Condensation of 2-((Alkylthio)(aryl)methylene)malononitrile with 1,2-Aminothiol as a Novel Bioorthogonal Reaction for Site-Specific Protein Modification and Peptide Cyclization

Article abstract of DOI:10.1021/jacs.9b11875

Site-specific modification of peptides and proteins has wide applications in probing and perturbing biological systems. Herein we report that 1,2-aminothiol can react rapidly, specifically and efficiently with 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) under biocompatible conditions. This reaction undergoes a unique mechanism involving thiol-vinyl sulfide exchange, cyclization, and elimination of dicyanomethanide to form 2-aryl-4,5-dihydrothiazole (ADT) as a stable product. An 1,2-aminothiol functionality can be introduced into a peptide or a protein as an N-terminal cysteine or an unnatural amino acid. The bioorthogonality of this reaction was demonstrated by site-specific labeling of not only synthetic peptides and a purified recombinant protein but also proteins on mammalian cells and phages. Unlike other reagents in bioorthogonal reactions, the chemical and physical properties of TAMM can be easily tuned. TAMM can also be applied to generate phage-based ADT-cyclic peptide libraries without reducing phage infectivity. Using this approach, we identified ADT-cyclic peptides with high affinity to different protein targets, providing valuable tools for biological studies and potential therapeutics. Furthermore, the mild reaction conditions of TAMM condensation warrant its use with other bioorthogonal reactions to simultaneously achieve multiple site-specific modifications.

Full text of DOI:10.1021/jacs.9b11875

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Article
Condensation of 2-((alkylthio)(aryl)methylene)malononitrile
with 1,2-aminothiol as a novel bioorthogonal reaction for
site-specific protein modification and peptide cyclization
Xiaoli Zheng, Zhuoru Li, Wei Gao, Xiaoting Meng, Xuefei Li,
Louis Y. P. Luk, Yibing Zhao, Yu-Hsuan Tsai, and Chuanliu Wu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b11875 • Publication Date (Web): 28 Feb 2020
Downloaded from pubs.acs.org on February 28, 2020
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Condensation of 2-((alkylthio)(aryl)methylene)malononitrile with
1,2-aminothiol as a novel bioorthogonal reaction for site-specific
protein modification and peptide cyclization
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Xiaoli Zheng,†,# Zhuoru Li,†,# Wei Gao,†,# Xiaoting Meng,†,‡,# Xuefei Li,‡ Louis Y. P. Luk,‡ Yibing
Zhao,† Yu-Hsuan Tsai,‡,* and Chuanliu Wu†,*
†The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry
of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen,
361005, P.R. China
‡School of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
ABSTRACT: Site-specific modification of peptides and proteins has wide applications in probing and perturbing biological systems.
Herein we report that 1,2-aminothiol can react rapidly, specifically and efficiently with 2-((alkylthio)(aryl)methylene)malononitrile
(TAMM) under biocompatible conditions. This reaction undergoes a unique mechanism involving thiol-vinyl sulfide exchange,
cyclization and elimination of dicyanomethanide to form 2-aryl-4,5-dihydrothiazole (ADT) as a stable product. An 1,2-aminothiol
functionality can be introduced into a peptide or a protein as an N-terminal cysteine or an unnatural amino acid. The bioorthogonality
of this reaction was demonstrated by site-specific labeling of not only synthetic peptides and a purified recombinant protein but also
proteins on mammalian cells and phages. Unlike other reagents in bioorthogonal reactions, the chemical and physical properties of
TAMM can be easily tuned. TAMM can also be applied to generate phage-based cyclic peptide libraries without reducing phage
infectivity. Using this approach, we identified ADT-cyclic peptides with high affinity to different protein targets, providing valuable
tools for biological studies and potential therapeutics. Furthermore, the mild reaction condition of TAMM condensation warrants its
use with other bioorthogonal reactions to simultaneously achieve multiple site-specific modifications.
