C(sp 3 )-H activation and merging with DNA coding

Abstract

Disclosed is the palladium-catalyzed C(sp 3 )‑H arylation of aliphatic carboxylic acids, amides, and ketones with BNA-encoded aryl iodides in water, in addition, sequential C‑H arylation chemistry enables on-DNA synthesis of structurally diverse scaffolds containing enriched C(sp 3 ) features, chiral centers, cyclopropane, cyclobutane, and heterocycles. This new chemistry allows for the preparation of DNA-encoded library (BEL) technology that can significantly speed up hit identification in drug discovery as it enables protein affinity selection of millions or billions of molecules in a single experiment. The sequential functionalization of multiple C‑H bonds provides a unique pathway to generate diversity and complexity from simple starting materials. The use of water as solvent, the presence of DMA, and the extremely low DMA-encoded coupling partner concentration (0.001 M) previously hindered the development of DMA-encoded C(sp 3 )‑H activation reactions, but many of those obstacles have now been overcome.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Application Serial No. 62 / 940,063, filed November 25, 2019, the disclosure of which is incorporated herein by reference. Background Technology

[0003] Brenner and Lerner 1 The initial concept proposed was to use DNA sequences to encode each individual reagent in each reaction step during split-pool synthesis. With millions or billions of DEL molecules incubated together with a fixed target protein in the same mixture, affinity selection of DNA-encoded libraries (DELs) targeting the target protein can now be performed on the bench. 2 The application of DEL technology has led to the rapid identification of lead compounds for drug discovery. 3 .

[0004] Currently, improving hit rate and enhancing the drug-like properties of lead compounds are key concerns for constructing superior DELs. In this context, establishing various types of DEL-compatible organic transformations (using water as a solvent, in the presence of DNA, with a DNA substrate concentration of 1 mM) is crucial. 4 The development of on-DNA reactions faces several challenges. DNA backbone degradation can occur under conventional reaction conditions; metal catalysts can be poisoned by oligonucleotides; and dissolving DNA-tagged substrates requires at least 20% water as a co-solvent.

[0005] Over the past decade, advances in DNA compatibility reactions have focused on nucleophilic aromatic substitution (S&A). N Ar), crosslinking, cycloaddition, and click chemistry to construct C(sp) 2 )-C(sp 2 ) 5 CN 6 CO 7 SX key 8 and heterocycles 9 Considering the incorporation of C(sp) 3 Carbon centers to construct C(sp) 2 )-C(sp 3 )key 10 And to avoid the well-known trend of high molecular weight and lipophilicity, the C(sp) of simple fatty acids and ketones is reduced. 3Coupling of α-H bonds with DNA-encoded heteroaryl groups would be highly desirable. The availability of multiple CH bonds in various carboxylic acids and ketones offers a unique opportunity to expand the accessible chemical space for DEL.

[0006] The recent development by the inventors and their colleagues of a wide range of transformations of the β-CH bond demonstrates the potential for generating unprecedented diversity from simplicity. 11 , such as in Figure 1A The diagram schematically illustrates this. The appeal of constructing DNA-encoding libraries using CH arylation of carboxylic acid-derived substrates is evident from recent studies in which CH activation was carried out in an organic solvent and subsequently individual DNA tags were attached. However, this approach does not allow encoding each individual reagent in each reaction step during cleavage synthesis, thus limiting the number of available building blocks and consequently the size of the library. 12 ( Figure 1B ).

[0007] Conversely, using CH activation as an on-DNA coupling step allows for the construction of much larger libraries. It is noteworthy that, despite the use of a very strong directing group, C(sp) activation has so far been limited in the presence of DNA. 3 The )-H activation reaction has not yet been successful. 13 This paper considers the development of on-DNA C(sp) substrates of different classes. 3 )-H activation, which can be sequentially used to construct C(sp) enriched C(sp) 3 DEL characteristics, chiral centers, small rings and heterocycles, such as in Figure 1C The above is illustrated schematically, and specific examples are given below. Summary of the Invention

[0008] This invention contemplates a method for preparing an aqueous composition comprising a library having multiple different bifunctional molecules, and the library itself. The contemplated method includes the following steps: reacting a bifunctionalized linker molecule B having terminals A' and C' according to formula A'-B-C' in one or more aliquots of the aqueous composition. Terminal C' comprises a tethered iodinated aromatic ring moiety without a secondary ring nitrogen atom, and terminal A' comprises a precursor Z' that identifies the nucleotide sequence. The iodinated aromatic ring moiety of the linker terminal C' is reacted in one or more reactants C4-C... 16 β-C(sp) of aliphatic carboxylic acid, formamide, or masking ketone unit X' 3 )-H or γ-C(sp 3The -H position is reacted via palladium-catalyzed arylation. Different X's react in each aliquot, as do different nucleotide identifier sequence precursors Z', which react with the terminal A' of the linker to form a product containing Z with formula (I). nα -ABCX nα Aqueous compositions of bifunctional molecules.

[0009] In formula (I), n is the position identifier of X and Z and is an integer from 1 to 10, preferably from 1 to 6, and more preferably from 2 to 5, such that when n is 1, X and Z are located at the position closest to connector B, and “α” identifies one or more specific reactions of C4-C. 16 An aliphatic carboxylic acid, formamide, or masking ketone unit X and one or more corresponding labeled paired DNA sequences Z in the resulting bifunctional molecule. Each Z' or Z is paired with and labeled with a specific X' or X. Optionally, the aqueous aliquot sample compositions thus formed, containing substantially equal amounts of bifunctional molecules, are mixed to form a single composition containing substantially equal numbers (amounts) of bifunctional molecules.

[0010] The carboxylic acid, formamide, or masking ketone functional group X in one or more aliquots of an aqueous composition containing one or more bifunctional molecules of formula (I) nα Reaction with one or more iodinated aromatic ring moieties (each other iodinated aromatic ring moiety used herein is also identical or different from the one present at the terminal C') W'. Reaction with one or more precursor nucleotide sequence identifiers Y' and Z. nα The reaction is to form one or more compounds containing formula (II)Y nβ -Z nα -ABCX nα -W nβ An aqueous composition of bifunctional molecules. A “β”-labeled bifunctional molecule of formula (II) with one or more specific reactive chemical groups W and one or more corresponding paired DNA sequence identifiers Y.

[0011] Optionally, the aqueous aliquots of the reactants thus formed, comprising substantially equal amounts of bifunctional molecules, are mixed to form a single composition comprising substantially equal amounts of bifunctional molecules. Each Y' or Y is paired with and identified by a specific W' or W.

[0012] One or more iodine-substituted aromatic ring moieties W present in an aqueous composition containing a bifunctional molecule of formula (II) or an aliquot thereof nβ In one or more C4-C reactions that are the same as or different from previous reactions 16 β-C(sp) of aliphatic carboxylic acid, formamide, or masking ketone unit V'3 )-H or γ-C(sp 3 The -H position is arylated via palladium-catalyzed reaction. This causes one or more precursor nucleotide sequence identifiers T' and Y to react. nγ The reaction forms one or more inclusion formulas (III)T nγ -Y nβ -Z nα -ABCX nα -W nβ -V nγ A composition of bifunctional molecules.

[0013] Each T' or T is paired with and identified by a specific V' or V. Additionally, γ identifies one or more specific C4-C atoms in the bifunctional molecule. 16 An aliphatic carboxylic acid, formamide, or masking ketone unit V and one or more corresponding marker paired DNA sequences Y.

[0014] Regarding the reaction sequence outlined above, it should be understood that at least one set of reaction steps includes reacting multiple aliquots of an aqueous composition containing bifunctional molecules individually with reactants that react differently from the bifunctional molecules in another aliquot in this step, and then combining the resulting aliquots to form an aqueous composition comprising a mixture of different bifunctional molecules constituting a library of said bifunctional molecules in the aqueous composition.

[0015] When referring to reactants or bifunctional molecules, it should also be understood that Z, A, C, X, Y, W, T, and V are the corresponding reaction forms of Z', A', C', X', Y', W', T', and V'.

[0016] Combining two or more aliquots containing a type III bifunctional molecule to form an aqueous composition containing a mixture of bifunctional molecules, thereby forming an aqueous composition containing a library. The library thus formed can be separated from the aqueous composition by methods such as lyophilization, precipitation with an organic solvent, chromatography, and other methods known to those skilled in the art.

[0017] It should also be noted that the synthesis of the considered bifunctional molecular library does not need to be stopped after the three reaction step cycles shown above, but can continue for several more reaction cycles following the pattern described above. Preferably, 2 to about 7 cycles, more preferably 3 to about 5 reaction cycles.

[0018] Libraries with multiple different bifunctional molecules were also considered, namely, bifunctional molecules of formula (III), Z, A, C, X, Y, W, T, and V. In the library of formula III functional molecules, Z, A, C, X, Y, W, T, and V have the same meanings as previously provided, as do the subscripts nγ, nβ, and nα. Therefore, T nγ -Y nβ -Z nα -A is a mixture of polynucleotide sequences, where specific identifiers mark the iodine-substituted aromatic ring moiety and C4-C of the reaction. 16 Aliphatic carboxylic acids, formamides, or masking ketone units, which constitute the reactive building blocks CX in a mixture of bifunctional molecules in the library. nα -W nβ -V nγ .

[0019] Preferably, adapter B contains an oligonucleotide sequence (approximately 18 to 22 base pairs (bp)) long enough to serve as a primer for repeated polymerase chain reactions (PCR), and also contains elements that allow cleavage of T. ni -Y ni -Z n The -A identifier is a polynucleotide sequence that is the recognition sequence of a predetermined restriction endonuclease. Preferably, each identifier oligonucleotide sequence is a double-stranded DNA and the specific identifier T... ni -Y ni -Z n -A polynucleotide sequences are also double-stranded DNA.

[0020] Preferably, each specific identifier double-stranded DNA oligonucleotide contains two restriction endonuclease recognition sites, one of which is located on either side of the specific identifier nucleotide sequence of the reactant used for synthesis. If the same endonuclease recognition site is located on either side of all oligonucleotides present at a given position in a bifunctional molecule, cleavage with that endonuclease yields a number of identifier DNA oligomers that can be separated and identified using mass spectrometry.

[0021] Technicians can also mix the target protein with a bifunctional molecule library or sublibrary to capture those bound library molecules, and then perform a PCR reaction on any target-bound bifunctional molecule, followed by sequencing of the replicated DNA.

[0022] Preferably, in some embodiments, each aromatic ring moiety comprises a six-membered ring, while in other embodiments, each aromatic ring moiety preferably comprises a five-membered ring. In still other embodiments, both the five- and six-membered aromatic ring moiety are present in the bifunctional molecular library. In some preferred embodiments, each aromatic ring moiety is carbocyclic, while in other preferred embodiments, each aromatic ring moiety is heterocyclic. In still other preferred embodiments, at least one aromatic ring moiety is carbocyclic and at least one aromatic ring moiety is heterocyclic.

[0023] In further optimization, C4-C 16 At least one of the aliphatic carboxylic acids, formamides, or masking ketones contains a 3- or 4-membered ring bonded to a carbonyl carbon atom, and the reacted 3- or 4-membered ring contains a β-C(sp) at the position where the arylation bond is formed. 3 )-H.

[0024] The following text also discloses information about C4-C. 16 A method for aqueous arylation of carboxylic acids, formamides, or masking ketones at the β or γ position of the carbonyl carbon atom. Attached Figure Description

[0025] In the accompanying drawings that form part of this disclosure,

[0026] Figure 1A , 1B 1C and 1C demonstrate the diversity of CH activation utilized in DNA-coding libraries (DELs). Figure 1A This demonstrates the synthesis of combinatorial chemistry libraries via sequential CH activation. 11 A schematic diagram, Figure 1B It is used to obtain DEL 12 A schematic diagram of Off-DNA CH activation, and Figure 1C The on-DNA CH activation sequence for obtaining DEL as described herein is schematically illustrated. An asterisk indicates the presence of a chiral center at the atom. TBHP, tert-butylhydrogen peroxide; Boc, tert-butyloxycarbonyl group; DG, direction group; and This indicates a connector that contains a bound DNA sequence and a bond.

[0027] Figure 2A and 2B The utility of multiple CH activations in the synthesis of DEL is shown, and the further addition of compound 1 (which itself is formed by CH activation) to form compound 1' is shown. Figure 2B An amide formation reaction between a DNA-bonded linker group amine and an aromatic carboxylic acid is shown, demonstrating the reliability of the on-DNA CH reaction of the present invention.

[0028] Figure 3A and 3B The diagram shows the linker (DNA-NH-) and C4-C bonded to the amine-terminated portion of the iodine-substituted aromatic ring. 16 β-C(sp) of carboxylic acids, formamides, or masking ketones 3 Aqueous arylation products and yields were obtained between H and H. This provides information on the process. Figure 3A and 3B The standard conditions for the reaction, including the general reaction, reactants, catalyst, oxidant and their concentrations, as well as the reaction temperature and duration, are shown in Table 1. Unless otherwise stated, the conditions in item 1 of Table 1 are used as the yields for the reaction. Figure 3A and Figure 3B The standard conditions for the reactions shown in both are as follows. Figure 3A This illustrates a single iodine-substituted aromatic ring moiety bonded to an amine-capped linker (DNA-NH-) and C4-C activated by several different formamides. 16 The products and yields of the reaction of carboxylic acids, each having a β-C (sp) relative to the carbonyl carbon atom of formamide. 3 The chiral ligand L1 shown in Table 1 exists in yields of )-H and has a substituent R at the α-position relative to both the nitrogen atom of the formamide group and the carbon atom of the carboxyl group. Figure 3A The reaction shown in the figure does not exist in its yield. Figure 3B The reaction is shown in the figure. Figure 3B Neopentanoic acid or 1-butylcyclopropane-1-carboxylic acid A23 is shown. Reaction products and yields with several different iodine-substituted aromatic ring moieties (with different aromatic ring structures and different iodine substituent positions). Exceptions: Compound 2: corresponding A, 500 mM; for compounds 3 to 13, 18, 19 and 39 to 45: AgTFA, 300 mM; corresponding A, 300 mM; 36 h; for compound 15: Ag3PO4, 200 mM; corresponding A, 300 mM; 36 h; for compound 21: corresponding A, 300 mM; 36 h; for compound 16: AgOTf, 300 mM; corresponding A, 300 mM; 36 h; for compound 17: H2O / DMA (6 / 1).

[0029] Figure 4A , 4B 4C shows the amine-capped linker (DNA-NH-) at the iodine-substituted aromatic ring portion of the DNA strand with C4-C. 16 β-C(sp) of carboxylic acids 3 Aqueous arylated products and yields obtained between )-H. Figure 4A It shows the provision of Figure 4B and 4CThe standard conditions for the reaction include the reactants, catalyst, oxidant and their concentrations, as well as the temperature and duration of the reaction. Figure 4B The diagram shows a single iodine-substituted aromatic ring moiety bonded to an amine-capped linker (DNA-NH-) with several different C4-C atoms. 16 The products and yields of carboxylic acid reactions, each of which has a β-C (sp...) 3 It has a substituent R at the α position relative to both the carboxyl carbon atom and the formamide nitrogen atom. Figure 4C The product shows the arylated DNA-chain aromatic moiety. Figure 4B Compound 68 shown is amidated with glycine methyl ester in pH 5.5 buffer for approximately 16 to 18 hours (overnight) at room temperature to form compound 73. DMTMM = 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride.

