Patent Publication Number: US-2023151047-A1

Title: Linker structures with minimal scar for enzymatic synthesis

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. application Ser. No. 17/162,546, filed Jan. 29, 2021, which claims the benefit of and priority to U.S. Provisional Application No. 63/131,700, filed Dec. 29, 2020, the entire contents of which are incorporated herein by reference. 
    
    
     BACKGROUND 
     Cleavable linker molecules are used in a variety of biotechnology applications. Linkers couple two molecular entities together with a strong, typically covalent, bond. Cleavage of a linker allows the two entities to separate from each other. The separation can be used to release a molecular entity from attachment to a substrate, trigger a reaction, generate a signal, release a blocking group, or for another purpose. There are many types of linkers and many ways to cleave linkers. For example, linkers may be cleaved by exposure to an acid or a base, exposure to specific chemicals, exposure to light, exposure to heat, exposure to electric current, etc. Linkers may be used in organic solvents or in aqueous solutions. 
     SUMMARY 
     This disclosure provides structures for electrochemically-cleavable linkers that cleave in solvents at electrochemical potentials below the redox potential of the solvents. The linkers may be used to connect a nucleotide to any of multiple different types of bound groups. For example, the linkers may be used to connect a nucleotide to a peptide, a linked nucleotide, a fluorophore, or a water-soluble group, etc. The linkers include at least one cleavable group that may be a methoxybenzyl alcohol, an ester, a propargyl thioether, a trichloroethyl ether, a pyrrolidinone-type safety-catch motif, a paramethoxy aniline, or a ketal. The cleavable group may also optionally include an extension that may be carbonyl group, an alkane group, or an alkene group. The linkers may also optionally include a bound group attachment group that connects the bound group to the linker. The linkers may also optionally include one or more flexible extensions such as polyethylene glycol (PEG). The linkers also include a nucleotide attachment group that attaches the linker to the nucleotide. 
     This disclosure also provides a technique for cleaving a linker in a solvent by creating an electrode potential in the solvent that is less than the redox potential of solvent which results in cleavage of a bond in the linker. Multiple applications for the electrochemically-cleavable linkers provided in this disclosure are also described. For example, the linkers may be used to tether a blocking group to a nucleotide for enzymatic nucleotide synthesis (e.g., terminal deoxynucleotide transferase (TdT) synthesis) or to tether a fluorophore to a nucleotide for use in sequencing-by-synthesis. In some implementations, microelectrodes of an electrode array may be used to create location-specific changes in the electrochemical microenvironment that trigger cleavage of the linkers only on the surface of the electrode array where the electrodes are activated. 
     This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter nor is it intended to be used to limit the scope of the claimed subject matter. The term “techniques,” for instance, may refer to system(s) and/or method(s) as permitted by the context described above and throughout the document. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The Detailed Description is set forth with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The use of the same reference numbers in different figures indicates similar or identical items. 
         FIG.  1    shows a generalized structure of an electrochemically-cleavable linker. Lowercase Greek letters represent the points of connection between the portions of the linker. 
         FIG.  2    shows structures of electrochemically-cleavable linkers that illustrate four different types of cleavable groups. 
         FIG.  3    shows structures of electrochemically-cleavable linkers that illustrate three different types of cleavable groups. 
         FIG.  4    shows the structure of a first example linker with a methoxybenzyl alcohol cleavable group. 
         FIG.  5    shows the structure of a second example linker with a dimethoxybenzyl alcohol cleavable group. 
         FIG.  6    shows the structure of a third example linker with a trimethoxybenzyl alcohol cleavable group. 
         FIG.  7    shows the structure of a fourth example linker with an ester cleavable group. 
         FIG.  8    shows the structure of a fifth example linker with a propargyl thioether cleavable group. 
         FIG.  9    shows the structure of a sixth example linker with a trichloroethyl ester cleavable group. 
         FIG.  10    shows the structure of a seventh example linker with a trimethoxy benzyl (TMB) cleavable group. 
         FIG.  11    shows the structure of an eighth example linker with a pyrrolidinone-type safety-catch motif. 
         FIG.  12    shows the structure of a nineth example linker with a paramethoxy aniline cleavable group. 
         FIG.  13    shows the structure of a tenth example linker with a ketal cleavable group. 
         FIG.  14    shows example points of attachment to bases of nucleotides and scars left on nucleotide bases following cleavage of a linker. 
         FIG.  15    shows a time series of steps in enzymatic nucleotide synthesis using single nucleotides attached to protecting groups by a linker. 
         FIG.  16    is a flow diagram of an illustrative process for enzymatic nucleotide synthesis. 
         FIG.  17    is a cyclic voltammogram trace showing change in current as the electrode potential is varied in a buffered solution containing a linker with Structure  1  shown in  FIG.  4   . 
         FIG.  18    is a cyclic voltammogram trace showing change in current as the electrode potential is varied in a buffered solution containing a linker with Structure  3  shown in  FIG.  6   . 
         FIG.  19    is a cyclic voltammogram trace showing change in current as the electrode potential is varied in a buffered solution containing a linker with Structure  6  shown in  FIG.  9   . 
         FIG.  20    is a cyclic voltammogram trace showing change in current as the electrode potential is varied in a buffered solution containing a linker with Structure  6  as shown in  FIG.  9    in the presence of vitamin B 12  mediator 
         FIG.  21    is a cyclic voltammogram trace showing change in current as the electrode potential is varied in a buffered solution containing a photo-cleavable linker used to attach TdT to a nucleotide for enzymatic nucleotide synthesis. 
         FIG.  22    is a cyclic voltammogram trace showing change in current as the electrode potential is varied in a buffered solution containing a chemically-cleavable linker used to attach TdT to a nucleotide for enzymatic nucleotide synthesis. 
         FIG.  23    shows the steps of synthesizing an illustrative electrochemically-cleavable linker. 
     
    
    
