An orthogonally protected CycloTriVeratrylene (CTV) as a highly pre-organized molecular scaffold for subsequent ligation of different cyclic peptides towards protein mimics

Article abstract of DOI:10.1016/j.bmc.2017.05.038

The synthesis of a semi-orthogonally protected CycloTriVeratrilene (CTV) scaffold derivative as well as the sequential introduction of three different peptide loops onto this molecular scaffold via Cu(I)-catalyzed azide alkyne cycloaddition towards a medium-sized protein mimic is described. This approach for the construction of medium-sized protein mimics is illustrated by the synthesis of a paratope mimic of the monoclonal antibody Infliximab (Remicade) and provides access to a range of highly pre-organized molecular constructs bearing three different peptide segments. This approach may find wide applications for development of protein-protein interaction disruptors as well as synthetic vaccines.

Full text of DOI:10.1016/j.bmc.2017.05.038

Accepted Manuscript
An Orthogonally Protected CycloTriVeratrylene (CTV) as a Highly Pre-organ-
ized Molecular Scaffold for Subsequent Ligation of Different Cyclic Peptides
towards Protein Mimics
Ondřej Longin, Helmus van de Langemheen, Rob M.J. Liskamp
PII:
S0968-0896(17)30455-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.038
BMC 13756
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 March 2017
8 May 2017
17 May 2017
Please cite this article as: Longin, O., van de Langemheen, H., Liskamp, R.M.J., An Orthogonally Protected
CycloTriVeratrylene (CTV) as a Highly Pre-organized Molecular Scaffold for Subsequent Ligation of Different
Cyclic Peptides towards Protein Mimics, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.05.038
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Leave this area blank for abstract info.
An orthogonally protected
CycloTriVeratrylene (CTV) as a highly pre-
organized molecular scaffold for subsequent
ligation of different cyclic peptides towards
protein mimics
Ondřej Longin, Helmus van de Langemheen, Rob M.J. Liskamp*
School of Chemistry, Joseph Black Building, University of Glasgow, University Avenue, Glasgow G12 8QQ (UK)
OH
HO
MeO
MeO
NH2
OMe
C
HO
Ac
O
N
N
S
C
NH2
O
C
C
S
O
N
N
Ac
S
S
O
O
C
O
N
NH2
N
N
S
TIPS
O
N
N
N
C
N
N
O
N
N
N
Ac
S
N
TES
O
MeO
OMe
O
O
MeO
N
O
O
MeO
N
O
OMe
OMe
O

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
An Orthogonally Protected CycloTriVeratrylene (CTV) as a Highly Pre-organized
Molecular Scaffold for Subsequent Ligation of Different Cyclic Peptides towards
Protein Mimics
,a
Ondřej Longina, Helmus van de Langemheena and Rob M.J. Liskamp∗
a
School of Chemistry, Joseph Black Building, University of Glasgow, University Avenue, Glasgow G12 8QQ (UK)
E-Mail: Robert.liskamp@glasgow.ac.uk
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The synthesis of a (semi)orthogonally protected CycloTriVeratrilene (CTV) scaffold derivative
as well as the sequential introduction of three different peptide loops onto this molecular
scaffold via Cu(I)-catalyzed azide alkyne cycloaddition towards a medium-sized protein mimic
is described. This approach for the construction of medium-sized protein mimics is illustrated by
the synthesis of a paratope mimic of the monoclonal antibody Infliximab (Remicade®) and
provides a convenient access to a range of highly pre-organised molecular constructs bearing
three different peptide segments. This approach may find wide applications for development of
protein-protein interaction disruptors as well as synthetic vaccines.
Available online
Keywords:
Protein mimic
Peptide cyclisation
Orthogonal functionalization
Molecular scaffold
Synthetic antibody
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Thus, a scalable (re)synthesis approach for any individual hit is
absolutely essential. So far, we have developed semi-
orthogonally protected TAC, ATAC and TACO scaffolds,1,3,4
Protein mimics form a category of medium sized molecules,
which might have a large impact because they could become
viable medium-sized molecule alternatives of biologics such as
antibodies and vaccines. Thus, they may have advantages closer
to those of smaller molecules such as better bio-availability and
stability and be less immunogenic. We have provided bases for
realization of molecular construction of protein mimics by (1) the
development of syntheses of different scaffolds for attachment of
(cyclic) peptides,1-4 (2) synthetic approaches for attachment of
different (cyclic) peptides to scaffolds,5-10 and (3) the generation
of collections of the resulting protein mimics.9,11,12 The latter
aspect is especially note worthy since design of an optimal
protein mimic is often hardly possible even when a significant
amount of structural data is available, so there has to be a
versatile approach to prepare collections or libraries of protein
mimics, which can be screened to find protein mimic "hits"9,12
For this purpose a non-orthogonally protected scaffold is used,
providing direct access to the required unprotected protein
mimics, which can be screened as mixtures or as single
compounds, followed by MS-identification of the hit(s).11,12
However, this/these hit(s) then have to be re-synthesized using
the an orthogonally protected scaffold to obtain appreciable
quantities of single protein mimic compounds for validation of
P4
O
P4
O
P4
O
O
O
O
N
N
P3
N
N
N
N
P1
P1
P3
P1
P3
O
N
N
R
N
P2
ATAC scaffold
P2
P2
TACO scaffold
TAC scaffold
Figure 1. Orthogonally protected TAC1,3,13,14, ATAC3, and TACO4 scaffolds
which can be applied for the convenient synthesis of selected
protein mimics (Figure 1). Other important orthogonally
protected molecular scaffolds include the RAFT-peptide (Mutter
and co-workers15), cholic acid derivatives (Savage and co-
workers16), cyclic β-peptide and penta-erythrityltetramine
derivatives (Lönnberg and co-workers17,18), a pentaerythritol
derivative (De Clerq and co-workers19) as well as a cyclicpeptide
derivative by Eichler and co-workers.20
t—he—hit—and subsequent structural and biological activity studies
∗ Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: author@university.edu

In the past we have used the highly pre-organized
CycloTriVeratrylene (CTV) scaffold successfully for
yield (Scheme 2). Moreover, the di-THP derivative 9 was easily
separable from mono and tri-THP-ethers 10, which could then be
converted back to CTV 6 in a moderate yield. Subsequently, di-
THP ether 9 was alkylated to give 11 followed by removal of the
THP-protecting group leading to mono-propargyl ether 7 in a
good (70% over 2 steps) yield.
development of effective collagen mimics.2 More recently,
preparation of collections of gp120 protein mimics, using these
different scaffolds, showed that CTV-scaffolded gp120 epitope
mimics were the most powerful protein mimics (IC50-values of
the gp120-CD4 interaction in ELISA of 1.7 and 2.2 µM).12
Therefore, we felt it was necessary to develop an orthogonally
protected CTV derivative, so that the selected protein mimics can
be conveniently re-synthesized. In this research we describe the
synthesis of the highly pre-organized semi-orthogonally
protected CTV scaffold as well as an example of sequential
introduction of three different cyclic peptides towards individual
protein mimics.
Scheme 2. Synthesis of mono-propargyl CTV 7 via di-THP-CTV ether 9
Next, silyl protected alkynes were introduced onto the mono
alkylated scaffold 7 (Scheme 3). Since the TIPS-group is more
stable, alkyne bromide 21 was introduced first onto the propargyl
1M HCl/MeOH
58 - 74%
MeO
HO
MeO
R1O
MeO
HO
HO
MeO
MeO
HO
O
O
O
2. Results and discussion
O
O
p-TsOH.H2O
CHCl3
+
Similar to the recently described semi-orthogonal alkyne
functionalized TAC-scaffold 1 (Figure 2), we wished to develop
a semi-orthogonally alkyne functionalized CTV scaffold 2.
