HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

Article abstract of DOI:10.1002/anie.201607657

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post-translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

Full text of DOI:10.1002/anie.201607657

Angewandte
Communications
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International Edition: DOI: 10.1002/anie.201607657
German Edition: DOI: 10.1002/ange.201607657
Peptide Synthesis
Hot Paper
HF-Free Boc Synthesis of Peptide Thioesters for Ligation and
Cyclization
Richard Raz, Fabienne Burlina, Mohamed Ismail, Julian Downward, Jiejin Li,
Stephen J. Smerdon, Martin Quibell, Peter D. White, and John Offer*
Abstract: We have developed a convenient method for the
direct synthesis of peptide thioesters, versatile intermediates for
peptide ligation and cyclic peptide synthesis. The technology
uses a modified Boc SPPS strategy that avoids the use of
anhydrous HF. Boc in situ neutralization protocols are used in
combination with Merrifield hydroxymethyl resin and TFA/
TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS
to post-translational modifications that are compatible with the
milder cleavage conditions, demonstrated here with the syn-
thesis of the phosphorylated protein CHK2. Peptide thioesters
give easy, direct, access to cyclic peptides, illustrated by the
synthesis of cyclorasin, a KRAS inhibitor.
P
eptide thioesters are key precursors for the synthesis of
Scheme 1. Applications of peptide thioesters. A) Native chemical liga-
proteins[1] and cyclic peptides[2] (Scheme 1). The demand for
peptide thioesters has increased with the success of native
chemical ligation (NCL). One of the greatest obstacles to
using NCL is the challenging synthesis of peptide thioesters.
Initially, peptide thioesters were prepared directly by tert-
butyloxycarbonyl (Boc) solid phase peptide synthesis (SPPS)
facilitated by the stability of the thioester bond to trifluoro-
acetic acid (TFA), used for Boc deprotection cycles.[3] The
major limitation of Boc SPPS is its requirement for anhydrous
HF for the deprotection and cleavage of the peptide from the
resin. HF requires specialized apparatus and training.[4] This
tion for the coupling of peptides and proteins by linking two
unprotected segments, one a peptide thioester. B) Head-to-tail cycliza-
tion of peptides containing both an N-terminal cysteine and a
C-terminal thioester.
has restricted Boc SPPS to a few laboratories experienced
with handling this extremely hazardous reagent.[5] In addition,
HF is not compatible with the incorporation of many post-
translational modifications. 9-Fluorenylmethoxycarbonyl
(Fmoc) SPPS has become the method of choice for the
routine synthesis of peptides in a large part due to its
avoidance of HF.[6] There has consequently been a consider-
able effort to develop robust Fmoc SPPS methods for the
synthesis of peptide thioesters.
[*] Dr. R. Raz, Dr. M. Ismail, Dr. J. Downward, Dr. J. Li, Dr. S. J. Smerdon,
M. Quibell, Dr. J. Offer
The Francis Crick Institute
1 Midland road, London, NW1 1AT (UK)
E-mail: john.offer@francis.crick.ac.uk
The direct synthesis of peptide thioesters by Fmoc SPPS is
complicated by the reactivity of the thioester bond. Coupling
a fully protected peptide fragment to a thiol in solution,
followed by deprotection remains a popular approach.[7]
Additionally, many ingenious, indirect approaches have
been developed. There are two types: safety catch, examples
of which include sulfonamide,[8] N-acylurea,[9] hydrazide/
azide;[10] and acyl shift, either O,S[11] or N,S.[12]
Nevertheless, Boc SPPS possesses a number of advantages
compared to Fmoc SPPS: higher solubility of Boc amino acids
promotes faster coupling;[13] TFA fully solvates the peptide–
resin at each deprotection cycle, preventing peptide–resin
aggregation and enabling the synthesis of long peptides;[14] in
comparison to Fmoc SPPS, there is much less aspartimide
formation.[15] In addition Boc deprotection is always com-
plete, in contrast to Fmoc where partial deprotection during
aggregation is problematic. Because of these attendant
benefits a combination of Boc SPPS and native chemical
ligation (NCL) has enabled the synthesis of many proteins of
sufficient quality to be crystallized for structural studies.[16]
Dr. F. Burlina
Sorbonne Universitꢀs, UPMC Univ Paris 06, ENS, CNRS
Laboratoire des Biomolꢀcules (LBM)
Paris (France)
and
Dꢀpartement de Chimie, ENS, PSL Research University, UPMC
Univ Paris 06, CNRS, LBM
Paris (France)
Dr. P. D. White
Merck Chemicals
Padge Road, Beeston, Notts, NG9 2JR (UK)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201607657.
