Tri-Orthogonal Scaffolds for the Solid-Phase Synthesis of Peptides

Article abstract of DOI:10.1002/ejoc.201800601

Multi-orthogonal scaffolds can be useful for the attachment of several different compounds to the same central skeleton. Such compounds can find applications in the development of protein mimics because of their potential to mimic several distant epitopes

Full text of DOI:10.1002/ejoc.201800601

A Journal of
Accepted Article
Title: New tri-orthogonal scaffolds for the solid-phase synthesis of
peptides
Authors: Jan Pícha, Benjamin Fabre, Miloš Buděšínský, Jan Hajduch,
Mehdi Abdellaoui, and Jiri Jiracek
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800601
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800601
Supported by

10.1002/ejoc.201800601
European Journal of Organic Chemistry
New tri-orthogonal scaffolds for the solid-phase synthesis of peptides
Pícha J. #, Fabre B.#, Buděšínský M., Hajduch J., Abdellaoui M., Jiráček J.*
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, v.v.i.,
Flemingovo n. 2, 16610 Praha 6, Czech Republic.
E-mail: jiracek@uochb.cas.cz
Homepage: https://www.uochb.cz/web/structure/184.html
# These authors contributed equally to the study.
Supporting information for this article is available on the www under http://dx.doi.org/ and
consists of: Experimental Section for the synthesis of compounds 1-12 (including Scheme S1),
additional analytical data (1H and 13C NMR and IR) for compounds 13-30 and compound 39,
description of the thermal cleavage of the Fmoc protecting group (including Scheme S2) and
HPLC chromatograms for compounds 16, 29, 30, 38, 39, 45, 51 and 57 (Figures S1-S8).
Graphical Abstract
Three tripodal scaffolds were developed and tested for the step-by-step synthesis of three
different peptides on the same scaffold by the solid-phase synthesis. Such compounds with
three differents peptides attached to a central core could mimic large proteins and find
applications in modulating protein-protein interactions.
Key Topic: multifunctional scaffolds, solid-phase peptide synthesis
Abstract
Multi-orthogonal scaffolds can be useful for the attachment of several different compounds to
the same central skeleton. Such compounds can find applications in the development of protein
1
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
mimics for their potential to mimic several distant epitopes in the protein structure. Based on
the structure of our previously published scaffold (Vaněk et al., Eur. J. Org. Chem. 17, 3689-
3701, 2015), we developed three new tri-orthogonal variants of this scaffold, which are suitable
for the solid-phase synthesis of three different peptides on the same skeleton. We combined
different chemical moieties for the phased attachment of amino acids to the scaffolds: Fmoc-
or Alloc-protected amine, free or TIPS-protected alkyne or azido group. We prepared and
characterized several model compounds and compared the suitability of new scaffolds for a
peptide synthesis. All three scaffolds provided peptides with satisfactory yields and purities,
making them suitable for a synthesis of libraries of compounds.
Introduction
The majority of biological processes in living organisms is mediated by actions of
peptides and proteins, which often interact with other peptides and proteins, creating
complicated networks of protein-protein interactions (PPIs).[1] Targeting PPIs with specific
inhibitors or activators can provide potentially useful tools for the treatment of various
disorders.[2] However, targeting PPIs with small molecules is extremely difficult, because
protein interaction surfaces are usually large, exposed to solvent, shallow and featureless and
noncontiguous.[3],[4] For this reason, many important proteins involved in PPIs are classified as
“undruggable targets”.[5] However, the majority of free energy for protein–protein interactions
is often mediated by what are called protein hot spots, which are groups of a few residues that
confer to a majority of free energy for the interaction.[6] Simultaneous targeting of a few
separated hot spots in a protein of interest could result in the potent inhibition or activation of
some biological event.
For this purpose, we previously developed two tripodal scaffolds, which enable stepwise
attachment of three different moieties to individual scaffold arms by Cu(I)-catalyzed click
chemistry (scaffold I)[7] or by a reaction of aldehydes with a hydrazide group of scaffold Ia[8]
(Figure 1). Recently, we synthesized a combinatorial library of 1ꢀ000 compounds based on the
structure of scaffold I modified with 30 different azides. The library was tested for binding to
the receptor for insulin. We identified few weak insulin receptor binders and activators.[9]
Nevertheless, it was clear that much larger compound diversity and complexity would be
necessary for the discovery of more potent insulin receptor binders. In this respect, amino acids
represent convenient types of building blocks for the modification of tripodal scaffold arms,
because they offer sufficient structural variety. There are only a few reports about the stepwise
2
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
solid-phase synthesis of three different peptides on specifically and orthogonally protected
molecular scaffolds of different chemical origins (Figure 1).[10]
In general, solid-phase peptide synthesis starts with the attachment of the first amino
acid by its carboxylic group to a reactive moiety on the solid support. The amino group
represents a convenient reactive partner for the carboxylate. For this reason, our general plan
consisted of the adaptation of the structure of scaffold I to a tri-orthogonal solid-phase peptide
synthesis, i.e. the introduction of amino groups to individual arms of scaffold I. In addition, we
designed and synthesized new scaffolds II-IV (Figure 1). Finally, we synthesized model
peptides and compared the suitability of each scaffold for the solid-phase peptide synthesis
(SPPS).
Results
Synthesis of scaffolds
The synthesis of 16 (scaffold II) from trimesic acid is shown in Scheme 1 and was
inspired by our previous work.[7] Briefly, during the first two reaction steps (steps a and b in
Scheme 1), both thermodynamic aspects (excess of acid and slow addition of the amine) and
kinetic factors (addition of the amine at 0 °C) affected significantly the yield of the reaction as
unwanted multi-acylations occurred. During the introduction of the third Fmoc-protected amino
“arm”, we expected the primary amine of salts 4 or 5 to be quickly generated, as the reaction
mixture contained an excess of DIPEA. Such primary amine could cleave the Fmoc protecting
group. To avoid this, we tested different modes of addition of the Fmoc-protected amines (in
solution or as a solid, in one or several portions), two different salts (trifluoroacetate 4 or
chlorohydrate 5), two different solvents (CH3CN or DMF) and an alternative DIC-mediated
chemistry, which does not require a base. We obtained the best yield (88ꢀ%) when salt 4 was
added in one portion to the PyBroP pre-activated acid 14 in CH3CN. Finally, the tert-butyl
protecting group in compound 15 was removed by treatment with 50 % TFA/DCM to afford
scaffold II (16).
For the synthesis of scaffold III (29) and scaffold IV (30), we chose a different strategy
(Scheme 2). Coupling the first arm to free trimesic acid leads to a mixture of products; unreacted
trimesic acid, desired monoamide, di- and triamide by-products and not easily removable
tripyrrolidinophosphinoxid. Previously, in the case of intermediate 13 (Scheme 1), this problem
was successfully solved by extensive and repetitive washings and triturations. Here, this work-
up was bypassed by introducing suitable protecting groups.
3
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
O
O
O
NH
O
HN
O
NH
O
NH2
NHAlloc
HN
O
OTHP
NHFmoc
HN
O
OTBDPS
NH
HN
O
ODMPM
N3
N3
NHivDde
Virta's
group
Scaffold from
Scaffolds from Madder's
group
H
N
O
O
O
O
HO
N
N
C
N
N
F2
H
NBSo
Fmoc
TES
N
N
H
O
Alloc
TIPS
Scaffold from Liskamp's
I
scaffold
group
O
O
O
O
O
H
N
HO
HO
N
N
H
Fmoc
TIPS
C
4 N
N
H
F2
TES
Fmoc
O
H
N
N
H
O
N
O
H
II 16
scaffold
Ia
scaffold
(
)
O
O
O
O
H
N
H
HO
HO
N
4 N
N
Alloc
4 N
N
Alloc
H
H
O
O
H
N
H
N3
N
N
Fmoc
O
N
H
Fmoc
O
H
IV
scaffold
30
III 29
scaffold
(
)
(
)
Figure 1. Scaffolds developed by other groups, scaffolds from our previous works (scaffolds I
and Ia) and new scaffolds prepared in this work (scaffolds II-IV).
