Chemical Protein Synthesis Using a Second-Generation N-Acylurea Linker for the Preparation of Peptide-Thioester Precursors

Article abstract of DOI:10.1021/jacs.5b03504

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C-terminal peptide-thioesters, key intermediates in NCL. We have developed an N-acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into N-acylurea peptides. Here, we introduce a new N-acylurea linker bearing an o-amino(methyl)aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired N-acyl-N′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer. (Chemical Equation Presented).

Full text of DOI:10.1021/jacs.5b03504

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Article
Chemical protein synthesis using a second generation N-
acylurea linker for the preparation of peptide-thioester precursors.
Juan B. Blanco-Canosa, Brunello Nardone, Fernando Albericio, and Philip E. Dawson
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b03504 • Publication Date (Web): 15 May 2015
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Chemical protein synthesis using a second generation N-
acylurea linker for the preparation of peptide-thioester pre-
cursors.
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Juan B. BlancoꢀCanosa,a Brunello Nardone,b Fernando Albericio,a,c Philip E. Dawsond
aInstitute for Research in Biomedicine (IRB Barcelona), Chemistry and Pharmacology programme, Baldiri Reixac
10, Barcelona 08028 (Spain)
bUniversity of Salerno, Departments of Chemistry and Biology, Via Giovanni Paolo II 132, Fisciano 84084 (Italy)
cUniversity of Barcelona, Department of Organic Chemistry, Martí i Franqués 1ꢀ11, Barcelona 08028 (Spain)
dThe Scripps Research Institute (TSRI), Department of Chemistry 10550 N. Torrey Pines Road, La Jolla 92037
(USA)
KEYWORDS. Native Chemical Ligation – thioesters – acylurea – microwave peptide synthesis.
ABSTRACT: The broad utility of Native Chemical Ligation (NCL) in protein synthesis has fostered a search for methꢀ
ods that enable the efficient synthesis of C-terminal peptideꢀthioesters, key intermediates in NCL. We have developed
an N-acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange
yielding thioester peptides, and subsequently NCL reaction. However, the synthesis of some glycine rich sequences
revealed limitations, such as diacylated products that can not be converted into Nꢀacylurea peptides. Here, we introꢀ
duce a new N-acylurea linker bearing an o-amino(methyl)aniline (MeDbz) moiety that enables in a more robust peptide
chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under differꢀ
ent coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and
desired N-acylꢀN’ꢀmethylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20
natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the
MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker
can be applied to the synthesis of cysteine rich proteins such the cyclotides Kalata B1 and MCoTIꢀII through a one
cyclization/folding step in the ligation/folding buffer.
valine,9 proline,10 lysine,11 arginine,12 threonine,13 and
1. Introduction.
likely could be extended to the remaining amino acids
Chemical Protein Synthesis (CPS) enables the synꢀ
thesis of proteins with precise control over the final
synthetic product, allowing the access to a wide diversiꢀ
ty of postꢀtranslational modifications (PTMs) including
glycoꢀ, phosphoꢀ, lipoꢀ and cyclic proteins.1 Native
Chemical Ligation (NCL) has been shown to be the
most generally applied approach for the chemoselecꢀ
tive assembly of unprotected peptides in aqueous soluꢀ
tion.2 NCL relies on the selective reaction of a Cꢀ
terminal peptide thioester and a Nꢀterminal cysteine
fragment. In the absence of a cysteine, the Nꢀterminal
residue can be replaced by a cysteineꢀsurrogate with a
thiol group in ß or γꢀposition, which can be further
desulfurized. This later approach was first demonstratꢀ
ed for Ala and Abu ligation sites (ß and γꢀ position,
respectively) and the generality of the strategy to other
amino acids was noted,3 suggesting a general apꢀ
proach for NCL at residues other than cysteine. Thus,
NCL has been applied to alanine,3 phenylalanine,4
leucine,5 glutamic acid,6 aspartic acid,7 glutamine,8
using a similar logic (Scheme 1). Furthermore, the use
α
of N -auxiliaries enabled the extension of NCL to glyꢀ
α
cine,14 and in principle these N -auxiliaries could be
utilized for ligations with other residues. Although this
ligation is very attractive, it is sterically very demanding
α
and the utilization of N -auxiliaries is still principally
constrained to XaaꢀGly junctions (Xaa = any amino
acid).
Common to all NCL family reactions is the synthesis
of Cꢀterminal peptide thioesters which can be obtained
in BocꢀSolid Phase Peptide Synthesis (BocꢀSPPS) in a
straightforward manner using mercaptoalkyl acid linkꢀ
ers such as mercaptopropionic or mercapto acetic
acids.15 However, the lability of the thioester bond unꢀ
der the basic/nucleophilic conditions used in Fmoc
chemistry (20% Piperidine/DMF) fostered the developꢀ
ment of alternative strategies. For example, activation
followed by thioesterification of the Cꢀterminal amino
acid
on
fully
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Scheme 1. Native Chemical Ligation and extended NCL using βꢀmercapto amino acid derivatives.
protected peptides,16 backbone amide linker (BAL),17
metalꢀassisted thiolysis of PAM/Wang resins,18 the
Kenner’s sulfonamide safetyꢀcatch linker19 and the aryl
hydrazine handle.20 More recently, a variety of different
methods designed to undergo N to S21 or O to S22 acyl
shift under acidic or during NCL conditions, or the utiliꢀ
zation of the transfer active ester approach using terꢀ
minal hydrazide peptide precursors.23
Previously, we disclosed a new approach based on
Nꢀacylꢀbenzimidazolinones or Nꢀacylureas24 (Nbz,
Scheme 2). Starting from 3,4ꢀdiaminobenzoic acid,
selective acylation of one of the amines yields an o-
aminoanilide peptide 1. Following chain assembling,
acylation with pꢀnitrophenyl chloroformate and cyclizaꢀ
tion under basic conditions affords the Nꢀacylurea proꢀ
tected peptide, 2. Acidolytic cleavage of the resin and
concomitant amino acid side chain deprotection yields
the Nꢀacylurea peptide 3, which can be further transꢀ
thioesterified (4) or utilized directly in NCL in presence
of a thiol catalyst (5). We and others have used the Nꢀ
acylurea method for the synthesis of proteins or protein
domains. For example, we have prepared the second
type 1 repeat of Thrombospondinꢀ1 (TSR2),25 a protein
that has shown a potent anti angiogenic activity. Elseꢀ
where, the Nbz strategy has been applied to the synꢀ
thesis of ubiquitinated peptide chains and Cꢀterminal
ubiquitinated hydrazide peptides,26 salicylaldehyde
ester precursors for serine and threonine ligation,27
cyclotides,28 human αꢀsynuclein,29 combinatorial asꢀ
sembly of affibody molecules,30 a phosphorylated fragꢀ
ment of the MDM2 E3 ubiquitin ligase,31 the collagenꢀ
ous domain of the Adiponectin hormone,32 a 44ꢀmer
histoneꢀderived fragment,33 the HIVꢀ1 Rev protein,34 the
Nꢀterminal domain of the Anthrax Lethal Factor,35 glyꢀ
coproteins,36 and phosphorylated and acetylated fragꢀ
ments of the histone H2B.37
Scheme 2. Key intermediates in the synthesis of Cꢀ
terminal peptide Nꢀacylureas. P = protecting group.
Nevertheless, it has been reported that in glycine rich
sequences acylation of the unprotected o-aminoanilide
can occur, especially in the presence of excess
base.33,38 Although reversible protection of the oꢀ
aminoanilide peptide with alloc group can address the
problem,33 an alternative, more powerful and elegant
approach would be to furnish each amine of the 3,4ꢀ
diaminoaryl linker with different reactivity. This apꢀ
proach would avoid extra protection and deprotection
steps and also produce a single isomer of the Nꢀ
acylurea product. Here we report the application to
peptide synthesis, peptide ligation and cyclic protein
synthesis of a second generation of o-aminoaniline
linker, the oꢀamino(methyl)aniline (Scheme 3, MeDbz)
in which a methyl group is introduced onto the paraꢀ
amino group. Following chain elongation the linker can
be transformed into a mildly activated Nꢀacylurea, (Nꢀ
acylꢀN’-methylꢀbenzimidazolinone
methylurea) (MeNbz) that shows a higher resistance to
or
NꢀacylꢀN’-
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acylation during SPPS compared to the first generation
of the Nbz linker.
(Figure S3). Conversely, the synthesis of the pentaGly
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following alloc removal after incorporation of the Cꢀ
terminal Gly still shows some overacylated products
(Figure S4). These results are consistent with a low
level of acylation during the chain elongation steps.
2. Results.
2.1 Synthesis of Fmoc-MeDbz (8).
5
Synthesis of 8, was achieved following reported proꢀ
cedures39 in 3 steps in a gram scale. Starting from 3ꢀ
fluoroꢀ4ꢀnitrobenzoic acid, 6, nucleophilic addition of
methylamine yields the 3ꢀnitroꢀ4ꢀ(methylamino)benzoic
acid, 7. After reduction of the nitro group through cataꢀ
lytic hydrogenation over Pd/C, chemoselective protecꢀ
tion of the primary aryl amine with FmocꢀCl yields the
expected Fmocꢀ3ꢀaminoꢀ4ꢀ(methylamino)benzoic acid
(Fmoc-MeDbz) (8, 71% overall yield, Scheme 3). Alꢀ
ternatively, the reduction of the nitro aromatic group
can also be carried out on solid phase using SnCl2.
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Scheme 3. Synthesis of Fmoc-MeDbz (8).
2.2 Synthesis and evaluation of the MeDbz
chemoselectivity in a polyglycine peptide: Gly5
sortase tag.
