Native chemical ligation in dimethylformamide can be performed chemoselectively without racemization

Article abstract of DOI:10.1002/psc.2401

Native chemical ligation of unprotected peptides in organic solvents has been previously reported as a fast, efficient, and suitable method for coupling of hydrophobic peptides. However, it has not been determined whether the reaction can be carried out without possible side reactions or racemization. Here, we present a study on the chemoselectivity of this method by model reactions designed to test the reactivity of Arg and Lys side chains as well as that of α-amino groups. A possible racemization of the C-terminal amino acid of the N-terminal peptide was also investigated. The results show that ligation in organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups. Furthermore, no racemization of the C-terminal amino acid was observed if both educts were added simultaneously. Thus, native chemical ligation can be performed either in aqueous buffer systems or in organic solvents paving the way for the synthesis of larger hydrophobic peptides and/or membrane proteins.

Full text of DOI:10.1002/psc.2401

Research Article
Received: 29 November 2011
Revised: 4 January 2012
Accepted: 10 January 2012
Published online in Wiley Online Library: 19 March 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2401
Native chemical ligation in dimethylformamide
can be performed chemoselectively
without racemization
Marc Dittmann,a Muheeb Sadek,a,b Ralf Seidela and Martin Engelharda*
Native chemical ligation of unprotected peptides in organic solvents has been previously reported as a fast, efficient, and
suitable method for coupling of hydrophobic peptides. However, it has not been determined whether the reaction can be
carried out without possible side reactions or racemization. Here, we present a study on the chemoselectivity of this method
by model reactions designed to test the reactivity of Arg and Lys side chains as well as that of a-amino groups. A possible
racemization of the C-terminal amino acid of the N-terminal peptide was also investigated. The results show that ligation in
organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups. Furthermore, no
racemization of the C-terminal amino acid was observed if both educts were added simultaneously. Thus, native chemical
ligation can be performed either in aqueous buffer systems or in organic solvents paving the way for the synthesis of larger
hydrophobic peptides and/or membrane proteins. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: native chemical ligation in organic solvents; peptide synthesis; hydrophobic peptides; racemization
of possible side reactions has not been addressed so far. For
a general application of NCL in organic solvents, it is manda-
Introduction
Native chemical ligation (NCL) is based on a thioester-
mediated chemoselective reaction of two unprotected
peptide segments resulting in a native peptide bond at the
ligation site [1]. To undergo NCL, the N-terminal peptide has
to possess a C-terminal thioester, whereas the C-terminal
peptide contains an N-terminal Cys. During the past 15 years,
NCL has been developed into a routinely used tool for the
synthesis of proteins soluble in aqueous buffers [2] that even
allowed the synthesis and chemical modification of proteins
of up to 304 amino acids [3]. However, ligation of hydropho-
bic peptides and/or proteins such as membrane proteins
is hampered by their low solubility and their tendency to
aggregate under standard aqueous conditions [4,5]. Various
attempts have been published to overcome these difficulties
utilizing aqueous buffer systems. For example, ligations have
been carried out in the presence of organic solvents, denatur-
ants such as urea or guanidinium salts, and/or detergents
such as DDM, SDS, and DPC [6–11]. However, a more general
method is not yet available.
tory that the ligation has to proceed chemoselectively in the
presence of other nucleophilic groups. Furthermore, during
activation of the a-COOH of the unprotected N-terminal pep-
tide, organic bases might trigger racemization at this site. Here,
we demonstrate that NCL in organic solvents is chemoselective
and occurs without racemization.
Materials and Methods
Boc-L-Leu-Pam resin was obtained from NeoMPS (Strasbourg,
France). Boc-protected amino acids and HBTU were purchased
from Merck Chemicals (Nottingham, UK). DCM and DMF
were obtained from Applied Biosystems (Darmstadt, Germany).
TFA was purchased from Roth (Karlsruhe, Germany). All other
chemicals were obtained from Sigma Aldrich (Taufkirchen,
Germany) with the highest purity available.
