Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries

Article abstract of DOI:10.1039/c3ob27470e

A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(i) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC₅₀ value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

Full text of DOI:10.1039/c3ob27470e

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This is an Accepted Manuscript, which has been through the RSC Publishing peer
Organic &
review process and has been accepted for publication.
Biomolecular
Accepted Manuscripts are published online shortly after acceptance, which is prior
to technical editing, formatting and proof reading. This free service from RSC
Publishing allows authors to make their results available to the community, in
citable form, before publication of the edited article. This Accepted Manuscript will
be replaced by the edited and formatted Advance Article as soon as this is available.
Chemistry
www.rsc.org/obc
Volume 8 | Number 3 | 7 February 2010 | Pages 481–716
To cite this manuscript please use its permanent Digital Object Identifier (DOI®),
which is identical for all formats of publication.
More information about Accepted Manuscripts can be found in the
Information for Authors.
Please note that technical editing may introduce minor changes to the text and/or
graphics contained in the manuscript submitted by the author(s) which may alter
content, and that the standard Terms & Conditions and the ethical guidelines
that apply to the journal are still applicable. In no event shall the RSC be held
responsible for any errors or omissions in these Accepted Manuscript manuscripts or
any consequences arising from the use of any information contained in them.
ISSN 1477-0520
PERSPECTIVE
FULL PAPER
Laurel K. Mydock and
AlexeiV. Demchenko
Shuji Ikeda et al.
Hybridization-sensitive fluorescent
DNA probe with self-avoidance ability
Mechanism of chemical O-glycosylation:
from early studies to recent discoveries
1477-0520(2010)8:3;1-H
www.rsc.org/obc
Registered Charity Number 207890

Page 1 of 10
Organic & Biomolecular Chemistry
View Article Online
51x17mm (300 x 300 DPI)

Organic & Biomolecular Chemistry
Page 2 of 10
Dynamic Article Links ►
Journal Name
View Article Online
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
ARTICLE TYPE
Scaffold optimization in discontinuous epitope containing protein
mimics of gp120 using smart libraries
Gwenn E. Mulder, H (Linda). C. Quarles van Ufford, Jeroen van Ameijde, Arwin J. Brouwer, John A.
W. Kruijtzer and Rob M. J. Liskamp*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A diversity of protein surface discontinuous epitopes mimics is now rapidly and efficiently accessible.
Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide
range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major
10 challenge. Using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general
and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three
different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a
selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the
cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120
15 mimic with an IC50 value of 1.7 µM. The approach described here provides a rapid and highly
reproducible access to clean, smart libraries of very complex bio-molecular construct representing protein
mimics for use as synthetic vaccines and beyond.
Introduction
One of the great challenges of this decade in drug discovery is
20 development of protein mimics to be obtained by
chemosynthesis, biosynthesis or a combination of both.1 In order
to be successful in mimicry of proteins and generate molecular
constructs that can interact with for example a partner protein, in
many cases two characteristics of protein mimics will be
25 important. Firstly, binding has to occur to relatively large surface
areas with often relatively flat profiles in many protein-protein
interfaces. Secondly, these surfaces often have a modular
architecture, and consist of multiple epitopes within the protein.2
In our research we are particularly interested in the development
30 of protein mimics containing discontinuous epitopes, that are
capable of interacting with these modular protein surfaces.
Previously, we have described protein mimics of cystatin B and
the whooping cough protein pertactin, which contained different
Figure 1: Detailed view of the discontinuous CD4 binding site of HIV
linear peptide sequences as mimics of the corresponding
35 discontinuous epitopes. However, peptide cyclization is likely to
improve the affinity of discontinuous epitope mimics.3-9 In
addition, cyclic peptides are chemically and enzymatically more
stable than their linear counterparts.5,6 Therefore, we have
focused on the introduction of cyclic peptides onto suitable
40 scaffold molecules.10,11 Although we have developed a method
for the sequential introduction of discontinuous epitopes onto a
suitable (TAC) scaffold, the question arises then whether the
ideal protein mimic has been synthesized. Important issues to be
addressed include: (i) are the cyclic peptides, which mimic the
45 epitope loops, of the right size? (ii) is the arrangement of the
cyclic peptides in the mimic an adequate representation of the
gp120, and schematic representation of a possible method to mimic this
binding site using cyclic peptides on a molecular scaffold.
situation in the protein complex? (iii) does the scaffold provide
the proper flexibility or rigidity? (iv) does the scaffold provide a
50 good platform for the relative involvement of the different loops
of the discontinuous epitope? Some of these issues were
addressed in our recent method for the preparation of “smart”
libraries.11 Libraries of scaffolded cyclic peptides mimicking the
discontinuous epitopes of HIV-gp120 were synthesized, resulting
55 in protein mimics that can interact with the CD4 receptor (Figure
1). From this library, the best member can be selected followed
by re-synthesis.3
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

Page 3 of 10
Organic & Biomolecular Chemistry
peptide segments may be most effective for inclusion in a
synthetic discontinuous epitope mimic. From the very start of this
View Article Online
10 project, we realized that in view of size and complexity,
prediction and/or modelling of the molecular constructs aimed at,
is very difficult. Therefore, generation of collections or libraries
is an attractive avenue, if not an essential approach, to uncover
potentially promising molecules.
15 Recently, we have described a method for conjugation of up to
three different cyclic peptides onto a TriAzaCyclophane (TAC)
scaffold using the Cu(I)-catalyzed azide-alkyne cycloaddtion
(CuAAC) to afford clean and reproducible (“smart”) libraries.11
The resulting protein mimics were evaluated in a competitive
20 ELISA with HIV gp120 at micromolar concentrations. The most
potent gp120 mimic was capable of competing with HIV-gp120
for CD-4-binding with an IC50-value of 16.8 µM. In the research
presented here, we have selected three alternative scaffold
molecules that all differ in flexibility/rigidity. These scaffold
25 molecules were all used for the preparation of smart libraries and
investigation of the ability of the resulting protein mimics to
compete with HIV-gp120 for CD4-binding. Finally, cyclic
peptides on the scaffold molecule that resulted in the most potent
Figure 2: Preorganization of epitopes by different scaffold molecules
In the research presented here, we report results on the
investigation of the influence of the scaffold. In our research we
aim at the development of protein mimic of gp120 to be used to
prevent (a) attachment of HIV-gp120 to the CD4-receptors of
cells and (b) using this mimic as a synthetic vaccine.12,13 Based
on the X-ray crystal structure of the gp120-CD4 protein
complex,14 we10 and others15 have determined which interacting
5
Scheme 1: Synthesis of cyclic azidopeptides and click chemistry to form gp120 discontinuous epitope mimics
2 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Organic & Biomolecular Chemistry
Page 4 of 10
protein mimics were varied, thereby obtaining an additional
normal silica column chromatography. This was highly
improvement of activity.
