Screening of Three Transition Metal-Mediated Reactions Compatible with DNA-Encoded Chemical Libraries

Article abstract of DOI:10.1002/hlca.201900033

The construction of DNA-encoded chemical libraries (DECLs) crucially relies on the availability of chemical reactions, which are DNA-compatible and which exhibit high conversion rates for a large number of diverse substrates. In this work, we present our

Full text of DOI:10.1002/hlca.201900033

DOI: 10.1002/hlca.201900033
FULL PAPER
Screening of Three Transition Metal-Mediated Reactions Compatible
with DNA-Encoded Chemical Libraries
Nicholas Favalli,a Gabriele Bassi,a Tania Zanetti,a Jörg Scheuermann,a and Dario Neri*a
a
Institute of Pharmaceutical Sciences, ETH Zürich, CH-8093 Zürich, Switzerland, e-mail: neri@pharma.ethz.ch
The article is dedicated to Prof. Dr. François Diederich, on the occasion of his Farewell Symposium
The construction of DNA-encoded chemical libraries (DECLs) crucially relies on the availability of chemical
reactions, which are DNA-compatible and which exhibit high conversion rates for a large number of diverse
substrates. In this work, we present our optimization and validation procedures for three copper and palladium-
catalyzed reactions (Suzuki cross-coupling, Sonogashira cross-coupling, and copper(I)-catalyzed alkyne-azide
cycloaddition (CuAAC)), which have been successfully used by our group for the construction of large encoded
libraries.
Keywords: DNA-encoded chemical libraries, Suzuki coupling, Sonogashira coupling, CuAAC reaction, copper,
palladium, DNA-compatible reaction.
screening experiment) can be directly retrieved from
the results of a DNA sequencing experiment, since
Introduction
The encoding of individual small organic molecules each library member is encoded by a distinctive DNA
with distinctive DNA fragments, serving as amplifiable fragment.[12,22,23] The construction of large DNA-
identification barcodes, allows the construction and encoded chemical libraries crucially relies on the
screening of chemical libraries of unprecedented availability of ‘robust’ reactions, which can be per-
size.[1–11] Libraries containing billions of compounds formed in water, are DNA-compatible,[24–26] and which
from a relatively small number of building blocks (e.g., accept a large number of structurally diverse sub-
few thousand chemical compounds) can be con- strates. One of the most widely used reactions for the
structed using a variety of different experimental construction of DNA-encoded chemical libraries is the
approaches, such as pool-and-split methodologies,[12] formation of amide bonds, since excellent procedures
DNA-templated chemical reactions with preformed are available,[24,27] and since thousands of building
oligonucleotide derivatives,[13–15] or the use of en- blocks (e.g., amines and carboxylic acids) can be
coded self-assembling strategies.[16–21] The attractive- purchased from commercial sources at moderate
ness of encoding chemical compounds with DNA costs. However, even a robust reaction such as amide
fragments relate to the possibility of constructing and bond formation with DMT-MM methodology displays
storing large libraries as a mixture, that can be acceptable conversion yields only for approximately
interrogated by affinity capture procedures on immo- 44% of carboxylic acids. For these reasons, improved
bilized proteins of interest, followed by decoding, methodologies are constantly being explored.[27]
using high-throughput DNA sequencing.[12,22,23] In-
In this work, we aimed at implementing and
deed, the identity and relative frequency of each optimizing copper and palladium-catalyzed reactions,
compound in the library (e.g., before and after a which could be readily used for the construction of
DNA-encoded chemical libraries.[28–31] We focused on
Suzuki cross-coupling, Sonogashira cross-coupling, and
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/hlca.201900033
copper(I)-catalyzed
alkyne-azide
cycloaddition
(CuAAC). There have been recent investigations on the
Helv. Chim. Acta 2019, 102, e1900033
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Helv. Chim. Acta 2019, 102, e1900033
Figure 1. a) Palladium-catalyzed Suzuki cross-coupling reactions between aryl halides (iodide and bromide) - DNA conjugates and
boronic acids or esters. b) UPLC chromatograms registered at 260 nm of starting material (1) and the deconvoluted ESI-TOF mass
spectrum of the corresponding peak (4.47). c) Chromatogram of crude Suzuki reaction between 1 and phenylboronic acid (B3) and
the deconvolution of ESI-TOF mass spectrum of the product (4.89); R=N3, 2-azido-3-(4-iodophenyl)propanamide; R1 =H, pinacol
ester. Ar: all structures of the tested boronic acid were reported in the Supporting Information, Table S1.