Synthesized peptides were analyzed by RP-HPLC on a
Beckman (Krefeld, Germany; System Gold; modules 126, 168
Recently, we have described NCL of model peptides in organic
solvents under purely anhydrous conditions (Figure 1) [12]. In
this work, it has been shown that DMF readily solubilizes hydro-
phobic peptides, and in the presence of a base, like TEA as well as
thiol additives, a fast and efficient NCL is observed. To improve the
solubility of hydrophobic products, different other additives were
also tested (e.g. a-cyclodextrin, sodium trifluoroacetate, and
lithium chloride [14,15]). Best results regarding solubility and
yields were obtained with thiophenol in the presence of LiCl.
Under these conditions, the reaction was already completed in
less than 3 h.
*
Correspondence to: Martin Engelhard, Max Planck Institute of Molecular
Physiology, Otto Hahn Str. 11, 44227 Dortmund, Germany. E-mail: martin.
engelhard@mpi-dortmund.mpg.de
a Max Planck Institute of Molecular Physiology, Otto Hahn Str. 11, 44227
Dortmund, Germany
b Present address: Institut für Organische und Biomolekulare Chemie, Georg-
August-Universität Göttingen, Tammannstr. 2, 37077 Göttingen, Germany
Abbreviations used: DDM, n-dodecyl-b-D-maltoside; DPC, dodecylphosphocho-
line; HBTU, O-benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluorophosphate;
Leu, L-leucyl; MPAA, (4-carboxylmethyl) thiophenol; NCL, native chemical ligation;
SLeu, ÀSCH2COLeu; TEA, triethylamine; TFA, trifluoroacetic acid.
Although these results demonstrate that NCL can be
performed in organic solvents with high yields, the question
J. Pept. Sci. 2012; 18: 312–316
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

NCL IN ORGANIC SOLVENTS
Figure 1. General scheme of NCL reactions in organic solvents (thioester = ÀSCH2COLeu) [12]. The sequences of the peptides were derived from
transmembrane-spanning helices of the archaeal transducer protein NpHtrII [13].
and 508) dual-pump high-pressure mixing system, 214 nm
UV and 280 nm detection, and Bischoff C18 column
crude product mixture was analyzed by HPLC on a C18
column (detection wavelength 214 nm). The amount of
educts, aryl thioester, and products were determined by
integration of peak areas using 32 Karat Software (Beckman
Coulter). Because the extinction coefficients of the aryl
thioesters were considerably higher than those of the alkyl
thioesters, the values for the aryl thioesters were divided by
an empirical determined factor of 1.9. To determine the rela-
tive amount of each compound in the reaction mixture,
the corrected peak areas were divided by the number of pep-
tide bonds. Fractions of reactants were finally calculated by
dividing the corrected peak areas by the sum of integrated
educt and product areas.
a
(250 Â 4.6 mm, 5 mm). The molecular masses of the peptides
were determined by ESI-MS on an ESI-MS Finnigan LCQ Advan-
tage MAX (San Jose, USA) apparatus. The preparative purifica-
tions were carried out on a Bischoff C18 column (250 Â 20 mm,
5 mm) using a Waters System (Eschborn, Germany; Controller
600, Dual l Absorbance detector 2487) equipped with a fraction
collector.
Peptide Synthesis
All peptides were synthesized manually using Boc-chemistry,
in situ neutralization, and HBTU activation protocols on
a 0.2 mmol synthetic scale [16]. The N-terminal peptides
Ala-Val-Gln-Glu-Ala-SLeu and Ala-SLeu were synthesized on a
resin that generates a C-terminal thioester after HF cleavage
[17]. The peptides were deprotected and cleaved off from
the resin by anhydrous HF and p-cresol as scavenger for 1 h
at 0 ꢀC. Peptides were purified by HPLC on a preparative
C18 column using linear gradients from buffer A (0.1% (v/v)
TFA in water) to B (0.08% (v/v) TFA in acetonitrile). Fractions
were analyzed by ESI-MS and HPLC on an analytical C18
column. Mass spectrometric data of synthesized peptides are
summarized in Table 1.