     DETAILED DESCRIPTION 
     This disclosure provides electrochemically-cleavable linkers that may be used for various applications in liquid solvent environments. Solvents include organic solvents such acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, and methanol. Solvents also include aqueous solvents such as a potassium phosphate buffer and water. Many electrochemically-cleavable linkers are unsuitable for use in certain solvents because the electrode potential required to cleave the linker exceeds the redox potential of the solvent. Thus, the energy from the electrode potential is partially or wholly used to reduce or oxidize the molecules of the solvent rather than to cleave the linkers. 
     Aqueous environments are particularly challenging to work in because there is only a narrow electrochemical window prior to electrolysis of water. Generation of a sufficient electric potential in an aqueous environment (e.g., about −1.23 V under standard conditions) will cause electrolysis of water releasing oxygen gas at the anode and hydrogen gas at the cathode. The actual voltage at which electrolysis of water occurs depends on the specific conditions of the electrochemical cell such as the pH, temperature, and type of electrode. Aqueous environments include water and aqueous solutions as well as mixtures of aqueous solutions and organic solvents. 
     The electrolysis potential of a solvent in a given electrochemical cell can be identified by testing of a “blank” sample that has only the solvent without a linker. If a blank sample exhibits electrolysis at a voltage that is higher than a sample containing a linker, then it can be appreciated that cleavage of the linker will occur at a voltage lower than the redox potential of the solvent. 
     The linkers provided in this disclosure include cleavable groups with oxidation or reduction potentials of less than the redox potential of the solvents in which they are used. In some applications, the solvent may be an aqueous solvent and the redox potential may be the hydrolysis potential of water. The linkers may also include additional groups that increase the water solubility of the linkers. The incorporation of polar functional groups, such as the alcohol, amine, amide, carboxylic acid, sulfonic acid, and phosphate groups, which either ionize or are capable of relatively strong intermolecular forces of attraction with water (hydrogen bonding) are used to increase water solubility. 
     Electrochemically-cleavable linkers are cleaved by addition of electrons to a bond in the linker. The electrons may be generated by activating an electrode in the proximity of the bond in the linker that is to be cleaved. This may be referred to as a “directly mediated cleavage” in which activation of the electrode, or other change in the local environment, directly causes cleavage of a bond in the linker. Directly electrochemically-cleavable linkers may include a methoxybenzyl alcohol, an ester, a propargyl thioether, a trichloroethyl ether, a pyrrolidinone-type safety-catch motif, a paramethoxy aniline, or a ketal that when released can trigger an inter-molecular fragmentation reaction thereby cleaving the linker. 
     Another technique for inducing cleavage due to change in the local conditions may be referred to as “indirectly mediated cleavage.” With indirectly mediated cleavage the change in the local conditions caused by activation of an electrode activates an auxiliary molecule which in turn causes cleavage of a bond in the linker. For example, electrochemical generation of a base can promote hydrolysis and cleavage of an ester, and electrochemically generated Pd 0  can promote cleavage of a propargyl thioether. Also, electron transfer agents such as ceric ammonium nitrate, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, or similar are mediator molecules that can be oxidized electrochemically and then facilitate electron transfer to a methoxybenzyl alcohol resulting in cleavage. Vitamin B 12  is an example of a mediator molecule that can be reduced electrochemically and then facilitate electron transfer to a trichloroethyl ether resulting in cleavage. 
     If electrochemically-cleavable linkers are anchored to a substrate rather than free in solution, changing the voltage microenvironment on the surface of the substrate can selectively cleave some but not all of the linkers. An electrode array that includes multiple spatially-addressable electrodes may be used to selectively cleave electrochemically-cleavable linkers anchored to the surface of the electrode array. Each electrode may be independently addressable allowing the creation of arbitrary and variable voltage microenvironments across the surface of the electrode array. The microelectrode density may be approximately 1000 microelectrodes/cm 2 , approximately 10,000 microelectrodes/cm 2 , or a different density. One example of an electrode array is provided in Bo Bi et al.,  Building Addressable Libraries : The Use of“Safety-Catch”  Linkers on Electrode arrays,  132 J. Am. Chem. Soc. 17,405 (2010). One example of a suitable electrode array with microelectrodes is provided in U.S. patent application Ser. No. 16/435,363 filed on Jun. 7, 2019, with the title “Reversing Bias in Polymer Synthesis Electrode Array.” 
     The electrodes may be embedded in solid material from which the electrode array is formed. The electrodes in an electrode array may be implemented using complementary metal-oxide-semiconductor (CMOS) integrated circuits. CMOS circuits use a combination of p-type and n-type metal-oxide-semiconductor field-effect transistors (MOSFETs) to implement logic gates and other digital circuits. The MOSFETs may be made through any conventional manufacturing process including, but not limited to, a triple-well process or a silicon-on-insulator (SOI) process. Although CMOS logic can be implemented with discrete devices for demonstrations, commercial CMOS products are integrated circuits composed of up to billions of transistors of both types, on a rectangular piece of silicon of between 10 and 400 mm 2 . A series of controllable gates/transistors implemented with CMOS circuits can be controlled to inject charge at any location on the electrode array. 
     One use for the electrochemically-cleavable linkers of this disclosure is to attach a blocking group to nucleotides for enzymatic nucleotide synthesis. Enzymatic nucleotide synthesis is a technique for synthesizing polynucleotides using template-independent polymerases such as TdT and tRNA nucleotidyltransferase. Enzymatic nucleotide synthesis is performed in aqueous environments in contrast to traditional phosphoramidite synthesis that is performed in the organic solvent acetonitrile. 
     The polymerase TdT adds nucleotides indiscriminately to the 3′ hydroxyl group at the 3′ end of single-stranded DNA. Because TdT performs unregulated synthesis, use of this enzyme to create a polynucleotide with a pre-specified arbitrary sequence requires regulation and control of the TdT activity. One way to force single-nucleotide addition is to attach a blocking group to each nucleotide so that once it is incorporated is not possible for the enzyme to add additional nucleotides until the blocking group is removed. The TdT enzyme itself may be attached to a nucleotide with the linker so that the enzyme acts as its own blocking group. See Sebastian Palluck et al., De novo DNA synthesis using polymerase-nucleotide conjugates, 36(7) Nature Biotechnology 645 (2018) and WO 2017/223517 A1. The linkers used by Palluck et al. are not electrochemically-cleavable linkers but rather they are photo-cleavable linkers and chemically-cleavable linkers. 
     Currently known template-independent polymerases include TdT and tRNA nucleotidyltransferase. TdT includes both the full-length wild-type enzyme, as well as modified enzymes that are truncated or internally modified. One example of modified TdT is provided in U.S. Pat. No. 10,059,929. An example of truncated TdT is provided in U.S. Pat. No. 7,494,797. Thus, template-independent polymerase as used herein includes full-length wild-type, truncated, or otherwise modified TdT, tRNA nucleotidyltransferase, and any subsequently discovered or engineered polymerases that can perform template-independent synthesis of polynucleotides. Template independent polymerase as used herein does not encompass modifications of TdT or tRNA nucleotidyltransferase that render those enzymes incapable of performing template-independent nucleotide polymerization. 
     Another use for electrochemically-cleavable linkers is to attach fluorophores to nucleotides for sequencing-by-synthesis. A different colored fluorophore may be conjugated to each variety of nucleotide (e.g., cytosine (C), guanine (G), adenine (A), or thymine (T)) using the linkers provided in this disclosure. The color of the fluorescence may be detected indicating which nucleotide has been incorporated into a growing DNA strand. This application differs from conventional sequencing-by-synthesis techniques in that the linkers are cleaved electrochemically rather than through use of a chemical cleavage agent. 
     As used herein, polynucleotides, also referred to as oligonucleotides, include both DNA, RNA, and hybrids containing mixtures of DNA and RNA. DNA includes nucleotides with one of the four natural bases cytosine (C), guanine (G), adenine (A), or thymine (T) as well as unnatural bases, noncanonical bases, and/or modified bases. RNA includes nucleotides with one of the four natural bases cytosine, guanine, adenine, or uracil (U) as well as unnatural bases, noncanonical bases, and/or modified bases. Nucleotides include both deoxyribonucleotides and ribonucleotides covalently linked to one or more phosphate groups. 
       FIG.  1    shows a schematic representation of an illustrative structure of a linker  100 . The linker  100  is electrochemically-cleavable at a lower electrochemical potential than the redox potential of the solvent in which the linker is present. The linker  100  may also be water soluble. The linker  100  may be used to attach a bound group or “P group” 102 to a nucleotide  104 . The nucleotide  104  may be a DNA or RNA nucleotide with any of the canonical bases— adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U)—or an artificial or non-canonical base. The nucleotide  104  is attached to one, two, or preferably three phosphate groups. Connecting the P group  102  to the nucleotide  104  are a “Y group” 106, a “L 1  group” 108, a “C 1  group” 110, a “C 2  group” 112, a “L 2  group” 114, and a “L 3  group” 116. The Y group  106  is a bound group attachment group that attaches the P group  102  the linker  100 . The L 1  group  108  and the L 2  group  114  are flexible extensions. The C 1  group  110  is a cleavable group. The C 2  group  112  is an extension of the cleavable group. The L 3  group  116  is a nucleotide attachment group that attaches the nucleotide  104  to the linker  100 . 
     The P group  102 , Y group  106 , and C 2  group  112  are optional. One of the L 1  108 and L 2  groups  114  may be omitted or both may be included. Connections between the groups are represented by the lower-case Greek letters α, β, χ, δ, ε, ϕ, and γ. 
     The bound group that is attached to the nucleotide  104  by the linker  100 , also called the P group  102 , may be a peptide, a linked nucleotide, a fluorophore, or a water-soluble group. As used herein, “peptide” may be either a single peptide or a polypeptide. Polypeptides are two or more amino acids linked in a chain with the carboxyl group of each acid being joined to the amino group of the next by a bond of the type —OC—NH—. Polypeptides include enzymes which are proteins that catalyze biochemical reactions. Examples of enzymes that may be attached as a bound group “P” 102 include DNA polymerases and RNA polymerases such as TdT and tRNA nucleotidyltransferase. 
     As used herein, a “linked nucleotide” may be any DNA or RNA nucleotide including oligonucleotides having two or more nucleotides with a canonical or non-canonical base. In some implementations, the linked nucleotide is complementary to the nucleotide  104  at the other end of the linker. Thus, the nucleotide  104  can form Watson-Crick base pairing with the linked nucleotide. In some implementations, the linked nucleotide may be a nucleotide without a triphosphate group. Lack of the triphosphate group can prevent a polymerase such as TdT from incorporating the linked nucleotide into a DNA strand. 
     The linked nucleotide may also be a universal base. A universal base is an artificial nucleotide base that fits inside a DNA double-stranded helix and forms hydrogen bonding with any other base. For example, the universal base may be deoxyinosine (e.g., 2′-deoxyisoinosine, 7-deaza-2′-deoxyinsoine, and 2-aza-2′-dexosyinosine), isocarbostyril nucleoside derivatives, or 8-aza-7-deazaadenine. These and other examples of universal bases are discussed in David Loakes, The Applications of Universal DNA Base Analogues, 29(12) Nucleic Acids Research 2437 (2001) and the references cited therein. If the linked nucleotide includes a universal base it may also hybridize with the nucleotide  104 . 
     A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups or planar or cyclic molecules with several 7E bonds. The fluorophore may be, for example, fluorescein (e.g. fluorescein amidite), rhodamine (e.g., Rhodamine 6G, Rhodamine 123, or Rhodamine B), cyanine which refers to a synthetic dye family belonging to polymethine group and includes streptocyanines or open chain cyanines, hemicyanines, and closed chain cyanines (e.g., Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7 available from GE healthcare); coumarin (2H-chromen-2-one) among the various coumarin laser dyes are coumarins 480, 490, 504, 521, 504T, and 521T; or boron-dipyrromethene (BODIPY) which is composed of dipyrromethene complexed with a disubstituted boron center, typically BF 2 . Cleavage of the linker  100  may release the fluorophore generating a detectable fluorescent signal. 
     As used herein, a “water-soluble group” is any organic (i.e., carbon containing) chemical moiety that is both itself water soluble and if present as the P group  102  causes the linker  100  as a whole structure to be water soluble. One illustrative water-soluble group is glutathione. Many polypeptides and enzymes are also water-soluble groups. A linked nucleotide, especially a nucleotide triphosphate, may be a water-soluble group. 
     The Y group  106 , if present, is a bioconjugation group that is selected based on the structure of the P group  102  so that the Y group  106  can form a covalent bond to one or more atoms in the P group  102 . Options for the Y group  106  are shown in the following table. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Bound Group Attachment Group Structures (Y group 106). 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 α-S-β 
               
