Solid-Phase-Based Synthesis of Ureidopyrimidinone-Peptide Conjugates for Supramolecular Biomaterials

Article abstract of DOI:10.1055/s-0035-1560520

Supramolecular polymers have shown to be powerful scaffolds for tissue engineering applications. Supramolecular biomaterials functionalized with ureidopyrimidinone (UPy) moieties, which dimerize via quadruple hydrogen-bond formation, are eminently suitable for this purpose. The conjugation of the UPy moiety to biologically active peptides ensures adequate integration into the supramolecular UPy polymer matrix. The structural complexity of UPy-peptide conjugates makes their synthesis challenging and until recently low yielding, thus restricted the access to structurally diverse derivatives. Here we report optimization studies of a convergent solid-phase based synthesis of UPy-modified peptides. The peptide moiety is synthesized using standard Fmoc solid-phase synthesis and the UPy fragment is introduced on the solid-phase simplifying the synthesis and purification of the final UPy-peptide conjugate. The convergent nature of the synthesis reduces the number of synthetic steps in the longest linear sequence compared to other synthetic approaches. We demonstrate the utility of the optimized route by synthesizing a diverse range of biologically active UPy-peptide bioconjugates in multimilligram scale for diverse biomaterial applications. 1 Introduction 2 Divergent Synthesis 3 Convergent Synthesis 4 UPy-Amine Strategy 5 UPy-Carboxylic Acid Strategy 6 Conclusion.

Full text of DOI:10.1055/s-0035-1560520

SYNLETT
0
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© Georg Thieme Verlag Stuttgart · New York
2015, 26, 2707–2713
letter
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Letter
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Solid-Phase-Based Synthesis of Ureidopyrimidinone–Peptide
Conjugates for Supramolecular Biomaterials
Isja de Feijtera,b,1
PG
PG
Olga J. G. M. Goora,b,1
Simone I. S. Hendriksea,b
Marta Comellas-Aragonèsa,b
Serge H. M. Söntjensc
Sabrina Zaccariaa,b
H2N
PG
1. UPy-COOH building block, HATU,
DIPEA, DMF (or DIMAc), r.t., 2 h
Peter P. K. H. Fransena,b
Joris W. Peetersc
Lech-Gustav Milroya,b,2
Patricia Y. W. Dankers*a,b,2
2. TFA–H2O–TIS (95:2.5:2.5% v/v), r.t., 4 h
3. RP-HPLC
O
O
H
N
H
N
H
O
N
N
O
3N
H
N
H
O
N
H
3,6
6,12
NH
O
O
a Institute for Complex Molecular Systems, Eindhoven University of
Technology, P.O. Box 513,5600 MB Eindhoven, The Netherlands
p.y.w.dankers@tue.nl
b Laboratory of Chemical Biology, Department of Biomedical
Engineering, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
c SyMO-Chem B.V., Het Kraneveld 4, 5612 AZ Eindhoven,
The Netherlands
Received: 14.08.2015
Accepted after revision: 13.10.2015
Published online: 03.11.2015
1 Introduction
The concept of tissue engineering was introduced in the
1980s,3 and presently comprises two approaches: the ex
vivo approach, in which cells are cultivated within a (syn-
thetic) scaffold before being implanted, and the other in
vivo or in situ approach, where a scaffold is implanted that
stimulates the self-healing capacity of the body. In both cas-
es the scaffold provides mechanical support to the sur-
rounding tissue and also has to be engineered to deliver the
necessary biological signals – for example, growth factors,
proteins, or peptide sequences derived from proteins – for
the surrounding cells to proliferate, migrate, and differenti-
ate.4–6 Short peptides are preferred for such purposes as
they are easy to synthesize to scale and are at present easier
to modify in a site-selective manner to introduce multiple
identical or different groups, typically without loss of func-
tion. Furthermore, peptides are generally more resistant to
chemical processing than proteins, which also facilitates
their integration into biomaterials. Polymer-based scaffolds
of varying degrees of mechanical strength have been gener-
ated to meet the specific mechanical demands in vivo. For
both classes – stiff and soft – a variety of covalent7–10 and
noncovalent11–17 polymer networks have been explored. In
contrast to covalently cross-linked scaffolds, the dynamic
nature of supramolecular cross-linking provides a more