Results and Discussion
In the previous work, two reaction conditions were found
suitable for NCL in organic solvents, which differ in the thiol
additive, namely, thiophenol or (4-carboxylmethyl) thiophenol
(MPAA). Because both thiols lead to fast reactions and high
yields, these conditions were analyzed for possible side reac-
tions. Amino acids whose side chains might react with acti-
vated thioesters are Lys and Arg. Additionally, free N-terminal
amino groups could also react. As an example, potential side
reactions for Lys during NCL are depicted in Figure 2.
A second serious problem concerns a decrease of the pKa of
the a-CH group adjacent to the C-terminal carboxyl group
during its activation that could lead to racemization. In the
following text, experimental results are presented addressing
these possible side reactions.
Ligation Experiments in Organic Solvents
Peptides (~3.5 mM) were dissolved in DMF or DMF in the
presence of 0.18 M LiCl. A thiol additive was added in various
concentrations (see Results and Discussion section). TEA
was added to a final concentration of 20 mM. The reaction
mixtures were stirred at 40 ꢀC. At each time point, an aliquot
(3 mL) was withdrawn and the reaction quenched by adding
20 mL of 50% acetonitrile in water (0.1% TFA). After reduction
of possible S–S bridges by tris(2-carboxyethyl) phosphine, the
Side Chain or N-Terminal Directed Reactions
To test whether nucleophilic amino groups can react directly with
C-terminal thioesters, we synthesized two C-terminal peptides
lacking an N-terminal Cys. These peptides contained either an
N-terminal Ala (Ala-Val-Ser-Ala-Ile-Leu) or an N-terminal Lys
(Lys-Val-Ser-Ala-Ile-Leu). The reaction of Ala-Val-Ser-Ala-Ile-Leu
or Lys-Val-Ser-Ala-Ile-Leu with Ala-Val-Gln-Glu-Ala-SLeu could
lead to possible products 3, 4, and 5 (Figure 2).
Table 1. Mass spectrometric data of peptides
Peptide
Theoretical
[M + H]+
Experimental
[M + H]+
In a first set of experiments, the reactions were carried out in
DMF using concentrations of 3.5 mM for each peptide in the
presence of 20 mM TEA, 0.3 M thiophenol, and 0.18 M LiCl. After
quenching the reaction with 50% acetonitrile in water (0.1%
TFA) at various time points, the crude product mixture was
analyzed by HPLC on a C18 column. HPLC traces from typically
ligation experiments are shown in Figure 3.
The ligation between Ala-Val-Gln-Glu-Ala-SLeu with Cys-Val-
Ser-Ala-Ile-Leu yields already after 1 min the transesterification
product Ala-Val-Gln-Glu-Ala-SPh (Figure 3a; peak at 20.8 min).
After 10 min, this reaction is almost completed. Concomitantly,
the final product Ala-Val-Gln-Glu-Ala-Cys-Val-Ser-Ala-Ile-Leu
(peak at 21.3 min) is formed. On the other hand, by replacing
Cys-Val-Ser-Ala-Ile-Leu by Lys-Val-Ser-Ala-Ile-Leu (Figure 3b),
Ala-Val-Gln-Glu-Ala-SLeu
Cys-Val-Ser-Ala-Ile-Leu
Ala-Val-Ser-Ala-Ile-Leu
Lys-Val-Ser-Ala-Ile-Leu
Ala-SLeu
704.8
605.7
573.2
630.8
277.4
221.3
292.4
292.4
1104.3
704.4
605.5
573.7
630.7
277.3
221.1
292.3
292.3
1104.2
Cys-Val
Ala-Cys-Val
D-Ala-Cys-Val
Ala-Val-Gln-Glu-Ala-Cys-Val-Ser-
Ala-Ile-Leu
Ala-Val-Gln-Glu-a-Cys-Val-Ser-Ala-
Ile-Leu
1104.3
1104.0
J. Pept. Sci. 2012; 18: 312–316 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

DITTMANN ET AL.
H
N
O
N
H
NH2
H2N
H
N
O
H2N
SR
4
O
H
N
O
H
N
O
base, aryl thiol
DMF
NH2
N
H
NH2
SR
+
+
O
H2N
H2N
3
1
2
H
N
O
N
H
O
H2N
5
Figure 2. Possible side reactions of NCL in organic solvents. Possible side reactions of the guanidinium group of Arg are not depicted.