implementation of these reactions on substrates Figure 1 illustrates the reaction of a p-iodophenyl
coupled to double-stranded DNA fragments.[24,32–40] derivative, coupled to an amino-tagged single-
However, it is often convenient to construct libraries stranded oligonucleotide comprising 14 bases, with
using single-stranded DNA fragments, as these molec- phenyl boronic acid. The purity and identity of the
ular assemblies can be expanded using encoding self- DNA conjugates is typically monitored by UPLC/MS,
assembling chemistry (ESAC)[16–21] or used for innova- thus allowing an experimental determination of the
tive screening techniques (e.g., interaction-dependent performance of the reaction. Throughout the manu-
PCR[41] or the hybridization with complementary script, this methodology has been used for the
oligonucleotide-photocrosslinker conjugates.[42,43] In calculation of conversion yields and for the character-
principle, the DNA-compatibility of a reaction could be ization of optimal reaction conditions.
different for single- and double-stranded DNA deriva-
tives, due to the different shielding of the bases.
As a first step, we explored conversion yields for
the reaction of various boronic acids with iodophenyl
or bromophenyl propionic acid derivatives on single-
stranded DNA, using Pd(OAc)2 as catalyst (Table 1).
Overall, conversion yields were better for the iodo-
phenyl derivative, which convinced us to focus on
iodinated aromatic compounds for further investiga-
Results and Discussion
Suzuki Cross-Coupling
We first focused on Suzuki coupling procedures, since tions.
these transformations are broadly used in Medicinal
We then explored different sources and equivalents
Chemical research for the formation of CÀ C bonds.[44] of palladium complexes, as well as temperature
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equivalents did not result in further improvements.
The optimal reaction condition was tested on 32
boronic acids (Figure 2 and Supporting Information,
Table 1. Suzuki cross-coupling performed on aryl iodide and
aryl bromide DNA conjugates. The reactions were carried out at
°
60 C for three hours using 0.4 equiv. of Pd(OAc)2 – TPPTS
complex as catalyst. The reported conversions were calculated
by integrating UPLC chromatograms of crude reactions regis-
tered at 260 nm.
Reactants vs. Substrate
0%
88%
0%
0%
>98%
60%
>98%
>98%
40%
40%
>98%
70%
50%
0%
70%
0%
conditions (Table 2). We obtained the best results
using Pd(OAc)2, using at least 0.4 equivalents of
palladium complexes. Reactions were often satisfac-
tory with as little as 0.2 equivalents. The use of >0.4
Figure 2. Screening results of Suzuki cross coupling reactions
between 32 boronic acids and a modified 14-mer oligonucleo-
tide with a p-iodo-phenylalanine scaffold. Red, yellow, and
green colors correspond to reactions with poor, intermediate,
or good conversions. R: N3, 2-azido-3-(4-iodophenyl)propana-
mide. Ar: all structures of the tested boronic acid were reported
in Supporting Information, Table S1.
Table 2. Screening of different catalysts, substrates, and reac-
tion conditions for Suzuki ‘on-DNA’ cross-couplings. Ar: all
structures of the tested boronic acid were reported in
Supporting Information, Table S1. The test reactions were carried
out on an amino-modified oligonucleotide coupled to 2-azido-
3-(4-iodophenyl)propanoic acid (X=I) or 2-azido-3-(4-bromo-
phenyl)propanoic acid (X=Br).
Table S1). In 72% of the cases, conversion yields were
>75%, while 6% of the boronic acids exhibited
conversion yields between 40 and 75%. Importantly,
22% of the substrates exhibited conversions lower
than 40%. These boronic acids were typically aliphatic
boronates, acrylboronates, p-pyridinylboronic acid de-
rivatives, and compounds which are not soluble in
DMA/water. Indeed, methyl ester derivatives were
deprotected yielding the corresponding carboxylic
acids (Supporting Information, Table S1).