Gly5 is a 5ꢀmer peptide employed in the sortase labeꢀ
ling technology40 that recently has found application in
the sortaseꢀassisted synthesis of peptide/protein thioeꢀ
sters.41 Owing to its exclusive glycine sequence, it renꢀ
ders a challenging model peptide for testing the roꢀ
bustness of the MeDbz linker. In order to confirm previꢀ
ous results,33,38 we decided to investigate the synthesis
of FmocꢀGly5ꢀNbzꢀR (9) on the original Dbz linker under
strong acylating conditions: 10ꢀfold excess each of
FmocꢀGlyꢀOH and HBTU and 20 eq of base. (Note: the
originally described acylation conditions for Dbz utilized
HOBt to buffer excess base). Under these conditions,
significant amounts of branched peptides were obꢀ
served (54%, method A section 4.2, and Figures 1a
and S1).
Figure 1. HPLC traces of synthetic 9: 9aꢀ9e = diacylated
products. Coupling conditions: 1st FmocꢀGly 4 eq, HBTU 4
eq, DIEA 4 eq, 40 min; then, a) FmocꢀGly 10 eq, HBTU 10
eq (0.5 M in DMF), DIEA 20 eq, 1h at rt; b) FmocꢀGly 4
eq, HBTU (4 eq, 0.2 M)/HOBt (4 eq, 0.2 M), DIEA 6 eq, 1h
at rt; c) structures of the peptides 9 and 9a-e (see Figure
S1 for detailed structures).
The introduction of the NꢀMe group on the oꢀamino
group of the linker should significantly decrease the
sensitivity of the linker towards undesired acylation.
Gratifyingly, the utilization of the MeDbz moiety and 5ꢀ
fold excess of FmocꢀGly and HBTU activation afforded
the desired peptide FmocꢀGly5ꢀMeNbzꢀR (10) with no
traces of side products (Figure 2a, method C in sec-
tion 4.3). Assaying harsher conditions such as a 10ꢀ
fold excess of amino acid and basic conditions, i.e. 20ꢀ
fold excess of DIEA (Figure S5), and the utilization of
microwave irradiation at either 75 C or 90 C (Figure S6
and Figure 2b, respectively) did not generate signifiꢀ
cant (< 1%) diacylated peptides. Together, these reꢀ
sults show that it is very challenging to couple even an
unhindered amino acid such as FmocꢀGly due to the
steric and electron withdrawing properties of the oꢀ
amino(methyl)anilide, which becomes deactivated by
the anilide and the amide groups in oꢀ and pꢀpositions,
respectively.42 Critically, the N-methylaniline moiety is
still reactive enough towards acylation with chloroforꢀ
mate derivatives (5 eq in CH2Cl2, 1h), and is able to
This pentaGly peptide proved to be generally chalꢀ
lenging on the Dbz linker. For example, at 4 eq Fmocꢀ
GlyꢀOH/HBTU/DIEA Figure S2) or under mildly acidic
conditions (4 eq each FmocꢀGly, HBTU, HOBt, and 6
eq DIEA Figure 1b, method B in section 4.2) some
diacylated products were detected (16 and 12% diacylꢀ
ated products, respectively) representing 2ꢀ4% acylaꢀ
tion per Gly residue coupled. Nevertheless, this low
sideꢀacylation percentage has not resulted in any imꢀ
pairment in the synthesis of Nbz peptides possessing
Gly residues at the Cꢀterminal position, such as in the
case of the Cꢀterminal GG sequence of ubiquitinꢀ
derived Nbz peptides.26
To control for the possibility that the overꢀacylation
could arise from the coupling of the first glycine, the
free amine was protected with alloc after introduction of
the FmocꢀDbz moiety on the resin, and removed followꢀ
ing the pentaglycine assembling.33 As expected, this
route yielded one major compound corresponding to
the desired monoacylated FmocꢀGly5ꢀNbzꢀArg peptide
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undergo cyclization (0.5 M in DMF, 30 min) to yield Nꢀ TFA. In order to facilitate the introduction of the Fmocꢀ
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acylꢀN’-methylurea peptides.
MeDbzꢀOH, a glycine spacer was coupled to the resin
in the first place. Coupling of the FmocꢀMeDbzꢀOH
moiety was accomplished using a 5ꢀfold excess with
regard to the number of mmol of resin, HBTU as actiꢀ
vating agent (5ꢀfold excess) and DIEA as base (5.5
foldꢀexcess) in DMF at a final concentration around
0.33 M. Following introduction of FmocꢀMeDbz, the Cꢀ
terminal residue was introduced either with HBTU or
HATU. Nevertheless, the utilization of HATU, or a simiꢀ
lar strong activating agent such as PyAOP or COMU,
led to complete acylation of the amino acid with no
need for double coupling (Scheme 4). Although not
strictly necessary for all amino acids, the utilization of
5ꢀfold eq of amino acid, HATU and basic conditions (10
eq of DIEA), at a concentration around 0.4ꢀ0.5 M afꢀ
forded reliable loadings >99%. However, in order to
avoid the epimerization of some racemization prone
residues, such as His43 and Cys44 (check section 2.4),
a neutral protocol based on DIC/HOOBt is epimerizaꢀ
tion free. A significant advantage of MeDbz linker over
the AllocꢀDbz strategy is the complete acylation of Val
and Ile, whereas in the allocꢀDbz approach only loadꢀ
ings of 47 and 33% were achieved, respectively.33 The
next amino acids were coupled under standard condiꢀ
tions: 5 eq of Xaa, 5 eq of HBTU (0.5 M in DMF) and
5.5 eq of DIEA. The last residue was introduced with Nꢀ
terminal Boc protection. Following acylation with pꢀ
nitrophenyl chloroformate in dichloromethane, cyclizaꢀ
tion was carried out in a solution of 0.5 M or 1 M DIEA
(in DMF) for 10ꢀ30 min. After cleavage, crude peptides
could be recovered in yields and purities typically highꢀ
er than 90% (Table S1 and Figure S7).
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Figure 2. HPLC traces of synthetic 10. Coupling condiꢀ
tions: a) 5 eq FmocꢀGly, 5 eq HBTU (0.5 M in DMF), 5.5
eq DIEA, 1h at rt. b) 1st FmocꢀGly as in a), next 10 eq
FmocꢀGly, 10 eq DIC, 10 eq Oxyma Pure, 3 min at 90 ºC
following MW1 setup (see experimental section 4.3 for
microwave irradiation characteristics).
2.3 Synthesis of VSYRAXaa-MeNbz-G (11A-Y)
model peptides with different C-terminal amino
acids attached to the MeDbz linker.
Having confirmed the superior characteristics of the
MeDbz linker, its general applicability in peptide synꢀ
thesis and NCL was demonstrated through preparing a
group of 20 model peptides VSYRAXaaꢀMeNbzꢀG
(11A-Y, the letter depicts the amino acid Xaa at the C-
terminus) including all twenty Cꢀterminal amino acids
attached to the MeDbz moiety, on a polysterene resin
functionalized with a Rink linker that is cleavable with
Scheme 4. Synthetic route to peptides 11A-Y. PS = polysterene.
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The carbamylation reaction could also be carried out
in other solvents including 1,2ꢀdichloroethane, THF,
α,α,αꢀtrifluorotoluene, 2,2,2ꢀtrifluoroethanol, hexꢀ
afluoroisopropanol or in DMF which is known as a betꢀ
ter swelling solvent for peptide resins.34 Alternatively,
intramolecular acylurea formation was accomplished in
a second step in solvents such as DCM or DMSO, or
using bases such as NꢀMethylmorpholine, although in
these cases the cyclization has a slower kinetics.45
Mercaptophenylacetic acid (MPAA).48 This allowed
straightforward monitoring of the ligation kinetics. A
short screening of different thiols (considering both the
kinetics and water solubility) led to the choice of MPAA
and (4ꢀHydroxy)thiophenol (MPOH) as the optimal thiol
catalysts for NCL with acylurea peptides, both displayꢀ
ing similar kinetics. However, we found that MPOH is
less expensive than MPAA, is less acidic, shares its
lack of odor and is soluble up to at least 200 mM at pH
7.0. So, for the model ligation reactions we chose
MPOH as the thiol catalyst.48
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2.4 Assessing the epimerization on Cys and His.
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The ligation reaction is highly dependent on pH, meꢀ
diating the desired conversion of the acylurea to a thiꢀ
oester, transthioesterication (typically rate determining
for NCL), while minimizing the potential side products
of epimerization and hydrolysis of the thioester. Ligaꢀ
tions were carried out in the presence of guanidine
hydrochloride as denaturing agent and TCEP to mainꢀ
tain a reducing environment and concentration of the
MPOH additive. We undertook a study of the rate of the
ligation in 3 different pH buffers: 5.8, 7.0 and 8.2 (Fig-
ure 3a). The results show that the optimal pH for the
ligation is around 7.0. At pH 8.2, the percentage of
hydrolysis increases and the ligation rate is not signifiꢀ
cantly improved. At lower pH, the rate of the ligation is
significantly slower. Interestingly, the relative rate of
ligation is reduced to a larger extent than that of hyꢀ
drolysis resulting in an increase in the hydrolysis obꢀ
served in the final product. As with the Dbz linker, if
desired, the Nꢀacylurea peptide could be fully exꢀ
changed with a soluble alkyl thiol such as MESNA for
longꢀterm storage.
