10.1002/anie.202003676
Angewandte Chemie International Edition
RESEARCH ARTICLE
atom and cleaved (see ESI Section 1.11, 1.12 for details). These
properties allow the detection in solution or even when bound to
the solid support, thus enabling for the first time a real-time
monitoring of both the coupling and the deprotection step.
Assessment of fluorescence at each coupling and deprotection
step during the synthesis of a pentapeptide H-LVAIG-NH2 was
performed by excitation at 280 nm and emission at 340 nm
(Figure 4, ESI section 1.12). The fluorescence allows
distinguishing between the reaction steps. After Smoc
deprotection, the fluorescence adjusts itself on a baseline value
with a specific auto-fluorescence. After coupling of an Nα-Smoc
amino acid, the fluorescence increases (Figure 4). The amino
acids show intrinsically different fluorescence; this could be
compensated by a normalisation that takes the quantum yield of
each amino acid into account.
Author Contributions
SK initiated, designed and coordinated the project; SK, CU, NK:
designed the experiments, performed all experiments and
analysis; OA, SK: wrote the manuscript; RM: performed NMR
studies and acquisition. OA and HK advised on all aspects. All
authors discussed the results and commented on the manuscript.
Conflict of Interests
The authors declare competing financial interests. S.K., H.K,
and C.U. are the founders of Sulfotools GmbH, a small chemical
company interested in aqueous peptide synthesis. N.K. is an
employee of Sulfotools GmbH. O.A., S.K., H.K, and C.U. are
named inventors on a patent application (WO 2016 050764)
filed by the Technische Universität Darmstadt and Sulfotools
GmbH on the aqueous peptide synthesis methodology
described in this work. R.M. declares no competing financial
interest.
Conclusion
To summarize, we developed the working concept of efficient
aqueous solid-phase peptide synthesis and demonstrated its
applicability to the synthesis of 22 biologically active peptides. To
make access to aqueous peptide synthesis, coupling efficiency
was assessed in water-based systems applying respective Nα-
Smoc amino acids and using different activation approaches. In
our hands, several water-compatible activating additives were
found appropriate, with EDC-HCl 37, Oxyma 39 and HOPO 40
being the most efficient ones. Our experiments showed that
although coupling of amino acids in pure water gave reasonable
yields and purity of peptides, the addition of organic co-solvents
enhanced coupling performance significantly. Additional studies
on enantiomeric composition showed no increased racemization
levels during the ASPPS process. Ionic properties of the Smoc
protecting group gave rise to an elegant approach towards a
reliable purification of synthetic peptides. Already Merrifield
showed that a mono-sulfonated Fmoc derivative could be applied
to peptide isolation with IEC.[47] This method was further optimized
and integrated as capping strategy into the ASPPS-based peptide
assembly. To that end, all the by-products originating from
incomplete couplings are labelled with charged sulfo-tags and can
be easily removed upon successive IEC after cleavage from solid
support. Our studies showed that sulfo-tag capping could also be
applied to Fmoc-SPPS. This method allows tailoring of
purification strategies depending on required peptide purity grade.
Moreover, the same method could be used to refine the waste
water (Section ESI 1.8, Figure S71).
Keywords: peptide synthesis • solid phase peptide synthesis
SPPS • sustainable chemistry • Smoc protecting group • water
based synthesis
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