straightforward way to functionalize polymers through in-
troduction of biological cues and markers conjugated to
complementary supramolecular monomers.18–21 Further-
more, the use of specific interactions facilitates precise hi-
erarchical self-organization and self-optimization,22–24 and
can be used to tune the rate of biodegradation.25 Supramo-
DOI: 10.1055/s-0035-1560520; Art ID: st-2015-s0639-l
Abstract Supramolecular polymers have shown to be powerful scaf-
folds for tissue engineering applications. Supramolecular biomaterials
functionalized with ureidopyrimidinone (UPy) moieties, which dimerize
via quadruple hydrogen-bond formation, are eminently suitable for this
purpose. The conjugation of the UPy moiety to biologically active pep-
tides ensures adequate integration into the supramolecular UPy poly-
mer matrix. The structural complexity of UPy–peptide conjugates
makes their synthesis challenging and until recently low yielding, thus
restricted the access to structurally diverse derivatives. Here we report
optimization studies of a convergent solid-phase based synthesis of
UPy-modified peptides. The peptide moiety is synthesized using stan-
dard Fmoc solid-phase synthesis and the UPy fragment is introduced on
the solid-phase simplifying the synthesis and purification of the final
UPy–peptide conjugate. The convergent nature of the synthesis reduc-
es the number of synthetic steps in the longest linear sequence com-
pared to other synthetic approaches. We demonstrate the utility of the
optimized route by synthesizing a diverse range of biologically active
UPy–peptide bioconjugates in multimilligram scale for diverse biomate-
rial applications.
1
2
3
4
5
6
Introduction
Divergent Synthesis
Convergent Synthesis
UPy–Amine Strategy
UPy–Carboxylic Acid Strategy
Conclusion
Key words modular approach, quadruple hydrogen bonding, pep-
tides, solid phase, biofunctionalization, bioactive materials
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lecular biomaterials based on polymers, cross-linked
through the fourfold hydrogen bonding of ureidopyrimidi-
none (UPy) moieties, have shown promising results as scaf-
folds in cardiovascular26 and renal11,27 regenerative medi-
cine. The introduction of extracellular matrix (ECM) de-
rived biological cues into these materials can be realized by
the noncovalent incorporation of UPy-functionalized bioac-
tive peptides. In this way, the properties of the supramolec-
ular biomaterial can be rapidly optimized through straight-
forward mixing of different peptide combinations at differ-
ent ratios.
UPy functionalization of peptides has previously been
performed via reaction of the N-terminus of the peptide
with a carbonyldiimidazole-activated methylisocytosine,
yielding an UPy–peptide without spacer between the UPy
and the first amino acid (e.g., UPy–peptide 1 in Figure 1),20
or via a UPy–hexyl isocyanate synthon (e.g., UPy–peptide 2
in Figure 1).28,29 The introduction of an oligoethylene glycol
(OEG) based linker group via an oxime ligation strategy
(UPy–peptide 3 in Figure 1) further increased the incorpo-
ration efficiency and accessibility of the peptides within the
biomaterial.30 However, for the production of functional
materials a fine-tuning of the amphiphilic properties of the
materials is desirable and can be achieved through the in-
troduction of a mixture of hydrophobic and hydrophilic
spacer groups within the UPy–peptide conjugates.
Here we describe the efficient solid-phase synthesis of
UPy-functionalized peptide conjugates with different
lengths of hydrophobic and hydrophilic linkers for use in
supramolecular biomaterial applications. The synthesis of
the UPy–peptide conjugates was explored using three strat-
egies – one divergent and two convergent; the advantages
and disadvantages of each strategy are discussed here. Our
data suggests that a convergent strategy, using amide-cou-
pling chemistry to introduce a suitable UPy-derived linker
molecule at the N-terminus of the peptide, is the most suit-
able for the scaled-up synthesis of UPy–peptide conjugates.
This convergent strategy in particular has enabled access to
a broad range of conjugates and should find extensive use
in the preparation of supramolecular biomaterials.