               
                 α-NH 2 -β 
               
               
                   
               
            
           
         
       
     
     In Table 1, a represents a point of attachment to the P group  102  and β represents a point of attachment to the L 1  group  108  or the C 1  group  110  if the L 1  group  108  is omitted. 
     The L 1  group  108 , if present, creates space between the P group  102  and the C 1  group  110  and provides flexibility to the structure of the linker  100 . Options for the L 1  group  108  are shown in the following table. The L 1  group  108  may include one or more structures from Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Flexible Extension Structures (L 1  group 108). 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     In Table 2, n is an integer which is between 1-30, 1-20, 1-10, or 1-5; R 1  and R 2  are each independently hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms; β represents a point of attachment to the Y group  106 , if present, or to the or P group  102 ; and χ represents a point of attachment to the C 1  group  110 . The orientation of the L 1  groups may be reversed. That is, β can represent a point of attachment to the C 1  group  110  and χ can represent a point of attachment to the Y group  106 , if present, or to the or P group  102 . Examples of suitable flexible extension structures include polyethylene glycol (PEG) and methylene. For example, the flexible extension structure may be a PEG trimer but it could also be a longer or shorter PEG structure. 
     The C 1  group  110  is a cleavable group that decomposes in response to application of an electrode potential that is less than the redox potential of the solvent such as less than the hydrolysis potential of water. Options for the C 1  group  110  are shown in the following table. 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Cleavable Group Extension Structures (C 1  group 110). 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 X 1  is 1 to 4 ring substituents consisting of a hydrogen, a hydroxyl group, an ether group with an alkyl group having 1 to 3 carbon  atoms, an amine group which is unsubstituled or substituted with one or two alkyl groups having 1 to 2 carbon atoms, an alkyl group  having 1 to 2 carbon atoms, or a halogen. X 2  is hydrogen, a methyl group, an ethyl group, or an isopropyl group. 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 R 3  is a tert-butyl, allyl, benzyl, methoxybenzyl, dimethoxybenzyl,  trimethoxybenzyl, nitrobenzyl, fluorenylmethoxycarbonyl,  cyanoethyl, or trichloroethyl group. 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 R 4  and R 5  are both separately hydrogen or gem dimethyl or a substituted or unsubstituled straight or branched alkyl group having 1 to 6 carbon atoms. X 1  is 1 to 4 ring substituents consisting of a hydrogen, a hydroxyl group, an ether group with an alkyl group having 1 to 3 carbon atoms, an amine group which is unsubsliluted or substituted with one or two alkyl groups having 1 to 2 carbon atoms, an alkyl group having 1 to 2 carbon atoms, or a halogen. 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 R 6  is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms. 
               