INTRODUCTION
utilization efficiency and confers minimum perturbation to
native proteins.4 Consequently, this functionality has been used
for site-specific protein labeling, semi-synthesis, and peptide
Site-specific modification is a powerful strategy for studying
the structure and function of peptides and proteins.1 To study
polypeptides within their natural environment, various site-
specific bioorthogonal reactions have been developed,
including ketone condensations, alkyne-azide cycloadditions,
Staudinger ligations, and Diels-Alder cycloadditions.2 Despite
the significant effort made, there are limitations associated with
currently available technologies. Complex reagents that are
expensive or difficult to prepare hamper potential applications;
also, bulky labels often disrupt the polypeptide structure and
hence function. Furthermore, the options for multiple
modifications by using mutually orthogonal labeling systems
are rather limited3. Importantly, many of the labeling systems
are not compatible for live-cell labeling due to their slow
reaction kinetics or lack of specificity or biocompatibility. Here,
we present our effort of developing a specific, rapid and
versatile labeling system involving a minimum functionality
(i.e., 1,2-aminothiol) easy to be introduced into chemically
synthesized and recombinantly produced polypeptides and a
reaction partner easily synthesized and tuned for its chemical
and physical properties.
5
cyclization.2c, Native chemical ligation relies on the 1,2-
6
aminothiol group of a N-terminal Cys residue.5e, Though
proven to be successful in protein synthesis, the reaction
requires a labile thioester motif which is unstable in biological
systems. 2-Cyanobenzothiazole can be used in 1,2-aminothiol
labeling, and this reaction has been developed for protein
5a,
7
labeling and functional macrocycle construction.2c,
1,2-
Aminothiol can also react with benzaldehyde bearing an ortho-
boronic acid substituent to promote formation of the
corresponding thiazolidine derivative.5c However, aryl
boronates are susceptible to rapid oxidation by hydrogen
peroxide produced by living cells.8 Novel biocompatible, robust
and orthogonal 1,2-aminothiol labeling system with simple pre-
requisite capable of modifying both large proteins and short
peptides is still needed.
Here,
we
report
the
2-
((alkylthio)(aryl)methylene)malononitrile
(TAMM)9
condensation for 1,2-aminothiol labeling. In the TAMM
reaction, a vinyl sulfide group is replaced by the thiol group of
the 1,2-aminothiol (Scheme 1). Subsequently, Michael addition
of the amino group takes place to form a thiazolidine, which
induces the elimination of dicyanomethanide to afford 2-aryl-
4,5-dihydrothiazole (ADT). This reaction can be used for site-
1,2-Aminothiol is a unique binucleophile that can be readily
incorporated into a protein. Its small size warrants high atomic
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specific labeling in vitro as well as modification of proteins on
the surface of live mammalian cells and phages without
affecting their viability and infectivity. Furthermore, the
TAMM condensation can be combined with phage display to
identify high-affinity cyclic peptides against different targets.
Together, these experiments highlight the versatility of the
TAMM chemistry and its potentials in future chemical and
synthetic biology research.
Page 2 of 7
reactions. To our delight, reaction of p1 with 2 equivalents of
1a afforded only the desired product (ADT-p1) in quantitative
yield (Figure 1b), indicating the bioorthogonality of this
reaction. The second-order rate constant in phosphate buffer
(pH 7.4) was calculated to be 4.2 M-1s-1 (Figure S4), a value that
is comparable to that reported for the reaction of 1,2-aminothiol
with 2-cyanobenzothiazole.2c This was further confirmed by
reacting p1 with 1a (2 eq) and 6-methoxy-2-
cyanobenzothiazole (2 eq) at the same time, affording ADT-p1
in about 60% yield (Figure S5). The bioorthogonality was
further confirmed using peptide p2 containing both an N-
terminal and internal Cys residues. Exclusive formation of the
desired ADT product at the N-terminal Cys residue was
observed when excess Ac-Cys was added into the reaction
mixture to suppress the thiol-vinyl sulfide exchange of 1a with
the internal Cys residue (Figure 1c and S6). The reaction
efficiency of p2 with 1a is also comparable to the reaction with
6-methoxy-2-cyanobenzothiazole (Figure S5).