One of the major drawbacks of the Fmoc approach
for the solid phase synthesis of peptide thioesters is the
racemization of the Cꢀterminal amino acid during the
coupling or activationꢀthioesterification steps 47
In parꢀ
.
ticular, special care must be taken on His43 and Cys.44
Model studies on both amino acids on the MeDbz linker
showed that epimerization levels were below 0.1%
when the linker was loaded using carbodiimide activaꢀ
tion in presence of additives such as HOOBt: 5 eq of
FmocꢀaaꢀOH, 5 eq of DIC and 5 eq of HOOBt with
preactivation time of 3 min. More specifically, Fmocꢀ
Cys(Trt)ꢀOH could be coupled under several different
conditions with no detectable epimerization (Figure
S8): HATU/DIEA (1:1.1), DIC/Oxyma (1:1), DIC/HOOBt
(1:1). However, the acylation of FmocꢀHis(Trt)ꢀOH
turned out to be more troublesome, and from the couꢀ
pling mixtures tested only DIC/HOOBt gave no detectꢀ
able levels of racemization (Figure S9).46
2.5 Optimization of the Native Chemical Ligation
conditions.
Figure 3b shows the reaction progress of VSYRALꢀ
MeNbzꢀG and a slight excess of CRAFS (1.5ꢀ2ꢀfold
excess) in presence of increasing concentrations of
MPOH (0ꢀ200 mM). Several conclusions can be drawn
from these reactions: i) as expected, the rate of the
ligation increases with the concentration of MPOH, but
ii) concentrations above 100 mM do not significantly
With the availability of the 20 model peptides we unꢀ
dertook a systematic study using the Nꢀterminal cysteꢀ
ine peptide CRAFS. First, ligations were performed
varying pH and concentration of thiol catalyst, choosing
Leu (11L) as a case study, a residue that using previꢀ
ously studied mercaptoproprionyl thioesters gives a
moderately fast reaction (completion in 2 h) in presꢀ
ence of high concentrations (200 mM) of 4ꢀ
speed
up
Figure 3. Kinetics of the formation of 12L: a) influence of the pH, b) influence of the concentration of 4ꢀMercaptophenol.
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the rate of the ligation. This suggests an optimal MPOH The ligation rates fell into 3 different groups: i) Pro,
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concentration around 100 mM, iii) hydrolysis is a comꢀ
petitive process, especially when the reaction is carried
out with no catalyst or at MPOH concentrations below
100 mM. For example, in the absence of MPOH, only a
~50% of the fraction ligated could be achieved after 10
h. These observations are consistent with hydrolytic
sensitivity of acylurea peptides compared to thioester
peptides.24 Therefore, attaining a rapid intermolecular
MeNbz –> S transfer is important to generate the hyꢀ
drolytically stable thioester peptide,49 which is hydrolytiꢀ
cally stable at pH 7.0 yet highly reactive towards Nꢀ
terminal cysteine peptides. From these model ligation
experiments, it is observed that a critical step in the
ligation of Cꢀterminal acylurea and Nꢀterminal cysteine
peptides is the intermolecular N to S acyl transfer to the
thiol additive. In order to achieve a high exchange rate,
thiols with a pKa around 7 (principally aryl thiols) with
good water solubility (~ 100 mM) are the optimal cataꢀ
lyst.
which gives the slowest rate (88% conversion in 48 h,
Figure 4b), ii) ßꢀbranched amino acids such as Val, Ile
that achieve near a > 95% conversion within 20 h (Fig-
ure 4b), and iii) amino acids where the ligation is comꢀ
plete in ≤ 5 h (Figure 4a). When compared to the reꢀ
ported kinetics of mercaptopropionyl thioesters15a some
divergences are clearly seen, which can be explained
based on the lower solubility of thiophenol (the saturatꢀ
ing concentration in the guanidine buffer is around 30
mM) that results in a slower transthioesterification, in
addition to the lower reactivity and competitive transꢀ
thioesterification of the alkyl mercaptoproprionyl leaving
group. This explanation is also consistent with the inꢀ
creased ligation displayed by preformed aryl thioeꢀ
sters.48,50
9
10
11
12
13
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15
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59
60
Intramolecular reactions involving the side chain of
the Cꢀterminal amino acid can affect the utility of NCL.
Consistent with the observations of Dang et al,51 Asn,
Gln were ligated efficiently, with no interfering reactions
of the side chains resulting in unreactive or isomeric
products. The ligation with Cꢀterminal Glu peptides,
yielded only traces, (~ 2%) of the undesired γꢀlinked
peptide at pH 7.0 (Figure 5a).51 However, ligations with
Asp result in rapid ligation but with significant quantities
of the βꢀlinked peptide in a ratio 1.8:1 (Asp:isoAsp, Fig-
ure 5b) presumably as a result of the anhydride forꢀ
mation with the Asp side chain and subsequent thiol
attack to either carbonyl group.52,53
2.6 Assaying the Native Chemical Ligation with
VSYRAXaa-MeNbz model peptides.
The ligation buffer conditions of 0.2 M sodium phosꢀ
phate, 0.1 M MPOH, 6 M guanidine hydrochloride, 20
mM TCEP, pH 7.0, were selected for subsequent studꢀ
ies. To evaluate the general utility of the linker, an exꢀ
haustive study of the ligation between peptides 11 (A to
Y) and CRAFS was performed to afford the ligated
peptides 12 (A to Y). These peptides included the fastꢀ
est Cꢀterminus, glycine and the slowest Cꢀterminus,
proline as observed previously with alkylꢀthioester pepꢀ
tides using thiophenol/benzyl mercaptan as catalysts
(3:1).15a
Thus, the repertoire of amino acids that are viable for
NCL can be extended to any given residue in the thioeꢀ
ster fragment, with the only exception of Cꢀterminal Asp
which would require orthogonal protecting group and
subsequent removal after NCL.52
Figure 4. Kinetics of the formation 12A-Y peptides using MPOH (100 mM) as catalyst at pH 7.0, room temperature. Note that
the fraction ligated for Asp includes 12D and 12isoD.
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1
2
3
4
5
6
7
8
9
Figure 5. NCL traces of: a) 11E (black) and 11isoE (red), b) 11D (black) and 11isoD (red) peptides. Reactions were carried out
at rt and 2 mM concentration of MeNbz peptides, 2.4ꢀ2.7 mM of CRAFS in 6M guanidine buffer (pH 7.0) containing 20 mM of
TCEP and 100 mM MPOH.
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60
2.7 Synthesis of cyclotides Kalata B1 and MCoTI-
on a Tentagel resin we observe, in addition to the exꢀ
pected linear KB1, a product with the same extra 56
units of mass (Figure 6b). Changing the composition of
the cleavage cocktail (TFA, PhOH, TIS, EDT, H2O) and
increasing the concentration of EDT or adding some
II.
Cyclotides are naturally occurring ribosomal proteins
isolated from plants with a headꢀtoꢀtail cyclic structure
and an intramolecular cystine knot arrangement, also
known as cyclic cystine knot (CCK).54 Cyclotides can
be classified in two major groups: Möbius if they have a
cisꢀPro configuration in loop 5, and Bracelet.55 Another
important class of cyclic proteins bearing a cystine knot
motif comprises the family of squash and Momordica
cochinchinensis trypsin inhibitors MCoTIꢀI/II.56 Cycloꢀ
tides have a strong resistance to proteolytic and chemꢀ
ical degradation and a broad spectrum of biological
activities. For example, Kalata B1 is thought to have
uterotonic properties and has been shown to display
antiꢀHIV, antiꢀmicrobial and insecticidal activity, whereꢀ
as MCoTIꢀII is one of the most potent trypsin inhibitor
known. Due to the variability in the loop size and seꢀ
quence, it is possible to replace some of the loops by
other non native sequences, without affecting the final
folding, and thus enabling the utilization of these cycloꢀ
tides as scaffolds for the grafting of bioactive peptide
drugs.57
t
specific Bu scavengers such as 1,2ꢀDimethylindole did
not decrease significantly the percentage of this byꢀ
product. The utilization of the PEGꢀPS resin together
with pseudoproline dipeptides only led to a slight imꢀ
provement of the quality of the crude peptide (Figure
6c).
SPPS of cyclotides represents a synthetic challenge
due to the presence of 6 cysteine residues that, apart
from their higher tendency to racemize during the couꢀ
pling,44 require careful conditions for the final cleavage
in order to avoid undesired sideꢀreactions such as alꢀ
t
kylation with Bu groups which was observed as a sigꢀ
nificant adduct in a previous synthesis of this protein
using the Dbz approach28. The complexity of this proꢀ
tein made it a rigorous test of synthesis, activation and
cleavage conditions for the MeDbz linker on a variety of
commonly used solid supports.
Figure 6. a) Amino acid sequence and disulfide knot arꢀ
rangement (yellow lines) of KB1; the black arrow denotes
the Cꢀterminal residue of the linear synthesis; b) HPLC
trace of the synthesis of linear KB1-MeNbz-G on Tentagel
resin; c) HPLC trace of the synthesis of linear KB1-
MeNbz-G on Tentagel resin using the pseudoproline diꢀ
peptide GT.
2.7.1 Kalata B1 (KB1).
The linear synthesis of cyclotides can be started at
any given amino acid after a cysteine residue. Although
there is a GlyꢀCys junction, we decide to cyclize at a
4Valꢀ5Cys point to show that hindered and slow reacting
amino acids are also effective cyclization points for the
synthesis of cyclotides (Figure 6a). The reported synꢀ
thesis of Kalata B1 using the Nbz linker carried out on
a PEGꢀPS resin Tentagel showed, apart from the exꢀ
pected peptide, a large percentage of an adduct with
an extra mass of 56 units corresponding to the tertꢀ
Butyl group, likely due to the alkylation of the Trp or
some Cys residue.28a When we synthesized linear KB1
Another type of solid support is the fully pegylated
ChemMatrix resin, which displays good swelling propꢀ
erties in a wide range of solvents including DMF,
CH2Cl2 and H2O. Synthesis on this resin in a 0.1 mmol
scale using an excess of amino acid of 10ꢀfold to enꢀ
sure complete acylation led to a good crude purity
(65%, Figure 7a, method D in section 4.5), and reꢀ
covery yield (73%), but still contained significant tertꢀ
butylated sideꢀproducts. Similar results were accomꢀ
plished under microwave conditions using a coupling
temperature of 75 ºC (Figure S11) and even when
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resynthesizing a circular permutation of the sequence
~20% (Figure 8b). It is interesting to note that the cyꢀ
to facilitate a 8Thrꢀ9Cys ligation site (Figure S12).
clic folded peptide displays a more hydrophobic charꢀ
acter than the cyclic peptide, due to that the hydrophoꢀ
bic residues are exposed to the solvent.59
1
2
3
4
5
6
7
8
In order to characterize the alkylated residue (likely a
Cys or Trp), we introduced a different combination of
protecting groups: in the first case, the trityl for 20Thr
and 22Ser in loops 4 and 5 that are next to 23Trp
(CTCSW, Figure S13), and in the second place the
TFA stable phenylacetamidomethyl (Phacm) for the
protection of the all Cys residues except the Nꢀterminal.