               
                   
               
            
           
         
       
     
     In Table 3, χ represents a point of attachment to the L 1  group  108 , the Y group  106 , or the P group  102  and δ represents a point of attachment to the C 2  group  112 , the L 2  group  114 , or the L 3  group  116 . 
     The C 2  group  112  is an extension of the C 1  group  110  that may be present or omitted. Options for the C 2  group  112  are shown in the following table. 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Cleavable Group Extension Structures (C 2  group 112). 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 R 7  is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms. 
               
               
                   
               
            
           
         
       
     
     In Table 4, 8 represents a point of attachment to the C 1  group  110  and c represents a point of attachment to the L 2  group  114  or the L 3  group  116 . 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Flexible Extension Structures (L 2  group 114). 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     In Table 5, n is an integer which is between 1-30, 1-20, 1-10, or 1-5; R 8  and R 9  are hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms; ε represents a point of attachment to the C 2  group  112 , if present, or to the C 1  group  110 ; and φ represents a point of attachment to the L 3  group  116 . 
     The L 3  group  116  is a nucleotide attachment group that connects the linker  100  to the nucleotide. Options for the L 3  group  116  are shown in the following table. 
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Nucleotide Attachment Group Structures (L 3  group 116). 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     In Table 6, φ represents a point of attachment to the C 1  group  110 , the C 2  group  112 , or the L 2  group  114  and γ represents a point of attachment to the nucleotide. In some implementations, the L 3  group  116  attaches to the base of the nucleotide. If the base is a purine base (i.e., adenosine or guanine), the L 3  group  116  may attach to the number 7 nitrogen of the purine base. If the base is a pyrimidine base (i.e., cytosine, thymine, or uracil) the L 3  group  116  may attach to the number 5 carbon of the pyrimidine base. 
       FIG.  2    shows generalized structures of linkers with four different examples of specific cleavable groups. All of the linkers shown in  FIG.  2    are specific examples of the general linker structure of linker  100  shown in  FIG.  1   . A methoxybenzyl alcohol linker  200  has a methoxybenzyl alcohol as the C 1  group  110  with p being 1, 2, or 3. Without being bound by theory, it is believed that increasing number of oxygen groups attached to the benzyl ring reduces the electrode potential necessary to cleave the linker. An ester linker  202  has an ester group as the C 1  group  110 . A propargyl thioether linker  204  has a propargyl thioether group as the C 1  group  110 . A trichloroethyl ether linker  206  has a trichloroethyl ether group as the C 1  group  110 . 
       FIG.  3    shows generalized structures of linkers with three different examples of specific cleavable groups. All of the linkers shown in  FIG.  3    are specific examples of the general linker structure of linker  100  shown in  FIG.  1   . A pyrrolidinone-type safety-catch motif linker  300  has carbamate coupled to a tri methoxybenzyl group as the C 1  group  110 . A paramethoxy aniline  302  has an ester group as the C 1  group  110 . A ketal linker  304  has a ketal group as the C 1  group  110 . 
       FIG.  4    illustrates an example linker  400  referred to as Structure  1  that has a methoxybenzyl alcohol C 1  group  110 . This linker  400  is a specific example of the methoxybenzyl alcohol linker  200  shown in  FIG.  2   . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  400 , the flexible extension L 2  group  114  is omitted. 
       FIG.  5    illustrates an example linker  500  referred to as Structure  2  that has a dimethoxybenzyl alcohol C 1  group  110 . This linker  500  is a specific example of the methoxybenzyl alcohol linker  200  shown in  FIG.  2   . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate but this is merely illustrative and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  500 , the flexible extension L 2  group  114  is omitted. 
       FIG.  6    illustrates an example linker  600  referred to as Structure  3  that has a trimethoxybenzyl alcohol C 1  group  110 . This linker  600  is a specific example of the methoxybenzyl alcohol linker  200  shown in  FIG.  2   . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  600 , the flexible extension L 2  group  114  is omitted. 
       FIG.  7    illustrates an example linker  700  referred to as Structure  4  that has an ester C 1  group  110 . This linker  700  is a specific example of the ester linker  202  shown in  FIG.  2   . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  700 , the flexible extension L 2  group  114  is omitted. 
       FIG.  8    illustrates an example linker  800  referred to as Structure  5  that has a propargyl thioether C 1  group  110 . This linker  800  is a specific example of the propargyl thioether linker  204  shown in  FIG.  2   . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  800 , the flexible extension L 2  group  114  is omitted. 
       FIG.  9    illustrates an example linker  900  referred to as Structure  6  that has a trichloroethyl ester C 1  group  110 . This linker  900  is a specific example of the trichloroethyl ester linker  206  shown in  FIG.  2   . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  900 , the flexible extension L 2  group  114  is omitted. 
       FIG.  10    illustrates an example linker  1000  referred to as Structure  7  that has a trimethoxy benzyl (TMB) C 1  group  110 . The P group  102  is shown as glutathione but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  1000 , the flexible extension L 2  group  114  is omitted. 
       FIG.  11    illustrates an example linker  1100  referred to as Structure  8  that has pyrrolidinone-type safety-catch motif C 1  group  110 . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  1100 , the cleavable group extension C 2  group  112  and the flexible extension L 2  group  114  are omitted. 
       FIG.  12    illustrates an example linker  1200  referred to as Structure  9  that has a paramethoxy aniline C 1  group  110 . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  1200 , the cleavable group extension C 2  group  112  and the flexible extension L 2  group  114  are omitted. 
       FIG.  13    illustrates an example linker  1300  referred to as Structure  10  that has a ketal C 1  group  110 . The P group  102  is shown as glutathione, but this is merely illustrative, and glutathione may be replaced with any of the other options for the P group  102 . The nucleotide  104  is shown as deoxyribose thymine triphosphate, but this is merely illustrative, and deoxyribose thymine triphosphate may be replaced with any other nucleotide  104 . In this example linker  1300 , the cleavable group extension C 2  group  112  and the flexible extension L 2  group  114  are omitted. 
       FIG.  14    shows examples of points of attachment of linkers to bases of nucleotides and the scars left after cleavage of a linker. The nucleotide bases thymine/uracil  1400 , cytosine  1402 , adenine  1404 , and guanine  1406  are shown with R representing the point of attachment to a linker and “sugar” representing the sugar group of the nucleotide. In these examples, the linker is connected to the number 7 nitrogen of the purine bases (i.e., adenosine or guanine) and the number 5 carbon of the pyrimidine bases (i.e., cytosine, thymine, or uracil). 
     Examples of the L 3  group  116  following cleavage of the linker are shown as structures  1408 ,  1410 ,  1412 ,  1414 ,  1416 , and  1418 . Any of these structures may be present in place of the R group shown on the nucleotide bases  1400 ,  1402 ,  1404 , and  1406 . Examples structures in which cytosine is shown attached to each of the examples of the L 3  group  116  are shown  1420 ,  1422 ,  1424 ,  1426 ,  1428 , and  1430 . Cytosine is merely illustrative and may be replaced with any of the other nucleotide bases. These structures  1420 ,  1422 ,  1424 ,  1426 ,  1428 , and  1430  represent the scars left on a nucleotide after cleavage of a linker. The scar terminates in a hydroxyl group or an amide group. If the nucleotide  104  is part of an oligonucleotide the scar may be removed, if necessary, through polymerase chain reaction (PCR) amplification. Copies of the single-stranded DNA created by PCR amplification can be created using nucleosides that do not have scars. Thus, none of the population of double-stranded DNA molecules created by PCR would have scars except for the single molecule that incorporates the original nucleotide. 
       FIG.  15    shows an example time series of steps in enzymatic nucleotide synthesis. A configuration at a first time point  1500  shows an electrode array  1502  containing multiple microelectrodes  1504 (A),  1504 (B), . . . ,  1504 (N) coated with single-stranded oligonucleotides  1506  capped by blocking groups  1508  attached to a terminal nucleotide via a linker  1510 . The linker  1510  may be any of the electrochemically cleavable linkers provided in this disclosure. The blocking group  1508  is one option for the P group  102  shown in  FIG.  1   . The blocking group  1508  prevents template-independent polymerases such as TdT from adding more than a single nucleotide during each round of synthesis. 