advantageous compared to preparative HPLC as a purification
method, since yields were much higher and larger quantities
View Article Online
60 could be purified in a single run. The purity of the cyclic peptides
was assessed after removal of the side chain protecting groups,
and was very good (>95%) for two of the three peptides. The
largest cyclic peptide 4b and elongated version of peptide 4c (see
experimental section) were purified by preparative HPLC to
Results and discussion
Scaffold selection
5
Three additional scaffold molecules were selected for synthesis of
protein mimics containing discontinuous epitopes of HIV gp120.
Since up to three different cyclic peptides have to be introduced 65 ensure a high quality precursor for subsequent attachment to the
onto the scaffold molecule, all selected scaffolds were trivalent
and equipped with alkyne functionalities for conjugation to the
scaffolds.
10 cyclic peptides using CuAAC. As is schematically indicated in
figure 2 by arrows indicating the relative movements of the
peptide loops, their orientation and extent of pre-organization is
clearly dependent on the character of the scaffold. Alkyne
containing triamine 5 may be considered as the flexible/open
15 analogue of the TAC-scaffold, and should reveal the
(dis)advantage of a more organized (TAC)scaffold. Alkyne
containing dendrimer building block16 7 allows in principle
organization of the peptide loops roughly in one plane, which is
not the case with the TAC or triamine scaffold. Alkyne-
20 functionalised CycloTriVeratrylene (CTV) 9 will furnish the
most pre-organized system, which is indicative from the use of
CTV for the organization of peptide chains into a triple helix
mimic and parallel organization of other ligands.17
Library synthesis of the discontinuous epitope containing
protein mimics
70 Libraries of discontinuous epitope containing protein mimics 6, 8
and 10 were synthesized using CuAAC reactions. Scaffold
molecules 5, 7 or 9 were incubated with equimolar mixtures of
cyclic azidopeptides 4a-c and subjected to CuAAC following the
procedure that was described previously for conjugation of cyclic
75 peptides to the TAC scaffold 11.11 Reaction conditions involved
the use of CuSO4 and sodium ascorbate in the presence of
TBTA.21,22 Full conversion of starting materials was observed for
reactions on all three scaffold molecules after only 20 minutes
reaction time in the microwave, and very clean reaction mixtures
80 containing only desired products were obtained, as was
confirmed with LC-MS. This clearly demonstrates the general
applicability and reliability of this method for generation of smart
libraries of discontinuous epitope mimics. The resulting reaction
mixtures were purified with preparative HPLC to isolate the
25 Epitope selection and synthesis of cyclic peptide loops
containing an azido handle
The HIV gp120 epitopes that were selected for this and previous 85 individual pure epitope mimics.11 In this way, in a single click
studies9-11,15,18
−
365SGGDPEI371
,
425NMWQEV430 and
experiment followed by one HPLC run, a library containing
practically all combinations of epitope mimics was obtained.
454LTRDGGN460 − represent conserved regions of the otherwise
30 highly variable gp120. In addition, these sequences contain the
primary contact residues of gp120 (D368, E370, W427, and
D457) for its interaction with CD4, as was determined from the
crystal structure of core gp120 in complex with an extracellular
two-domain fragment of CD4.14,19 Although the interacting loops
35 are located relatively far away in the primary structure of the
protein, the crystal structure clearly shows the proximity of the
selected epitopes in the folded, receptor-bound state. These
epitopes are therefore suitable candidates for preparation of the
corresponding peptide loops followed by their attachment to a
40 scaffold in order to mimic the protein binding site.
Evaluation of binding affinity of the gp120 protein mimics
90 To evaluate the relative capacity of the scaffold molecules for
positioning and pre-organizing the discontinuous epitopes of HIV
gp120, the three different libraries of protein mimics were
screened for activity in a competitive ELISA experiment. CD4-
coated plates were used, and the ability of gp120 mimics to
95 compete with recombinant monomeric gp120(IIIB) for binding
to CD4 was assessed. The amounts of captured gp120 were
detected by peroxidase-conjugated murine anti-gp120 MAb. The
maximum signal was determined by detection of only gp120
without addition of any protein mimic, and the background
100 absorption was determined by performing the assay without
addition of gp120 or a protein mimic. The latter values were
subtracted from the competition experiment values and the
activity was expressed as a percentage of the maximum attainable
effect (figure 3).
105 In addition to evaluation of the library members of the
discontinuous epitope protein mimics, binding of the individual
unscaffolded cyclic peptides was investigated. At the highest
concentration (1 mg/mL) tested, no inhibition of gp120-binding
was observed by any of the separate cyclic peptides. This clearly
110 demonstrated the necessity of the presence of at least two peptide
loops in one molecular construct. Moreover, an unscaffolded
equimolar mixture of the three peptide loops did not show any
binding to CD4, which unambiguously confirmed the
Synthesis of cyclic azidopeptides containing these epitopes was
performed on 2-chlorotrityl chloride resin, since peptides can be
cleaved from this resin without affecting their side chain
protecting groups. Moreover, cleavage of peptides from this resin
45 yields
a peptide with a C-terminal carboxylic acid that
subsequently can be used for head-to-tail cyclization. To avoid
racemization during the cyclization reaction, glycine was chosen
as the C-terminal amino acid for preparation of each peptide loop.
The azide handle for CuAAC was introduced by incorporation of
50 Fmoc-azidolysine.20
After completion of their synthesis (Scheme 1) and removal of
the N-terminal Fmoc-group, peptides were cleaved from the resin
and cyclized in solution using BOP as a coupling reagent
(Scheme 1). Cyclization reactions were performed in high
55 dilution and monitored by analytical HPLC. After aqueous
workup, the protected cyclic peptides could be purified using
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

Page 5 of 10
Organic & Biomolecular Chemistry
Dynamic Article Links ►
Journal Name
View Article Online
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
ARTICLE TYPE
Figure 3: Inhibition of gp120 binding of gp120 discontinuous epitope protein mimics in competitive ELISA. Libraries of protein mimics based on four
different scaffold molecules were evaluated. Left: First screening of two newly synthesized libraries 6 and 8 at 250 µg/mL and comparison with results
of the previously published TAC-scaffolded library at a concentration of 1 mg/mL. Right: Evaluation of all newly synthesized libraries 6, 8 and 10, at a
concentration of 125 µg/mL. The results of the TAC-based library 12 at 1 mg/mL were included as a reference.
