RESUMEN

Block-truncated poly(phosphodiester)s are digital macromolecules storing binary information that can be decoded by MS/MS sequencing of individual blocks released as primary fragments of the entire polymer. As such, they are ideal species for the serial sequencing methodology enabled by MS-(CID)-IMS-(CID)-MS coupling, where two activation stages are combined in-line with ion mobility spectrometry (IMS) separation. Yet, implementation of this coupling still requires efforts to achieve IMS resolution of inner blocks, that can be considered as small oligomers with α termination composed of one nitroxide decorated with a different tag. As shown by molecular dynamics simulation, these oligomers adopt a conformation where the tag points out of the coil formed by the chain. Accordingly, the sole nitroxide termination was investigated here as a model to reduce the cost of calculation aimed at predicting the shift of collision cross-section (CCS) induced by new tag candidates and extrapolate this effect to nitroxide-terminated oligomers. A library of 10 nitroxides and 7 oligomers was used to validate our calculation methods by comparison with experimental IMS data as well as our working assumption. Based on conformation predicted by theoretical calculation, three new tag candidates could be proposed to achieve the +40 Å2 CCS shift required to ensure IMS separation of oligomers regardless of their coded sequence.

RESUMEN

A library of phosphoramidite monomers containing a main-chain cleavable alkoxyamine and a side-chain substituent of variable molar mass (i.e. mass tag) was prepared in this work. These monomers can be used in automated solid-phase phosphoramidite chemistry and therefore incorporated periodically as spacers inside digitally-encoded poly(phosphodiester) chains. Consequently, the formed polymers contain tagged cleavable sites that guide their fragmentation in mass spectrometry sequencing and enhance their digital readability. The spacers were all prepared via a seven steps synthetic procedure. They were afterwards tested for the synthesis and sequencing of model digital polymers. Uniform digitally-encoded polymers were obtained as major species in all cases, even though some minor defects were sometimes detected. Furthermore, the polymers were decoded in pseudo-MS3 conditions, thus confirming the reliability and versatility of the spacers library.

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A major step towards reliable reading of information coded in the sequence of long poly(phosphodiester)s was previously achieved by introducing an alkoxyamine spacer between information sub-segments. However, MS/MS decoding had to be performed manually to safely identify useful fragments of low abundance compared to side-products from the amide-based alkoxyamine used. Here, alternative alkoxyamines were designed to prevent side-reactions and enable automated MS/MS sequencing. Different styryl-TEMPO spacers were prepared to increase radical delocalization and stiffness of the structure. Their dissociation behavior was investigated by EPR and best results were obtained with spacers containing in-chain benzyl ring, with no side-reaction during synthesis or sequencing. Automated decoding of these polymers was performed using the MS-DECODER software, which interprets fragmentation data recorded for each sub-segment and re-align them in their original order based on location tags.

RESUMEN

Light-induced alteration of macromolecular information plays a central role in biology and is known to influence health, aging and Darwinian evolution. Here, we report that light can also trigger sequence variations in abiotic information-containing polymers. Sequence-coded poly(phosphodiester)s were synthesized using four phosphoramidite monomers containing either photo-sensitive or photo-inert substituents. These monomers allow different sequence manipulations. For instance, using two light-cleavable monomers containing o-nitrobenzyl ether and o-nitroveratryl ether motifs, photo-erasable digital polymers were prepared. These polymers can be decoded by tandem mass spectrometry but become unreadable after UVA exposure. The opposite behavior, i.e. photo-revealable sequences, was obtained with polymers made of two isobaric monomers containing light-cleavable o-nitrobenzyl ether and light-inert p-nitrobenzyl ether substituents. Furthermore, when the latter two monomers were used in conjunction with a third monomer bearing a light-inert OH group, site-directed photo-mutations were induced in synthetic polymers. This was used herein to change the meaning of binary sequences.

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RESUMEN

In order to improve their MS/MS sequencing, structure of sequence-controlled synthetic polymers can be optimized based on considerations regarding their fragmentation behavior in collision-induced dissociation conditions, as demonstrated here for two digitally encoded polymer families. In poly(triazole amide)s, the main dissociation route proceeded via cleavage of the amide bond in each monomer, hence allowing the chains to be safely sequenced. However, a competitive cleavage of an ether bond in a tri(ethylene glycol) spacer placed between each coding moiety complicated MS/MS spectra while not bringing new structural information. Changing the tri(ethylene glycol) spacer to an alkyl group of the same size allowed this unwanted fragmentation pathway to be avoided, hence greatly simplifying the MS/MS reading step for such undecyl-based poly(triazole amide)s. In poly(alkoxyamine phosphodiester)s, a single dissociation pathway was achieved with repeating units containing an alkoxyamine linkage, which, by very low dissociation energy, made any other chemical bonds MS/MS-silent. Structure of these polymers was further tailored to enhance the stability of those precursor ions with a negatively charged phosphate group per monomer in order to improve their MS/MS readability. Increasing the size of both the alkyl coding moiety and the nitroxide spacer allowed sufficient distance between phosphate groups for all of them to be deprotonated simultaneously. Because the charge state of product ions increased with their polymerization degree, MS/MS spectra typically exhibited groups of fragments at one or the other side of the precursor ion depending on the original α or ω end-group they contain, allowing sequence reconstruction in a straightforward manner. Graphical Abstract ᅟ.