Pd(II) Source Time
Temp. Aryl halides Equiv. of Pd[a]
°
Pd(OAc)2
PdCl2
PdCl2(PPh3)2
[PdCl(allyl)]2
10 min 50 C
ArÀ I
0.2
0.4
1.0
1.5
2.0
4.0
°
1 h 60 C
2 h 65 C
4 h 70 C
ArÀ Br
°
°
24 h
In conclusion, we found that the Suzuki reaction
could reliably be implemented with iodophenyl DNA
[a]
Equivalents with respect to DNA–aryl halide conjugate (1).
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derivatives, using Pd(OAc)2 as catalyst (0.4 equiv.,
°
60 C, 3–24 h) with aromatic, heteroaromatic, and
vinylboronic acids.
Sonogashira Cross-Coupling
In full analogy to the optimization of Suzuki reaction,
we first studied the choice of catalyst (type and
equivalents) and of temperature conditions (Table 3).
Table 3. Screening of different catalysts, substrates, and reac-
tion conditions for Sonogashira ‘on-DNA’ cross-couplings. R: all
structures of the tested alkynes are reported in Supporting
Information, Table S2. The test reactions were carried out on an
amino-modified oligonucleotide coupled to 2-acetamido-3-(4-
iodophenyl)propanoic acid.
Pd(II) Source Cu(II) Source Time
Temp. Equiv. of Pd[a]
°
Pd(OAc)2
PdCl2
PdCl2(PPh3)2 Cu(OTf)2
[PdCl(allyl)]2 Cu(OAc)2L
Copper-free
Cu(OAc)2
Cu(SO4)2
10 min 50 C
0.2
0.5
1.0
1.5
2.0
4.0
°
1 h 60 C
°
2 h 65 C
°
4 h 70 C
24 h
[a]
Equivalents with respect to DNA-aryl halide conjugate (3).
Various palladium and copper complexes were tested,
alone and in combination. The best conversions were
obtained for a copper-free reaction using one equiv-
°
alent of [PdCl(allyl)]2 at 70 C for three hours. We have
°
observed that the presence of copper at 60 and 70 C
may cleave and degrade the DNA products. An
example of Sonogashira cross coupling performed
with the described conditions was reported in Figure 3.
The reaction was carried out on a single-strand DNA
derivative (compound 3) with alkyne A34. The LCÀ MS
traces show an excellent conversion and side products
were not observed.
Figure 3. Palladium-catalyzed Sonogashira cross-coupling reac-
tion between aryl iodide 3 and 4-chloro-2-ethynyl-1-methox-
ybenzene (A34). a) TOF MS spectra and b) UPLC chromatogram
registered at 260 nm of the crude reaction. c) Deconvolution of
MS peak of the product (5.13).
Figure 4 shows representative coupling results for
some of the 44 alkynes that were tested. For 43% of
the reagents, >75% conversion was achieved. A
conversion between 40% and 75% was observed for
30% of the alkynes, whereas 25% of the compounds aryl-bromides (A6) and compounds with poor water-
exhibited a conversion <40%. ‘Difficult’ alkynes solubility (A3, A36, and A64). Aromatic aryl-bromides
included structures that were more prone to decom- (A32) and aromatic aryl-chlorides (A62, A18, and A34)
position. These alkynes were typically amines (A102, exhibit excellent conversions. Side products of over-
A33, A118, A119, A93, A23, and A95), heteroaromatic
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In order to perform reactions in aqueous solution,
with a considerable saving in the amounts of reagents
to be used, we synthesized a water-soluble pre-
catalyst obtained by mixing copper (II) acetate with a
potassium salt of 6 (potassium 4,4’,4’’-[nitrilotris
(methylene-1H-1,2,3-triazole-4,1-diylmethylene)]triben-
zoate). The complex was subsequently reduced in situ
by sodium ascorbate obtaining the Cu(I) catalyst which
promoted efficient conversions, with a broad scope of
substrates (Figure 5, Table 4). The importance of keep-
Table 4. A comparison between pseudo-solid phase CuAAC
and the optimized water-compatible CuAAC conditions. The
test reactions were carried out on an amino-modified oligonu-
cleotide coupled to 2-azido-3-(4-iodophenyl)propanoic acid.
