Optically verified polymer synthesis

Abstract

Compositions and methods for optically-verified, sequence-controlled polymer synthesis are described.

Claims

1. A template-free method of synthesizing a polymer chain of a pre-determined sequence of polymers, the method comprising the steps of: a) immobilizing a polymer chain seed molecule on a solid support; b) optionally verifying immobilization of the seed molecule by fluorescent detection means; c) contacting the immobilized seed molecule with a monomer unit comprising a polymer building block component of interest, wherein the polymer building block component comprises a fluorescent-labelled, removable or cleavable protecting group selected from the group consisting of a boronate analog, a silyl group or a silyl analog, under conditions and with reagents suitable for the addition of the monomer unit/polymer building block to the immobilized seed molecule; d) removing excess monomer units and reagents not added to the immobilized seed molecule; e) verifying the addition of the monomer unit/polymer building block to the seed molecule by detecting the presence or absence of the fluorescent-labeled protecting group of the polymer building block of the monomer unit, wherein the detection of the fluorescent label indicates the addition of the monomer unit/polymer building block to the seed molecule, thereby extending the seed molecule polymer chain by one polymer building block of interest; f) removing the fluorescent-labelled protecting group of the polymer building block of the monomer unit added to the seed molecule; and repeating steps c) through f) to synthesize the polymer chain.

2. The method of claim 1, wherein the monomer unit comprises a boronate analog comprising an N-methyliminodiacetic acid (MIDA) group, wherein the methyl of the MIDA is replaced by a linker comprising a fluorophore.

3. The method of claim 1, wherein the monomer unit comprises an analog of triisopropyl silyl chloride.

4. The method of claim 1, wherein in step c) the monomer unit comprising the building block is chemically coupled to the immobilized molecule.

5. The method of claim 1 wherein polymer chain comprises two fluorophores, wherein the first fluorophore is linked to the protecting group of a monomer unit and the second fluorophore is located within a monomer unit incorporated in the polymer chain.

6. The method of claim 5 wherein one fluorophore is an acceptor fluorophore and the other fluorophore is a donor fluorophore resulting in energy transfer between the two fluorophores and the method comprises means to measure the distance between the two monomer units during the extension of the polymer chain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request. Of the drawings:

(2) FIG. 1: A schematic representation of FRET between two fluorophores, one coupled to a fixed position in the molecular chain and one attached to the terminal unit, which can be used to measure changes in the distance between polymer units during synthesis.

(3) FIG. 2: A representative synthesis of a fluorophore-labeled MIDA-boronate analog for iterative synthesis.

(4) FIG. 3: An iterative cross-coupling strategy using MIDA boronates.

(5) FIGS. 4A and B: FIG. 4A shows an iterative cross-coupling strategy using click chemistry. FIG. 4B shows an iterative cross-coupling strategy using Sonagashira couplings.

(6) FIG. 5: A representative synthesis for a fluorescent analog of triisopropyl silyl chloride.

(7) FIGS. 6 A and B: Example PNA macrocycles containing a fluorophore that can be used for DNA-templated polymer synthesis. FIG. 6A shows a fluorophore labeled PNA. FIG. 6B shows a fluorophore labeled blocking group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(8) The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

(9) As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

(10) It will be understood that although terms such as “first” and “second” are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, an element discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present invention.

(11) Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

(12) Iterative Synthesis Chemistries

(13) Immobilization of Seed Molecule

(14) A “seed” molecule is immobilized on a substrate as described above. The chemistries for this immobilization may enable the later chemical removal of this seed molecule and the final oligomer chain. Methods for immobilization of the seed molecule can include, but are not limited to, acid-labile linkers (e.g. azidomethyl-methyl maleic anhydride, silyl ethers) or photocleavable linkers (e.g. orthonitrobenzyl alcohols). Upon completion of synthesis suitable linkers will be cleaved “on demand” by selective chemical, electrochemical, or photochemical methods that will not affect the stability of the grown oligomer chain (e.g, orthogonal chemistries).

(15) Synthesis of a Fluorophore-Labeled MIDA Boronate

(16) The synthesis as shown in FIG. 2 allows for flexibility with linker selection for the nitrogen in the iminodiacetic acid ligand as well as fluorophore selection. MIDA (N-methyliminodiacetic acid) analogs where the methyl is replaced by a linker fluorophore could be constructed by alkylation of an amino alcohol 2 with tert-butyl bromoacetate 1 under established conditions. [2] The resulting free alcohol is either converted to an azide or alkyne-substituted ether using Mitsunobo conditions to afford compounds 4 or 5. [3] Compound 5 can also be formed by first converting compound 3 to the corresponding tosylate and then substituting with the alkynyl-alcohol in the presence of a base (e.g., sodium hydride). Cu-catalyzed click chemistry will then be used to install a fluorophore, yielding compounds 6 or 7. Many “clickable” fluorophores conjugated to either azides or alkynes are commercially available from companies such as Click Chemistry Tools or ThermoFisher Scientific. Finally, removal of the tert-butyl groups and boronic ester exchange with commercially available boronic acids will yield fluorophore-labeled MIDA boronate analogs 8 or 9 [4]. Alternatively, steps 3 and 4 could be reversed (generating the MIDA boronate analogs first, followed by click chemistry for fluorophore introduction). Click chemistry of MIDA boronates is known. [5]