Thus, trimesic acid was esterified with methanol to give trimethylester 17, which was
converted to 18 by a saponification of only one methoxycarbonyl moiety. Reaction between 18
and salt 1 afforded intermediate 19, which was easily isolated by a silica gel column
4
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
chromatography. Both ester functionalities were removed by basic hydrolysis to give diacid 20.
The second “arm” was attached by amidation with amines 7 or 9 to give the desired compounds
21 or 23, as well as the by-products 22 or 24. The yields were satisfactory: 77ꢀ% for 21 and
55ꢀ% for 23.
O
O
O
HOOC
COOH
HOOC
COOH
4N
OH
b
a
O
COOH
acid
N
O
N
O
H
H
TES
TES
14
13
Trimesic
O
O
O
O
NHFmoc
O
HO
NHFmoc
c
4 N
N
4 N
N
d
H
H
O
O
N
H
O
N
H
O
TES
TES
II 16
scaffold
15
(
)
O
O
H
N
H
FmocHN
FmocHN
Cl
NH3
NH
3 CF3COO
5
4
O3S
7
Scheme 1: Reagents, conditions, yields: (a) NH2CH2C≡C-TES, TEA, PyBroP, DMF, 0ꢀ°C to rt
for 16 h (53ꢀ%); (b) 7, TEA, PyBroP, DMF, 0ꢀ°C to rt for 16 h (40ꢀ%); (c) 4, DIPEA, PyBroP,
CH3CN, rt, 5 h (88ꢀ%); (d) TFA, DCM, rt, 1.5 h (82ꢀ%).
The third arm was attached similarly. Surprisingly, however, reaction of 21 with
trifluoroacetate salt 4 gave trifluoroacetamide 27 and starting acid 21. Treatment of 4 with an
excess of TEA released the corresponding free amine, which reacts faster with trifluoroacetate
species than with carboxylate group of 21. When trifluoroacetate salt 4 was replaced with
chlorohydrate 5, expected products 25 and 26 were isolated. We assumed that the presence of
a bulky Fmoc moiety caused the low yields observed during the third coupling. For this reason,
we used the Boc derivative 2 instead of the Fmoc derivative 5. However, the yields were similar.
A plausible explanation could be the steric hindrance of the two-arm intermediates 21 and 23.
TFA-mediated deprotection of acido-labile tert-butyl group afforded the desired scaffold III
(29) and scaffold IV (30). Compound 29 was obtained, also starting from 28; both acido-labile
protecting groups were removed in one step, followed by the introduction of the Fmoc group
5
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
instead of the Boc group. HPLC analyses of scaffolds II, III and IV confirmed their high
chemical purity (see chromatograms in the Supporting Information).
H
O
O
O
OH
O
OH
N
O
NHAlloc
b
a
c
O
O
O
O
O
O
O
O
O
O
OH
Trimesic
OH
acid
O
O
O
O
19
17
18
H
N
H
N
H
O
O
NHAlloc
NHAlloc
N
O
NHAlloc
e or
d
f
+
O
O
O
O
O
O
O
O
O
O
O
OH
N
N
N
OH
R
R
R
O
OH
4
4
4
20
R
R
21
23
22
24
N3
N3
H
F
N
3C
i
NHFmoc
g
h
27
O
H
N
H
N
O
O
NHAlloc
NHAlloc
O
O
O
O
O
O
O
O
NH
N
NH
N
BocHN
R
FmocHN
R
4
4
R
R
28
25
26
j
k
N3
H
N
NHAlloc
NHBoc
O
SO
SO
3 H3N
3 H3N
NHAlloc
1
2
O
O
O
NH
N
FmocHN
R
OH
O
4
H
N
H
N3
R
O
29
III
scaffold
O3S
30 scaffold IV
9
N3
Scheme 2. Reagents, conditions, yields: (a) MeOH, H2SO4, reflux, overnight (91ꢀ%); (b)
NaOH, methanol and water, rt, overnight (76ꢀ%); (c) 1, PyBroP, TEA, DMF, rt, overnight
6
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
(67ꢀ%); (d) NaOH, MeOH and water, rt, overnight (92ꢀ%); (e) 7, PyBroP, TEA, DMF, rt,
overnight (77ꢀ% for 21, 33ꢀ% for 22); (f) 9, PyBroP, TEA, DMF, rt, overnight (55ꢀ% for 23,
12ꢀ% for 24); (g) 5, PyBroP, DIPEA, DMF, rt, 4.5 h (20ꢀ% for 25, 29% for 26); (h) 4, PyBroP,
TEA, DMF, rt, overnight (39ꢀ%); (i) 2, PyBroP, TEA, DMF, rt, overnight (33ꢀ%); (j) TFA,
DCM, rt, 3.5 h (85ꢀ% for 29, 90ꢀ% for 30); (k) TFA, DCM, rt, 3.5 h, then Fmoc-Osu, NaHCO3,
water, dioxane, rt, overnight (42ꢀ% over two steps).
Synthesis of peptides on the scaffolds
For the synthesis of peptides on the scaffolds, we first examined the sequential clicking
of azides 11 and 12 to the resin-bound scaffold I as shown in Scheme 3. These azides possess
the Fmoc-protected amino group, which enables the synthesis of peptides directly on the resin-
bound scaffold. For the solid-phase synthesis, we used the PEG-based ChemMatrix resin[11]
with a highly acido-labile Ramage linker.[12] We used only half-maximum loading to the resin,
to limit the possible steric hindrance which we had previously encountered.[7]
The synthesis of the scaffold I-attached tripeptides proceeded without any problem with
apolar amino acids (compounds 38 and 39 in Scheme 3), but gave multi-component mixtures
with amino acids containing side-chain protections (Asp(tBu), Lys(Boc), His(Trt) etc.). We
found that tert-butyl ammonium fluoride (TBAF), used for the deprotection of the
triisopropylsilyl (TIPS) group, cleaves the protecting groups from the amino acids on the first
two arms. We did not find any convenient alternative TIPS-cleaving agent to fluoride (TBAF)
and therefore investigated peptide syntheses within scaffolds II, III and IV (Figure 1) with the
TIPS-alkynyl arm replaced with a Fmoc-amino group.
Next, as shown in Scheme 4, we synthetized compound 45 on the resin-bound scaffold
II. After loading scaffold II on the resin with PyBrop/DIPEA chemistry, we acetylated the
unreacted amines of the resin and obtained the resin-bound compound 40. The first model
dipeptide (Ile-Asp) was then built on the Fmoc-amino group-containing arm of the scaffold
using HATU/DIPEA activation. Thereafter, the N-terminal amines were acetylated with acetic
anhydride to give the resin-bound compound 41. Azide 11 was then selectively clicked to the
free alkyne arm via CuAAC protocol[13] to afford the resin-bound compound 42. The second
peptide (Met-Lys) was synthesized and capped as described for the first arm to give the resin-
bound compound 43.