ꢁ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
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We therefore decided to explore a modified Boc SPPS
route to peptide thioesters that used a milder acid for final
cleavage. This approach would have the following benefits:
the direct synthesis of peptide thioesters with a single, safer,
cleavage step; the possibility of parallel synthesis and small-
scale cleavages and greater compatibility with post-transla-
tional modifications. The superior quality of peptides syn-
thesised by Boc SPPS was the main factor.[13,17]
There have been many attempts to substitute HF with
trifluoromethanesulfonic acid (TFMSA).[18] However, in
contrast to HF, it is difficult to remove as it is not volatile,
and residual reagent can cause degradation of the peptide
product. A two-step deprotection has been evolved to address
this problem.[19]
TFA/TMSBr (trimethylsilyl bromide) mixtures can cleave
benzyl-based side-chain protection from Asp/Glu/Ser/Thr/
Lys/Tyr/Cys and Mts from arginine.[20] As demonstrated in the
elegant work of the Yajima group TFA/TMSBr has many
notable properties as a cleavage reagent: it is volatile, more so
than TFA, and therefore easily removed by sparging; it
reduces any methionine sulfoxide formed during synthesis;
and benzyl deprotection of aspartyl residues is not accom-
panied by aspartimide formation.[20] It is routinely used in
Fmoc SPPS when stronger deprotection conditions are
required.[21]
Unfortunately, TFA/TMSBr is not a sufficiently strong
acid to achieve complete peptide cleavage from methylben-
zylhydrylamine (MBHA) and 4-(hydroxymethyl)phenylace-
tamidomethyl (PAM) resins and deprotect all the standard
side-chain protection of classical Boc SPPS. However, these
limitations could potentially be overcome by combining TFA/
TMSBr cleavage with Merrifield hydroxymethyl resin, Boc
in situ neutralization protocols[14] and changing some of the
side-chain protection for that identified by the Yajima group
as most compatible: Arg to Mts, Cys to Mob and Merrifieldꢀs
original choice of benzyl for Asp and Glu (Table S1 in the
Supporting Information (SI)). Cyclohexyl had been intro-
duced primarily because of concerns over aspartimide
formation during HF cleavage with benzyl protection,[15b]
not so problematic with TFA/TMSBr cleavage.[20]
The use of Merrifield resin for Boc SPPS was largely
abandoned when 4-(Hydroxymethyl)phenylacetamidomethyl
(PAM) and 4-methylbenzylhydrylamine (MBHA) resins were
introduced, primarily because of reported peptide cleavage
by TFA over the prolonged (20 min) TFA cleavage cycles.[22]
However, contemporary Boc SPPS in situ neutralization
protocols with their shorter, typically, 2 ꢁ 1 min treatment
with TFA per cycle[14] are more suitable. Merrifield resin had
also been associated with other side reactions, notably
capping by trifluoroacetylation and formation of deletion
sequences caused by the presence of aldehyde impurities on
the resin.[23] However, these problems were identified many
years ago, before improvements in the chemical purity of
commercial resins and before the adoption of HBTU and
other uronium coupling agents that do not couple trifluoro-
acetic acid. Consequently, a re-examination of Boc SPPS on
modern preparations of Merrifield resin, with in situ neutral-
ization protocols was timely.
