Solid-Phase Synthesis of β-Amino Ketones Via DNA-Compatible Organocatalytic Mannich Reactions

Article abstract of DOI:10.1021/acscombsci.7b00151

One-bead-one-compound (OBOC) libraries constructed by solid-phase split-and-pool synthesis are a valuable source of protein ligands. Most OBOC libraries are comprised of oligoamides, particularly peptides, peptoids, and peptoid-inspired molecules. Further diversification of the chemical space covered by OBOC libraries is desirable. Toward this end, we report here the efficient proline-catalyzed asymmetric Mannich reaction between immobilized aldehydes and soluble ketones and anilines. The reaction conditions do not compromise the amplification of DNA by the PCR. Thus, this chemistry will likely be useful for the construction of novel DNA-encoded libraries by solid-phase synthesis.

Full text of DOI:10.1021/acscombsci.7b00151

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Article
Solid-Phase Synthesis of #-Amino Ketones Via DNA-
Compatible Organocatalytic Mannich Reactions
Nam Tran Hoang, and Thomas Kodadek
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00151 • Publication Date (Web): 09 Jan 2018
Downloaded from http://pubs.acs.org on January 10, 2018
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SolidꢀPhase Synthesis of βꢀAmino Ketones
Via DNAꢀCompatible Organocatalytic Mannich Reactions
Nam Tran-Hoang and Thomas Kodadek*
Department of Chemistry
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The Scripps Research Institute
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30 Scripps Way, Jupiter, Florida 33458
*
To whom correspondence should be addressed. Eꢀmail: kodadek@scripps.edu
Keywords: Mannich reaction, solidꢀphase synthesis, organocatalysis, one bead one
compound libraries, DNAꢀencoded libraries
Abstract
Oneꢀbeadꢀoneꢀcompound (OBOC) libraries constructed by solidꢀphase splitꢀandꢀpool
synthesis are a valuable source of protein ligands. Most OBOC libraries are comprised
of oligoamides, particularly peptides, peptoids and peptoidꢀinspired molecules. Further
diversification of the chemical space covered by OBOC libraries is desirable. Towards
this end, we report here the efficient prolineꢀcatalyzed asymmetric Mannich reaction
between immobilized aldehydes and soluble ketones and anilines. The reaction
conditions do not compromise the amplification of DNA by the PCR. Thus, this
chemistry will likely be useful for the construction of novel DNAꢀencoded libraries by
solidꢀphase synthesis.
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Introduction
An important first step in many probe/drug discovery campaigns is a high
throughput screen against a target of interest. Traditional highꢀthroughput screening
(HTS) is expensive and requires elaborate infrastructure. Thus, there is continuing
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interest in the development of faster and cheaper ways to screen large numbers of
compounds. For example, Houghten and colleagues have demonstrated the power of
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ꢀ3
positional scanning libraries for this purpose. Another popular format has been one
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bead one compound (OBOC) libraries created by solidꢀphase split and pool synthesis.
These libraries can be mined for protein ligands using binding assays with labeled
5ꢀ16
proteins or even cells.
While the identification of protein ligands in OBOC libraries
was slowed by nagging technical problems, most of these issues have now been
17ꢀ20
solved.
In most OBOC screening efforts, the structures of the molecules on beads
scored as hits had to be characterized de novo, usually by tandem mass spectrometry
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, 21ꢀ22
and/or Edman sequencing
after cleavage of the compound from the bead. This
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limited OBOC libraries to mostly oligoamides, such as peptides, peptoids,
and
24ꢀ29
peptoidꢀlike conformationally constrained species.
This limitation can be
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circumvented by encoding strategies, such as Lam’s one bead two compound strategy,
in which the synthetic history of a molecule displayed on the bead surface is encoded
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by a peptide or peptoid created in the proteinꢀinaccessible interior of the bead. More
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recently, DNAꢀencoding technology has been applied to OBOC libraries, which has
the advantage of allowing much smaller beads, such as 10 ꢁm TentaGel microspheres,
to be employed for library construction. This is because the encoding tags on the beads
scored as hits are amplified by PCR prior to sequencing. Thus, very little material is
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needed. 10 ꢁm beads pass readily through a flow cytometer, vastly increasing the
number of beads that can be analyzed and thus the size of the libraries that can be
employed. This advance has highlighted the need to expand the chemical diversity of
DNAꢀencoded OBOC libraries beyond oligoamides and related molecules. We are
particularly interested in exploring solidꢀphase reactions that produce conformationally
restricted chiral centers, since there is general agreement that molecules with more
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“
threeꢀdimensionality” will be more selective ligands than the highly aromatic,
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hydrophobic species that dominate many typical screening collections.