7
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
O
O
O
O
O
O
HO
C
N
N
N
H
C
N
N
H
F2
H
b
a,
F2
TES
TES
O
N
O
N
H
H
TIPS
TIPS
scaffold I
31
c
O
O
N
O
O
O
O
N
C
N
N
H
C
N
N
H
H
F2
H
TES
F2
b
TES
d,
N
N
O
N
N
N
N
O
33
N
H
H
N
TIPS
HN
Fmoc
TIPS
HN
32
F
O
F
AcHN
e
O
O
N
O
O
O
O
N
C
N
N
H
C
N
N
H
N
N
N
N
H
F2
H
F2
NH
Fmoc
N
NH
N
A
NHAc
A
N
N
N
N
O
b
d,
O
N
N
O
35
N
N
H
H
TIPS
HN
TIPS
HN
34
F
F
O
O
F
F
AcHN
AcHN
f
O
O
O
O
O
O
N
C
F2
N
N
N
C
N
N
H
N
N
N
N
H
H
H
F2
NH
O
N
NH
O
N
A
A
c
NHAc
NHAc
A
A
N
N
N
N
O
N
N
O
N
H
N
N
N
H
NH
Fmoc
N
HN
HN
37
36
F
F
O
O
F
F
AcHN
AcHN
g
d,
O
O
O
:
11
With azide
H2N
C
N
N
H
N
N
N3
F2
FmocHN
NH
O
N
N
A
11
NHAc
A
N
N
N
O
N
N
H
HN
N
O
AcHN
N3
NHFmoc
F
F
O
O
HN
38
12
G
G
NH2
O
O
O
:
12
With azide
H2N
C
N
N
H
N
N
F2
O
N
O
H
AcHN
F
N
N
N
HN
F
O
O
39
N
H
N
A
A
N
N
O
NHAc
O
N
O
G
HN
G
NH2
Scheme 3. Reagents and conditions: (a) Ramage ChemMatrix (2 equiv), DIPEA, PyBroP,
DMF, rt, 5 h; (b) 5ꢀ% Ac2O (v/v), 5ꢀ% DIPEA (v/v) in NMP/DCM (1:1), rt, 2×15 min; (c) Azide
11 or 12 (5 equiv), CuSO4.5H2O (0.1 equiv), sodium ascorbate (0.5 equiv), DMF/H2O (6:4),
rt, 16 h; (d) Peptide synthesis (HATU/DIPEA); (e) Azide 11 or 12 (5 equiv), CuSO4.5H2O (5
equiv), sodium ascorbate (10 equiv), DMF/H2O (6:4), rt, 3×16 h; (f) TBAF (5 equiv) in DMF,
8
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
3×1 h; (g) TFA/H2O, scavengers, rt, 2×1 h. Amino acids are shown in one-letter codes: A for
alanine, F for phenylalanine and G for glycine.
We previously reported the one-pot TES-deprotection/CuAAC of a TES-protected
alkyne using high-copper(I) loadings.[7] Here we employed this strategy and transformed the
TES-alkyne group of compound 43 to the triazole 44 in one step. Next, we removed remaining
copper ions[14] by washing the resin with a solution of 20% 4-methylpiperidine in DMF. The
TES-deprotection with copper is slow and we obtained only a 70ꢀ% conversion after 16 h of the
reaction. After repeating the reaction for a further 16 h, the conversion increased to about 90ꢀ%.
Eventually, a third treatment allowed the full conversion (> 98%). Next, we built the third
peptide (His-Trp) on the resin-bound compound 44, and then cleaved the compound from the
resin altogether with side chain protecting groups, using a TFA/scavengers mixture. Finally,
the compound 45 was isolated from the crude mixture by HPLC.
Scaffold III has the TES-alkyne arm replaced by an Alloc-protected amine. This
substitution significantly shortens the synthesis on scaffold III. The synthesis of the first two
arms of 49 (Scheme 5) was the same as in the case of scaffold II. The Alloc-protecting group
was removed from 49 by treatment with Pd(PPh3)4 and in the presence of (CH3)2NH.BH3
scavenger, according to the literature.[15] The last dipetide (Trp-His) was built on the third arm
of 50 and, after acidic cleavage from support and simultaneous removal of all protecting groups,
we isolated the final product 51.
The synthesis of compound 57 (Scheme 6) was similar to the preparation of compound
51 (Scheme 5). The only different step (Step d) was the reduction of the azide. To reduce the
azido group, we first used phosphines: triphenyl-, tributyl- or tris(2-carboxyethyl)phosphine
(TCEP). Triphenylphosphine was unreactive, tributylphosphine led to complex reaction
mixtures, but TCEP gave a pure product after 2 h of reaction in a mixture of THF and water.
We also tried gaseous hydrogen sulfide as an alternative reducing agent. Hydrogen sulfide gas
was blown into the syringe containing the resin in a pyridine/H2O mixture. After three
treatments of 2 h each, the azido group was cleanly reduced to the corresponding amine.
9
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
O
O
O
O
H
N
HO
N
N
N
N
4
4
H
H
a,b
TES
Fmoc
TES
O
O
H
H
N
N
N
N
Fmoc
O
O
H
H
II 16
40
scaffold
(
)
c,b
O
O
O
O
H
N
H
N
N
N
N
4
N
4
H
H
d
TES
H
O
TES
D NHAc
O
H
N
N
N
D
NHAc
N
H
N
N
I
O
N
N
I
O
Fmoc
H
H
O
O
42
41
c,b
O
O
H
N
N
N
4
H
TES
O
H
NHAc
D
N
N
N
I
N
O
N
HN
O
H
O
K
43
AcHN M
e
O
O
H
N
N
N
4
N
N
H
H
N
Fmoc
N
O
O
H
N
N
N
D
NHAc
N
I
O
N
HN
H
K
44
AcHN M
O
f
c,
O
O
NH2
H2N
N
N
W H
4
N
N
H
N
H
N
O
O
H
N
N
N
D
NHAc
N
I
O
N
HN
O
H
O
K
45
AcHN M
Scheme 4. Reagents and conditions: (a) Ramage ChemMatrix, DIPEA, PyBroP, DMF, rt, 5 h;
(b) 5ꢀ% Ac2O (v/v), 5ꢀ% DIPEA (v/v) in NMP/DCM (1:1), rt, 2×15 min; (c) Peptide synthesis
(HATU/DIPEA); (d) Azide 11 (5 equiv), CuSO4.5H2O (0.1 equiv), sodium ascorbate (0.5
equiv), DMF/H2O (6:4), rt, 16 h ; (e) Azide 11 (5 equiv), CuSO4.5H2O (5 equiv), sodium
ascorbate (10 equiv), DMF/H2O (6:4), rt, 3×16 h; (f) TFA/H2O, scavengers, rt, 2×1 h. Amino
acids are shown in one-letter codes: D for aspartic acid, I for isoleucine, H for histidine, M for
methionine, K for lysine and W for tryptophane.
10
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
O
O
O
O
H
H
N
H
N
N
HO
N
N
Alloc
N
N
H
Alloc
4
4
H
a,b
O
O
H
H
N
N
N
Fmoc
N
Fmoc
O
O
H
H
III 29
46
scaffold
(
)
c,b
O
O
O
O
H
N
H
H
H
N
N
N
N
4
N
Alloc
N
N
H
Alloc
4
d
H
O
O
H
H
N
N
N
D
NHAc
D
NHAc
N
N
I
O
N
H
I
O
N
H
O
O
FmocHN
47
48
c,b
O
O
O
O
H
H
H
N
NH2
N
N
N
N
N
N
Alloc
4
4
e
H
H
O
O
H
H
NHAc
D
I
N
NHAc
D
N
N
N
N
I
N
N
O
N
N
O
O
N
O
H
H
O
O
M
M
AcNH
K
N
AcNH
K
N
50
49
H
H
c,f
O
O
H
N
H2N
W
N
N
H NH2
4
H
O
O
H
NHAc
D
N
N
I
N
N
O
N
O
H
O
M
AcNH
K
N
51
H
Scheme 5. Reagents and conditions: (a) Ramage ChemMatrix, DIPEA, PyBroP, DMF, rt, 5 h;
(b) 5ꢀ% Ac2O (v/v), 5ꢀ% DIPEA (v/v) in NMP/DCM (1:1), rt, 2×15 min; (c) Peptide synthesis
(HATU/DIPEA); (d) Azide 11 (5 equiv), CuSO4.5H2O (0.1 equiv), sodium ascorbate (0.5
equiv), DMF/H2O (6:4), rt, 16 h; (e) Pd(PPh3)4 (0.15 equiv), (CH3)2NH.BH3 (20 equiv), NMP,
N2 purging, rt, 1.5 h; (f) TFA/H2O, scavengers, rt, 2×1 h.
