis of
thioesters using Fmoc chemistry was enabled by revising the Fmoc
deprotection strategy through the replacement of piperidine with
less nucleophilic bases.6 Nevertheless, aminolysis of the thioester
linkage, especially for the first few cycles and racemization of the
C-terminal amino acid residue remain problematic. Considerable
efforts have been applied for the development of methods for the
synthesis of latent thioester precursors, which are compatible with
standard Fmoc chemistry. These methods include the activation of
protected peptides in solution or on resins,7 the use of thiol labile
safety-catch sulfonamides,8a–c acylurea,8d pyroglutamyl imides,8e
peptide azide,8f and peptidyl-N-acetylguanidine,8g and the genera-
tion of thioesters through O?S9 or N?S10 acyl transfer.
In our efforts to develop novel and convenient Fmoc-based
methods for the preparation of peptide thioesters, we are particu-
larly focused on the N?S acyl transfer reaction.10 The process is
mechanistically reminiscent of intein-mediated protein splicing12
and is essentially the reverse of peptide ligation. Under normal
conditions, the ligation reaction is favored over the thioester for-
mation process. Several methods have been developed to drive
the process in the direction of thioester formation. One of the
strategies to promote N?S acyl transfer is to make this happen
at a tertiary amide rather than a secondary amide under mild
acidic conditions. In such a system, the reverse re-ligation process
is slowed down as a protonated secondary amine is involved,
which enables the transiently presented thioester intermediate to
be captured by either excess thiol additives to generate a thioester,
or by an in situ cysteine peptide to form the ligation product di-
rectly. Based on this rationale, we proposed that a peptide carrying
a C-terminal 2-pyrrolidinemethanethiol (PMT) unit could serve as
a thioester equivalent and ligate in situ with a cysteine peptide via
N?S acyl transfer. The PMT linker can be easily prepared on
large-scale quantity from commercially available H-(L)-prolinol.
The synthesis of PMT peptides is fully compatible with standard
Fmoc solid-phase peptide synthesis (SPPS) on a 2-chlorotrityl
chloride (2-ClTrt) resin.
To test our proposal, we first synthesized the PMT linker and
loaded it onto 2-ClTrt resin (Scheme 1). To synthesize the linker,
H-(L)-prolinol (1) was first protected with Boc anhydride. After
activating the hydroxyl group of the alcohol 2 with methanesulfo-
nyl chloride (MsCl), the obtained mesyl ester 3 was substituted
with potassium thioacetate through an SN2 reaction. The acetyl
⇑
Corresponding author. Tel.: +65 63162867; fax: +65 67953021.
E-mail address: cfliu@ntu.edu.sg (C.-F. Liu).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.05.013

3778
R. Yang et al. / Tetrahedron Letters 54 (2013) 3777–3780
(Boc)2O, Et3N
CH2Cl2
synthesis and peptide ligation. Under low-power microwave irra-
MsCl, Et3N
CH2Cl2
diation, the ligation rate between 6 and 7 was dramatically accel-
erated. After 7 h, the yield had reached 93%. For the ligation
between 6 and 7, two side products with a mass of 634.45 and
676.4, respectively, were observed on many occasions (peak d
and e in Fig. 2). They were likely derived from the cysteinyl-pep-
tide 7 as they were also present when other PMT peptides with dif-
ferent C-terminal residues were ligated with 7. The nature of these
side products is unknown.
O
quantitative
O
OH
OH
>99%
S
N
Boc
N
Boc
N
H
O
3
2
1
KSAc,
DMF, 83%
NaOH, H2O,
MeOH, 95%
TFA, TIS,
tBuSH, CH2Cl2
S
N
SH
N
O
Boc
Boc
5
4
To optimize the pH values for PMT-mediated ligation, the liga-
tion between 6 and 7 at different pH (4, 5, 6, and 7) was compared
(Supplementary data, Figs. S5 and S6). The ligation was performed
in the presence of MMA under microwave irradiation. After 7 h, the
ligation yields at pH 4, 5, 6, and 7 were 81%, 93%, 87%, and 67%,
respectively. These results indicated that our ligation system pre-
ferred mild acidic conditions (pH 5–6). However, the reaction at
pH 7 seemed to give a lower amount of the two side products men-
tioned above.
SH
S
Cl
+
N
N
Cl
H2
H2
Scheme 1. Chemical synthesis of the PMT linker and loading of the linker onto
2-ClTrt resin.