Of course, a central issue in the development of new reaction schemes in this
format is compatibility with the encoding DNA. Enough DNA on each bead must remain
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amplifiable at the end of the library synthesis to allow facile identification of hits.
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Therefore, we were attracted to organocatalytic methods,
which generally proceed
under mild conditions. In this report, we focus on the prolineꢀcatalyzed Mannich reaction,
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pioneered by List and coꢀworkers.
While Mannich reactions have been used for
these efforts did not employ organocatalysis or
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solidꢀphase synthesis previously,
produce optically enriched products. We demonstrate that addition of a variety of
soluble ketones and anilines with a resinꢀbound aldehyde proceeds with good yield and
stereoselectivity in many cases. The process is shown to have little effect on the
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amplifiability of DNA using a quantitative PCR assay and thus will likely be of utility for
the construction of DNAꢀencoded OBOC libraries.
Results and Discussion
TentaGel microspheres (90 ꢁm; 0.29 mmol loading per gram) were modified with
a disulfideꢀcontaining linker (Figure 1) to allow the release of compounds from the solid
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support under reductive conditions (treatment with TCEP)
to avoid potential
racemization of the Mannich product under the usual acidic cleavage conditions. The
amino groups in the linker were acylated with 4ꢀformyl benzoic acid using Oxyma
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coupling conditions (Figure 1). The aldehydeꢀdisplaying resin (50 mg) was incubated
for five minutes at room temperature with solution of 4ꢀmethoxyaniline (7 mg; 4 eq.) in
1
45 ꢁL DMSO, then a preꢀsonicated solution of (S)ꢀproline catalyst (1.67 mg, 1 eq.) and
1ꢀmethoxypropanꢀ2ꢀone (13.5 L; 10 eq.) in 145 ꢁL DMSO was added to reaction vessel
without removal of the excess aniline. After incubating for 18 hours at room temperature
with gentle shaking, the beads were washed thoroughly to remove excess reactants.
The disulfide linkage was reduced using TCEP (16.63 mg; 4 eq.) and NaHCO (19.49
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mg; 16 eq.) in 290 ꢁL H O). The organic products were extracted with dichloromethane,
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dried with MgSO , and analyzed by liquid chromatographyꢀmass spectrometry (LCꢀMS).
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Two peaks having the mass of the expected Mannich products were observed (Supp.
Figure 4a), suggesting that they are either diastereomers or regioisomers. To
distinguish between these possibilities, each compound was purified by HPLC and
analyzed by 1HꢀNMR spectroscopy (Supp. Figure 4). These spectra indicated that the
only regioisomer produced is the one in which the methoxyꢀsubstituted carbon acts as
the nucleophile. Furthermore, the NMR spectra argued that the syn isomer is the major
product, having the smaller 3.0 Hz vicinal coupling constant at chiral centers, instead of
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.6 Hz for the anti isomer. This result is analogous to that obtained in the solutionꢀphase
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Mannich reaction using similar substrates.
The order of elution of the anti and syn
isomers was also consistent with the order of elution of the known anti and syn isomers
produced in the solution phase reaction, (Supp. Figure 4b and Supp. Figure 3b).
Assuming the diastereomers have similar molar extinction coefficients at 254 nm, the
diastereomeric ratio (d.r.) is 79:21 (syn:anti). Chiral LC analysis of the cleaved mixtures
from Mannich reactions catalyzed by (R)ꢀproline and (S)ꢀproline indicated that the
enantiomer excess (e.e.) for the syn diastereomer is 81%, while the value for the antiꢀ
diastereomer is 56% (Supp. Figure 4b).