Patricia Y. W. Dankers, PhD PhD, is an associate professor in Supra-
molecular Biomaterials, in the Department of Biomedical Engineering
and the Institute for Complex Molecular Systems at the Eindhoven Uni-
versity of Technology (TU/e), The Netherlands. Her particular research
interests are on the design and synthesis of bioinspired functional bio-
materials. In her research she combines supramolecular chemistry with
biology and medicine. Her main goal is to translate these biomaterials
to applications in the field of regenerative medicine, with a focus on
cardiovascular and renal applications. She studied chemistry at the Rad-
boud University Nijmegen. In 2002 she started her first PhD in Natural
Sciences at TU/e studying supramolecular biomaterials under the super-
vision of Prof. Dr. Bert Meijer. In a second PhD in Medical Sciences at the
University of Groningen she worked on kidney regenerative medicine
together with Prof. Dr. Marja J. A. van Luyn (2013). Additionally, she
worked for the company SupraPolix in Eindhoven. In 2010 she per-
formed research in the Institute for BioNanotechnology in Medicine at
Northwestern University Chicago in the group of Prof. Dr. Samuel I.
Stupp. She is a Veni laureate (2008) and received an ERC starting grant
(2012). She has been awarded various (EU) grants and awards, such as
the DSM Science & Technology Award, and the Pauline van Wachem
Award for the best thesis in biomaterials research and tissue engineer-
ing. From 2011–2013 she has been a member of the first ‘Jonge Ge-
zondheidsraad’, the Dutch Health Council. Since 2015 she is a member
of ‘De Jonge Akademie’ of the Royal Academy of Sciences, the Nether-
lands (KNAW). Currently, she is the chairman of the Netherlands Society
of Biomaterials and Tissue Engineering (NBTE). Additionally, she consid-
ers the promulgation of science to society a very important topic.
Therefore, she recently developed and initiated a teaching program at
primary schools about performing research. Besides scientific research
her interests involve playing the clarinet and enjoying life with her two
little sons and family.
O
H
N
H
H
H
N
H
N
O
N
N
N
O
O
N
N
N
N
GRGDS
GRGDS
3 H
H
2
NH
O
O
NH
O
2
1
O
O
H
N
H
N
H
H
N
O
N
N
GGGGRGDS
N
N
O
O
N
6
7
3 H
H
H
NH
O
O
O
3
Figure 1 UPy–peptide designs, exemplified by the ECM-derived (GGG)GRGDS sequence, previously used for the functionalization of UPy-based bio-
materials.20,29,30
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2 Divergent Synthesis
azole-N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HBTU) and N,N-diisopropylethylamine [DIPEA, conditions
(iv) a)] yielded a mixture of the desired product 7 and a
cross-linked byproduct 6. The chemoselectivity observed in
the EDC case is most likely due to protonation of the second
amine group. Nevertheless, a double-coupling strategy us-
ing EDC and DMAP was needed to ensure complete conver-
sion to the monocoupled product. Thereafter, the hydro-
phobic spacer was introduced as the Fmoc-protected amino
dodecanoic acid 8 using HBTU and DIPEA, followed by
Fmoc-deprotection. The UPy moiety was subsequently cou-
pled onto the resin-bound amine by using an excess of
UPy–hexyl isocyanate28 (UPy-C6-NCO) to afford the UPy-
functionalized GGGGRGDS, GGGPHSRN, and GGGYIGSR
peptide conjugates (Table 1, 9a–c) after resin cleavage and
deprotection of peptide side-chain groups using a mixture
of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and
H2O (95:2.5:2.5 v/v%). Overall yields of 5–12% over 23 steps
were obtained for the three peptide sequences relevant for
cell adhesion and migration.33,34 Purities of typically >95%
were obtained after purification by reversed-phase prepar-
ative high-pressure column chromatography (RP-HPLC).
A solid-phase-synthesis strategy is known to signifi-
cantly increase the efficiency of the synthesis by avoiding
complicated workup procedures, reducing reaction times
through the use of excess reagents, and by simplifying the
purification due to washing steps.31 Furthermore, a diver-
gent strategy is an effective way to build and generate
structurally diverse compounds, especially peptide-based
libraries. For these reasons, a divergent synthesis route was
initially investigated to prepare three different UPy–peptide
conjugates with hydrophobic (C12) and hydrophilic OEG
spacers on a Rink amide resin in a filter-equipped syringe.
First, the peptide sequence 4 was prepared by standard sol-
id-phase Fmoc peptide synthesis (SPPS, Scheme 1).32 After
removal of the Fmoc-group, the N-terminus of the peptide
was reacted with succinic anhydride and pyridine to intro-
duce a carboxylic acid group 5, which permitted the cou-
pling of an OEG diamine using amide-coupling chemistry.