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Scheme 1. Mechanism of TAMM condensation
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CN
EtS
CN
1
(
)
R
R
O
NH2
S
O
R
CN
R
O
NH2
NH
S
N
O
CN
O
S
N
AcHN
CN
R
SH
SH
O
R
1b
(
: R = OH)
AcHN
CN
CN
S
1a
( : R = OH)
RESULTS AND DISCUSSION
This work originated from our longstanding interest in
developing dynamic vinyl sulfide bonds10 using electron-
deficient styrenes (e.g. TAMM) as the Michael acceptors.11
While TAMM (e.g. 1) undergoes thiol-vinyl sulfide exchange
with thiol-containing molecules, such as acetylcysteine (Ac-
Cys) to afford 1a (Scheme 1), the reaction of 1 with Cys
surprisingly formed a product with a mass of 66 Da lower than
originally expected. Further 1H and 13C NMR characterizations
Figure 1. TAMM is a versatile motif for bioorthogonal reaction
with 1,2-aminothiol. Chromatograms show absorbance at 280 nm.
(a) ADT product 1b (m/z for [M+1]+ expected: 208.0432, found:
208.0427) can be formed by reaction of Cys with either 1 or 1a in
phosphate buffer (pH 7.4). (b, c) Reaction of the designated peptide
(50 μM) with 1a (100 μM, 2 eq) in phosphate buffer (pH 7.4) for 2
h. (b) Peptide p1 as the substrate to form ADT-p1 (m/z for [M+1]+
expected: 749.3188, found: 749.3218). (c) Peptide p2 as the
substrate in the presence of Ac-Cys (250 μM) to form ADT-p2 (m/z
for [M+1]+ expected: 909.3494, found: 909.3605).
revealed
the
product
as
2-phenyl-4-carboxyl-4,5-
dihydrothiazole (1b), suggesting irreversible elimination of
dicyanomethanide and the formation of dihydrothiazole (Figure
S1). Although dihydrothiazole derivatives can be synthesized
through reactions of 1,2-aminothiols with aryl nitriles,12
thionoesters or dithioesters,13 these reactions have not been used
for bioorthogonal labeling as they require either high
temperature or labile reagents. On the other hand, in the TAMM
condensation, both the reagent and the product are very stable
(Figure S2), and the reaction can proceed either in the presence
of a base (e.g., NaHCO3) or in phosphate buffer (Figure S3). In
addition, the exchangeability of the thiol leaving group in
TAMM endows the reagent with a facile tunable nature in its
chemical and physical properties. For example, a carboxyl
functionality (e.g. 1a) can be installed on the styrene scaffold to
enhance the water solubility of TAMM. Specifically, 1a can be
isolated in high yield by reaction of 1 with Ac-Cys (Figure 1a).
Similar to 1, reaction of 1a with Cys also led to formation of
ADT product 1b.
To investigate the bioorthogonality and reaction kinetics of
the TAMM condensation, we examined the reaction of peptide
p1 in phosphate buffer (pH7.4). Peptide p1 was chosen as a
model substrate as it contains not only an 1,2-aminothiol
functionality at its N-terminal Cys but also a Lys residue of
which the side-chain amino group can potentially cause side
Figure 2. Site-specific labeling of a purified protein in vitro. (a)
Structure of compounds. (b) Reaction scheme and mass spectra.
Reagents and conditions: (i), 200 mM methoxyamine, 50 mM
phosphate buffer (pH 6.0), 150 mM NaCl, 2 mM TCEP; (ii), 100
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μM 2-Rh, 50 mM phosphate buffer (pH 7.4), 150 mM NaCl, 500
μM TCEP.
Figure 3. Site-specific labeling of a cell-surface protein on
mammalian cells. (a) Generation of a N-terminal Cys for TAMM
condensation by proteolysis. (b) Structure of 2-BDP. (c) Confocal
microscopy images showing TEV-dependent labeling. Scale bars
represent 20 µm.
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1,2-Aminothiol can also be added into a protein by genetic
code expansion for TAMM labeling.5a To demonstrate this
feasibility, His-tagged green fluorescent protein (GFP)
containing a Nε-L-thiaprolyl-L-lysine (ThzK) residue at the
150th amino acid residue was prepared (Figure 2). Treatment of
GFP(150-ThzK) with methoxyamine converted the
thiazolidine into 1,2-aminothiol to yield GFP(150-CysK),
which can be modified with a TAMM-rhodamine conjugate (2-
Rh). The success in fluorescent labeling was evident by both
mass spectroscopy (Figure 2) and in-gel fluorescence analysis
(Figure S6). In addition, GFP(150-CysK) can also be labeled
with other TAMM-fluorophore conjugates (Figure S7).