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10.1002/ejoc.201800601
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O
O
O
O
H
H
N
H
N
HO
N
N
N
Alloc
N
N
Alloc
4
4
H
H
a,b
O
O
H
H
N3
N
N3
N
N
Fmoc
N
Fmoc
O
O
H
H
IV
52
Scaffold
(30)
c,b
O
O
O
N
O
H
H
H
N
H
N
d
N
N
N
N
Alloc
N
H
Alloc
4
4
H
O
O
H
H
N
NH2
N
D
NHAc
N3
D
O
NHAc
N
I
O
N
H
I
O
H
O
54
53
c,b
O
O
O
O
H
H
N
H
N
N
NH2
e
N
N
H
Alloc
NHAc
N
N
4
4
H
O
O
O
H
N
H
D
AcHN
K
NH
I
AcHN
K
NH
N
D
I
NHAc
M
N
O
M
N
O
H
H
O
O
O
55
56
c,f
O
O
H
N
H2N
W
O
NH2
N
N
H
4
H
O
O
H
N
AcHN
K
NH
D
NHAc
M
N
I
O
H
O
57
Scheme 6. Reagents and conditions: (a) Ramage ChemMatrix, DIPEA, PyBroP, DMF, rt, 5 h;
(b) 5ꢀ% Ac2O (v/v), 5ꢀ% DIPEA (v/v) in NMP/DCM (1:1), rt, 2×15 min; (c) Peptide synthesis
(HATU/DIPEA); (d) TCEP, H2O/THF (2:1), rt, 2 h (e) Pd(PPh3)4 (0.15 equiv), (CH3)2NH.BH3
(20 equiv), NMP, N2 bubbling, rt, 1.5 h; (f) TFA/H2O, scavengers, rt, 2×1 h.
Discussion
Here we describe the development of three tripodal scaffolds compatible with the solid-
phase peptide synthesis. Despite their similar structures, their preparation differed. Scaffold II
was prepared by a direct and sequential amidation of trimesic acid, adapting our protocol for
the synthesis of scaffold I.[7] We believe that the presence of a bulky and lipophilic TES group
in scaffold II resulted in very different solubilities of its monoamidate and di- or triamidate
derivatives in aqueous or organic media. Consequently, we were able to isolate the monoamide
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13 from the crude mixture by successive extractions and washings. On the contrary, during the
synthesis of scaffolds III and IV, we were not able to isolate monoamidation products from the
crude reaction mixtures. Scaffolds III and IV required the full esterification, followed by the
mono-saponification of trimesic acid.
Scaffold I is not compatible with SPPS, because the fluoride TBAF agent used to
deprotect the TIPS group also cleaves the acid-labile protections of the amino acids’ side-
chains. Some research groups have already reported the cleavage of carbamate protecting
groups with TBAF (reviewed in Ref.[16]) For instance, Coudert and co-workers reported the
complete deprotection of tert-butyl carbamates at room temperature in a few hours.[17] Gea et
al. faced a similar problem when they performed the deprotection of a tert-butyldiphenylsilyl
ether from a tripodal scaffold bearing protected amino acids.[10c] There, TBAF caused the
cleavage of acid-labile side-chain protections. They eventually succeeded with
tris(dimethylamino)sulfonium (trimethylsilyl)difluoride, one of the few sources of anhydrous
fluoride. We chose to abandon the TIPS-alkyne group and preferred the use of a Fmoc-amino
moiety in scaffolds II, III and IV. The solid-phase synthesis on scaffold II was straightforward
and we obtained compound 45 in an acceptable yield (30%, after HPLC purification).
As discussed above, the one-pot TES deprotection/CuAAC was slow and, for that
reason, we preferred to incorporate an Alloc-amino moiety to scaffold III. We tested different
allyl scavengers, p-toluenesulfinic acid,[18] phenylsilane and borane dimethylamine
complexes[15] for the catalytic deallylation with tetrakis(triphenylphosphine)palladium. The
borane dimethylamine complex gave the best result, yielding the pure product in 1.5 h. Finally,
we eventually modified scaffold III by replacing the alkyne arm by an azido moiety in scaffold
IV. As explained above, the reduction with tributylphosphine or triphenylphosphine in
THF/water mixture failed, but the TCEP worked well.
This means that an important factor for success could be the water solubility of the
phosphine. Other groups also failed at reducing a solid-supported azide with
triphenylphosphine because the reactions stopped after the formation of the iminophosphorane
intermediate.[10g, 10h] They eventually succeeded by using the more reactive trimethylphosphine.
In a different approach, we used gaseous hydrogen sulfide as the reducing agent. The reaction
was slower than with TCEP, but was also highly satisfactory. Nevertheless, we prefer the TCEP
approach, which is more convenient and safer.
Finally, we compared the solid-phase syntheses on scaffolds II-IV, using three model
dipeptides (Ile-Asp, Met-Lys and His-Trp). We selected these amino acids to effectively test
our methodology as they represent bulky amino acids, are prone to oxidations or alkylations,
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10.1002/ejoc.201800601
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and contain protecting groups. We were also curious whether Met would poison the Pd(0)
catalyst used during the deallylation. We considered three criteria that we judged relevant for
our future combinatorial processes: purity, time of the synthesis and feasibility of procedures.
The HPLC chromatograms of the crude compounds 45, 51 and 57 are shown in Figure 2. The
HPLC purity (integration at 218 nm) of the desired product (P) in the crude mixtures varied
from 53ꢀ% (for compound 51) to 63ꢀ% (for compound 57). In all three crude reaction mixtures,
we obtained a major and similar side product (S) that was identified as an oxidation product
(M+16 by mass spectrometry), probably resulting from the partial oxidation of methionine,
which is not surprising. Scaffolds II and IV gave more pure crude products (compounds 45 and
57, respectively), whereas the crude compound 51 prepared from scaffold III contained almost
30% of the oxidation side product. In general, even if all three scaffolds provide products of
satisfactory purities, scaffolds II and IV clearly represent the more optimal tools for further
applications in hit discovery and combinatorial applications.
Figure 2. RP-HPLC chromatograms of the crude compounds 45 (in green), 51 (in blue) and 57
(in red). The peaks of the desired products are labeled as P and the peaks of major side-products,
which were identified as oxidation products, are labeled as S.
Our general strategy was to replace the slow CuAACs and TES deprotection by two
faster reactions: Pd-catalysed deallylation and azide reduction. Besides its slowness in the solid
phase (presumably due to the slow diffusion of the active species within the resin), the CuAAC
is hardly compatible with an automatic synthesizer. On the contrary, the azido reduction by
phosphine can be automated,[10g] which is an advantage regarding planned combinatorial
processes. Here a possible limitation of scaffold IV appears; the Pd-catalyzed deallylation
cannot be automated, because the reaction must be performed using fresh and homogenous
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solutions of palladium or borane and because the resin suspension has to be stirred by a strong
nitrogen flow.