Figure 3. HPLC analysis of the NCL at different time points. Peaks were collected, analyzed with ESI-MS, and assigned. (a) Ligation with Cys-Val-Ser-Ala-
Ile-Leu and (b) ligation with Lys-Val-Ser-Ala-Ile-Leu as C-terminal peptide (SLeu = ÀSCH2COLeu).
only Ala-Val-Gln-Glu-Ala-SPh is generated indicating that
primary and e-amino groups are not reacting or are forming
only in minor yields.
group is inert towards reaction with N-terminal thioester (data
not shown). It should be noted that some sequences might
contain a high number of positive charges. In this case, a care-
ful increase of base should be considered. As a summary, NCL
can be performed chemoselectively in organic solvents without
the need for protection of side chain functional groups.
To determine whether such side reactions do occur, more
flat gradients were used. Figure 4 summarizes the results. Data
were processed as described in the Materials and Methods
section. The fraction of educts and products was plotted
against the reaction time at 0 and 2 h. It is evident that small
amounts (<2%) of the thioester are reacting with primary
amines in both experimental systems using thiophenol or
MPAA. On the other hand, the classical NCL with an N-terminal
Cys is kinetically favored so that side products are not formed
(detection limit <0.1%). Under these conditions, a yield of
about 50% of the final NCL product is obtained after 2 h reac-
tion time. The conversion of Ala-Val-Gln-Glu-Ala-SLeu to the
corresponding aryl thioester can be observed in all three
experiments. It should be noted that dimerization product 4
has not been detected in the HPLC chromatograms. However,
careful mass spectrometric analysis of the crude reactions
mixture indicated minute amounts.
Epimerization Test
Another potential side reaction of NCL in organic solvents con-
cerns epimerization during the ligation reaction. This unwanted
side reaction might arise in the presence of a base (e.g. TEA) as
a result of a lower pKa of the a–C–H bond of the C-terminal
thioester.
To analyze this question, thiophenol and MPAA were
used respectively for the synthesis of a short tripeptide. The
N-terminal educt was an Ala-thioester and the C-terminal
educt the dipeptide Cys-Val. The two possible epimers
(D-Ala-Cys-Val and L-Ala-Cys-Val); can be separated by HPLC
on a C18 column with a standard water/acetonitrile buffer
system (Figure 5, trace a). As can be seen in both examples
using MPAA and thiophenol as activating agents, no D-epimer
is detected (detection limit is <0.1%).
In an additional set of experiments, the guanidinium group
of Arg was tested as possible reaction partner. With the use
of both thiol reagents (thiophenol and MPAA), commercially
available N-terminal Z-protected Arg was allowed to react with
the N-terminal peptide (Ala-Val-Gln-Glu-Ala-SLeu). No reaction
product could be detected indicating that also the guanidinium
To estimate whether epimerization can occur at all, Ala-SLeu
was preincubated with thiophenol or MPAA and base for 2 h
(Table 2). Subsequently, the Cys-Val peptide was added and
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 312–316

NCL IN ORGANIC SOLVENTS
Figure 4. NCL in DMF. Plotted are the fractions of reaction compounds. Their sum is normalized to 1. (a) Reaction conditions: 0.3 M thiophenol, 0.18 M
LiCl in DMF; (b) reaction conditions: 20 mM MPAA in DMF. (SLeu = ÀSCH2COLeu; X = Cys, Ala, or Lys). The yield (determined from three independent
experiments) for the synthesis of Ala-Val-Gln-Glu-Ala-Cys-Val-Ser-Ala-Ile-Leu (4A left panel) was 46.1 Æ 2.6%.
It is clearly evident that relatively low amounts of the
D-epimer (D-Ala-Cys-Val) are formed, if the thiol additive does
not exceed 20 mM. Higher concentrations (0.3 M thiophenol or
0.3 M thiophenol/benzyl mercaptan) lead to a considerable in-
crease of D-epimer. This result can be explained by the high
excess of the thiol additive, which leads to a fast and almost
complete transesterification within 10 min (Figure 3) leaving
enough time for racemization. Obviously, arylthioester is quite
susceptible to racemization, but this reaction can be avoided
by reducing the thiol concentration and mixing the reactants
simultaneously.