1.0
1-1-3*
1-1-3
0.5
0.0
0.0
0.5
1.0
1.5
2.0
2.5
Log (concentration ug/mL)
Figure 4. Results of competitive ELISA for all CTV-based mimics. Normalized values for inhibition of gp120-binding to CD4 are displayed, where
0 is full inhibition and 1 is no inhibition. Left: All obtained mimics were screened at four concentrations in twofold serial dilutions, starting at 125
µg/mL. Middle: The four most potent mimics were tested in higher dilutions in order to determine IC50 values. Right: Comparison of CTV-
scaffolded mimics carrying 'original' and elongated cyclic peptides. IC50 value of [1-1-3*] 1.7 µM ± 0.48.
individual library member. Results of this experiment are shown
20 in figure 4 and IC50 values of the most potent protein mimics are
displayed in table 1. The experiment showed that the [1-1-3]-
construct was most potent protein mimic in binding to CD4 with
an IC50 value of 2.2 µM, which corresponds to an almost tenfold
increased activity compared to the most potent TAC-scaffolded
25 mimic.11 Also, this most potent CTV-scaffolded mimic carries a
different combination of cyclic peptides, that is [1-1-3] than the
most active TAC-scaffolded protein mimic, that is [2-2-3]. This
shows that not only the character, that is the nature of the amino
acid residues in the peptide loops is important, but also their
30 orientation in space is of crucial importance and has to be taken
into consideration when designing an optimal discontinuous
epitope protein mimic.
Table 1: IC50-values and mass values found for the most active
CTVscaffolded mimics
Loop
IC50 (µM)
[M+H]+
[M+H]+found
combination
calculated
1-1-3
1-3-3
1-2-3
2-3-3
2.20 ± 0.35
7.49 ± 1.42
6.20 ± 1.72
3.39 ± 0.54
3438.6210
3580.6728
3881.8453
4023.8970
3438.39
3580.33
3881.01
4022.43
requirement of a molecular scaffold for assembly of the different
epitope loops leading to an effective discontinuous epitope
containing protein mimic. For evaluation of the activity of the
libraries, libraries 6, 8, and 10 of gp120 discontinuous epitope
protein mimics, were screened at 125 and 250 µg/mL in an initial
ELISA experiment. This first experiment revealed significant
10 inhibitory activity differences between the libraries based on
different scaffold molecules (Figure 2). At a concentration of 250
µg/mL most protein mimics were able to inhibit binding of gp120
by more than 60%. At a concentration of 125 µg/mL however, the
activity of especially the triamine-scaffolded protein mimics
15 dropped, while all CTV-scaffolded protein mimics still showed
almost complete inhibition at this concentration.
5
Optimization of cyclic peptides
35 It was decided to investigate if the peptide loops of the most
successful CTV-protein mimic could be further optimized leading
to an even better discontinuous epitope mimic.
Perusal of the crystal structure of the gp120-CD4 complex
showed that epitopes 454LTRDGGN460 (loop 1, Figure 5) and
40 365SGGDPEI371 (loop 3, Figure 5) are part of larger loop-like
structures. This suggested that enlargement of the cyclic peptides
and conjugation to the CTV-scaffold leading to additional gp120
Based on these results, the library of CTV-scaffolded protein
mimics was tested in higher dilutions to identify the most active
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 4

Organic & Biomolecular Chemistry
Page 6 of 10
different trivalent scaffold molecules, resulting in clean
collections (6, 8 and 10) of HIV-gp120 discontinuous epitope
View Article Online
protein mimics, in order to investigate the necessity of orientation
and extent of pre-organization. Screening for their ability to
40 compete with HIV-gp120 for binding to CD4 led to CTV-
scaffold protein mimic [1-1-3] having an IC50 value of 2.2 µM.
An additional improvement of this activity to an IC50 value of 1.7
µM could be reached for [1-1-3*] by enlargement of peptide loop
3 to 3*. Results were very reproducible and the synthetic
45 approach is reliable and broadly applicable, since it allows
variation of each of components of the protein mimics. In our
opinion, this demonstrates the potential of this approach for the
generation of protein mimics of other proteins containing
discontinuous binding sites, possibly ultimately leading to the
50 generation of synthetic antibodies.
Experimental section
General information
Figure 4: Top: Crystal structure of HIV gp120. Epitopes for mimicry
indicated in blue (loop 1), pink (loop 2), and green (loop 3). The amino
acids that were selected for extension of the loops are indicated in red.
Bottom: CTV scaffold carrying initial (left) and extended (right) loops.
Unless stated otherwise, chemicals were obtained from
commercial sources and used without further purification. Peptide
55 grade DiPEA, CH2Cl2, NMP, TFA and HPLC grade solvents
were purchased from Biosolve B.V. (Valkenswaard, The
Netherlands). Fmoc-protected amino acids were purchased from
GL Biochem Ltd. (Shanghai, China). Sidechain protecting groups
for amino acids were as follows: Ser(tBu), Asp(OtBu),
60 Glu(OtBu), Thr(tBu), Asn(Trt), Trp(Boc), Gln(Trt), Lys(Boc),
Arg(Pbf).
2-Chlorotrityl chloride resin (100-200 mesh, 1% DVB, 1.0-1.6
mmol/g) was purchased from Iris Biotech GmbH and was used
for synthesis of the cyclic peptides. Solid phase peptide synthesis
65 was carried out in plastic syringes with a polyethylene frit (20
µm) obtained from Screening Devices B.V. The resin loading was
determined by measuring the UV absorbance of the piperidine-
dibenzofulvene adduct (λmax 300 nm).23
protein mimics (Figure 5) might lead to an improved activity of
these compounds. Based on the crystal structure the epitope
454LTRDGGN460 (loop 1) was extended at its C-terminus with
five amino acids, resulting in 454LTRDGGNSNNEI465. Loop 3 or
365SGGDPEI371 was extended with two amino acids on its N-
terminus, giving 363QSSGGDPEI371 (Figure 5). The modified
cyclic peptides were synthesized according to the procedure
described above and depicted in scheme 1, starting with a C-
terminal glycine residue incorporation of an azidolysine residue
10 for attachment to the scaffold molecule. Since we specifically
wanted to investigate the influence of the enlarged loops 1 and 3,
that is loops 1* and loops 3*, only these were introduced onto the
CTV-scaffold 9 and a library of discontinuous epitope protein
mimics was generated by CuAAC. In a first experiment, a
15 combination of loop 1 and loop 3* was conjugated to CTV.