[0030] Figures 5A to 5D The diagram shows the iodine-substituted aromatic moieties of DNA strands and those containing β-C (sp...) 3 )-H and γ-C(sp 3 The results of the arylation reaction of masking ketones with )-H groups. Except as described below for compounds with specific numbers, the reactions were carried out using the standard conditions shown in Table 3 below. More specifically, Figure 5A The yield of aqueous arylation products obtained at the β-position of the carbonyl carbon atom of a masked ketone, which is previously and reversibly masked as an oxime (whose hydroxyl group is bonded to either of the two exemplary directional groups DG1 and DG2), is shown. Figure 5B The yields of aqueous arylated products obtained at the β-position relative to similar masking ketone carbonyl carbon atoms (which react with iodinated aromatic ring moieties of various different chains having iodine groups at different ring positions) are shown. Figure 5C The deblocking reaction is shown to remove the DG1 ketone carbonyl blocking group from compound 83 to form compound 103. Figure 5D A schematic flow chart is shown of the considered synthetic process involving multiple CH activation reactions to construct DEL diversity through multiple CH activation and reaction. Figure 5E A representative synthesis of compound 106 is shown, with the following conditions for steps [a), b), c), ..., etc.:

[0031] a)A1( 1000 equivalent), Pd(OAc)2 (10 equivalent), L1 ( a) 20 equivalents), Ag2CO3 (300 equivalents), NaOAc (150 equivalents), H2O / DMA / HFIP (8 / 1 / 1), 80℃, 16 hours; b) 4-Iodobenzylamine (300 equivalents), DMTMM (300 equivalents), phosphate buffer (pH 5.5), room temperature, 16 hours; c) C10 ( 300 equivalent), Pd(OAc)2 (40 equivalent), L8 ( d) Aniline (500 equivalents), AgTFA (500 equivalents), NaOAC (150 equivalents), H2O / DMA (9 / 1), 80℃, 20 hours; d) Aniline (500 equivalents), acetone (300 equivalents), phosphate buffer (pH 6.5), 50℃, 24 hours. Detailed Implementation

[0032] This invention considers a method for preparing an aqueous composition comprising a library having multiple different bifunctional molecules, as well as the library itself and the method of preparation thereof. The main reaction for preparing the library under consideration is a palladium(II)-catalyzed arylation reaction carried out in an aqueous medium. The arylation reaction under consideration forms a carbon-carbon (CC) bond between the reaction substrate and an aromatic or heteroaromatic iodide.

[0033] In the reaction under consideration, the iodine group of the iodinated aromatic ring moiety, which does not contain a secondary ring nitrogen atom, is subjected to C4-C... 16 The previous β-C(sp) of aliphatic carboxylic acid, formamide, or masking ketone unit 3 )-H or γ-C(sp 3 The )-H position of the bond is substituted, thereby forming a new C-C bond. Palladium-catalyzed arylation reactions between similar reactants have been reported in the literature, but they were carried out in organic solvents without the addition of water. This is considered the first report of such a reaction in an aqueous medium, and also the first report of its use in the preparation of bifunctional molecules and libraries containing them.

[0034] The method considers the following steps: reacting a bifunctionalized linker molecule B having terminals A' and C' according to formula A'-B-C' in one or more aliquots of an aqueous composition. Terminal C' comprises a bonded (chain-linked) iodinated aromatic ring moiety without a secondary ring nitrogen atom, and terminal A' comprises an identifier nucleotide sequence precursor Z'. The iodinated aromatic ring moiety of the linker terminal C' is reacted in one or more reactants C4-C 16 β-C(sp) of aliphatic carboxylic acid, formamide, or masking ketone unit X' 3 )-H or γ-C(sp 3The -H position is reacted via palladium-catalyzed arylation. Different X's react in each aliquot, as do different nucleotide identifier sequence precursors Z', which react with the terminal A' of the linker to form a product containing Z with formula (I). nα -ABCX nα Aqueous compositions of bifunctional molecules.

[0035] In formula (I), n is the position identifier of X and Z and is an integer from 1 to 10, preferably from 1 to 6, and more preferably from 2 to 5, such that when n is 1, X and Z are located at the position closest to connector B, and “α” identifies one or more specific reactions of C4-C. 16 An aliphatic carboxylic acid, formamide, or masking ketone unit X and one or more corresponding labeled paired DNA sequences Z in the resulting bifunctional molecule. Each Z' or Z is paired with and labeled with a specific X' or X. Optionally, the aqueous aliquot sample compositions thus formed, containing substantially equal amounts of bifunctional molecules, are mixed to form a single composition containing a substantially equivalent mixture of bifunctional molecules.

[0036] The masking ketones under consideration are compounds in which the carbonyl group is protected and does not react during arylation, but can be readily deprotected to provide the carbonyl group again. Exemplary masking carbonyl groups include oximes, hydrazones, and ketals. When used as an oxime, the hydroxyl group itself is protected from reaction by forming an ether bond, as shown below.

[0037] Available Pd(II) catalysts are well known in the art. Exemplary catalysts include PdCl2, Pd(TFA)2, Pd(Piv)2, [PdCl(C3H5)]2, PdCl2(PPh3)2, Pd(PPh3)4, Pd2(dba)3, [PdCl2(MeCN)2], [Pd(OTf)2·4MeCN], and [Pd(BF4)2·4MeCN]. Among these catalysts, Pd(TEA)2, Pd(Piv)2, and Pd(OAc)2 are currently preferred. The catalysts considered are used in catalytic amounts. Based on the molar number of the reaction substrate, this amount is typically from about 5 to about 40 molar percentages, and more preferably from about 10 to about 20 molar percentages.

[0038] Exemplary basic salts include NaOAc, Na2CO3, NaHCO3, Na2HPO4, Na2HPO4, NaH2PO4, Na3PO4, KHCO3, KOAc, K2CO3, K3PO4, K2HPO4·3H2O, Li2CO3, and Cs2CO3. Among these salts, NaOAc and Li2CO3 typically provide the highest yields and are preferred.

[0039] After the arylation reaction is complete, a scavenger, such as sodium diethyldithiocarbamate trihydrate, is typically added to the reaction mixture to aid in the recovery of the palladium catalyst. For each amount of iodine-substituted aromatic ring moiety in the chain, approximately 70 to approximately 90 equivalents of scavenger are used to achieve maximum palladium recovery and the presence of the initial DNA.

[0040] The reaction medium under consideration optionally, but preferably, includes ligands that promote both CH cleavage and subsequent functionalization steps. Effective CH functionalization typically requires a synergistic relationship between the ligand and the substrate coordinated to the metal center.

[0041] Although some reactions are not typically required, the presence of ligand molecules generally increases the yield of the desired product. The ligand is typically present in the reaction composition at a molar percentage of about 10 to about 30 based on the substrate moles. Preferably, the ligand is present at about 20 molar percentage. Alternatively, the ligand is present in an amount of about 1.5 to about 4 times the molar amount of the palladium(II) catalyst.

[0042] The palladium-catalyzed arylation reaction mixture under consideration is maintained at a temperature of about 70°C to about 100°C for a period of time sufficient for electrophilic insertion and formation of the arylation product. More preferably, the temperature is about 75°C to about 90°C. The reaction time is typically about 15 to about 30 hours, with about 18 to about 25 hours being common.

[0043] The carboxylic acid, formamide, or masking ketone functional group X in one or more aliquots of an aqueous composition containing one or more bifunctional molecules of formula (I) nα Reaction with one or more iodine-substituted aromatic ring moieties W' (which may be the same as or different from those present at the terminal C') that do not contain a secondary ring nitrogen atom. One or more precursor nucleotide sequence identifiers Y' and Z. nα To form one or more containing formula (II)Y nβ -Z nα -ABCX nα -W nβ An aqueous composition of bifunctional molecules. The aqueous aliquots of the resulting bifunctional molecules are optionally mixed to form a single composition comprising a mixture of approximately equal amounts of bifunctional molecules. Each Y' or Y is paired with and identified by a specific W' or W. "β" identifies one or more specific reactive chemical groups W and corresponding one or more paired DNA sequence identifiers Y in the bifunctional molecules of formula (II).

[0044] One or more iodine-substituted aromatic ring moieties W present in an aqueous composition containing a bifunctional molecule of formula (II) or an aliquot thereof nβIn one or more C4-C reactions that are the same as or different from previous reactions 16 β-C(sp) of aliphatic carboxylic acid, formamide, or masking ketone unit V' 3 )-H or γ-C(sp 3 The -H position is arylated via palladium-catalyzed reaction. This causes one or more precursor nucleotide sequence identifiers T' and Y to react. nγ The reaction forms one or more inclusion formulas (III)T nγ -Y nβ -Z nα -ABCX nα -W nβ -V nγ A composition of bifunctional molecules.

[0045] Each T' or T is paired with and identified by a specific V' or V. Additionally, γ identifies one or more specific C4-C atoms in the bifunctional molecule. 16 An aliphatic carboxylic acid, formamide, or masking ketone unit V and one or more corresponding marker paired DNA sequences Y.

[0046] Regarding the reaction sequence outlined above, it should be understood that at least one set of reaction steps includes reacting multiple aliquots of an aqueous composition containing bifunctional molecules individually with reactants that are different from those reacted in another aliquot in this step, and then combining the resulting aliquots to form an aqueous composition comprising a mixture of different bifunctional molecules constituting a library of said bifunctional molecules in the aqueous composition.

[0047] The connector is also referred to as a "head base" in this document. The following shows an exemplary head base used herein, along with two abbreviations frequently used herein. and DNA-NH2:

[0048]

[0049] The amino group -NH2 shown is a functional group that can be used to react with the iodine-substituted aromatic moiety and chain it to the remainder of the head group, while the DNA sequence can be easily extended by known oligonucleotide synthesis methods.

[0050] The preferred double-stranded oligonucleotide identifier Z' is used to identify a specific X' arylated at position "n" in an organic residue "string" added to the linker. The Greek letter accompanying each subscript "n" represents one to 40 different chemical structures and a corresponding oligonucleotide sequence identifying each of those structures. In preferred practice, one to about 20 different chemical structures are used at each position "n," along with the same number of identifier DNA sequences at the same position "n" on the other side of the linear bifunctional molecule relative to B. More preferably, one to about 10 different chemical structures are used, along with the same number of identifier DNA sequences present at each position in the bifunctional molecule.

[0051] In a preferred embodiment, the nucleotide sequence of the restriction endonuclease is located flanking both upstream and downstream of the X' marker sequence so that the positional identifier identified by the endonuclease can be easily removed to identify one or more of its reactant-marker nucleotide sequences. Preferably, the same endonuclease sequence is present on both sides of the marker sequence, so only one enzyme is needed to remove the marker sequence at a given position in the bifunctional molecule.

[0052] It should be noted that each aqueous arylation is preferably at the β-C (sp) of the carbonyl carbon relative to the carboxylic acid, formamide, or masking ketone unit. 3 Performed at position H.

[0053] In the bifunctional molecule of formula III, each T' or T is paired with and identified by a specific V' or V, while Y ni Zn, A, B, C, X n and W ni As defined above.

[0054] Combining two or more aliquots of a bifunctional molecule of formula III to form an aqueous composition comprising a mixture of bifunctional molecules, thereby forming an aqueous composition comprising a library. The library thus formed can be separated from the aqueous composition by means such as lyophilization, precipitation with an organic solvent, and other methods known to those skilled in the art.

[0055] It should also be noted that the synthesis of the considered bifunctional molecular library does not need to be stopped after the three reaction step cycles shown above, but can continue for several more reaction cycles following the pattern described above. Preferably, 2 to about 7 cycles, more preferably 3 to about 5 reaction cycles.

[0056] When multiple distinct residues are desired at a given position in the sequence, the aqueous composition containing the bifunctional molecule is divided into at least as many aliquots as the number of distinct reactants to be added, the identifier DNA sequence of each reactant, and the flanking position identifiers (if desired).

[0057] If it is desired that the next adjacent position be occupied by the mixture, the aliquot of each reaction is further divided into at least the number of reactants used in this step. In each newly divided aliquot, the members of the next reactant group are reacted individually with the growing bifunctional molecule, as are the DNA sequences that identify each one and the flanking restriction site sequences that identify the specific position (if desired).

[0058] The following uses different syntheses to illustrate the above variants and other parallel syntheses (whose products were combined and then partitioned):

[0059] Brenner and Lerner, Proc Natl Acad Sci, USA 89:5381-5383 (June 1992), U.S. Patent Nos. 5,573,905, 5,723,598 and 6,060,596, and Houghten, Proc Natl Acad Sci, USA 82:5131-5135 (1985), Houghten et al., Biotechniques, 4(6):522-528 (1986), U.S. Patent Nos. 4,631,211 and 5,763,193

[0060] Each of their publicly available contents is incorporated through citation.

[0061] If a mixture is not expected to be the next possible site, all previously reacted aliquots are combined to provide a single composition containing mixed bifunctional molecules. These mixed bifunctional molecules can then be reacted with individual reactants and used or recycled for later use without further reaction.

[0062] Therefore, T nγ -Y nβ -Z nα -A is a polynucleotide sequence in which a specific identifier identifies the bifunctional molecule in the library that constitutes CX. nα -W nβ -V nγ The aromatic ring moiety and C4-C of the reaction building blocks 16 Aliphatic carboxylic acid, formamide, or masking ketone unit. Preferably, linker B comprises an oligonucleotide sequence containing a predetermined recognition sequence for a restriction endonuclease that allows cleavage of T... nγ -Ynβ -Z nα -A identifier is a polynucleotide sequence.

[0063] Preferably, each identifier oligonucleotide sequence is a double-stranded DNA and a specific identifier T. nγ -Y nβ -Z nα -A polynucleotide sequences are also double-stranded DNA. More preferably, each specific identifier double-stranded DNA oligonucleotide contains two restriction endonuclease recognition sites, one on either side of the specific identifier nucleotide sequence used in synthesis. Another preferred embodiment is a C4-C bonded to the aromatic ring partial arylation. 16 Aliphatic carboxylic acids, formamides, or masking ketones are affected by the β-C(sp) of a previously present carbonyl group relative to the carboxylic acid, formamide, or masking ketone. 3 Bonded at the -H position.

[0064] In some embodiments, it is preferred that each aromatic ring moiety comprises a six-membered ring, while in other embodiments, it is preferred that each aromatic ring moiety comprises a five-membered ring. In still other embodiments, both the five- and six-membered aromatic ring moiety are present in a library of bifunctional molecules. In some preferred embodiments, each aromatic ring moiety is carbocyclic, while in other preferred embodiments, each aromatic ring moiety is heterocyclic. In still other preferred embodiments, at least one aromatic ring moiety is carbocyclic and at least one aromatic ring moiety is heterocyclic.

[0065] In further optimization, C4-C 16 At least one of the aliphatic carboxylic acids, carbamates, or masking ketones contains a 3- or 4-membered ring bonded to a carbonyl carbon atom, and the reacted 3- or 4-membered ring contains a β-C(sp) at the position where the arylation bond is formed. 3 )-H.

[0066] The following also discloses C4-C transitions at the β or γ position of the carbonyl carbon atom in carboxylic acids, formamides, or masking ketones. 16 A method for the aqueous arylation of aliphatic carboxylic acids, formamides, or masking ketones.