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Proteolytic process is another approach to generate 1,2-
aminothiol functionality for TAMM labeling.2c We therefore
explore this strategy to label cell-surface proteins on
mammalian cells with TAMM-fluorophore conjugates. For
example, TEV protease can recognize ENLYFQC sequence and
cleave the amide bond between Gln and Cys, generating a N-
terminal Cys residue. Accordingly, a plasmid expressing a
fusion protein containing a N-terminal extracellular-targeting
sequence, followed by the TEV recognition sequence
ENLYFQC, fluorescent protein mCherry and a transmembrane
domain, was constructed (Figure 3a). Live transfected HEK
cells were incubated with the labeling solution containing both
TEV protease and TAMM-fluorophore conjugate 2-BDP
(Figure 3b) for 30 minutes. Subsequent washing removed the
unreacted conjugate before imaging. Live-cell fluorescence
imaging in the mCherry channel confirmed the membrane
expression of the fusion protein (Figure 3). In addition,
fluorescence from 2-BDP clearly superimposed with mCherry
fluorescence (Pearson’s correlation coefficients = 0.92),
indicating site-specific fluorescent labeling. This was further
confirmed by in-gel fluorescence (Figure S8). Using this
approach, we were able to selectively modify cell-surface
proteins with different TAMM-fluorophore conjugates (Figure
S8).
Figure 4. TAMM-mediated peptide cyclization. (a) Structure of
ClAc-3. (b) Cyclization of a peptide bearing a N-terminal Cys and
an internal Cys residues. (c) Reaction of p2 (50 μM) and ClAc-3
(100 μM) in phosphate buffer (pH 7.4) in the presence of Ac-Cys
(200 μM) to form cADT-p2 (m/z for [M+2]2+ expected: 518.1998,
found: 518.1125).
The TAMM reaction can be applied to labeling N-terminal
cysteine, and this is further demonstrated by applying it in a
coupled reaction that generates cyclic peptides. TAMM
containing a chloroacetyl group (e.g. ClAc-3, Figure 4a) could
be applied to cyclize peptides and proteins containing both an
N-terminal and an internal Cys residues. This is because the
chloroacetyl group is considered inert to intermolecular
reactions with thiols.14 Nevertheless, after the N-terminal
TAMM condensation, it can undergo intramolecular reaction
with the thiol group on the internal Cys residue (Figure 4b) as
the proximity effect can accelerate reactions up to 105 folds.15
To avoid the unwanted exchange reaction between internal Cys
residue and the vinyl sulfide functionality on the TAMM
reagent, as well as possible oxidation of Cys residues, 4
equivalents of Ac-Cys and TCEP were included in the reaction
mixtures. Under this condition, model peptides, p2 and p3, of
different loop lengths were cyclized in high yields upon
treatment with ClAc-3 to afford the corresponding ADT-cyclic
peptides (Figures 4b and S9). The cyclization primed by
TAMM condensation has advantages in terms of
biocompatibility and specificity compared to other crosslinking
reagents with two highly reactive groups to free thiols, such as
1,3-bis(bromomethyl)benzene (BBMB).3a, 16
Encouraged by the success in cell-surface protein labeling
and peptide cyclization, we decided to combine TAMM
condensation and phage display to screen ADT-cyclic peptides
binding to different protein targets. We designed a library of
peptides displayed at the N-terminus of pIII protein on M13
phages.17 After cleavage of the signal sequence by the
endogenous enzyme in E. coli,18 the peptide-pIII fusions will
contain an N-terminal and an internal Cys residues separated by
nine random amino acids encoded by NNK, forming the CX9C
library, where X is any amino acid (Figure 5a). Display of
peptides with nine randomized residues can generate quality
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peptide libraries with sufficient content (>109) and reasonable
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coverage of sequence space.
The CX9C library was then used for screening high-affinity
cyclic peptide ligands to different protein targets. Anti-
apoptotic protein Bcl-2 was selected as the first target, which is
closely associated with cancer.22 A truncated Bcl-2/Bcl-xL
chimera was expressed as a fusion protein with a Sumo-His-
tag.23 This protein was biotinylated and immobilized on
magnetic beads with streptavidin, to which the ADT-cyclic
phages were applied. After three rounds of panning, 30 phage
clones were randomly picked and sequenced, giving 12 unique
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We first used a biotin-containing TAMM (Biotin-3) to
optimize reaction conditions for TAMM condensation. Under
the optimized condition, phages were first treated with 1 mM
TCEP for 30 min to reduce disulfide bonds, followed by
addition of 1 mM Biotin-3 and 2 mM Ac-Cys for 2-h incubation.