MeO
MeO
MeO
MeO
R2
(+)
(+)
(+)
O
O
O
(30 %)
6
9
10
R1=H, R2=H
or
Br
R1=THP, R2=THP
Cs2CO3
MeCN
MeO
O
MeO
O
O
HO
O
1M HCl/MeOH
MeO
MeO
MeO
Figure 2. Recently developed orthogonally protected TAC-scaffold13 1 and
the orthogonally protected (+)-CTV scaffold 2.
MeO
HO
However, in contrast to the TAC-scaffold,1,3 1 orthogonality
of protection cannot be achieved during synthesis of the CTV
scaffold21 6 (Scheme 1) itself, but has to be introduced post-
synthesis, thus, after completion of its synthesis. First, it was
attempted to mono-functionalize the CTV scaffold 6 with
propargyl bromide (14). This gave only low yields of CTV
propargyl ether 7, together with di-alkylated derivative 8 and
starting material, which were hardly separable.
(+)
(91%)
7
O (+)
(77%)
O
11
7
CTV derivative 7.
Along with the desired mono-TIPS
derivative 12 the di-TIPS side product 13 was formed. Because
of the formation side product 13 and only 1.05 eq. of 21 used in
the reaction, some starting material 7 remained unreacted and
was recovered (25%). Finally, the mono-TIPS derivative 12 was
alkylated with TES-protected alkyne bromide 15 to give the
desired semi-orthogonally protected CTV scaffold derivative 2 in
a good yield (72%) (Scheme 3).
MeO
O
OH
OH
Pd/C 10%
HClO4
Br
HClO4
O
(aq. 65%)
K2CO3
Scheme 3. Completion of the synthesis of the semi-orthogonally protected
CTV scaffold 2.
acetone,
65o
MeOH
0°C => RT
EtOH/dioxane
65°C
OMe
OMe
MeO
OH
O
MeO
The preparation of the required silyl protected alkyne
bromides 15 and 21 is shown in Scheme 4. First, a direct
silylation of propargyl bromide 14 with TES-Cl was attempted
(Scheme 4).
(87%)
4
3
O
(+)
(52%)
5
MeO
HO
HO
MeO
MeO
HO
MeO
MeO
O
O
Br
MeO
O
MeO
O
MeO
O
R1
O
HO
Cs2CO3
+
TIPS
TIPS
+
TIPS
(21)
Cs2CO3, MeCN
0°C => RT
MeO
MeO
MeO
HO
O
O
Br
MeO
MeO
MeO
MeO
O
HO
MeO
HO
(+)
(52%)
(+)
(14%)
(+)
MeO
HO
MeO
3
(3 %)
(+)
(+)
(+)
O
6
7
8
(43%)
(23%)
7
12
13
MeO
Scheme 1. Synthesis of CTV scaffold 6 and mono-alkylation attempt.
TIPS
(15)
TES
O
O
Br
TIPS
Therefore, it was attempted to temporarily protect two
phenolic hydroxy functionalities and subsequently alkylate the
remaining free hydroxy group. Attempts to protect two hydroxy
functionalities as bis-esters or bis-silylethers were unsuccessful.
Nevertheless, the desired temporary protection was achieved
using THP protecting group, which afforded the desired di-THP
derivative 9 from CTV 6 under mild conditions in a decent 30%
Cs2CO3, MeCN
MeO
MeO
(+)
O
(72%)
2
TES

chemoselective cyclization using polar hinge 22 leading to cyclic
peptides 26-28 (Scheme 5). The azide functionality in the hinge
22 allowed ligation of the resulting cyclic peptides by Cu(I)-
catalyzed azide alkyne cycloaddition (CuAAC) onto the CTV
scaffold derivative 2 in a similar manner as was described
previously for semi-orthogonally protected TAC scaffold 1.13,14
However, no microwave irradiation was necessary and the
solvent mixture was changed to i-PrOH/DMF/H2O 4/3/1, v/v/v
for complete dissolution of the CTV scaffold derivative 2. Thus,
first cyclic peptide 26 was ligated to 2 via CuAAC to give the
CTV scaffold containing one peptide loop (29), in a
1) n-BuLi, THF
-78°C
TES
TES
Br
Br
+
2) TES-Cl
-78°C=>RT
(37%)
(16%)
16
1416
15
O
1) n-BuLi, THF
-78°C
O
O
Br
Br2,Ph3P
p-TsOH
HO
O
O
DCM, 0°C
DCM
2) TES-Cl or TIPS-Cl
-78°C=>RT
0°C=>RT
(97%)
17
18
PG
PG
19: PG = TES: 88%
20: PG = TIPS: 86%
15: PG = TES: 88%
21: PG = TIPS: 86%
Scheme 4. Synthesis of silyl protected alkyne bromides 15 and 21.
Scheme 5. Sequential ligation of cyclic peptides 26, 27, 28 onto the
semi-orthogonally protected CTV scaffold 2.
However, the target TES-protected alkyne bromide 15 was
obtained in a low yield partly due to formation of the substitution
by-product 16. Here too, the THP group offered a remedy.
Hence, the desired silyl protected alkyne bromides 15 and 21
were prepared starting from propargyl alcohol 17 which was
protected as a THP ether 18. Following the protection, the free
alkyne of the THP ether 18 was silylated with either TES-Cl or
TIPS-Cl to give corresponding TES and TIPS protected
analogues 19 and 20 respectively. Lastly, bromination and
concomitant removal of the THP ethers of the silyl protected
alkynes 19 and 20 led to the formation of the desired silyl
protected alkyne bromides 15 and 21.
good yield (70%) (Scheme 5). Next, the TES protecting
group was mildly removed using AgNO3 leading to deprotected
scaffold derivative 30 (68%) and cyclic peptide 27 was ligated
via CuAAC to afford the CTV scaffold containing two peptide
loops i.e. 31 (40%). The last peptide loop 28 was introduced by
CuAAC after removal of the TIPS protecting group (TBAF·3H2O
in DMF, 42%) to complete the molecular construction of the
three peptide loops containing protein mimic 33 of Infliximab in
40% yield.
O
O
Br
Br
N
O
N
22
N3
N
TFA/H2O/EDT/TIS
Ac
C
C
NH2
SPPS
95/5/2.5/2.5
H
N
O
O
Ac
Ac
C
C
H2N
NH2
C
C
S
S
NH4HCO3 (20 mM, aq)/MeCN
N
O
N
PG
PG
STrt
SH
3/1
STrt
SH
N
1 mM
N3
pH = 7.9
26 - 28
NH2
NH2
NH
HN
HN
O
NH
NH
N
O
O
S
HO
O
N
H
OH
O
OH
H
N
H
N
HN
O
NH2
O
NH
NH
N
HO
O
N
H
H
O
O
NH
NH
O
NH
NH
NH
O
HN
H2N
N
H
O
N
H
O
O
N
H
O
N
H
O
O
NH
HN
HN
N
H2N
HN
HN
O
O
HN
O
O
HO
O
O
HO
O
S
S
NH2
NH
O
NH
S
NH2
O
O
O
O
Ac
Ac
C
Ac
NH2
NH2
C
NH2
C
C
C
C
S
NH2
S
S
O
O
O
O
O
O
O
N
N
N
O
N
O
HN
HN
N
O
N
O
S
S
S
S
S
S
N
O
N
N
O
N
N
O
N
N
O
N
O
N
N
N
N
N3
N3
N3
N3
N3
N3
O
26
27
28
NH2
Ac
C
O
N
N
S
C
TIPS
NH2
O
C
C
S
O
N
Ac
S
TES
S
N
N
O
O
O
MeO
C
O
O
N
MeO
NH2
1. AgNO3 : 30 (68%)
2. 27, CuAAC
1. TBAF: 32 (42%)
2. 28, CuAAC
OMe
N
S
O
26, CuAAC
O
N
N
29, (70%)
31, (40%)
CTV-two
N
C
N
N
O
N
N
CTV-one
Ac
N
S
peptide loop
peptide loops
N
O
MeO
OMe
O
N
O
2
N
OMe
O
33 (40%)
To illustrate the potential of the semi-orthogonally protected
CTV scaffold 2 for the construction of protein mimics, three
cyclized peptides obtained by using our recently described polar
hinge10, were sequentially introduced in a convenient "click and
cleavage" approach. The peptide segments represent CDR-loops
of the monoclonal antibody Infliximab (Remicade®), which is a
powerful tumor necrosis factor α (TNF α) inhibitor. The amino
acid sequences of these peptides were derived from the X-ray
structure of an Infliximab-TNFα-trimer complex22 After their
solid phase syntheses, the N,C-terminal dicysteine containing
peptides were cleaved and deprotected, followed by
3. Conclusion
We have described the construction of an orthogonally
protected molecular scaffold, which is probably the most pre-
organized scaffold presently available for the construction of
protein mimics. Moreover, owing to the protection group strategy
desired different peptide segments can be introduced
corresponding to different epitopes, paratopes, protein hot-spots
etc., which offers a great potential for applications of this CTV
scaffold including antibody mimics and synthetic vaccines.