[0067] The iodinated aromatic ring moiety containing β-C(sp) without secondary ring nitrogen atoms is substituted with iodine. 3 )-H or γ-C(sp 3 The reactants of )-H (i.e., C4-C) 16 Aliphatic carboxylic acids, formamides or masking ketones), catalytic amounts of palladium(II) catalysts, silver salts, basic alkali metal salts, and optionally Pd in ​​an aqueous medium 2+The ion-interacting ligands are dissolved or dispersed in an aqueous medium to form an aqueous reaction medium. The aqueous reaction medium is maintained at a temperature of about room temperature (about 20°C) to about 100°C, and preferably about 70°C to about 100°C, for a period of about 15 to about 30 hours to provide a product containing an aromatic ring moiety free of secondary ring nitrogen atoms bonded at ring positions formally occupied by iodine substituents and C4-C6 bonds. 16 β-C(sp) of aliphatic carboxylic acids, formamides, or masking ketones 3 )-H or γ-C(sp 3 The previous positions occupied by )-H.

[0068] In a preferred embodiment, it contains β-C(sp) 3 )-H or γ-C(sp 3 C4-C of )-H 16 The aliphatic carboxylic acid, formamide, or masking ketone is present in an aqueous reaction medium in a molar excess of about 10:1 to about 1200:1 relative to the iodinated aromatic moiety. In another preferred embodiment, the silver salt and the basic alkali metal salt are present in the aqueous reaction medium in a molar ratio of about 2:1 to about 4:1, and the molar ratio of the silver salt to the iodinated aromatic moiety is about 100:1 to about 400:1.

[0069] In another preferred embodiment, the ligand is present in an amount of about 1.5 to about 4 times the molar amount of the palladium(II) catalyst. Preferred ligands are selected from one or more of the following:

[0070]

[0071] In another preferred embodiment, the iodine-substituted aromatic ring moiety without secondary ring nitrogen atoms comprises a single 5- or 6-membered aromatic ring, itself linked to a DNA-containing adapter described as "DNA-", and selected from one or more of the following:

[0072]

[0073] In some preferred embodiments, β-C(sp) is included. 3 )-H or γ-C(sp 3 The masking ketones of )-H are selected from one or more of the following:

[0074]

[0075]

[0076] result

[0077] Since carboxylic acids and (hetero)aryl iodides are common building blocks of DNA-coding libraries, the coupling of aryl iodides to free carboxylic acids in the DNA chain was first investigated via Pd-catalyzed β-CH arylation. Besides the need to adapt the chemistry to aqueous conditions, significant challenges arose, particularly the limited stability of DNA to low pH and heat, the potential to interfere with DNA base reactivity, and the high dilution of DNA chain components. Nevertheless, it was decided to develop this chemistry around free carboxylic acids.

[0078] Having β-C(sp 3 )-H or γ-C(sp 3 Some illustrative examples of )-H available for carboxylic acids are shown in the table below.

[0079]

[0080] Based on previous CH arylation work 14 Following the guidance, palladium sources, ligands, silver sources, bases, and cosolvents were screened as shown in Tables 1 and 2.

[0081] Optimized conditions yielded product compound 1 in 78% yield without diarylation or triarylation of neopentanoic acid (Entry 1, Table 1). Palladium, silver, and heat were mandatory (Entries 2 to 4, Table 1). Ligands were not required (Entry 5, Table 1), but amino acid-derived ligands improved the yield, and ligand L1 was found to be optimal.

[0082] Table 1

[0083]

[0084]

[0085] Due to the use of excessive amounts of silver salt and palladium, the major byproduct protodeiodination (e.g., compound S1b) is always present to a very small extent. Compound S1b, which does not contain iodide, withstands the reaction conditions and recovers completely, confirming that the coupling occurs at the aryl iodide rather than at some undetermined location on the DNA tag (entry 6, Table 1). The dithiocarbamate, due to its stronger coordination with palladium, releases DNA from the DNA-palladium complex, resulting in an insoluble complex.

[0086] After each reaction, the mixture was incubated with sodium diethyldithiocarbamate trihydrate to remove palladium. DNA recovery levels were significantly affected by the dithiocarbamate scavenger loading.

[0087] examine Figure 3AThe results shown indicate that a variety of carboxylic acids adjacent to a quaternary carbon atom are suitable for this chemistry, including those containing a hydroxyl group, ether, or fluorine (compounds 2 to 14). However, acids adjacent to a secondary or tertiary carbon atom react in yields of <30% (compounds 15 to 16). Importantly, cyclopropanecarboxylic acids and cyclobutanecarboxylic acids (expected as isosteres of olefins) are competent coupling partners (compounds 17 to 21).

[0088] It is well known that the pd-catalyzed β-CH arylation mechanism only produces the cis stereoisomer. LCMS analysis could not determine whether the chiral ligand L1 exhibited any absolute stereoinduction.

[0089] Figure 3B The results shown demonstrate that the substitution mode of aryl iodides is flexible (compounds 22 to 28, 39 to 44). Although previously published reaction conditions in organic solvents do not allow for the coupling of heteroaryl iodides... 24,25 However, it is thought that the tolerance of DNA conditions (containing nitrogen-containing heterocyclic bases) should allow for the coupling of heteroaryl iodides (e.g., pyridine and pyrazole). This is indeed the case; heteroaryl iodides successfully react with carboxylic acids (compounds 29 to 38, 42 to 44) under the same reaction conditions. Since aryl iodides and heteroaryl iodides react under the same conditions, they can both be present in the considered DEL split-cell synthesis.

[0090] As shown in part of Table 1 above, for Figure 3A and 3B The standard reaction conditions are based on the presence of β-C (sp) in 1000 equivalent amounts. 3 )-H or γ-C(sp 3 The reaction of α-H with carboxylic acid begins with 10 nanomoles (nmol) of DNA-linked aromatic iodide. This reaction occurs in the presence of 10 equivalents of Pd(Ac)₂, 120 equivalents of the ligand, 300 equivalents of Ag₂CO₃, and 150 equivalents of NaOAC dissolved or dispersed in a medium containing water, dimethylacetamide, and hexafluoro-2-propanol in an 8:1:1 ratio. The reaction is carried out at 80°C for 16 hours.

[0091] Unless otherwise stated, those conditions are used as standard conditions. For compound 2: carboxylic acid is present in 500 equivalents. For compounds 3 to 14, 18, 19 and 39 to 44: AgTFA, 300 equivalents; carboxylic acid, 300 equivalents, 36 hours. For compound 15: Ag3PO4, 200 equivalents; carboxylic acid, 300 equivalents; 36 hours. For compound 21: carboxylic acid, 300 equivalents; 36 hours. For compound 16: AgOTf, 300 equivalents; carboxylic acid, 300 equivalents; 36 hours. For compound 17: H2O / DMA (6 / 1).

[0092] Table 2

[0093]

[0094]

[0095] A re-evaluation of the silver source, base, and co-solvent in the absence of ligands led to optimized conditions providing compound 45 in 69% yield (Entry 1, Table 2). Palladium is essential for this transformation (Entry 2, Table 2). The silver salt and base are not strictly required, but have a significant effect on the yield (Entries 3 to 9, Table 2). Surprisingly, this reaction was found to proceed well at room temperature as well (Entry 10, Table 2).

[0096] Amides derived from cyclopropane carboxylic acids or cyclobutane carboxylic acids and a wide range of α-amino acids react readily at both heating and room temperature (compounds 45 to 56), which... Figure 4A This can also be seen in the yields shown. pd-catalyzed arylation proceeds only at the β-CH bond from the amide; attempted coupling with Ac-L-Val-OH failed to yield the arylated product compound 59, indicating that α-arylation did not occur. The two LC peaks of equal mass are due to the generation of diastereomers following CH arylation. To demonstrate this, a representative off-DNA reaction was performed to synthesize product compound 51. As expected, a mixture of diastereomers was observed, but the proportion was slightly lower due to the different reaction temperatures.

[0097] While attention was focused on amides derived from α-amino acids and those containing cyclopropyl or cyclobutyl rings (desired as isosteres of olefins), the chemical reactions can be extended to other alkylcarboxylic acids (compounds 57 and 58) and β-amino acids (compound 60). Different aromatic substitution patterns on the aryl iodides of the DNA chain are tolerable (compounds 61 to 65), as in... Figure 4B As seen in the yield figures.

[0098] Similar to the β-CH arylation of carboxylic acids, the DNA tolerance conditions used for amide arylation can also be applied to heteroaryl iodides such as pyridine and pyrazole (compounds 66 to 72) used for coupling DNA strands. If desired, the carboxylic acid of the product can be further modified (compound 73), as shown in... Figure 4C .

[0099] DNA-compatible C(sp) compounds for carboxylic acids and amides have been developed. 3 )-H arylation, attention has turned to C(sp) arylation, which is similar to DNA compatibility. 3 )-H arylation utilizes ketones. Ketones are useful monomers for constructing DELs because they can be processed in one step via reductive amination. Previous work using aminooxyacetic acid as a removable orientation group to recruit palladium activates the β-CH bond of ketones. 16 Guided by [the relevant authority], a DNA-tolerant version of this response was created and optimized.

[0100] In Table 3 and Figures 5A to 5D The optimized reaction shown yielded the arylated product compound 83 in 62% yield (Entry 1, Table 3, below). This reaction requires palladium and heat (Entries 2 and 3, Table 3) and is strongly influenced by silver salts (Entries 4 to 6, Table 3); the effects of ligands and bases are less significant (Entries 7 and 8, Table 3). Ligand L8 was also found to reduce DNA degradation and provide a clearer LC trace. Although large excesses of palladium are generally associated with DNA tag degradation, this chemistry yielded higher yields at 20 mM Pd / L (Entry 1, 40 equivalents) than at 30 equivalents (Entry 9, Table 3).

[0101] Table 3

[0102]

[0103]

[0104] The above conditions affect β-CH arylation in different contexts, including on acyclic ketone derivatives (compounds 74-77, 84-89), at positions adjacent to simple or complex rings (compounds 78-82, 90, 91), and on simple or complex rings (compounds 83, 92), as by... Figure 5A The yields shown are as described. The ketones to be arylated are allowed to have a wide range of functional groups, including esters, ethers, acetals, and amides.

[0105] Ketone derivatives with β-quaternary ammonium centers can also be γ-arylated (compound 93), such as... Figure 5A As shown in the image. Figure 5BThe yields shown demonstrate that aryl iodides of DNA lineage accept different substitution modes (compounds 94 to 97) and that heteroaryl iodides can react under the same conditions (compounds 98 to 102).

[0106] The ability to convert oxime ethers back to ketones is crucial for carrying out this chemical reaction in the construction of DEL. These oxime ethers were found to be readily hydrolyzed in the presence of aniline and acetone (compounds 83 to 103), possibly through equilibrium shift with aniline and capture of free aminooxyacetic acid with acetone. 17 This reaction is in Figure 5C As shown in the image.

[0107] These C(sp) 3 Each of the C(sp)-H activation reactions, as a new cleavage in the DEL synthesis, can be incorporated into a unique structural motif. Multiple C(sp) groups in the DEL synthesis... 3 The combination of β-H activations can further enhance diversity. Therefore, a multi-step DNA synthesis consisting of β-CH arylation of carboxylic acids, amide formation, β-CH arylation of masking ketones, and ketone deprotection was initiated to demonstrate how these CH-activated chemistry combinations can be used to develop a large DEL of various drug-like compounds. Representative analogue synthesis was conducted in... Figure 5D As shown in the image.

[0108] Therefore, the pyridyl iodide compound S18 is coupled with neopentanoic acid to form intermediate compound 31, and the amide is coupled with p-iodobenzylamine, thus establishing a second CH activation event. Coupling with a masked cyclobutyl ketone yields oxime ether compound 105, whose hydrolysis shows that ketone compound 106 has an overall yield of 6% in the four steps, as shown in the figure. Figure 5E As shown in the image.

[0109] The above is just one of many sequences that technicians can design by combining a series of CH arylation steps in sequence with common DNA-coding library construction steps such as amide formation or reductive amination. Importantly, the ability to perform CH arylation using aryl iodides of the DNA lineage means that full-power split-cell combinatorial synthesis is possible.

[0110] To evaluate the compatibility of CH activation chemistry with DNA synthesized by DEL, selected chemically modified on-DNA analogs (CH arylation products 1, 45, and 83) and their starting aryl iodide analog S1a were enzymatically ligated with 65-mer dsDNA, resulting in oligomers of approximately equal length to the coding tag constructed by 3 cycles of DEL. All four ligation reactions proceeded successfully, indicating that the chemistry had no significant effect on coding reproducibility. To determine the amount of amplifiable DNA remaining after exposure to CH activation conditions, the ligation products from 1, 45, and 83 were amplified by PCR and compared with those from S1a (the untreated control). All three reactions showed satisfactory PCR activity (60% to 80% amplifiable DNA remaining). Furthermore, Sanger sequencing reads confirmed the integrity of their nucleotide sequence structure.

[0111] Since purification is an inherently difficult process in cleavage synthesis, the reactivity required for DNA-compatible reactions must be free of unidentified byproducts that would complicate analysis. In this case, the major byproducts generated by our reaction platform consist only of the starting material or its original dehalogenated derivatives. Finally, we were able to obtain all products in moderate and overall useful yields via our on-DNA CH arylation platform; above the 25% threshold considered feasible in DEL synthesis. 10f It is also encouraging that we were able to achieve 60% to 80% DNA recovery from qPCR experiments, which is higher than the acceptable threshold of 30% that is considered feasible in these process practices. 4c In summary, these promising results further demonstrate our DEL-compatible C(sp) 3 The feasibility of the )-H activation platform enables practitioners to quickly generate structural complexity and diversity in a modular manner.

[0112] In summary, DEL-compatible C(sp) compounds for carboxylic acids, amides, and ketones have been developed. 3 )-H activation reaction. Ligands are crucial for reactivity under DEL conditions. These schemes are related to C(sp) coupling partners of small-ring and heterocyclic compounds, which are expected to improve drug-like properties. 3 -H bond compatibility. C(sp) used for the order of DEL synthesis 3 )-H activation is used to construct a structure containing high C(sp) 3 The chemical diversity of the )-H characteristic provides unique tools.

[0113] 1. General Information

[0114] Equipment and chemicals

[0115] use Modular heating blocks (64 wells) heat PCR tubes to run DNA reactions. A 10K variable speed mini centrifuge (BT604) was purchased from BTLab Systems. Samples were presented to HPLC-MS using 9mm threaded polypropylene vials and caps. Hexafluoroisopropanol (HFIP) was purchased from Oakwood. N,N-Dimethylacetamide (DMA) was obtained from Honeywell. N,N-Dimethylformamide (DMF) and acetonitrile (CH3CN) were obtained by passing previously degassed solvents through an activated alumina column. All reactions were performed using deionized water. Pd(OAC)2 was purchased from Strem Chemicals, Inc. Ag2CO3, AgOAc, AgTFA, and Ag3PO4 were obtained from Sigma-Aldrich. All other reagents were purchased at the highest commercial quality and were ready for use without further purification. Ligands were prepared according to previously published protocols, except for some which are now commercially available. Iodine-substituted heteroaromatic acids were obtained from Pfizer. Carboxylic acids, amides, and ketones with directional groups were synthesized according to previously published protocols, except for some which are also commercially available. DMTMM = 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride.

[0116] DNA headpiece material

[0117] The DNA header (5'- / 5Phos / GAGTCA / iSp9 / -iUniAmM / -iSp9 / -TGACTCCC-3', as shown below) was obtained from Biosearch Technologies, Petaluma, CA. The abbreviated DNA header is shown below.

[0118]

[0119] DNA sample analysis

[0120] DNA concentration:

[0121] Assuming a total DNA recovery rate of 100% after the reaction, the DNA sample to be analyzed by HPLC-MS was prepared as 0.1 mM in H2O.