Using a biotin-streptavidin pull-down assay,19 this one-pot
procedure reproducibly gave > 90% yield for TAMM
condensation (Figure 5b). In contrast, there is no significant
difference in pull-down efficiency of (i) phages without
treatment of Biotin-3 (i.e. blank control as signals were resulted
from non-specific binding to streptavidin), (ii) phages without
pretreatment with TCEP, (iii) phages treated with TCEP and
ClAc-3 before the addition of Biotin-3, and (iv) phages without
an N-terminal Cys residue (e.g. GCXCX5CX5C, from N-to-C
terminus; termed GC4 library).
amino acid sequences with
a consensus sequence of
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CPXRYXWDXXC (Figures 6a and S10). However, drastically
different results were obtained from the disulfide-cyclic phages
(no treatment of TCEP and ClAc-3, Figure 5a) and the linear
phages (only treated with TCEP but not ClAc-3, Figure 5a)
under the same conditions. Different consensus sequences
found in the two controls highlights that the ADT-cyclization
enables the display of unique cyclic conformations on phage
surface that are not available in conventional linear and
disulfide-cyclic peptide libraries. The binding of three phage
clones (CB-4, CB-5, CB-7) to Bcl-2 was tested using ELISA,
and TAMM-mediated cyclization was shown to be crucial for
binding of phages to Bcl-2 (Figure 6b).
Chemically cyclized peptide libraries are particularly useful
20
for selecting lead peptides by phage display.16c,
A
conventional crosslinking reagent, BBMB, can be used, but it
causes >2 orders of magnitude reduction in phage infectivity
(Figure 5c), likely due to nonspecific crosslinking of native
cysteine residues in phage protein pIII.16c, 21 This consequently
decreases the library size and hampers the chance of isolating a
“hit”. To our delight, cyclization using ClAc-3 has no effect on
phage infectivity (Figure 5c). This highlights the advantage of
TAMM condensation in constructing cyclic peptide libraries.
Figure 6. TAMM-mediated cyclization enables selection of
ADT-cyclic peptides with high binding affinity. (a) WebLogo24
showing consensus sequences obtained from the panning of
phages with either ADT-cyclic, disulfide-cyclic or linear
peptide structure against Bcl-2. (b) Binding of three phage
clones (CB-4, CB-5, CB-7) to immobilized Bcl-2. Bound
phages were detected with an HRP-conjugated anti-M13
antibody. (c) Binding of ADT-cyclic, disulfide cyclic or linear
CB-7-F to Bcl-2 determined using a fluorescence polarization
assay. (d) Binding of cADT-CB-4/5/7 to Bcl-2 by competition
with cADT-CB-7-F in a fluorescence polarization assay.
Figure 5. Cyclization of peptides displayed on the surface of
phages using ClAc-3. (a) Scheme for the experimental operations.
(b) Using streptavidin pull-down assay to characterize the TAMM
condensation efficiency. Phages can only be pulled down
efficiently with Biotin-3 when they have a free N-terminal Cys
residue. Control phages (GC4) have an N-terminal Gly residue. (c)
Infectivity of phages under different conditions. buffer-1: 10 mM
PBS (pH 7.4); buffer-2: 20 mM NH4HCO3 (pH 8.0), 5 mM EDTA.
The three peptides were then chemically synthesized and
cyclized using ClAc-3. To quantify the binding affinity, a
lysine-conjugated fluorescein (F) was attached to the C-
terminus of peptide cADT-CB-7. The dissociation constant (Kd)
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of the resulting cADT-CB-7-F to Bcl-2 was determined to be
orthogonality to existing strategies for cyclic peptide
construction27 and can be combined with recent advance of
genetic code expansion in phage display,28 generating peptide
libraries with more complex topologies.