(17) Iterative Cross-Coupling with MIDA Boronate

(18) An immobilized molecule with a terminal boronic acid as shown in FIG. 3 is reacted with a MIDA boronate with a blocked boronic acid that has a pendant fluorophore on the blocking group. In the presence of a Pd catalyst, base, and polar solvent, the halide on the MIDA boronate reacts with the boronic acid through a Suzuki-Mayaura cross-coupling reaction to form a carbon-carbon bond. The addition can then be fluorescently verified, and then the fluorophore and the blocking group on the boronic acid removed by treatment with NaOH and THF, leaving a boronic acid group available for the subsequent addition.

(19) Alternative Fluorophore-Labeled Building Blocks

(20) Other potential iterative chemistries would be an iterative click reaction wherein each step is blocked by capping the alkyne with a fluorescent silyl group. A similar concept could be applied to iterative Sonagashira chemistries. (See FIGS. 4A and B). Although the fluorescent silyl protecting group Bistert-butyl chloro(pyren-1-ylmethoxy)silane has been used, it has limitations in that the excitation/emission wavelengths are fixed and still in the far UV range (346/390 nm, respectively). [6]

(21) A fluorescent analog of triisopropyl silyl chloride, the synthesis for which is shown in FIG. 5, could be used with either an iterative click chemistry, Sonagashira chemistry, or the macrocycles. A silyl group with a handle for conjugation could be prepared by Grignard addition of a masked ketone (e.g., generic structure 2) to diispropylchlorosilane 1. [7] Chlorination of the silylating agent could be carried out using a number of conditions known to those skilled in the art, an example being tert-butyl hypochlorite [8] or CuCl2/CuI [7]. The silyl chloride can added to an alkyne to generate the bifunctional building block by activation the terminal alkyne with an organometallic agent, such as a lithium reagent. Unmasking the ketone and forming the oxime through condensation with a hydroxylamine-conjugated fluorophore (commercially available from ThermoFisher Scientific) would yield the fluorescently tagged building block.

(22) Alternative Iterative Cross-Coupling

(23) An immobilized molecule with a terminal alkyne is reacted with a monomer containing an azide and a silyl-protected alkyne with a pendant fluorophore in the presence of a Cu catalyst. The addition can then be fluorescently verified and then the fluorophore and the silyl blocking group are removed through the introduction of fluoride ions, leaving an alkyne group available for the subsequent addition.

(24) Alternatively, an immobilized molecule with a terminal alkyne is reacted with a monomer containing a halide and a silyl-protected alkyne with a pendant fluorophore in the presence of a Pd and a Cu catalyst through a Sonagashira coupling reaction. The addition can then be fluorescently verified and then the fluorophore and the silyl blocking group are removed through the introduction of fluoride ions, leaving an alkyne group available for the subsequent addition.

(25) Templated Synthesis

(26) Synthesis of Fluorescent Macrocycles

(27) The macrocycles containing the polymer building block and the PNA can be synthesized as described in [1]. The silyl/fluorophore protected alkyne can be synthesized as shown in FIG. 5. For the incorporation of a non-terminating fluorophore, a fluorophore containing amino acid can be incorporated during the synthesis of the PNA complex, prior to formation of the macrocycle (FIGS. 6A and B).

REFERENCES

(28) [1] Nature Chemistry 2013, 5, 282-292. [2] For an example, see Tetrahedron 2017 73, 2468. [3] For Mitsunobo conditions to install azides, see Org. Process Res. Dev. 2011, 15, 1116; for Mitsunobo chemistry on iminodiacetic acid substrates, see Chem. Commun. 2005, 13, 1784. [4] Example of using halide-substituted boronic acids to synthesize MIDA boronates: Aldrichimica Acta 2009, 42, 17-27 [5] Journal of Organic Chemistry 2011, 76, 10241-10248. [6] Nucleotides & Nucleosides, and Nucleic Acids, 2005, 24, 1345 [7] Organic & Biomolecular Chemistry 2003, 1, 3758. [8] Synthetic Communications 1999, 29, 3499.

(29) The entire contents of the publications, patents and patent applications described herein are incorporated by reference in their entirety.

(30) While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims