MERGING C(sp3)-H ACTIVATION WITH DNA-ENCODING

Abstract

Palladium-catalyzed C(sp3)—H arylation of aliphatic carboxylic acids, amides and ketones with BNA-encoded aryl iodides in water is disclosed, Furthermore, sequential C—H arylation chemistry enabled the on-DNA synthesis of structurally-diverse scaffolds containing enriched C(sp3) character, chiral centers, cyclopropane, cyclobutane, and heterocycles. That new chemistry permits preparation of a DNA—encoded library (BEL) technology that can dramatically expedite hit identification in drug discovery owing to its ability to perform protein affinity selection with millions or billions of molecules in a single experiment. The sequential functionalization of multiple C—H bonds provides an unique avenue for creating diversity and complexity from simple starting materials. The use of water as solvent, the presence of DMA, and the extremely low concentration of DMA-encoded coupling partners (0.001 M) have previously hampered the development DMA-encoded C(sp3)—H activation reactions, but many of those hurdles have now been overcome.

Description

BACKGROUND ART

The concept of using DNA sequences to encode each single reagent in every reaction step during the split-pool synthesis was originally proposed by Brenner and Lerner¹. DNA-encoded library (DEL) affinity selection against target proteins can now be performed at the benchtop, with millions or billions of DEL molecules incubated with the immobilized target protein in the same mixture². The application of DEL technology has led to rapid identification of lead compounds for drug discovery³.

Currently, increasing the hit rate as well as improving the drug-like properties of the lead compounds is a primary concern for constructing superior DELs. In this context, establishing diverse types of organic transformations compatible with DEL technology (water as solvent, presence of DNA, 1 mM concentration of DNA substrate) is of pivotal importance⁴. Development of on-DNA reactions faces a number of distinct challenges. DNA backbone degradation could occur under conventional reaction conditions; metal catalysts could be poisoned by oligonucleotides; at least 20% water is required as co-solvent for dissolving DNA-tagged substrates.

Over the past decade, advances in DNA-compatible reactions focused on nucleophilic aromatic substitution (S_N,Ar), cross-coupling, cycloaddition, and click chemistry to construct C (sp²)-C(sp²)⁵, C—N⁶, C—O⁷, S—X bonds⁸ and heterocycles³. Considering the well-known trends for incorporating C(sp³) carbon centers to build C (sp²)-C(sp³) bonds¹⁰ and avoiding high molecular mass and lipophilicity, coupling C(sp³)—H bonds of simple aliphatic acids and ketones with DNA-encoded heteroaryls will be highly desirable. The availability of multiple C—H bonds of a wide range of carboxylic acids and ketones offers a unique opportunity to expand the accessible chemical space for DELs.

The recent development of a wide range of transformations of β—C—H bonds by the inventor and his co-workers demonstrates the potential for creating unprecedented diversity from simplicity¹¹ as are illustrated schematically in the panel of FIG. 1A. The attractiveness of using C—H arylation of carboxylic acid derived substrates to build DNA-4. encoded libraries is evident from a recent study where C—H activation reactions were performed in organic solvent and the DNA tags were subsequently attached individually. However, this approach does not allow encoding each single reagent in every reaction step during the split-pool synthesis, thus limiting the number of available building blocks and thereby the size of the library¹² (FIG. 1B).

In contrast, using C—H activation as a coupling step on DNA allows much larger libraries to be constructed. Notably, despite the use of a very powerful directing group, C(sp³)—H activation reactions have not been successful in the presence of DNA thus far¹³. Development of on-DNA C (sp³)—H activation of different classes of substrates is contemplated herein that can be employed sequentially to build DELs with enriched C(sp³) character, chiral centers, small rings and heterocycles as shown schematically in FIG. 1C, and specifically exemplified hereinafter.

SUMMARY OF THE INVENTION

The present invention contemplates a method for preparing an aqueous composition containing a library having a plurality of different bifunctional molecules, as well as a library itself. A contemplated method contemplates the steps of: reacting a bifunctional linker molecule B having termini A′ and C′ according to the formula A′-B-C′ that is present in one or more aliquots of aqueous compositions. Terminus C′ contains a bonded (tethered) iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms, and terminus A′ contains an identifier nucleotide sequence precursor, Z′. The linker terminal C′ iodo-substituted aromatic ring moiety is reacted by palladium-catalyzed arylation at a β—C(sp³)—H or γ-C(sp³)—H position of one or more reactant C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units, X′. A different X′ is reacted in each aliquot, as is a different nucleotide identifier sequence precursor, Z′, with terminus A′ of the linker, to form an aqueous composition containing bifunctional molecules having the Formula (I), Z_nα—A—B—C—X_nα.