To evaluate the effect of the thiol additives, three different thi-
ols were tested for the ligation between 6 and 7 at pH 5.0 and
microwave irradiation (Supplementary data, Figs. S7 and S8). It
was found that the ligation rate was similar when MMA or 4-mer-
captophenyl acetic acid (MPAA) was used. Both reactions were
nearly complete within 7 h. However, the ligation with MPAA
showed less clean HPLC profiles and more side products, possibly
originating from 7, were observed. When sodium 2-mercaptoeth-
ylsulfonate (MESNa) was used, the yield was only 66% after 7 h.
These results showed that MMA was a better thiol additive for
our ligation system.
group of compound 4 was removed by saponification to give N-
protected PMT 5. The overall yield of compound 5 was 78%, there-
by allowing the possibility of preparing the linker in large quantity.
For the loading of the linker to the 2-ClTrt resin, compound 5 was
deprotected, dissolved in dry CH2Cl2 and added to the resin which
had been pretreated with 30% TFA in CH2Cl2. The loading was com-
plete within 1 h (see Supplementary data for the details of the
loading process). Fmoc-Gly-OH was then coupled onto the resin.
After removing the Fmoc group, a quantitative ninhydrin test13
was performed with the resin to determine its substitution, which
was 0.52 mmol/g. The substitution was comparable to many of the
commercial resins and was suitable for SPPS. A model peptide, H-
LSTEG-PMT (6) was synthesized with the PMT-2-ClTrt resin fol-
lowing standard Fmoc-chemistry. After cleavage, the analytical
HPLC of the crude peptide 6 showed a relatively clean product.
The minor side product with a mass of 56 Da higher was due to
the S-alkylation of 6 by a t-butyl cation during cleavage. The ana-
lytical HPLC and ESI-MS of purified 6 are shown in Figure 1.
To test whether PMT peptides can be used for chemical ligation,
a study using 6 and H-CAKAFA-NH2 (7) was performed. Peptides 6
In summary, the optimal conditions for PMT-mediated ligations
are microwave irradiation, MMA as the thiol additive and pH 5–6.
The ligation of 6 and 7 under these optimized conditions is shown
in Figure 2.
To test whether the newly developed PMT ligation works for
other non-glycine C-terminal residues, peptides H-LSTEX-PMT
(X = A, L, F, S, and V) were synthesized in the same way as peptide
6. These peptides were ligated with peptide 7 with MMA as the
thiol additive at pH 5.0 under microwave irradiation (Table 1). Ex-
cept for b-branched Val, the PMT peptides with the other four C-
terminal residues ligated efficiently with peptide 7. These ligations
were complete within 12 h with yields between 80% and 90%. The
ligation between the Val peptide and 7 was slower, and only
reached 29% yield after 12 h.
(1.6 mg, 16.6 mM) and 7 (3.3 mg, 34 mM) were dissolved in 160 lL
of ligation buffer containing 6 M guanidine hydrochloride
(GdnÁHCl), 0.2 M NaOAc, 25 mM tris(2-carboxyethyl)phosphine
(TCEP), and 2% v/v methyl mercaptoacetate14 (MMA), pH 5.0. The
above ligations at various temperatures were compared (Supple-
mentary data, Figs. S3 and S4). The reaction proceeded slowly at
room temperature and the yield was 45% after 24 h. At 37 and
42 °C, the ligation yield reached 83% and 90%, respectively, after
24 h. This indicated that significant activation energy might be re-
quired for N?S acyl transfer to occur. It is well-known that micro-
wave irradiation can promote dramatically the rate of peptide
Figure 2. (A) The C18 analytical HPLC of the ligation between 6 and 7 under the
optimized conditions. Peak a: peptide 7; peak b: peptide 6; peak c: ligation product,
H-LSTEGCAKAFA-NH2; peaks d and e indicating molecular masses of 634.45 and
676.4 as detected by ESI-MS, respectively, are unidentified; asterisk peaks: buffer
content and non-peptide materials. Gradient: 40% of buffer B in buffer A for 40 min.
(B) ESI-MS of the ligation product. Monoisotopic mass, calcd: 1095.54; observed:
[M+H]+ = 1096.43.
Figure 1. (A) C18 analytical HPLC of purified 6. Gradient: 40% of buffer B (90%
aqueous acetonitrile containing 0.045% TFA) in buffer A (H2O containing 0.045%
TFA) for 40 min. (B) ESI-MS of 6. Monoisotopic mass, calcd: 604.29; observed:
[M+H]+ = 605.57.

