Exploration of a Au(i)-mediated three-component reaction for the synthesis of DNA-tagged highly substituted spiroheterocycles

Article abstract of DOI:10.1039/c7ob02347b

We demonstrate a Au(i)-mediated three-component reaction to DNA-tagged highly substituted 6-oxa-1,2-diazaspiro[4.4]nonanes from either DNA-coupled aldehydes, hydrazides, or alkynols. The choice of the starting material coupled to the DNA tag was critial for the purity of the product as the DNA-aldehyde conjugate yielded the purest products, whereas the alkynol- and hydrazide conjugates returned complex product mixtures. The reaction was compatible with thymine-, cytosine-, and, surprisingly, with adenine-DNA, while guanine-containing DNA strands were degraded under the reaction conditions.

Full text of DOI:10.1039/c7ob02347b

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Exploration of a Au(I)-mediated three-component reaction for the
synthesis of DNA-tagged highly substituted spiroheterocycles
Received 00th January 20xx,
Accepted 00th January 20xx
Mateja Klika Škopić,a Suzanne Willems,a,b Bernd Wagner,a Justin Schieven,a Norbert Krause,a and
Andreas Brunschweiger*a
DOI: 10.1039/x0xx00000x
We demonstrate a Au(I)-mediated three-component reaction to DNA-tagged highly substituted 6-oxa-1,2-
diazaspiro[4.4]nonanes from either DNA-coupled aldehydes, hydrazides, or alkynols. The choice of the starting material
coupled to the DNA tag was critial for the purity of the product as the DNA-aldehyde conjugate yielded the purest
products, whereas the alkynol- and hydrazide conjugates returned complex product mixtures. The reaction was
compatible with thymine-, cytosine-, and, surprisingly, with adenine-DNA, while guanine-containing DNA strands were
degraded under the reaction conditions.
www.rsc.org/
Introduction
Genetically tagged collections of drug-like compounds (Fig.
1A), called DNA-encoded libraries (DELs), have emerged as a
valuable addition to the arsenal of technologies for the
identification of small organic molecules binding to a target
protein.1 In contrast to discrete screening libraries of small
molecules, DELs are much more efficiently handled as large
compound mixtures. These are screened by an efficient, generic
assay based on the principle of selection. The whole encoded
library is incubated as a mixture with a target protein
immobilized on a surface, washing steps enrich compounds
binding to the protein. Protein binders are then eluted, and
detected by sequencing of their DNA tag. DELs have yielded
many bioactive compounds, some of them are useful chemical
biology probes, and a few attained status as drug candidates.1
Several encoding strategies have been developed by academic,
and industrial research groups for drug discovery.2 Commonly,
encoded libraries are synthesized by combinatorial cycles of
alternated synthesis, and encoding steps tracking the synthesis.
Synthesis methods employed for the synthesis of these libraries
must give high yields, display a broad scope, tolerate water as
(co)-solvent, and must preserve the genetic information.3
Workhorse reactions meeting all these requirements are e.g.
carbonyl reactions, nucleophilic substitution of reactive halides,
and C-C-cross coupling reactions. All of these reactions couple
building blocks, yielding in effect rather planar and stretched
out structures.4 Accessing DNA-tagged three-dimensional
Fig. 1 Previous and current work towards expanding the chemical space of genetically
tagged libraries. A) Generic structure of a DNA-encoded compound. B) The TiDEC
approach to a DNA-encoded library of pyrazolines. C) Investigations in the suitability of
a Au(I)-catalyzed spirocycle synthesis for encoded library synthesis.
a.
Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-
Hahn-Str. 6, 44227 Dortmund, Germany. E-mail: andreas.brunschweiger@tu-
dortmund.de
b.
present address: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz
1, 45470 Mülheim an der Ruhr, Germany.
Electronic Supplementary Information (ESI) available: detailed synthesis
procedures and analytical data of all compounds. See DOI: 10.1039/x0xx00000x
chemical matter from simple, and readily available starting
materials to provide DELs with shape diversity is currently a
This journal is © The Royal Society of Chemistry 20xx
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major challenge in the field. Diverse (partially) saturated catalysis giving access to highly substituted pyrazVoiel(winAr)teicsle fOrnolimne
scaffolds are attractive library constituents as they project their simple starting materials by a Au(I)D-mOIe: d10ia.1t0e3d9/Ca7nOnBu0l2a3t4io7Bn
substituents into space.5
reaction for DEL-synthesis (Fig. 1B).