[0122] analyze:

[0123] A 1 μL DNA solution was analyzed at 60 °C on a Waters I-Class LC system, Waters BEH C18, in (A) water and (B) methanol, with 114 mM HFIP and 14 mM Et3N (0.3 mL / min, 10% to 26% B, over 10 min). Yield was determined by calculating the percentage of UV absorbance corresponding to the product peak at 260 nm, neglecting possible differences in UV absorption coefficients between DNA products and assuming 100% mass recovery. Peak identification was performed using ESI [M]. 3- The ions are identified.

[0124] Deconvolution:

[0125] Using MassLynx TM The V4.1 software is used for data visualization and integration.

[0126] Yield calculation:

[0127] Ignoring differences in UV coefficients across all DNA products and assuming a total DNA recovery of 100%, the yield of DNA products was determined from the UV absorption trace (260 nm) peak area using the following formula:

[0128]

[0129] MS deconvolution:

[0130] Given the observed multi-charge (negative) mass, the triple-charge mass was determined as the base peak in all cases. The observed m / z can be calculated as m / z = [M] / z - 1.00794.

[0131] 2. Ligand structures L1 to L13

[0132]

[0133] Ligands L3, L5, L6 through L13 were purchased from Sigma-Aldrich, Nova Biochem, TCI, and Combi-Blocks Inc. Ligand L1 14b L2 18 L4 19 The synthesis was based on previous reports.

[0134] 3. Preparation of DNA-conjugated aryl iodides

[0135] 3.1 General procedures for using aryl iodides for DNA conjugation 1

[0136]

[0137] Material

[0138] Head: 20mM, in water

[0139] Sodium carboxylate: 1.0 M, in water [add 1 mmol of acid to 1.0 mL of NaOH (40 mg) aqueous solution]

[0140] 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM): 1.0 M in water (294.7 mg DMTMM dissolved in 1 mL H2O)

[0141] Borate buffer: 100 mM, in H2O

[0142] General Operation 1

[0143] 1) For the head solution (400 nmol, 20, keep at room temperature for 3 hours).

[0144] 2) Add 5M NaCl to the mixture and store at -20°C. Then store the mixture in a refrigerator at -20°C for more than 30 minutes.

[0145] 3) Centrifuge the sample at 10,000 rpm for 7 minutes at 4°C in a microcentrifuge. Discard the supernatant and dry the precipitate under vacuum. Then dissolve the precipitate in deionized N,N- and heat at 70°C for 12 hours.

[0146] 4) Cool to room temperature, add 5M NaCl sequentially, and store the resulting mixture at -20°C for 30 minutes. Centrifuge the mixture at 10,000 rpm for 7 minutes at 4°C, then remove the supernatant and dry the precipitate under vacuum. The precipitate can be used for the next study without further purification.

[0147] 5) HPLC-

[0148] (10mM),

[0149] Test samples with a concentration of 0.1 mM were prepared.

[0150] 3.2 Structure of S1 to S23

[0151]

[0152] Iodine-substituted aromatic acids S1 to S13 were purchased from Sigma-Aldrich, Oakwood, TCI, and Combi-Blocks Inc. Heteroaromatic acids S14 to S23 were obtained from Pfizer.

[0153] 3.3 LC trace and mass characterization of S1 to S23

[0154] S1a's LC trace and mass

[0155]

[0156] Follow general procedure 1.

[0157] Yield: 85%

[0158] Precision quality: 5178.8211

[0159] The triple charge mass [M] / 3-1.00794, calculated value 1725.2658; observed value 1725.2783.

[0160]

[0161] Follow general procedure 1.

[0162] Yield: 83%

[0163] Precision quality: 5052.9244

[0164] The mass of the triple charge [M] / 3-1.00794, calculated value 1683.3002; observed value 1683.3007.

[0165]

[0166] Follow general procedure 1.

[0167] Yield: 83%

[0168] Precision quality: 5178.8211

[0169] The triple charge mass [M] / 3-1.00794, calculated value 1725.2658; observed value 1725.2783.

[0170]

[0171] Follow general procedure 1.

[0172] Yield: 78%

[0173] Precision quality: 5192.8367

[0174] The triple charge mass [M] / 3-1.00794, calculated value 1729.9376; observed value 1729.9423.

[0175]

[0176] Follow general procedure 1.

[0177] Yield: 74%

[0178] Precision quality: 5164.8054

[0179] The triple charge mass [M] / 3-1.00794, calculated value 1720.5939; observed value 1720.6038.

[0180]

[0181] Follow general procedure 1.

[0182] Yield: 69%

[0183] Precision quality: 5178.8211

[0184] The triple charge mass [M] / 3-1.00794, calculated value 1725.2658; observed value 1725.2783.

[0185]

[0186] Follow general procedure 1.

[0187] Yield: 67%

[0188] Precision quality: 5198.7664

[0189] The mass of the triple charge [M] / 3-1.00794, calculated value 1731.9142; observed value 1731.9187.

[0190]

[0191] Follow general procedure 1.

[0192] Yield: 83%

[0193] Precision quality: 5164.8054

[0194] The triple charge mass [M] / 3-1.00794, calculated value 1720.5939; observed value 1720.6038.

[0195]

[0196] Follow general procedure 1.

[0197] Yield: 76%

[0198] Precision quality: 5178.8211

[0199] The triple charge mass [M] / 3-1.00794, calculated value 1725.2658; observed value 1725.2783.

[0200]

[0201] Follow general procedure 1.

[0202] Yield: 71%

[0203] Precision quality: 5198.7664

[0204] The mass of the triple charge [M] / 3-1.00794, calculated value 1731.9142; observed value 1731.9187.

[0205]

[0206] Follow general procedure 1.

[0207] Yield: 85%

[0208] Precision quality: 5164.8054

[0209] The triple charge mass [M] / 3-1.00794, calculated value 1720.5939; observed value 1720.6038.

[0210]

[0211] Follow general procedure 1.

[0212] Yield: 75%

[0213] Precision quality: 5178.8211

[0214] The triple charge mass [M] / 3-1.00794, calculated value 1725.2658; observed value 1725.2783.

[0215]

[0216] Follow general procedure 1.

[0217] Yield: 71%

[0218] Precision quality: 5182.7960

[0219] The triple charge mass [M] / 3-1.00794, calculated value 1726.5907; observed value 1726.6053.

[0220]

[0221] Follow general procedure 1.

[0222] Yield: 55%

[0223] Precision quality: 5182.7960

[0224] The triple charge mass [M] / 3-1.00794, calculated value 1726.5907; observed value 1726.6053.

[0225]

[0226] Follow general procedure 1.

[0227] Yield: 57%

[0228] Precision quality: 5165.8007

[0229] The mass of the triple charge [M] / 3-1.00794, calculated value 1720.9256; observed value 1720.9265.

[0230]

[0231] Follow general procedure 1.

[0232] Yield: 65%

[0233] Precision quality: 5165.8007

[0234] The mass of the triple charge [M] / 3-1.00794, calculated value 1720.9256; observed value 1720.9265.

[0235]

[0236] Follow general procedure 1.

[0237] Yield: 79%

[0238] Precision quality: 5165.8007

[0239] The mass of the triple charge [M] / 3-1.00794, calculated value 1720.9256; observed value 1720.9265.

[0240]

[0241] Follow general procedure 1.

[0242] Yield: 59%

[0243] Precision quality: 5209.8269

[0244] The triple charge mass [M] / 3-1.00794, calculated value 1735.6010; observed value 1735.6018.

[0245]

[0246] Follow general procedure 1.

[0247] Yield: 56%

[0248] Precision quality: 5165.8007

[0249] The mass of the triple charge [M] / 3-1.00794, calculated value 1720.9256; observed value 1720.9265.

[0250]

[0251] Follow general procedure 1.

[0252] Yield: 77%

[0253] Precision quality: 5168.8116

[0254] The mass of the triple charge [M] / 3-1.00794, calculated value 1721.9293; observed value 1721.9290.

[0255]

[0256] Follow general procedure 1.

[0257] Yield: 76%

[0258] Precision quality: 5168.8116

[0259] The triple charge mass [M] / 3-1.00794, calculated value 1721.9293; observed value 1721.9459.

[0260]

[0261] Follow general procedure 1.

[0262] Yield: 69%

[0263] Precision quality: 5168.8116

[0264] The mass of the triple charge [M] / 3-1.00794, calculated value 1721.9293; observed value 1721.9290.

[0265]

[0266] Follow general procedure 1.

[0267] Yield: 67%

[0268] Precision quality: 5154.7847

[0269] The mass of the triple charge [M] / 3-1.00794, calculated value 1717.2536; observed value 1717.2589.

[0270]

[0271] Follow general procedure 1.

[0272] Yield: 69%

[0273] Precision quality: 5170.7618

[0274] The triple charge mass [M] / 3-1.00794, calculated value 1722.5793; observed value 1722.5918.

[0275] 4. Experimental section for on-DNA CH arylation of free carboxylic acids

[0276] 4.1 Substrate structures of free carboxylic acids A1 to A38

[0277]

[0278] Carboxylic acids are obtained from commercial sources or synthesized following literature procedures. 14b,20

[0279] 4.2 Condition Optimization

[0280] Table 4. Evaluation of A1 concentration

[0281]

[0282]

[0283] Table 5. Evaluation of bases

[0284]

[0285]

[0286] Table 6. Evaluation of Pd / L concentration

[0287]

[0288]

[0289] Table 7. Evaluation of Standard Conditions

[0290]

[0291]

[0292] a Only S1b was fully recovered.

[0293] 4.3 DNA Recovery Research

[0294] Table 8. Evaluation of DNA Recovery

[0295]

[0296]

[0297] a The total DNA recovery rate was calculated using S1a as the standard.

[0298] 4.4 General Procedures for On-DNA CH Arylation of Free Carboxylic Acids 2

[0299] Condition A

[0300]

[0301] Material

[0302] DNA-conjugated aryl iodide S: 10 mM, in H2O

[0303] Carboxylic acid A: 3M, in DMA (Note: High concentrations can increase the overall volume).

[0304] L1: 200 mM in hexafluoroisopropanol (HFIP) (4.4 mg in Pd(OAc)2)

[0305] NaOAc: 1.5M, in H2O

[0306] Sodium diethyldithiocarbamate trihydrate (scavenger): 1M in H2O (225.3 mg in 1.0 mL H2O)

[0307] operate

[0308] 1) Add Pd(OAc)2-dried, carboxylic acid A DNA-conjugated aryl iodide S (10 nmol, 1 NaOAc) to the prepared AgTFA (300 equivalents, 0.66 mg).

[0309] d. Finally, L1 solution (20

[0310] The reaction mixture was then heated at 80°C for 36 hours.

[0311] 2) Cool the reaction mixture to room temperature.

[0312] The resulting mixture was then heated at 80°C for 30 minutes.

[0313] 3) Cool the reaction mixture to room temperature and add 5M NaCl solution (10% by volume). Store the mixture in a refrigerator at -20°C for more than 30 minutes.

[0314] 4) Centrifuge the sample at 10,000 rpm for 7 minutes in a microcentrifuge. Discard the supernatant and dry the precipitate under vacuum. Redissolve the resulting DNA precipitate in H2 and centrifuge at 10,000 rpm for 2 minutes in a microcentrifuge. Analyze using An-MS.

[0315] Condition B:

[0316]

[0317] Material

[0318] S1a: 10mM, in H2O

[0319] Cyclopropaneformic acid (A22): 3M, in H2O

[0320] L1: 200mM, in HFIP

[0321] Pd(OAc)2: 100mM, in HFIP

[0322] NaOAc: 1.5M, in H2O

[0323] Sodium diethyldithiocarbamate trihydrate (scavenger): 1M, in H2O

[0324] operate

[0325] Add Pd(OAc)2, L1 solution (20

[0326] -Dry, add Ag2CO3 (300 equivalents, 0.63 mg), then A22 (1000 equivalents, 3.3 mg).

[0327] S1a (10 nmol, 1 NaOAc aqueous solution (150)

[0328] Vortex. Heat the reaction mixture at 80°C for 16 hours.

[0329] 2) Cool the reaction mixture to room temperature, 9

[0330] The resulting mixture was then heated at 80°C for 30 minutes.

[0331] 3) Cool the reaction mixture to room temperature and add 5M NaCl solution (10% by volume). Store the mixture in a refrigerator at -20°C for more than 30 minutes.

[0332] 4) Centrifuge the sample at 10,000 rpm for approximately 7 minutes in a microcentrifuge. Discard the supernatant and dry the precipitate under vacuum. Redissolve the resulting DNA precipitate in H2 and centrifuge at 10,000 rpm for 2 minutes in a microcentrifuge. Analyze using An-MS.

[0333] 4.5 Scope and Limitations of Free Carboxylic Acids

[0334] Table 9. Scope and limitations of free carboxylic acids

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341] a Use A2 (500mM); b Follow condition B; c Using A26 (1000mM), 16 hours. ND = Not detected.

[0342] 4.6 Trace and mass characterization of LC from 1 to 51

[0343] 1 LC trace and mass

[0344]

[0345] Use A1 (1000mM) following general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0346] Yield: -

[0347] Ratio (product / deiodide / aryl iodide): 66 / 2 / 4

[0348] Precision quality: 5152.9766

[0349] The triple charge mass [M] / 3-1.00794, calculated value 1716.6509; observed value 1716.6649.

[0350] 2 LC trace and mass

[0351]

[0352] Use A2 (500mM) following general procedure 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0353] Yield: -

[0354] Ratio (Product / Deiodide / Aryl Iodide): 53 / 6 / 24

[0355] Precision quality: 5166.9927

[0356] The mass of the triple charge [M] / 3-1.00794, calculated value 1721.3230; observed value 1721.3342.

[0357] 3 LC trace and mass

[0358]

[0359] Use A3 to follow general operation 2 (condition A).

[0360] Yield: -

[0361] Ratio (product / deiodide / aryl iodide): 61 / 3 / 4

[0362] Precision quality: 5181.0081

[0363] The mass of the triple charge [M] / 3-1.00794, calculated value 1725.9948; observed value 1725.9928.

[0364] 4 LC trace and mass

[0365]

[0366] Use A4 to follow general operation 2 (condition A).

[0367] Yield: -

[0368] Ratio (product / deiodide / aryl iodide): 48 / 5 / 6

[0369] Precision quality: 5195.0238

[0370] The triple charge mass [M] / 3-1.00794, calculated value 1730.6667; observed value 1730.6748.

[0371] 5 LC trace and mass

[0372]

[0373] Use A5 following general operation 2 (condition A).

[0374] Yield: -

[0375] Ratio (Product / Deiodide / Aryliodide): 41 / 10 / 18

[0376] Precision quality: 5195.0238

[0377] The triple charge mass [M] / 3-1.00794, calculated value 1730.6667; observed value 1730.6748.

[0378] 6 LC trace and mass

[0379]

[0380] Use A6 to follow general operation 2 (condition A).

[0381] Yield: -

[0382] Ratio (Product / Deiodide / Aryliodide): 27 / 12 / 10

[0383] Precision quality: 5214.9925

[0384] The triple charge mass [M] / 3-1.00794, calculated value 1737.3229; observed value 1737.3254.

[0385] 7 LC trace and mass

[0386]

[0387] Use A7 to follow general operation 2 (condition A).

[0388] Yield: -

[0389] Ratio (product / deiodide / aryl iodide): 16 / 4 / 5

[0390] Precision quality: 5168.9718

[0391] The mass of the triple charge [M] / 3-1.00794, calculated value 1721.9827; observed value 1721.9800.

[0392] 8 LC trace and mass

[0393]

[0394] Use A8 following general operation 2 (condition A).