In this research we have described a versatile construction of a
mimic (33) of the anti-TNFα monoclonal antibody Infliximab
(Remicade®) containing three different cyclic peptides
corresponding to three CDR-loops of this antibody. These cyclic
peptides were introduced in "click and cleavage" approach, in
which sequentially a peptide loop was ligated, followed by
cleavage of the protecting group and repeating this procedure. In
establishing this procedure the mono and dipeptide loop
constructs were purified and characterized, but we think that this
procedure has possibilities for a fast and easy preparation of
protein mimics in a "kit-like" manner with only purification after
the last stage.
CH2Cl2 and acetonitrile were obtained from Fischer Scientific
(Loughborough, United Kingdom). Solid phase peptide synthesis
was performed on a PTI Tribute-UV peptide synthesizer.
Lyophilizations were performed on a Christ Alpha 2-4 LDplus
apparatus. Analytical high pressure liquid chromatography
(HPLC) was carried out on a Shimadzu instrument comprising a
communication module (CBM-20A), autosampler (SIL-20HT),
pump modules (LC-20AT), UV/Vis detector (SPD-20A) and
system controller (Labsolutions V5.54 SP), with a Phenomenex
Gemini C18 column (110 Å, 5 µm, 250 × 4.60 mm). UV
measurements were recorded at 214 and 254 nm, using a standard
protocol: 100% buffer A (acetonitrile/H2O 5:95 with 0.1% TFA)
for
2 min followed by a linear gradient of buffer B
Under present investigation is the biological evaluation of the
obtained bio-molecular construct. Although a strong biological
activity is of course hoped for, realistically, probably libraries of
this antibody mimic have to be prepared first, before finding hits
with decent biological activities. However, this will be possible
using our earlier described combinatorial approach for obtaining
collections of discontinuous epitopes11, now to be applied to the
paratope of Infliximab. Promising hits can then be re-synthesized
for validation and on a larger scale using the orthogonally
protected CTV scaffold described here.
(acetonitrile/H2O 95:5 with 0.1% TFA) into buffer A (0-100%)
over 30 min at a flow rate of 1.0 mL·min-1. Purification of
peptidic compounds was performed on an Agilent Technologies
1260 infinity preparative system using UV detector with a
Phenomenex Gemini C18 column (110 Å, 10 µm, 250 × 20 mm).
Auto-collection of fractions was based on the UV measurements
at 214 nm, using either 100% buffer A or 95% buffer A with 5%
buffer B for 5 min followed by and linear gradient of buffer B
into buffer A (specified for each compound) over 65 min at a
flow rate of 12.5 mL·min-1 using the same buffers as described
for analytical HPLC. Liquid chromatography mass spectrometry
(LCMS) was carried out on a Thermo Scientific LCQ Fleet
quadrupole mass spectrometer with a Dionex Ultimate 3000 LC
using a Dr. Maisch Reprosil Gold 120 C18 column (110 Å, 3 µm,
150 × 4.0 mm), using a 0-100% linear gradient of buffer B into
buffer A and the same flow rate and buffers as described for
analytical HPLC.
Finally, it is important to realize that also other (bio)
molecular constructs e.g. different carbohydrate constructs,
nucleic acid can be selectivity ligated to this semi-orthogonally
protected CTV scaffold, which may open up a plethora of
applications.
4. Experimental part
4.1. General information
All reagents and solvents were used as received. 1H NMR and
the 13C NMR spectra were recorded on Bruker 400 MHz
Spectrospin spectrometer (400 MHz, 100 MHz) in CDCl3.
Chemical shifts (δ) are reported in parts per million (ppm)
relative to trimethylsilane (TMS, 0.00 ppm) or CDCl3
(7.26 ppm). Splitting patterns are designated as singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m). TLC was carried
out on silica gel plates (Merck 60F254) and the visualization was
performed by both, the UV detection (254 nm) and staining
solutions (cerium molybdenate, potassium permanganate)
followed by heating. For column chromatography, silica gel
Geduran® Si 60 (40 - 63 μm) was used. All chemicals and
solvents were purchased from regular commercial sources in
analytical or HPLC grade and were not further purified. Dry
solvents (THF, DCM) were dispensed from Pure Solv™ 500
Solvent Purification System and other dry solvents (acetonitrile)
were prepared from commercially available HPLC grade solvents
by removal of residual water with 4Å molecular sieves overnight.
HRMS-ESI was recorded on Bruker microTOFq High Resolution
Mass Spectrometer in a positive mode. EI-MS was recorded on
Jeol MSTATION JMS-700 in a positive mode.
4.2. Scaffold synthesis and derivatization
1-O-allyl-vanillyl alcohol (4) Compound 4 was prepared
according to a modified literature procedure.21 To the solution of
vanillyl alcohol 3 (55.0 g, 356.8 mmol) in acetone (82 mL),
K2CO3 (49.8 g, 360.3 mmol) and allyl bromide (34.0 mL,
392.5 mmol) were added. The resulting mixture was stirred under
reflux for 4.5 h. Afterwards, acetone was removed under reduced
pressure and the residue was partioned between CH2Cl2 (500 mL)
and water (400 mL). The organic phase was then dried over
MgSO4, and filtered. Crude 4 (65.1 g, 94.0%) was obtained upon
removal of CH2Cl2 under reduced pressure. The crude product
was crystallized from MTBE/n-hexane affording 4 (60.3 g,
1
87.0%) as white crystals. Rf = 0.32 (n-hexane/EtOAc 6/4). H
NMR (400 MHz, CDCl3): δ = 1.94 (s, 1H), 3.89 (s, 3H), 4.61 -
4.63 (m, 4H), 5.29 (dd, J = 1.4 Hz, 10.5 Hz, 1H), 5.41 (dd, J =
1.5 Hz, 17.3 Hz, 1H), 6.04 - 6.14 (m, 1H), 6.86 (s, 2H), 6.94 (s,
1H). 13C NMR (100 MHz, CDCl3): δ = 55.9, 65.2, 70.0, 110.9,
113.5, 117.9, 119.3, 133.3, 134.1, 147.5, 149.6. HRMS-ESI: m/z
calcd for C11H14NaO3 [M+Na]+, 217.0835; found, 217.0834.This
compound has been also previously reported.21
Tris(O-allyl) CTV (5) Compound 5 was prepared according to
a literature procedure21 in the following manner. Compound 4
(57.4 g, 295.5 mmol) was dissolved in MeOH (340 mL) and the
solution was cooled to 0°C resulting in the formation of a
precipitate. Next, upon the dropwise addition of 60% (aq) HClO4
(171 mL) the precipitate dissolved and the solution became pink.