R. Yang et al. / Tetrahedron Letters 54 (2013) 3777–3780
3779
Table 1
The ligation yield (%) between H-LSTEXaa-PMT peptides and H-CAKAFA-NH2
a
Entry
Xaa
1.5 h
3 h
7 h
12 h
1
2
3
4
5
Ala
Leu
Phe
Ser
Val
21
n.d.
32
24
n.d.
35
39
60
40
12
65
68
85
65
20
81
86
90
82
29
a
Reaction conditions: H-LSTEXaa-PMT (15 mM) and H-CAKAFA-NH2 (30 mM) in
the buffer containing 6 M GdnÁHCl, 0.2 M NaOAc, 25 mM TCEP and 2% v/v MMA, pH
5 under microwave irradiation. The ligation yield is calculated based on HPLC
analysis and the consumption of the PMT peptides. n.d. = not determined.
The conditions for PMT ligation and thioester ligation are rela-
tively orthogonal to each other. Under thioester ligation condi-
tions, PMT peptides prefer the amide form and ligate slowly with
cysteine peptides. Therefore, we proposed a one-pot three-seg-
ment N-to-C sequential ligation strategy15 involving thioester
and PMT peptides. The first step was the thioester-mediated liga-
tion between the N-terminal thioester and central cysteinyl PMT
segment performed at room temperature under mild alkaline con-
ditions. The second ligation was the PMT-mediated ligation be-
tween the first-step ligation product and a C-terminal cysteine
peptide. To test the proposal, a model sequential ligation was per-
formed. For the first ligation, 4.3 mg (14.2 mM) of H-FKLAKF-
SCH2CH2CONH2 (8) and 3.6 mg (14.1 mM) of H-CLSTEG-PMT (9)
Figure 4. (A) C8 analytical HPLC monitored semi-synthesis of H4K16Ac. Peak a:
peptide 10; peak b: H4 C-terminal domain; peak c: ligation product. B) MALDI-TOF
MS of the ligation product. Average isotopic mass calcd: 11253.02, [M+H]+ found:
11252.50. (C) MALDI-TOF MS of the S-alkylated product, H4K16Ac. Average isotopic
mass calcd: 11296.09, [M+H]+ found: 11299.05.
we reported the semi-synthesis of H4K16Ac through a traditional
NCL/S-alkylation approach.17 Herein, we synthesized H4K16Ac
through PMT-mediated ligation in a similar manner. First, the H4
N-terminal PMT peptide (10) containing residues 1–19 and
K16Ac was synthesized on a PMT-2-ClTrt resin using Fmoc chem-
istry. In a typical ligation reaction, 3.4 mg of peptide 10 (4 mM) and
were dissolved in 360
lL of buffer containing 6 M GdnÁHCl, 0.2 M
phosphate, 25 mM TCEP, and 2% v/v MMA, pH 8.0. After 2 h at
room temperature, the ligation was complete (Fig. 3A). Without
isolating the ligation product or changing the thiol additive, the
second ligation was initiated by adding 7 mg of peptide 7 to the
mixture and adjusting the pH to 5.5 with dilute hydrochloric acid.
After 5 h under microwave irradiation, the ligation was complete,
to give 7 mg of the final ligation product after purification (isolated
yield 72%). The second step of ligation was also performed at 42 °C.
The reaction was almost complete after 14 h.
5 mg of C-terminal domain (1.33 mM) were dissolved in 400 lL of
ligation buffer containing 6 M GdnÁHCl, 0.2 M NaOAc, 25 mM TCEP,
and 2% v/v MMA, pH 5.0. The ligation proceeded efficiently under
microwave irradiation. After 1 h, over 26% of the C-terminal do-
main was consumed based on C8 analytical HPLC. After 5 h, the
ligation yield reached 67% (Fig. 4). The ligation mixture was sepa-
rated by C4 semi-preparative HPLC and 2.4 mg of ligation product
was obtained in an isolated yield of 40% (calculated based on the C-
terminal domain). To convert the cysteine residue at the ligation
site into a lysine analog, the full length H4 was alkylated with 2-
bromoethylamine according to the previously reported procedure
(Fig. 4).17 2 mg of the final H4K16Ac was obtained (83% yield).