[0395] Yield: -

[0396] Ratio (Product / Deiodination / Aryl Iodide): 28 / 3 / 11

[0397] Precision quality: 5193.0081

[0398] The triple charge mass [M] / 3-1.00794, calculated value 1729.9948; observed value 1729.9934.

[0399] 7 LC trace and mass

[0400]

[0401] Use A9 following general operation 2 (condition A).

[0402] Yield: -

[0403] Ratio (product / deiodide / aryl iodide): 39 / 4 / 6

[0404] Precision quality: 5207.0238

[0405] The triple charge mass [M] / 3-1.00794, calculated value 1734.6667; observed value 1734.6637.

[0406] 8 LC trace and mass

[0407]

[0408] Use A10 following general operation 2 (condition A).

[0409] Yield: -

[0410] Ratio (Product / Deiodide / Aryl Iodide): 61 / 5 / 0

[0411] Precision quality: 5237.0344

[0412] The triple charge mass [M] / 3-1.00794, calculated value 1744.6702; observed value 1744.6731.

[0413] 9 LC trace and mass

[0414]

[0415] Use A11 to follow general operation 2 (condition A).

[0416] Yield: -

[0417] Ratio (product / deiodination / aryl iodide): 41 / 11 / 3

[0418] Precision quality: 5243.0238

[0419] The triple charge mass [M] / 3-1.00794, calculated value 1746.6667; observed value 1746.6749.

[0420] 10 LC trace and mass

[0421]

[0422] Use A12 to follow general operation 2 (condition A).

[0423] Yield: -

[0424] Ratio (product / deiodide / aryl iodide): 39 / 9 / 4

[0425] Precision quality: 5287.0500

[0426] The mass of the triple charge [M] / 3-1.00794, calculated value 1761.3421; observed value 1761.3392.

[0427] 11 LC trace and mass

[0428]

[0429] Use A13 to follow general operation 2 (condition A).

[0430] Yield: -

[0431] Ratio (product / deiodide / aryl iodide): 52 / 4 / 5

[0432] Precision quality: 5211.0187

[0433] The mass of the triple charge [M] / 3-1.00794, calculated value 1735.9983; observed value 1736.0114.

[0434] 12 LC trace and mass

[0435]

[0436] Use A14 to follow general operation 2 (condition A).

[0437] Yield: -

[0438] Ratio (product / deiodination / aryl iodide): 54 / 2 / 10

[0439] Precision quality: 5213.0144

[0440] The triple charge mass [M] / 3-1.00794, calculated value 1736.6635; observed value 1736.6769.

[0441] 13 LC trace and mass

[0442]

[0443] Use A15 following general operation 2 (condition A).

[0444] Yield: -

[0445] Ratio (product / deiodide / aryl iodide): 42 / 5 / 6

[0446] Precision quality: 5263.0112

[0447] The mass of the triple charge [M] / 3-1.00794, calculated value 1753.3291; observed value 1753.3391.

[0448] 14 LC trace and mass

[0449]

[0450] Use A16 to follow general operation 2 (condition A).

[0451] Yield: -

[0452] Ratio (product / deiodide / aryl iodide): 52 / 3 / 12

[0453] Precision quality: 5193.0081

[0454] The triple charge mass [M] / 3-1.00794, calculated value 1729.9948; observed value 1730.0104.

[0455] 15 LC trace and mass

[0456]

[0457] Using A17 follows general operation 2 (condition A), except that Ag3PO4 is used instead of AgTFA.

[0458] Yield: -

[0459] Ratio (Product / Deiodide / Aryliodide): 24 / 7 / 5

[0460] Precision quality: 5124.9455

[0461] The mass of the triple charge [M] / 3-1.00794, calculated value 1707.3072; observed value 1707.3116.

[0462] LC trace and mass

[0463]

[0464] Using A18 follows general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0465] Yield: -

[0466] Ratio (Product / Deiodide / Aryliodide): 24 / 6 / 6

[0467] Precision quality: 5138.9612

[0468] The mass of the triple charge [M] / 3-1.00794, calculated value 1711.9791; observed value 1711.9851.

[0469] LC trace and mass

[0470]

[0471] Using A19 follows general operation 2 (condition A), except that AgOAc is used instead of AgTFA.

[0472] Yield: -

[0473] Ratio (product / deiodination / aryl iodide): 24 / 17 / 3

[0474] Precision quality: 5152.9768

[0475] The triple charge mass [M] / 3-1.00791, calculated value 1716.6510; observed value 1716.6649.

[0476] LC trace and mass

[0477]

[0478] Using A20 follows general operation 2 (condition A), except that AgOAc is used instead of AgTFA.

[0479] Yield: -

[0480] Ratio (Product / Deiodide / Aryl Iodide): 20 / 16 / 17

[0481] Precision quality: 5166.9925

[0482] The mass of the triple charge [M] / 3-1.00794, calculated value 1721.3229; observed value 1721.3342.

[0483] 16 LC trace and mass

[0484]

[0485] Using A21 follows general operation 2 (condition A), except that AgOTf is used instead of AgTFA.

[0486] Yield: -

[0487] Ratio (Product / Deiodide / Aryliodide): 22 / 27 / 14

[0488] Precision quality: 5206.9486

[0489] The triple charge mass [M] / 3 - 1.00794, calculated value 1734.6416; observed value 1734.6637.

[0490] 17 LC trace and mass

[0491]

[0492] Use A22 to follow general operation 2 (condition B).

[0493] Yield: -

[0494] Ratio (product / deiodide / aryl iodide): 35 / 9 / 7

[0495] Precision quality: 5136.9455

[0496] The mass of the triple charge [M] / 3-1.00794, calculated value 1711.3072; observed value 1711.3074.

[0497] 18 LC trace and mass

[0498]

[0499] Use A23 to follow general operation 2 (condition A).

[0500] Yield: -

[0501] Ratio (Product / Deiodide / Aryliodide): 40 / 3 / 21

[0502] Precision quality: 5193.0081

[0503] The triple charge mass [M] / 3-1.00794, calculated value 1729.9948; observed value 1729.9934.

[0504] 19 LC trace and mass

[0505]

[0506] Use A24 following general operation 2 (condition A).

[0507] Yield: -

[0508] Ratio (Product / Deiodide / Aryl Iodide): 34 / 0 / 29

[0509] Precision quality: 5255.0238

[0510] The triple charge mass [M] / 3-1.00794, calculated value 1750.6667; observed value 1750.6652.

[0511] LC trace and mass

[0512]

[0513] Using A25 follows general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0514] Yield: -

[0515] Ratio (product / deiodide / aryl iodide): 18 / 15 / 6

[0516] Precision quality: 5164.9768

[0517] The triple charge mass [M] / 3-1.00794, calculated value 1720.6510; observed value 1720.6547.

[0518] 20 LC trace and mass

[0519]

[0520] Use A26 (1000mM) following general procedure 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0521] Yield: -

[0522] Ratio (product / deiodide / aryl iodide): 33 / 23 / 5

[0523] Precision quality: 5150.9612

[0524] The triple charge mass [M] / 3-1.00794, calculated value 1715.9791; observed value 1715.9694.

[0525] 21 LC trace and mass

[0526]

[0527] Using A27 follows general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0528] Yield: -

[0529] Ratio (product / deiodination / aryl iodide): 34 / 15 / 18

[0530] Precision quality: 5178.9925

[0531] The mass of the triple charge [M] / 3-1.00794, calculated value 1725.3229; observed value 1725.3293.

[0532] 22 LC trace and mass

[0533]

[0534] Use S2 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0535] Yield: -

[0536] Ratio (product / deiodide / aryl iodide): 46 / 31 / 4

[0537] Precision quality: 5152.9768

[0538] The triple charge mass [M] / 3-1.00794, calculated value 1716.6510; observed value 1716.6649.

[0539] LC trace and mass

[0540]

[0541] Use S3 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0542] Yield: -

[0543] Ratio (Product / Deiodide / Aryl Iodide): 57 / 3 / 15

[0544] Precision quality: 5166.9925

[0545] The mass of the triple charge [M] / 3-1.00794, calculated value 1721.3229; observed value 1721.3342.

[0546] LC trace and mass

[0547]

[0548] Use S10 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0549] Yield: -

[0550] Ratio (Product / Deiodide / Aryliodide): 57 / 8 / 23

[0551] Precision quality: 5138.9612

[0552] The mass of the triple charge [M] / 3-1.00794, calculated value 1711.9791; observed value 1711.9851.

[0553] 25 LC trace and mass

[0554]

[0555] Use S4 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0556] Yield: -

[0557] Ratio (product / deiodide / aryl iodide): 35 / 24 / 3

[0558] Precision quality: 5138.9612

[0559] The mass of the triple charge [M] / 3-1.00794, calculated value 1711.9791; observed value 1711.9851.

[0560] 26 LC trace and mass

[0561]

[0562] Use S6 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0563] Yield: -

[0564] Ratio (product / deiodide / aryl iodide): 35 / 22 / 3

[0565] Precision quality: 5172.9222

[0566] The triple charge mass [M] / 3-1.00794, calculated value 1723.2995; observed value 1723.3057.

[0567] 27 LC trace and mass

[0568]

[0569] Use S7 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0570] Yield: -

[0571] Ratio (Product / Deiodide / Aryl Iodide): 50 / 5 / 22

[0572] Precision quality: 5138.9612

[0573] The mass of the triple charge [M] / 3-1.00794, calculated value 1711.9791; observed value 1711.9851.

[0574] 28 LC trace and mass

[0575]

[0576] Use S9 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0577] Yield: -

[0578] Ratio (Product / Deiodide / Aryliodide): 41 / 11 / 18

[0579] Precision quality: 5172.9222

[0580] The triple charge mass [M] / 3-1.00794, calculated value 1723.2995; observed value 1723.3057.

[0581] 29 LC trace and mass

[0582]

[0583] Use S14 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0584] Yield: -

[0585] Ratio (product / deiodide / aryl iodide): 42 / 10 / 2

[0586] Precision quality: 5139.9564

[0587] The mass of the triple charge [M] / 3-1.00794, calculated value 1712.3109; observed value 1712.3240.

[0588] 30 LC trace and mass

[0589]

[0590] Use S15 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0591] Yield: -

[0592] Ratio (product / deiodide / aryl iodide): 53 / 7 / 4

[0593] Precision quality: 5139.9564

[0594] The mass of the triple charge [M] / 3-1.00794, calculated value 1712.3109; observed value 1712.3240.

[0595] 31 LC trace and mass

[0596]

[0597] Use S18 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0598] Yield: -

[0599] Ratio (product / deiodide / aryl iodide): 41 / 7 / 2

[0600] Precision quality: 5139.9564

[0601] The mass of the triple charge [M] / 3-1.00794, calculated value 1712.3109; observed value 1712.3070.

[0602] 32 LC trace and mass

[0603]

[0604] Use S16 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0605] Yield: -

[0606] Ratio (Product / Deiodide / Aryliodide): 30 / 18 / 28

[0607] Precision quality: 5139.9564

[0608] The mass of the triple charge [M] / 3-1.00794, calculated value 1712.3109; observed value 1712.3240.

[0609] 33 LC trace and mass

[0610]

[0611] Use S17 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0612] Yield: -

[0613] Ratio (product / deiodination / aryl iodide): 35 / 13 / 11

[0614] Precision quality: 5183.9827

[0615] The triple charge mass [M] / 3-1.00794, calculated value 1726.9863; observed value 1726.9968.

[0616] 34 LC trace and mass

[0617]

[0618] Use S19 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0619] Yield: -

[0620] Ratio (Product / Deiodide / Aryliodide): 35 / 24 / 17

[0621] Precision quality: 5142.9673

[0622] The triple charge mass [M] / 3-1.00794, calculated value 1713.3145; observed value 1713.3240.

[0623] 35 LC trace and mass

[0624]

[0625] Use S20 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0626] Yield: -

[0627] Ratio (product / deiodide / aryl iodide): 22 / 43 / 3

[0628] Precision quality: 5142.9673

[0629] The triple charge mass [M] / 3-1.00794, calculated value 1713.3145; observed value 1713.3240.

[0630] 36 LC trace and mass

[0631]

[0632] Use S21 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0633] Yield: -

[0634] Ratio (Product / Deiodide / Aryliodide): 23 / 19 / 30

[0635] Precision quality: 5142.9673

[0636] The triple charge mass [M] / 3-1.00794, calculated value 1713.3145; observed value 1713.3240.

[0637] 37 LC trace and mass

[0638]

[0639] Use S22 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0640] Yield: -

[0641] Ratio (Product / Deiodide / Aryliodide): 51 / 7 / 0

[0642] Precision quality: 5128.9405

[0643] The triple charge mass [M] / 3-1.00794, calculated value 1708.6389; observed value 1708.6487.

[0644] 38 LC trace and mass

[0645]

[0646] Use S23 and A1 (1000mM) to follow general operation 2 (condition A), except that Ag2CO3 is used instead of AgTFA.

[0647] Yield: -

[0648] Ratio (Product / Deiodide / Aryliodide): 46 / 12 / 0

[0649] Precision quality: 5144.9176

[0650] The mass of the triple charge [M] / 3-1.00794, calculated value 1713.9646; observed value 1713.9681.

[0651] 39 LC trace and mass

[0652]

[0653] Use S2 and A23 to follow general operation 2 (condition A).

[0654] Yield: -

[0655] Ratio (product / deiodination / aryl iodide): 38 / 3 / 14

[0656] Precision quality: 5193.0081

[0657] The triple charge mass [M] / 3-1.00794, calculated value 1729.9948; observed value 1729.9934.

[0658] 40 LC trace and mass

[0659]

[0660] Use S5 and A23 to follow general operation 2 (condition A).

[0661] Yield: -

[0662] Ratio (product / deiodination / aryl iodide): 24 / 16 / 3

[0663] Precision quality: 5193.0081

[0664] The triple charge mass [M] / 3-1.00794, calculated value 1729.9948; observed value 1729.9934.

[0665] 41 LC trace and mass

[0666]

[0667] Use S7 and A23 to follow general operation 2 (condition A).

[0668] Yield: -

[0669] Ratio (product / deiodination / aryl iodide): 33 / 3 / 12

[0670] Precision quality: 5178.9925

[0671] The mass of the triple charge [M] / 3-1.00794, calculated value 1725.3229; observed value 1725.3293.

[0672] 42 LC trace and mass

[0673]

[0674] Use S16 and A23 to follow general operation 2 (condition A).

[0675] Yield: -

[0676] Ratio (Product / Deiodination / Aryl Iodide): 21 / 3 / 25

[0677] Precision quality: 5179.9877

[0678] The triple charge mass [M] / 3-1.00794, calculated value 1725.6546; observed value 1725.6526.

[0679] 43 LC trace and mass

[0680]

[0681] Use S20 and A23 to follow general operation 2 (condition A).

[0682] Yield: -

[0683] Ratio (Product / Deiodide / Aryliodide): 21 / 17 / 0

[0684] Precision quality: 5182.9986

[0685] The triple charge mass [M] / 3-1.00794, calculated value 1726.6583; observed value 1726.6564.

[0686] 44 LC trace and mass

[0687]

[0688] Use S21 and A23 to follow general operation 2 (condition A).

[0689] Yield: -

[0690] Ratio (Product / Deiodide / Aryliodide): 20 / 12 / 11

[0691] Precision quality: 5182.9986

[0692] The triple charge mass [M] / 3-1.00794, calculated value 1726.6583; observed value 1726.6564.

[0693] LC trace and mass

[0694]

[0695] Use S22 and A23 to follow general operation 2 (condition A).

