Side reactions in the SPPS
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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