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Sequence-defined peptide triazole nucleic acids (PTzNA) were synthesized by means of a solid-phase orthogonal "AB+CD" iterative strategy. In this approach, AB and CD building blocks containing carboxylic acid (A), azide (B), alkyne (C), and primary amine (D) functions are assembled together by successive copper-catalyzed azide-alkyne cycloaddition (CuAAC) and acid-amine coupling steps. Different PTzNA genetic sequences were prepared using a library of eight building blocks (i.e., four AB and four CD building blocks).

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A new orthogonal solid-phase iterative strategy is proposed for the synthesis of sequence-coded polymers. This approach relies on the use of two successive chemoselective steps: (i) phosphoramidite coupling, and (ii) radical-radical coupling. These repeated steps can be performed using two different types of building blocks, i.e. a phosphoramidite monomer that also contains an alkyl bromide and a hydroxy-functionalized nitroxide. The phosphoramidite and the hydroxy group are reacted in step (i), thus leading to a phosphite that is oxidized in situ into a phosphate bond. The alkyl bromide is activated by copper bromide in step (ii) to afford a carbon-centered radical that is spin-trapped in situ by the nitroxide. The iterative repetition of these steps allow synthesis of uniform polymers, as evidenced by high-resolution electrospray mass spectrometry. Moreover, binary information could be easily implemented in the polymers using different types of phosphoramidite monomers in step (i). Interestingly, it was found that the formed information-containing polymers are very easy to sequence by tandem mass spectrometry due to the presence of easily cleavable alkoxyamine bonds formed in step (ii).

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Soluble polystyrene supports with optimal molecular structures for iterative phosphoramidite chemistry were prepared by atom-transfer radical polymerization (ATRP) and subsequent chain-end modification steps. The controlled radical polymerization of styrene was first performed in the presence of an 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino-functional ATRP initiator. Soluble supports of different molecular weight were prepared. Size-exclusion chromatography and NMR analysis indicated formation of well-defined polymers with controlled chain lengths and narrow dispersity. After synthesis, the bromo ω end group of the ATRP polymer was removed by dehalogenation in the presence of tributyltin hydride, and the Fmoc protecting group of the α moiety was subsequently cleaved with piperidine. The resulting α-primary amine was afterwards treated with a linker containing a carboxyl group, a cleavable ester site, and a dimethoxytrityl-protected hydroxyl group to afford ideal soluble supports for phosphoramidite chemistry. NMR analysis indicated that these chain-end modifications were quantitative. The supports were tested for the synthesis of a non-natural sequence-defined oligophosphates. High-resolution ESI-MS analysis of the cleaved oligomers indicated formation of uniform species, and thus confirmed the efficiency of the ATRP-made soluble polymer supports. In addition, the synthesis of a thymidine-loaded soluble support was achieved.

RESUMEN

A simplified strategy for preparing non-natural information-containing polymers is reported. The concept relies on the successive ligation of oligomers that contain minimal sequence motifs. It was applied here to the synthesis of digitally-encoded poly(triazole amide)s, in which propyl and 2-methyl propyl motifs are used to code 0 and 1, respectively. A library of four oligo(triazole amide)s containing the information dyads 00, 01, 10, and 11 was prepared. These oligomers contain two reactive functions, that is, an alkyne and a carboxylic acid. Thus, they can be linked to another with the help of a reactive spacer containing azide and amine functions. Using two successive chemoselective steps, that is, azide-alkyne Huisgen cycloaddition and carboxylic acid-amine coupling, monodisperse polymers can be obtained. In particular, the library of dyads permits the implementation of any desired sequence using a small number of steps. As a proof-of-concept, the synthesis of molecular bytes 00000000 and 00000110 is described.

RESUMEN

A library of eight sequence-defined model oligomers, whose sequence is based on a (0,1) binary code, is prepared through chemoselective repeating cycles of amidification and copper-assisted alkyne-azide cycloaddition reactions from a non-modified Wang resin. This library is constructed from two AB (A = acid, B = alkyne) building blocks, i.e., 4-pentynoic acid and 2-methyl-4-pentynoic acid acting, respectively, as non-coding (0) and coding (1) monomer, and 1-amino-11-azido-3,6,9-trioxaundecane as complementary CD (C = amine, D = azide) spacer building block. In particular, encoded triads are synthesized by consecutive covalent attachment of five building blocks (i.e., three coding/non-coding monomers and two spacers). In this communication, optimal protocols for the synthesis of the targeted oligomers are reported along with their full characterization by (1) H NMR, MALDI-TOF mass spectrometry, and size-exclusion chromatography. It is found that all possible encoded triads (i.e., eight possibilities) could be synthesized using this approach. Indeed, monodisperse sequence-defined oligomers are prepared and characterized in all cases.

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Charged macromolecules with controlled microstructures were prepared. Well-defined non-ionic precursors were first synthesized by sequence-controlled radical polymerization of tert-butyl 4-vinyl benzoate with various N-substituted maleimides. Afterwards, these macromolecules were hydrolyzed into polyanions.

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Nucleic acid-templated reactions leading to a fluorescent product represent an attractive strategy for the detection and imaging of cellular nucleic acids. Herein we report the use of a Staudinger reaction to promote the reduction of profluorescent azidorhodamine. The use of two cell-permeable GPNA probes, one labeled with the profluorescent azidorhodamine and the other with trialkylphosphine, enabled the detection of the mRNA encoding O-6-methylguanine-DNA methyltransferase in intact cells.

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