Reaction
Conditions
Pseudo-solid
phase CuAAC
Solution-phase
CuAAC
Pre-catalyst
Reducing agent
CuSO4 +TBTA
sodium
ascorbate
Cu(OAc)2 +6
sodium
ascorbate
0.1
Equiv. of Cu[a]
Solvent(s)
>10
DMSO:tBuOH:H2O
mixtures
triethylamine
r.t.
400
24 h
H2O
Base
K2CO3
°
Temperature
Equiv. of Alkyne[a]
Reaction time
35 C
40
3 h
[a]
Equivalents with respect to azido-DNA conjugate (1).
Figure 4. Screening results of Sonogashira cross coupling
reactions between 44 alkynes and a modified 14-mer oligonu-
cleotide with a p-iodo-phenyl moiety. Red, yellow, and green
colors correspond to reactions with poor, intermediate, or good
conversions. R: all structures of the tested alkynes were
reported in Supporting Information, Table S2.
ing Cu(I) in stable complexes has previously been
recognized,[48] as Cu(I) ions may promote DNA
cleavage.[48–50]
Figure 6 shows representative results for the reac-
tion of an azide derivative on single-stranded DNA
with 115 different terminal alkynes. For 65% of the
reaction were not observed (Figures 2 and 4 and reagents, a conversion >75% was observed. Only 9%
Supporting Information, Table S2).
of the alkynes exhibited a conversion between 40%
and 75%, while 29% of the reagents showed a worse
performance (Figure 6, Supporting Information, Ta-
ble S3). Conversions lower than 40% were often
Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC)
CuAAC is a frequently used reaction for the construc- observed for hydrophobic compounds which do not
tion of DNA-encoded chemical libraries,[37,45,46] be- readily dissolve in water and for structures (e.g.,
cause of its robustness and of the broad availability of methyl esters or carboxylic acids).
azides and terminal alkynes.
Previous activities of our laboratory featured the
use of pseudo-solid phase ‘on-DNA CuAAC’ chemistry.
Conclusions
The reactions were performed using organic solvents
t
(DMSO and BuOH mixtures) and the conventional Suzuki coupling, Sonogashira coupling, and copper-
TBTA (1-(1-benzyltriazol-4-yl)-N,N-bis[(1-benzyltriazol- catalyzed azide-alkyne cycloaddition reactions were
4-yl)methyl]methanamine) as copper (I) ligand.[47]
extensively tested with ca. 200 substrates. We estab-
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Figure 5. a) Copper-catalyzed azido-alkyne cycloaddition (CuAAC) reactions between an azido modified oligonucleotide and
alkynes. b) UPLC chromatogram of crude CuAAC reaction between 1 and 4-ethynyl-2-fluoropyridine and the deconvolution of ESI-
TOF MS spectra of the product (4.66). UPLC chromatogram and MS spectra of starting material 1 is reported in Figure 1b. Ar: 4-
Iodophenyl; R: all structures of the tested alkynes were reported in Supporting Information, Table S1.
lished conditions for all three reactions, which worked
well with the majority of the building blocks.
It has often been assumed that double-stranded
DNA may be more suitable for library construction, as
Similar to previous studies with amide bond the heteroduplex could potentially contribute to the
forming reactions,[27] it is extremely difficult to identify integrity of the DNA bases. In our experience, how-
experimental procedures, which yield conversions ever, single-stranded DNA structures preserve their
>75% for all possible substrates.