Spirocycles are archetypes of three-dimensional structures, and
well represented in natural products, yet they are compared to
more simple (hetero)cyclic structures much less intensely
explored in drug research.12 Here, we systematically
investigated a Au(I)-mediated three-component reaction to
Table 1 Reaction optimization for the conversion of hexT-1a to spirocycle hexT-4a by
Au(I)-mediated three-component reaction. a) Conditions see table, eq. relative to the
DNA, room temperature, tris(2,4-di-tert-butylphenyl)phosphite]gold chloride, AgSbF6;a
b) aq. NH3/MeNH2. R: para-(aminocarbonylmethyleneoxy)phenyl. Wavy bond to hexT:
5‘-(C6)-amino-linker, bold bond: connection to solid phase, filled circle: solid phase.
hexT-6-oxa-1,2-diazaspiro[4.4]nonane conjugates
4
from
readily available aldehydes , hydrazides
1
2
, and alkynols 3 13
.
Additionally, the connectivity between hexT and spirocycle,
and the tolerance of different DNA sequences to optimized
conditions for the Au(I)-mediated reaction were explored (Fig.
1C).
Results and discussion
The synthesis of the hexT-diazaoxaspironane conjugate hexT-
4a was explored with the controlled pore glass (CPG) solid
support-bound hexT-aldehyde conjugate 1a
,
the non-
entry
solvent
cat.
(eq.)
10
10
50
100
250
250
250
250
-
250
250
250
250
250
250
2a
(eq.)
100
500
500
500
500
500
500
-
500
500
500
500
500
500
500
3a
(eq.)
200
time
[h]
20
20
20
20
14
18
20
20
20
14
14
14
14
14
14
4a
[%]b
-
5
[%]b
< 5
< 5
< 5
< 5
35
20
< 5
-
symmetrically substituted hydrazide 2a, and the alkynol 3a
using tris(2,4-di-tert-butylphenyl)phosphite]gold chloride and
AgSbF6 as catalyst (Table 1). Following reaction, the DNA
conjugates were cleaved from the solid phase with a mixture of
aqueous ammonia/methylamine, HPLC-purified, and analyzed
by HPLC and MALDI MS.
1c
2 c
3 c
4 c
5 c
6 c
7 c
8
THF
THF
THF
THF
THF
THF
THF
THF
THF
MeOH
MeCN
DMF
C2H4Cl2
CH2Cl2
toluene
1000
1000
1000
1000
1000
1000
-
1000
1000
1000
1000
1000
1000
1000
-
40
50
55
75
90
-
9
-
-
-
11
12
13
14
15
16
90
45
75
55
70
50
45
-
10
10
15
a Reaction performed on a 30 nmol scale; b conversion estimated by HPLC analysis
of the crude product; c note that these conjugates contain a PEG(4) linker.
They can be incorporated into DELs through functionalized
scaffolds,6 or by development of DNA-compatible synthesis
methods. Examples for the latter are the Diels-Alder reaction, a
tertiary amino effect reaction to spirocycles, a few heterocycle-
forming reactions, and the metathesis reaction, reflecting the
strong interest in expanding DEL synthesis methods.7 Metal-
catalysis is a vibrant research field in organic chemistry,
enabling synthesis of ever more diverse structures from simple
starting materials.8 However, many metal ion catalysts,
displaying Lewis acid and oxidizing properties, are interacting
or even reacting with purine nucleosides in a DNA strand,
causing eventually depurination of DNA.9 Thus, they usually
demand the chemically much more stable PNA for tagging.10
We recently described an encoding strategy called TiDEC
(oligothymidine initiated DNA-encoded chemistry), which
initiates DEL synthesis with a chemically surprisingly stable
hexathymidine adapter “hexT”.11 The hexT adapter
oligonucleotide tolerated the harsh reaction conditions of Au(I)
Fig. 2 Synthesis and comparison of the reference ref-hexT4a with hexT4a (Table 1). A)
Synthesis of the control ref-hexT-4a; a) HATU, DIPEA, DMF, room temperature, 4
hours; b) aq. NH3/MeNH2. B) HPLC analysis of ref-hexT-4a. C) HPLC analysis of hexT-4a.