Once HClO4 was added, the reaction mixture was allowed to
warm up to RT and stirred for 16.5 h during which white
precipitate gradually formed. Afterwards, the reaction mixture
was diluted with CH2Cl2 (570 mL) and washed with water
(7 × 570 mL) till the neutral pH was reached, which can be also
recognized by the change of the color of the organic layer from
pink to yellow. The organic phase was then dried over MgSO4,
Fmoc-amino acids were obtained from Activotec (Cambridge,
United Kingdom) and N,N,N′,N′-Tetramethyl-O-(6-chloro-1H-
benzotriazol-1-yl)uranium hexafluorophosphate (HCTU) was
obtained from Matrix Innovation (Quebec, Canada). Tentagel
S RAM resin (particle size 90µm, capacity 0.25 mmol.g-1) was
obtained from IRIS Biotech (Marktredwitz, Germany). Methyl
tert-butyl ether (MTBE), n-hexane (HPLC grade) and TFA were
obtained from Aldrich (Milwaukee, USA). DMF (Peptide grade)
was obtained from VWR (Lutterworth, United Kingdom).
Piperidine and DiPEA were obtained from AGTC Bioproducts
(Hessle, United Kingdom), and 1,2-ethanedithiol (EDT) was
obtained from Merck (Darmstadt, Germany). HPLC grade

filtered, concentrated under reduced pressure to give pale yellow
solid which was suspended in Et2O (100 mL) and stirred for 2 h
before being filtered off. The filtrate was washed with Et2O
(3 × 30 mL) and dried on high vacuum. Compound 5 (27.1 g,
52.0%) was obtained as a white solid. Rf = 0.47 (n-hexane/EtOAc
6/4). 1H NMR (400 MHz, CDCl3): δ = 3.54 (d, J = 13.9 Hz), 3.86
(s, 9H), 4.56 - 4.66 (m, 6H), 4.77 (d, J = 13.7 Hz, 3H), 5.28 (dd,
J = 1.4 Hz, 10.5 Hz, 3H), 5.39 (dd, J = 1.6 Hz, 17.3 Hz, 3H),
6.04 - 6.13 (m, 3H), 6.82 (s, 3H), 6.88 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ = 36.3, 55.9, 70.0, 113.5, 115.5, 117.3,
131.6, 132.2, 133.6, 146.6, 148.0. HRMS-ESI: m/z calcd for
C33H36NaO6 [M+Na]+, 551.2404; found, 551.2391.This
compound has been also previously reported.21
[M+Na]+, 599.2615; found, 599.2592. Along with the product 9,
a mixture of 10 was obtained as a yellow foam. For analytical
purposes 1H NMR spectra of pure Mono(O-THP) CTV-diOH and
Tri(O-THP) CTV were measured before the compounds were
mixed for recovery of compound 6. Mono(O-THP) CTV-diOH:
1H NMR (400 MHz, CDCl3): δ = 1.56 - 1.71 (m, 3H), 1.81 - 2.06
(m, 3H), 3.47 - 3.59 (m, 4H), 3.82 - 3.89 (m, 9.5H), 4.00 - 4.05
(m, 0.5H), 4.65 - 4.71 (m, 3H), 5.17 (s, 0.5H), 5.42 (s, 0.5H),
5.56 (s, 2H), 6.78 - 6.91 (m, 5H), 7.12 (s, 1H). Rf = 0.20
1
(n-hexane/EtOAc 1/1). Tri(O-THP) CTV: H NMR (400 MHz,
CDCl3): δ = 1.52 - 1.69 (m, 9H), 1.79 - 2.03 (m, 9H), 3.47 - 3.58
(m, 6H), 3.80 - 3.90 (m, 10.5H), 3.98 - 4.04 (m, 1.5H), 4.68 -
4.72 (m, 3H), 5.18 (m, 1.5H), 5.39 (m, 1.5H), 6.84 (s, 3H), 7.11 -
7.13 (m, 3H). Rf = 0.52 (n-hexane/EtOAc 1/1).
CTV-triOH (6) Compound 6 was prepared according to a
modified literature procedure.21 To the solution of 5 (26.0 g,
49.2 mmol) in dioxane/EtOH (130 mL/230 mL), 10% Pd/C
(5.4 g), 65% (aq) HClO4 (5.4 mL) were added and the resulting
reaction mixture was stirred under nitrogen atmosphere at 65°C
for 24 h and then for 48 h at RT. Next, catalyst was filtered off
and washed with dioxane (50 mL) and CH2Cl2 (200 mL). The
filtrate was then washed with water (2 × 700 mL, 2 × 500 mL),
aqueous layer was extracted with CH2Cl2 (100 mL) and the
combined organic layer was dried over MgSO4, filtered,
concentrated under reduced pressure to ca 100 mL, and allowed
to crystallize. The crystals were filtered off, washed with cold
CH2Cl2 (3 × 30 mL) and dried on a high vacuum to afford 6
(10.5 g, 52.1%) as white crystals. Rf = 0.18 (n-hexane/EtOAc
1/1). 1H NMR (400 MHz, CDCl3): δ = 3.50 (d, J = 13.9 Hz), 3.85
(s, 9H), 4.72 (d, J = 13.7 Hz, 3H), 5.41 (s, 3H), 6.79 (s, 3H), 6.88
(s, 3H). 13C NMR (100 MHz, CDCl3): δ = 36.3, 56.1, 112.3,
115.5, 131.2, 132.5, 144.2, 145.3. HRMS-ESI: m/z calcd for
C24H24NaO6 [M+Na]+, 431.1465; found, 431.1464.This
compound has been also previously reported.23
Recovery of CTV-triOH (6) To the suspension of side products
10 in MeOH (13 mL per 1 g of mixture), 1M HCl (2.6 mL per
1 g of mixture) was added and the reaction mixture was stirred
for 2 h during which a white precipitate was formed. Afterwards,
MeOH was removed under reduced pressure and the creamy
residue was dissolved in EtOAc/MeOH (40/1, v/v, 130 mL per
1 g of mixture) and washed with water/brine (1/1, v/v, 40 mL per
1 g of mixture). The aqueous layer was extracted with EtOAc
(25 mL per 1 g of mixture) and the combined organic layer was
washed with brine (25 mL per 1 g of mixture), dried over
MgSO4, filtered, and concentrated under reduced pressure to a
low volume. White precipitate was formed during removal of
solvents. The suspension was kept at +4°C overnight before the
white precipitate was filtered off and washed with EtOAc to give
compound 6 ( typically between 58% - 74%).
Di(O-THP)-O-propargyl CTV (11) To the solution of
compound 9 (1.0 g, 1.7 mmol) in acetonitrile (17 mL) Cs2CO3
(0.6 g, 1.9 mmol) was added, followed by the addition of
propargyl bromide (80% in toluene) (0.23 mL, 2.1 mmol) and the
resulting reaction mixture was stirred for 2.5 h. Afterwards,
acetonitrile was removed under reduced pressure and the viscous
residue was diluted with EtOAc (100 mL) and washed with water
(100 mL). Aqueous layer was extracted with EtOAc (50 mL) and
combined organic layer was washed with brine (50 mL), dried
over MgSO4, filtered, and EtOAc was removed under reduced
Di(O-THP) CTV-OH (9), Mono(O-THP) CTV-diOH and
Tri(O-THP) CTV (10)
To a suspension of CTV 6 (5.0 g, 12.4 mmol) in CHCl3
(124 mL), DHP (2.3 mL, 25.4 mmol) and p-TsOH monohydrate
(0.02 g, 0.1 mmol) were added. After 2 h of stirring, during
which the reaction mixture became pink, additional DHP
(1.2 mL, 12.3 mmol) was added, which resulted in the formation
of a solution which was stirred for an additional 1 h. Afterwards,
the reaction mixture was diluted with Et2O (125 mL) followed by
immediate addition of saturated NaHCO3 (50 mL). Direct
addition of CHCl3 or saturated NaHCO3 to reaction mixture was
attempted too but lead to partial decomposition in certain cases.