First, the stability of Merrifield resin linked peptides to
TFA was reinvestigated. For this, we used hydroxymethyl
resin derivatized with Fmoc-Gly because Gly is one of the
more acid labile residues[22] and Fmoc provides a good
reporter to monitor loss. The rate of Fmoc-Gly cleavage by
neat TFA was monitored directly by following the release of
Fmoc by UV (Figure 1B). Less than 5% amino acid cleavage
from the resin was observed after 500 min TFA treatment.
The experiment was repeated with Fmoc-Gly attached to
mercaptopropionicacidleucine (MPAL) linker (Figure 1B)
used for thioester synthesis. Chain loss was considerably
slower with the MPAL linker presumably because of the
comparably more hindered terminal leucine residue. In both
cases the loss of amino acid was in general agreement with
previous measurements.[22] Although there was chain loss with
the MPAL linker, at 1% over 500 min, it was suitable for
in situ neutralization cycles (Figure 1B).
Next, we investigated the use of Boc in situ neutralization
cycles on Merrifield resin to see if the notorious capping and
deletion reactions occur under these conditions. Elastin
1 (Table 1) has been synthesised recently by Kent and co-
workers and gave us a direct comparison of yield and purity to
current, optimized, Boc-SPPS.[24] The resin was derivatized
with PBr3 and Fmoc-valine to obviate racemization.[25] Syn-
thesis was carried out manually with in situ neutralization Boc
cycles and the peptide cleaved with TFA/TMSBr/thioanisole/
EDT (1:0.05:0.05:0.025) at room temperature for 1 h. The
Table 1: Yields of peptides synthesized via modified Boc SPPS.
Peptide Sequence
Crude
Isolated
yield [%][a] yield [%][b]
1
2
3
4
5
6
7
8
H-PGVGPGVGV-OH
H-GCCSDPRCRYRCR-OH
H-LAPAV-MPAL
H-LAPAA-MPAL
H-LAPAG-MPAL
H-LYRAF-MPAL
H-LAPAG-MPAA
H-LAPAA-MPAA
H-LAPAQ-MPAA
H-LAPAV-MPAA
H-LAPAT-MPAA
H-LAPAW-MPAA
H-LYRAI-MPAA
H-LYRAL-MPAA
98
77
92
90
86
81
68
84
83
89
86
83
91
82
57
66
81
21
54
50
22
34
16
31
25
36
36
26
28
23
23
24
16
9
10
11
12
13
14
15
16
17
H-LETVSpTQELY-MPAA
H-LKAQADIYKA-MPAA
H-dAlaArgArgArgdNalArgPhe(4-F) 84
dNleGlnTrpThr-MPAA
18
19
H-CdYVYNTRSGWRWYT-MPAA
H-AEQH(DNP)KIVMETVPLKAQA
DIYKA-MPAA
88
51
28
14
20
21
22
H-LEDLRQQLQQAEEALVAKQE
LI-MPAA
H-LEDLRQQLQQAEEALVAKQELI
DKL-MPAA
H-LEDLRQQLQQAEEALVAKQELI
DKLKEEA-MPAA
80
68
64
19
13
10
[a] Yield calculated from resin loading and weight of crude, unpurified
peptide. [b] Isolated yields calculated on dried weight of purified peptide
(including TFA salts).