Wavy bond to hexT: 5‘-(C6)-amino-linker, bold bond: connection to solid phase, filled
circle: solid phase, r.t.: retention time.
In a first series of reactions run in THF at room temperature for
20 hours, equivalents of reactants and catalyst were explored
(Table 1, entries 1-4, and 7, see also Table S1, and ESI for
extended information, and HPLC traces of all crude reactions).
Increasing the excess of hydrazide 2a to 500 equivalents,
2 | J. Name., 2012, 00, 1-3
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alkynol 3a to 1000 equivalents, and catalyst to 250 equivalents performing the reaction in the presence of the sVoieliwdArstiuclpe pOonlrinte-
were in our hands critical for product yield. Shorter reaction bound DNA has any impact on the diastDeOreI:o1m0.e10ri3c9/rCa7tOioB0o2f34th7Be
times (entries 5,6) returned a second product with the mass of products. Comparison of the HPLC traces of compounds hexT-
the plausible azomethine imine intermediate hexT-5 which we 4a and ref-hexT-4a suggests that the hexT DNA did not impact
found stable during cleavage of DNA with concentrated the diastereomeric ratio of the product (Fig. 2B,C).
aqueous ammonia and HPLC purification (ESI, Fig. S8).
Among the solvents that were investigated (entries 11-16), the
polar aprotic MeCN gave similar results to THF, whereas the
reaction yielded predominantly, if not exclusively the
azomethine imine intermediate hexT-5 in all non-polar
solvents, in the polar aprotic solvent DMF, and also in the polar
protic MeOH. These results are in agreement with previous
observations.13 Control reactions either without catalyst or
without reactants returned the starting material peaks in the
HPLC analysis as expected (entries 8-9).
Table 2 Scope of the reaction yielding hexT-6-oxa-1,2-diazaspiro[4.4]nonane conjugates
hexT-4. a) Reaction condition No. 7 (Table 1); b) AMA (aqueous ammonia (30 %)/
aqueous methylamine (40 %), 1:1, vol/vol), 30 min, room temperature). R: para-
(aminocarbonylmethyleneoxy)phenyl. Filled circle denotes solid support (controlled
pore glass, CPG); wavy bond to hexT: 5'-(C6)-amino-linker.
2
mass calc.
entry
hexT
3
n
1
mass founda
R1
R2
2392.7
2394.5
1
4a
t-Boc
a
2302.6b
2
3
4
4b
4c
4d
t-Boc
Ac
a
a
b
1
1
2
2305.1b
2363.7
2366.2c
2406.8
2410.3
t-Boc
Fig. 3 Synthesis of the hexT-6-oxa-1,2-diazaspiro[4.4]nonane conjugate 4e. A) Reaction
scheme of the spirocycle formation. The synthesis route starts from the solid support
bound hexT-aldehyde 1a, which is reacted with carbazate 7 to hexT-hydrazide 2d, the
hexT hydrazide conjugate is then reacted with benzaldehyde 1b and pentynol 3a to the
spirocycle 4e. Reagents and conditions: a) NaBH3CN, aqueous 3-(N-
morpholino)propanesulfonic acid buffer, room temperature, overnight; b) 250 eq.
tris(2,4-di-tert-butylphenyl)phosphite]gold chloride, 250 eq. AgSbF6, 1000 eq. 1b, 1000
eq. 3a, THF, room temperature, 20 hours; c) aq. NH3/MeNH2, room temperature, 30
min. B) HPLC trace of the crude reaction mixture showing formation of numerous
a
b
measured by MALDI-MS;
loss of the photolabile 3,4-methylendioxy-6-
c
nitrobenzyl group upon irradiation in the mass spectrometer; phthalimide
protective group removed in the product.
