Side reactions in the SPPS of Cys-containing peptides

Article abstract of DOI:10.1007/s00726-013-1471-7

Alkylation of sensitive amino acids during synthesis of biologically important peptides is a common and well-documented problem in Fmoc-based strategy. Herein, we probed for the first time an unexpected S-alkylation of Cys-containing peptides that occur during the final TFA cleavage of peptides from the Wang solid support. Through a battery of approaches (NMR, UV and LC-MS) the formed by-product was assigned as the alkylation of the cysteine sulfydryl group by the p-hydroxyl benzyl group derived from the acidic Wang linker decomposition. Factors affecting this side reaction were monitored and a protocol that minimizes the presence of the by-product is reported.

Full text of DOI:10.1007/s00726-013-1471-7

Amino Acids (2013) 44:1357–1363
DOI 10.1007/s00726-013-1471-7
ORIGINAL ARTICLE
Side reactions in the SPPS of Cys-containing peptides
•
Panagiotis Stathopoulos Serafim Papas Charalambos Pappas Vassilios Mousis
•
•
•
•
Nisar Sayyad Vassiliki Theodorou Andreas G. Tzakos Vassilios Tsikaris
•
•
Received: 19 September 2012 / Accepted: 12 February 2013 / Published online: 5 March 2013
Ó Springer-Verlag Wien 2013
Abstract Alkylation of sensitive amino acids during
synthesis of biologically important peptides is a common
and well-documented problem in Fmoc-based strategy.
Herein, we probed for the first time an unexpected
S-alkylation of Cys-containing peptides that occur during
the final TFA cleavage of peptides from the Wang solid
support. Through a battery of approaches (NMR, UV and
LC–MS) the formed by-product was assigned as the
alkylation of the cysteine sulfydryl group by the p-hydro-
xyl benzyl group derived from the acidic Wang linker
decomposition. Factors affecting this side reaction were
monitored and a protocol that minimizes the presence of
the by-product is reported.
as for the Wang resin (Fig. 1b), resulting in side chain alkyl-
ation of indole ring of Trp-containing peptides (Giraud et al.
1999; Mutulis et al. 2003; Atherton et al. 1988).
To our knowledge, such by-products have not been
reported for cysteine-containing peptides, although they are
prone to oxidation and alkylation by cations produced during
the cleavage process. Cys-containing peptides are very
important biomolecules due to the inherent reactivity of the
thiol group, allowing them to participate in an array of pro-
cesses ranging from redox reactions, metal ion binding, post-
translational modifications, disulfide and thioether bond
formation, to name some (Bauhuber et al. 2009; Papas et al.
2007; Torres and Gait 2012; Mand et al. 2012; Mann et al.
´
2010; Jancso et al. 2011). In our laboratory, using the Wang
Keywords Solid phase peptide synthesis Á Wang resin
decomposition Á Cysteine Á S-alkylation Á TFA cleavage
resin for the synthesis of various Cys-containing peptides
with the Fmoc strategy, we observed both the expected
peptide and formation of a by-product (Fig. 2), similar to the
case of Trp-containing peptides (Fig. 1b). The percentage of
this formed by-product varied up to 35 % (data not shown).
This finding prompted us to explore the nature of this
by-product, as also the influence of the position of Cys on
the peptide sequence. A suggested mechanism for such
by-product formation is also provided. The possibility of
minimizing its formation, by changing resin substitution
and composition of the cleavage mixture was explored.
Introduction
Decomposition of the resin linkers upon the TFA cleavage of
the peptides in the Fmoc strategy is a common and well-doc-
umented phenomenon. This was observed to be the case both
for the Rink amide resin (Fig. 1a), resulting in C-terminal
alkylated amide by-products (Stathopoulos et al. 2006), as well
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-013-1471-7) contains supplementary
material, which is available to authorized users.