To analyze whether these results hold true also for longer
peptides, Ala-Val-Gln-Glu-Ala-SLeu and Cys-Val-Ser-Ala-Ile-Leu
were used for NCL. The reference compounds Ala-Val-Gln-
Glu-L-Ala-Cys-Val-Ser-Ala-Ile-Leu and Ala-Val-Gln-Glu-D-Ala-
Figure 5. Racemization of NCL in DMF studied by the synthesis of the
tripeptide Ala-Cys-Val: (a) D-Ala-Cys-Val and L-Ala-Val-Cys synthesized by
Cys-Val-Ser-Ala-Ile-Leu were synthesized by SPPS. These two
epimers were separated on HPLC using a phosphate/acetonitrile
buffer system (Figure 6, trace a). Formation of the epimers during
ligation of Ala-Val-Gln-Glu-Ala-SLeu with Cys-Val-Ser-Ala-Ile-Leu
was analyzed after the reaction was stopped at 2 h.
SPPS; (b) NCL with MPAA (20 mM); (c) NCL with thiophenol (0.3 M thiophe-
nol). The peaks at 17.5 and 25 min were analyzed by ESI-MS, but no
mass could be identified. The all L-peptide is labeled with L and the
D-Ala1 epimer with D.
It is evident that only the all L-epimer is observed (detection
limit is <0.1%) in agreement with the results obtained from the
formation of the tripeptide Ala-Cys-Val. It should be noted that
higher thiophenol concentrations do not lead to epimerization
(Figure 6, trace c).
the reaction was stopped after 1 h by the addition of 0.1% TFA/
water. After removal of organic solvent under reduced pressure
the reaction mixture was analyzed by HPLC.
Table 2. Racemization studied by the synthesis of the tripeptide
Ala-Cys-Val. TEA (20 mM) was used in all experiments
Concluding Remarks
Reaction condition
L-Ala-Cys-Val D-Ala-Cys-Val
In this work, we could demonstrate that NCL in DMF proceeds
chemoselectively. Other functional amino groups present
in the peptide chain (N-terminal amino group, Lys and Arg)
are inert under these conditions. Importantly, NCL in organic
solvents leads to the all L-epimer if both educts and thiol
additives are mixed simultaneously. These results and the
observation that the reaction proceeds quite fast and in high
yields [12] open the way for the ligation of hydrophobic pep-
tides and, ultimately, for the synthesis of membrane proteins.
(%)
(%)
20 mM MPAA
97
97
89
64
56
53
3
3
20 mM thiophenol
20 mM thiophenol/0.18 M LiCl
0.3 M thiophenol
11
36
44
47
0.3 M thiophenol/0.3 M benzyl mercaptan
0.3 M thiophenol/0.18 M LiCl
J. Pept. Sci. 2012; 18: 312–316 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

DITTMANN ET AL.
5 Olschewski D, Becker CFW. Chemical synthesis and semisynthesis of
membrane proteins. Mol. Biosyst. 2008; 4: 733–740.
6 Kochendoerfer GG, Salom D, Lear JD, Wilk-Orescan R, Kent SBH,
DeGrado WF. Total chemical synthesis of the integral membrane
protein influenza A virus M2: role of its C-terminal domain in
tetramer assembly. Biochemistry 1999; 38: 11905–11913.
7 Valiyaveetil FI, MacKinnon R, Muir TW. Semisynthesis and folding
of the potassium channel KcsA. J. Am. Chem. Soc. 2002; 124:
9113–9120.
8 Sato T, Saito Y, Aimoto S. Synthesis of the C-terminal region of opioid
receptor like 1 in an SDS micelle by the native chemical ligation: effect
of thiol additive and SDS concentration on ligation efficiency. J. Pept.
Sci. 2005; 11: 410–416.