In order to confirm formation of the spirocycle conjugate
hexT-4a, we synthesized and fully characterized a reference
spirocycle
6 that was then coupled by amide formation to 5’-
products
besides
the
target
spirocycle
4e.
R:
para-
amino-linker-modified hexT yielding the reference compound
ref-hexT-4a (Fig. 2A, and ESI Fig S12). Both compounds
hexT-4a and ref-hexT-4a showed the same mass in the
MALDI analysis, and more importantly, the HPLC trace of the
reference hexT-spirocycle conjugate mirrored the product of the
Au(I)-mediated spirocycle synthesis (Fig. 2B,C) lending
credibility to the formation of the spirocycle conjugate hexT-4a
suggested by reaction mechanism (Table 1), HPLC analysis,
and matching mass spectrum. As the Au(I)-catalyzed reaction
furnishes diastereomers, we were curious to learn whether
(aminocarbonylmethyleneoxy)phenyl. Wavy bond to hexT: 5‘-(C6)-amino-linker, bold
bond: connection to solid phase, filled circle: solid phase.
Next, the product scope of the reaction towards a projected
library synthesis was explored:
a hydrazide with the
photocleavable 3,4-methylenedioxy-2-nitro-benzyl protective
group 2b that is removed upon UV-irradiation at 365 nm, a
hydrazide that is substituted with an acetyl group instead of
Boc, and a protected amino-substituted benzyl group 2c, and
also the hexinol 3b all furnished the target hexT spirocycle
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conjugates 4b-d (Table 2, Table S2, see ESI for MALDI MS
spectra of all products).
View Article Online
DOI: 10.1039/C7OB02347B
Multi-component reactions open up the very attractive
opportunity to exploit each reactant as connection to the DNA.
Thus, library diversity can be efficiently accessed with one
chemical transformation, and overlapping sets of reactants.
Therefore, we next investigated the feasibility of conjugating
the hydrazide to the hexT adapter oligonucleotide, and
converting this compound to the target spirocycle by the Au(I)-
mediated reaction.
Table 3 Reaction optimization for the conversion of the hexathymidine-hydrazide
conjugate hexT-2d to spirocycle hexT-4e by Au(I)-mediated three-component reaction
(see Fig. 3).a
entry
solvent
cat.
(eq.)
250
250
250
250
250
250
1b
(eq.)
500
500
500
500
500
500
3a
time
[h]
20
20
20
14
20
20
4e
[%]b
15
35
25
20
40
5
(eq.)
1000
1000
1000
1000
1000
1000
1
2
3
4
5
6
MeOH
DMF
MeCN
THF
THF
1,2-DCE
a reaction performed on a 30 nmol scale; b conversion estimated by HPLC analysis
of the crude reaction.
Fig. 4 Synthesis of the hexT-6-oxa-1,2-diazaspiro[4.4]nonane conjugate 4j from hexT-
alkynol 3c. A) reaction scheme of the spirocycle formation, reagents and conditions: a)
30 nmol scale, 250 eq. tris(2,4-di-tert-butylphenyl)phosphite]gold chloride, 250 eq.
AgSbF6, 1000 eq. 1b, 500 eq. 2e, MeCN, 45°C, 17 hours; b) aq. NH3/MeNH2, room
temperature, 30 min. B) HPLC trace of the crude reaction mixture showing formation of
the water addition product hexT-12 as major product, and the target spirocycle 4j
closely eluting with several side products. PMB: para-methoxybenzyl. R: para-
(aminocarbonylmethyleneoxy)phenyl. Wavy bond to hexT: 5‘-(C6)-amino-linker, bold
bond: connection to solid phase, filled circle: solid phase.