2
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Figure 1. A) Direct synthesis of peptide thioesters by a modified Boc protocol illustrated for MPAA thioester. i) PBr3/DCM; ii) MPAA/DIEA/DMF;
iii) TFA/TES/H2O; iv) Boc SPPS; v) TFA/TMSBr/thioanisole/EDT (1:0.05:0.05:0.025), 1 h. DCM=dichloromethane, DIEA=diisopropylethylamine,
DMF=dimethylformamide. B) Time course of Fmoc-Gly-OH and Fmoc-Gly-MPAL release from Merrifield resin with TFA, monitored by Fmoc
absorbance at 301 nm. C) Analytical HPLC and MALDI-TOF MS of crude elastin synthesized with in situ neutralization Boc cycles on
hydroxymethyl resin. D) Analytical HPLC and MALDI-TOF MS of crude peptide thioester LAPAV-MPAL.
cleavage mixture was sparged under a stream of nitrogen and
the peptide precipitated from ice-cold ether. No further
peptide was recovered after a repeat cleavage of the resin.
This simple, safe procedure contrasted to the lengthy HF
cleavage and represented a considerable time saving. Success
was reflected in the impressive final isolated yield 81%,
comparable with 61% of Dang et al.[24] Inspection of the
analytical HPLC of the crude peptide (Figure 1C) revealed
no deletion sequences or capping. The high yield and
simplicity of cleavage encouraged us to explore a more
challenging target.
We chose a well-characterized test peptide, a-conotoxin
RgIA 2 (Table 1). Its synthesis has been described in detail.[4a]
It features a challenging sequence, rich in Arg and Cys. We
substituted the Tosyl protection of Arg with Mts and the Meb
protection of Cys with Mob. The side-chain protecting group
selection is shown in Table S1 (SI). Analytical HPLC of the
crude material compared well with that obtained from
conventional Boc SPPS (Figure S1, SI).[4a]
Having proved the strategy with peptide acids we wanted
to test peptide thioesters. Initially, model thioester peptides
were prepared using standard MPAL linker with short test
sequences. The use of a spacer residue such as leucine before
mercaptopropionic acid is proven to increase the final
yield,[3a] although it is also used without.[19b] Several examples
were synthesized and gave products of satisfactory purity by
inspection of HPLC (Figure 1D) and yield (Table 1).
NCL often makes use of the more kinetically activated
thiophenyl esters.[25] They have been synthesized on-resin
with a mercaptophenylacetic acid (MPAA) linker with
conventional Boc SPPS.[26] A series of MPAA peptide
thioesters were synthesized here using or procedure (Fig-
ure 1A). The weight gain of the resin was recorded to
measure any possible chain loss either from TFA lability or
instability of the thioester bond to the coupling conditions
(Table 1). The weight gain suggested that losses were low and
the crude product were of generally excellent quality
(Figure 2). The weight gain was a better guide than isolated
yield as the latter was very variable depending on efficient
loading of the linker, solubility of the peptide and the
equipment used for purification. Examples were chosen to
test compatibility with all the amino acids (Table 1).
One of the advantages of this method is its greater
compatibility with post-translational modifications compared
to conventional Boc SPPS. We illustrated the scope of the
technique by the preparation of phosphorylated CHK2
protein. CHK2 is a serine/threonine kinase which upon
activation by phosphorylation on Thr-68 plays a central role
in DNA damage response.[27] Furthermore, it is involved in
cell cycle checkpoint activation, apoptosis, viral infectivity
and other pathways.[28] The C-terminal portion bearing an N-
terminal cysteine was prepared by expression. The N-terminal
peptide has been previously synthesized by Fmoc SPPS using
the sulfonamide safety catch method.[29] Phosphorylated
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threonine was added to the synthesis as the building block
Boc-Thr-(PO3Me2)-OH. A 2.5-fold excess of phosphorylated
MPAA thioester 15 was ligated with truncated CHK2 protein
(100 mm concentration) in phosphate buffer (200 mm; pH 7.0).