Interestingly, the use of 1-ethyl-3-(3-dimethyllaminopro-
pyl)carbodiimide (EDC) and dimethylaminopyridine
[DMAP, conditions (iv) b)] in this case gave exclusively the
desired product 7, whereas activation with o-benzotri-
H
i, ii
Fmoc
N
Fmoc-NH-GGGGR(Pbf)GD(t-Bu)S(t-Bu)
8 cycles
4
O
i, iii
H
O
N
GGGGRGDS
N
O
19
H
iv a, viii
O
HO
GGGGR(Pbf)GD(t-Bu)S(t-Bu)
N
O
H
H
O
5
N
GGGGRGDS
N
iv b
O
H
O
6
H
H2N
N
GGGGR(Pbf)GD(t-Bu)S(t-Bu)
O
N
H
19
O
7
v
O
O
H
H
N
N
GGGGR(Pbf)GD(t-Bu)S(t-Bu)
O
N
H
O
N
H
5
19
O
O
O
8
vi–ix
O
H
N
H
N
H
N
O
N
O
N
N
H
O
NH-GGGGRGDS
O
3 H
5
19
NH
O
O
9a
Scheme 1 Divergent synthesis of UPy–peptide conjugates on a Rink amide resin, as exemplified by GGGGRGDS peptide conjugate 9a. Reagents and
conditions: (i) 20% (v/v%) piperidine–NMP, 2 × 5 min, r.t.; (ii) Fmoc-protected amino acid, HBTU, DIPEA, NMP, 30 min, r.t.; (iii) succinic anhydride, pyri-
dine, DMF, 24 h, r.t.; (iv) a) PEG(18)-diamine, HBTU, DIPEA, DMF 24 h, r.t.; b) PEG(18)-diamine, EDC, DMAP, DMF, 2 × 24 h, r.t.; (v) 12-(Fmoc-amino)do-
decanoic acid, HBTU, DIPEA, DMF 24 h, r.t.; (vi) 20% (v/v%) piperidine–DMF, 30 min r.t.; (vii) UPy-C6-NCO,28 CHCl3, 48 h, r.t.; (viii) TFA–H2O–TIS
(95:2.5:2.5 v/v%) 4 h, r.t.; (ix) RP-HPLC (H2O–MeCN–0.1% TFA).
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, 2707–2713

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Table 1 Summary of the UPy–Peptide Conjugates and Yields Obtained via Divergent and Convergent Strategies
Synthetic strategy
Divergent
Compound
Peptide sequence (R)a
Yield (%)
9a
9b
9c
GGGGRGDS
GGGPHSRN
GGGYIGSR
12
5
O
O
O
O
O
O
O
O
H
H
N
H
N
H
N
O
N
N
R
R
R
8
N
N
H
O
O
O
O
O
N
H
5
19
3 H
NH
O
O
O
O
O
O
O
Convergent 10
17a
17b
17c
17d
GGRGDS
GGPHSRN
GSGDRG
GGDGEA
21
20
32
20
H
H
N
H
N
H
N
O
N
N
N
N
3 N
H
N
H
N
H
2
5
NH
O
O
O
O
O
11
18a
18b
GGRGDS
19
33
GGGPHSRN
H
N
H
N
H
N
H
N
O
O
O
O
N
N
H
N
H
6
11
3 H
NH
O
12
19a
19b
19c
GGRGDS
50
GGRGDSb
c(KRGDf)b,c
57
H
N
H
N
H
N
H
N
56d
O
3 N
H
N
H
R
3
6
NH
O
13
20a
20b
GGRGDSb
GGDGEAb
42
42
O
H
H
N
H
N
O
N
N
R
N
N
H
N
H
6
3 H
12
NH
a UPy linker is attached to N-terminal amino group.
b Coupled using HATU.
c Solution-phase chemistry.
d Yield of the protected UPy–peptide.
3 Convergent Synthesis
UPy building blocks 10–13 (Figure 2), were synthesized ac-
cording to an efficient and scalable solution-phase protocol
in yields ranging from 11–25% over 6–10 steps [see the
Supporting Information (SI) for experimental conditions
and compound characterization]. The difference between
these UPy building blocks lies in their chain length and in
the composition of the hydrophilic and hydrophobic spac-
ers. Compounds 10–13 were then coupled to the resin-
bound peptide using amide-coupling chemistry at the step
prior to resin cleavage. The two convergent approaches ex-
plored (Scheme 2), differed at the point of connection:
while the first approach – UPy–amine strategy – intro-
duced the UPy building blocks 10 and 11 through activation
of a resin-bound acid functionality, the second approach –
UPy–carboxylic acid strategy – required pre-activation of
compounds 12 or 13 before coupling to the peptide N-ter-
minus.