This approach to the target spirocycle would be more attractive
from a library synthesis perspective as it benefits from the
abundant availability of aldehyde building blocks. We first
synthesized the hexT-hydrazide conjugate hexT-2d from the
aldehyde hexT-1a by reductive amination with tert-butyl
carbazate (Figs. 3, and S3, ESI). This conjugate hexT-2d was
then reacted with benzaldehyde 1b, and pentynol 3a to furnish
hexT-spirocycle 4e under several reaction conditions (Fig. 3A,
Tables 3 and S3, ESI). Again, long reaction times, large excess
of reactants and THF as solvent turned out to be required for
product formation (Tables 3, and S3, ESI). However,
conversion rates to the hexT-spirocycle 4e were in our hands
much lower than those to hexT-spirocycle 4a, and in contrast to
the latter, we obtained 4e as part of a complex compound
mixture in each reaction (Fig. 3B). This product mixture
contained the co-eluting Boc-cleavage products hexT-8, and
the imine hexT-9 which resulted from incomplete reduction in
that required HPLC purification, calling the utility of this
approach for library synthesis in question.
Table
4 Reaction optimization for the conversion of the hexythymidine-alkynol
conjugate hexT-3c to spirocycle hexT-4j by Au(I)-mediated three-component reaction
(see Fig. 4).a
entry
solvent
cat.
T
[°C]
45
25
45
50
55
45
45
45
45
45
45
45
time
[h]
17
17
17
17
17
17
17
17
41
17
17
17
add.b
4j
[%]c
-
1
2
3
4
5
6
7
8
9
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
THF
Au(I)/Ag(I)
Au(I)/Ag(I)
Au(I)/Ag(I)
Au(I)/Ag(I)
Au(I)/Ag(I)
Au(I)
-
the preceding step; the azomethine imine side product hexT-10
,
MS 5Å
MS 5Å
MS 5Å
MS 5Å
MS 5Å
MS 5Å
MS 5Å
MS 5Å
MS 5Å
MS 5Å
MS 5Å
-
which formed in analogous manner to the side product hexT-5
observed in the reaction to hexT-4a; a side-product that we
tentatively assigned the structure of the Mannich-type product
hexT-11, as a similar side-product formed in the synthesis of
pyrazolines11 (see also Fig. S19, ESI); and the target product 4e
which eluted as a distinct peak with nearly the same retention
time as hexT-4a (Fig. S20, ESI). The side products were
characterized with control experiments, and by MALDI
analysis (see ESI). Next, the scope of the reaction was explored
with a few aldehydes. Substituted electron-rich and electron-
poor aromatic, a heterocyclic, and an aliphatic aldehyde all
furnished the target hexT-conjugates 4f-i (Table S4, ESI).
However, we obtained in each reaction a compound mixture
40
30
25
-
Ag(I)
-
Au(I)/Ag(I)
Au(I)/Ag(I)
Au(I)/Ag(I)
Au(I)/Ag(I)
Au(I)/Ag(I)
30
10
25
trace
-
THF
10
11
12
C2H4Cl2
toluene
DMF
a reaction performed on a 30 nmol scale with 1000 eq. of aldehyde 1b, 500 eq. of
hydrazide 2e, and 250 eq. of tris(2,4-di-tert-butylphenyl)phosphite]gold chloride,
AgSbF6; b additive; c conversion estimated by HPLC analysis of the crude reaction.
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To complete the walk around the three component reaction, all tested conditions complex product mixtures from which the
View Article Online
DOI: 10.1039/C7OB02347B
we coupled an alkynol (19, Fig. S4, ESI) to the hexT furnishing target spirocycles had to be isolated by careful ion-pair HPLC
hexT-3c, and reacted this hexT-conjugate with benzaldehyde purification, and we were not able to completely suppress the
1b and hydrazide 2e to the hexT-spirocycle 4j at elevated addition of water to the starting material hexT-3c. Thus,
temperature of 45 °C (Fig. 4A).13 Initial efforts to obtain the starting library synthesis from the hexT-alkynol conjugate
target molecule were thwarted by the formation of the water hexT-3c is according to these results not a viable option.
adduct hexT-12 as sole product (Table 4, entry 1, see extended
Next, we were curious to learn about the tolerance of
Table S5, ESI). The water adduct can be explained by the different DNA sequences to the optimized reaction conditions
Au(I)-mediated formation of a highly reactive cyclic enol established for the Au(I)-mediated synthesis of hexT-
ether13 from the alkynol hexT-3c which is hydrolysed by the diazaoxaspirononane 4a
.