     In one implementation, the enzyme TdT itself may be used as the blocking group  1008 . A technique for using TdT as a blocking group to force single-nucleotide addition with enzymatic nucleotide synthesis is described in Sebastian Palluck et al., De novo  DNA synthesis using polymerase - nucleotide conjugates,  36(7) Nature Biotechnology 645 (2018) and WO 2017/223517 A1. 
     The blocking group  1508  may, in some implementations, be a linked nucleotide, including a short oligonucleotide (e.g., 2-10 bp), that is complementary to the nucleotide at the 3′ end of the oligonucleotide  1506 . The complementary relationship may result in hybridization which can cause the two nucleotides joined by the linker  1510  to form a loop or hairpin structure. This prevents addition of other nucleotides onto the 3′ end of the oligonucleotide  1506 . As an alternative to a complementary nucleotide, the linked nucleotide may be a nucleotide that includes one or more universal bases. The universal bases can hybridize with any other nucleotide in oligonucleotide  1506  and may form a similar loop or hairpin structure. 
     A second time point  1512 , shows selective deblocking of some but not all of the oligonucleotides  1506  attached to the surface of electrode array  1502 . Activation of one of the microelectrodes such as  1504 (B) selectively triggers cleavage of the linkers  1510  attached to that microelectrode  1504 (B) without cleaving linkers  1510  attached to any of the other microelectrodes  1504  on the surface of the electrode array  1502 . Cleavage of the linkers  1510  releases the blocking groups  1508  so that nucleotide extension may occur on the deblocked oligonucleotides. If the blocking group  1508  is a complementary nucleotide or nucleotides with a universal base, the hydrogen bonds responsible for base pairing are unlikely to be sufficiently strong to keep the nucleotide hybridize to the oligonucleotide strand  1506  once the linker  1510  is cleaved. However, the temperature of the electrodes cell may be elevated to promote disassociation. 
     A third time point  1514 , shows single nucleotide extension of the unblocked oligonucleotides  1506  by addition of nucleotide-blocking group structures  1516  with an electrochemically-cleavable linker  1510  that attaches the nucleotide to the blocking group  1508 . The nucleotides included in the nucleotide-blocking group structures  1516  are joined to the end of the unblocked oligonucleotides by the action of a polymerase or ligase. This process may then be repeated with selective addition of single nucleotides at locations on the electrode array  1502  that are selectively deblocked by activation of the corresponding microelectrodes  1504 . By repeating this series of steps, multiple different oligonucleotides may be synthesized on the surface of the electrode array by repeated single-nucleotide addition. 
       FIG.  16    shows a process  1600  for enzymatic nucleotide synthesis. Process  1600  may be implemented using any of the linkers shown in  FIGS.  1 - 13   .  FIG.  15    illustrates some of the steps of process  1600 . 
     At operation  1602 , single-stranded oligonucleotides are attached to the surface of an electrode array. This results in the creation of an electrode array that is covered with a plurality of oligonucleotides. The surface of the electrode array is inside of an electrochemical cell. The electrochemical cell is filled with an aqueous solution such as a buffered solution for use with a template-independent polymerase. The oligonucleotides are single-stranded molecules with a length of between about 3-30 nucleotides. A template-independent polymerase uses the polynucleotides as a starting point for enzymatic polynucleotide synthesis by adding additional nucleotides to the 3′ terminal nucleotides at the end of the original, bound oligonucleotides. 
     At operation  1604 , one or more microelectrodes on the electrode array are identified. The microelectrodes may be identified by a computer system that tracks the sequence in which the microelectrodes have been activated and that controls activation of the microelectrodes according to programmatic instructions. The programmatic instructions may be designed to synthesize multiple oligonucleotides with specific, predetermined sequences according to techniques known to those of skill in the art. 
     At operation  1606 , an electrode potential is selectively created at one or more of the microelectrodes. The change in electrode potential may positive or negative and has a magnitude that is less than the hydrolysis potential of water. The electrode potential cleaves the linkers attached to blocking groups on the 3′ ends of the oligonucleotides attached to the surface of the electrode array. The electrode potential in the proximity of other electrodes that are not activated does not change or changes only to a degree that does not cause cleavage of the linkers. This provides selective deblocking of some but not all of the nucleotides attached to the surface of the electrode array. 
     At operation  1608 , a wash solution is delivered to the surface of the electrode array. The wash solution may be flowed across the entire surface of the electrode array. This washing step can remove any of the blocking groups that are in solution following cleavage of the linkers. The wash solution may be water without added salts or an aqueous solution that contains at least one of a salt or a buffer. The buffer may be any one of a number of aqueous buffers that are compatible with polymerases and single-stranded nucleotides such as PBS or tris-buffered saline (TBS). 
     At operation  1610 , the surface of the electrode array is contacted with a predetermined nucleotide attached to a blocking group via an electrochemically-cleavable linker with a cleavage potential less than the hydrolysis potential of water. The electrochemically-cleavable linker may be the linker  100  shown in  FIG.  1   . For example, the nucleotide may be one of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), or uridine triphosphate (UTP). The nucleotide may be provided in a nucleotide solution that contains only a single type of nucleotide and a template-independent polymerase in an appropriate buffer solution. During operation  1610  the template-independent polymerase adds the nucleotide to the deblocked 3′ ends of the oligonucleotides. 
     The blocking group is an entity that when attached to the nucleotide prevents a polymerase from adding additional nucleotides to nucleotide that is linked to the blocking group. The blocking group may be the polymerase enzyme itself, such as TdT, or a linked nucleotide that hybridizes to the nucleotide added on to the end of an oligonucleotide attached to the surface of the electrode array. The linked nucleotide may be a complementary nucleotide or a nucleotide with a universal base either of which can hybridize to the nucleotide thereby preventing the action of the polymerase. 
     At operation  1612 , a wash solution is delivered to the surface of the electrode array. The wash solution may remove unreacted nucleotides and enzymes. This prevents the incorporation of an incorrect nucleotide during a subsequent cycle of synthesis. The wash solution may be without added salts or an aqueous solution that contains at least one of a salt or a buffer. The buffer may be any one of a number of aqueous buffers that are compatible with polymerases and single-stranded nucleotides such as PBS or TBS. The wash solution used at operation  1612  may be the same as the wash solution used at operation  1608 . 
     Process  1600  may iteratively repeat by returning to operation  1604  until oligonucleotides with the desire sequences are fully synthesized. Each subsequent iteration of synthesis may add a different nucleotide at a different set of locations depending on which oligonucleotides have been unblocked by activation of specific electrodes. Repeating this process while varying the nucleotide added and the locations at which the blocking groups are removed makes it possible to create multiple different oligonucleotide sequences with single-base specificity using a template-independent polymerase. 
     Examples 
     The following examples show the results of cyclic voltammetry experiments performed on electrochemical cells containing various linkers. Cyclic voltammetry is an electrochemical technique that measures the current that develops in an electrochemical cell under conditions where voltage is in excess of that predicted by the Nernst equation. Cyclic voltammetry is performed by cycling the potential of a working electrode and measuring the resulting current against a reference electrode which maintains a constant potential. 
     In a cyclic voltammetry experiment, the working electrode potential is ramped linearly versus time. Unlike in linear sweep voltammetry, after the set potential is reached in a CV experiment, the working electrode&#39;s potential is ramped in the opposite direction to return to the initial potential. The current at the working electrode is plotted versus the applied voltage (i.e., the potential of the working electrode) to give the cyclic voltammogram trace. Techniques for performing cyclical voltammetry experiments are known to those of ordinary skill in the art and may be found in Skoog, D.; Holler, F.; Crouch, S.  Principles of Instrumental Analysis  (2007) and Kissinger, P. T., Heineman, W. R.,  Cyclic Voltammetry,  60 J. of Chem. Education, 702 (1983). In all the following examples electrode potential is measured in volts (V) relative to a reference electrode and the current is measured in amperes (A). 