Considering the various parameters, scaffold IV appears to be the most suitable one for
potential combinatorial synthesis in the solid phase. However, scaffold II could enable an
alternative diversity of the structures of the final compounds by using different non-peptide
azides during the two CuAACs. This could also be useful, e.g. for introducing more rigidity to
final compounds.
In that respect, we are aware that peptide compounds derived from scaffolds II-IV will
have a somewhat flexible behavior. In order to examine this, we assigned the proton and carbon
NMR spectra of the compound 39 and measured intra-molecular NOEs as well. We observed
important line broadening, as well as a lack of inter-arm NOEs. These observations suggest a
rather extended conformation of the three arms of compound 39, with fairly important
conformational freedom. In the context of drug discovery, such a flexible scaffold allows for
the exploration of a fairly large chemical space. On the other hand, they can suffer from
unfavorable entropic effects, which can decrease the overall binding energy to the target. For
this reason, we are currently developing more constrained tripodal scaffolds. We believe that
both approaches (flexible vs constrained scaffolds) could be complementary when starting a hit
discovery process.
Overall, we developed three variants of tripodal scaffolds, which enable an efficient
stepwise synthesis of three different peptides on the same scaffold. Here we may mention that
we also attempted to modify scaffold I with pre-prepared tripeptides containing azido-amino
acids.[19] This strategy was lengthy and rather unsuccessful because the CuAAC with azido-
peptides was inefficient, especially on the second and third arms. This underlines the advantage
of the peptide synthesis directly on the scaffolds. The methodology could also be useful for a
facile synthesis of potential proteomimetics in the form of combinatorial libraries.
In the accompanying article,[20] all three new scaffolds were further validated by the
preparation of new tripodal compounds with tetra to hexapeptides derived from the insulin
structure, or octapeptides derived from previously developed insulin peptide mimetics.[21] The
compounds were tested for their ability to bind and activate receptors for insulin and IGF-1.
Experimental Section
General. The conditions and methods for purification and analyses of compounds as well as
NMR and IR analytical data and HPLC chromatograms are provided in the Supporting
information.
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For the synthesis of compounds 1-12, refer to the Supporting information (Scheme S1
and below). For compounds 16, 25, 29, a thermal cleavage of the Fmoc protection group[22] was
observed at 90ꢀ°C and details are shown in the Scheme S2.
5-((3-(triethylsilyl)prop-2-yn-1-yl)carbamoyl)isophthalic acid (13): PyBrop (29.3 g, 62.8
mmol, 1.4 equiv) was added to a solution of trimesic acid (56.6 g, 269.3 mmol, 6 equiv) and
TEA (43.8 mL, 314.2 mmol, 7 equiv) in DMF (300 mL) at 0ꢀ°C. A solution of 3-
(triethylsilyl)prop-2-yn-1-amine[7] (7.6 g, 44.9 mmol, 1 equiv) in DMF (150 mL) was added
dropwise (exothermic process). After complete addition of the amine, the ice bath was removed
and the resulting mixture was stirred overnight at room temperature. TLC analysis performed
on a pretreated plate with gaseous NH3 in the system (IPA : conc. aq. ammonia : water 7 : 1 :
2) showed completion of the reaction (Rf = 0.73). The solvent was evaporated at 70 °C in vacuo
and the solid residue was suspended in an AcOH-acetone mixture (60 mL + 240 mL). The
resulting slurry was stirred for 20 minutes and filtered through an S3 frit. The solid was
transferred back to the round bottom flask, stirred in acetone (50 mL) and filtered as above. The
solid (mostly unreacted trimesic acid) was then discarded and the filtrate was evaporated in
vacuo to give a pale yellow residue. This residue was partitioned between Et2O (500 mL) and
DMF-H2O (1:1, 500 mL in total), and the aqueous layer was further extracted with Et2O (5 x
200 mL). The combined organic layers were then washed successively with 50ꢀ% aqueous DMF
(3 x 300 mL) to remove the rest of the unreacted trimesic acid and brine (300 mL), dried over
anhydrous sodium sulfate, evaporated in vacuo, co-evaporated with 100 mL of MeOH, and
evaporated to dryness. The crude product was obtained as a pale yellow semi-solid (19.0 g),
containing the desired monoamide 13, the diamide and tri(pyrrolidin-1-yl)phosphine oxide
(recognized by 1H NMR). The solid was crushed, transferred to a flask containing 300 mL of
toluene, and heated to reflux for 1 h under intensive stirring. After cooling overnight at 5ꢀ°C,
the slurry was filtered through an S4 frit, and the solid was washed with a small amount of
hexane and dried to dryness to give the monoamide 13. Analysis by NMR and HPLC revealed
complete disappearance of phosphinoxide and diamide. Yield 8.6 g, 53 %. White solid. HRMS
(m/z): calcd for C18H22O5NSi (M+H)+ 360.1273, found 360.1276.
3-((5-(tert-butoxy)-5-oxopentyl)(prop-2-yn-1-yl)carbamoyl)-5-((3-(triethylsilyl)prop-2-
yn-1-yl)carbamoyl)benzoic acid (14): PyBrop (6.8 g, 14.6 mmol, 1.1 equiv) was added to a
solution of monoamide 13 (4.8 g, 13.3 mmol, 1 equiv) and Triethylamine (11.1 mL, 79.7 mmol,
6 equiv) in DMF (40 mL) at 0 °C. A solution of the tosylate salt 7 (5.3 g, 13.9 mmol, 1.05
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equiv) in DMF (40 mL) was added dropwise. After complete addition, the ice bath was removed
and the resulting mixture was stirred overnight at room temperature. A TLC plate, pretreated
with gaseous NH3, showed in the system (ethyl acetate : acetone : ethanol : water 4 : 1 : 1 :1)
completion of reaction (Rf = 0.60). The solvent was then evaporated at 70ꢀ°C in vacuo and the
residue was partitioned between Et2O (150 mL) and an AcOH-H2O mixture (10 mL + 120 mL).
The aqueous layer was further extracted with Et2O (2 x 100 mL). The combined organic layers
were washed successively with H2O (100 mL) and brine (100 mL), dried over anhydrous
sodium sulfate and evaporated to dryness to give 12.4 g of brown oil. The crude product was
purified by flash chromatography on silica gel (elution with a linear gradient of 1ꢀ%
AcOH/EtOAc (v/v) in toluene) to give the diamide 14 as a white foam. Yield 2.96 g, (40 %).
HRMS (m/z): calcd for C30H42O6N2NaSi (M+Na)+ 577.2704, found 577.2705.
tert-butyl
5-(3-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)-N-
(prop-2-yn-1-yl)-5-((3-(triethylsilyl)prop-2-yn-1-yl)carbamoyl)benzamido)pentanoate
(15): DIPEA (3.10 mL, 17.8 mmol, 4.0 equiv) was added to a solution of diamide 14 (2.96 g,
5.3 mmol, 1.2 equiv) in CH3CN (40 mL) in a water bath at room temperature, followed by
PyBrop (3.10 g, 6.7 mmol, 1.5 equiv). The resulting solution was stirred for 10 minutes and
trifluoroacetate amine 4 (1.41 g, 4.4 mmol, 1.0 equiv) was added in one portion. The resulting
mixture was stirred at room temperature for 5 h. TLC analysis performed in the system (ethyl
acetate : toluen 1 :1) on (Rf = 0.39) showed completion. The solvent was evaporated at 40 °C
in vacuo and the residue was partitioned between ethyl acetate (100 mL) and 2 % AcOH/H2O
(v/v, 100 mL in total). The organic layer was then washed successively with saturated NH4Cl
(80 mL), H2O (80 mL) and brine (80 mL), dried over anhydrous sodium sulfate and evaporated
to dryness to give 7.4 g of brown oil. The crude product was purified by flash chromatography
on silica gel (elution with a linear gradient of ethyl acetate in toluene) to give triamide 15 as a
white foam. Yield 3.2 g (88 %). HRMS (m/z): calcd for C47H58O7N4NaSi (M+Na)+ 841.3967,
found 841.3968.