9
Only in this later approach, which allowed the linear
partially protected Kalata B1 in good analytical quality
(75%) and recovery yield (77%), showed minimum
amounts of tert-butylated peptides (Figure 7b), thus
indicating that some of the Cys residues were acting as
an internal scavenger and capturing a tBu group.58
10
11
12
13
14
15
16
17
18
19
20
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22
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48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. a) Cyclization of KB1-MeNbz-G crude syntheꢀ
sis; b) oneꢀpot cyclizationꢀfolding of KB1-MeNbz-G crude
synthesis; c) MALDIꢀTOF spectra of cyclic KB1 (cKB1)
(left) and folded KB1 (right).
2.7.2 MCoTI-II.
The linear synthesis of MCoTIꢀII was performed folꢀ
lowing the same strategy of KB1, chosen the cyclizaꢀ
tion point at the 28Tyr residue (Figure 9a). In order to
avoid aspartimide formation in the AspꢀGly sequence
during the Fmoc removal, the Fmocꢀ(Hmb)GlyꢀOH
amino acid was used for the temporal protection of the
amide bond with the TFA labile Hmb (2ꢀHydroxyꢀ4ꢀ
methoxybenzyl) protecting group. The linear MCoTI-II-
MeNbz-G peptide was recovered in an 81% yield and
an analytical crude purity around 70% (Figure 9b).
Following the same, single cyclizationꢀfolding approach
for KB1, the linear precursor was correctly folded afꢀ
fording the MCoTIꢀII cyclotide in good overall yields
(22%, Figure 10a). Notably, the folded MCoTIꢀII is
more hydrophilic than the cyclic peptide (Figure 10b),
indicating that in this case the hydrophilic residues
have a more contact surface with the solvent.
Figure 7. HPLC trace of the synthesis of linear KB1-
MeNbz-G (a) and KB1(Phacm)5-MeNbz-G (b) syntheꢀ
sized both on ChemMatrix resin; c) ESIꢀMS spectra of the
main peak of the KB1-MeNbz-G synthesis (left), and KB1-
(Phacm)5-MeNbz-G crude (right). (Note that the linear
synthesis started at Val in position 4).
Following the linear synthesis, the crude peptide was
cyclized using the above conditions optimized for NCL
(Figure 8a), and then folded in a (NH4)2CO3:iPrOH (1:1)
buffer at pH 8.5 in presence of the GSH:GSSG (2
mM:0.4 mM) redox system.59 As reported for linear
cyclotides synthesized using either Boc or Fmoc chemꢀ
istry,59b,60 the cyclization and folding could be carried out
in one single step in the folding buffer at pH = 7.3 ꢀ 7.5
affording the folded Kalata B1 in recovered yield of
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proteins with PTMs, incorporation of nonꢀnatural amino
1
acids or siteꢀspecific labeling. By enabling the producꢀ
tion of homogeneous forms of these macromolecules,
precise structure determinations, and structureꢀactivity
relationships of enzymes, substrates and ligand binding
proteins have been interrogated. Native Chemical
Ligation has become the reaction of choice for CPS,
especially because its wide compatibility with biological
macromolecules. NCL relies in the condensation beꢀ
tween two unprotected peptides, a Cꢀterminal thioester
and another Nꢀterminal cysteine, in aqueous buffer at
neutral pH and room temperature. From a synthetic
point of view, the critical element of utilizing NCL is the
efficient production of C-terminal peptideꢀthioesters,
which can be easily prepared in BocꢀSPPS using merꢀ
captoalkyl linkers. Complementary Fmocꢀbased methꢀ
ods for the synthesis of peptideꢀthioesters continue to
be developed although it has been challenging to attain
suitable approaches that are robust for chain elongaꢀ
tion by SPPS while maintaining efficient Cꢀterminal
activation, avoiding epimerization and maintaining pepꢀ
tide solubility.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Figure 9. a) amino acid sequence and disulfide knot arꢀ
17
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59
60
rangement (yellow lines) of MCoTI-II (the black arrow
denotes the Cꢀterminal residue of the linear synthesis); b)
HPLC trace of the synthesis of linear MCoTI-II-MeNbz-G
on ChemMatrix resin. Inset, ESIꢀMS of the product correꢀ
sponding to the main peak.
We hypothesized that Cꢀterminal Nꢀacylurea pepꢀ
tides61 could act as precursors for thioester peptides
and undergo exchange with thiols to yield a Cꢀterminal
thioester for NCL. Using oꢀaminoanilide linkers based
on diaminobenzoic acid, we found that Nꢀacylurea
peptides undergo fast Nꢀacylurea –> thiol exchange
under NCL conditions, and consequently allow their
utilization for CPS. This approach has been widely
used for the synthesis of proteins, including proteins
with PTMs. However, under strongly basic conditions
and in Gly rich sequences, the acylation of the unproꢀ
tected oꢀaminoanilide, can give rise to branched side
products that cannot not be further converted in Nꢀ
acylurea peptides.33,38
In order to analyze this side reaction in a systematic
manner, we designed a pentaglycine containing pepꢀ
tide 9, and under basic coupling conditions, indeed
observed a high ratio of doublyꢀacylated products.
While these products could be significantly reduced
using a more acidic protocol and a lower excess of
FmocꢀGly during coupling. Nevertheless, a robust and
universally applicable Dbz linker should be compatible
with all coupling conditions used for SPPS while mainꢀ
taining the simplicity of the Dbz linker with respect to
activation with mild chloroformate derivatives and
avoidance of unnecessary protecting groups.
The 3ꢀaminoꢀ4ꢀ(methylamino)benzoic acid (MeDbz)
linker was designed to enable chemoselectivity beꢀ
tween the two arylꢀamines by Nꢀalkylation. Acylation at
the unhindered primary aromatic amine would facilitate
chain elongation, while diꢀacylation at the Nꢀmethylated
position would be significantly reduced. Critical to the
approach was maintaining efficient formation of the
desired Nꢀacylurea (MeNbz) product on resin using
nitrophenyl chloroformate. Gratifyingly, the MeDbz
linker showed superior performance in the synthesis of
a pentaglycine sequence 10 when compared to the
Dbz moiety (peptide 9). The oꢀ(methylamino)anilide
functional group is significantly more resistant towards
acylation during amino acid couplings, even withstandꢀ
Figure 10
. a) oneꢀpot cyclizationꢀfolding of MCoTI-II-
MeNbz-G crude synthesis (peak at 18.1 min is the exꢀ
pected MCoTI-II product); b) cyclization of MCoTI-II-
MeNbz-G crude synthesis (peak at 25.3 min matches the
cyclic MCoTI-II product, cMCoTI-II); c) ESIꢀMS spectra of
cMCoTI-II (left), and folded MCoTI-II (right).
3. Discussion.
Efficient methods for ligating unprotected peptides in
aqueous solution has greatly facilitated the chemical
synthesis and semisynthesis of proteins, including
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ing microwave coupling conditions with temperatures to tail cyclic structure and a cystine knot topology. Cyꢀ
1
2
3
4
5
6
7
8
up to 90 C. Critically, the secondary oꢀ
(methylamino)anilide is sufficiently reactive towards
chloroformate, and the resulting Nꢀmethylcarbamate
undergoes efficient cyclization under basic conditions
to afford the desired Cꢀterminal MeNbz peptide. While
this activation can be successfully implemented with
many solvents and bases, for large peptides we sugꢀ
gest a twoꢀstep protocol in which the MeNbz resin is
treated with nitrophenyl chloroformate (10 eq either in
CH2Cl2 or ClCH2CH2Cl, 0.25 M) for 2 h, followed by
washing and treatment with 1 M DIEA in DMF for 1 h.
Since reactions on large peptides are often strongly
affected by sequence dependent aggregation effects,
we recommend a small test cleavage to confirm full
activation of the MeDbz resin.
clotides own a high chemical and proteolytic stability,
and some of their different loops hold a significant flexꢀ
ibility that allows the exchange by non native cyclotide
sequences of approximately the same length. This
versatility, the oral bioavailability and the stability are
fundamental advantages for the grafting of small pepꢀ
tide motifs with potential therapeutic use, and thus why
it is conceived the utilization of the cyclotides as scafꢀ
folds in drug design.