In Formula (I), n is a position identifier for X and Z, and is an integer from 1 to 10, preferably 1 to about 6, and more preferably 2 to about 5, such that when n is 1, X and Z are located most proximal to the linker B, and “α” identifies one or more specific reacted C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units X, and the corresponding, paired one or more identifying paired DNA sequences, Z, in the thus formed bifunctional molecule. Each Z′ or Z is paired with and identifies a particular X′ or X, respectively. Optionally, reacted aqueous aliquot compositions containing approximately equal amounts of bifunctional molecules so formed are admixed to form a single composition containing a mixture of bifunctional molecules in approximately equal numbers (amounts).

The carboxylic acid, carboxamide or masked ketone functionality present in one or more aliquots of the one or more aqueous compositions that contain bifunctional molecules of Formula (I), X_nα, is reacted with one or more iodo-substituted aromatic ring moieties, W′, that is free of secondary ring nitrogen atoms as is every further iodo-substituted aromatic ring moiety used herein, and is the same or different from that present at terminus C′. One or more precursor nucleotide sequence identifiers, Y′, reacts with Z_nα to form one or more aqueous compositions containing bifunctional molecules having the Formula (II), Y_nβ—Z_nα—A—B—C—X_nα—W_nβ identifies one or more specific reacted chemical groups W and the corresponding one or more paired DNA sequence identifiers of Y in the bifunctional molecule of Formula (II).

Reacted aqueous aliquot compositions so formed containing approximately equal amounts of bifunctional molecules are optionally admixed to form a single composition containing an approximately equal mixture of bifunctional molecules. Each Y′ or Y is paired with and identifies a particular W′ or W, respectively.

The one or more iodo-substituted aromatic ring moieties, W_nβ, present in the aqueous composition or aliquots thereof containing a bifunctional molecule of Formula (II) is reacted by palladium-catalyzed arylation at the β—C(sp³)—H or γ—C(sp³)—H position of one or more C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone unit that is the same or different from that reacted previously, V′. One or more precursor nucleotide sequence identifiers T′ is reacted with Y_nγ to form one or more compositions containing bifunctional molecules of Formula (III), T_nγ—Y_nβ—Z_nα,—A—B—C—X_nα—W_nβ—V_nγ.

Each T′ or T is paired with and identifies a particular V′ or V, respectively. Additionally, γidentifies one or more specific C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units V and the corresponding one or more identifying paired DNA sequences of Y in the bifunctional molecule.

It is to be understood in regard to the above outlined reaction sequence, that at least one set of reaction steps includes a plurality of aliquots of an aqueous composition that contains bifunctional molecules that individually reacted with a reactant different from that reacted with bifunctional molecules in another aliquot at that step, followed by combining the aliquots produced to form an aqueous composition containing admixture of a plurality of different bifunctional molecules that constitute a library of said bifunctional molecules in an aqueous composition.

It is also to be understood in reference to reactants or bifunctional molecules that Z, A, C, X, Y, W, T and V are reacted forms of the corresponding Z′, A′, C′, X′, Y′ W′, T′ and V′.

Two or more of the aliquots containing bifunctional molecules of Formula III are combined to form an aqueous composition containing an admixture of bifunctional molecules, thereby forming an aqueous composition containing the library. A library so formed can be isolated by separation from the aqueous composition as by lyophilization, precipitation with an organic solvent, chromatography and other means well known to skilled workers.

It is still further to be noted that the synthesis of a contemplated library of bifunctional molecules need not stop after three cycles of reaction steps as illustrated above, but can continue for several further cycles of reaction that follow the pattern set out above. Two cycles to about 7 are preferred, with 3 to about 5 reaction cycles being more preferred.

A library having a plurality of different bifunctional molecules having the Formula (III) bifunctional molecules of Formula (III), Z, A, C, X, Y, W, T and V are is also contemplated. In a library of bifunctional molecules of Formula III, Z, A, C, X, Y, W, T and V have the same meanings as previously provided, as do the subscripted symbols nγ, nβ and nα. Thus, T_nγ—Y_nβ—Z_nα-A is a mixture of polynucleotide sequences in which the specific identifiers identify the reacted iodo-substituted aromatic ring moieties and C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units that constitute the reacted building blocks of C—X_nα—W_nβ—V_nγ in a mixture of bifunctional molecules of a library.

Preferably, linker B includes an oligonucleotide sequence long enough to be a primer for duplication polymerase chain reaction (PCR), about 18-22 base pairs (bp), and that also contains the recognition sequence of a predetermined restriction endonuclease that permits cleavage of the polynucleotide sequence of the T_ni—Y_ni—Z_n—A identifiers. It is further preferred that each identifier oligonucleotide sequence be double stranded DNA and that the specific identifier T_ni—Y_ni—Z_n—A polynucleotide sequence also be double stranded DNA.