5-(3-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)-N-(prop-2-yn-1-
yl)-5-((3-(triethylsilyl)prop-2-yn-1-yl)carbamoyl)benzamido)pentanoic acid 16 (Scaffold
II): Trifluoroacetic acid (3 mL) was added to a solution of triamide 15 (3.20 g, 3.91 mmol) in
DCM (3 mL) at 0ꢀ°C. The reaction mixture was stirred for 2 h at room temperature and the
solvents were evaporated at 40ꢀ°C in vacuo. The residue was purified by flash chromatography
on silica gel (elution with a linear gradient of 10ꢀ% MeOH/CHCl3 (v/v) in CHCl3) to give a pale
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yellow foam. This foam was lyophilized from 1,4-dioxane to afford 16 as a white solid. The
RP-HPLC analysis of compound 16 is shown in the Supporting Information. Yield 2.40 g (82
%). RP-HPLC (Method A): 95.8 %. HRMS (m/z): calcd for C43H50O7N4NaSi (M+Na)+
785.3341, found 785.3343.
Trimethyl 1, 3, 5-benzenetricarboxylate (17): Trimesic acid (30 g, 0.143 mol) with
concentrated sulfuric acid (6 mL) was refluxed overnight in methanol (350 mL). After cooling
at -20ꢀ°C, the mass of crystals was filtered and washed with 500 mL of chilled methanol.
Crystallization from methanol afforded a pure product. Yield 32.6 g (91ꢀ%). White solid, m.p.
140-143ꢀ°C (methanol) Rf = 0.84 (50ꢀ% toluene / 50ꢀ% ethyl acetate). HRMS (EI) calc for
C12H12O6 [M]+ 252.0634, found: 252.0632.
3,5-bis(Methoxycarbonyl)benzoic acid (18): A partial saponification was performed by the
reaction of 17 (32.4 g, 0.128 mol) with 1M aqueous NaOH solution (4.6 g, 0.115 mol) in
methanol (2 L), using the protocol described in the literature.[23] Yield 23.3 g (76ꢀ%). White
solid, m.p. 136-137ꢀ°C (ethyl acetate – petroleum ether) Rf = 0.69 (ethyl acetate methanol
acetone water 6/2/1/0.5). HRMS (ESI) calc for C11H10O6Na [M+Na]+ 261.0370, found:
261.0370.
Dimethyl-5-(2-(((allyloxycarbonyl)amino)ethyl)carbamoyl)-1,3-benzendicarboxylate
(19): TEA (18 mL, 128.8 mmol) was added in one portion to a solution of salt 1 (10.2 g, 32.2
mmol), 17 (7.7 g, 32.2 mmol) and PyBroP (21.2 g, 45.1 mmol) in DMF (200 mL). The mixture
was allowed to react overnight at room temperature. 500 mL of water was added and the product
was extracted with ethyl acetate (4 x 150 mL). The combined organic phases were washed with
water (150 mL), brine (3 x 150 mL), dried over anhydrous sodium sulphate, filtrated and
evaporated. The brown residue was purified by flash chromatography, using a linear gradient
of ethyl acetate in toluen. Evaporation of the fractions containing the product afforded a solid,
which was crystalized from a mixture of ethyl acetate-hexane. Yield 7.6 g (67ꢀ%). White solid,
m.p. 132-134 °C. Rf = 0.69 (ethyl acetate). HRMS (ESI) calc for C17H21O7N2 [M+1]+ 365.1343,
found: 365.1344.
5-(2-(((allyloxycarbonyl)amino)ethyl)carbamoyl)-1,3-benzendicarboxylic
acid
(20):
Diester 19 (7 g, 19.2 mmol) was suspended in methanol (30 mL) and NaOH (3.1 g ; 76.9 mmol)
in water (50 mL) was added. The mixture was stirred overnight at room temperature. Thereafter,
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1M HCl was added to the clear solution until pH value was approx. ~ 6. Methanol was
evaporated and the flask with the resulting aqueous solution was immersed in an ice bath, with
the pH adjusted by 1 M HCl to pH ~ 1. The slurry was kept at 5ꢀ°C for 30 minutes. The
precipitate was filtered in a Büchner funnel, washed with 100 mL of chilled water and dried
under a deep vacuum over P2O5. Yield 6 g (92ꢀ%). White solid, m.p. 252-255 °C. Rf = 0.66
(conc. ammonia isopropyl alcohol water 6/2/1). HRMS (ESI) calc for C15H15O7N2 [M+1]+
335.0885, found: 335.0886.
3-((((2-Allyloxycarbonyl)amino)ethyl)carbamoyl)
-
3
-
(((5-(tert-butoxy)-5-
oxopentyl)prop-2-yn-1-yl)carbamoyl)benzoic acid (21)
1-((((2-Allyloxycarbonyl)amino)ethyl)carbamoyl)
-
3,5-bis(((5-(tert-butoxy)-5-
oxopentyl)prop-2-yn-1-yl)carbamoyl)benzene (22): PyBroP (8.2 g, 17.6 mmol) and TEA (15
mL, 112 mmol) were added within 10 minutes to a solution of 20 (5.4 g, 16 mmol) in DMF
(150 mL), followed by 7 (6.1 g, 16 mmol), which was added in three equal portions. The
reaction mixture was stirred overnight at room temperature. Afterwards, 300 mL of water was
added. The resulting mixture was acidified with glacial acetic acid until pH 5 was reached. The
aqueous phase was extracted with ethyl acetate (4 x 150 mL). The combined organic layers
were washed with water (100 mL), brine (3 x 100 mL), and dried over anhydrous sodium
sulfate. Evaporation of the filtrate afforded a brown residue, which was subjected to flash
chromatography. Less polar, unwanted diamide 22 was eluated with ethyl acetate; the desired
product 21 was then eluted with a linear gradient of ethyl acetate / MeOH / acetone (6 : 2 :1) in
ethyl acetate.
21. Yield 6.6 g (77ꢀ%). Bright yellow oil. Rf = 0.49 (ethyl acetate : MeOH : acetone : water 6 :
2 : 1 : 0.5). HRMS (ESI) calc for C27H34O8N3 [M+1]+ 528.2351, found: 528.2342.
22. Yield 3.8 g (33ꢀ%). Colorless oil. Rf = 0.76 (ethyl acetate). HRMS (ESI) calc for
C39H54O9N4Na [M+Na]+ 745. 3783, found: 745.3785.
3-((((2-Allyloxycarbonyl)amino)ethyl)carbamoyl) - 5 - ((2-azidoethyl-5-(tert-butoxy)-5-
oxopentyl)carbamoyl)benzoic
acid
(23)
and
1-((((2-
Allyloxycarbonyl)amino)ethyl)carbamoyl)
-
3,5-bis((2-azidoethyl-5-(tert-butoxy)-5-
oxopentyl)carbamoyl)benzene (24): Compounds 23 and 24 were prepared by a reaction of 20
(3.6 g, 10.7 mmol), PyBroP (5.5 g, 11.8 mmol), 9 (4.4 g, 10.7 mmol) and TEA (10.4 mL, 74.9
mmol) in DMF (100 mL), using the protocol described for 21 and 22.
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23: Yield 3.3 g (55ꢀ%). Colorless oil. Rf = 0.61 (ethyl acetate : MeOH : acetone : water 6 : 2 :
1 : 0.5). HRMS (ESI) calc for C26H35O8N6 [M+1]+ 559.25219 found: 559.2524.