The ligation was monitored by gel electrophoresis and was
complete after 60 min and the further addition of another 2.5-
fold of peptide 15 (Figure 3B). Phosphorylated CHK2
protein dimerized confirming successful ligation (Fig-
ure 3D).[29]
Another application of peptide thioesters is for the
synthesis of cyclic peptides. With many leads being identified
from the screening of natural products and selection tech-
nologies[30] chemistry remains the bottleneck to their large-
scale preparation. Peptide thioesters are particularly adept at
cyclization by NCL.[2a] We demonstrated this with the syn-
thesis of 18 (Table 1) which contained a good range of
residues including tryptophan. Side-chain unprotected tryp-
tophan was used in the synthesis, a standard procedure for
peptide thioester synthesis by Boc SPPS. The peptide was
dissolved in phosphate buffer containing MPAA. Cyclization
monitored by analytical HPLC was complete after 15 min
(Figure S22, SI).
Figure 2. Crude HPLC traces and MALDI-TOF MS of MPAA thioesters
synthesized using Boc protocols on Merrifield hydroxymethyl resin.
The peptides where cleaved with TFA/TMSBr/thioanisole/EDT
(1:0.05:0.05:0.025) for 1.5 h at room temperature and precipitated
from Et2O. A) LAPAV-MPAA and B) LEDLRQQLQQAEEALVAKQELI-
MPAA.
Peptide thioesters have been successfully cyclized in the
absence of N-terminal cysteine. Recently, peptide thiophenyl
esters were shown to cyclize.[2c] Independently, the Houghten
group cyclized peptide MPAL thioesters.[2b] We decided to
repeat the conditions of Houghten and co-workers but using
Figure 3. Synthesis and application of an MPAA thioester phosphopeptide. A) Analytical HPLC (20–60% B=CH3CN/H2O/TFA (90:10:0.1) in
30 min) and MALDI-TOF MS of the purified phosphopeptide 15. B) Time course of the ligation of 15 with CHK2(73-538)S73C. The lower band is
CHK2(73-538)S73C and the upper band is the ligated protein. After 60 min, approximately 50% ligation was observed. Additional peptide was
added at the 30 min and 60 minute time points, and the ligation proceeded to >90% conversion. The ligation product contains the residues 63–
538 with a phosphorylated Thr-68 and a Ser-73 to Cys mutation. C) Ligation of phosphopeptide 15 to CHK2(73-538)S73C and dimerization of
phosphoprotein. D) Gel-filtration profiles of the purified unligated CHK2(73-538)S73C and ligated pT68CHK2. Indicating dimerization for ligated
material, small amount of dimerization was also present for the unligated protein.
4
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Figure 4. Synthesis, cyclization and characterization of cyclorasin 9A5. A) Analytical HPLC of crude linear cyclorasin 9A5 precursor synthesized as
an activated MPAA thioester. Insert shows MALDI-TOF MS of the purified product. B) Analytical HPLC and MALDI-TOF MS of purified cyclized
cyclorasin 9A5, 24. The crude linear peptide (1 mm) was cyclized by adding to a mixture of MeCN:H2O (7:1) and imidazole. The reaction was
monitored to completion by analytical HPLC, cyclorasin 9A5 was purified by semi-preparative HPLC. C) COSY NMR spectrum of cyclorasin 9A5.
COSY showing four distinct Arg H(d)–H(e) correlations, indicating head-to-tail topology. D) Binding study of cyclorasin 9A5 with wild-type KRAS
protein. Microscale thermophoresis studies were performed in triplicate showing a Kd of 587 nm (see Figure S24 in the SI for biophysical
characterization).
MPAA thioesters. The target we chose was the KRAS Acknowledgements
inhibitor cyclorasin 9A5.[31] The analytical HPLC of the
This work was supported by the Francis Crick Institute which
receives its core funding from cancer research UK
(FC001070, FC001156), the UK Medical Research Council
(FC001070, FC001156) and the Wellcome Trust (FC001070,
FC001156) and by funding from the European Research
Council Advanced Grant RASTARGET.
crude linear peptide 17 was very clean (Figure 4A) consid-
ering it possessed four arginines and a tryptophan.