9 Clayton D, Shapovalov G, Maurer JA, Dougherty DA, Lester HA,
Kochendoerfer GG. Total chemical synthesis and electrophysiological
characterization of mechanosensitive channels from Escherichia coli
and Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. 2004; 101:
4764–4769.
10 Lahiri S, Brehs M, Olschewski D, Becker CF. Total chemical synthesis of
an integral membrane enzyme: diacylglycerol kinase from Escherichia
coli. Angew. Chem. Int. Ed. 2011; 50: 3988–3992.
11 Musiol HJ, Dong S, Kaiser M, Bausinger R, Zumbusch A, Bertsch U,
Moroder L. Toward semisynthetic lipoproteins by convergent
strategies based on click and ligation chemistry. Chembiochem
2005; 6: 625–628.
12 Dittmann M, Sauermann J, Seidel R, Zimmermann W, Engelhard M.
Native chemical ligation of hydrophobic peptides in organic solvents.
J. Pept. Sci. 2010; 16: 558–562.
Figure 6. Racemization of NCL in DMF studied by the synthesis of Ala-
Val-Gln-Glu-Ala-Cys-Val-Ser-Ala-Ile-Leu after a reaction time of 2 h. (a)
Ala-Val-Gln-Glu-D-Ala-Cys-Val-Ser-Ala-Ile-Leu and Ala-Val-Gln-Glu-L-Ala-
Cys-Val-Ser-Ala-Ile-Leu synthesized by SPPS. The all L-peptide is labeled
with L and the D-Ala5 epimer with D; (b) NCL with MPAA (20 mM MPAA,
20 mM TEA in DMF, 40 ꢀC); (c) NCL with thiophenol (0.3 M thiophenol,
20 mM TEA, 0.18 M LiCl in DMF, 40 ꢀC). Peptides were separated by HPLC
on a C18 column using linear gradients from buffer A (5 mM disodium
hydrogen phosphate, pH 8 in water) to B (5 mM disodium hydrogen phos-
phate, pH 8 in 60% (v/v) acetonitrile/40% (v/v) water).
Acknowledgements
13 Klare JP, Chizhov I, Engelhard M. Microbial rhodopsins: scaffolds
for ion pumps, channels, and sensors. In Bioenergetics Results and
Problems in Cell Differentiation (45) Bioenergetics: Energy Conservation
and Conversion, Schäfer G, Penefsky H (eds). Springer-Verlag: Berlin
Heidelberg, 2008; 73–122.
We thank the ‘Volkswagenstiftung’ and the ‘Studienstiftung des
deutschen Volkes’ for their financial support.
14 Seebach D, Thaler A, Beck AK. Solubilization of peptides in non-polar
organic solvents by the addition of inorganic salts: facts and implica-
tions. Helv. Chim. Acta 1989; 72: 857–867.
15 Robey FA. Selective and facile cyclization of N-chloroacetylated
peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent
systems. J. Pept. Res. 2000; 56: 115–20.
References
1 Dawson PE, Muir TW, Clark-Lewis I, Kent SBH. Synthesis of proteins by
native chemical ligation. Science 1994; 266: 776–779.
2 Kent SBH. Total chemical synthesis of proteins. Chem. Soc. Rev. 2009;
38: 338–351.
3 Kumar KSA, Bavikar SN, Spasser L, Moyal T, Ohayon S, Brik, A. Total
chemical synthesis of a 304 amino acid K48-linked tetraubiquitin
protein. Angew. Chem. Int. Ed. 2011; 50: 6137–6141.
16 Schnölzer M, Alewood P, Jones A, Alewood D, Kent SBH. In situ
neutralization in Boc-chemistry solid phase peptide synthesis. Int. J.
Pept. Protein Res. 1992; 40: 180–193.
4 Johnson ECB, Kent SBH. Towards the total chemical synthesis of inte-
gral membrane proteins: a general method for the synthesis of hydro-
phobic peptide-a-thioester building blocks. Tetrahedron Lett. 2007;
48: 1795–1799.
17 Hackeng TM, Griffin JH, Dawson PE. Protein synthesis by
native chemical ligation: expanded scope by using straight-
forward methodology. Proc. Natl. Acad. Sci. USA 1999; 96:
10068–10073.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 312–316