[0696] Yield: -

[0697] Ratio (Product / Deiodide / Aryliodide): 18 / 7 / 0

[0698] Precision quality: 5168.9718

[0699] The mass of the triple charge [M] / 3-1.00794, calculated value 1721.9827; observed value 1721.9800.

[0700] 5. Experimental section on on-DNA CH arylation of amides

[0701] 5.1 Substrate structures of amides B1 to B16

[0702]

[0703] The amides were obtained from commercial sources or synthesized following literature procedures. 21

[0704] 5.2 Condition Optimization

[0705] Table 10. Evaluation of Ag salts

[0706]

[0707] 1 AgOAc 46 2 <![CDATA[AgNO2]]> 12 3 AGTFA 46 4 <![CDATA[Ag2CO3]]> 42 5 AgOTs 35

[0708] Table 11. Evaluation of bases

[0709]

[0710] 1 NaOAc 46 2 <![CDATA[Na2CO3]]> 55 3 <![CDATA[K2CO3]]> 41 4 <![CDATA[K3PO4]]> 31 5 <![CDATA[K2HPO4·3H2O]]> 18 6 <![CDATA[Li2CO3]]> 69 7 <![CDATA[Cs2CO3]]> 41

[0711] Table 12. Evaluation of Standard Conditions

[0712]

[0713] 1 none 69 2 <![CDATA[No Pd(OAc)₂]]> 0 3 No AgOAc 24 4 <![CDATA[Li2CO3]]> 35 5 Room temperature instead of 80℃ 60

[0714] 5.3 General procedures for on-DNA CH arylation of amides 3

[0715]

[0716] Material

[0717] DNA-conjugated aryl iodide S: 10 mM, in H2O

[0718] Amide B: 2M, in DMA

[0719] Pd(OAc)2: 200mM, in HFIP

[0720] Sodium diethyldithiocarbamate trihydrate (scavenger): 1M, in H2O

[0721] operate

[0722] Add Pd(OAc)2- and dry.

[0723] AgOAc (300 equivalents, 0.5 mg), amide B

[0724] DNA-conjugated aryl iodide S (10 nmol, 1 L) Li₂CO₃

[0725] The mixture is added. The mixture is vortexed. The reaction mixture is heated at 80°C for 20 hours.

[0726] Add the cleaning agent and reheat the mixture at 80°C for 30 minutes.

[0727] 3) Cool to room temperature, 5M NaCl

[0728] so

[0729] Store in a refrigerator at -20°C for more than 30 minutes.

[0730] 4) Centrifuge the sample at 10,000 rpm for approximately 10 minutes using a microcentrifuge. Discard the supernatant and dry the precipitate under vacuum. Redissolve the DNA precipitate in H2.

[0731] In a microcentrifuge at 10,000 rpm. One al

[0732] The sample was obtained and analyzed by HPLC-MS.

[0733] 5.4 General Procedures for Dipeptide Synthesis 4

[0734]

[0735] Material

[0736] 68: Approximately 5 mM, in H2O

[0737] Glycine methyl ester hydrochloride: 1M, in H2O

[0738] DMTMM: 1M, in H2O

[0739] pH 5.5 phosphate buffer: 0.2M NaH2PO4, in H2O

[0740] operate

[0741] Add 22 to solution 68

[0742] 5.5 Phosphate buffer, glycine methyl ester

[0743] Temperature overnight (16 to 18 hours).

[0744] 2) Add 5M NaCl solution (10% by volume) and cold ethanol (2.5 times by volume, ethanol stored at -20°C). Store the mixture in a refrigerator at -20°C for 1 hour.

[0745] 3) Centrifuge the sample at 10,000 rpm for approximately 10 minutes using a microcentrifuge. Discard the supernatant and dry the precipitate under vacuum. Redissolve the DNA precipitate in H2.

[0746] The sample was aliquoted (50 μL) in a microcentrifuge at 10,000 rpm and analyzed by HPLC-MS.

[0747] 5.5 Trace and quality characterization by LC from 52 to 80

[0748] 45 LG trace and mass

[0749]

[0750] Use B1 following general procedure 3.

[0751] Yield: -

[0752] Ratio (Product / Deiodide / Aryl Iodide): 59 / 0 / 3

[0753] Precision quality: 5221.9983

[0754] The mass of the triple charge [M] / 3-1.00794, calculated value 1739.6582; observed value 1739.6648.

[0755] Using B1 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0756] Yield: -

[0757] Ratio (product / deiodide / aryl iodide): 51 / 4 / 5

[0758] Precision quality: 5221.9983

[0759] The mass of the triple charge [M] / 3-1.00794, calculated value 1739.6582; observed value 1739.6648.

[0760] 46 LC trace and mass

[0761]

[0762] Use B2 following general procedure 3.

[0763] Yield: -

[0764] Ratio (product / deiodide / aryl iodide): 56 / 3 / 0

[0765] Precision quality: 5236.0140

[0766] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0767] Using B2 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0768] Yield: -

[0769] Ratio (product / deiodination / aryl iodide): 71 / 1 / 10

[0770] Precision quality: 5236.0140

[0771] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0772] 47 LC trace and mass

[0773]

[0774] Use B3 following general procedure 3.

[0775] Yield: -

[0776] Ratio (product / deiodide / aryl iodide): 72 / 2 / 6

[0777] Precision quality: 5262.0296

[0778] The mass of the triple charge [M] / 3 - 1.00794, calculated value 1753.0019; observed value 1752.9962.

[0779] Using B3 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0780] Yield: -

[0781] Ratio (Product / Deiodide / Aryl Iodide): 55 / 6 / 28

[0782] Precision quality: 5262.0296

[0783] The mass of the triple charge [M] / 3 - 1.00794, calculated value 1753.0019; observed value 1752.9962.

[0784] 48 LC trace and mass

[0785]

[0786] Use B4 following general procedure 3.

[0787] Yield: -

[0788] Ratio (Product / Deiodide / Aryliodide): 29 / 10 / 0

[0789] Precision quality: 5219.9827

[0790] The mass of the triple charge [M] / 3-1.00794, calculated value 1738.9863; observed value 1738.9816.

[0791] Using B4 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0792] Yield: -

[0793] Ratio (product / deiodination / aryl iodide): 46 / 4 / 10

[0794] Precision quality: 5219.9827

[0795] The mass of the triple charge [M] / 3-1.00794, calculated value 1738.9863; observed value 1738.9816.

[0796] 49 LC trace and mass

[0797]

[0798] Use B5 following general procedure 3.

[0799] Yield: -

[0800] Ratio (product / deiodide / aryl iodide): 56 / 3 / 4

[0801] Precision quality: 5248.0140

[0802] The triple charge mass [M] / 3-1.00794, calculated value 1748.3301; observed value 1748.3356.

[0803] Using B5 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0804] Yield: -

[0805] Ratio (product / deiodide / aryl iodide): 35 / 9 / 41

[0806] Precision quality: 5248.0140

[0807] The triple charge mass [M] / 3-1.00794, calculated value 1748.3301; observed value 1748.3356.

[0808] 50 LC trace and mass

[0809]

[0810] Use B6 following general procedure 3.

[0811] Yield: -

[0812] Ratio (product / deiodide / aryl iodide): 48 / 6 / 5

[0813] Precision quality: 5193.9670

[0814] The mass of the triple charge [M] / 3-1.00794, calculated value 1730.3144; observed value 1730.3170.

[0815] Using B6 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0816] Yield: -

[0817] Ratio (Product / Deiodide / Aryl Iodide): 51 / 1 / 29

[0818] Precision quality: 5193.9670

[0819] The mass of the triple charge [M] / 3-1.00794, calculated value 1730.3144; observed value 1730.3170.

[0820] 51 LC trace and mass

[0821]

[0822] Use B7 following general procedure 3.

[0823] Yield: -

[0824] Ratio (product / deiodide / aryl iodide): 63 / 3 / 0

[0825] Precision quality: 5236.0140

[0826] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0827] Using B7 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenger.

[0828] Yield: -

[0829] Ratio (product / deiodination / aryl iodide): 66 / 2 / 13

[0830] Precision quality: 5236.0140

[0831] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0832] 52 LC trace and mass

[0833]

[0834] Use B8 following general procedure 3.

[0835] Yield: -

[0836] Ratio (Product / Deiodide / Aryliodide): 72 / 0 / 0

[0837] Precision quality: 5207.9827

[0838] The triple charge mass [M] / 3-1.00794, calculated value 1734.9863; observed value 1734.9877.

[0839] Using B8 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0840] Yield: -

[0841] Ratio (Product / Deiodide / Aryl Iodide): 70 / 0 / 16

[0842] Precision quality: 5207.9827

[0843] The triple charge mass [M] / 3-1.00794, calculated value 1734.9863; observed value 1734.9877.

[0844] 53 LC trace and mass

[0845]

[0846] Use B9 following general procedure 3.

[0847] Yield: -

[0848] Ratio (product / deiodide / aryl iodide): 62 / 2 / 0

[0849] Precision quality: 5236.0140

[0850] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0851] Using B9 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0852] Yield: -

[0853] Ratio (product / deiodide / aryl iodide): 70 / 5 / 9

[0854] Precision quality: 5236.0140

[0855] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0856] 54 LC trace and mass

[0857]

[0858] Use B10 following general procedure 3.

[0859] Yield: -

[0860] Ratio (product / deiodide / aryl iodide): 72 / 2 / 3

[0861] Precision weight: 5250.0296

[0862] The triple charge mass [M] / 3-1.00794, calculated value 1749.0019; observed value 1749.0034.

[0863] Using B10 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0864] Yield: -

[0865] Ratio (Product / Deiodide / Aryl Iodide): 19 / 2 / 55

[0866] Precision weight: 5250.0296

[0867] The mass of the triple charge [M] / 3 - 1.00794, calculated value 1749.0019; observed value 1749.0034

[0868] 55 LC trace and mass

[0869]

[0870] Use B11 following general procedure 3.

[0871] Yield: -

[0872] Ratio (product / deiodination / aryl iodide): 44 / 4 / 5

[0873] Precision quality: 5248.0140

[0874] The triple charge mass [M] / 3-1.00794, calculated value 1748.3301; observed value 1748.3356.

[0875] Using B11 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenger.

[0876] Yield: -

[0877] Ratio (product / deiodide / aryl iodide): 40 / 9 / 10

[0878] Precision quality: 5218.0140

[0879] The triple charge mass [M] / 3-1.00794, calculated value 1748.3301; observed value 1748.3356.

[0880] 56 LC trace and mass

[0881]

[0882] Use B12 following general procedure 3.

[0883] Yield: -

[0884] Ratio (product / deiodide / aryl iodide): 63 / 3 / 2

[0885] Precision quality: 5248.0140

[0886] The triple charge mass [M] / 3-1.00794, calculated value 1748.3301; observed value 1748.3356.

[0887] When using B12, follow general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0888] Yield: -

[0889] Ratio (product / deiodide / aryl iodide): 68 / 6 / 8

[0890] Precision quality: 5248.0140

[0891] The triple charge mass [M] / 3-1.00794, calculated value 1748.3301; observed value 1748.3356.

[0892] 57 LC trace and mass

[0893]

[0894] Use B13 following general procedure 3.

[0895] Yield: -

[0896] Ratio (Product / Deiodide / Aryliodide): 43 / 6 / 22

[0897] Precision quality: 5272.0140

[0898] The triple charge mass [M] / 3-1.00794, calculated value 1756.3301; observed value 1756.33413.

[0899] 58 LC trace and mass

[0900]

[0901] Use B14 following general procedure 3.

[0902] Yield: -

[0903] Ratio (product / deiodide / aryl iodide): 30 / 2 / 43

[0904] Precision quality: 5291.9593

[0905] The mass of the triple charge [M] / 3-1.00794, calculated value 1762.9785; observed value 1762.9895.

[0906] The use of B14 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0907] Yield: -

[0908] Ratio (Product / Deiodide / Aryl Iodide): 13 / 6 / 58

[0909] Precision quality: 5291.9593

[0910] The mass of the triple charge [M] / 3-1.00794, calculated value 1762.9785; observed value 1762.9895.

[0911] 60 LC trace and mass

[0912]

[0913] Use B16 following general procedure 3.

[0914] Yield: -

[0915] Ratio (product / deiodide / aryl iodide): 37 / 5 / 2

[0916] Precision quality: 5236.0140

[0917] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0918] Using B16 follows general procedure 3, except that the reaction is carried out at room temperature and quenched with a scavenging agent.

[0919] Yield: -

[0920] Ratio (product / deiodide / aryl iodide): 54 / 15 / 5

[0921] Precision quality: 5236.0140

[0922] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[0923] 61 LC trace and mass

[0924]

[0925] Use S7 and B3 following general operation 3.

[0926] Yield: -

[0927] Ratio (Product / Deiodide / Aryl Iodide): 50 / 2 / 0

[0928] Precision quality: 5262.0296

[0929] The triple charge mass [M] / 3-1.00794, calculated value 1753.0019; observed value 1753.0133.

[0930] 62 LC trace and mass

[0931]

[0932] Use S9 and B3 following general operation 3.

[0933] Yield: -

[0934] Ratio (Product / Deiodide / Aryliodide): 55 / 5 / 0

[0935] Precision quality: 5295.9906

[0936] The triple charge mass [M] / 3-1.00794, calculated value 1764.3223; observed value 1764.3309.

[0937] 63 LC trace and mass

[0938]

[0939] Use S13 and B3 to follow general operation 3.

[0940] Yield: -

[0941] Ratio (product / deiodination / aryl iodide): 51 / 3 / 0

[0942] Precision quality: 5280.0202

[0943] Triple charge mass [M] / 3–1.00794, calculated value 1758.9988; observed value 1759.0026.

[0944] 64 LC trace and mass

[0945]

[0946] Use S12 and B3 following general operation 3.

[0947] Yield: -

[0948] Ratio (product / deiodide / aryl iodide): 52 / 6 / 1

[0949] Precision quality: 5280.0202

[0950] The triple charge mass [M] / 3-1.00794, calculated value 1758.9988; observed value 1759.0026.

[0951] 65 LC trace and mass

[0952]

[0953] Use S3 and B3 following general operation 3.

[0954] Yield: -

[0955] Ratio (Product / Deiodide / Aryl Iodide): 50 / 1 / 0

[0956] Precision quality: 5290.0609

[0957] The triple charge mass [M] / 3-1.00794, calculated value 1762.3457; observed value 1762.3534.

[0958] 66 LC trace and mass

[0959]

[0960] Use S18 and B3 to follow general operation 3.

[0961] Yield: -

[0962] Ratio (product / deiodide / aryl iodide): 45 / 2 / 0

[0963] Precision quality: 5263.0249

[0964] The mass of the triple charge [M] / 3-1.00794, calculated value 1753.3337; observed value 1753.3391.

[0965] 67 LC trace and mass

[0966]

[0967] Use S15 and B3 following general operation 3.

[0968] Yield: -

[0969] Ratio (product / deiodide / aryl iodide): 42 / 1 / 3

[0970] Precision quality: 5263.0249

[0971] The mass of the triple charge [M] / 3-1.00794, calculated value 1753.3337; observed value 1753.3391.

[0972] 68 LC trace and mass

[0973]

[0974] Use S16 and B3 to follow general operation 3.

[0975] Yield: -

[0976] Ratio (product / deiodide / aryl iodide): 53 / 2 / 3

[0977] Precision quality: 5263.0249

[0978] The mass of the triple charge [M] / 3-1.00794, calculated value 1753.3337; observed value 1753.3391.

[0979] LC trace and mass of 69

[0980]

[0981] Use S17 and B3 following general operation 3.