For this experiment aldehyde
massive amounts of water generated by the condensation of conjugates of six different solid phase-bound DNA sequences
aldehyde 1b and hydrazine 2b. Therefore, all further reactions were synthesized: hexC-1a hexTC-1a hexA-1a hexG-1a,
,
,
,
were performed adding the water scavengers MgSO4 or hexACT-1a, and hexACGT-1a. These were reacted with each
molecular sieves to the reaction mixture. Under these 500 eq. of hydrazide 2a and 1000 eq. of pentynol 3a, and 250
conditions, formation of a product corresponding to the mass of eq. of Au(I) and AgSbF6 to furnish DNA-6-oxa-1,2-
the hexT-spirocycle conjugate hexT-4j was detected (entry 3). diazaspiro[4.4]nonane conjugates. In addition to the hexT-
However, product yields were modest, and made an conjugate hexT-4a, we were able to isolate distinct product
investigation of the reaction conditions necessary. Running the peaks for the oligopyrimidine conjugates hexC-4a and hexTC-
reaction at 25°C returned unreacted starting material, increasing 4a. The nucleosidic bond of both pyrimidine nucleosides is
the temperature to 50°C or 55°C (entries 2-5), and also much more stable than that of the purines, and thus the
prolonged reaction time at 45°C resulted in lower product pyrimidines tolerate incubation with Lewis acids better. As
formation due degradation (entries 9). Among the solvents that expected, incubating high concentrations of the Lewis acidic,
were tested, acetonitrile, THF, and dichloroethane yielded the oxidizing agents Au(I) and Ag(I) with the hexaguanine
spiroheterocycle (entries 3, 8, 10), whereas no or only traces of sequence hexG-1a
, and also with the DNA sequence
the product were detected in toluene and DMF. Thus, the hexACGT-1a that contained all four nucleobases had
spirocycle formation displayed a narrow window with respect disastrous consequences for the DNA (Fig S30, ESI). However,
to reaction conditions, and it required the extra effort to keep surprisingly, the hexaadenine-aldehyde conjugate hexA-1a
the solvent dry in order to reduce formation of the water adduct tolerated the conditions of Au(I) catalysis (Table 1) and was
hexT-12
.
converted
to
the
corresponding
6-oxa-1,2-
diazaspiro[4.4]nonane conjugate hexA-4a
designed a DNA sequence that was devoid of the nucleobase
guanine, synthesized the aldehyde conjugate of this sequence
.
We therefore
Table 4 Tolerance of different DNA sequences to the Au(I)-mediated three-component
reaction. Reagents and conditions: a) 30 nmol scale, 250 eq. tris(2,4-di-tert-
butylphenyl)phosphite]gold chloride, 250 eq. AgSbF6, 500 eq. 2a, 1000 eq. 3a, THF,
room
temperature,
20
hours;
b)
aq.
NH3/MeNH2.
R:
para-
hexACT-1a
spirocycle hexACT-4a
, and indeed reacted it successfully to the
(aminocarbonylmethyleneoxy)phenyl. Wavy bond to DNA: 5‘-(C6)-amino-linker, bold
bond: connection to solid phase, filled circle: solid phase.
.
entry
sequence
5’-3’
DNA-4a
mass calc.
2392.8
2302.7
2347.7
2446.9
2371.7
2542.8
2421.7
DNA-4a
mass founda
2394.5
2304.7
2351.9
2448.7
2374.2
-
1
2
3
4
5
6
7
4a-TTTTTT
4a-CCCCCC
4a-TCTCTC
4a-AAAAAA
4a-TCATCT
4a-GGGGGG
4a-CAGTCG
-
a measured by MALDI-MS.