Next, the aqueous layer was extracted with Et2O (100 mL) and
the combined organic layer was dried over MgSO4, filtered, and
solvents were removed under reduced pressure. The crude
pressure. The crude product was purified by
a column
chromatography (n-hexane/EtOAc 9/1 to n-hexane/EtOAc 7/3).
Compound 11 (0.8 g, 76.8%) was obtained as a white foam.
Rf = 0.51 (n-hexane/EtOAc 1/1). 1H NMR (400 MHz, CDCl3):
δ = 1.53 - 1.69 (m, 6H), 1.80 - 2.05 (m, 6H), 2.39 (s, 1H),
3.47 - 3.57 (m, 5H), 3.80 - 3.83 (m, 10H), 3.98 - 4.03 (m, 1H),
4.67 - 4.73 (m, 5H), 5.17 - 5.20 (m, 1H), 5.40 (s, 1H), 6.83 - 6.84
(m, 2H), 6.87 (s, 1H), 7.01 (d, J = 5.8 Hz, 1H), 7.12 - 7.15 (m,
2H). 13C NMR (100 MHz, CDCl3): δ = 18.7, 19.0, 25.3, 30.4,
30.5, 36.4, 36.5, 55.8, 56.0, 56.2, 56.3, 56.4, 56.6, 56.7, 57.0,
61.9, 62.2, 75.8, 79.1, 97.1, 97.2, 98.1, 98.2, 98.3, 113.4, 113.5,
113.8, 114.1, 114.6, 114.7, 114.8, 116.4, 116.5, 116.6, 118.6,
118.9, 119.0, 119.6, 119.7, 119.8, 131.3, 131.4, 131.5, 131.6,
131.7, 131.9, 132.0, 132.1, 132.2, 132.3, 133.1, 133.2, 133.8,
133.9, 134.0, 145.0, 145.1, 145.2, 145.3, 148.4, 148.5, 148.6,
148.9, 149.1. HRMS-ESI: m/z calcd for C37H42NaO8 [M+Na]+,
637.2772; found, 637.2755.
product was purified by
a
column chromatography
(n-hexane/EtOAc 2.5/1 to n-hexane/EtOAc 1/1). Compound 6
(2.1 g, 29.9%) was obtained as a yellow foam. Rf = 0.33
1
(n-hexane/EtOAc 1/1). H NMR (400 MHz, CDCl3): δ = 1.52 -
1.74 (m, 6H), 1.79 - 1.95 (m, 4H), 1.99 - 2.08 (m, 2H), 3.47 -
3.59 (m, 5H), 3.81 - 3.91 (m, 10H), 3.99- 4.06 (m, 1H), 4.69 -
4.75 (m, 3H), 5.17 - 5.19 (m, 1H), 5.40 - 5.45 (m, 1H), 5.47 (s,
1H), 6.81 (d, J = 3.1 Hz, 1H), 6.84 (d, J = 1.9 Hz, 1H), 6.85 (s,
1H), 6.90 (dd, J = 1.8 Hz, 5.7 Hz, 1H), 7.11- 7.15 (m, 2H).
13C NMR (100 MHz, CDCl3): δ = 18.8, 19.0, 19.1, 25.4, 30.4,
30.6, 36.4, 36.5, 55.9, 56.0, 56.3, 56.4, 56.5, 56.6, 62.0, 62.3,
97.2, 97.3, 98.2, 98.3, 112.3, 113.9, 114.1, 114.3, 114.4, 115.5,
115.6, 115.7, 115.8, 118.7, 119.0, 119.5, 120.0, 120.1, 131.5,
131.6, 131.7, 131.9, 132.0, 132.1, 132.3, 132.4, 133.1, 133.3,
133.9, 134.0, 144.1, 144.2, 144.9, 145.0, 145.1, 145.3, 145.4,
148.6, 148.7, 149.1. HRMS-ESI: m/z calcd for C34H40NaO8
O-Propargyl CTV-diOH (7) Compound 11 (790 mg,
1.29 mmol) was suspended in MeOH (20 mL) and then 1M HCl
(1 mL) was added. The reaction mixture was stirred for 1 h
during which the suspension turned into a yellow solution.
Afterwards, MeOH was removed under reduced pressure and the
viscous residue was diluted with EtOAc (40 mL) and washed
with water (30 mL). Next, the aqueous phase was extracted with
EtOAc (20 mL) and the combined organic layer was washed with

brine (30 mL), dried over MgSO4, filtered, and EtOAc was
removed under reduced pressure. The crude product was purified
by a column chromatography (CHCl3 to CHCl3/MeOH 50/1).
Compound 7 (524 mg, 91.4%) was obtained as a white foam.
2H). 13C NMR (100 MHz, CDCl3): δ = 4.2, 7.4, 11.1, 18.6, 36.5,
56.0, 56.1, 57.0, 58.5, 58.6, 58.7, 75.7, 79.1, 89.2, 90.3, 101.8,
102.5, 113.5, 113.6, 114.0, 116.7, 117.9, 131.5, 131.6, 133.4,
133.5, 133.7, 145.4, 145.9, 146.0, 148.5, 148.6, 148.8.
HRMS-ESI: m/z calcd for C48H64NaO6Si2 [M+Na]+, 815.4134;
found, 815.4102.
1
Rf = 0.25 (n-hexane/EtOAc 1/1). H NMR (400 MHz, CDCl3):
δ = 2.44 (t, J = 2.4 Hz, 1H), 3.49 - 3.56 (m, 3H), 3.84 (s, 3H),
3.86 (s, 6H), 4.70 - 4.77 (m, 5H), 5.39 (s, 1H), 5.41 (s, 1H), 6.79
(s, 1H), 6.84 (s, 1H), 6.85 (s, 1H), 6.89 (s, 1H), 6.90 (s, 1H), 7.01
(s, 1H). 13C NMR (100 MHz, CDCl3): δ = 56.8, 57.9, 76.4, 78.0,
79.9, 113.0, 113.1, 114.4, 116.3, 117.4, 131.9, 132.5, 132.9,
133.3, 134.3, 144.9, 145.9, 146.0, 149.2. HRMS-ESI: m/z calcd
for C27H26NaO6 [M+Na]+, 469.1622; found, 469.1603.
4.3. Preparation of silyl alkyne bromides
O-THP Propargyl alcohol (18) Compound 18 was prepared
according to a literature procedure.24 To the solution of propargyl
alcohol 17 (2.6 mL, 44.6 mmol) and p-TsOH monohydrate
(0.09 g, 0.5 mmol) in CH2Cl2 (45 mL) DHP (4.3 mL, 46.8 mmol)
was added dropwise at 0°C. After stirring for 5 min at 0°C, the
reaction mixture was allowed to warm up to RT and was stirred
for 1 h. Next, the reaction mixture was washed with saturated
NaHCO3 (30 mL) and the aqueous layer was extracted with
CH2Cl2 (45 mL). The combined organic layer dried over MgSO4,
filtered, and concentrated under reduced pressure to give
compound 18 (6.1 g, 97.2%) as a yellow oil. Rf = 0.30
(n-hexane/Et2O 95/5). 1H NMR (400 MHz, CDCl3):
δ = 1.50 - 1.67 (m, 4H), 1.70 - 1.89 (m, 2H), 2.41 (t, J = 2.4 Hz,
1H), 3.52 - 3.57 (m, 1H), 3.82 - 3.87 (m, 1H), 4.27 (ddd,
J = 2.4 Hz, 11.3 Hz, 15.8 Hz, 2H), 4.83 (t, J = 3.4 Hz, 1H).