Cyclization in acetonitrile in the presence of an aqueous
imidazole solution gave good results (Figure S23, SI). The
head-to-tail connectivity of cyclorasin 9A5 was confirmed by
NMR spectroscopy (Figure 4C). The binding was character-
ized by microscale thermophoresis studies (Figure 4D).
Labeled KRAS-GTPgS interacts with cyclorasin 9A5 with
an affinity of 0.6 mm similar to the values measured previously
with FITC-labeled peptide[31] and the dissociation constant
between cyclorasin and wild-type KRAS was measured
(Figure 4D and SI).
Keywords: Boc-SPPS · cyclic peptide · peptide ligation ·
peptide thioester · post-translational modification
In summary we have reported a direct Boc SPPS approach
for the synthesis of peptide thioesters with a TFA/TMSBr
cleavage replacing HF treatment. This is a simple, practical
method with a gentler cleavage step, compatible with many
post-translational modifications. The avoidance of HF makes
Boc SPPS much more accessible. The in situ neutralization
cycles, effective for overcoming difficult sequences provide
peptides of high purity, difficult to achieve by Fmoc SPPS
without the often problematic use of backbone protection.[6]
[1] a) P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. Kent, Science
1994, 266, 776 – 779; b) P. E. Dawson, S. B. Kent, Annu. Rev.
Biochem. 2000, 69, 923 – 960.
[2] a) L. Zhang, J. P. Tam, J. Am. Chem. Soc. 1997, 119, 2363 – 2370;
b) Y. Li, A. Yongye, M. Giulianotti, K. Martinez-Mayorga, Y.
Yu, R. A. Houghten, J. Comb. Chem. 2009, 11, 1066 – 1072; c) P.
Agrigento, F. Albericio, S. Chamoin, I. Dacquignies, H. Koc, M.
Eberle, Org. Lett. 2014, 16, 3922 – 3925.
[3] a) H. Hojo, Y. Kwon, Y. Kakuta, S. Tsuda, I. Tanaka, K. Hikichi,
S. Aimoto, Bull. Chem. Soc. Jpn. 1993, 66, 2700 – 2706; b) T. M.
Hackeng, J. H. Griffin, P. E. Dawson, Proc. Natl. Acad. Sci. USA
1999, 96, 10068 – 10073.
Angew. Chem. Int. Ed. 2016, 55, 1 – 7
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
[4] a) M. Muttenthaler, F. Albericio, P. E. Dawson, Nat. Protoc.
2015, 10, 1067 – 1083; b) J. Tam, R. Merrifield, The Peptides,
Vol. 9 (Eds.: S. Udenfriend, J. Meienhofer), Academic Press,
New York, 1987, pp. 185 – 254; c) S. Sakakibara, Y. Shimonishi,
Y. Kishida, M. Okada, H. Sugihara, Bull. Chem. Soc. Jpn. 1967,
40, 2164 – 2167.
[16] S. B. Kent, Chem. Soc. Rev. 2009, 38, 338 – 351.
[17] a) M. Beyermann, M. Bienert, Tetrahedron Lett. 1992, 33, 3745 –
3748.
[18] a) J. P. Tam, W. F. Heath, R. B. Merrifield, J. Am. Chem. Soc.
1986, 108, 5242 – 5251; b) J. M. Stewart, Methods Enzymol. 1997,
289, 29 – 44; c) G. N. Jubilut, E. M. Cilli, M. Tominaga, A.
Miranda, Y. Okada, C. R. Nakaie, Chem. Pharm. Bull. 2001, 49,
1089 – 1092.
[19] a) M. Murakami, R. Okamoto, M. Izumi, Y. Kajihara, Angew.
Chem. Int. Ed. 2012, 51, 3567 – 3572; Angew. Chem. 2012, 124,
3627 – 3632; b) M. Izumi, M. Murakami, R. Okamoto, Y.
Kajihara, J. Pept. Sci. 2014, 20, 98 – 101; c) Z. P. Gates, J. R.
Stephan, D. J. Lee, S. B. Kent, Chem. Commun. 2013, 49, 786 –
788.