9
Synthesis of cyclotides has been previously accomꢀ
plished, either using Boc or Fmoc chemistry, but with
no satisfactory results. In Fmoc chemistry, the main
problems arise from the number of cysteines, which
apart from a higher tendency to racemize, have the
10
11
12
13
14
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58
59
60
t
propensity to capture Bu cations during the cleavage
step. Preliminary synthesis of KB1 carried out on a
polystyrene resin gave poor analytical LCꢀMS profiles,
with deletions in some amino acids and a high abunꢀ
The high resistance towards diꢀacylation also faciliꢀ
tates the introduction of the Cꢀterminal amino acid,
which previously required mild acylation conditions that
was tailored to the individual amino acid. Loading the
MeDbz linker with a 5ꢀfold excess of amino acid /
HATU / excess DIEA, allows efficient incorporation of
most amino acids. However, for His and Cys a more
conservative protocol based on activation with DIC and
HOOBt was employed in order to avoid the chance of
racemization. Using these conditions we did not obꢀ
serve epimerization in the final peptideꢀMeNbz pepꢀ
tides or their ligation products.
t
dance of Bu adducts. Chain elongation on a more
hydrophilic polymer such as PEGꢀpolysterene resin
(Tentagel) led to an improvement of the crude purity,
but a significant sideꢀproduct was observed, with a
t
mass consistent with a Bu adduct. Shifting to a fully
pegylated resin, such as ChemMatrix, the desired KB1ꢀ
MeNbzꢀG was obtained following cleavage in an anaꢀ
t
lytical purity around 65%, although the butylated sideꢀ
product was still present. Changing the protecting
group scheme on 20Thr and 22Ser (tBu –> Trt) or swapꢀ
ping the cyclization point did not significantly reduce
this sideꢀproduct. However, the choice of the Phacm as
cysteine protecting group yielded a linear crude peptide
with no major sideꢀproducts, which let us to surmise
that likely one of the cysteine residues was scavenging
NCL conditions were optimized to suppress the
MeNbz hydrolysis and maximize ligation rate. We find
that a pH of 7.0 and a thiol concentration (4ꢀ
mercaptophenol) of 100 mM is generally suitable for
peptide ligation reactions. We observed a direct deꢀ
pendence of the ligation rate with the concentration of
the thiol additive which suggests that the Nꢀacylurea –>
thiol transthioesterification is rate determining when the
additive is below 100 mM. These optimized conditions
were further applied to the ligation of model peptides
corresponding to all 20 amino acids at the C-terminal
position. Similar to previous, the peptides could be
grouped into three different categories based on ligaꢀ
tion rate: i. 17 out of 20 gave ligations in a 5 h time
frame, ii. the ßꢀbranched amino acids valine and isoꢀ
leucine reacted more slowly, needing ~20 h to achieve
conversions above 90%, iii. proline, which requires
around 48 h to achieve conversions near to 90%. While
ligation reactions at Asp gave a significant ratio of the
ßꢀlinked isomer, likely as a consequence of anhydride
formation, the analogous reaction with Glu resulted in
only traces of isomeric products. Interestingly, a comꢀ
parison of the kinetics between the Pheꢀ
t
the Bu group. Nevertheless, using a oneꢀstep apꢀ
proach in a GSH:GSSG buffer, it was possible to perꢀ
form the cyclization and folding of the linear KB1ꢀ
MeNbzꢀG yielding the fully folded KB1 in recovered
overall yields around 20%. Following the same apꢀ
proach, MCoTIꢀIIꢀMeNbzꢀG was cyclized and folded in
a similar yield. Interestingly, KB1 and MCoTIꢀII have
very different HPLC elution profiles with regard to their
reduced cyclic precursors: whereas folded KB1 is reꢀ
markably more hydrophobic, native MCoTIꢀII is more
hydrophilic, reflecting the side chains that are presentꢀ
ed towards solvent when the disulfide rich hydrophobic
core is formed. These different hydrophobic characterꢀ
istics of the cyclotides may be significant when designꢀ
ing engineered cyclotides with biomedical applications.
In summary, we have developed a second generaꢀ
tion of the Nꢀacylurea linker, the MeNbz, with signifiꢀ
cant practical advantages over the first generation Nbz
linker. Importantly, MeDbz is resistant to multiple acylaꢀ
tion cycles of FmocꢀGly, forcing coupling conditions
with HATU and excess base and, remarkably, is comꢀ
patible with coupling at elevated temperatures of 90 C
(microwave). While the linker is robust towards undeꢀ
sired acylation, the peptideꢀMeDbz linker can be transꢀ
formed to the desired MeNbz (Nꢀacylurea) in a straightꢀ
forward manner using nitrophenyl chloroformate. Imꢀ
portantly, the resulting mildly activated peptideꢀMeNbz
products are competent for ligation by NCL in the presꢀ
ence of thiol additives. The MeDbz linker represents an
mercaptopropionyl
thioester
and
the
PheꢀN-
methylacylurea, both at a concentration of 2 mM and
using the standard ligation conditions (100 mM MPOH,
20 mM TCEP, pH 7.0, rt in 6 M guanidine buffer) yieldꢀ
ed similar ligation rates (Figure S19) showing that both
display similar reactivity.62
Since the primary use of these Cꢀterminally activated
peptides is for the synthesis of complex proteins by
NCL, we synthesized two members of the cyclotide
family: Kalata B1 and MCoTIꢀII. Cyclotides are a speꢀ
cial class of PTM proteins found in plants with a head
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Journal of the American Chemical Society
important step forward towards the development of a
Method B: following exactly the same protocol as in
method A until the introduction of the first glycine, then
the next 4 FmocꢀGly amino acids (0.2 mmol per couꢀ
1
2
3
4
5
6
7
8
rapid and efficient Chemical Protein Synthesis technolꢀ
ogy, because with the current fastꢀSPPS technologies
which includes, for instance, rapid flowꢀbased peptide
synthesis63 and microwave heating,64 it is easy to enviꢀ
sion that 30 to 40ꢀmer MeNbz peptides can be syntheꢀ
sized in under 5 h, and then assembled by NCL,
speeding enormously the synthesis of proteins by
chemical means.
pling cycle) were coupled using
a solution of
HBTU/HOBt (0.2 M in DMF, 0.2 mmol HBTU, 0.2 mmol
HOBt) and DIEA (0.3 mmol) during 1 h. Next, the pepꢀ
tide was acylated with pꢀnitrophenyl chloroformate,
cyclized under basic treatment and cleaved from the
resin using acydolitic conditions.
9
4. Experimental section.
4.3 Synthesis of Fmoc-Gly5-MeNbz-R (10).
10
11
12
13
14
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16
17
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59
60
4.1 Synthesis of Fmoc-MeDbz (8).
Method C: starting with 0.1 mmol of Rinkꢀamide
functionalized resin, following coupling of Arg and Dbz,
FmocꢀGly (0.5 mmol) was dissolved in a HBTU solution
(0.5 M, 0.5 mmol, 1 mL) and DIEA (0.096 mL, 0.55
mmol). The amino acid was preactivated for 30 s, and
then added to the resin. The resulting suspension was
bubbled with a gentle stream of N2, and after 1 h the
resin was filtered and washed with DMF. The next
FmocꢀGly residues were incorporated using 0.5 mmol
of amino acid activated with a solution of HBTU 0.5 M
in DMF (0.5 mmol of HBTU) and DIEA (0.096 mL, 0.55
mmol). Coupling times were 1 h. Following last glycine,
the o-(methylamino)anilide peptide was acylated with a
solution of pꢀnitrophenyl chloroformate (0.5 mmol) in
CH2Cl2 (2 mL) during 60 min. Then, the resin was filꢀ
tered, washed with CH2Cl2 and treated with a solution
of DIEA (0.5 M in DMF) during 15 min. After washing
the resin with DMF, CH2Cl2 and finally dried, it was
cleaved using the standard cleavage cocktail.
To a mixture of 4ꢀFluoroꢀ3ꢀnitrobenzoic acid (10 g,
0.054 mol) in MeOH (150 mL) was added a MeNH2
solution (40% in MeOH, 40 mL). The resulting mixture
was stirred at room temperature overnight, and then
poured into 150 mL of water. The reaction was acidified
with aqueous HCl (37%) until a yellow solid crashed
out from the solution. The product was collected by
filtration, washed with H2O and finally dried out. Next, it
was dissolved in MeOH (500 mL) and hydrogenated
over 500 mg of Pd/C (10%) overnight. The catalyst was
separated by filtration, and the solvent removed under
reduced pressure in a rotary evaporator. The 3ꢀaminoꢀ
4ꢀ(methylamino)benzoic acid was reꢀdissolved in a
H2O/CH3CN mixture (1:1, 100 mL) containing DIEA (9
mL, 0.052 mol) and a solution of FmocꢀCl (12.8 g,
0.049 mol) in CH3CN (50 mL) poured dropwise. Followꢀ
ing completion of the reaction, the CH3CN was reꢀ
moved and the product collected by filtration, washed
with CH3CN, and dried out under vacuum (15 g, 71%).
1HꢀNMR (400 MHz, [DMSOꢀD6], 25ºC): 2.79 (s, 3H),
4.30ꢀ4.40 (m, 3H), 6.62 (t, J = 6.6 Hz, 1H), 7.30ꢀ7.40
(m, 2H), 7.45 (t, J = 7.4 Hz, 2H), 7.63ꢀ7.80 (m, 3H),
7.92 (d, J = 7.9 Hz, 2H), 8.73 (s, 1H), 12.13 (s 1H). 13Cꢀ
NMR (100 MHz, [DMSOꢀD6], 25ºC): 29.5, 46.7, 66.0,
108.8, 116.7, 120.1, 122.0, 125.3, 127.1, 127.7, 140.7,
143.8, 154.7, 167.3
Method MW1: starting with 0.1 mmol of Rinkꢀamide
resin, and using the same protocol as in method C until
the introduction of the first Gly residue (FmocꢀGlyꢀ
MeDbzꢀArg(Pbf)ꢀresin), the next 4 FmocꢀGly residues
were introduced under microwave conditions using the
following setup:
i. coupling: FmocꢀGly (1.0 mmol, 2.5 mL, 0.4 M in
DMF), DIC (1.0 mmol, 2.0 mL, 0.5 M in DMF) and Oxꢀ
yma Pure (1.0 mmol, 1 mL, 1.0 M in DMF) were added
to the resin following this order; [FmocꢀGly, DIC, Oxyꢀ
ma Pure]final = 0.18 M. The resulting suspension was
heated under microwave irradiation: first 20 s at 75 ºC,
power = 170 watts; then, 3 min at 90 ºC, power = 50
watts.