Subsequently, a combination of loop 1* and loop 3 was attached
to the CTV scaffold. Finally, both enlarged loops 1* and 3* were
introduced. The resulting collections of discontinuous epitope
mimics, together with the original CTV-scaffolded protein
20 mimics carrying combinations of loop 1 and 3, were evaluated
with competitive ELISA. This experiment revealed that
elongation of loop 1 did not affect the binding of the [1*-1*-3] or
[1*-3-3] mimics in any way.
5
All components used in gp120-capture ELISA experiments were
70 purchased from ImmunoDiagnostics, Inc. (Woburn, MA).
Reactions were performed at room temperature. Solution phase
reactions were monitored by TLC analysis and Rf-values were
determined on Merck pre-coated silica gel 60 F-254 (0.25 mm)
plates. Spots were visualized with UV-light, and by heating plates
75 dipped
in
ninhydrine,
Cl2/N,N,N,N’-tetramethyl-4,4’-
diaminodiphenylmethane (TDM)24 or a KMnO4 solution. Column
chromatography was performed using Silica-P Flash silica gel (60
Å, particle size 40-63 µm; Silicycle). Microwave reactions were
performed in a Biotage Initiator (300W) reactor in sealed vessels
80 suitable for reaction volumes of 0.5-2 mL.
Substitution of loop 3 with its enlarged version however,
25 modestly improved the activity of the resulting CTV-scaffolded
protein mimics (Figure 3, rightside graph). The clean synthesis of
these discontinuous epitope protein mimics, now containing
further modified loops, demonstrates the potential of this
approach for screening and optimizing the activity of protein
30 mimics.
1H NMR experiments were conducted on a 300 MHz Varian G-
300 spectrometer, and chemical shifts are given in ppm (δ)
relative to TMS (0.00 ppm). 13C NMR spectra were recorded at
75 MHz at a Varian G-300 spectrometer and chemical shifts are
85 given in ppm (δ) relative to CDCl3 (77 ppm).
Analytical HPLC was accomplished on a Shimadzu-10Avp
(Class VP) system using UV-detector operating at 214 and 254
nm. The mobile phase was 0,1% trifluoroacetic acid (TFA) in
CH3CN/H2O 5:95 (buffer A) and 0,1% trifluoroacetic acid (TFA)
90 in CH3CN/H2O 95:5 (buffer B). For analysis of protected (cyclic)
peptides the mobile phase was 0,1% trifluoroacetic acid (TFA) in
Conclusions
In summary, we have developed
a rapid, versatile and
reproducible method for the generation of libraries of
discontinuous epitope protein mimics. CuAAc of a mixture of up
35 to three cyclic peptides was successfully applied on three
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

Page 7 of 10
Organic & Biomolecular Chemistry
CH3CN/H2O 20:80 (buffer A) and 0,1% trifluoroacetic acid
reagents HBTU and HOBt and 4 equivalents of DiPEA. The resin
(TFA) in CH3CN/iPrOH/H2O 50:45:5 (buffer B). A Phenomenex 60 was washed with NMP (3 × 10 mL, 2 min) and CH2Cl2 (3 × 10
Gemini C18 column (110 Å, 5 µm, 250×4.60 mm) was used at a
flow rate of 1 mL min-1 using a standard protocol: 100% buffer A
for 1 min, then a linear gradient of buffer B (0-100% in 30 min).
Purification of the peptide-containing compounds was performed
mL, 2 min) after every coupling step. The couplinVgiewsteAprtsicleanOdnline
deprotection steps were monitored using the Kaiser test or
bromophenol blue test in case of secondary amines.25
5
After coupling of the final Fmoc-amino acid, acetylation was
on a Prep LCMS-QP8000α HPLC system (Shimadzu) using a 65 carried out to protect unreacted residues, and prevent them from
Phenomenex Gemini C18 column (10 µm, 110 Å, 250×20 mm) at
participation in the cyclization reaction. The resin was treated
with 10 mL capping solution containing Ac2O (4.72 mL, 42.7
mmol), DiPEA (2.18 mL, 22.8 mmol) and HOBt (0.23 g, 1.7
mmol) in NMP (100 mL) for 2 × 15 min, followed by washing
70 steps with NMP (3 × 10 mL, 2 min), CH2Cl2 (3 × 10 mL, 2 min)
and NMP (3 × 10 mL, 2 min). Finally, the N-terminal Fmoc-
group was removed using the conditions mentioned earlier, to
obtain the linear sidechain-protected azidopeptides 2a-c.
a flow rate of 12.5 mL min-1 using a standard protocol: 100%
10 buffer A for 5 min followed by a linear gradient of buffer B (0-
55% in 100 min) using the same buffers as described for
analytical HPLC.
Electrospray ionization mass spectrometry (ESI-MS) was
performed on a Shimadzu LCMS-QP8000 single quadrupole
15 bench-top mass spectrometer operating in a positive ionization
mode or a Thermo- Finnigan LCQ Deca XP Max ion trap mass
Cleavage of peptides from solid support and cyclization:
spectrometer. Analytical LC-MS was preformed on Thermo- 75 The side chain-protected linear azidopeptides 2a-c were cleaved
Finnigan LCQ Deca XP Max. MALDI-TOF-MS spectra were
recorded on a Kratos Analytical (Shimadzu) AXIMA CFR mass
20 spectrometer using α-cyano-4-hydroxycinnamic acid (CHCA) or
sinapic acid as a matrix and human ACTH (18-39) or bovine
from the resin using a solution of TFE in CH2Cl2 (2:8 (v/v)), 10
mL for each gram of resin, 1 hour reaction time. The liquid was
filtered from the resin beads and concentrated in vacuo. The
residue 3a, 3b or 3c was dissolved in CH2Cl2 or DMF, depending
insulin oxidized
B chain as references. High resolution 80 on the solubility of the peptides 3a-c, with a final peptide
electrospray ionization (ESI) mass spectra were measured on a
Micromass LCT mass spectrometer calibrated with CsI. All
25 reported mass values are monoisotopic.
concentration of 1mg/mL. To this solution 1.2 equivalents of
BOP and 2.4 equivalents of DiPEA were added and the reaction
mixture was stirred at room temperature. Progress of the
cyclization reaction was monitored with analytical HPLC.
