Racemization Studies - CEM Gmbh

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Application Note BIO-0003
Microwave Synthesis: Racemization Studies
INTRODUCTION
One potential issue in peptide synthesis is
racemization of cysteine and histidine. This
is a concern for microwave peptide syntheis
because of the elevated temperatures
during deprotection and coupling. Studies
have shown that lowering the temperature
to 50 °C during the coupling of histidine and
cysteine will decrease racemization.
MATERIALS AND METHODS
Reagents
All Fmoc amino acids were obtained from
Novabiochem and contained the following
side chain protecting groups: Asn(Trt),
Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt),
Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu),
Thr(tBu), Trp(Boc), and Tyr(tBu). O-
(Benzotriazol-1-yl)-N,N,N’,N’tetramethyluronium
hexafluorophosphate
(HBTU), N-hydroxybenzotriazole (HOBt),
benzotriazol-1-yl-N-oxytris(pyrrolidino)phosphonium
hexaflourophosphate (PyBOP), and Rink
amide MBHA resin were also obtained from
Novabiochem. Diisopropylethyamine
(DIEA), N-methylmorpholine (NMM),
collidine (TMP), piperidine, piperazine,
trifluoroacetic acid (TFA), thioanisole, 1,2ethanedithiol
(EDT), and phenol were
obtained from Sigma Aldrich.
Dichloromethane (DCM), N,N-
Dimethylformamide (DMF), Nmethylpyrrolidone
(NMP), anhydrous ethyl
ether, acetic acid, HPLC grade water and
acetonitrile were obtained from VWR.
Peptide Synthesis:
VYWTSPFMKLIHEQCNRADG-NH2
A peptide containing all 20 amino acids was
synthesized under a variety of conditions
using the CEM Liberty automated
microwave peptide synthesizer on 0.152 g
Rink amide MBHA resin (0.66 meq/g
substitution). Deprotection was performed
in two stages with an initial deprotection of
30 s followed by 3 min at 50 W with a
maximum temperature of 80 °C (5 min
followed by 15 min at 0 W for conventional
synthesis). Coupling reactions were
performed in the presence of a 5-fold molar
excess of 0.2 M Fmoc-protected amino
acids dissolved in DMF with various types of
activation. Coupling reactions were 5 min at
40 W (30 min at 0 W for conventional
synthesis) with a maximum temperature of
80 °C. In latter experiments, coupling
conditions of cysteine and histidine were
altered to 2 min at 0 W followed by 4 min at
40 W with a maximum temperature of 50
°C. Cleavage was performed using 10 mL of
Reagent K (TFA/phenol/water/thioanisole/
EDT; 82.5:5:5:5:2.5) for 180 minutes.
Following cleavage, peptides were
precipitated out and washed using ice cold
anhydrous ethyl ether.
Peptide Analysis
All peptides were dissolved in 10% acetic
acid solution and lyophilized to dryness. The
peptide was analyzed using a Waters
Atlantis dC18 column (3µm, 2.1 × 100mm)
at 214nm. Separation was achieved by
gradient elution of 5-60% solvent B (solvent

A=0.05% TFA in water; solvent B=0.025%
TFA in acetonitrile) over 60 min at a flow
rate of 0.5 ml/min. Racemization analysis of
amino acids was performed by C.A.T. GmbH
& Co. using a published GC-MS method that
involves hydrolysis of the peptide in 6 N
DCl/D2O.
RESULTS
In order to optimize the peptide purity by
lowering the racemization of histidine and
cysteine during microwave enhanced
synthesis, multiple deprotection and
cleavage solutions were tested (Table 1).
For experiments where the coupling
temperature of Cys and His were changed,
the remaining amino acids in the peptide
were coupled at 80 °C.
Conventional synthesis yields less than 1.5%
d-enantiomer for all amino acids, yet crude
purity is only 68.4% because the
conventional synthesis results in numerous
amino acid deletions. A microwave
enhanced synthesis with piperidine
deprotection and HBTU/DIEA activation
indicates the sequence is susceptible to
racemization at histidine (9.4% d-His) and
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Application Note BIO-0003
cysteine (4.48% d-Cys) residues.
Racemization levels of other amino acids
during microwave synthesis were
comparable to those of conventional
synthesis (data not shown). The addition of
HOBt to the activator or substituting PyBOP
produced similar degrees of racemization as
HBTU activation. Lowering the temperature
from 80 °C to 50 °C during the coupling of
cysteine and histidine reduces the
racemization of His from >8.00% to 1.59%
and Cys from >3.96% to 3.16%.
CONCLUSION
The increased racemization associated with
microwave synthesis can be decreased by
lowering the temperature to 50 °C during
the coupling of histidine and cysteine. After
the histidine or cysteine has been coupled
there is no further increase in racemization
throughout the peptide chain synthesis.
Microwave synthesis, along with lower
coupling temperatures for histidine and
cysteine, leads to increased crude purity
and reduced synthesis time.
REFERENCES
Palasek S, Cox Z, Collins J. J. Pept. Sci. 2007, 13, 143-148.
Table 1. Racemization of amino acids measured by GC-MS after hydrolysis with 6 N DCl/D2O
Conventional Microwave
Deprotection A, 25 °C A, 80 °C B, 80 °C B, 80 °C C, 80 °C C, 80 °C C, 80 °C C, 80 °C
Coupling D, 25 °C D, 80 °C E, 80 °C F, 80 °C D, 80 °C G, 80 °C H, 50 °C H, 25 °C (0 W)
D-Cys 1.09