ESIꢀMS: calcd. (C23H21N2O4, M + H) 389.4, found
389.1
4.2 Synthesis of Fmoc-Gly5-Nbz-R (9).
Method A: starting with 0.05 mmol of Rinkꢀamide
functionalized resin, following coupling of Arg and Dbz,
FmocꢀGly (0.2 mmol) and HBTU (0.2 mmol) were disꢀ
solved in DMF (1 mL) and DIEA (0.035 mL, 0.2 mmol)
was added. The amino acid was preactivated for 30 s,
and then added to the resin. The resulting suspension
was bubbled with a gentle stream of N2, and after 40
min the resin was filtered and washed with DMF. The
next FmocꢀGly residues were incorporated using 0.5
mmol of amino acid activated with a solution of HBTU
0.5 M in DMF (0.5 mmol of HBTU) and DIEA (1.0
mmol). Coupling times were 1 h. Following last glycine,
the o-(methylamino)anilide peptide was acylated with a
solution of pꢀnitrophenyl chloroformate (0.25 mmol) in
CH2Cl2 (1 mL) during 1 h. Then, the resin was filtered,
washed with CH2Cl2 and treated with a solution of DIEA
(0.5 M in DMF) during 15 min. After washing the resin
with DMF, CH2Cl2 and finally dried, it was cleaved using
the standard cleavage cocktail (TFA/TIS/H2O,
95:2.5:2.5).
ii. fmoc removal: first 30 s at 75 ºC, power= 155
watts; next, 90 s at 90 ºC, power = 30 watts.
Following last Gly, the FmocꢀGly5ꢀMeDbzꢀR peptide
was acylated with pꢀnitrophenyl chloroformate, cyclized
under basic conditions to yield, after acydolitic cleavꢀ
age, the FmocꢀGly5ꢀMeNbzꢀR peptide.
4.4 Synthesis of model MeNbz peptides (11A-Y).
The following protocols (aꢀc) describe the incorporaꢀ
tion on solid phase of the first amino acid after the
MeDbz linker. HATU is used as a general coupling
agent. Nevertheless, in order to avoid racemization in
some amino acids such as His and Cys a different
protocol is recommended. Moreover, it is not strictly
necessary the utilization of excess of base for all amino
acids, especially those that are not βꢀbranched. Addiꢀ
tionally, FmocꢀGly is easily incorporated using HBTU.
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The coupling conditions refer to 0.1 mmol of resin
(RinkꢀPS, 0.69 mmol/g).
Manual synthesis (method D): next residues after
1
the Cꢀterminal were coupled using 1.0 mmol of Fmocꢀ
aa preactivated during 30 s with a solution of HBTU 0.5
M (1.0 mmol of HBTU) and DIEA (0.191 mL, 1.1
mmol). Coupling times were typically 30 min. For the
Fmoc removal, a solution of piperidine (20% in DMF)
was added to the resin and the suspension shaken for
10 min. The Nꢀterminal cysteine was introduced as
BocꢀCys(Trt)ꢀOH. Following last amino acid, the resin
was washed with CH2Cl2 and a solution of pꢀnitrophenyl
chloroformate (201 mg, 1.0 mmol) in CH2Cl2 (4 mL)
was added and the resulting suspension shaken for 2
h. Following carbamylation, cyclization to yield the
acylurea peptide was carried out by treatment with a
basic solution of DIEA (1 M in DMF) during 1 h. Finally
the peptidylꢀresin was washed with CH2Cl2 and dried
under vacuum. Cleavage with cocktail ‘R’ (TFA, TIS,
H2O, EDT; 92.5:2.5:2.5:2.5), with or without 1,2ꢀ
Dimethylindole (2.5%), yielded the linear KB1ꢀMeNbzꢀ
G, KB1(Phacm)5ꢀMeNbzꢀG and MCoTIꢀIIꢀMeNbzꢀG
cyclotides. KB1ꢀMeNbzꢀG: 430 mg resin –> 133 mg
crude (73%); ESIꢀMS: calcd. (C128H197N39O42S6, M+ averꢀ
age) 3146.58, found 3145.7. KB1(Phacm)5ꢀMeNbzꢀG:
582 mg resin –> 250 mg crude (77%); ESIꢀMS: calcd.
(C173H242N44O47S6, M+ average) 3882.46, found 3882.5;
MCoTIꢀIIꢀMeNbzꢀG: 653 mg resin –> 266 mg crude
(81%); ESIꢀMS: calcd. (C149H241N51O48S6, M+ average)
3707.24, found 3707.0.
2
3
4
5
6
7
8
9
a) introduction of Gly: FmocꢀGly (149 mg, 0.5 mmol)
and HBTU (189 mg, 0.5 mmol) were dissolved in DMF
(1 mL) and DIEA (0.096 mL, 0.55 mmol) was added.
After 30 s, the solution was added to the resin and
gently stirred under a N2 stream for 40 min. Then, the
resin was washed with DMF and the peptide elongation
continued using standard conditions.
b) introduction of His and Cys: FmocꢀHis/Cys(Trt)ꢀ
OH (0.5 mmol) and HOOBt (82 mg, 0.5 mmol) were
dissolved in DMF (1 mL) and DIC (0.077 mL, 0.50
mmol) was added. After 3 min, the solution was added
to the resin and gently stirred under a N2 stream for 90
min. Following coupling, the resin was washed with
DMF and the peptide elongation continued following
standard conditions.
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c) coupling of the remaining amino acids: Fmocꢀaa
(0.5 mmol) and HATU (190 mg, 0.5 mmol) were disꢀ
solved in DMF (1 mL) and DIEA (0.191 mL, 1.1 mmol)
was added. After 30 s, the solution was added to the
resin and gently stirred under a N2 stream for 90 min.
After coupling, the resin was washed with DMF and the
chain elongation carried on following standard condiꢀ
tions.
Coupling of the VSYRA sequence following the C-
terminal amino acid: the Fmoc/Bocꢀaa (0.5 mmol) was
dissolved in a HBTU solution (0.5 M in DMF, 1.0 mL)
and DIEA (0.096 mL, 0.55 mmol) was added. After 30
s, the solution was added to the resin and gently stirred
under a N2 stream for 30 min. The last Val was coupled
as BocꢀVal. Fmoc removal was carried out with a piperꢀ
idine solution (20% in DMF) for 5 min. Following last
Automated microwave synthesis of KB1 (method
MW2): following manual couplings of Gly, MeDbz linker
and the Cꢀterminal Val residue, the remaining amino
acids were introduced under microwave conditions
using a 10ꢀfold excess of amino acid (1.0 mmol) and
HBTU/DIEA (1.0:1.2 mmol, respectively). The final
concentration of Fmocꢀaa and HBTU was 0.125 M, and
for DIEA 0.15 M. The coupling cycles were as followꢀ
ing:
residue, the resin was washed with DMF and CH2Cl2
,
and a solution of p-nitrophenyl chloroformate (100 mg,
0.5 mmol) in CH2Cl2 (2 mL) was added to the resin and
the resulting mixture shaken for 1 h. Next, the solution
was removed and the resin washed with CH2Cl2. A
solution of DIEA (0.5 M or 1 M in DMF) was added to
the resin and stirred under a N2 stream. βꢀbranched
residues (Val and Ile) were treated with DIEA 1 M for
30 min. For the remaining amino acids, the cyclization
step was carried out in a solution of DIEA 0.5 M for 15ꢀ
30 min.
i. coupling: a solution of Fmocꢀaa (0.2 M, 5 mL),
HBTU (0.5 M, 2 mL) and DIEA (1.2 M, 1 mL) were
added to the resin following this order, and the resulting
suspension was heated up under microwave irradiation
during 5 min at 75 ºC with a power = 27 watts. Cys was
coupled at 50 ºC keeping identical the rest of the paꢀ
rameters.
Arg double coupling: 1st coupling, T = 25 ºC, power =
0 watts, t = 30 min; then, T = 75 ºC, power = 27 watts, t
= 5 min. 2nd coupling: T = 75 ºC, power = 27 watts, t = 5
min.
Following acylurea formation, the resin was washed
with CH2Cl2, dried under vacuum and cleaved using the
standard cleavage solution for 1 h. Then, the TFA pepꢀ
tide containing solution was concentrated in a rotavaꢀ
por, and precipitated over Et2O, spun out and the solid
redissolved in a H2O/CH3CN solution and lyophilized.
Typical recovery yields of crude peptides were above
90% and analytical crude purities ranged from 90 to
95%.
ii. fmoc removal: 1st deprotection, t = 30 s, T = 75 ºC,
power = 55 watts; 2nd deprotection, t = 180 s, T= 75 ºC,
power = 65 watts.
KB1ꢀMeNbzꢀG: 452 mg resin –> 142 mg crude
(73%).
Cyclization of linear cyclotides (cKB1 and cMCoTI-
II): the crude linear peptides were dissolved in the ligaꢀ
tion buffer (6 M guanidine, 0.2 M sodium phosphate, 50
mM MPOH, 20 mM TCEP, pH 7.0) at a final concentraꢀ
tion around 1 mM. The progress of the reaction was
monitored by LCꢀMS, and after completion of the inꢀ
tramolecular ligation, the cyclic cyclotides were purified
by reverseꢀphase preparative HPLC. cKB1: 103 mg
crude –> 30 mg (33%); MALDIꢀTOF: calcd.
4.5 Synthesis of cyclotides.
The linear synthesis of the cyclotides KB1 and MCoꢀ
TIꢀII was carried out on a ChemMatrix solid support
(0.52 mmol/g) on a 0.1 mmol scale. Following coupling
of Gly and MeDbz linker, the Cꢀterminal amino acid
was introduced using the conditions reported above for
each specific residue.