The microtiterplate reader used in the ELISA experiments was a
BioTek µQuant (Beun de Ronde, Abcoude, The Netherlands). 85 Depending on the sequence and the solvent, reaction times varied
Software used for data analysis was the Full Mode-KC4 version
3.4.
from 16 hours to three days. When full conversion was observed
with analytical HPLC, the reaction mixture was concentrated in
vacuo and the residue 4a, 4b or 4c was dissolved in EtOAc. The
organic phase was washed with a 1N KHSO4 solution, water, 5%
90 NaHCO3 solution and brine, dried over Na2SO4 and concentrated
in vacuo.
30
General procedure for solid phase peptide synthesis
Synthesis of all cyclic peptides was performed on 2-chlorotrityl
chloride resin using conventional Fmoc/tBu chemistry.23 Solvents
used for synthesis were dried on molsieves (4Å), 10 mL solvent
Side-chain deprotection: The purified cyclic peptides 4a-c
(vide infra) were treated with a solution (10 mL per 0.25 mmol)
of TFA/TIS/H2O (95:2.5:2.5 (v/v/v)). The reaction mixture was
95 stirred during 3 hours, followed by precipitation of the peptides
from MTBE/hexane 1:1 (v/v) at -20◦C. The precipitates were
35 per gram of resin was used.
Immobilization of the first monomer: Resin (1 g, 1.0-1.6
mmol) was swelled in CH2Cl2 (3 × 2 min). To the resin, a solution
of 2 mmol Fmoc-amino acid and 5 mmol DiPEA dissolved in
CH2Cl2 was added and the mixture was shaken during 1 hour at
40 room temperature. After that, the reaction vessel was drained and
the resin was washed with DMF (2 × 2 min), followed by
t
dissolved in BuOH/H2O 1:1 (v/v) and lyophilized. The purity of
the peptides was analyzed with analytical HPLC and the peptides
were characterized with mass spectrometry.
100 Purification of cyclic peptides 4a-c
treatment (2
× 10 min) with 10 mL of a mixture of
CH2Cl2/MeOH/DiPEA (80:15:5 (v/v/v)) to quench any remaining
reactive 2-chlorotrityl chloride. The resin was washed again with
45 DMF (3× 2 min) and CH2Cl2 (3 × 2 min) and dried in vacuo. The
loading efficiency was determined by photometric quantification
Compound 4a, loop 1: K(N3)LTRDGGNG
The crude protected cyclic peptide was purified by column
chromatography in CH2Cl2/EtOH 95:5. The product was obtained
as a yellow oil. Rf: 0.67 (CH2Cl2/EtOH 9:1).
of the absorbance of the dibenzofulvene-piperidine adduct at 300 105 Synthesis on 0.5 mmol scale. Yield after deprotection of side
nm.
Linear peptide synthesis: The resin was washed with NMP (3
50 × 10 mL per gram of resin, 2 min). The N-terminal Fmoc
protecting group was removed by 20% piperidine in NMP (3 × 10
mL, 8 min) followed by washing steps with NMP (3 × 10 mL, 2 110
min), CH2Cl2 (3 ×10 mL, 2 min) and NMP (3
chains: 106 mg (23%).
[M+H]+ monoisotopic calculated for C36H60N16O13: 925.4599,
[M+2H]2+calculated: 463.2336, HRMS, [M+2H]2+ found:
463.2316, HPLC: Rt= 13.84 min, purity 94.5%.
Compound 4b, loop 2: INMWQEVGKAK(N3)G
× 10 mL, 2 min). The amino acid coupling mixtures were
55 prepared by dissolving 4 equivalents of amino acid, 4 equivalents
of HOBt and HBTU and 8 equivalents of DiPEA in NMP and
The crude protected cyclic peptide was purified with column
chromatography in CH2Cl2/MeOH 95:5. The product was
obtained as a yellow oil. Rf: 0.59 (CH2Cl2/MeOH 9:1). Synthesis
coupled during 60 min. Fmoc-azidolysine was coupled overnight 115 on 0.25 mmol scale. Yield after deprotection of sidechains: 210
using 2 equivalents of amino acid, 2 equivalents of the coupling
mg (61%). HPLC analysis shows some small impurities,
6 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Organic & Biomolecular Chemistry
Page 8 of 10
therefore the product is purified another time with preparative
MeOH/acetone/AcOH 95:5:1 to CH2Cl2/acetone/AcOH 90:10:1
HPLC. Pure product was obtained as a white powder. Yield: 40 60 when the product started eluting. Pure product was obtained as a
mg (12%).
colourless oil that crystallized into a white solid.VYiewielAdrt:ic7le.9O1nlinge
(94%). Rf: 0.35 (CH2Cl2/acetone/AcOH 90:10:1). H-NMR (300
1
[M+H]+ monoisotopic calculated for C60H93N19O16S: 1368.6841,
[M+2H]2+calculated: 684.8457, HRMS [M+2H]2+ found:
684.8446, HPLC: Rt= 17.51 min, purity= 95.3%
5
MHz, CDCl3): δ 1.26-1.32 (m, 2H, Lys-δCH2), 1.46-1.49 (m, 1H,
Lys-γCH2), 1.58-1.65 (m, 1H, Lys-γCH2), 1.65-1.80 (m, 1H, Lys-
65 βCH2), 1.85-2.01 (m, 1H, Lys-βCH2), 3.26-3.30 (t, 2H, Lys-εCH2),
4.20-4.24 (t, 1H, Lys-αCH), 4.42-4.44 (d, 2H, Fmoc-CH2), 4.55
(br.s, 1H, Fmoc-CH), 5.26-5.29 (d, 1H, NH), 7.23-7.43 (m, 4H,
Fmoc-ARCH), 7.57-7.60 (d, 2H, Fmoc-ARCH), 7.75-7.77 (d, 2H,
Fmoc-ARCH)
Compound 4c, loop 3: SGGDPEIVTK(N3)G
The crude protected cyclic peptide was purified with column
10 chromatography using a gradient of CH2Cl2/MeOH 97:3 to 9:1.