Materials and methods
P. Stathopoulos Á S. Papas Á C. Pappas Á V. Mousis Á
N. Sayyad Á V. Theodorou Á A. G. Tzakos (&) Á V. Tsikaris (&)
Department of Chemistry, Section of Organic Chemistry and
Biochemistry, University of Ioannina, 45110 Ioanina, Greece
e-mail: atzakos@cc.uoi.gr
Reagents
Fmoc amino acid derivatives, 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU),
and 1-Hydroxybenzotriazole (HOBt) were purchased
from Neosystem Laboratoire (Strasbourg, France).
V. Tsikaris
e-mail: btsikari@cc.uoi.gr
123

1358
P. Stathopoulos et al.
Linker
Resin
Trp-containing peptide
(B)
O
C
(A)
Peptide
OMe
NH
O
CH2
AAn
AA1
O
CH2
......
CH
CO
......
NH
CH2
O
CH2
R
N
OMe
scavengers
+
TFA
TFA
AAn
......
NH
AA1
......
OH
CH
CO
O
C
Peptide
CH2
NH
OH
O
C
Peptide
NH2
+
N
OH
H
Structure of by- product
Structure of by-product
Structure of target peptide
Fig. 1 Possible by-product formation in Rink Amide a and Wang b resins
Linker
Resin
Cys-containing peptide
AAn
NH
AA1
O
CH2
......
O
CH2
CH
CH2
S
CO
......
R
scavengers
TFA
+
AAn
NH
......
AA1 OH
......
AAn
NH
AA1 OH
......
CH
CO
CH
CH2
S
CO
......
CH2
+
OH
H
S
Structure of by-product
Structure of target peptide
Fig. 2 TFA cleavage of a Cys-containing peptide from the Wang resin
4-(Hydroxymethyl) phenoxymethyl-linked polystyrene
(Wang) resin and Fmoc-L-Ala-Wang resin were obtained
from GL Biochem (Shanghai, China) and Neosystem
Laboratoire (Strasbourg, France) respectively. TIS, EDT,
N,N-diisopropylethylamine (DIEA), TFA, 1,3-dimethoxy-
benzene (DMB), and piperidine were Merck–Schuchardt
(Darmstadt, Germany) products and used without further
purification. N,N-dimethylformamide (DMF) distillated
over ninhydrin and stored under preactivated molecular
sieves 4E, dichloromethane and the gradient degree high-
performance liquid chromatography (HPLC) solvents ace-
tonitrile and methanol were purchased from Labscan
(Dublin, Ireland).
Amino acids were introduced protected as Fmoc-
Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Trp(Boc)-
OH. Fmoc deprotection steps were carried out with 20 %
piperidine in DMF (v/v) for 15 min. Coupling reactions
of Fmoc amino acids were performed in DMF using a
molar ratio of amino acid/HBTU/HOBt/DIEA/resin
(3:3:3:6:1). Reactions were monitored with the color
Kaiser test (Sarin et al. 1981).
Cleavage and deprotection
The typical cleavage protocol included the following: ali-
quots of the dry peptide resin were placed into a rotating
reaction vessel and the tested cleavage mixture was added
in a ratio of 20 ml/g peptide resin. After 3 h stirring, the
resin was filtered and washed with TFA. The combined
filtrates were concentrated under reduced pressure. Hexane
was added and the resulted solution was reconcentrated.
This procedure was performed twice. The peptide was
Peptide synthesis
The peptides shown in Table 1 were synthesized manu-
ally with standard Fmoc a-protection (Fields and Noble
1990) first introduced by (Carpino and Han 1970, 1972).