       FIG.  17    shows a cyclic voltammogram trace for 20 mM of a linker  1700  with a cleavable group that is the same as in Structure  2  shown in  FIG.  5    compared to a blank electrochemical cell containing the same solvent. The linker  1700  differs from Structure  2  because it does not include the groups P, C 2 , L 2 , L 3 , or a nucleotide. The electrode potential was generated with a 12.5 mm 2  platinum working electrode and a platinum counter electrode using a 10 mV/s sweep rate. The linker  1700  exhibits a pronounced anodic peak indicating cleavage of the linker. There is no similar anodic peak potential in the blank sample. 
       FIG.  18    shows a cyclic voltammogram trace for 20 mM of a linker  1800  with a cleavable group that is the same as in Structure  3  shown in  FIG.  6    compared to a blank electrochemical cell containing the same solvent. The linker  1800  differs from Structure  3  because it does not include the groups P, C 2 , L 2 , L 3 , or a nucleotide. The electrode potential was generated with a 12.5 mm 2  platinum working electrode and a platinum counter electrode using a 10 mV/s sweep rate. The sample containing the linker  1800  exhibits an anodic peak potential at before the asymptotic increase. 
       FIG.  19    shows a cyclic voltammogram trace measured in μA for 20 mM of a 2,2,2-trichloroethanol linker  1900  that includes the same cleavable group as Structure  6  shown in  FIG.  9    compared to a blank electrochemical cell containing the same solvent. The electrode potential was generated with a 20 mm 2  carbon working electrode and a carbon counter electrode using a 5 mV/s sweep rate. A subtle reduction peak indicating cleavage of the linker is identifiable as an asymptotic point on the slope of the linker sample. There is not a distinct peak because trichloroethanol reduces through a multi-step reaction that appears as a broad shoulder in the cyclic voltammogram trace. 
       FIG.  20    shows a cyclic voltammogram trace for 20 mM 2,2,2-trichloroethanol linker  2000  and 20 mM vitamin B 12  mediator compared to a blank electrochemical cell containing only the solvent and the vitamin B 12  mediator. The electrode potential was generated with a 12.5 mm 2  platinum working electrode and a platinum counter electrode. The relevant features of the curve are an increase in current and shift in solvent reduction voltage in the sample containing the linker  2000 . 
       FIG.  21    shows a cyclic voltammogram trace for 13 mM of the photocleavable linker  2100  from Palluck et al. compared to a blank electrochemical cell containing the same solvent. The electrode potential was generated with a 12.5 mm 2  platinum working electrode and a platinum counter electrode using a 100 mV/s sweep rate. The trace does not show a reduction wave prior to the onset of reduction of the blank sample indicating that the linker is not cleaved. 
       FIG.  22    shows a cyclic voltammogram trace for 20 mM of the dibutyl disulfide linker  2200  from Palluck et al. compared to a blank electrochemical cell containing the same solvent. The electrode potential was generated with a 12.5 mm 2  platinum working electrode and a platinum counter electrode using a 100 mV/s sweep rate. The trace does not show a reduction wave prior to the onset of solvent reduction indicating that the linker is not cleaved. 
     Illustrative Linker Synthesis 
       FIG.  23    shows an example series of four synthetic steps used to make an example linker with the features described in this disclosure. A similar synthetic process may be used to make any of the other linkers provided in this disclosure. Persons of ordinary skill in the art will readily understand how to modify the process described below to generate other linkers such as, for example, linkers with Structure  1 , Structure  2 , Structure  3 , Structure  4 , Structure  5 , or Structure  6 . Examples of suitable techniques may be found in Francis Carey &amp; Robert Giuliano, Organic Chemistry (11 th  ed. 2020) and Peter G. M. Wuts,  Green&#39;s Protective Groups in Organic Synthesis  (5 th  ed. 2014). 
     Step 1: a) A solution of triethylene glycol (6.64 mL, 2 eq) and triethylamine (16.6 mL, 4.8 eq) in tetrahydrofuran (THF) (50 mL) was cooled in an ice/water bath. A solution of methanesulfonyl chloride (1.93 mL, 1 eq) in THF (8 mL) was added dropwise, and the reaction mixture was allowed to warm to room temperature overnight. The volatiles were removed in vacuo. The residue was suspended in EtOH and concentrated in vacuo. 
     b) The crude mesylate was dissolved in EtOH (60 mL). Sodium azide (3.25 g, 2 eq) was added, and the resulting suspension was refluxed overnight. The volatiles were removed in vacuo. The residue was diluted with half-saturated brine, washed with three portions of cyclohexane, then extracted with four portions of dichloromethane (DCM). The combined DCM layers were dried over MgSO 4  and concentrated in vacuo. The crude material was purified by flash column chromatography on silica gel, eluting with a gradient from 0-8% MeOH in CDCl 3  to give 2.16 g pale yellow oil (49%). 
     Step 2: a) Methanesulfonyl chloride (1.04 mL, 1.1 eq) was added dropwise to a solution of the product generated in step 1 (2.16 g, 1 eq) and triethylamine (5.14 mL, 3 eq) in DCM (45 mL). The resulting suspension was stirred for 1 h. The reaction mixture was diluted with DCM, washed with water, 1 M HCl (aq), and saturated NaHCO 3 , dried over Na 2 SO 4 , and concentrated in vacuo. 
     b) The crude mesylate was dissolved in acetonitrile (50 mL). 4-hydroxy-3-methoxybenzyl alcohol (2.28 g, 1.2 eq) and potassium carbonate (2.55 g, 1.5 eq) were added, then the resulting mixture was refluxed overnight. The reaction mixture was cooled to room temperature and filtered, washing with EtOH, and the filtrate concentrated in vacuo. The crude material was purified by flash column chromatography on silica gel, eluting with a gradient from 0-6% MeOH in CDCl 3  to give 3.07 g pale yellow, viscous oil (80%).  1 H NMR (300 MHz, CDCl 3 ) δ=6.94-6.81 (m, 3H), 4.62 (d, J=5.5 Hz, 2H), 4.18 (dd, J=6.0, 5.0 Hz, 2H), 3.91-3.86 (m, 2H), 3.87 (s, 3H), 3.77-3.72 (m, 2H), 3.70-3.66 (m, 4H), 3.38 (t, J=5.0 Hz, 2H). 
     Step 3: A solution of the product generated in step 2 (1.0 g, 1 eq) in DMF (16 mL) was cooled in an ice/water bath. Sodium hydride (60 wt % in mineral oil) (0.32 g, 2.5 eq) was added, and the resulting suspension was stirred for 30 min. Propargyl bromide (80 wt % in toluene) was added, and the resulting solution was allowed to warm to room temperature overnight. The reaction mixture was quenched with saturated NH 4 C 1  (aq), diluted further, and extracted with three portions of DCM. The combined organic layers were dried over MgSO 4  and concentrated in vacuo. The crude residue was purified by flash column chromatography, eluting with DCM to provide 0.84 g yellow oil (75%).  1 H NMR (300 MHz, CDCl 3 ) δ=6.95-6.84 (m, 3H), 4.54 (s, 2H), 4.22-4.16 (m, 2H), 4.15 (d, J=2.5 Hz, 2H), 3.89 (t, J=5.0 Hz, 2H), 3.86 (s, 3H), 3.77-3.72 (m, 2H), 3.71-3.66 (m, 4H), 3.38 (t, J=5.0 Hz, 2H), 2.47 (t, J=2.5 Hz, 1H). 
     Step 4: Cuprous iodide (2.4 mg, 0.2 eq) and triethylamine (50.1 μL, 7.2 eq) were added to a degassed solution of 5-iodo-2′-deoxyuridine-5′triphosphate tetratriethylamine salt (50 mg, 1 eq) and the product generated in step 3 (35 mg, 2 eq) in a 2:1 mixture of H 2 O and acetonitrile. An independently degassed solution of palladium(II) acetate (1.1 mg, 0.1 eq) and 3,3′,3″-phosphanetriyltris(benzenesulfonic acid) trisodium salt (14.2 mg, 0.5 eq) in a 2:1 mixture of H 2 O and acetonitrile was added, and the resulting mixture was shaken at 65° C. for 1 h. The reaction mixture was cooled to room temperature and filtered, washing with deionized water. The filtrate was purified by ion exchange chromatography on diethylaminoethyl cellulose (DEAE) Sephadex, eluting with a gradient from 0-1 M TEAB buffer to give the desired product. 
     Illustrative Embodiments 
     The following clauses described multiple possible embodiments for implementing the features described in this disclosure. The various embodiments described herein are not limiting nor is every feature from any given embodiment required to be present in another embodiment. Any two or more of the embodiments may be combined together unless context clearly indicates otherwise. As used in this document “or” means and/or. For example, “A or B” means A without B, B without A, or A and B. As used herein, “comprising” means including all listed features and potentially including addition of other features that are not listed. “Consisting essentially of” means including the listed features and those additional features that do not materially affect the basic and novel characteristics of the listed features. “Consisting of” means only the listed features to the exclusion of any feature not listed. 
     Clause 1. A compound having a structure P—Y-L 1 -C 1 -C 2 -L 2 -L 3 -nucleotide, wherein: 
     P is a bound group that is that is a peptide, a linked nucleotide, a fluorophore, or a water-soluble group; 
     Y is a bound group attachment group with the structure 
     
