Replication of synthetic recognition-encoded oligomers by ligation of trimer building blocks

The development of methods for replication of synthetic information oligomers will underpin the use of directed evolution to search new chemical space. Template-directed replication of triazole oligomers has been achieved using a covalent primer in conjunction with non-covalent binding of complementary building blocks. A phenol primer equipped with an alkyne was first attached to a benzoic recognition unit on a mixed sequence template via selective covalent ester base-pair formation. The remaining phenol recognition units on the template were then used for non-covalent binding of phosphine oxide oligomers equipped with an azide. The efficiency of the templated CuAAC reaction between the primer and phosphine oxide building blocks was investigated as a function of the number of H-bonds formed with the template. Increasing the strength of the non-covalent interaction between the template and the azide lead to a significant acceleration of the templated reaction. For shorter phosphine oxide oligomers intermolecular reactions compete with the templated process, but quantitative templated primer elongation was achieved with a phosphine oxide 3-mer building block that was able to form three H-bonds with the template. NMR spectroscopy and molecular models suggest that the template can fold, but addition of the phosphine oxide 3-mer leads to a complex with three H-bonds between phosphine oxide and phenol groups, aligning the azide and alkyne groups in a favourable geometry for the CuAAC reaction. In the product duplex, 1H and 31P NMR data confirm the presence of the three H-bonded base-pairs, demonstrating that the covalent and non-covalent base-pairs are geometrically compatible. A complete replication cycle was carried out starting from the oligotriazole template by covalent attachment of the primer, followed by template-directed elongation, and hydrolysis of the the ester base-pair in the resulting duplex to regenerate the template and liberate the copy strand. We have previously demonstrated sequence-selective oligomer replication using covalent base-pairing, but the trimer building block approach described here is suitable for replication of sequence information using non-covalent binding of the monomer building blocks to a template.


General experimental details.
All the reagents and materials used in the synthesis of the compounds described below were bought from commercial sources, without prior purification.Dry THF and CH2Cl2 were obtained from a solvent purification system (Pure Solv™, Innovative Technology, Inc.).Anhydrous DMF was purchased from Sigma-Aldrich.Thin layer chromatography was carried out using with silica gel 60F (Merck) on glass plates.Flash chromatography was carried out on an automated system (Combiflash Rf+ or Combiflash Rf Lumen) using prepacked cartridges of silica (25μ PuriFlash® columns).All NMR spectroscopy was carried out on a Bruker 400 MHz DPX400, 400 MHz AVIII400, 500 MHz DCH cryoprobe or 500 MHz TCI Cryoprobe spectrometer using the residual solvent as the internal standard.All chemical shifts (δ) are quoted in ppm and coupling constants given in Hz.Splitting patterns are given as follows: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet).FT-IR spectra were measured on a Bruker Alpha spectrometer equipped with an ATR cell.UPLC analysis of samples was performed using Waters Acquity H-class UPLC coupled with a single quadrupole Waters SQD2.Acquity UPLC CSH C18 column, 130 Å, 1.7 µm, 2.1 mm x 50 mm or Acquity UPLC BEH C8 column, 130 Å, 1.7 μm, 2.1 mm x 50 mm were used as UPLC columns.The conditions of the UPLC method are as follows: gradients of water + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B) as specified in each case.Flow rate: 0.6 ml/min; Column temperature of 40 o C; Injection volume of 2 µL.The signal was monitored at 254 nm.HRMS analysis was performed in an Agilent walk up 6230 LC/TOF using a gradient from 5 to 100% of acetonitrile (0.25% formic acid) in water (0.25% formic acid) over 6 minutes.

Synthesis of 4-mer template and loading of primer.
The synthesis of the 4-mer template and the corresponding primer-loaded derivative is highlighted in Scheme S1.From 2-mer 1 [S1] , the CuAAC coupling with protected monomer 2 [S1] and subsequent TBAF-mediated deprotection yielded 3-mer 3. CuAAC coupling of 3 with monomer 4 [S1] gave rise to 4-mer template 5. Primer 6 [S1] was directly loaded onto the template by ester coupling and the primer-loaded derivative 7 was obtained in low yield due to the preferential formation of the corresponding lactone.

Templating reaction using primed template 7, 3-mer 8 and 1-mer 9
Compound 7 is used as template for the template-directed synthesis of its complementary duplex from the alkyne functionality provided by the primer.Two different azides are used: 8 is the complementary phosphine oxide 3-mer while 9 is the non-complementary one.CuAAC reaction with 7 leads to the formation of two products: 10 is the non-templated product from the reaction of 7 and 9 while 11 is the templated product from the reaction of 7 and 8 (Scheme S2.The template effect provided by H-bonding between the phenol groups in the template and the phosphine oxides of the 3-mer is studied using the simple alkyne S1 as control (Scheme S3).

Scheme S2.
Reaction schemes for the template-directed synthesis of the sequence complementary duplex from 7 using 1-mer 9 and 3-mer 8.

Scheme S3.
Control reaction where no template effect is possible.

Synthesis of templated product 11 (for characterization purposes)
A solution of 7 (2.

Hydrolysis of duplex 11 providing copy 12
Basic hydrolysis of the ester bond in duplex 11 afforded the corresponding complementary copy (12) along with the starting template 5, as shown in scheme S4. Figure S2 shows the corresponding UPLC traces for the hydrolysis reaction and isolation of template and copy.

Full replication cycle from template 5
The full replication cycle from template 5 is illustrated in Scheme S5.The first step involves covalent attachment of the primer 6 by ester coupling to give the primed template 7. The template-directed CuAAC reaction was then carried out using a mixture of the complementary phosphine oxide 3-mer 8 and a competing phenol 1-mer 9.The final step is the cleavage of the covalent ester base-pair in the isolated duplex to release the template and the copy strand.The two oligomers were separated and the pure copy 12 was fully characterized.

Scheme S4 .
Scheme S4.Cleavage of the ester in 11 to give access to copy 12 along with template 5.
Scheme S5.Full replication cycle from template 5.