123

Side reactions in the SPPS
1359
Table 1 Percentage of side products (SP) depending on the cysteine position, resin substitution, and cleavage mixtures
Entry
Peptide analogues
Substitution
Cleavage mixture (3 h)
SP (%)a
1
Ac-Ala-Arg(Pbf)-Cys(Trt)-Wang
Ac-Ala-Arg(Pbf)-Cys(Trt)-Wang
Ac-Ala-Arg(Pbf)-Cys(Trt)-Wang
Ac-Ala-Arg(Pbf)-Cys(Trt)-Wang
Ac-Ala-Arg(Pbf)-Cys(Trt)-Wang
Ac-Arg(Pbf)-Cys(Trt)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Cys(Trt)-Arg(Pbf)-Ala-Wang
Ac-Trp(Boc)-Arg(Pbf)-Ala-Wang
Ac-Trp(Boc)-Arg(Pbf)-Ala-Wang
0.70
0.70
0.70
0.32
0.32
0.60
0.79
0.79
0.40
0.40
0.40
0.40
0.40
0.40
0.40
0.40
94 %TFA, 2.5 %H2O, 2.5 %EDT, 1 %TIS
94 %TFA, 2.5 %H2O, 2.5 %EDT, 1 %TIS and 10 eq L-Trp
80 %TFA, 18 %EDT, 1 %TIS, 1 %H2O
94 %TFA, 2.5 %H2O, 2.5 %EDT, 1 %TIS
95 %TFA, 2.5 %TIS, 2.5 %H2O
16.4
14.3
5.0
2
3
4
10.8
61.6
9.2
5
6
94 %TFA, 2.5 %H2O, 2.5 %EDT, 1 %TIS
94 %TFA, 2.5 %H2O, 2.5 %EDT, 1 %TIS
95 %TFA, 2.5 %TIS, 2.5 %H2O
7
4.3
8
21.3
3.9
9
94 %TFA, 2.5 %H2O, 2.5 %EDT, 1 %TIS
89 %TFA, 2.5 %H2O,2.5 %EDT, 1 %TIS, 5 %DMB
92.5 %TFA, 2.5 %TIS, 5 %DMB
10
11
12
13
14
15
16
a
2.0
25.7
18.5
44.1
3.3
90 %TFA, 2.5 %TIS, 2.5 %H2O, 5 %DMB
95 %TFA, 2.5 %TIS, 2.5 %H2O
95 %TFA, 2.5 %TIS, 2.5 %EDT
94 %TFA, 2.5 %H2O, 2.5 %EDT, 1 %TIS
95 %TFA, 2.5 %H2O, 2.5 %TIS
2.2
15.1
The percentages represent the relative peak intensities estimated from the related ESI–MS spectra
precipitated with cold diethyl ether, filtered, dissolved in
2 N acetic acid, and lyophilized.
flow rate of 6 ll/min. The ionization source conditions
were as follows: capillary voltage, 3.5 kV; drying gas
temperature, 325 °C; trap drive 50; skimmer, 40 V; nitro-
gen flow and pressure, 5 L min-1 and 15 psi, respectively.
Maximum accumulation time of ion trap and the number of
MS repetitions to obtain the MS average spectra were set at
30 and 3 ms, respectively. All hardware components were
controlled by Agilent Chemstation Software.
NMR spectroscopy
All the NMR spectra were recorded on a Bruker AV-500
spectrometer equipped with a cryoprobe. The NMR sam-
ples were prepared by dissolving the solid material in
1
DMSO-d6 at a concentration of 5 mM. 2D H-1H COSY,
2D 1H-1H TOCSY (80 ms mixing time), 2D 1H-1H
1
ROESY, 2D H-1H NOESY (300 ms mixing time) exper-
Results and discussion
iments were performed on the isolated S-alkylated
by-product of the Ac-Ala-Arg-Cys-OH at DMSO-d6 and
298 K using standard Bruker pulse sequences. Diffusion
ordered NMR spectroscopy measurements were recorded
at 292 K using the bipolar pulse longitudinal eddy current
delay (BPPLED) pulse sequence. More specific, 16
BPPLED spectra with 16 K data points were collected and
the eddy current delay was set to 5ms. The pulse gradient
was increased from 2 to 95 % of the maximum gradient
strength using a linear ramp. After Fourier transformation
and baseline correction, the diffusion dimension was trea-
ted with the Topspin 2.1 suite.