13C NMR (100 MHz, CDCl3): δ = 19.0, 25.3, 30.2, 53.9, 61.9,
74.0, 79.7, 96.8. MS-CI: m/z calcd for C8H12O2 [M+H]+, 141;
found, 141. This compound has been also previously reported.25
O-Propargyl-O-(TIPS)propargyl
CTV-OH
(12),
O-Propargyl bis[O-(TIPS)propargyl] CTV (13) Cs2CO3 (181 mg,
0.56 mmol) was added to the solution of compound 7 (226 mg,
0.51 mmol) in dry acetonitrile (20 mL). Next, a solution of
compound 21 (146 mg, 0.53 mmol) in dry acetonitrile (3 mL)
was added dropwise. The reaction mixture was stirred under
nitrogen atmosphere for 20 h. Afterwards, acetonitrile was
removed under reduced pressure and the residue was suspended
in Et2O (40 mL) and washed with 1M KHSO4 (40 mL). The
aqueous layer was extracted with Et2O (20 mL) and the
combined organic layer washed with brine (40 mL), dried over
MgSO4, filtered, and Et2O was removed under reduced pressure.
The crude product was purified by a column chromatography
(CH2Cl2 to CH2Cl2/MeOH 100/1) to afford compound 13 (93 mg,
23.2%) as a pale yellow solid. Rf = 0.87 (n-hexane/EtOAc 1/1).
1H NMR (400 MHz, CDCl3): δ = 1.02 (m, 42H), 2.45 (t,
J = 2.4 Hz, 1H), 3.50 - 3.57 (m, 3H), 3.83 - 3.84 (m, 9H),
4.57 - 4.86 (m, 9H), 6.83 (s, 2H), 6.85 (s, 1H), 7.00 (s, 1H), 7.14
(s, 1H), 7.16 (s, 1H). 13C NMR (100 MHz, CDCl3): δ = 11.1,
18.6, 36.5, 56.0, 56.2, 57.0, 58.6, 75.7, 79.1, 89.2, 102.5, 113.5,
113.9, 116.6, 117.6, 117.9, 131.5, 131.7, 133.3, 133.6, 145.3,
145.9, 146.0, 148.5, 148.8. HRMS-ESI: m/z calcd for
C51H70NaO6Si2 [M+Na]+, 857.4603; found, 857.4569. Compound
12 (139 mg, 42.9%) was obtained as a yellow amorphous solid.
O-THP (TIPS)Propargyl alcohol(20)Compound 20 was
prepared according to a modified literature procedure.26
A
solution of compound 18 (1.0 g, 7.1 mmol) in dry THF (20 mL)
was cooled to -78°C. Next, n-BuLi (3.0 mL, 7.5 mmol, 2.5M in
hexane) was added dropwise and the reaction mixture was stirred
under nitrogen atmosphere at -78°C for 1 h before TIPS-Cl
(1.7 mL, 7.9 mmol) was added. The reaction mixture was then
allowed to warm up to RT and stirred under nitrogen atmosphere
for additional 4 h before it was quenched by addition of saturated
NH4Cl (15 mL). The reaction mixture was extracted with Et2O
(2 × 25 mL), dried over MgSO4, filtered, and Et2O was carefully
removed under reduced pressure with water bath kept at RT. The
crude product was purified by a column chromatography
(n-hexane to n-hexane/Et2O 99/1) to afford 20 (1.8 g, 85.9%) as a
1
Rf = 0.62 (n-hexane/EtOAc 1/1). H NMR (400 MHz, CDCl3):
δ = 1.01 (m, 21H), 2.44 (m, 1H), 3.47 - 3.54 (m, 3H), 3.83 (s,
9H), 4.58 - 4.84 (m, 7H), 5.44 (d, J = 9.4 Hz, 1H), 6.79 - 6.85 (m,
3H), 6.89 (d, J = 6.8 Hz, 1H), 6.99 (d, J = 6.3 Hz, 1H), 7.13 (d,
J = 3.3 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ = 11.1, 18.6,
36.4, 56.0, 56.1, 56.2, 57.0, 57.1, 58.6, 75.6, 75.7, 79.1, 79.2,
89.2, 102.6, 112.1, 112.4, 113.5, 113.7, 113.9, 115.7, 116.6,
116.8, 117.9, 131.1, 131.3, 131.4, 131.6, 131.7, 131.9, 132.4,
133.6, 133.7, 144.2, 145.3, 145.4, 145.9, 148.5, 145.7, 145.8.
HRMS-ESI: m/z calcd for C39H48NaO6Si [M+Na]+, 663.3112;
found, 663.3092. Starting material, compound 7, (57 mg, 25.2%)
was recovered.
1
clear oil. Rf = 0.44 (n-hexane/Et2O 95/5). H NMR (400 MHz,
CDCl3): δ = 1.07 (m, 21H), 1.51 - 1.65 (m, 4H), 1.71 - 1.87 (m,
2H), 3.50 - 3.55 (m, 1H), 3.83 - 3.89 (m, 1H), 4.31 (ddd,
J = 2.4 Hz, 6.3 Hz, 16.1 Hz, 2H), 4.91 (t, J = 3.4 Hz, 1H).