The product was obtained as colorless oil. Rf: 0.61
(CH2Cl2/MeOH 9:1)
70 13C-NMR (75 MHz, CDCl3): δ 22.40, 28.32, 31.82, 47.11, 51.02,
53.48, 67.11, 119.99, 125.01, 127.06, 127.74, 141.30, 143.60,
156.06, 176.71
Synthesis on 0.25 mmol scale. Yield after deprotection of side
chains: 56 mg (21%).
15 [M+H]+ monoisotopic calculated for C44H70N14O17: 1067.5116,
[M+2H]2+calculated: 534.2594, HRMS [M+2H]2+ found:
534.2652, HPLC: Rt= 15.52 min, purity= 100%
Synthesis of the scaffold molecules
Linear triamine scaffold 5
75 4-pentynoic acid (2.47 g, 25.15 mmol) was dissolved in 30 mL
DCM. The solution was stirred and cooled with an ice bath. BOP
(11.12 g, 25.15 mmol) and DiPEA (8.56 mL, 50.30 mmol) were
added to the solution, followed by addition of bis(3-
aminopropylamine (1.08 mL, 7.6 mmol). The reaction mixture
80 was allowed to warm up to room temperature and was stirred
overnight, after which the mixture was concentrated in vacuo.
The residue was dissolved in EtOAc and washed three times with
1N KHSO4, with water, three times with 1N NaHCO3 and with
brine. The organic phase was dried over Na2SO4 and concentrated
85 in vacuo.
Cyclic peptide LTRDGGNSNNEIK(N3)G, loop 1*
20 The crude protected cyclic peptide was purified with column
chromatography using a gradient of CH2Cl2/EtOH 95:5 to 9:1.
The product was obtained as colorless oil. Rf: 0.56 (CH2Cl2/EtOH
9:1)
Synthesis was performed on 0.25 mmol scale. Yield after
25 deprotection of side chains: 48 mg (13%).
[M+H]+ monoisotopic calculated for C58H95N23O23: 1482.70,
found 1483.01
HPLC: Rt= 15.33 min, purity= 100%
The product was purified by column chromatography using 3%
MeOH in DCM. When the product started to elute, 10% MeOH
in DCM was used. Pure product 5 was obtained as a yellow oil
that slowly crystallized into a light yellow solid. Yield: 2.71 g
30 Cyclic peptide QSSGGDPEIVTK(N3)G, loop 3*
The crude protected cyclic peptide was purified with column
chromatography in CH2Cl2/MeOH 95:5. The product was
obtained as a yellow oil. Rf: 0.54 (CH2Cl2/MeOH 9:1). Synthesis
on 0.25 mmol scale. Yield after deprotection of side chains: 190
35 mg (42%). HPLC analysis shows some small impurities,
therefore the product is purified another time with preparative
HPLC. Pure product was obtained as a white powder. Yield: 9,8
mg (3%).
1
90 (93%). Rf: 0.61 (DCM/MeOH 9:1). H-NMR (300 MHz, CDCl3,
TMS): δ 1.62-1.68 (m, 2H, CH2CH2CH2), 1.80-1.85 (m, 2H,
CH2CH2CH2), 1.98 (t, 2H, alkyne-H), 2.04 (t, 1H, alkyne-H),
2.39-2.44 (m, 4H, C(O)CH2CH2), 2.50-2.56 (m, 8H, C(O)CH2
and C(O)CH2CH2), 3.16-3.22 (q, 2H, NHCH2), 3.30-3.35 (m, 4H,
95 NHCH2 and NCH2), 3.42 (t, 2H, NCH2), 5.98 (s, 1H, NH), 6.77
(s, 1H, NH)
[M+H]+ monoisotopic calculated for C52H83N17O21: 1282.60,
40 found 1282.49
13C-NMR (75 MHz, CDCl3): δ 14.74, 14.92, 27.41, 29.33, 31.88,
35.44, 35.79, 36.96, 42.48, 45.44, 69.25, 82.94, 171.10, 171.40,
171.59
HPLC: Rt= 14.68 min, purity= 100%
Fmoc-L-ε-azidolysine20
100
Dendrimer building block 7
Fmoc-Lys(Boc)-OH (10 g, 21.32 mmol) was dissolved in 100 mL
45 CH2Cl2. The solution was stirred during 1 hour after which a
white suspension was obtained. The solvents were evaporated
and the residue was dissolved in 100 mL water and 200 mL
MeOH. CuSO4 (53.2 mg, 0.21 mmol) was added and the pH of
the solution was adjusted to 8.5 with K2CO3. Imidazole sulfonyl
50 azide HCl salt (5.36 g, 25.59 mmol, synthesized according to the
procedure by Stick et al.5) was added and the pH of the reaction
mixture was maintained at 8.5 by addition of K2CO3. The clear
blue solution was stirred overnight, after which MeOH was
evaporated and the aqueous solution was acidified to pH 2 by
55 addition of 2N HCl. The aqueous phase was extracted four times
with 100 mL CH2Cl2 and the combined CH2Cl2 fractions were
dried over Na2SO4 and concentrated in vacuo. The resulting
yellow oil was purified with column chromatography in
Dendrimer 7 was synthesized starting from its hydrochloric acid
salt precursor, which was prepared following a previously
described procedure.26
105 To a suspension of this hydrochloride salt precursor (930 mg, 2.0
mmol), 4-pentynoic acid (588 g, 6.0 mmol) and BOP (2.93 g, 6.6
mmol) in CH2Cl2 (20 mL) was added DiPEA (3.39 mL, 19.5
mmol). The mixture was stirred at room temperature for 16h and
concentrated in vacuo. The residue was dissolved in ethyl acetate
110 and washed with 1 M KHSO4 (twice), 1 M NaOH (twice) and
brine. The organic layer was dried over Na2SO4 and concentrated
in vacuo to a volume of approximately 20 mL. Precipitation after
addition of hexanes, followed by filtration afforded the product as
a white solid (951 mg, 80%). Rf = 0.44 (MeOH/CH2Cl2, 5/95).