It is also preferred that each of the specific identifier double stranded DNA oligonucleotides contains two restriction endonuclease recognition sites, one on either side of the specific identifier nucleotide sequence of the reactant used in synthesis. If the same endonuclease recognition site is placed on either side of all of the oligonucleotides present at a given position of the bifunctional molecules, cleavage with that endonuclease provides a number of identifier DNA oligos that can be separated and identified using mass spectral analysis.

A skilled worker can also admix a target binding protein with a bifunctional molecule library or sublibrary, capture those library molecules that bind and then conduct a per reaction on any target-bound bifunctional molecule, followed by sequencing of the replicated DNA.

It is further preferred in some embodiments that each aromatic ring moiety contain a six-membered ring, whereas it is preferred in other embodiments that each aromatic ring moiety contains a five-membered ring. In still other embodiments, both 5—and 6-membered aromatic ring moieties are present in a library of bifunctional molecules. In some preferred embodiments, each aromatic ring moiety is carbocyclic, whereas in other preferred embodiments each aromatic ring moiety is heterocyclic. In further preferred embodiments, at least one aromatic ring moiety is carbocyclic and at least one aromatic ring moiety is heterocyclic.

In still further preferences, at least one of the C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone contains a 3- or 4-membered ring bonded to the carbonyl carbon atom, and the reacting 3- or 4-membered ring contained a β—C(sp³)—H at the position of arylation bond formation.

A method of carrying out an aqueous arylation reaction of a C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone at a position β—or γ— to the position of the carboxylic acid, carboxamide or masked ketone carbonyl carbon atom is also disclosed hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure,

FIGS. 1A, 1B and 1C illustrate the exploitation of diversity of C—H activation in a DNA-encoded library (DEL). FIG. 1A illustrates a schematic synthesis of combinatorial chemical libraries through sequential C—H activation¹¹, FIG. 1B is a schematic representation of Off-DNA C—H activation for access to DEL¹², and FIG. 1C schematically illustrates sequential on-DNA C—H activation for access to DEL as described herein. Asterisks indicate the presence of a chiral center at the atom. TBHP, tert-butyl hydroperoxide; Boc, tert-butyloxycarbonyl group; DG, directing group; and

represents a linker including a bonded DNA sequence and a bond.

FIGS. 2A and 2B illustrate the utility of multiple C—H activation in DEL synthesis. illustrating a further addition to Compound 1 that itself was formed by C—H activation to form Compound 1′. FIG. 2B shows the amide-forming reaction between a DNA-bonded linker group amine an aromatic carboxylic acid that illustrates the reliability of the present on-DNA C—H reaction.

FIGS. 3A and 3B illustrate aqueous arylation products and yields obtained between an amine-terminated tethering linker (DNA—NH—) bonded to an iodo-substituted aromatic ring-moiety and the β—C(sp³)—H of a C₄-C₁₆ carboxylic acid, carboxamide or masked ketone. The general reaction, reactants, catalysts, oxidants and their concentrations, as well as temperature and duration of reactions, providing standard conditions under which the reactions of FIGS. 3A and 3B were carried out are shown in Table 1. Unless otherwise noted, condition of entry 1 of Table 1 was used as the standard condition in the reactions whose yields are shown in both of FIGS. 3A and 3B. FIG. 3A illustrates products and yields for the reaction of a single iodo-substituted aromatic ring-moiety bonded to an amine-terminated linker (DNA—NH—) and several different carboxamide-activating C₄-C₁₆ carboxylic acids that each have a β—C(sp³)—H relative to the carboxamide carbonyl carbon atom and having a substituent R at the α-position relative to both the carboxamido nitrogen atom and the carboxyl carbon atom. A chiral ligand, L1, shown in Table 1 was present for reactions whose yields are shown in FIG. 3A and absent in reactions whose yields are shown in FIG. 3B. FIG. 3B illustrates the reaction products and yields of pivalic acid or 1-butylcyclopropane-1-carboxylic acid, A23

with several different iodo-substituted aromatic ring moieties having different aromatic ring structures and different positioning of the iodo substituent. Exceptions:Compound 2:corresponding A, 500 mM; for Compounds 3-13, 18, 19 and 39-45: AgTFA, 300 mM; corresponding A, 300 mM; 36 hours; for Compound 15: Ag₃PO₄, 200 mM; corresponding A, 300 mM; 36 hours; for Compound 21:corresponding A, 300 mM; 36 hours; for Compound 16: AgOTf, 300 mM; corresponding A, 300 mM; 36 hours; for Compound 17: H₂O/DMA (6/1).