24: Yield 1 g (12ꢀ%). Colorless oil. Rf = 0.43 (ethyl acetate). HRMS (ESI) calc for
C37H56O9N10Na [M+Na]+ 807.41239 found: 807.4125.
Tert-butyl 5-3-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)-5-((2-
(((allyloxy)carbonyl)amino)ethyl)carbamoyl)-N-(prop-2-yn-1-yl)benzamido)pentanoate
(25): PyBroP (7.3 g, 15.7 mmol) and DIPEA (7.2 ml, 41.6 mmol) were added to the solution
of 21 (6.6 g, 12.5 mmol) in DMF (100 mL), followed by the addition of 5 (3.3 g, 10.4 mmol)
in one portion. The reaction mixture was stirred for 4.5 h at room temperature. Thereafter, water
(200 mL) was added, and the resulting solution was acidified with 1 M solution of citric acid
until pH 5 was reached. The aqueous phase was extracted with ethyl acetate (4 x 150 mL) and
the combined organic layers were washed with water (100 mL), brine (3 x 100 mL) and dried
over anhydrous sodium sulfate. Evaporation of the filtrate afforded a crude product, which was
purified by flash chromatography, using a linear gradient of ethyl acetate / MeOH / acetone (6
: 2 :1) in ethyl acetate. Yield 1.9 g (20ꢀ%). White foam. Rf = 0.47 (ethyl acetate). HRMS (ESI)
calc for C44H51O9N5Na [M+Na]+ 816.3579, found: 816.3580.
tert-butyl 5-(3-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)-5-((2-
(((allyloxy)carbonyl)amino)ethyl)carbamoyl)-N-(2-azidoethyl)benzamido)pentanoate
(26): Intermediate 26 was prepared as described for 25 by a reaction of 23 (2.8 g, 5 mmol),
PyBroP (3.5 g, 7.5 mmol), DIPEA (3.5 g, 20 mmol) and 5 (1.6 g, 5 mmol) in DMF (100 mL).
Yield 1.2 g (29ꢀ%). White foam. Rf = 0.42 (ethyl acetate). HRMS (ESI) calc for C43H52O9N8Na
[M+Na]+ 847.37495, found: 847.3752.
Using the protocol described above, the reaction of 21 (2.5 g, 4.7 mmol), PyBroP (6.6 g, 14.1
mmol), TEA (2.6 ml, 18.8 mmol) and 4 (1.9 g, 4.7 mmol) in DMF (100 mL) gave the starting
compound 21 and N-trifluoroacetyl-N´-(9H-fluoren-9-yl)methoxy)carbonyl)ethylenediamine
27. Yield 0.7 g (39ꢀ%). Colorless crystals, m. p. 198-199ꢀ°C. Rf = 0.68 (ethyl acetate : toluen 50
: 50). Anal. calc. (%): C 60.32, H 4.53, 7.40 N, 15.06 F. Found (%): C 60.35, 4.63 H, 7.16 N,
14.80 F. HRMS (ESI) calc for C19H18O3N2F3 [M+1]+ 379.12640, found: 379.1266.
Tert-butyl
5-(3-((2-(((allyloxy)carbonyl)amino)ethylcarbamoyl)-5-((2-((tert-
butoxycarbonyl)amino)ethyl)carbamoyl)-N-(prop-2-yn-1-yl)benzamido)pentanoate (28):
Boc-protected intermediate 28 was prepared as described for 25 by the reaction of 21 (6.6 g,
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12.5 mmol), PyBroP (17.5 g, 37.5 mmol), TEA (7 mL, 50 mmol) and 2 (8.3 g, 25 mmol) in
DMF (70 mL). Yield 2.8 g (33ꢀ%). White foam. Rf = 0.41 (ethyl acetate). HRMS (ESI) calc for
C34H49O9N5Na [M+Na]+ 694.3423, found: 694.3425.
5-(3-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)-5-((2-
(((allyloxy)carbonyl)amino)ethyl)carbamoyl)-N-(prop-2-yn-1-yl)benzamido)pentanoic
acid
29
(Scaffold
III)
and
5-(3-((2-((((9H-fluoren-9-
yl)methoxy)carbonyl)amino)ethyl)carbamoyl)-5-((2-
(((allyloxy)carbonyl)amino)ethyl)carbamoyl)-N-(2-azidoethyl)benzamido)pentanoic acid
30 (Scaffold IV): 25 (1.8 g, 2.3 mmol) or 26 (1.1 g, 1.33 mmol) was stirred in DCM (3 mL)
and TFA (6 mL) for 3.5 h at room temperature. TLC analysis revealed the full deprotection of
the tert-butyl protecting group. The volatile material was evaporated under reduced pressure
and the residual brown oil was purified by flash chromatography with a linear gradient of ethyl
acetate / MeOH / acetone (6 : 2 :1) in ethyl acetate. The product was dissolved in 10 mL dioxane
and lyophilized. The RP-HPLC analyses of scaffold III (29) and scaffold IV (30) are shown
in the Supporting Information.
Analogously, 28 (2.8 g, 4.2 mmol) was stirred DCM (3 mL) and TFA (6 mL). After 3 h at room
temperature, the solvents were removed by evaporation. The residual oil was emulgated in a
solution of NaHCO3 (1.4 g ; 8.4 mmol) in water (50 mL). The flask was placed in an ice-cooling
bath. A solution of Fmoc-Osu (1.4 g ; 4.2 mmol) in dioxane (20 mL) was added dropwise under
vigorous stirring. The reaction mixture was stirred overnight at room temperature. 1M citric
acid was then added until pH ≈ 3 was reached. The reaction mixture was extracted with ethyl
acetate (3 x 50 mL) and the combined organic phases were washed in brine (3 x 50 mL), dried
over anhydrous sodium sulfate and filtered. The solvent was evaporated and the resulting brown
oil was subjected to flash chromatography with a linear gradient of ethyl acetate / MeOH /
acetone (6 : 2 :1) in ethyl acetate. The title compound was dissolved in 10 mL dioxane and
lyophilized to afford 1.3 g (42ꢀ%) of the required product as a white solid.
29. Yield 1.4 g (85ꢀ%). White solid. Rf = 0.71 (ethyl acetate : MeOH : acetone 6 : 2 : 1). RP-
HPLC (Method A): purity = 96ꢀ%. HRMS (ESI) calc for C40H43O9N5Na [M+Na]+ 760.2953,
found: 760.2954.
30. Yield 0.9 g (90ꢀ%). White solid. Rf = 0.69 (ethyl acetate : MeOH : acetone 6 : 2 : 1). RP-
HPLC (Method A): 98 %. HRMS (ESI) calc for C43H44O9N8Na [M+Na]+ 791.3124, found:
791.3126.
21
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10.1002/ejoc.201800601
European Journal of Organic Chemistry
General description for the solid-phase synthesis of 25, 38, 39, 45, 51 and 57
Peptide synthesis was performed manually in a PP syringe equipped with a Teflon frit.
Loading to the resin (31, 40, 46, 52): In a 10 mL-fritted polypropylene syringe, Ramage
ChemMatrix® resin (213 mg, 100 µmol - Aldrich 727792, lot # BCBV2122, loading = 0.47
mmol/g) with free amino groups was swelled in MeOH, DCM, and DMF (10 minutes each, 5
mL of each solvent). The resin was then washed with 3×3 mL DMF. A solution of scaffold I,
scaffold II (16), scaffold III (29) or scaffold IV (30) (50 µmol, 1.0 equiv), PyBroP (47 mg,
100 µmol, 2.0 equiv), and DIPEA (26 µL, 150 µmol, 3.0 equiv) in DMF (1.5 mL) was added
to the resin. The syringe was rotated at room temperature for 5 h. The resin was successively
washed with 3×3 mL of DMF, MeOH, DCM and NMP/DCM (1:1). The unreacted amino
groups of the resin were capped by two treatments of 5 % Ac2O (v/v), 2 % DIPEA (v/v) in
NMP/DCM (1:1) (2.5 mL and 15 min for each treatment). The resin was washed with 3×3 mL
of NMP/DCM (1:1), MeOH, DCM and DMF to give the resin-bound compound 31, 40, 46 and
52.