integrity in the reactions described in this manuscript
The construction of DNA-encoded chemical libra- and in other reactions (data not shown). It is
ries can often afford to be performed using lower particularly convenient to use single-stranded DNA for
conversion rates (e.g., >40%), since bioactive com- library construction, since n oligonucleotides are
pounds are readily identified in screening procedures required for the encoding of n building blocks. The
also when products are not completely pure.[12,15,51–53] use of double-stranded DNA fragments typically
Nonetheless, scientists aim at constantly improve requires the use of twice the number of oligonucleo-
library purity, in order to have consistent correlations tides, as well as the need for a linker that bridges and
between enrichment factors in screening experiments stabilizes the heteroduplex.[13] Single-stranded DNA
and affinity measurements at the hit validation libraries are conveniently encoded using splint ligation
stage.[46,54]
The pie charts presented in the article, describing be removed at the end of the encoding step.[17,45]
the cumulative results of conversion rates for Suzuki Single-stranded DNA-encoded chemical libraries
procedures, with short oligonucleotides that can easily
coupling, Sonogashira coupling, and copper-catalyzed containing millions of compounds can be easily
azide-alkyne cycloaddition reactions, provide a strong synthesized.[37,45,46,55] They can be converted into
rationale for the quantitative evaluation of model double-stranded DNA using Klenow polymerization
reactions prior to library construction. Building blocks procedures.[12,45,46,52,54] Alternatively, the pairing of two
which show poor conversions may be replaced by single-stranded DNA-encoded chemical libraries leads
other molecules, which give better yields. The data to ESAC libraries of very large size.[16,17,19–21] The total
contained in Supporting Information, Tables S1–S3 may number of compound combinations in ESAC libraries
serve as reference data base for other groups, who corresponds to the product of the sizes of the
may wish to use the same experimental conditions for individual libraries.
library construction.
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Experimental Section
General Experimental Information
All reagents and solvents used were of analytical
grade. Buffers were prepared with ultrapure water
(Merck Millipore Quantum® TEX). All chemicals were
purchased from Acros, Alfa Aesar, Sigma-Aldrich, or TCI.
The 5’-aminomodified-14mer oligonucleotide (5’-C6-
amino-GGAGCTTCTGAATT, MS=4473 Da) used for
tests was purchased by LGC Biosearch Technologies. All
boronic acid solutions were purchased from Apollo
Scientific and alkynes solutions were purchased from
Enamine. Purifications were carried out on a semi-
preparative HPLC with Waters XTerra® Shield RP18
(125 Å, 5 μm) using 100 mM triethylammonium acetate
(TEAA pH=7.0)/acetonitrile (MeCN) as mobile phase
(flow=4.0 mL/min). Databases (Supporting Informa-
tion, Table S1–S3) were managed with Instantjchem
18.
Synthesis of 1 and 1b (General Procedure for ‘on-DNA’
amide coupling formation; Procedure 1)
200 mM 2-azido-3-(4-iodophenyl)propanoic acid (8; for
the synthesis of 1) and 2-azido-3-(4-bromophenyl)
propanoic acid (9; for the synthesis of 1b) were
activated by adding 100 mM 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide (EDC) in DMSO (0.95 equiv.)
and 100 mM N-hydroxysuccinimide (S-NHS) in DMSO/
water 2:1 (1.5 equiv.). The resulting mixtures were
°
agitated for 20 min at 30 C. The 5’-aminomodified
oligonucleotide
(5’-C6-amino-GGAGCTTCTGAATT,
Figure 6. Screening results of CuAAC coupling reactions be-
tween 116 alkynes and a modified 14-mer oligonucleotide with
a phenylalanine based-scaffold bearing an azido group. Red,
yellow, and green colors correspond to reactions with poor,
intermediate, or good conversions. Ar: 4-Iodophenyl; R: all
structures of the tested alkynes are reported in Supporting
Information, Table S3.
MS=4473 Da, 1–500 nmol, 1 mM) in 50 mM TEA·HCl
buffer (pH=10) was subsequently added to 100 equiv.
of the corresponding activated S-NHS ester. The
°
reactions were heated at 37 C for 12 h, and the
products were precipitated with ethanol and purified
by HPLC (Supporting Information, Figure S1).
Synthesis of 2 (optimized procedure for ‘on-DNA’
Suzuki cross-coupling)
While efficient reaction conditions could be found
for Suzuki coupling, Sonogashira coupling, and copper-
catalyzed alkyne-azide cycloadditions, the field of The catalyst solution was prepared by mixing 20 μL of
DNA-encoded chemistry will crucially rely on similar 10 mM palladium (II) acetate in N,N-dimethylacetamide
studies on other reactions, in order to expand the (DMA), 100 μL of 100 mM trisodium 3,3’,3-phosphine-
chemical diversity that can be used for library screens. triyltribenzenesulfonate (TPPTS) in water and 480 μL of
While dozens of DNA-compatible reactions have been water, resulting in a 0.33 mM solution of Pd(0)-TPPTS
proposed,[24] the scope of those reactions with a complex. To a 0.1 mM solution of oligonucleotide 1
sufficiently broad set of building blocks is often (2 nmol, 20 μL) in water were subsequently added
missing.