1
115 Mp = 133 °C. H NMR (500 MHz, CDCl3): δ = 2.04 (m, 9H, 3×
OCH2CH2, 3× ≡-H), 2.43 (m, 6H, 3× C(O)CH2), 2.52 (m, 6H, 3×
Journal Name, [year], [vol], 00–00 | 7
This journal is © The Royal Society of Chemistry [year]

Page 9 of 10
Organic & Biomolecular Chemistry
C(O)CH2CH2), 3.50 (m, 6H, 3× NHCH2), 3.89 (s, 3H, OCH3),
solution (4.49 µmol, 1.12 mg) and 100 µL of the sodium
4.11 (m, 6H, 3× OCH2), 6.69, 6.85 (2bt, 3H, 3× NH), 7.25 (s, 2H, 60 ascorbate stock solution (8.99 µmol, 1.78 mg) were added to the
View Article Online
Ph-C2,6-H). 13C NMR (500 MHz, CDCl3): δ = 14.8, 14.9
(C(O)CH2CH2), 29.0, 30.1 (OCH2CH2), 35.1, 35.2 (C(O)CH2),
37.0, 37.1 (NHCH2), 52.2 (OCH3), 66.8, 71.7 (OCH2), 69.1, 69.2
(C≡C-H), 83.0 (C≡C-H), 107.7 (Ph-C2,6), 125.3 (Ph-C1), 141.0
solution, followed by addition the TBTA solution (1.10 µmol,
0.60 mg). The peptide solutions were combined and added to the
reaction mixture. Depending on the amount of DMF that was
used to dissolve the peptides, H2O was added to obtain a 3:2
5
(Ph-C4), 152.2 (Ph-C3,5), 166.4 (CO2Me), 171.3, 171.4 (NHC=O). 65 DMF/H2O ratio and 1-1.5 mL total volume. The microwave
[M+H]+ monoisotopic calculated for C32H41N3O8: 596.2966,
found: 596.45.
vessel was sealed and allowed to react in the microwave at 80°C
during 25 minutes.
10
After reaction, a sample was taken from the reaction mixture for
LC-MS analysis. Based on this analysis, the individual
70 components were purified with preparative HPLC.
CTV derivative 9 was prepared in two steps starting from
vanillyl alcohol, by alkylation with propargyl bromide followed
by conversion into the CTV-derivative.
1-O-propargyl-vanillylalcohol: Vanillyl alcohol (3.08 g, 20.0
HIV-1 gp120 capture ELISA
15 mmol) and propargyl bromide (80 % in toluene, 2.16 mL, 20
mmol) was dissolved in acetone (40 mL). After addition of
K2CO3 (4.15 g, 30 mmol) the reaction mixture was stirred at
reflux temperature overnight. Evaporation of the solvent was
followed by addition of water (75 mL) and CH2Cl2 (75 mL),
20 separation of the layers, drying of the organic phase (Na2SO4)
and evaporation of the solvent. The alkylated vanillyl alcohol was
obtained in 93% (3.57 g) as a white solid and used without
further purification.
Recombinant HIV-1IIIB gp120 (rgp120) protein capture ELISA
was performed according to the manufacturer’s instructions
75 (ImmunoDiagnostics, Inc., Woburn, MA). Firstly, various
concentrations of free ligand rgp120 in sample buffer (up to 2
µg/ml, in a 2-fold dilution series) were added to a CD4-coated
plate in the absence of test compounds. After a 60-min incubation
at room temperature, the amounts of captured rgp120 were
80 detected by peroxidase-conjugated murine anti-gp120 MAb.29
We determined that optimal OD450-values were obtained when a
final rgp120 concentration of 1 µg/ml was used. When optimal
concentrations were determined, competition experiments were
performed. Test compounds diluted in sample buffer (50 µL 0,1%
1H-NMR (300 MHz, CDCl3) d 1.73 (br. s, 1H, OH), 2.50 (t, 1H,
25 3J 2.3 Hz, propargyl-CH), 3.88 (s, 3H, CH3), 4.63 (2H, CH2OH),
3
4.76 (d, 2H, J 2.3 Hz, propargyl-CH2), 6.88 (m, 1H, ArH), 6.95
(d, 1H, 3J 2.0 Hz, ArH), 7.01 (d, 1H, 3J 8.3Hz, ArH)
13C-NMR (75.5 MHz, CDCl3) d 55.7 (OCH3), 56.7 (propargyl- 85 BSA in PBS) and 2% DMSO were added to the CD4-coated
CH), 65.0 (CH2OH), 75.7 (propargyl-CH2), 78.5 (propargyl-Cq),
30 110.8, 114.3, 119.0, 135.0, 146.1, 149.7 (ArC)
plate, immediately followed by addition of 50 µL 2 µg/ml rgp120
(final concentration 1 µg/mL). After 4 hours incubation at room
temperature, the plate was washed with wash buffer (0,1% Tween
20 in PBS) followed by incubation with peroxidase-conjugated
90 murine anti-gp120 MAb to detect the amounts of captured
rgp120. Any unbound material was washed away using wash
buffer, and plates were developed by adding 100 µL/well
substrate solution (0,1 mg/mL TMB in 0.1 N NaOAc buffer pH
5.5, containing 0.003% H2O2). The reaction was stopped by
(O-propargyl)3-CTV 9: After dissolving 1-O-propargyl-
vanillylalcohol (3.57 g, 18.6 mmol) in methanol (20 mL), the
o
reaction mixture was cooled to 0 C and perchloric acid (11.0
mL) was added dropwise and then stirred overnight at room
35 temperature. CH2Cl2 (100 mL) was added and the crude material
was washed with 1M NaOH (aq) until the organic layer was no
longer acidic. After drying on Na2SO4 and evaporation of the
solvents, the crude material was triturated with Et2O, yielding 2 95 adding 100 µL 4N sulphuric acid. Absorbances (ODs) were read
(2.01 g, 21%) as an off-white solid.
at 450 nm using a microtiterplate reader. All assays were
performed in duplicate and were independently repeated at least
two times.
40 1H-NMR (300 MHz, d6-DMSO) d 3.42 (3H, s, propargyl-CH),
3.50 (3H, d, 3J 13.5 Hz, Ar-CHH’), 3.74 (9H, s, OCH3), 4.71 (9H,
m, Ar-CHH’ + propargyl-CH2), 7.11 (6H, 2 x s, ArH).
13C-NMR (75.5 MHz, d6-DMSO) d 35.4 (Ar-CH2), 56.0 (OCH3),
56.3 (propargyl-CH), 78.0 (propargyl-CH2), 79.9 (propargyl-Cq),
45 114.1, 116.1, 131.8, 133.3, 145.1, 148.0 (ArC).
100 Acknowledgements
This research was (partly) financed by Chemical Sciences of The
Netherlands Organization for Scientific Research (NWO).