[20] N. Fujii, A. Otaka, N. Sugiyama, M. Hatano, H. Yajima, Chem.
Pharm. Bull. 1987, 35, 3880 – 3883.
[21] W. C. Chan, P. D. White, Fmoc Solid Phase Peptide Synthesis
(Eds.: W. C. Chan, P. D. White), Oxford University Press,
Oxford, 2000, pp. 41 – 76.
[5] D. Lowe, http://blogs.sciencemag.org/pipeline/archives/2004/03/
03/, “Things I wonꢀt touch (1)”. Access date 07/07/2016.
[6] R. Behrendt, P. White, J. Offer, J. Pept. Sci. 2016, 22, 4 – 27.
[7] a) S. Flemer, J. Pept. Sci. 2009, 15, 693 – 696; b) F. El Oualid, R.
Merkx, R. Ekkebus, D. S. Hameed, J. J. Smit, A. de Jong, H.
Hilkmann, T. K. Sixma, H. Ovaa, Angew. Chem. Int. Ed. 2010,
49, 10149 – 10153; Angew. Chem. 2010, 122, 10347 – 10351.
[8] a) R. Ingenito, E. Bianchi, D. Fattori, A. Pessi, J. Am. Chem. Soc.
1999, 121, 11369 – 11374; b) Y. Shin, K. A. Winans, B. J. Backes,
S. B. Kent, J. A. Ellman, C. R. Bertozzi, J. Am. Chem. Soc. 1999,
121, 11684 – 11689; c) S. Mezzato, M. Schaffrath, C. Unverzagt,
Angew. Chem. Int. Ed. 2005, 44, 1650 – 1654; Angew. Chem.
2005, 117, 1677 – 1681; d) F. Burlina, C. Morris, R. Behrendt, P.
White, J. Offer, Chem. Commun. 2012, 48, 2579 – 2581.
[9] a) J. B. Blanco-Canosa, P. E. Dawson, Angew. Chem. Int. Ed.
2008, 47, 6851 – 6855; Angew. Chem. 2008, 120, 6957 – 6961;
b) J. B. Blanco-Canosa, B. Nardone, F. Albericio, P. E. Dawson,
J. Am. Chem. Soc. 2015, 137, 7197 – 7209; c) J. X. Wang, G. M.
Fang, Y. He, D. L. Qu, M. Yu, Z. Y. Hong, L. Liu, Angew. Chem.
Int. Ed. 2015, 54, 2194 – 2198; Angew. Chem. 2015, 127, 2222 –
2226.
[10] a) J.-S. Zheng, S. Tang, Y.-C. Huang, L. Liu, Acc. Chem. Res.
2013, 46, 2475 – 2484; b) G. M. Fang, Y. M. Li, F. Shen, Y. C.
Huang, J. B. Li, Y. Lin, H. K. Cui, L. Liu, Angew. Chem. Int. Ed.
2011, 50, 7645 – 7649; Angew. Chem. 2011, 123, 7787 – 7791;
c) G. M. Fang, J. X. Wang, L. Liu, Angew. Chem. Int. Ed. 2012,
51, 10347 – 10350; Angew. Chem. 2012, 124, 10493 – 10496.
[11] P. Botti, M. Villain, S. Manganiello, H. Gaertner, Org. Lett. 2004,
6, 4861 – 4864.
[12] a) H. Hojo, Y. Onuma, Y. Akimoto, Y. Nakahara, Y. Nakahara,
Tetrahedron Lett. 2007, 48, 25 – 28; b) K. Sato, A. Shigenaga, K.
Tsuji, S. Tsuda, Y. Sumikawa, K. Sakamoto, A. Otaka, Chem-
BioChem 2011, 12, 1840 – 1844; c) N. Ollivier, J. Dheur, R.