Fig. 5 Synthesis of a hexTC-tagged β-carboline hexTC-15 from the corresponding
tryptophane conjugate hexTC-14 by trifluoroacetic acid-catalyzed Pictet-Spengler
reaction. Reagents and conditions : a) HATU, DIPEA, DMF, room temperature, 4 hours;
b) piperidine, DMF; c) 2 % trifluoroacetic acid, dichloromethane, room temperature, 18
hours; d) aq. NH3/MeNH2, room temperature, 30 min. Wavy bond to hexTC: 5‘-(C6)-
The reaction gave the target products with benzaldehydes,
and cyclopropyl carboxaldehyde, and with several
unsymmetrically substituted hydrazides. The latter could be
substituted with different benzyl groups, and with a tert-Boc or amino-linker, bold bond: connection to solid phase, filled circle: solid phase.
an acetyl group (Table S6, ESI). However, we obtained under
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We attribute the differential stability of the adenine- versus the following small molecule synthesis.11 Here, we demonstrate the
View Article Online
DOI: 10.1039/C7OB02347B
guanine-containing sequences under conditions of the Au(I)- synthesis of hexathymidine DNA conjugates of the 6-oxa-1,2-
mediated reaction to the lower redox potential of guanine diazaspiro[4.4]nonane scaffold,
a fully saturated, highly
versus adenine,14 and the higher susceptibility of guanine to substituted spirocyclic structure, from simple, readily available
electrophilic attack, which is documented e.g. for the reaction aldehydes, hydrazides, and alkynols by a Au(I)-mediated
of cisplatin with DNA.15
reaction. Reactions that employ three or even more reactants
Having established that both pyrimidine nucleobases were offer the opportunity to append any one of these to the DNA,
stable under the conditions of the Au(I)-mediated thereby varying the connectivity between small molecule and
spirocyclization reaction, we revisited the previously published tag. We appended any of the three reactants required for
hexT-compatible Au(I)-mediated annulation reaction yielding synthesis of the target 6-oxa-1,2-diazaspiro[4.4]nonane to the
highly substituted pyrazol(in)es (Fig. 1B), and the acid- hexT adapter oligonucleotide, and optimized reaction
catalyzed Pictet-Spengler reaction.11 Coupling Fmoc-protected conditions for product formation. However, it turned out that
tryptophan to the 5’-aminolinker modified hexTC yielded the DNA-aldehyde conjugate hexT-1a was the only viable
hexTC-14 which was N-deprotected on solid support, and option for library synthesis, yielding the target spirocycle with
reacted with benzaldehyde 1b under catalysis by the strong minimal formation of side products (Fig. 2). The corresponding
protic acid TFA. Gratifyingly, the reaction furnished the target reaction employing the hydrazide conjugate hexT-2d returned a
β-carboline hexTC-15 (Figs 5, and S31/32 ESI). In a further complex product mixture (Fig. 3) due to massive formation of
experiment para-ethynylbenzoic acid 16 was coupled to hexTC degradation and side products. Also, the alkynol hexT-3c gave
furnishing the conjugate hexTC-17
.
the target product in low yields, mainly due to addition of water
to the intermediate cyclic enol ether,13 and as a mixture making
tedious purification necessary which precludes parallel
synthesis of larger numbers of compounds. Next, several DNA
sequences were tested for their compatibility with the Au(I)-
mediated reaction. While both pyrimidine bases tolerated the
reaction conditions, the guanine-containing sequences were
degraded to
a degree that precluded product analysis.
Surprisingly, we found the adenine-containing hexA, and
consequently the sequence hexATC sufficiently stable to allow
for product isolation. In conclusion, this study prompts an
investigation in the tolerance of the four DNA nucleobases to
diverse reaction conditions, which may eventually be exploited
by the design of novel DNA-tagging strategies.
Conflicts of interest
There are no conflicts to declare.
Fig. 6 Synthesis of a hexTC-tagged pyrazoline hexTC-18 from the alkyne conjugate
hexTC-17, isobutyraldehyde 1g and the N-benzyl- and N´-Boc-substituted hydrazide 2a
by a Au(I)-mediated annulation reaction. Reagents and conditions: a) HATU, DIPEA,
Acknowledgements
We are grateful for financial support by the BMBF grant
1316053.
DMF, room temperature,
4 hours; b) 30 nmol scale, 250 eq. tris(2,4-di-tert-
butylphenyl)phosphite]gold chloride, 250 eq. AgSbF6, 1000 eq. 1g, 1000 eq. 2a, MeCN,
50°C, 20 hours; c) aq. NH3/MeNH2, room temperature, 30 min. Wavy bond to hexTC: 5‘-
(C6)-amino-linker, bold bond: connection to solid phase, filled circle: solid phase.
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