Though clearly applicable to the synthesis of structural-
ly diverse UPy–peptide conjugates bearing different linker
lengths and amphiphilic character, the disappointingly low
yields for 9a–c prompted the exploration of other conver-
gent synthetic strategies. In particular, a scalable, more effi-
cient synthesis of structurally diverse UPy–peptide conju-
gates bearing the same hydrophobic and hydrophilic linker
groups was sought. This is not possible in the divergent
strategy where the entire synthesis needs to be performed
from start to end.
Therefore, two different convergent approaches were
investigated with a view to increasing the overall yield and
efficiency of the synthesis. For both approaches, the pep-
tides were first synthesized by standard Fmoc SPPS on a
Rink amide resin. In contrast to the divergent approach, the
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O
amine
NH2
H
H
N
H
N
O
N
N
N
N
H
O
2
5
3 H
NH
O
O
10
short
O
H
N
H
N
H
O
N
N
O
NH2
N
N
H
O
11
6
3 H
NH
O
O
11
long
acid
OH
O
H
H
H
O
N
N
N
N
O
N
H
N
H
O
6
3
3
NH
O
O
O
short
12
O
O
H
N
H
N
H
O
N
N
O
N
H
N
H
O
OH
3
6
12
NH
O
O
long
13
Figure 2 Amine-terminated (10, 11) and carboxylic acid terminated (12, 13) UPy building blocks bearing different spacer lengths for convergent UPy–
peptide synthesis
O
O
H
H
N
H
H
GGRGDS
O
N
N
N
O
N
N
N
O
N
6
11
3 H
H
H
NH
O
O
O
18a
H
Fmoc
N
iv, v, vi
O
i, ii
a. UPy-amine
strategy
GGR(Pbf)GD(t-Bu)S(t-Bu)
HO
O
6 cycles
N
H
i, iii
O
FmocNHGGR(Pbf)GD(t-Bu)S(t-Bu)
O
H
N
O
b. UPy-carboxylic
acid strategy
H
H
N
N
N
O
GGRGDS
N
N
O
12
N
i, vii, v, viii
6
3 H
H
H
NH
O
O
20a
Scheme 2 Convergent synthesis of UPy–peptide conjugates on a Rink amide resin, as exemplified by GGRGDS peptide conjugates 18a via UPy–amine
strategy and 20a via UPy–carboxylic acid strategy. (i) piperidine–NMP (20% v/v), 2 × 5 min, r.t.; (ii) Fmoc-protected amino acid, HBTU, DIPEA, NMP, 30
min, r.t.; (iii) succinic anhydride, pyridine, DMF, 16 h, r.t.; (iv) UPy building block 11, PyBOP, DIPEA, DMF 2 × 16 h, r.t.; (v) TFA–H2O–TIS (95:2.5:2.5 v/v)
4 h, r.t.; (vi) RP-HPLC (H2O–MeCN–0.1% TFA); (vii) UPy building block 13, HATU, DIPEA, DMF 2 h, r.t.; viii) TFA–H2O–TIS–EDT (94:2:2:2 v/v) 4 h, r.t.
4 UPy–Amine Strategy
the resin using a mixture of TFA, TIS, and H2O (95:2.5:2.5
v/v), and purified by preparative RP-HPLC. This resulted in
the successful formation of fibronectin (GGRGDS, GGPHS-
RN, and scrambled control peptide GSGDRG)34 and collagen
I (GGDGEA)35 derived peptide conjugates 17a–d in yields of
20–32% for building block 10. Similar yields were obtained
for UPy–peptide conjugates 18a and 18b, where the UPy
building block 11 was equipped with a longer OEG spacer.