Mhidia, A. Blanpain, O. Melnyk, Org. Lett. 2010, 12, 5238 – 5241;
d) F. Burlina, G. Papageorgiou, C. Morris, P. D. White, J. Offer,
Chem. Sci. 2014, 5, 766 – 770; e) D. Macmillan, A. Adams, B.
Premdjee, Isr. J. Chem. 2011, 51, 885 – 899.
[22] A. Mitchell, B. Erickson, M. Ryabtsev, R. Hodges, R. Merrifield,
J. Am. Chem. Soc. 1976, 98, 7357 – 7362.
[23] a) S. B. Kent, A. R. Mitchell, M. Engelhard, R. Merrifield, Proc.
Natl. Acad. Sci. USA 1979, 76, 2180 – 2184; b) S. Kent, D.
Alewood, P. Alewood, M. Baca, A. Jones, M. Schnolzer, R.
Epton, Innovation and Perspectives in Solid Phase Synthesis
(Ed.: R. Epton) Intercept, Andover, 1992, pp. 1 – 27.
[24] B. Dang, B. Dhayalan, S. B. Kent, Org. Lett. 2015, 17, 3521 –
3523.
[25] D. Bang, B. L. Pentelute, S. B. Kent, Angew. Chem. Int. Ed. 2006,
45, 3985 – 3988; Angew. Chem. 2006, 118, 4089 – 4092.
[26] D. Bang, B. L. Pentelute, Z. P. Gates, S. B. Kent, Org. Lett. 2006,
8, 1049 – 1052.
[27] J.-Y. Ahn, X. Li, H. L. Davis, C. E. Canman, J. Biol. Chem. 2002,
277, 19389 – 19395.
[28] L. Zannini, D. Delia, G. Buscemi, J. Mol. Cell Biol. 2014, 6, 442 –
457.
[29] J. Li, I. A. Taylor, J. Lloyd, J. A. Clapperton, S. Howell, D.
MacMillan, S. J. Smerdon, J. Biol. Chem. 2008, 283, 36019 –
36030.
[30] a) A. Tavassoli, S. J. Benkovic, Nat. Protoc. 2007, 2, 1126 – 1133;
b) R. Maini, S. Umemoto, H. Suga, Curr. Opin. Chem. Biol.
2016, 34, 44 – 52.
[31] P. Upadhyaya, Z. Qian, N. G. Selner, S. R. Clippinger, Z. Wu, R.
Briesewitz, D. Pei, Angew. Chem. Int. Ed. 2015, 54, 7602 – 7606;
Angew. Chem. 2015, 127, 7712 – 7716.
[13] S. B. H. Kent, Annu. Rev. Biochem. 1988, 57, 957 – 990.
[14] M. Schnçlzer, P. Alewood, A. Jones, D. Alewood, S. B. Kent, Int.
J. Pept. Protein Res. 1992, 40, 180 – 193.
[15] a) R. Behrendt, S. Huber, P. White, J. Pept. Sci. 2016, 22, 92 – 97;
b) J. P. Tam, T. W. Wong, M. W. Riemen, F. S. Tjoeng, R. B.
Merrifield, Tetrahedron Lett. 1979, 42, 4033 – 4036.
Received: August 7, 2016
Published online: && &&, &&&&
6
www.angewandte.org
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Peptide Synthesis
R. Raz, F. Burlina, M. Ismail,
J. Downward, J. Li, S. J. Smerdon,
M. Quibell, P. D. White,
J. Offer*
&&&& — &&&&
Boc synthesis made easy: Peptide thio-
esters can be directly and conveniently
synthesised by Boc chemistry using
a combination of mild acid deprotection
(trifluoroacetic acid/trimethylsilyl bro-
HF-Free Boc Synthesis of Peptide
Thioesters for Ligation and Cyclization
mide) and Merrifield resin. This method
was applied to the preparation of cyclic
peptides and a phosphorylated protein.
Angew. Chem. Int. Ed. 2016, 55, 1 – 7
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!