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0.128 μM by fluorescence polarization. Neither the disulfide-
cyclic CB-7-F nor its linear counterpart exhibits a measurable
binding affinity (Figure 6c). The affinity of non-fluorescent
cADT-CB-4/5/7 to Bcl2 was measured in competition with
cADT-CB-7-F by monitoring fluorescence polarization. All
three peptides exhibited a submicromolar affinity to Bcl-2
(Figure 6d), whereas disulfide-cyclic and linear CB-4/5/7
showed negligible affinity (Figure S10). In addition, two
peptides that were most abundantly enriched from selections of
linear and disulfide-cyclic libraries respectively were
synthesized and labeled with fluorescein for affinity
characterization and comparison. While the disulfide-cyclic
peptide exhibits negligible binding to Bcl-2, the linear peptide
can bind to Bcl-2 with a dissociation constant of 0.062 μΜ
(Figure S11). The high affinity of the linear peptide suggests the
possibility of selecting new cyclic peptide ligands capable of
binding with Bcl-2 protein by varying the structure or length of
ADT crosslinks. These results, taken together, also encourage
further studies on both structures of peptide-protein complexes
and screening of ADT-cyclic peptide libraries with new
crosslink structures. Currently, development of peptide ligands
and therapeutics to Bcl-2 family proteins has been an emerging
frontier.25 Available peptide binders to Bcl-2 are mainly stapled
ASSOCIATED CONTENT
Supporting Information
Experimental details for the chemical synthesis, cloning, biological
1
experiments; H and 13C NMR spectra of all new compounds; in-
9
gel fluorescence images of proteins; sequences of selected peptides.
This material is available free of charge via the Internet at
http://pubs.acs.org).
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AUTHOR INFORMATION
Corresponding Author
*tsaiy5@cardiff.ac.uk
*chlwu@xmu.edu.cn
Author Contributions
#X.Z., Z.L., W.G., X.M: These authors contributed equally.
Notes
The authors declare no competing financial interests.
peptides of ≥20 residues derived from the Bcl-2 homology
25b
domains.25a,
These peptides have an α-helical structure
ACKNOWLEDGMENT
stabilized through hydrocarbon crosslinkers. While displaying
no sequence homology to the previously reported stapled
peptides,25a our ADT-cyclic peptides represent interesting
alternatives for future studies due to their smaller size and ease
of preparation.
We are grateful for the financial support from the National Natural
Science Foundation of China (21822404, 91853112 and
21675132), the Fundamental Research Funds for the Central
Universities (20720180034), the Royal Society (IES\R1\180220),
the Program for Changjiang Scholars and Innovative Research
Team in University (IRT_17R66), and the Foundation for
Innovative Research Groups of the National Natural Science
Foundation of China (21521004). We thank Dr Yi-Lin Wu for the
helpful discussions.
The general applicability of the TAMM-mediated cyclization
in phage display was further demonstrated by using two other
proteins, Keap1 and Mdm2, as the immobilized targets. Both
proteins are potential therapeutic targets for various diseases,
including cancers, autoimmunity diseases, and inflammatory
diseases.26 After selections, consensus sequences were obtained
for each target (Figure S12), and the identified ADT-cyclic
peptides showed submicromolar binding affinities (Figures
S13). For selection of cyclic peptide ligands towards many
other targets of therapeutic interest, both the structure of ADT-
crosslinker and the number of randomized amino acid residues
can be fine-tuned for optimization.
REFERENCES
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CONCLUSION
In summary, we have described a novel condensation reaction
for the labeling of 1,2-aminothiol functionality on peptides and
proteins in vitro as well as on cell and phage surfaces. TAMM
condensation represents a useful alternative to existing
approaches for protein labeling. It is compatible with
physiological conditions and proceeds with a high degree of
specificity and efficiency. On the other hand, TAMM
conjugates containing a chloroacetyl group can cyclize peptides
with an N-terminal and an internal Cys residues. This strategy
is applicable to phage display technique without reducing phage
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valuable tools for biological studies and potential therapeutics.
Moreover, it provides a new perspective of designing
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Journal of the American Chemical Society
Table of Content
1
2
3
4
5
1,2-aminothiol
TAMM
ADT
R3S
NC
R2
SH
S
N
R2
R1
R1
CN
NH2
condensation
6
O
O
7
8
O
O
9
S
N
H
N
HN
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
S
N
S
C
X
X
X
X
X
X
X
X
X
C
7
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