       
         
         
             
             
         
       
     
     wherein α represents a point of attachment to P and β represents a point of attachment to L 1  or C 1 ; 
     L 1  is optionally a flexible extension with the one or more of the structures: 
     
       
         
         
             
             
         
       
     
     wherein n is 1-20, R 1  is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein β represents a point of attachment to Y or P and χ represents a point of attachment to C 1 ; 
     C 1  is a cleavable group with the structure: 
     
       
         
         
             
             
         
       
     
     wherein X 1  is 1 to 4 ring substituents consisting of a hydrogen, a hydroxyl group, an ether group with an alkyl group having 1 to 3 carbon atoms, an amine group which is unsubstituted or substituted with one or two alkyl groups having 1 to 2 carbon atoms, an alkyl group having 1 to 2 carbon atoms, or a halogen; X 2  is hydrogen, a methyl group, an ethyl group, or an isopropyl group; R 3  is a tert-butyl, allyl, benzyl, methoxybenzyl, dimethoxybenzyl, trimethoxybenzyl, nitrobenzyl, fluorenylmethoxy carbonyl, cyanoethyl, or trichloroethyl group, R 4  and R 5  are both separately hydrogen or gem dimethyl or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms; and R 6  is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms; 
     C 2  is optionally an extension of C 1  with the structure: 
     
       
         
         
             
             
         
       
     
     wherein R 7  is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, 8 represents a point of attachment to C 1  and ε represents a point of attachment to L 2  or L 3 ; 
     L 2  is optionally flexible extension with the structure: 
     
       
         
         
             
             
         