13C NMR (100 MHz, CDCl3): δ = 11.2, 18.6, 19.2, 25.4, 30.4,
54.7, 62.2, 87.0, 96.3, 103.4. HRMS-ESI: m/z calcd for
C17H32NaO2Si [M+Na]+, 319.2064; found, 319.2049. This
compound has been also previously reported.27
O-Propargyl-O-(TIPS)propargyl-O-(TES)propargyl CTV (2)
To the suspension of compound 12 (303 mg, 0.47 mmol) in dry
acetonitrile (5 mL) Cs2CO3 (162 mg, 0.50 mmol) was added
followed by the addition of the solution of compound 15
(121 mg, 0.52 mmol) in dry acetonitrile (0.5 mL). The resulting
mixture was stirred for 5.5 h and then acetonitrile was removed
under reduced pressure. The viscous residue was suspended in
Et2O (40 mL) and washed with water (30 mL). The aqueous layer
was extracted with Et2O (30 mL) and the combined organic layer
washed with brine (20 mL), dried over MgSO4, filtered, and Et2O
was removed under reduced pressure. The crude product was
purified by a column chromatography (n-hexane/Et2O 3/1 to n-
hexane/Et2O 2/1). Compound 2 (269 mg, 71.7%) was obtained as
a white foam. Rf = 0.60 (n-hexane/EtOAc 3/1). 1H NMR
(400 MHz, CDCl3): δ = 0.58 (m, 6H), 0.94 (m, 9H), 1.02 (m,
21H), 2.45 (m, 1H), 3.50 - 3.57 (m, 3H), 3.84 (m, 9H), 4.56 --
4.85 (m, 9H), 6.83 - 6.85 (m, 3H), 7.00 (s, 1H), 7.09 - 7.15 (m,
(TIPS)Propargyl bromide (21) Compound 21 was prepared
according to a modified literature procedure.26 Br2 (130 μL,
2.53 mmol) was added dropwise at 0°C to a solution of Ph3P
(697 mg, 2.66 mmol) in dry CH2Cl2 (10 mL) and the solution was
stirred under nitrogen atmosphere for 30 min during which a
white precipitate was formed. Next, compound 20 (750 mg,
2.53 mmol) was added dropwise and the reaction mixture was
stirred under nitrogen atmosphere at 0°C for additional 5 h during
which the white precipitate dissolved. The reaction mixture was
then diluted with water (15 mL) and extracted with n-hexane
(2 × 20 mL). The combined organic layer was washed with
saturated NaHCO3 (10 mL), dried over MgSO4, filtered, and
n-hexane was carefully removed under reduced pressure with
water bath kept at RT. The crude product was purified by a
column chromatography using (n-hexane) to afford 21 (682 mg,
1
98.0%) as a clear oil. Rf = 0.69 (n-hexane). H NMR (400 MHz,

CDCl3): δ = 1.07 (m, 21H), 3.95 (s, 2H). 13C NMR (100 MHz,
CDCl3): δ = 11.2, 15.0, 18.5, 89.2, 101.9. This compound has
been also previously reported.27
was washed with DMF (6 x 30 sec). After the last amino acid
coupling, the Fmoc group was cleaved using the normal
deprotection conditions (described above) and the resulting free
N-terminus was acetylated by treatment of the resin bound
peptide with acetic anhydride (250 µL) and DiPEA (10 equiv for
the 0.1 mmol scale and 8 equiv for the 0.25 mmol scale) in DMF
using the standard coupling times (described above). After the
last step the resin was washed with DMF (5 x 30 sec), DCM
(5 x 30 sec), dried over a nitrogen flow for 10 min, followed by
the cleavage of the resin-bounded peptide. Cleavage and global
deprotection was achieved by treatment of the resin with
TFA/H2O/TIS/EDT (10 mL for the 0.25 mmol scale and 5 mL
for the 0.1 mmol scale, 90/5/2.5/2.5, v/v/v/v) for 3 hours. Next,
the peptide was precipitated by dropwise addition of the TFA
mixture to a cold (4 °C) solution of MTBE/n-hexane (1/1, 90 mL
for the 0.25 mmol scale and 45 mL for the 0.1 mmol scale). After
centrifugation (3500 rpm, 5 min) the supernatant was decanted
and the pellet was re-suspended in MTBE/n-hexane (1/1, v/v)
and centrifuged again. Finally, the pellet was washed twice with
MTBE/n-hexane 50 mL (1/1, v/v), each time collected by
centrifugation, dissolved in t-BuOH/H2O (1/1, v/v) and
lyophilized to yield the crude linear peptide. Hence following
peptides were obtained:
O-THP (TES)Propargyl alcohol (19) Compound 19 was
prepared according to a modified literature procedure.27
A
solution of compound 18 (1.0 g, 7.1 mmol) in dry THF (20 mL)
was cooled to -78°C. Next, n-BuLi (3.0 mL, 7.5 mmol, 2.5M in
hexane) was added dropwise and the reaction mixture was stirred
under nitrogen atmosphere at -78°C for 1 h before TES-Cl
(1.3 mL, 7.9 mmol) was added. The reaction mixture was then
allowed to warm up to RT and stirred under nitrogen atmosphere
for additional 4 h before it was quenched by addition of saturated
NH4Cl (15 mL). The reaction mixture was extracted with Et2O
(2 × 25 mL), dried over MgSO4, filtered, and Et2O was carefully
removed under reduced pressure with water bath kept at RT. The
crude product was purified by a column chromatography using
(n-hexane to n-hexane/Et2O 99/1) to afford 19 (1.6 g, 87.9%) as a
1
clear oil. Rf = 0.42 (n-hexane/Et2O 95/5). H NMR (400 MHz,
CDCl3): δ = 0.57 - 0.64 (m, 6H), 0.95 - 1.02 (m, 9H), 1.50 - 1.67
(m, 4H), 1.70 - 1.87 (m, 2H), 3.50 - 3.55 (m, 1H), 3.82 - 3.88 (m,
1H), 4.29 (s, 2H), 4.86 (t, J = 3.4 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ = 4.3, 7.4, 19.1, 25.4, 30.3, 54.7, 62.0, 82.3, 96.5,
102.7. HRMS-ESI: m/z calcd for C14H26NaO2Si [M+Na]+,
277.1594; found, 277.1584. Although compound 8 has been also
previously reported,28 no spectroscopic data of compound 8 are
available.
Peptide 1·2TFA (23), Ac-CRSKSIYC-NH2, tR = 13.6 min.
LCMS-ESI : average mass calcd [M]+: 1000.2; found: 1000.5.
Peptide 2·TFA (24), Ac-CSNHWMNC-NH2, tR = 15.4 min.
(TES)Propargyl bromide (15) Compound 15 was prepared
according to a modified literature procedure.26 Br2 (0.3 mL,
5.9 mmol) was added dropwise at 0°C to a solution of Ph3P
(1.6 g, 6.2 mmol) in dry CH2Cl2 (23.6 mL) and the solution was
stirred under nitrogen atmosphere for 30 min during which a
white precipitate was formed. Next, compound 19 (1.5 g,
5.9 mmol) was added dropwise and the reaction mixture was
stirred under nitrogen atmosphere at 0°C for additional 5 h during
which the white precipitate dissolved. The reaction mixture was
then diluted with water (15 mL) and extracted with n-hexane
(2 × 20 mL). The combined organic layer was washed with
saturated NaHCO3 (10 mL), dried over MgSO4, filtered, and
n-hexane was carefully removed under reduced pressure with
water bath kept at RT. The crude product was purified by a
column chromatography using (n-hexane) to afford 15 (1.1 g,
LCMS-ESI: average mass calcd [M]+: 1035.2; found: 1035.4.
Peptide 2·2TFA (25), Ac-CSHSWRWC-NH2, tR = 15.7 min.
LCMS-ESI: average mass calcd [M]+: 1105.3; found: 1105.5.
4.4.2. Cyclic peptides
All linear peptides were cyclized at the concentration of 1 mM
in the following way. The crude linear peptide and the
perhydro triazine hinge 22 were placed into a flask. Then,
acetonitrile was added followed by the addition of aqueous
solution of NH4HCO3 (20 mM, pH = 7.9) to form a 1/3
(acetonitrile/NH4HCO3 v/v) mixture. The progress of the
cyclization was checked by analytical HPLC after 30 min. In case
of all the peptides, 30 min were sufficient for the complete
cyclization. Next, acetonitrile was removed under reduced
pressure and the remaining aqueous solution/suspension was
lyophilized. The crude cyclic peptides were purified using
preparative HPLC. The fractions containing product were
combined and lyophilized yielding the desired cyclic peptides as
a white fluffy solid.
1
82.3%) as a clear oil. Rf = 0.66 (n-hexane). H NMR (400 MHz,
CDCl3): δ = 0.61 (q, J = 7.9 Hz, 6H), 0.99 (t, J = 7.8 Hz, 9H),
3.95 (s, 2H). 13C NMR (100 MHz, CDCl3): δ = 4.2, 7.3, 14.8,
90.1, 101.2. This compound has been also previously reported.29
hinge
Ac-CRSKSIYC-NH2
4.4. Peptide synthesis and preparation of synthetic antibody
Cyclic peptide·2TFA (26), Purified
using 5% to 30% of buffer B in buffer
4.4.1. Linear peptides
A. Average yield 22% (19 steps, 92%
per step). tR = 14.1 min. LCMS-ESI: average mass calcd [M]+:
1250.4; found: 1250.5.
General method for automated peptide synthesis: The peptides
were synthesized on a PTI Tribute-UV peptide synthesizer.
Tentagel S RAM resin (1.0 g, 0.25 mmol, 1.0 equiv or 400 mg,
0.1 mmol, 1.0 equiv) was allowed to swell (3 x 10 min).