Figure 1: Mannich reaction on TentaGel microsphere, with disulfideꢀcontaining linker for
cleavage using TCEP. Note that the addition of 4ꢀformyl benzoic acid and subsequent
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Mannich couplings likely occurs on the secondary amine in the linker as well. Since
this material is never released from the bead and analyzed, it is abbreviated as “R”.
The absolute configuration of the two enantiomers comprising the syn
diastereomer was assigned by similar order of elution in Chiral LC, in comparison to the
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analogous solution phase reaction (Supp. Figure 3b) where the product has been
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characterized by Xꢀray crystallography: (S, R) for the major enantiomer, (R, S) for the
minor enantiomer. As the reaction was performed on solid phase in small scale, a
metric was devised to relatively quantify reaction yield: conversion–the ratio of products’
peak area over total peak area of product and starting material at 254 nm. The
conversion for the reaction is 83%.
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Attempts to improve conversion and stereoselectivity of the reaction by modifying
the solvent and temperature were unsuccessful. Hence, the conditions described above
were used to explore the scope of the reaction with respect to the aniline and ketone
components. We focused on anilines that provide an additional handle for further
elaboration of the products since we are interested in the solidꢀphase synthesis of novel,
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conformationally constrained oligomers.
Using 1ꢀhydroxypropanꢀ2ꢀone as the
nucleophile, we found that metaꢀ and paraꢀhalogenated anilines work well, proceeding
with high conversion and good stereoselectivity (Table 1). Excellent conversion was
also observed with 4ꢀFmocꢀprotected aminoꢀ and aminomethylꢀsubstituted anilines.
Unfortunately, the latter reaction provided only a 54% e.e. The e.e. of the product
produced from 4ꢀFmocꢀprotected aminoꢀaniline could not be determined because the
enantiomers failed to separate on the chiral column. Orthoꢀsubstituted anilines did not
yield the desired Mannich products in high yield, perhaps because of steric hindrance.
Benzylamine was also investigated as a possible substitute for aniline. The conversion
was an acceptable 66%, but the diasteromeric ratio was poor, being close to 1:1 (Table
1). This may be acceptable for library preparation, where mixtures of stereoisomers can
be tolerated, but it would be important to have a more selective method for hit reꢀ
synthesis to validate which of the stereoisomers is the active compound in a screening
experiment.
The scope of the reaction with respect to the ketone component is described in
Table 2. Ketones with a hydroxyl or alkoxy substituent on the nucleophilic carbon were
the best substrates. Simple ketones coupled more sluggishly. Only about 30% of the
starting aldehyde was converted to products when acetone was employed in the
reaction, though the enantioselectivity was good. Chloroacetone gave a better
conversion (53%), with good stereoselectivity.
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Given the mild conditions of the Mannich reaction, we anticipated it would be
compatible with DNA encoding tags. To test this, we used an assay described by
Malone and Paegel, in which beadꢀdisplayed duplex DNA is subjected to the reaction
conditions and the amount of DNA that remains amplifiable is determined by
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quantitative PCR. We found that the in the presence of the reactants used in Figure 1,
87% of DNA remained amplifiable relative to a control sample not subjected to any
chemistry (Supp. Figure 23 and Supp. Table 6). This chemistry will therefore be of utility
in the synthesis of DNAꢀencoded OBOC libraries.
Finally, while we employed a disulfide linker in these studies to eliminate any
chance of racemization of the chiral center upon release of the product from the bead,
subsequent investigations of the stereochemical integrity of the Mannich products
demonstrated that this concern was unfounded. Exposure of the Mannich products to
the conditions used to free compounds from RAM resins (dilute trifouroacetic acid for 15
minutes) did not result in racemization of the product. Similarly, the standard conditions
used to remove the Fmoc group also did not racemize the stereocenters (data not
shown).