Identification of by-product formation in a
Cys-containing peptide model
To probe the nature of the Cys-containing by-products we
synthesized the cysteine-containing peptide model Ac-Ala-
Arg-Cys-OH. The synthesis occurred on Wang resin using
the Fmoc based methodology. After the final TFA cleavage
of peptide from the solid support using the standard
cleavage mixture (94 % TFA, 2.5 % EDT, 2.5 % H2O,
1 % TIS), and HPLC purification we isolated both the
desired peptide and a by-product, which exhibited an
increased mass by 106 Amu with respect to the target
peptide (Figure S2). Moreover, this by-product exhibited
an absorbance at 280 nm in the UV detector, despite the
fact that the studied peptide sequence was depleted of
aromatic residues (Fig. 3).
Electrospray mass spectroscopy (ESI–MS)
Electrospray mass spectra were obtained on a quadrupole
ion-trap mass spectrometer (Agilent Technologies, model
MSD trap SL). Samples were dissolved in the mixture
H2O/CH3CN/HCOOH (49:49:2) and injected into the ESI
source (Agilent Technologies, Karlsruhe, Germany) at a
Using ESI–MS we found that the relative percentage of
by-product (SP(%)) was *16.4 (Table 1). To unambigu-
ously determine the identity of the formed by-product we
123

1360
P. Stathopoulos et al.
214 nm
a
M/Z: 497.1
M/Z: 391.0
b
280 nm
Fig. 3 ESI-MS spectra and HPLC profiles at 214 and 280 nm of the
crude Ac-Ala-Arg-Cys-OH peptide (1) synthesized on the Wang
resin. The numbers in the graph denote: the desired peptide (a) and
the by-product with an increased weight of 106 Amu compared to the
target molecule (b)
used NMR spectroscopy (Fig. 4). On the basis of the
recorded 1H-NMR spectrum a p-hydroxy benzyl group was
found to be covalently attached on the thiol-group of Cys
(Fig. 4), similarly to the case of Trp-containing peptides
(Figs. 1b, 2). By lowering the temperature (from 298 to
292 K) a minor conformer could be observed (Figure S9).
Diffusion-ordered spectroscopy (DOSY) is a sensitive
NMR technique that allows the determination of self-dif-
fusion coefficients and provides information about the
given molecular species like shape, weight, size and assists
the determination and detection of intermolecular interac-
tions and conformational changes (Kontogianni et al. 2013;
Primikyri et al. 2012). From the recorded DOSY spectrum
(Figure S8 and ?Figure S9) we found that the minor
conformer presented the same diffusion coefficient to the
major conformer. The minor conformer could have resulted
possibly due to a cation-p interaction formed between the
side-chain of arginine and the p-hydroxy benzyl ring. The
accessibility of Cys containing peptides for alkylation was
further verified by the synthesis of the Ac-Ala-Arg-OH
peptide that did not resulted in any by-product formation
(Figure S1).
of Cys-containing peptides with the p-hydroxy benzyl
group seems to be sensitive to the position of Cys in the
peptide sequence. Specifically, the highest tendency of
by-product formation was observed when the cysteine
residue occupied the C-terminus of the peptide (entries 1, 4
and 5).
Influence of the Wang resin substitution on the
S-alkylated by-product formation
To probe the influence of the resin substitution on the
by-product formation, cleavage experiments were per-
formed for peptide analogues Ac-Ala-Arg-Cys-OH and
Ac-Cys-Arg-Ala-OH, having cysteine residue at the C- and
the N-terminus respectively,. In these experiments the
cleavage mixture was kept constant (94 %TFA, 2.5 %H2O,
2.5 %EDT, 1 %TIS), whereas the Wang resin substitution
was varied. As illustrated in Table 1, when the resin sub-
stitution was increased, the yield of the S-alkylated
by-product formation was also increased (entries 1, 4 and
7, 9 respectively).