[0982] Yield: -

[0983] Ratio (Product / Deiodide / Aryl Iodide): 56 / 9 / 0

[0984] Precision quality: 5307.0511

[0985] The triple charge mass [M] / 3-1.00794, calculated value 1768.0091; observed value 1768.0140.

[0986] 70 LC trace and mass

[0987]

[0988] Use S21 and B3 to follow general operation 3.

[0989] Yield: -

[0990] Ratio (product / deiodide / aryl iodide): 33 / 16 / 5

[0991] Precision quality: 5266.0358

[0992] The mass of the triple charge [M] / 3-1.00794, calculated value 1754.3373; observed value 1754.3339.

[0993] 71 LC trace and mass

[0994]

[0995] Use S22 and B3 following general operation 3.

[0996] Yield: -

[0997] Ratio (Product / Deiodide / Aryliodide): 39 / 3 / 0

[0998] Precision quality: 5252.0089

[0999] The triple charge mass [M] / 3-1.00794, calculated value 1749.6617; observed value 1749.6714.

[1000] 72 LC trace and mass

[1001]

[1002] Use S23 and B3 following general operation 3.

[1003] Yield: -

[1004] Ratio (product / deiodide / aryl iodide): 52 / 3 / 0

[1005] Precision quality: 5267.9860

[1006] The triple charge mass [M] / 3-1.00794, calculated value 1754.9874; observed value 1754.9857.

[1007] 73 LC trace and mass

[1008]

[1009] Use 68 following general operation 4.

[1010] Yield: -

[1011] Precision quality: 5334.0620

[1012] The triple charge mass [M] / 3-1.00794, calculated value 1777.0127; observed value 1777.0140.

[1013] 6. Experimental section for on-DNA CH arylation of ketones

[1014] 6.1 Substrate structures of ketones with directional groups (DG) at C1-C30

[1015]

[1016] Ketones containing directional groups (C1-C30) were synthesized as previously reported. 16a

[1017] 6.2 Condition Optimization

[1018] Table 13. Ligand Optimization

[1019]

[1020]

[1021] Table 14. Cosolvent Optimization

[1022]

[1023] 1 <![CDATA[H2O / DMA(1 / 1)]]> 28 2 <![CDATA[H2O / DMA(2 / 1)]]> 39 3 <![CDATA[H2O / DMF(2 / 1)]]> 35 4 <![CDATA[H2O / DMSO(2 / 1)]]> 26

[1024] Table 15. Optimization of Pd / L8 concentration

[1025]

[1026] 1 20 / 10 33 2 20 / 15 48 3 20 / 20 48

[1027] Table 16. Solvent Ratio Optimization

[1028]

[1029] 1 4 / 1 51 2 9 / 1 62

[1030] Table 17. Evaluation of Standard Conditions

[1031]

[1032]

[1033] a Only S1b was fully recovered.

[1034] 6.3 General procedures for on-DNA CH arylation of ketones 5

[1035]

[1036] Material

[1037] DNA-conjugated aryl iodide S: 10 mM, in H2O

[1038] Ketone C: 3M, in DMA (Note: High concentrations can increase the overall volume).

[1039] L8

[1040] Pd(OAc)2

[1041] NaOAc: 1.5M, in H2O

[1042] Sodium diethyldithiocarbamate trihydrate (scavenger): 1M, in H2O

[1043] operate

[1044] 1) Add Pd(OAc)2,-dried, ketone-C DNA-conjugated aryl iodide S (10 nmol, 1 L8(40)), NaOAc to the prepared AgTFA (500 equivalents, 1.1 mg).

[1045] Vortex the mixture. Heat the reaction mixture at 80°C for 20 hours.

[1046] Add the cleaning agent and reheat the mixture at 80°C for 30 minutes.

[1047] 3) Cool to room temperature, 5M NaCl

[1048] Store in a refrigerator at -20°C for more than 30 minutes.

[1049] 4) Centrifuge the sample at 10,000 rpm for approximately 7 minutes in a microcentrifuge. Discard the supernatant and dry the precipitate under vacuum. Redissolve the DNA precipitate in H2 and centrifuge for approximately 2 minutes.

[1050] The sample was obtained and analyzed by HPLC-MS.

[1051] 6.4 Restriction of Ketones

[1052]

[1053] 6.5 General Operations for Removing DG

[1054]

[1055] A solution of substrate 83 of DNA lineage (approximately 10 μL)

[1056] The reaction mixture was then heated at 50°C for 24 hours.

[1057] 2) Cool to room temperature and store the 5M NaCl mixture in a refrigerator at -20°C for more than 30 minutes.

[1058] 3) Centrifuge the sample at 10,000 rpm for approximately 7 minutes using a microcentrifuge. Discard the supernatant and dry the precipitate under vacuum. Redissolve the DNA precipitate in H2.

[1059] The sample was obtained and analyzed by HPLC-MS.

[1060] 6.6 Trace and mass characterization by LC from 89 to 119

[1061] 74 LC trace and mass

[1062]

[1063] Use C1 following general operation 5.

[1064] Yield: -

[1065] Ratio (product / deiodination / aryl iodide): 31 / 1 / 12

[1066] Precision quality: 5252.0453

[1067] The triple charge mass [M] / 3-1.00794, calculated value 1749.6738; observed value 1749.6714.

[1068] 75 LC trace and mass

[1069]

[1070] Use C2 following general procedure 5.

[1071] Yield: -

[1072] Ratio (product / deiodination / aryl iodide): 55 / 1 / 12

[1073] Precision quality: 5238.0296

[1074] The triple charge mass [M] / 3-1.00794, calculated value 1745.0019; observed value 1745.0151.

[1075] 76 LC trace and mass

[1076]

[1077] Use C3 following general operation 5.

[1078] Yield: -

[1079] Ratio (product / deiodination / aryl iodide): 38 / 1 / 25

[1080] Precision quality: 5252.0453

[1081] The triple charge mass [M] / 3-1.00794, calculated value 1749.6738; observed value 1749.6886.

[1082] 77 LC trace and mass

[1083]

[1084] Use C4 following general procedure 5.

[1085] Yield: -

[1086] Ratio (Product / Deiodide / Aryliodide): 47 / 3 / 0

[1087] Precision quality: 5224.0140

[1088] The triple charge mass [M] / 3-1.00794, calculated value 1740.3301; observed value 1740.3309.

[1089] 78 LC trace and mass

[1090]

[1091] Use C5 following general operation 5.

[1092] Yield: -

[1093] Ratio (Product / Deiodide / Aryl Iodide): 34 / 1 / 22

[1094] Precision quality: 5264.0453

[1095] The mass of the triple charge [M] / 3-1.00794, calculated value 1753.6738; observed value 1753.6821.

[1096] 79 LC trace and mass

[1097]

[1098] Use C6 following general operation 5.

[1099] Yield: -

[1100] Ratio (Product / Deiodide / Aryliodide): 17 / 7 / 39

[1101] Precision quality: 5336.0664

[1102] The triple charge mass [M] / 3-1.00794, calculated value 1777.6809; observed value 1777.6874.

[1103] 80 LC trace and mass

[1104]

[1105] Use C7 following general operation 5.

[1106] Yield: -

[1107] Ratio (product / deiodination / aryl iodide): 34 / 2 / 41

[1108] Precision quality: 5266.0245

[1109] The triple charge mass [M] / 3-1.00794, calculated value 1754.3336; observed value 1754.3339.

[1110] 81 LC trace and mass

[1111]

[1112] Use C8 following general operation 5.

[1113] Yield: -

[1114] Ratio (Product / Deiodide / Aryl Iodide): 29 / 2 / 8

[1115] Precision quality: 5321.0667

[1116] The triple charge mass [M] / 3-1.00794, calculated value 1772.6810; observed value 1772.6836.

[1117] 82 LC trace and mass

[1118]

[1119] Use C9 following general operation 5.

[1120] Yield: -

[1121] Ratio (product / deiodination / aryl iodide): 36 / 2 / 26

[1122] Precision quality: 5322.0507

[1123] The mass of the triple charge [M] / 3-1.00794, calculated value 1773.0090; observed value 1773.0112.

[1124] 83 LC trace and mass

[1125]

[1126] Use C10 following general procedure 5.

[1127] Yield: -

[1128] Ratio (product / deiodide / aryl iodide): 53 / 2 / 5

[1129] Precision weight: 5250.0296

[1130] The triple charge mass [M] / 3-1.00794, calculated value 1749.0019; observed value 1749.0034.

[1131] LC trace and mass

[1132]

[1133] Use C11 following general operation 5.

[1134] Yield: -

[1135] Ratio (product / deiodination / aryl iodide): 17 / 9 / 32

[1136] Precision quality: 5278.0609

[1137] The triple charge mass [M] / 3 - 1.00794, calculated value 1758.3457; observed value 1758.3500.

[1138] LC trace and mass

[1139]

[1140] Use C12 following general operation 5.

[1141] Yield: -

[1142] Ratio (Product / Deiodide / Aryliodide): 17 / 4 / 33

[1143] Precision quality: 5308.0715

[1144] The mass of the triple charge [M] / 3-1.00794, calculated value 1768.3492; observed value 1768.3584.

[1145] 84 LC trace and mass

[1146]

[1147] Use C13 following general operation 5.

[1148] Yield: -

[1149] Ratio (product / deiodination / aryl iodide): 36 / 2 / 13

[1150] Precision quality: 5224.0140

[1151] The triple charge mass [M] / 3-1.00794, calculated value 1740.3301; observed value 1740.3309.

[1152] LC trace and mass

[1153]

[1154] Use C14 following general procedure 5.

[1155] Yield: -

[1156] Ratio (Product / Deiodide / Aryliodide): 18 / 2 / 28

[1157] Precision quality: 5296.0351

[1158] The triple charge mass [M] / 3-1.00794, calculated value 1764.3371; observed value 1764.3481.

[1159] 85% LC trace and mass

[1160]

[1161] Use C15 following general procedure 5.

[1162] Yield: -

[1163] Ratio (product / deiodide / aryl iodide): 33 / 1 / 9

[1164] Precision quality: 5312.0453

[1165] The triple charge mass [M] / 3-1.00794, calculated value 1769.6738; observed value 1769.6847.

[1166] 86 LC trace and mass

[1167]

[1168] Use C16 following general operation 5.

[1169] Yield: -

[1170] Ratio (product / deiodide / aryl iodide): 38 / 0 / 6

[1171] Precision quality: 5238.0296

[1172] The triple charge mass [M] / 3-1.00794, calculated value 1745.0019; observed value 1744.9980.

[1173] 87 LC trace and mass

[1174]

[1175] Use C17 following general procedure 5.

[1176] Yield: -

[1177] Ratio (product / deiodination / aryl iodide): 32 / 1 / 21

[1178] Precision quality: 5286.0296

[1179] The triple charge mass [M] / 3-1.00794, calculated value 1761.0019; observed value 1761.0127.

[1180] LC trace and mass

[1181]

[1182] Use C18 following general procedure 5.

[1183] Yield: -

[1184] Ratio (Product / Deiodination / Aryl Iodide): 17 / 6 / 14

[1185] Precision quality: 5282.0194

[1186] The triple charge mass [M] / 3-1.00794, calculated value 1759.6652; observed value 1759.6725.

[1187] 88% LC trace and mass

[1188]

[1189] Use C19 following general procedure 5.

[1190] Yield: -

[1191] Ratio (product / deiodination / aryl iodide): 37 / 1 / 24

[1192] Precision quality: 5372.1028

[1193] The triple charge mass [M] / 3-1.00794, calculated value 1789.6930; observed value 1789.6906.

[1194] 89 LC trace and mass

[1195]

[1196] Using D20 follows general operation 5, except that Pd(OAc)2 (15mM) and L8 (15mM) are used in H2O / DMA (2 / 1).

[1197] Yield: -

[1198] Ratio (Product / Deiodination / Aryl Iodide): 29 / 2 / 10

[1199] Precision quality: 5328.0766

[1200] The mass of the triple charge [M] / 3-1.00794, calculated value 1775.0176; observed value 1775.0293.

[1201] 90% LC trace and mass

[1202]

[1203] C21 follows general operation 5, except that Pd(OAc)2 (15mM) and L8 (15mM) are used in H2O / DMA (2 / 1).

[1204] Yield: -

[1205] Ratio (product / deiodination / aryl iodide): 58 / 1 / 14

[1206] Precision quality: 5264.0453

[1207] The mass of the triple charge [M] / 3-1.00794, calculated value 1753.6738; observed value 1753.6821.

[1208] 91 LC trace and mass

[1209]

[1210] Use C22 following general procedure 5.

[1211] Yield: -

[1212] Ratio (product / deiodination / aryl iodide): 34 / 4 / 25

[1213] Precision quality: 5276.0453

[1214] The mass of the triple charge [M] / 3-1.00794, calculated value 1757.6738; observed value 1757.6803.

[1215] 92 LC trace and mass

[1216]

[1217] Use C23 following general operation 5.

[1218] Yield: -

[1219] Ratio (Product / Deiodination / Aryl Iodide): 29 / 1 / 19

[1220] Precision quality: 5302.0609

[1221] The triple charge mass [M] / 3-1.00794, calculated value 1766.3457; observed value 1766.3441.

[1222] LC trace and mass

[1223]

[1224] Use C24 following general procedure 5.

[1225] Yield: -

[1226] Ratio (product / deiodination / aryl iodide): 13 / 2 / 41

[1227] Precision quality: 5266.0609

[1228] The triple charge mass [M] / 3-1.00794, calculated value 1754.3457; observed value 1754.3511.

[1229] LC trace and mass

[1230]

[1231] Use C25 following general procedure 5.

[1232] Yield: -

[1233] Ratio (product / deiodination / aryl iodide): 8 / 2 / 52

[1234] Precision quality: 5252.0453

[1235] The triple charge mass [M] / 3-1.00794, calculated value 1749.6738; observed value 1749.6886.

[1236] 93% LC trace and mass

[1237]

[1238] Use C26 following general procedure 5.

[1239] Yield: -

[1240] Ratio (Product / Deiodide / Aryliodide): 19 / 2 / 30

[1241] Precision quality: 5306.0922

[1242] The triple charge mass [M] / 3-1.00794, calculated value 1767.6895; observed value 1767.6869.

[1243] LC trace and mass

[1244]

[1245] Use S4 and C10 following general operation 5.

[1246] Yield: -

[1247] Ratio (product / deiodination / aryl iodide): 23 / 6 / 2

[1248] Precision quality: 5236.0140

[1249] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[1250] LC trace and mass

[1251]

[1252] Use S5 and C10 following general operation 5.

[1253] Yield: -

[1254] Ratio (product / deiodination / aryl iodide): 22 / 10 / 4

[1255] Precision weight: 5250.0296

[1256] The triple charge mass [M] / 3-1.00794, calculated value 1749.0019; observed value 1749.0034.

[1257] 94 LC trace and mass

[1258]

[1259] Use S7 and C10 following general operation 5.

[1260] Yield: -

[1261] Ratio (product / deiodination / aryl iodide): 51 / 1 / 9

[1262] Precision quality: 5236.0140

[1263] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[1264] 95% LC trace and mass

[1265]

[1266] Use S9 and C10 following general operation 5.

[1267] Yield: -

[1268] Ratio (Product / Deiodination / Aryl Iodide): 26 / 3 / 16

[1269] Precision quality: 5269.9750

[1270] The triple charge mass [M] / 3-1.00794, calculated value 1755.6504; observed value 1755.6549.

[1271] 96% LC trace and mass

[1272]

[1273] Use S10 and C10 following general operation 5.