Furthermore, an irreversible side reaction was observed,
which would contribute to the overall low yields (Scheme
3). Analogous to aspartyl and asparaginyl cyclization, pre-
To enable coupling of UPy–amine building blocks 10
and 11, a carboxylic acid group was installed at the peptide
N-terminus by reacting the α-amino group with succinic
anhydride. Coupling of the functionalized peptide to UPy–
amine derivatives 10 or 11 (Figure 2) was achieved via the
activated ester, formed on treatment with benzotriazol-1-
yloxytripyrrolidinophosphonium
(PyBOP) in the presence of DIPEA. In the final step, the
UPy–peptide conjugate was deprotected and cleaved from
hexafluorophosphate
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sumably, the 1-hydroxybenzotriazol activated ester 14 un-
dergoes an intramolecular ring-closure reaction with the
amide at the peptide N-terminus, forming the succinimide
adduct of the peptide 15 on the resin while releasing the
benzotriazolate 16 into solution.33,36,37 Though this conver-
gent UPy–amine strategy is more practical than the diver-
gent route for the preparation of structurally diverse pep-
tide sequences bearing the same UPy linker group, we de-
cided to explore another convergent strategy to further
improve the yields.
building blocks 12 and 13, which were readily synthesized
with high overall yields. The convergent strategy enables a
fast optimization of the peptide sequence and an efficient
coupling of the preprepared UPy linker moiety, facilitating
the access to diverse libraries of UPy–peptide conjugates.
These can be readily mixed with the UPy-functionalized
polymeric material in different compositions and at differ-
ent ratios, allowing future application of these conjugates in
the biomedical field.
Acknowledgment
O
N
O–
O
N
N
O
We thank Ewelina Burakowska-Meise for initial experiments, and
Jolanda J. H. Spiering, Maarten H. Bakker and Joost L. J. van Dongen for
assistance with synthesis and analysis. Furthermore we gratefully ac-
knowledge E. W. Meijer, Henk M. Janssen, and Maartje M. C. Bastings
for scientific discussions. The research leading to these results has re-
ceived funding from the Ministry of Education, Culture and Science
(Gravity program 024.001.035), the Netherlands Organisation for Sci-
entific Research (NWO), the European Research Council (FP7/2007–
2013) ERC Grant Agreement number 308045, the Union’s Seventh
Framework Programme (FP/2007–2013) Grant Agreement number
310389, and Marie Curie Actions (VLPsiRNA-FP7-PEOPLE-2009-IOF-
254069). This research forms part of the Project P3.01 BioKid of the
research program of the BioMedical Materials institute, co-funded by
the Dutch Ministry of Economic Affairs. The financial contribution of
the Dutch Kidney Foundation is gratefully acknowledged.
N
Peptide
N
Peptide
N
N
N
H
O
O
14
15
16
Scheme 3 Proposed mechanism for the side reaction yielding the suc-
cinimide adduct of the peptide 15 related to the use of compounds 10
and 11
5 UPy-Carboxylic Acid Strategy38
Final yields of between 42–57% were achieved for the
UPy–peptide conjugates using a similar convergent strate-
gy, but which avoids undesirable succinimide formation. To
this end, carboxylic acid derived UPy building blocks 12
and 13 (Figure 2) were used for direct coupling to the pep-
tide N-terminus. Amide coupling was performed using Py-
BOP in the presence of DIPEA in DMF. After deprotection
and resin cleavage purification by preparative RP-HPLC
yielded the UPy–peptide conjugates in yields of up to 42%
for 20a,b. Yields of 50% were obtained for lower molecular
weight sequences such as 19a. Pleasingly, the yield could be
further improved to 57% for the UPy–GGRGDS conjugate
19b using HATU {1-[bis(dimethylamino)methylene]-1H-
1,2,3-triazolo-[4,5-b]pyridinium 3-oxid hexafluorophos-
phate} instead of PyBOP as activating agent. Furthermore,
the use of HATU circumvents the formation of a benzotri-
azole PyBOP adduct at the UPy–enolate tautomer,39,40
which could be readily converted into the desired product
(SI, Figure S16).
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560520.
S
u
p
p
ortiInfogrmoaitn
S
u
p
p
o
nrtogI
f
rmoaitn
References and Notes
(1) These authors contributed equally.
(2) These authors contributed equally.
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In addition to peptides on solid support, the UPy–car-
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© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, 2707–2713

2713
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allowed to swell in N,N-dimethylacetamide (DMAc) for 3 h,
rinsed with DMAc and combined with a solution of carboxylic
acid terminated UPy building block (0.15 mmol), DIPEA (0.3
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for 30 min. The resin was agitated in the reaction mixture for 90
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using preparative RP-HPLC-MS on a C18 column using a gradi-
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© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, 2707–2713