       
     
     wherein n is 1-20, R 8  and R 9  are hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein c represents a point of attachment to C 1  or C 2  and φ represents a point of attachment to L 3  and wherein at least one of L 1  or L 2  is present, and 
     L 3  is a nucleotide attachment group with the structure: 
     
       
         
         
             
             
         
       
     
     wherein φ represents a point of attachment to C 1 , C 2 , or L 2  and γ represents a point of attachment to the nucleotide. 
     Clause 2. The compound of clause 1, wherein P is present, Y is present, L 1  is present, C 2  is omitted, and L 2  is omitted. 
     Clause 3. The compound of the compound of any of clauses 1-2, wherein P is present and a peptide, wherein the peptide is an enzyme. 
     Clause 4. The compound of clause 3, wherein the enzyme is TdT. 
     Clause 5. The compound of any of clauses 1-2, wherein P is present and a linked nucleotide comprising at least one of DNA, RNA, or a synthetic nucleotide having a universal base. 
     Clause 6. The compound of any of clauses 1, 2, or 5, wherein P is present and a linked nucleotide that is complementary to the nucleotide. 
     Clause 7. The compound any of clauses 1-6, wherein L 2  is omitted. 
     Clause 8. The compound of any of clauses 1-7, wherein L 1  is present and is 
     
       
         
         
             
             
         
       
     
     and n is 2. 
     Clause 9. The compound of any of clauses 1-7, wherein L 1  is present and is 
     
       
         
         
             
             
         
       
     
     Clause 10. The compound of any of clauses 1-9, wherein C 1  is: 
     
       
         
         
             
             
         
       
     
     wherein X 1  is 1 to 4 ring substituents consisting of a hydrogen, a hydroxyl group, an ether group with an alkyl group having 1 to 3 carbon atoms, an amine group which is unsubstituted or substituted with one or two alkyl groups having 1 to 2 carbon atoms, an alkyl group having 1 to 2 carbon atoms, or a halogen and X 2  is hydrogen, a methyl group, an ethyl group, or an isopropyl group. 
     Clause 11. The compound of clause 10, wherein X 1  is hydrogen and X 2  is hydrogen. 
     Clause 12. The compound of clause 10, wherein X 1  is 2 methyl ether ring substituents and X 2  is hydrogen. 
     Clause 13. The compound of clause 12 having the structure: 
     
       
         
         
             
             
         
       
     
     Clause 14. The compound of any of clauses 1-9, wherein C 1  is: 
     
       
         
         
             
             
         
       
     
     and R 3  is a tert-butyl, allyl, benzyl, methoxybenzyl, dimethoxybenzyl, trimethoxybenzyl, nitrobenzyl, fluorenylmethoxycarbonyl, cyanoethyl, or trichloroethyl group. 
     Clause 15. The compound of clause 14, wherein R 3  is trimethoxybenzyl. 
     Clause 16. The compound of clause 15 having the structure: 
     
       
         
         
             
             
         
       
     
     Clause 17. The compound of any of clauses 1-9, wherein C 1  is: 
     
       
         
         
             
             
         
       
     
     and R 4  and R 5  are both separately hydrogen or gem dimethyl or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms. 
     Clause 18. The compound of clause 17, wherein X 1  is hydrogen, n is 1, R 4  is hydrogen, and R 5  is gem dimethyl. 
     Clause 19. The compound of clause 18 having the structure: 
     
       
         
         
             
             
         
       
     
     Clause 20. The compound of any of clauses 1-9, wherein C 1  is: 
     
       
         
         
             
             
         
       
     
     and R 6  is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms. 
     Clause 21. The compound of clause 20, wherein R 6  is methyl. 
     Clause 22. The compound of clause 21 having the structure: 
     
       
         
         
             
             
         
       
     
     Clause 23. The compound of any of clauses 1-15, 17, 18, 20, or 21 wherein L 3  is 
     
       
         
         
             
             
         
       
     
     Clause 24. The compound of any of clauses 1-12, 14, 15, 17, 18, 20, or 21, wherein 
     L 3  is 
     
       
         
         
             
             
         
       
     
     Clause 25. The compound of any of clauses 1-12 or 14-22, wherein L 3  is 
     
       
         
         
             
             
         
       
     
     Clause 26. The compound of any of clauses 1-25, wherein the nucleotide comprises a DNA nucleotide triphosphate or an RNA nucleotide triphosphate. 
     Clause 27. The compound of any of clauses 1-26, wherein the base of the nucleotide is a pyrimidine base and L 3  is attached to the number 5 carbon of the pyrimidine base or the base of the nucleotide is a purine base and L 3  is attached to the number 7 nitrogen of the purine base. 
     Clause 28. A method of enzymatic oligonucleotide synthesis comprising: selectively creating an electrode potential less than the hydrolysis potential of water at one or more microelectrodes on an electrode array thereby cleaving blocking groups from the ends of growing oligonucleotide strands; and contacting the surface of the electrode array with a predetermined nucleotide attached to a blocking group via an electrochemically-cleavable linker with a cleavage potential less than the hydrolysis potential of water. 
     Clause 29. The method of clause 28, wherein the electrochemically-cleavable linker is a compound of any of clauses 1-27. 
     Clause 30. A method of cleaving a linker in a solvent comprising: creating an electrode potential in the solvent that is less than the redox potential of the solvent, wherein the linker has a structure of a compound of any of clauses 1-27. 
     Clause 31. The method of clause 30, wherein the linker has a structure of: 
     
       
         
         
             
             
         
       
     
     Clause 32. The method of clause 30 or 31, wherein the solvent is an aqueous buffer, an organic solvent, or mixture of an aqueous buffer and organic solvent. 
     CONCLUSION 
     Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts are disclosed as example forms of implementing the claims. 
     The terms “a,” “an,” “the” and similar referents used in the context of describing the invention are to be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The terms “based on,” “based upon,” and similar referents are to be construed as meaning “based at least in part” which includes being “based in part” and “based in whole,” unless otherwise indicated or clearly contradicted by context. The terms “portion,” “part,” or similar referents are to be construed as meaning at least a portion or part of the whole including up to the entire noun referenced. As used herein, “approximately” or “about” or similar referents denote a range of ±10% of the stated value. 
     For ease of understanding, the processes discussed in this disclosure are delineated as separate operations represented as independent blocks. However, these separately delineated operations should not be construed as necessarily order dependent in their performance. The order in which the processes are described is not intended to be construed as a limitation, and unless other otherwise contradicted by context any number of the described process blocks may be combined in any order to implement the process or an alternate process. Moreover, it is also possible that one or more of the provided operations is modified or omitted. 
     Certain embodiments are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. Skilled artisans will know how to employ such variations as appropriate, and the embodiments disclosed herein may be practiced otherwise than specifically described. Accordingly, all modifications and equivalents of the subject matter recited in the claims appended hereto are included within the scope of this disclosure. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 
     Furthermore, references have been made to publications, patents, and/or patent applications throughout this specification. Each of the cited references is individually incorporated herein by reference for its particular cited teachings as well as for all that it discloses.