Deprotection of the Fmoc group was achieved by treatment of the
resin with 20% piperidine in DMF using the RV_top_UV_Xtend
protocol from the Tribute-UV peptide synthesizer followed by a
DMF washing step (5 x 30 sec). The Fmoc-protected amino acids
(with the 0.1 mmol scale 5 equiv was used and with the
0.25 mmol scale 4 equiv was used) were coupled using HCTU
(with the 0.1 mmol scale 5 equiv was used and with the
0.25 mmol scale 4 equiv was used) and DiPEA (with the
0.1 mmol scale 10 equiv was used and with the 0.25 mmol scale
8 equiv was used) in DMF, as a coupling system, with 2 min
pre-activation. The coupling time was 10 min when the peptide
was synthesized on a 0.1 mmol scale and 20 min when the
0.25 mmol scale was conducted. After every coupling the resin
Cyclic peptide 2·TFA (27),
Purified using 5% to 30% of
buffer B in buffer A. Average
hinge
Ac-CSNHWMNC-NH2
yield 20% (19 steps, 92% per step). tR = 15.3 min. LCMS-ESI:
average mass calcd [M]+: 1285.4; found: 1285.5.
Cyclic peptide 2·2TFA (28),.
Purified using 5% to 40% of buffer
B in buffer A. Average yield 11%
hinge
Ac-CSHSWRWC-NH2
(19 steps, 89% per step). tR = 16.0 min. LCMS-ESI: average
mass calcd [M]+: 1355.5; found: 1355.5.
4.4.3. Preparation of a synthetic antibody mimic

CTV Scaffold derivative (29) Semi-orthogonally protected
CTV scaffold 2 (5.2 mg, 6.50 µmol), cyclic peptide 26 (10.6 mg,
7.15 µmol), TBTA (1.0 mg, 1.95 µmol) were placed into a flask
and degassed DMF (975 µL) was added and the mixture was
stirred under nitrogen atmosphere until clear solution was
obtained. Next, degassed i-PrOH (1300 µL), deionized H2O
(290 µL) were added followed by the addition of 0.2M aqueous
sodium ascorbate (19.5 µL, 3.90 µmol) and 0.12M aqueous
CuSO4 (16.3 µL, 1.95 µmol). The reaction mixture was stirred
under nitrogen atmosphere and monitored by analytical HPLC
which showed complete conversion after 1 h. Then, the solvents
were removed with nitrogen stream giving red liquid residue
(ca 50 µL) which was diluted with buffer B (150 µL) and buffer
A (1200 µL). The resulting solution was centrifuged (4500 rpm,
5 min) and purified using 5% to 100% of buffer B in buffer A.
Scaffold derivative 29·2TFA (10.5 mg, 71.0%) was obtained as a
white fluffy solid after lyophilization. tR = 29.1 min. LCMS-ESI:
average mass calcd [M+2H]2+: 1022.8; found: 1022.5.
0.45 µmol), TBTA (0.1 mg, 0.25 µmol) were placed into a flask
and degassed DMF (130 µL) was added and the mixture was
stirred under nitrogen atmosphere until clear solution was
obtained. Next, degassed i-PrOH (60 µL), deionized H2O (15 µL)
were added followed by the addition of 0.2M aqueous sodium
ascorbate (2.5 µL, 0.49 µmol) and 0.12M aqueous CuSO4 (2.1
µL, 0.25 µmol). The reaction mixture was stirred under nitrogen
atmosphere and monitored by analytical HPLC which showed
complete conversion after 89 h. Then, the solvents were removed
with nitrogen stream giving red solid which was diluted with
buffer B (50 µL) and buffer A (460 µL). The resulting solution
was centrifuged (4500 rpm, 5 min) and purified using 5% to 80%
of buffer B in buffer A. Synthetic antibody 33·5TFA (0.8 mg,
40.0%) was obtained as a white fluffy solid after lyophilization.
tR = 16.9 min. LCMS-ESI: average mass calcd [M+3H]3+:
1472.3; found: 1472.1.
References and notes
CTV Scaffold derivative (30) To a solution of scaffold
derivative 29·2TFA (10.3 mg, 4.53 µmol) in degassed DMF (680
µL), degassed i-PrOH (910 µL), and deionized H2O (225 µL),
AgNO3 (3.9 mg, 22.65 µmol) was added and the reaction mixture
was stirred under nitrogen atmosphere and monitored by
analytical HPLC which showed complete conversion after 1 h.
Then, the solvents were removed with nitrogen stream giving
clear honey which was diluted with buffer A (1300 µL). The
resulting solution was centrifuged (4500 rpm, 5 min) and purified
using 5% to 100% of buffer B in buffer A. Scaffold derivative
30·2TFA (6.3 mg, 64.3%) was obtained as a white fluffy solid
1. Opatz, T.; Liskamp, R.M.J. Org. Lett. 2001, 3, 3499-3502.
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11. Mulder, G.E.; Kruijtzer, J.A.W.; Liskamp, R.M.J. Chem.
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12. Mulder, G.E.; van Ufford, H.C.Q.; van Ameijde, J.; Brouwer,
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after lyophilization. tR
=
24.9 min and 25.1 min
(diastereoisomers). LCMS-ESI: average mass calcd [M+2H]2+:
965.7; found: 965.5.
CTV Scaffold derivative (31) Scaffold derivative 30·2TFA (6.0
mg, 2.78 µmol), cyclic peptide 27 (4.3 mg, 3.06 µmol), TBTA
(0.4 mg, 0.83 µmol) were placed into a flask and degassed DMF
(415 µL) was added and the mixture was stirred under nitrogen
atmosphere until clear solution was obtained. Next, degassed
i-PrOH (555 µL), deionized H2O (125 µL) were added followed
by the addition of 0.2M aqueous sodium ascorbate (8.4 µL,
1.67 µmol) and 0.12M aqueous CuSO4 (7.0 µL, 0.83 µmol). The
reaction mixture was stirred under nitrogen atmosphere and
monitored by analytical HPLC which showed complete
conversion after 19 h. Then, the solvents were removed with
nitrogen stream giving red thick solid which was diluted with
buffer B (50 µL) and buffer A (470 µL). The resulting solution
was centrifuged (4500 rpm, 5 min) and purified using 5% to 90%
of buffer B in buffer A. Scaffold derivative 31·3TFA (4.0 mg,
40.4%) was obtained as a white fluffy solid after lyophilization.
tR = 21.7 min. LCMS-ESI: average mass calcd [M+3H]3+:
1072.6; found: 1072.4.
14. Werkhoven, P. R.; van de Langemheen, H.; van der Wal, S.;
Kruijtzer, J. A. W.; Liskamp, R. M. J. J. Pept. Sci. 2014 20,
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CTV Scaffold derivative (32) To a solution of scaffold
derivative 31·3TFA (3.8 mg, 1.07 µmol) in degassed DMF (430
µL) TBAF·3H2O (3.4 mg, 10.70 µmol) was added and the
reaction mixture was stirred under nitrogen atmosphere and
monitored by analytical HPLC which showed complete
conversion after 17.5 h. Then, DMF was removed with nitrogen
stream giving clear solid which was diluted with buffer A (515
µL). The resulting solution was centrifuged (4500 rpm, 5 min)
and purified using 5% to 80% of buffer B in buffer A. Scaffold
derivative 32·3TFA (1.5 mg, 41.7%) was obtained as a white
fluffy solid after lyophilization. tR = 18.1 min. LCMS-ESI:
average mass calcd [M+3H]3+: 1020.5; found: 1020.3.
2008-2016.
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Synthetic antibody mimic (33) CTV Scaffold derivative
32·3TFA (1.4 mg, 0.41 µmol), cyclic peptide 29 (0.7 mg,

Fraley, K. A.; Bingcang, A. L.; Latus, L. J.; Pommier, Y.; Burke,
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Supplementary Material
Supplementary material is included