60 μL of 200 mM Na2CO3 in water, 2.5 μL of catalyst
solution (0.8 nmol in Pd) and 10 μL of 200 mM ArB
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Scheme 1. Synthesis of 8 and 9. a) CuSO4 2%, K2CO3, MeOH, 24 h, r.t.
(OH)2 (Supporting Information, Table S1) in DMA. The sodium-L-ascorbate in water. The mixture was agitated
°
°
resulting mixture was agitated from 3 to 24 h at 60 C. at 35 C for 3 h.
Synthesis of 3
Synthesis of Compounds 8 and 9 (Scheme 1)
The azido oligonucleotide conjugate 1 (100 nmol) was The commercially available p-I- or p-Br-(D,L)-phenyl-
treated with 0.5 mL of 30 mM tris(2-carboxyethyl) alanine (5.4 mmol) was dissolved in dry methanol
phosphine (TCEP) in 500 mM Tris·HCl buffer (pH=7.4) (15 mL)
and
1H-imidazole-1-sulfonyl
azide
°
for 3 h at 35 C yielding the corresponding primary hydrochloride (1.36 g, 6.5 mmol), anhydrous potassium
amine 7 (Supporting Information, Figure S1). The ob- carbonate (1.86 g, 13.5 mmol), and anhydrous copper
tained product was acetylated by adding activated sulfate (40 mg, 0.02 mmol) were added, and the
acetic acid S-NHS ester (Procedure 1). The product was resulting mixture was stirred at room temperature for
precipitated and purified by HPLC (Supporting Informa- 24 h. The mixture was concentrated under reduced
tion, Figure S1).
pressure, and the products were extracted with AcOEt.
The pure compounds 8 and 9 (Scheme 1) were
obtained by silica gel chromatography with hexane/
AcOEt 7:3 as an eluent with 56% of yield.
Synthesis of 4 (optimized procedure for ‘on-DNA’
Sonogashira cross coupling)
1
The catalyst solution was prepared by mixing 100 μL
Data of Compound 8. H-NMR (400 MHz, (D6)DMSO):
of 10 mM allylpalladium (II) chloride dimer in DMA, 7.71–7.63 (m, 2 H); 7.13–7.04 (m, 2 H); 4.41 (dd, J=8.7,
100 μL of 100 mM TPPTS in water and 800 μL of water, 5.0, 1 H); 3.06 (dd, J=14.2, 5.0, 1 H); 2.89 (dd, J=14.2, 8.7,
resulting in a 1 mM solution of Pd(0)-TPPTS complex. 1 H).
To a 0.1 mM solution of oligonucleotide 3 (2 nmol, 20
μL) in water were subsequently added 50 μL of
1
Data of Compound 9. H-NMR (400 MHz, (D6)DMSO):
200 mM Na2CO3 in water, 2 μL of catalyst solution (2 7.53–7.42 (m, 2 H); 7.26–7.18 (m, 2 H); 4.36 (dd, J=8.1,
nmol in Pd) and 5 μL of 100 mM alkyne (Supporting 4.6, 1 H); 3.09 (dd, J=14.2, 4.6, 1 H); 2.84 (dd, J=14.2, 8.1,
Information, Table S2) in DMSO. The resulting mixture 1 H).
°
was agitated for 3 h at 70 C.
Synthesis of Cu(I) Ligand 6
Synthesis of 5 (optimized procedure for ‘on-DNA’
Synthesis of 10. The commercially available methyl 4-
CuAAC)
(bromomethyl)benzoate (3.0 g, 13.1 mmol) was dis-
All solvents were degassed in argon atmosphere. The solved in DMF (20 mL) and then NaN3 (8.5 g,
pre-catalyst solution was prepared by mixing 25 μL of 130.8 mmol) and water (2 mL) were added (Scheme 2).