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Journal of the American Chemical Society
(C117H185N35O39S6, M+ average) 2898.33, found 2898.2.
cMCoTIꢀII: 50 mg crude –> 19 mg (41%); ESIꢀMS:
calcd. (C138H229N47O45S6, M+ average) 3458.99, found
3458.5.
ligation of MeNbz peptides. This material is available free
of charge via the Internet at http://pubs.acs.org.
1
2
3
AUTHOR INFORMATION
4
5
6
7
8
9
Folding of cyclicꢀcyclotides: cKB1 and cMCoTIꢀII
were dissolved in a (NH4)2CO3 (0.2 M in H2O)/iPrOH
solution (1:1) containing GSH:GSSG (2 mM:0.4 mM) at
a final concentration around 0.2 mM and pH 8.5. The
folding reaction was monitored by LCꢀMS, and after
proper cystine formation, the solution was acidified with
aqueous HCl until pH 2ꢀ3, the iPrOH removed by rotary
evaporation and the folded cyclotides purified by reꢀ
verseꢀphase. KB1: 25 mg –> 16 mg (64%); MALDIꢀ
TOF: calcd. (C117H179N35O39S6, M+ average), 2892.28
found 2892.0. MCoTIꢀII: 13 mg –> 10 mg (77%) ESIꢀ
MS: calcd. (C138H223N47O45S6, M+ average) 3452.95,
found 3452.3.
Corresponding Author
* dawson@scripps.edu
ACKNOWLEDGMENT
The present work has been funded by the National Instiꢀ
tutes of Health R01 GM098871 (PED) Spanish Ministerio
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de Economía
y Competitividad through the grants
CTQ2012ꢀ31197 and RYCꢀ2011ꢀ09001 (JBBC).
REFERENCES
(1) a) Dawson, P. E.; Kent, S. B. Annu. Rev. Biochem.
2000, 69, 923ꢀ960; b) Kent, S. B. Chem. Soc. Rev. 2009, 38,
338ꢀ351; c) Payne, R. J.; Wong, C. H. Chem. Commun. 2010,
46, 21ꢀ43; d) Kan, C.; Danishefsky, S. J. Tetrahedron 2009,
65, 9047ꢀ9065; e) Unverzagt, C.; Kajihara, Y. Chem. Soc.
Rev. 2013, 42, 4408ꢀ4420.
(2) Dawson, P. E.; Muir, T. W.; ClarkꢀLewis, I.; Kent, S. B.
Science 1994, 266, 776ꢀ779.
(3) Yan, L. Z.; Dawson, P. E. J. Am. Chem. Soc. 2001, 123,
526ꢀ533.
(4) Crich, D.; Banerjee, A. J. Am. Chem. Soc. 2007, 129,
10064ꢀ10065.
(5) Harpaz, Z.; Siman, P.; Kumar, K. S.; Brik, A.
Chembiochem 2010, 11, 1232ꢀ1235.
(6) Cergol, K. M.; Thompson, R. E.; Malins, L. R.; Turner,
P.; Payne, R. J. Org. Lett. 2014, 16, 290ꢀ293.
(7) Thompson, R. E.; Chan, B.; Radom, L.; Jolliffe, K. A.;
Payne, R. J. Angew. Chem. Int. Ed. Engl. 2013, 52, 9723ꢀ
9727.
(8) Siman, P.; Karthikeyan, S. V.; Brik, A. Org. Lett. 2012,
14, 1520ꢀ1523.
(9) a) Haase, C.; Rohde, H.; Seitz, O. Angew. Chem. Int.
Ed. Engl. 2008, 47, 6807ꢀ6810; b) Chen, J.; Wan, Q.; Yuan,
Y.; Zhu, J.; Danishefsky, S. J. Angew. Chem. Int. Ed. Engl.
2008, 47, 8521ꢀ8524.
(10) Townsend, S. D.; Tan, Z.; Dong, S.; Shang, S.;
Brailsford, J. A.; Danishefsky, S. J. J. Am. Chem. Soc. 2012,
134, 3912ꢀ3916.
(11) Yang, R.; Pasunooti, K. K.; Li, F.; Liu, X. W.; Liu, C. F.
J. Am. Chem. Soc. 2009, 131, 13592ꢀ13593.
One step cyclizationꢀfolding of the linear cyclotides:
the crude linear cyclotides (KB1ꢀMeNbzꢀG and MCoTIꢀ
IIꢀMeNbzꢀG) were dissolved in a (NH4)2CO3 (0.2 M in
H2O)/iPrOH solution (1:1) containing GSH:GSSG (10
mM: 2.0 mM) at a final concentration around 0.5 mM
and a pH 7.5. After folding, the reaction was acidified
i
with HCl until pH 2ꢀ3, the PrOH removed by rotary
evaporation and the folded cyclotides purified by reꢀ
verseꢀphase. KB1: 125 mg crude –> 20 mg (18%).
MCoTIꢀII: 100 mg crude –> 20 mg (22%).
4.6 Protocol for ligation reactions at different
pHs, varying concentrations of MPOH and model
peptides.
A degassed aqueous solution of guanidine hydroꢀ
chloride (6 M), sodium phosphate (0.2 M), TCEP (20
mM) and the MPOH concentration of the assay, was
fitted to the desired pH with either an aqueous NaOH(aq)
(concentrated) or HCl (2 M in H2O) solution. The ligaꢀ
tion buffer was added to a mixture of MeNbz peptides
11A-Y and CRAFS to achieve a final concentration of 2
mM and 3ꢀ3.5 mM, respectively. The resulting solution
was stirred at room temperature, and at the indicated
times aliquots of the reaction (50 ꢁL) were taken and
quenched with a solution of hydroxylamine hydrochloꢀ
ride (50 ꢁL, 1 M in H2O, pH ~ 5.0). The mixtures were
analyzed by HPLC at 220 and 280 nm and all the sigꢀ
nals with a Tyr absorption profile were integrated at
280 nm to calculate the fraction of ligated peptide. The
same ligations were run in parallel until completion
without hydroxylamine quenching, and the kinetics and
product distribution analyzed obtaining similar results,
which supports the kinetics analysis (Figure S10).
(12) Malins, L. R.; Cergol, K. M.; Payne, R. J.
Chembiochem 2013, 14, 559ꢀ563.
(13) Chen, J.; Wang, P.; Zhu, J.; Wan, Q.; Danishefsky, S.
J. Tetrahedron 2010, 66, 2277ꢀ2283.
(14) a) Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23ꢀ26; b)
Offer, J.; Boddy, C. N.; Dawson, P. E. J. Am. Chem. Soc.
2002, 124, 4642ꢀ4646; c) Canne, L. E.; Bark, S. J.; Kent, S.
B. H. J. Am. Chem. Soc. 1996, 118, 5891ꢀ5896; d) Offer, J.
Biopolymers 2010, 94, 530ꢀ541.
(15) a) Hackeng, T. M; Griffin, J. H; Dawson, P. E. Proc.
Natl. Acad. Sci. USA 1999, 96, 10068ꢀ10073; b) Camarero, J.
A.; Adeva, A.; Muir, T. W. Lett. Pep. Sci. 1999, 7, 17ꢀ21; c)
Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111ꢀ117.
(16) a) Futaki, S.; Sogawa, K.; Maruyama, J.; Asahara, T.;
Niwa, M.; Hojo, H. Tet. Lett. 1997, 38, 6237ꢀ6240; b) Mezo, A.
R.; Cheng, R. P.; Imperiali, B. J. Am. Chem. Soc. 2001, 123,
3885ꢀ3891.
(17) Alsina, J.; Yokum, T. S.; Albericio, F.; Barany, G. J.
Org. Chem. 1999, 64, 8761ꢀ8769.
(18) Sewing, A.; Hilvert, D. Angew. Chem. Int. Ed. Engl.
2001, 40, 3395ꢀ3396.
(19) a) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B.
H.; Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999,
ASSOCIATED CONTENT
Supporting Information. NMR characterization of Fmoc-
MeDbz, LCꢀMS of the synthesis of pentaglycine peptides
9 and 10, model peptides 11A-Y, epimerization studies on
His and Cys, ligations with model peptides (12A-Y), anaꢀ
lytical LC of KB1-MeNbz-G synthesis, analytical LC of
pure cKB1 and cyclic cMCoTI-II, pure folded KB1 and
folded MCoTI-II, ligation kinetics of 11F and VSYRAF-
(SCH2CH2CO)-G with CRAFS, an expanded view of rate
of ligation with model peptides 11A-Y and CRAFS (Figure
4a), and a flow chart showing the synthetic route and
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Page 14 of 15
121, 11684ꢀ11689; b) Ingenito, R.; Bianchi, E.; Fattori, D.;
(42) Otaka, A.; Sato, K.; Ding, H.; Shigenaga, A. Chem.
Rec. 2012, 12, 479ꢀ490.
Pessi, A. J. Am. Chem. Soc. 1999, 121, 11369ꢀ11374.
(20) Camarero, J. A.; Hackel, B. J.; de Yoreo, J. J.;
Mitchell, A. R. J. Org. Chem. 2004, 69, 4145ꢀ4151.
(21) a) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.;
Melnyk, O. Org Lett 2010, 12, 5238ꢀ5241; b) 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. Engl. 2012, 51, 209ꢀ213; c) Sato, K.;
Shigenaga, A.; Tsuji, K.; Tsuda, S.; Sumikawa, Y.; Sakamoto,
K.; Otaka, A. Chembiochem 2011, 12, 1840ꢀ1844; d) Zheng,
J. S.; Chang, H. N.; Wang, F. L.; Liu, L. J. Am. Chem. Soc.
2011, 133, 11080ꢀ11083.
(22) a) Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H.