[1274] Yield: -

[1275] Ratio (product / deiodination / aryl iodide): 29 / 1 / 4

[1276] Precision quality: 5236.0140

[1277] The triple charge mass [M] / 3-1.00794, calculated value 1744.3301; observed value 1744.3309.

[1278] 97% LC trace and mass

[1279]

[1280] Use S3 and C10 following general operation 5.

[1281] Yield: -

[1282] Ratio (product / deiodide / aryl iodide): 43 / 1 / 4

[1283] Precision quality: 5264.0454

[1284] The mass of the triple charge [M] / 3-1.00794, calculated value 1753.6739; observed value 1753.6821.

[1285] 98% LC trace and mass

[1286]

[1287] Use S14 and C10 following general operation 5.

[1288] Yield: -

[1289] Ratio (product / deiodide / aryl iodide): 36 / 5 / 1

[1290] Precision quality: 5237.0092

[1291] The triple charge mass [M] / 3-1.00794, calculated value 1744.6618; observed value 1744.6731.

[1292] 99% LC trace and mass

[1293]

[1294] Follow the general operating procedure 5 for using S15 and C10.

[1295] Yield: -

[1296] Ratio (Product / Deiodide / Aryl Iodide): 38 / 3 / 0

[1297] Precision quality: 5237.0092

[1298] The triple charge mass [M] / 3-1.00794, calculated value 1744.6618; observed value 1744.6731.

[1299] 100 LC trace and mass

[1300]

[1301] Use S18 and C10 following general operation 5.

[1302] Yield: -

[1303] Ratio (Product / Deiodide / Aryliodide): 29 / 6 / 2

[1304] Precision quality: 5237.0092

[1305] The triple charge mass [M] / 3-1.00794, calculated value 1744.6618; observed value 1744.6731.

[1306] 101 LC trace and mass

[1307]

[1308] Use S16 and C10 following general operation 5.

[1309] Yield: -

[1310] Ratio (Product / Deiodide / Aryl Iodide): 29 / 6 / 26

[1311] Precision quality: 5237.0092

[1312] The triple charge mass [M] / 3-1.00794, calculated value 1744.6618; observed value 1744.6731.

[1313] LC trace and mass

[1314]

[1315] Using S19 and C10 follows general operation 5, the difference being the use of H2O / DMA (2 / 1).

[1316] Yield: -

[1317] Ratio (Product / Deiodination / Aryliodide): 13 / 10 / 29

[1318] Precision quality: 5240.0201

[1319] The mass of the triple charge [M] / 3 - 1.00794, calculated value 1745.6654; observed value 1745.6653.

[1320] 102 LC trace and mass

[1321]

[1322] Use S23 and C10 following general operation 5.

[1323] Yield: -

[1324] Ratio (Product / Deiodination / Aryliodide): 28 / 3 / 14

[1325] Precise quality: 5241.9704

[1326] The triple charge mass [M] / 3-1.00794, calculated value 1746.3155; observed value 1746.3156.

[1327] 103 LC trace and mass

[1328]

[1329] Use 83 following general operating procedure 6.

[1330] Yield: -

[1331] Precision quality: 5148.9819

[1332] The triple charge mass [M] / 3-1.00794, calculated value 1715.3194; observed value 1715.3248.

[1333] 7. Representative synthesis of compound 106

[1334] Step 1: First C(sp) 3 )-H activation

[1335]

[1336] 31 LC trace and mass See above.

[1337] Step 2: Amide

[1338]

[1339] 104 LC trace and mass

[1340] Use compound 31 and 4-iodobenzylamine following general procedure 4.

[1341] Yield: -

[1342] Precision quality: 5354.9160

[1343] The mass of the triple charge [M] / 3-1.00794, calculated value 1783.9641; observed value 1783.9607.

[1344] Step 3: The second C(sp) 3 )-H activation

[1345] 105 LC trace and mass

[1346] Use 104 and C10 following general operation 5.

[1347] Yield: -

[1348] Precision weight: 5426.1246

[1349] The triple charge mass [M] / 3-1.00794, calculated value 1807.7003; observed value 1807.7014.

[1350] Step 4: Remove DG

[1351]

[1352] 106 LC trace and mass

[1353] Use 105 following general operating procedure 6.

[1354] Yield: -

[1355] Precision quality: 5325.0769

[1356] The triple charge mass [M] / 3-1.00794, calculated value 1774.0177; observed value 1774.0288.

[1357] Quote:

[1358] 1. Brenner & Lerner, Proc. Natl. Acad. Sci. USA89, 5381-5383 (1992).

[1359] 2. (a) Goodnow, "A Brief History of the Development of CombinatorialChemistry and the Emerging Need for DNA-Encoded Chemistry". In A Handbook for DNA-Encoded Chemistry, wiley-Blackwell, 2014, pp 19-43; (b) Franzini et al., Acc.Chem.Res., 2014, 47, 1247; (c) Zimmermann&Neri, Drug Discov. Today, 2016, 21, 1828; (d) Lerner & Brenner, Angew. Chem., Int. Ed., 2017, 56, 1164; (e) Neri, ChemBioChem, 2017, 18, 827; (f) Shi et al. al., Bioorg.Med.Chem.Lett., 2017, 27, 361; (g) Satz, ACSMed.Chem.Lett., 2018, 9, 408; (h) Zhao et al., Expert Opin. Drug Discov., 2019, 14, 735.

[1360] 3.(a)Zhao et al.,Angew.Chem.,Int.Ed.,2014,53,10056;(b)Ding et al. al.,ACSMed.Chem.Lett.,2015,6,888;(c)Arico-Muendel,Med.Chem.Commun.,2016,7,1898;(d)Belyanskaya et al.,ChemBioChem,2017,18,837;(e)Neri&Lerner,Annu.Rev.Biochem.,2018,87,479;(f)Yuen et al.,J.Am.Chem.Soc.,2019,141,5169;(g)Cai et al.,J.Am.Chem.Soc.,2019,141,17057.

[1361] 4.(a)Satz et al.,Bioconjug.Chem.26,1623-1632(2015);(b)Franzini&Randolph,J.Med.Chem.,2016,59,6629,&(CSc)Malo Comb.Sci.,2016,18,182.

[1362] 5.(a)Ding&.Clark,ACS Comb.Sci.,2015,17,1;(b)Ding et al.,Bioconjug.Chem.,2016,27,2597;(c)Lu et al.,Bioconjug.Chem.,2017,28,1625,

[1363] 6.(a)Lu et al.,Bioconjug.Chem.28,1625-1629(2017);b)Ruff&Berst,Med.Chem.Commun.,2018,9,1188.

[1364] 7.Wang et al.,iScience,2019,15,307.

[1365] 8.(a)Probst et al.,Chem.Eur.J.,2018,24,1795;(b)Liu et al.,Angew.Chem.,Int.Ed.,2019,58,8029\et(c) al.,Angew.Chem.,Int.Ed.,2020,59,7377.

[1366] 9.(a)Li et al.,Org.Lett.,2018,20,7186;(b)Gerry et 1.,Org.Lett.,2019,21,1325;(c)Maet al.,Angew.Chem.,Int.Ed.,2019,58,9254;(d)J.Am.Chem.Soc.,2019,141,10546;(e)Potowski et al.,Chem.Sci.,2019,10,10481.

[1367] 10.(a) et al.,Chem.Med.Chem.,2018,13,2159;(b)Wang et al.,Proc.Natl.Acad.Sci.USA,2018,115,E6404;(c)Flood et al.,J.Am.Chem.Soc.,2019,141,9998;(d)Phelan et al.,J.Am.Chem.Soc.,2019,141,3723;(e)Badir et al.,Org.Lett.,2020,22,1046;(f)Ruff et al.,ACS Comb.Sci.,2020,22,120.

[1368] 11.(a)He et al.,Chem.Rev.,2017,117,8754;(b)Zhuang&Yu,Nature,2020,577,656.

[1369] 12.Gerry et al.,J.Am.Chem.Soc.,2019,141,10225.

[1370] 13.Wang et al.,Org.Lett.,2018,20,4764.

[1371] 14.(a)Chen et al.,Angew.Chem.,Int.Ed.,2017,56,1506;(b)Shen et al.,J.Am.Chem.Soc.,2018,1495.6454

[1372] 15.(a)Gong et al.,J.Am.Chem.Soc.,2014,136,16940;(b)Liu et al.,Angew.Chem.,Int.Ed.,2017,56,10924

[1373] 16. (a) Zhu et al., J.Am.Chem.Soc., 2017, 139, 16080; (b) Zhu et al., J.Am.Chem.Soc., 2018, 140, 3564.

[1374] 17. &Kool, Chem. Rev., 2017, 117, 10358.

[1375] 18.Zhuang et al., J.Am.Chem.Soc.2018 140, 10363-10367.

[1376] 19.Wu et al.,.Science 2017 355, 499-503.

[1377] 20.Park et al., ACS Catal.2018 8, 9292-9297.

[1378] 21.Dydio et al., J.Am.Chem.Soc.2011 133, 17176-17179.

[1379] Every patent, patent application, and article cited in this document is incorporated by reference. Nouns without quantifiers used herein refer to one or more grammatical objects (i.e., at least one). For example, “element” refers to one or more elements.

[1380] The foregoing description and embodiments are intended to be illustrative and not restrictive. Furthermore, other variations within the spirit and scope of the invention are possible and will be readily apparent to those skilled in the art.

Claims

1. A method for preparing an aqueous composition comprising a library containing a plurality of different bifunctional molecules, the method comprising: a) In one or more aliquots, under conditions sufficient to provide a first aqueous composition, a bifunctional linker molecule B having terminals A' and C' according to formula A'-B-C' is reacted with one or more reactant units X' in water, wherein: The terminal A' contains the identifier nucleotide precursor Z'; B is the connector; The terminal C' contains a bonded iodine-substituted aromatic ring moiety that does not contain a secondary ring nitrogen atom; Reactant unit X' is C4-C 16 Aliphatic carboxylic acids, amides, or masking ketones; The first aqueous composition includes formula Z nα -ABCX nα Bifunctional molecules, Where n is the position identifier of X and Z and is an integer from 1 to 10, such that when n is 1, X and Z are located at the position closest to the connector B; α identifies one or more specific reactant units X, and corresponding one or more paired DNA sequences Z in the bifunctional molecular reactant; and Each Z' or Z is paired with a specific X' or X and identified accordingly; The conditions include the β-C(sp) at the reactant unit X'. 3 )-H or γ-C(sp 3 Palladium-catalyzed arylation of the )-H position at the bonded iodine-substituted aromatic ring moiety, wherein different X's are present in each aliquot, and one or more nucleotide precursors Z' are added to the terminal A', with different Z's added to each aliquot; b) The C4-C present in one or more aliquots of the first aqueous composition of step a) 16 Aliphatic carboxylic acid, amide or masking ketone reactant group X nα Reacting with one or more iodine-substituted aromatic ring moieties W' that do not contain a secondary ring nitrogen atom, and reacting one or more nucleotide precursors Y' with Z nα The reaction forms the inclusion Y nβ -Z nα -ABCX nα -W nβ A second aqueous composition of bifunctional molecules, Each Y' or Y is paired with a specific W' or W and identified therefrom; Wherein β identifies one or more specific chemical groups W, and the corresponding one or more paired DNA sequences Y in the bifunctional molecule reactant; c) The iodine-substituted aromatic ring moiety W present in the aqueous composition or aliquot of the sample formed in step b) nβ Palladium-catalyzed arylation to C4-C 16 The β-C(sp) of aliphatic carboxylic acid, amide, or masking ketone reactant unit V' 3 )-H or γ-C(sp 3 The -H position, and the binding of the nucleotide precursor T' to Y nγ The reaction forms one or more compounds containing formula T nγ -Y nβ -Z nα -ABCX nα -W nβ -V nγ Aqueous compositions of bifunctional molecules; Each T' or T is paired with and identified by a specific V' or V; and γ identifies one or more reactive units V, and corresponding one or more paired DNA sequences T in the bifunctional molecular reactants; in: At least one of reaction steps a), b), and c) includes reacting a plurality of aliquots of an aqueous composition containing a bifunctional molecule individually with a reactant that is different from the reactant in another aliquot in this step, and then combining the resulting aliquots to form the library. Z, A, C, X, Y, W, T, and V are the reaction forms corresponding to Z', A', C', X', W', T', and V'; Each iodinated aromatic ring moiety containing a nitrogen atom without a secondary ring is attached at its terminal C' to a DNA-containing adapter described as "DNA-", wherein the iodinated aromatic ring moiety is selected from... ； And each C4-C 16 Aliphatic carboxylic acids, amides, or masking ketone reactant units are selected from 。 2. The method of claim 1, further comprising step d) of recovering the library from the aqueous composition.

3. A library of bifunctional molecules in an aqueous composition that can be prepared by the method of claim 1 or 2.

4. The library according to claim 3, wherein adapter B comprises an oligonucleotide containing a predetermined recognition sequence for a restriction endonuclease.

5. The library according to claim 4, wherein the oligonucleotide is double-stranded DNA.

6. The library according to claim 5, wherein T nγ Y nβ and Z nα Each one in it is a double-stranded DNA oligonucleotide.

7. The library of claim 6, wherein each of the double-stranded DNA oligonucleotides comprises two restriction endonuclease recognition sites, one of which is located on either side of a specific identifier nucleotide sequence of the reactant used for synthesis.

8. The library of claim 3, wherein each iodine-substituted aromatic moiety comprises a six-membered ring.

9. The library of claim 3, wherein each iodine-substituted aromatic moiety comprises a five-membered ring.

10. The library of claim 3, wherein each iodine-substituted aromatic moiety is carbocyclic.

11. The library of claim 3, wherein one of the iodine-substituted aromatic moieties is heterocyclic.

12. The library of claim 3, wherein each of the aromatic portions is a carbocyclic compound.

13. The library according to claim 3, wherein the C4-C 16 At least one of aliphatic carboxylic acids, amides, or masking ketones comprises a 3- or 4-membered ring bonded to a carbonyl group, wherein the 3- or 4-membered ring contains a β-C(sp) group at the position where the arylation bond is formed. 3 )-H.

14. A method for arylating a reactive group of a first compound at the β- or γ-position, comprising: The first compound, the iodine-substituted aromatic ring moiety without secondary nitrogen atoms, the palladium(II) catalyst, the silver salt, the basic alkali metal salt, and the optional ligands interacting with Pd(II) ions are dissolved or dispersed in an aqueous medium to provide a mixture. The mixture is maintained at a temperature of about 70°C to about 100°C for about 15 to about 30 hours to provide a second compound, which is arylated; The iodine-substituted aromatic ring moiety, which does not contain a secondary nitrogen atom, comprises a single 5- or 6-membered aromatic ring, which is itself linked to a DNA-containing adapter described as "DNA-" and is selected from one or more of the following: ； The reactive group is C4-C. 16 Aliphatic carboxylic acids, amides, or masking ketones, The masking ketone is selected from ； as well as The carboxylic acid is selected from 。 15. The method of claim 14, wherein the molar ratio of the first compound to the iodine-substituted aromatic ring moiety is from 10:1 to 1200:

1.

16. The method of claim 14, wherein the molar ratio of the silver salt to the basic alkali metal salt is 2:1 to 4:1, and the molar ratio of the silver salt to the iodine-substituted aromatic moiety is 100:1 to 400:

1.

17. The method according to claim 14, wherein the molar ratio of the ligand to the palladium(II) catalyst is from 1.5:1 to 4:

1.

18. The method of claim 14, wherein the ligand is selected from one or more of the following: 。 19. The method of claim 14, wherein the position is the β-position of the reactive group.

Images