42ꢀ43
In conclusion, the prolineꢀcatalyzed asymmetric Mannich reaction
has been
adapted to solidꢀphase synthesis. A variety of metaꢀ and paraꢀsubstituted anilines work
well in a reaction employing an immobilized benzaldehyde and soluble hydroxyacetone
(Table 1). The syn product is favored with the diastereoisomeric ratio ranging from
approximately 20:1 in the best case to 3:1 in the worst. The enantiomeric excess of the
syn product varies from 96% to 54%. The scope of the reaction with respect to the
ketone is somewhat limited. Only 1ꢀhydroxypropanꢀ2ꢀone and 1ꢀmethoxypropanꢀ2ꢀone
provided a high yield of Mannich products under these conditions, suggesting that a
hydroxy or alkoxy substituent is required at the nucleophilic carbon. Importantly, the
reaction conditions are tolerant of DNA, so this chemistry should be useful in the
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preparation of novel DNAꢀencoded OBOC libraries.
Experimental Section
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Linker synthesis and general solid-phase Mannich reaction protocol. TentaGel S NH2
resin (0.29 mmol per g loading) was used as the solid support. To 50 mg of DMFꢀ
equilibrated resin was added 217 ꢁL of 2M bromoacetic acid (BAA) (60 mg, 30 eq.) in
DMF and 217 ꢁL of 2M DIC (68 ꢁL, 30 eq.) in DMF (149.4 ꢁL). After vortexing briefly (≈
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seconds), the mixture was shaken gently for 5 minutes at 37°C. The supernatant was
separated from resin by applying vacuum. The resin was then washed three times with
DMF, three times with MeOH, and reꢀequilibrated in MeOH. 435 ꢁL of 1M cystamine
(97.96 mg, 30 eq.) in 2/1 v/v MeOH/DIEPA was then added to the resin, which was then
incubated for one hour at 37°C with gentle shaking. The resin was pelleted by applying
vacuum to remove supernatant, then washed three times with MeOH, three times with
DMF, and reꢀequilibrated in DMF.
To immobilize the aldehyde, 290 ꢁL of 0.2M 4ꢀformyl benzoic acid (8.71 mg), 0.2
M Oxyma (8.24 mg), 0.2M DIC (9.1 ꢁL) in DMF (280 L) (4 eq. each compared to resin
loading) were preꢀincubated for five minutes at room temperature. The solution was
then added to the resin and the beads were incubated for one hour at room temperature
with gentle shaking. After pelleting the beads, the supernatant was removed and the
beads were washed three times with DMF, three times with DMSO and then
equilibrated in DMSO.
The Mannich reaction was conducted by first adding 145 ꢁl of 0.4 M aniline (4 eq.)
in DMSO to the beads and incubating at room temperature for five minutes with gentle
shaking. During this time, a solution was prepared containing 1 M ketone (10 eq.) and
0
.1M Dꢀ or Lꢀproline (1.7 mg, 1 eq.) in a total volume of 145 ꢁl. This solution was
sonicated for five minutes, then added to the beads without draining the excess aniline.
The mixture was then shaken gently for 18 hours at room temperature. The beads were
then pelleted and washed three times with DMSO.
To release the products from the beads, the beads were first equilibrated in water.
Then 290 ꢁl of an aqueous solution containing 0.2M tris(2ꢀcarboxyethyl)phosphine
hydrochloride (TCEP−HCl) (16.63 mg, 4 eq.) and 0.8M NaHCO (19.49 mg, 16 eq.) was
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added to the beads, which were shaken gently for 1.5 hours at room temperature. The
beads were pelleted and both the bead and the supernatant was extracted with three
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times 500 ꢁl of CH Cl . The organic layer was separated and dried with MgSO . Finally,
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the liquid was filtered into a vial, the solution was flushed with argon and sealed until
analysis.
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Associated Content
Supporting Information
Full experimental details and characterization of the products are available free of
charge at http://pubs.acs.org.
Author Information
Corresponding author: Kodadek@scripps.edu
Funding
This work was supported by a grant from the National Institutes of Health (AG052813).
Acknowledgement
The authors thank Dr. Ted Kamenecka (TSRI) for his help with chiral chromatography;
Dr. Xiaohai Li and Dr. Michael Cameron (TSRI) (NIH grant 1S10OD010603) for their
help with HRMS; Dr. Jeremy Mason for useful discussions concerning interpretation of
NMR spectra; Vuong Dang, Kevin Pels, Paige Dickson, and Scott Simanski (TSRI) for
their help with the DNA damage assay and for technical advice.
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