°
10 mM Cu(OAc)2, 100 μL of 10 mM solution of 8 in The resulting mixture was heated at 80 C for 4 h and
200 mM K2CO3 and 2’325 μL of water, resulting in a then concentrated under reduced pressure. The crude
100 μM solution of Cu(II)-8 complex. To a 100 mM product was diluted with CH2Cl2 and washed with
solution of oligonucleotide 1 (10 nmol in 100 μL) in water. The organic phases were collected and concen-
water were subsequently added 0.1 μL of 50 mM trated. The pure product 10 was obtained by silica gel
K2CO3, 10 μL of pre-catalyst solution (1 nmol) and chromatography with hexane/AcOEt 8:2 as an eluent
1
40 μL of 10 mM alkyne (Supporting Information, Ta- with 96% of yield. H-NMR (400 MHz, CDCl3): 8.09–
ble S3) in DMSO. The solution was mixed and the 7.99 (m, 2 H); 7.40–7.34 (m, 2 H); 4.39 (s, 2 H); 3.90 (s, 3
catalyst was activated by adding 10 μL of 10 mM
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°
Scheme 2. Synthesis of 6. a) NaN3 (10 equiv.), DMF/H2O 9:1, 80 C, 4 h. b) tripropargylamine, CuI (2%), TBTA (2%), DMF, TEA, r.t., 48
°
h. c) 3 M LiOH, H2O/MeOH 1:1, 80 C, 1 h.
H). 13C-NMR (101 MHz, CDCl3): 166.59; 140.42; 130.10; H]+), 664.236 ([M+2 H]+), 665.238 ([M+3 H]+),
127.91; 119.99; 54.26; 52.18.
1325.370 ([2 M+H]+).
Synthesis of 11. All solvents were degassed in argon
atmosphere. Compound 10 (2.4 g, 12.6 mmol) was
dissolved in DMF (10 mL) and tripropargylamine
Acknowledgements
(4.1 mmol, 590 μL), CuI (48 mg, 0.25 mmol) and Financial support from the ETH Zürich, the Swiss
100 mg of tris(benzyltriazolylmethyl)amine (TBTA, National Science Foundation (Grants
132 mg, 0.25 mmol) were added. The mixture was 30030B_163479),Sinergia (CRS112_16099), and from
stirred at r.t. for 48 h. The solvent was evaporated the ERC Advanced Grant ‘ZAUBERKUGEL’ (Grant agree-
under reduce pressure, and the pure product 11 was ment 670603) is gratefully acknowledged. Instant
obtained by silica gel chromatography with CH2Cl2/ JChem (ChemAxon) was used for structure and data
1
MeOH 9:1 as an eluent with >98% of yield. H-NMR management (http://www.chemaxon.com).
(400 MHz, (D6)DMSO): 8.17 (s, 3 H); 7.93 (d, J=8.2, 5 H);
7.37 (d, J=8.3, 6 H); 5.70 (s, 6 H); 3.83 (s, 9 H); 2.54 (s, 6
H). 13C-NMR (101 MHz, (D6)DMSO): 166.27; 141.79;
Author Contribution Statement
130.05; 129.71; 128.35; 52.66; 40.91. ESI-TOF-MS (pos.):
623 ([M–3 N2]+), 651 ([M–2 N2]+), 679 ([M–N2]+), 706 N. F. designed and performed the experiments,
([M+H]+), 707 ([M+2 H]+), 727 ([M+Na]+), 1410 analyzed the data, and wrote the manuscript, G. B. and
(2 M+).
T. Z. performed the experiments, analyzed the data, J.
S. and D. N. conceived the project and wrote the
Synthesis of 6. The methyl ester 11 (1.0 g, manuscript.
1.41 mmol) was dissolved in MeOH (10 mL) and 3 M
LiOH water solution (30 mmol, 10 mL) was added. The
°
resulting suspension was stirred at 80 C for 1 h. The References
reaction was cooled and quenched with 3 M HCl. The
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7.87 (m, 6 H); 7.38–7.29 (m, 6 H); 5.67 (s, 6 H); 3.64 (s, 6
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Received January 31, 2019
Accepted March 4, 2019
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