In conclusion, we have developed a novel N-peptidyl PMT-
based method for chemical ligation. This novel method shares
many similarities with the peptidyl N,N-bis(mercaptoethyl)amide
(BMEA) method recently reported by Melnyk’s group10i and our
group.10j Both linkers can be easily synthesized and the prepara-
tions of these peptides are fully compatible with conventional
Fmoc-SPPS. Both ligation methods work efficiently under mild
acidic and microwave irradiation conditions. Both methods can
be applied to one-pot N-to-C sequential three-segment ligations
when they are used in combination with normal peptide thioes-
ters.14,15,18 Despite sharing many common properties, these two
methods do behave differently in certain aspects. For example, un-
like with BMEA peptides, we were unable to isolate the thioester
product when PMT peptides were treated for exchange with an ex-
cess thiol compound (data not shown). Nevertheless, the PMT-
based method is another example of using the N?S acyl transfer
principle for the Fmoc-solid phase synthesis of peptide thioester
precursors or equivalents. The method expands the repertoire of
chemical ligation methods and provides additional choice for pep-
tide and protein synthesis.
To demonstrate that the novel PMT-mediated ligation approach
can serve as a useful tool for protein synthesis, we applied the
strategy to the semi-synthesis of lysine 16 acetylated histone H4
(H4K16Ac). H4K16Ac is one of the important histone post-transla-
tional modifications and has been shown to be involved in the reg-
ulation of chromatin compaction and transcription.16 Previously,
Figure 3. (A) C18 analytical HPLC monitored one-pot N-to-C sequential ligation.
Peak a: peptide 8; peak b: peptide 9; peak c: H-FKLAKF-SCH2COOMe; peak d: first
step ligation product, H-FKLAKFCLSTEG-PMT (H-FG12-PMT); peak e: peptide 7;
peak f: second step ligation product, H-FKLAKFCLSTEGCAKAFA-NH2 (H-FA18-NH2);
peak g: the hydrolysis product, H-FG12-OH. B) The ESI-MS of H-FG12-PMT. [M+2H]2+
obsd: 722.00; monoisotopic mass calcd: 1441.75. (C) The ESI-MS of H-FA18-NH2.
[M+2H]2+ obsd: 967.75; monoisotopic mass calcd: 1933.00.
Acknowledgments
We thank the Ministry of Education and A⁄Star of Singapore
and Nanyang Technological University for financial support.

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R. Yang et al. / Tetrahedron Letters 54 (2013) 3777–3780
Ed. 2011, 50, 7645; (g) Okamoto, R.; Morooka, K.; Kajihara, Y. Angew. Chem., Int.
Supplementary data
Ed. 2012, 51, 191.
9. Warren, J. D.; Miller, J. S.; Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc. 2004,
126, 6576; (b) Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H. Org. Lett. 2004,
6, 4861; (c) Zheng, J.-S.; Cui, H.-K.; Fang, G.-M.; Xi, W.-X.; Liu, L. ChemBioChem
2010, 11, 511; (d) Eom, K. D.; Tam, J. P. Org. Lett. 2011, 13, 2610.
10. (a) Kawakami, T.; Sumida, M.; Nakamura, K.; Vorherr, T.; Aimoto, S. Tetrahedron
Lett. 2005, 46, 8805; (b) Ollivier, N.; Behr, J.-B.; El-Mahdi, O.; Blanpain, A.;
Melnyk, O. Org. Lett. 2005, 7, 2647; (c) Nagaike, F.; Onuma, Y.; Kanazawa, C.;
Hojo, H.; Ueki, A.; Nakahara, Y.; Nakahara, Y. Org. Lett. 2006, 8, 4465; (d) Ohta,
Y.; Itoh, S.; Shigenaga, A.; Shintaku, S.; Fujii, N.; Otaka, A. Org. Lett. 2006, 8, 467;
(e) Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y. Tetrahedron
Lett. 2007, 48, 25; (f) Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1903, 2007, 48;
(g) Tsuda, S.; Shigenaga, A.; Bando, K.; Otaka, A. Org. Lett. 2009, 11, 823; (h)
Kang, J.; Reynolds, N. L.; Tyrrell, C.; Dorin, J. R.; Macmillan, D. Org. Biomol. Chem.
2009, 7, 4918; (i) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O. Org.
Lett. 2010, 12, 5238; (j) Hou, W.; Zhang, X.; Li, F.; Liu, C.-F. Org. Lett. 2011, 13,
386; (k) Sharma, R. K.; Tam, J. P. Org. Lett. 2011, 13, 5176; (l) Zheng, J.-S.; Chang,
H.-N.; Wang, F.-L.; Liu, L. J. Am. Chem. Soc. 2011, 133, 11080.