Influence of the cleavage mixtures on the S-alkylated
by-product formation
Influence of cysteine position on the S-alkylated
by-product formation
Evaluation of the cleavage conditions that could minimize
the percentage of the S-alkylated by-product was estimated
by using various cleavage mixtures. Table 1 summarized
some of the results obtained from these experiments. From
entries 9–14 of Table 1 we conclude that the presence of
EDT in the cleavage mixture is crucial for suppression of
S-alkylated by-product formation, while DMB and H2O
don’t prevent significantly cysteine alkylation. The reduc-
tion of the by-product formation by EDT was confirmed
both for higher resin substitution (entries 7, 8) as also for
To evaluate the influence of cysteine position on the by-
product formation we synthesized and studied the peptide
variants Ac-Arg-Cys-Ala-OH and Ac-Cys-Arg-Ala-OH,
altering the position of cysteine from the C- to the N-ter-
minal end of the initial peptide model (Ac-Ala-Arg-Cys-
OH). The experimental conditions used for the synthesis of
the peptide analogues Ac-Arg-Cys-Ala-OH and Ac-Cys-
Arg-Ala-OH were similar with those of the peptide model
(Ac-Ala-Arg-Cys-OH). As showed in Table 1, alkylation
123

Side reactions in the SPPS
1361
Fig. 4 Selected region of the
1
500 MHz H-NMR spectrum of
the isolated S-alkylated
by-product of the Ac-Ala-Arg-
Cys-OH at DMSO-d6 at 298 K.
The structure of the relevant
compound is shown as inset and
assignment of specific protons is
illustrated on the spectrum. The
1
full H-NMR spectrum as also
the 2D 1H-1H COSY, 2D 1H-1H
TOCSY, 2D 1H-1H ROESY, 2D
1H-1H NOESY and DOSY
NMR spectra can be found in
Figures S3–S9
Fig. 5 Suggested intra-
molecular and inter-molecular
mechanisms for S-alkylated
by-product formation, for the
case that Cys is located in the
C-terminus. A relevant inter-
molecular mechanism could be
suggested also for the case when
Cys is not located on the C
terminus (Figure S10). The
Wang resin linker is illustrated
in blue color and the peptide in
red color. The polymer support
is shown in blue sphere (color
figure online)
S
O
Trt
O
N
H
O
TFA (H+)
H
H
S
H
O
H
O
Trt
H
O
S
Trt
O
N
N
H
H
O
O
H
S
H
OH
OH
H
S
O
H
S
OH
Trt
H2C
N
H
H2C
O
O
H
N
O
H
H
N
O
H
O
intramolecular
TFA (H+)
OH
H
O
S
S
O
N
H
O
H
OH
N
H
O
different position of cysteine residue in the peptide
sequence (entries 4, 5). These results emphasize the
importance of EDT in the cleavage mixture towards cys-
teine alkylkation, that it is independent both on the location
of cysteine, in the peptide sequence, as also on the resin
substitution. A reduction of the byproduct formation to
14.3 % was also observed when in the cleavage mixture
was added 10 eq. of L-Trp (94 %TFA, 2.5 %H2O,
2.5 %EDT, 1 %TIS, 10 eq L-Trp), though the higher
concentration of EDT provided better results (entries 2, 3).
123

1362
P. Stathopoulos et al.
Relevant reactivity of Cys- in respect to Trp-containing
peptides for the alkylated by-product formation
Conclusions
Constructively, we exploited the potential formation of
by-products for Cys containing peptides through Fmoc-
based peptide synthesis on Wang solid support. The
alkylation of Cys-containing peptides with the p-hydroxy
benzyl group from the Wang resin linker decomposition
was shown to be directly dependent of the position of Cys
in the peptide sequence, resin substitution and cleavage
mixture composition. The presence of EDT in the cleavage
mixture is suitable for suppression of Cys alkylated pep-
tides. Suggested intra- and inter-molecular mechanisms
that could describe the formation and explain the relevant
percentage of such side products are provided. It should be
noted that although these observed Cys alkylated peptides
can be thought of as a drawback in SPPS of Cys contained
peptides in future it might be proved as a useful approach
to sculpt ligands for novel drug targets (Janga and Tzakos
2009; Nagulapalli et al. 2012).
