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m/z ¼ 1075.52 for [M + H+ + 16]. Yields for crude peptides with
tyrosine residues were directly calculated from HPLC chro-
matogram at 274 nm by comparison with Boc-Tyr-OH as
standard.
Abbreviations
AcN
Boc
Acetonitrile
Butyloxycarbonyl
DBU
DBF
1,8-Diazabicyclo[5.4.0]undec-7-ene
Dibenzofulvene
Aspartimide and related side-products
Model aspartimide prone peptide VKDGYI was synthesized on
rink amide-AM resin at 50 mmole scale using 5% piperazine +
1% DBU + 1% FA in DMF as Fmoc deprotection solution. The
DCM Dichloromethane
DIC
DIPEA N,N-Diisopropylethylamine
N,N0-Diisopropylcarbodiimide
resin bound peptide was divided into 10 aliquots, each repre- DMF
Dimethylformamide
EtOH Ethanol
senting ꢃ5 mmoles of peptide. Each aliquot of resin bound
FA
Formic acid
peptide was incubated with 500 mL of a specic deprotection
ꢁ
HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo
[4,5-b]pyridinium-3-oxid-hexauorophosphate
HOAt 1-Hydroxy-7-azabenzotriazole
HOBt Hydroxybenzotriazole
Oxyma 2-Ethyl(hydroxyimino)cyanoacetate
PolyA Poly alanine
solution for 60 min at 50 C. Post-incubation, the peptide was
cleaved and TFA solution was analysed directly by UPLC-MS.
The desired peptide as well as aspartimide and related side-
products were identied from the chromatogram at 274 nm
and relative amounts were calculated using the MassLynx
soware (Waters); tR(desired peptide) ¼ 7.3–7.5 min, m/z ¼
693.50 for [M + H+]; tR(aspartimide) ¼ 7.85–8.0 min, m/z ¼
675.47 for [M + H+]; tR(piperazide, multiple peaks) ¼ 5.4–5.8
min, m/z ¼ 761.59 for [M + H+]; tR(a + b piperidide) ¼ 10.1 and
10.5 min, m/z ¼ 760.62 for [M + H+].
PolyQ Poly glutamine
rt
t-Bu
Tis
Room temperature
tert-Butyl
Triisopropylsilane
Compatibility with 2-chloro trityl chloride resin
Acknowledgements
Fmoc-Tyr(O-tBu) was coupled to 2-chlorotritylchloride resin
preloaded with Gly (loading 0.51 mmol gꢀ1) using standard
coupling method as above and Fmoc group was removed with
5% piperazine + 1% DBU + 1% FA. Reference protected dipep-
tide (Tyr(O-tBu)-Gly) was obtained by treating the resin with 5%
TFA in DCM for 1 h with continuous vortexing at 900 rpm. To
The authors thank IIT Gandhinagar for start-up grant and
Centre for Biomedical Engineering, IIT Gandhinagar for
providing nancial support. Authors also acknowledge Central
Facilities at IIT Gandhinagar for mass spectroscopy.
determine the premature cleavage of the dipeptide, the peptidyl References
resin was incubated with deprotection solution (5% piperazine
1 A. El-Faham and F. Albericio, Chem. Rev., 2011, 111, 6557–
6602.
+ 1% DBU + 1% FA) at rt. Aliquots were withdrawn at 1, 2, 4, 8
and 24 h and injected onto UPLC-MS for analysis. All chro-
matograms at 274 nm were analyzed for the appearance of
a peak at 274 nm (tR ¼ 7.6 min, m/z ¼ 295.15 [M + H+]) corre-
sponding to the prematurely cleaved protected dipeptide.
2 J. M. Collins, K. A. Porter, S. K. Singh and G. S. Vanier, Org.
Lett., 2014, 16, 940–943.
3 J. M. Collins, in Microwaves in Organic Synthesis, Wiley-VCH
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Cysteine epimerization studies
To determine the extent of epimerization, model tripeptide
NH2-Gly-Cys-Phe-OH was assembled on rink amide resin (0.6
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Fmoc was removed using 5% piperazine + 1% DBU + 1% FA.
The resin bound peptide was divided into 4 aliquots; each
containing ꢃ5 mg of peptide. Aliquots were incubated with 500
mL of specic deprotection solution for 60 min at 50 ꢁC. One
aliquot with no incubation served as negative control. Post-
5 J. D. Wade, M. N. Mathieu, M. Macris and G. W. Tregear, Lett.
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6 J. Hachmann and M. Lebl, J. Comb. Chem., 2006, 8, 149.
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´
´
8 S. E. Blondelle, B. Forood, R. A. Houghten and E. Perez-Paya,
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9 B. D. Larsen and a Holm, Int. J. Pept. Protein Res., 1994, 43, 1–9.
incubation, the peptide was cleaved and TFA solution was 10 R. Warrass, J. M. Wieruszeski, C. Boutillon and G. Lippens, J.
analysed directly by UPLC-MS. A positive control was obtained Am. Chem. Soc., 2000, 122, 1789–1795.
for this experiment by synthesizing tripeptide using HOAt/ 11 M. Dettin, S. Pegoraro, P. Rovero, S. Bicciato, a Bagno and
HATU/DIPEA as coupling reagent which resulted in 10.2% epi- C. Di Bello, J. Pept. Res., 1997, 49, 103–111.
merized peptide. The ratio of L-peptide to D-peptide was calcu- 12 F. Nissen, T. E. Kra, T. Ruppert, M. Eisenhut, U. Haberkorn
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min, m/z ¼ 325.38; tR (D-peptide) ¼ 9.78, m/z ¼ 325.38 for [M + 13 L. a. Carpino and G. Y. Han, J. Org. Chem., 1972, 37, 3404–
H+].
3409.
104424 | RSC Adv., 2015, 5, 104417–104425
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