General procedure for scaffold conjugation of cyclic peptides
by optimized CuAAc.27,28
10-fold stock solutions of CuSO4 (44.9 µmol, 11.2 mg in 1 mL
50 H2O) and sodium ascorbate (89.9 µmol, 17.8 mg in 1 mL H2O)
were prepared. TBTA21,22 was dissolved in 100 µL DMF.
Peptides were dissolved individually in the smallest possible
amount (100-500 µL) of DMF (4Å).
Solutions of three peptides in DMF were prepared: loop 1
55 (compound 4a, 7.49 µmol, 6.93 mg), loop 2 (compound 4b, 7.49
µmol, 10.25 mg) and loop 3 (compound 4c, 7.49 µmol, 7.99 mg).
Scaffold 5, 7 or 9 (7.49 µmol) was added to the microwave vessel
and dissolved in 100 µL DMF (4Å). 100 µL of the CuSO4 stock
Notes and references
105 Medicinal Chemistry & Chemical Biology, Utrecht Institute of
Pharmaceutical Sciences, Faculty of Science, Utrecht, The Netherlands.
Fax: 31 30 2536655; Tel: 31 30 2537307; E-mail: r.m.j.liskamp@uu.nl
† Electronic Supplementary Information (ESI) available: HPLC and MS
data of the used cyclic peptides and all used scaffolded compounds (6, 8,
110 10 and TAC-derived). See DOI: 10.1039/b000000x/.
1.
H. Yin and A. D. Hamilton, Angew Chem Int Ed Engl, 2005,
44, 4130–4163.
8 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Organic & Biomolecular Chemistry
Page 10 of 10
2.
3.
J. A. Robinson, S. DeMarco, F. Gombert, K. Moehle, and D.
Obrecht, Drug Discovery Today, 2008, 13, 944–951.
C. Heinis, T. Rutherford, S. Freund, and G. Winter, Nat Chem
Biol, 2009, 5, 502–507.
View Article Online
5 4.
5.
A. Angelini, L. Cendron, S. Chen, J. Touati, G. Winter, G.
Zanotti, and C. Heinis, ACS Chem. Biol., 2012, 7, 817–821.
V. Baeriswyl, H. Rapley, L. Pollaro, C. Stace, D. Teufel, E.
Walker, S. Chen, G. Winter, J. Tite, and C. Heinis,
ChemMedChem, 2012, 7, 1173–1176.
10 6.
S. Chen, J. Morales-Sanfrutos, A. Angelini, B. Cutting, and C.
Heinis, ChemBioChem, 2012, 13, 1032–1038.
J. E. Bock, J. Gavenonis, and J. A. Kritzer, ACS Chem. Biol.,
2012.
7.
8.
15
A. Gokhale, T. K. Weldeghiorghis, V. Taneja, and S. D.
Satyanarayanajois, J. Med. Chem., 2011, 54, 5307–5319.
D. J. Craik, J. E. Swedberg, J. S. Mylne, and M. Cemazar,
Expert Opin. Drug Discov., 2012, 7, 179–194.
C. Chamorro, J. A. W. Kruijtzer, M. Farsaraki, J. Balzarini, and
R. M. J. Liskamp, Chem. Commun., 2009, 821.
G. E. Mulder, J. A. W. Kruijtzer, and R. M. J. Liskamp, Chem.
Commun., 2012, 48, 10007.
P. Gilbert, M. Wang, T. Wrin, C. Petropoulos, M. Gurwith, F.
Sinangil, P. D’Souza, I. R. Rodriguez Chavez, A. DeCamp, M.
Giganti, P. W. Berman, S. G. Self, and D. C. Montefiori, J.
Infect. Dis., 2010, 202, 595–605.
9.
10.
20 11.
12.
25
13.
J. G. Schellinger, L. M. Danan-Leon, J. A. Hoch, A. Kassa, I.
Srivastava, D. Davis, and J. Gervay-Hague, J. Am. Chem. Soc.,
2011, 133, 3230–3233.
14.
30
P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski
and W. A. Hendrickson, Nature, 1998, 393, 648–659.
R. Franke, T. Hirsch, H. Overwin, and J. Eichler, Angew. Chem.
Int. Ed., 2007, 46, 1253–1255.
15.
16.
35 17.
18.
A. J. Brouwer and R. M. J. Liskamp, Eur. J. Org. Chem., 2005,
2005, 487–495.
E. T. Rump, D. T. S. Rijkers, H. W. Hilbers, P. G. de Groot,
and R. M. J. Liskamp, Chemistry, 2002, 8, 4613–4621.
R. Franke, T. Hirsch, and J. Eichler, J. Recept. Signal
Transduct. Res., 2006, 26, 453–460.
19.
P. D Kwong, R. Wyatt, S. Majeed, J. Robinson, R. W. Sweet, J.
Sodroski, and W. A. Hendrickson, Structure, 2000, 8, 1329–
1339.
40
20.
E. D. Goddard-Borger and R. V. Stick, Org. Lett., 2007, 9,
3797–3800.
21.
45
Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless,
and M. G. Finn, J. Am. Chem. Soc., 2003, 125, 3192–3193.
T. R. Chan, R. Hilgraf, K. B. Sharpless, and V. V. Fokin, Org.
Lett., 2004, 6, 2853–2855.
22.
23.
24.
50
G. B. Fields, Int. J. Peptide Protein Res., 1990, 35, 161–214.
E. Von Arx, M. Faupel, and M. Brugger, Journal of
Chromatography A, 1976, 120, 224.
25.
E. Kaiser, R. L. Colescott, C. D. Bossinger, and P. I. Cook,
Analytical Biochemistry, 1970, 34, 595.
26.
55 27.
28.
A. J. Brouwer, S. J. E. Mulders, and R. M. J. Liskamp, Eur. J.
Org. Chem., 2001, 2001, 1903–1915.
C. W. Tornøe, C. Christensen, and M. Meldal, J. Org. Chem.,
2002, 67, 3057–3064.
V. V. Rostovtsev, L. G. Green, V. V. Fokin, and K. B.
Sharpless, Angew. Chem. Int. Ed., 2002, 2596.
Q.-E. Yang, A. G. Stephen, J. W. Adelsberger, P. E. Roberts,
W. Zhu, M. J. Currens, Y. Feng, B. J. Crise, R. J. Gorelick, A.
R. Rein, R. J. Fisher, R. H. Shoemaker, and S. Sei, J. Virology,
2005, 79, 6122–6133.
29.
60
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 9