To probe the relevant reactivity of Cys- in respect
to Trp- containing peptides towards the alkylated
by-product formation, the Ac-Cys(Trt)-Arg(Pbf)-Ala-
Wang was compared to the Ac-Trp(Boc)-Arg(Pbf)-Ala-
Wang peptide resin, using the same experimental
cleavage conditions. Interestingly, the cysteine-containing
peptide provided almost three times higher yield of the
by-product in respect to the Trp-containing peptide,
highlighting the higher sensitivity of cysteine in respect
to the Trp residue (entries 13, 16). The beneficial role of
EDT in the cleavage mixture was further confirmed also
for the Trp-containing peptide. Absence of EDT from the
cleavage mixture resulted in 15.1 % yield for the Trp
alkylation in respect to 2.2 % obtained upon the presence
of EDT (entries 15, 16).
Intermolecular versus intramolecular mechanism
for S-alkylated by-product formation
Acknowledgments This study was supported by the Regional
Operational Programme (ROP) of Thessaly-Mainland Greece- Epirus
within the frame of NSRF for the period 2007-2013 (ESPA 2007-13)
and co-financed by the European Union (European Social Fund
?ESF) and Greek national funds through the Operational Program
‘‘Education and Lifelong Learning’’ of the National Strategic Refer-
ence Framework (NSRF)—Research Funding Program: THALIS
Investing in knowledge society through the European Social Fund.
Moreover, special thanks are given to the Mass Spectrometry Unit
and NMR center of University of Ioannina for providing access to the
LC–MS/MS and NMR facilities.
As mentioned before, a higher tendency of S-alkylated
by-product formation was observed when cysteine resi-
due occupied the C terminus of the peptide sequence.
Specifically, the relevant yields of the by-product for the
Ac-Cys-Arg-Ala-OH, Ac-Arg-Cys-Ala-OH and Ac-Ala-
Arg-Cys-OH peptides were determined as 4.3, 9.2 and
16.4 % respectively. Enhancement of the by-product
abundance when cysteine was approaching the Wang lin-
ker triggered the hypothesis that an intramolecular mech-
anism could be postulated. Indeed, a closer spatial
approximation of the thiol group of cysteine to the linker
could be in favour of the performance of this side-reaction.
To shed light on this hypothesis, and exclude the potential
existence of an intermolecular mechanism, we incubated
the purified peptides Ac-Ala-Arg-Cys-OH or Ac-Cys-Arg-
Ala-OH with Fmoc-L-Ala-Wang in the presence of a
standard cleavage mixture constituted of 94 %TFA,
2.5 %H2O, 2.5 %EDT and 1 %TIS. After 3 h stirring, the
TFA-scavengers solution was concentrated under reduced
pressure and the peptide was precipitated with cold diethyl
ether, filtered, dissolved in 2 N acetic acid, and lyophilized,
as mentioned before. From ESI–MS spectra obtained for
the two originally unmodified peptides Ac-Ala-Arg-Cys-
OH or Ac-Cys-Arg-Ala-OH we detected the molecular ion
both of the desired peptides (M/Z: 391) and of the corre-
sponding S-alkylated side products (M/Z: 497). The per-
centage of these by-products were *12 %. The above
results indicate that a concomitant participation of both an
intra- and an inter-molecular mechanism could co-exist in
the formation of these by-products (Fig. 5).
Conflict of interest The authors declare that they have no conflict
of interest.