11. (a) Swinnen, D.; Hilvert, D. Org. Lett. 2000, 2, 2439; (b) Brask, J.; Albericio, F.;
Jensen, K. J. Org. Lett. 2003, 5, 2951.
12. Chong, S.; Mersha, F. B.; Comb, D. G.; Scott, M. E.; Landry, D.; Vence, L. M.;
Perler, F. B.; Benner, J.; Kucera, R. B.; Hirvonen, C. A.; Pelletier, J. J.; Paulus, H.;
Xu, M.-Q. Gene 1997, 192, 271.
13. Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 117,
147.
14. MMA was used as a thiol additive for chemical ligation through N?S acyl
transfer, see: Yang, R.; Hou, W.; Zhang, X.; Liu, C.-F. Org. Lett. 2012, 14, 374.
15. For a recent review, see: Raibaut, L.; Ollivier, N.; Melnyk, O. Chem. Soc. Rev.
2012, 41, 7001.
16. Shogren-Knaak, M. A.; Ishii, H.; Sun, J.-M.; Pazin, M.; Davie, J. R.; Peterson, C. L.
Science 2006, 311, 844.
Supplementary data (experimental details and data for new
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.tetlet.2013.05.013.
References and notes
1. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776.
2. (a) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2, 2141; (b) Nilsson,
B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 1939, 2000, 2; (c) Nilsson, B. L.;
Kiessling, L. L.; Raines, R. T. Org. Lett. 2001, 3, 9.
3. Aimoto, S. Biopolymers 1999, 51, 247.
4. For recent reviews, see: (a) Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem.,
Int. Ed. 2008, 47, 10030; (b) Kang, J.; Macmillan, D. Org. Biomol. Chem. 1993,
2010, 8; (c) Mende, F.; Seitz, O. Angew. Chem., Int. Ed. 2011, 50, 1232.
5. (a) Tam, J. P.; Lu, Y.-A.; Liu, C.-F.; Shao, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
12485; (b) Hackeng, T. M.; Giffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 10068.
6. (a) Li, X. Q.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669; (b) Bu, X.
Z.; Xie, G. Y.; Law, C. W.; Guo, Z. H. Tetrahedron Lett. 2002, 43, 2419.
7. (a) Futaki, S.; Sogawa, K.; Maruyama, J.; Asahara, T.; Niwa, M.; Hojo, H.
Tetrahedron Lett. 1997, 38, 6237; (b) Alsina, J.; Yokumu, T. S.; Albericio, F.;
Barany, G. J. Org. Chem. 1999, 64, 8761; (c) Nakamura, K.; Hanai, N.; Kanno, M.;
Kobayashi, A.; Ohnishi, Y.; Ito, Y.; Nakahara, Y. Tetrahedron Lett. 1999, 40, 515;
(d) Li, L.; Wang, P. Tetrahedron Lett. 2007, 48, 29; (e) Lelièvre, D.; Barta, P.;
Aucagne, V.; Delmas, A. F. Tetrahedron Lett. 2008, 49, 4016; (f) Ficht, S.; Payne,
R. J.; Guy, R. T.; Wong, C. H. Chem. Eur. J. 2008, 14, 3620.
8. (a) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. R.
J. Am. Chem. Soc. 1999, 121, 11684; (b) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi,
A. J. Am. Chem. Soc. 1999, 121, 11369; (c) Mende, F.; Seitz, O. Angew. Chem., Int.
Ed. 2007, 46, 4577; (d) Blanco-Canosa, J. B.; Dawson, P. E. Angew. Chem., Int. Ed.
2008, 47, 6851; (e) Tofteng, A. P.; Sørensen, K. K.; Conde-Frieboes, K. W.; Hoeg-
Jensen, T.; Jensen, K. J. Angew. Chem., Int. Ed. 2009, 48, 7411; (f) Fang, G.-M.; Li,
Y.-M.; Shen, F.; Huang, Y.-C.; Li, J.-B.; Lin, Y.; Cui, H.-K.; Liu, L. Angew. Chem., Int.
17. Allahverdi, A.; Yang, R.; Korolev, N.; Fan, Y. P.; Davey, C. A.; Liu, C.-F.;
Nordenskiöd, L. Nucleic Acids Res. 2011, 39, 1680.
18. Ollivier, N.; Vicogne, J.; Vallin, A.; Drobecq, H.; Desmet, R.; El Mahdi, O.;
Leclercq, B.; Goormachtigh, G.; Fafeur, V.; Melnyk, O. Angew. Chem., Int. Ed.
2012, 51, 209.