Org. Lett. 2004, 6, 4861ꢀ4864; b) Warren, J. D.; Miller, J. S.;
Keding, S. J.; Danishefsky, S. J. J. Am. Chem. Soc. 2004,
126, 6576ꢀ6578.
(23) a) Fang, G. M.; Li, Y. M.; Shen, F.; Huang, Y. C.; Li, J.
B.; Lin, Y.; Cui, H. K.; Liu, L. Angew. Chem. Int. Ed. Engl.
2011, 50, 7645ꢀ7649; b) Wang, P.; Layfield, R.; Landon, M.;
Mayer, R. J.; Ramage, R. Tetrahedron Lett. 1998, 39, 8711ꢀ
8714; c) Wang, P.; Shaw, K. T.; Whigham, B.; Ramage, R.
Tetrahedron Lett. 1998, 39, 8719ꢀ8720; d) Wang, P.; Layfield,
R.; Landon, M.; Mayer, R.; Ramage, R. J. J. Peptide Res.
1999, 53, 673ꢀ677; e) Wang, P. Tetrahedron Lett. 2007, 48,
7313ꢀ7315.
1
2
3
4
5
6
7
8
(43) a) Jones, J. H.; Ramage, W. I. J. Chem. Soc., Chem.
Comm. 1978, 472ꢀ473; b) IsidroꢀLlobet, A.; Alvarez, M.;
Albericio, F. Chem. Rev. 2009, 109, 2455ꢀ2504.
(44) a) Han, Y.; Albericio, F.; Barany, G. J. Org. Chem.
1997, 62, 4307ꢀ4312; b) Angell, Y. M.; Alsina, J.; Albericio, F.;
Barany, G. J. Pept. Res. 2002, 60, 292ꢀ299.
(45) It is important to mention that, probably due to traces
amounts of H2O in the solvents, and under some conditions,
i.e. 1 M DBU in DMF can lead to complete hydrolysis of the
peptide.
(46) Valeur, E.: Bradley, M. Chem. Soc. Rev. 2009, 38,
606ꢀ631.
(47) Nagalingam, A. C.; Radford, S. E.; Warriner, S. L.
Synlett 2007, 2517ꢀ2520.
(48) Johnson, E. C.; Kent, S. B. J. Am. Chem. Soc. 2006,
128, 6640ꢀ6646.
(49) Jencks, W. P.; Cordes, S.; Carriuolo, J. J. Biol. Chem.
1960, 235, 3608ꢀ3614.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(50) Bang, D.; Pentelute, B. L.; Gates, Z. P.; Kent, S. B.
Org. Lett. 2006, 8, 1049ꢀ1052.
(51) Dang, B.; Kubota, T.; Mandal, K.; Bezanilla, F.; Kent,
S. B. J. Am. Chem. Soc. 2013, 135, 11911ꢀ11919.
(52) Villain, M.; Gaertner, H.; Botti, P. Eur. J. Org. Chem.
2003, 3267ꢀ3272.
(24) BlancoꢀCanosa, J. B.; Dawson, P. E. Angew. Chem.
Int. Ed. Engl. 2008, 47, 6851ꢀ6855.
(25) Tiefenbrunn, T. K.; BlancoꢀCanosa, J.; Dawson, P. E.
Biopolymers 2010, 94, 405ꢀ413.
(53) Interestingly, hydrolysis was only 15%, likely due to
the rapid thiolysis of the anhydride intermediate.
(54) Craik, D. J.; Daly, N. L.; Bond, T.; Waine, C. J. Mol.
Biol. 1999, 294, 1327ꢀ1336.
(26) a) Hemantha, H. P.; Brik, A. Bioorg. Med. Chem. 2013,
21, 3411ꢀ3420; b) Hemantha, H. P.; Bavikar, S. N.; Hermanꢀ
Bachinsky, Y.; HajꢀYahya, N.; Bondalapati, S.; Ciechanover,
A.; Brik, A. J. Am. Chem. Soc. 2014, 136, 2665ꢀ2673.
(27) Zhang, Y.; Xu, C.; Lam, H. Y.; Lee, C. L.; Li, X. Proc.
Natl. Acad. Sci. USA 2013, 110, 6657ꢀ6662.
(28) a) Gunasekera, S.; Aboye, T. L.; Madian, W. A.; Elꢀ
Seedi, H. R.; Goransson, U. Int. J. Pept. Res. Ther. 2013, 19,
43ꢀ54; b) Akcan, M.; Craik, D. J. Methods Mol. Biol. 2013,
1047, 89ꢀ101.
(29) Fauvet, B.; Butterfield, S. M.; Fuks, J.; Brik, A.;
Lashuel, H. A. Chem. Commun. 2013, 49, 9254ꢀ9256.
(30) Lindgren, J.; Ekblad, C.; Abrahmsen, L.; Karlstrom, A.
E. Chembiochem 2012, 13, 1024ꢀ1031.
(31) Zhan, C.; Varney, K.; Yuan, W.; Zhao, L.; Lu, W. J.
Am. Chem. Soc. 2012, 134, 6855ꢀ6864.
(32) Harris, P. W. R.; Brimble, M .A. Int. J. Pept. Res. Ther.
2012, 18, 63ꢀ70.
(33) Mahto, S. K.; Howard, C. J.; Shimko, J. C.; Ottesen, J.
J. Chembiochem 2011, 12, 2488ꢀ2494.
(34) Siman, P.; Blatt, O.; Moyal, T.; Danieli, T.; Lebendiker,
M.; Lashuel, H. A.; Friedler, A.; Brik, A. Chembiochem 2011,
12, 1097ꢀ1104.
(35) Pentelute, B. L.; Barker, A. P.; Janowiak, B. E.; Kent,
S. B.; Collier, R. J. ACS. Chem. Biol. 2010, 5, 359ꢀ364.
(36) Okamoto, R.; Mandal, K.; Ling, M.; Luster, A. D.;
Kajihara, Y.; Kent, S. B. Angew. Chem. Int. Ed. Engl. 2014,
53, 5194ꢀ5198.
(55) Rosengren, K. J.; Daly, N. L.; Plan, M. R.; Waine, C.;
Craik, D. J. J. Biol. Chem. 2003, 278, 8606ꢀ8616.
(56) a) Polanowski, A.; Wilusz, T.; Nienartowicz, B.;
Cieslar, E.; Slominska, A.; Nowak, K. Acta Biochim. Pol.
1980, 27, 371ꢀ382; b) Hernandez, J. F.; Gagnon, J.; Chiche,
L.; Nguyen, T. M.; Andrieu, J. P.; Heitz, A.; Hong, T. T.;
Pham, T. T.; Nguyen, D. L. Biochemistry 2000, 39, 5722ꢀ
5730; c) Heitz, A.; Hernandez, J. F.; Gagnon, J.; Hong, T. T.;
Pham, T. T.; Nguyen, T. M.; Nguyen, D. L.; Chiche, L.
Biochemistry 2001, 40, 7973ꢀ7983.
(57) Henriques, S. T.; Craik, D. J. Drug Discov. Today
2010, 15, 57ꢀ64; b) Garcia, A. E.; Camarero, J. A. Curr. Mol.
Pharmacol. 2010, 3, 153ꢀ163.
(58) Tiefenbrunn, T. K.; Dawson, P.E. Protein Sci. 2009,
18, 970ꢀ979.
(59) a) Daly, N. L.; Love, S.; Alewood, P. F.; Craik, D. J.
Biochemistry 1999, 38, 10606ꢀ10614; b) Gunasekera, S.;
Daly, N. L.; Clark, R. J.; Craik, D. J. Antioxid. Redox Signal.
2009, 11, 971ꢀ980.
(60) a) Thongyoo, P.; RoqueꢀRosell, N.; Leatherbarrow, R.
J.; Tate, E. W. Org. Biomol. Chem. 2008, 6, 1462ꢀ1470; b)
Park, S.; Gunasekera, S.; LetaꢀAboye, T.; Göransson, U. Int.
J. Pept. Protein Res. 2010, 16, 167ꢀ176.
(61) a) Sola, R.; Saguer, P.; David, M. L.; Pascal, R. J.
Chem. Soc., Chem. Comm. 1993, 1786ꢀ1788; b) Pascal, R.;
Chauvey, D.; Sola, R. Tetrahedron Lett. 1994, 35, 6291ꢀ6294.
(62) However, alkylꢀ and arylthioesters are more stable
than N-acylureas which slowly hydrolyze even at acidic pH ~
2. Despite this lower stability, its purification, isolation and
handling are perfectly possible.
(63) Simon, M. D.; Heider, P. L.; Adamo, A.; Vinogradov, A.
A.; Mong, S. K.; Li, X.; Berger, T.; Policarpo, R. L.; Zhang, C.;
Zou, Y.; Liao, X.; Spokoyny, A. M.; Jensen, K. F.; Pentelute,
B. L. ChemBioChem 2014, 15, 713ꢀ720.
(37) Chiang, K. P.; Jensen, M .S.; McGinty, R. K.; Muir, T.
W. Chembiochem 2009, 10, 2182ꢀ2187.
(38) White, P. D.; Behrendt, R. J. Pep. Sci. 2010, 16, 71ꢀ
72.
(39) Kuzniewski, C. N.; Gertsch, J.; Wartmann, M.;
Altmann, K. H. Org. Lett. 2008, 10, 1183ꢀ1186.
(40) Proft, T. Biotechnol. Lett. 2010, 32, 1ꢀ10.
(64) Collins, J. M.; Porter, K. A.; Singh, S. K.; Vanier, G. S.
Org. Lett. 2014, 16, 940ꢀ943.
(41) Ling, J. J.; Policarpo, R. L.; Rabideau, A. E.; Liao, X.;
Pentelute, B. L. J. Am. Chem. Soc. 2012, 134, 10749ꢀ10752.
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