Peptide synthesis (compounds 33, 35, 38, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57):
Fmoc deprotection: The resin was washed with 3×3 mL DMF and treated with 20 % 4-
methylpiperidine (v/v) in DMF (2.5 mL for each treatment, two treatments of 5 + 20 min). The
resin was washed with 3×3 mL DMF, DCM, MeOH and DMF.
Peptide coupling: The resin was washed with 3×3 mL NMP or DMF. To the resin was added a
solution of Fmoc-amino acid (3 equiv), HATU (3 equiv) and DIPEA (4 equiv) in NMP or DMF
(1.5 mL). The coupling was performed for 40 to 50 minutes at room temperature. The resin was
washed with 3×3 mL NMP or DMF and the coupling was repeated once as above. The resin
was finally washed with 3×3 mL of DMF, MeOH, DCM and DMF.
Capping: After completion of the first or the second arm of the scaffold, the N-terminal end of
the peptide was acetylated. The resin was washed with 3×3 mL NMP/DCM (1:1) and treated
with 5ꢀ% Ac2O (v/v), 5ꢀ% DIPEA (v/v) in NMP/DCM (1:1) (2.5 mL for each treatment, two
treatments of 15 min). The resin was washed with 3×3 mL of NMP/DCM (1:1), MeOH, DCM
and DMF.
CuAAC with a low copper load (32, 37, 42 and 48): The resin was washed with 3×3 mL DMF.
The following solutions were sequentially added to the resin: i) azide (250 µmol, 5.0 equiv) in
0.9 mL of DMF; ii) sodium ascorbate (50 µL of a freshly prepared 0.5 M aqueous solution, 25
µmol, 0.5 equiv) in 0.25 mL of H2O; iii) copper(II) sulfate pentahydrate (50 µL of a freshly
prepared 0.1 M aqueous solution, 5 µmol, 0.1 equiv) in 0.25 mL of H2O. The syringe was
22
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800601
European Journal of Organic Chemistry
agitated at room temperature for 16 h and the resin was washed with 3x3 mL of DMF/water
(1:1), MeOH, DCM and DMF to give the triazole compound.
One-pot TES-deprotection/CuAAC with a high copper load (34 and 44): The resin was washed
with 3×3 mL DMF. The following solutions were sequentially added to the resin: i) azide (250
µmol, 5.0 equiv) in 3 mL of DMF; ii) sodium ascorbate (99 mg, 500 µmol, 10 equiv) in 1 mL
of H2O; iii) copper(II) sulfate pentahydrate (62.5 mg, 250 µmol, 5 equiv) in 1 mL of H2O. The
syringe was agitated at room temperature for 16 h and the resin was washed with 3x3 mL
DMF/water (1:1), water, MeOH, DCM and DMF. The coupling was repeated once, using the
same conditions. The resin was finally washed with 3x3 mL DMF/water (1:1), water, MeOH,
DCM and DMF to give the triazole compound. After these washings, the resin still contained
some copper, which was washed out from the resin during the following Fmoc deprotection
with 20ꢀ% 4-methylpiperidine (v/v) solution in DMF.
Removal of Alloc protecting group (50 and 56): The resin was washed with 5 x 5 ml of N2-
degazed NMP and transferred with 4 ml of degazed NMP to a 15-ml polypropylene centrifuge
tube. A long needle was introduced tightly above the bottom of the tube and the slurry was
deoxygenated with a stream of dry nitrogen for 30 minutes. Pd(PPh3)4 (0.15 equiv) and
(CH3)2NH.BH3 (20 equiv) were added as solutions in degazed NMP (0.3 mL). The mixture was
allowed to react for 1.5 h at room temperature under vigorous bubbling of the dry nitrogen. The
resin was transferred back to the syringe, washed with 5 x 5 ml NMP, 3 x 5 ml 20ꢀ% 4-
methylpiperidine in DMF and 5 x 5 ml DMF.
Reduction of azide (54): The resin was washed 3 x 3 ml H2O/THF (2:1, v/v) and TCEP (20
equiv) and DIPEA (12 equiv) in 2 mL H2O/THF (2:1, v/v) were added. The syringe was rotated
for 2 h at room temperature. The resin was washed with 3x3 mL of mL H2O/THF (2:1, v/v),
MeOH, DCM and DMF.
Alternatively, gaseous hydrogen sulfide was aspirated in a syringe containing the resin in a
pyridine/H2O mixture (2:1, 2 mL). The syringe was left at room temperature for 2 h and the
resin was washed with 3x3 mL pyridine/H2O (2:1). This operation was repeated twice. The
resin was washed with 3x3 mL pyridine/H2O (2:1), MeOH, DCM and DMF. CAUTION:
hydrogen sulfide is a very toxic gas that should be handled with care and under a fume hood.
Cleavage from the resin (38, 39, 45, 51, 57): The resin was washed with 3×3 mL DCM,
transferred to a fritted glass reactor and dried under reduced pressure. The following cleavage
mixture was added to the reactor: 95ꢀ% TFA/H2O (10 mL) + 2ꢀ% TIS (v/v), + 2ꢀ% DODT (v/v),
+ 2ꢀ% thioanisole (v/v). Two cleavages of 1 h each were performed. The combined filtrates
were evaporated under reduced pressure at 40 °C. The oily residue was then triturated twice
23
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800601
European Journal of Organic Chemistry
with 25 mL Et2O. The resulting solid was dissolved in 40 % ACN/H2O and lyophilized. The
crude product was analyzed by RP-HPLC and purified on preparative RP-HPLC, as described
in the General section. The crude purity, the yield of the synthesis, the final purity and the
HRMS of compounds 38, 39, 45, 51 and 57 are given in Table 1. The RP-HPLC profiles of the
isolated product of compounds 38, 39, 45, 51 and 57 are shown in the Supporting Information.
Table 2: Purities of crude compounds 38, 39, 45, 51 and 57, yields after isolation by RP-HPLC,
purities of the isolated products and HRMS of purified compounds. Purities were determined
by integration of peaks at 218 nm.
Compound Purity of the
Crude
Purity of
HPLC
Yield
HR-MS
(calculated)
(M+H)+
HR-MS
(experimental)
after HPLC
purification
Compound
purified
compound
> 95ꢀ%
82 %
76 %
63 %
53 %
60 %
42 %
38 %
30 %
28 %
22 %
1349.6210
1481.6997
1492.7379
1411.7052
1330.6725
1349.6215
1481.7001
1492.7385
1411.7045
1330.6726
38
39
45
51
57
> 95ꢀ%
> 95ꢀ%
> 95ꢀ%
> 95ꢀ%
Acknowledgements
This work was supported by the European Regional Development Fund; OP RDE; Project:
“Chemical Biology for drugging undruggable targets (ChemBioDrug)” (No.
CZ.02.1.01/0.0/0.0/16_019/0000729), by the Grant Agency of the Czech Republic (Grant 14-
17305S), by the Academy of Sciences of the Czech Republic (Research Project RVO:6138963,
support to the Institute of Organic Chemistry and Biochemistry, and partially by the Medical
Research Council (Grant MR/K000179/1).
Keywords: Scaffold. Peptide synthesis. Solid-phase synthesis. Click chemistry.
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