References
Atherton E, Cameron LR, Sheppard RC (1988) Peptide synthesis. Part
10. Use of pentafluorophenyl esters of fluorenyl methoxycar-
bonylamino acids in solid phase peptide synthesis. Tetrahedron
44:843–857
¨
Bauhuber S, Hozsa C, Breunig M, Gopferich A (2009) Delivery of
nucleic acids via disulfide-based carrier systems. Adv Mater
21:3286–3306
Carpino LA, Han GY (1970) The 9-fluorenylmethoxycarbonyl
function, a new base-sensitive amino-protecting group. J Am
Chem Soc 92:5748–5749
Carpino LA, Han GY (1972) The 9-fluorenylmethoxycarbonyl amino-
protecting group. J Org Chem 37:3404–3409
Fields GB, Noble RL (1990) Solid phase peptide synthesis utilizing
9-fluorenylmethoxycarbonyl amino acids. Int. J Pept Protein Res
35:161–214
Giraud M, Cavelier F, Martinez J (1999) A side-reaction in the SPPS
of Trp-containing peptides. J Pept Sci 5:457–461
´
Jancso A, Szunyogh D, Larsen FH, Thulstrup PW, Christensen NJ,
Gyurcsik B, Hemmingsen L (2011) Towards the role of metal
ions in the structural variability of proteins: CdII speciation of a
123

Side reactions in the SPPS
1363
metal ion binding loop motif. Metallomic Integ Biomet Sci
3:1331–1339
Nagulapalli M, Parigi G, Yuan J, Gsponer J, Deraos G, Bamm VV,
Harauz G, Matsoukas J, de Planque MR, Gerothanassis IP, Babu
MM, Luchinat C, Tzakos AG (2012) Recognition pliability is
coupled to structural heterogeneity: a calmodulin intrinsically
disordered binding region complex. Structure 20:522–533
Papas S, Akoumianaki T, Kalogiros C, Hadjiarapoglou L, Theodor-
opoulos P, Tsikaris V (2007) Synthesis and antitumor activity of
peptide-paclitaxel conjugates. J Pept Sci 13:662
Janga Sarath Chandra, Tzakos Andreas (2009) Structure and organi-
zation of drug-target networks: insights from genomic
approaches for drug discovery. Mol BioSyst 5:1536–1548
Kontogianni V, Charisiadis P, Primikyri A, Pappas CG, Exarchou V,
Tzakos AG, Gerothanassis IP (2013) Hydrogen bonding probes
of phenol –OH groups Org. Biomol Chem 11:1013–1025
´
Mand HL, Norden B, Fant K (2012) Functionalization with C-termi-
Primikyri A, Kyriakou E, Charisiadis P, Tsiafoulis C, Stamatis H,
Tzakos AG, Gerothanassis IP (2012) Fine-tuning of the diffusion
dimension of –OH groups for high resolution DOSY NMR
applications in crude enzymatic transformations and mixtures of
organic compounds. Tetrahedron 68:6887–6891
Sarin VK, Kent SBH, Tamand JP, Merrifield RB (1981) Quantitative
monitoring of solid-phase peptide synthesis by the ninhydrin
reaction. Anal Biochem 117:147–157
Stathopoulos P, Papas S, Tsikaris V (2006) C-terminal N-alkylated
peptide amides resulting from the linker decomposition of the
Rink amide resin: a new cleavage mixture prevents their
formation. J Pept Sci 12:227–232
Torres AG, Gait MJ (2012) Exploiting cell surface thiols to enhance
cellular uptake. Trends Biotechnol 30:185–190
nal cysteine enhances transfection efficiency of cell-penetrating
peptides through dimer formation. Biochem Biophys Res
Commun 418:469–474
Mann RJ, Al-Sabah S, De Maturana RL, Sinfield JK, Donnelly D
(2010) Functional coupling of Cys-226 and Cys-296 in the
glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide
bond that is close to the activation pocket. Peptides
31:2289–2293
Mutulis F, Erdelyi M, Mutule I, Kreicberga J, Yahorava S, Yahorau
A, Borisova-Jan L, Wikberg JES (2003) 2-(p-Hydroxybenzyl)
indoles—by-products formed upon cleavage of indole deriva-
tives from carboxylated wang polymer—an NMR study. Mol-
ecules 8:728–734
123