FIGS. 4A, 4B and 4C illustrate aqueous arylation products and yields obtained between an amine-terminated linker (DNA—NH—) tethering an iodo-substituted aromatic ring-moiety and the β—C(sp³)—H of a C₄-C₁₆ carboxylic acid. FIG. 4A illustrates the reaction, reactants, catalysts, oxidants and their concentrations, as well as temperature and duration of reactions, providing standard conditions under which the reactions of FIGS. 4B and 4C were carried out. FIG. 4B illustrates products and yields for the reaction of a single iodo-substituted aromatic ring-moiety bonded to an amine-terminated linker (DNA—NH—) and several different C₄-C₁₆ carboxylic acids that each have a β—C(sp³)—H and having a substituent R at the α-position relative to both the carboxyl carbon atom and the carboxamido nitrogen atom. FIG. 4C illustrates the amidification of a product arylated DNA-tethered aromatic moiety, Compound 68 shown in FIG. 4B, and glycine methyl ester in pH 5.5 buffer at room temperature for about 16-18 hours (overnight) to form Compound 73. DMTMM=4-(4,6-dimethoxy-1,3,5-triazin-yl)-4-methylmorpholinium chloride.

FIGS. 5A through 5D illustrate results of arylation reactions of a DNA-tethered iodo-substituted aromatic moiety with masked ketones containing β—C(sp³)—H and γ—C(sp³)—H groups. The reactions were carried out using the standard conditions shown in Table 3 hereinafter except as noted below for particular numbered compounds. More specifically, FIG. 5A illustrates aqueous arylation product yields obtained at the β-position relative to the masked ketone carbonyl carbon atom that was previously and reversibly masked as an oxime whose hydroxyl group was bonded to either of two exemplary directing groups, DG1 and DG2. FIG. 5B illustrates aqueous arylation product yields obtained at the β-position relative to a similarly masked ketone carbonyl carbon atom reacted with various different tethered iodo-substituted aromatic ring moieties having the iodo group at varying ring positions. FIG. 5C illustrates a de-blocking reaction to remove the DG1 ketone carbonyl blocking group to form Compound 103 from Compound 83. FIG. 5D illustrates a schematic flow chart of a contemplated synthetic procedure involving multiple C—H activation reactions for building DEL diversity through multiple C—H activations and reactions. FIG. 5E illustrates a representative synthesis for Compound 106 in which conditions for the steps [a), b), c) . . . etc] were as follows: a) A1

1000 equiv), Pd(OAc)₂ (10 equiv), L1

20 equiv), Ag₂CO₃ (300 equiv), NaOAc (150 equiv), H₂O/DMA/HFIP (8/1/1), 80° C., 16 hours; b) 4-iodobenzylamine (300 equiv), DMTMM (300 equiv), phosphate buffer (pH 5.5), r.t. 16 hours; c) C10

300 equiv), Pd(OAc)₂ (40 equiv), L8

40 equiv), AgTFA (500 equiv), NaOAc (150 equiv), H₂O/DMA (9/1), 80° C., 20 hours; d) aniline (500 equiv), acetone (300 equiv), phosphate buffer (pH 6.5), 50° C., 24 hours.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention contemplates a method for preparing an aqueous composition containing a library having a plurality of different bifunctional molecules, as well as a library itself and a method of its manufacture. A principal reaction utilized in preparing a contemplated library is a palladium (II)-catalyzed arylation reaction that is carried out in an aqueous medium. A contemplated arylation reaction forms a carbon-to-carbon (C—C) bond between a reactive substrate and an aromatic or heteroaromatic iodide.

In a contemplated reaction, the iodo group of an iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms is replaced with a bond to the former β—C(sp³)—H or γ—C(sp³)—H position of a C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone unit, thereby forming a new C—C bond. Palladium-catalyzed arylation reactions between similar reactants have been reported in the literature, but were carried out in organic solvents that were free of added water. It is believed that this is the first report of carrying out such a reaction in an aqueous medium and also the first report of use of such a reaction in the preparation of bifunctional molecules and libraries containing same.

A method contemplates the steps of: reacting a bifunctional linker molecule B having termini A′ and C′ according to the formula A′-B-C′ that is present in one or more aliquots of aqueous compositions. Terminus C′ contains a bonded (tethered) iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms, and terminus A′ contains an identifier nucleotide sequence precursor, Z′. The linker terminal C′ iodo-substituted aromatic ring moiety is reacted by palladium-catalyzed arylation at a β—C(sp³)—H or γ—C(sp³)—H position of one or more reactant C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units, X′. A different X′ is reacted in each aliquot, as is a different nucleotide identifier sequence precursor, Z′, with terminus A′ of the linker, to form an aqueous composition containing bifunctional molecules having the Formula (I), Z_nα—A—B—C—X_nα.

In Formula (I), n is a position identifier for X and Z, and is an integer from 1 to 10, preferably 1 to about 6, and more preferably 2 to about 5, such that when n is 1, X and Z are located most proximal to the linker B, and “α” identifies one or more specific reacted C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units X, and the corresponding, paired one or more identifying paired DNA sequences, Z, in the thus formed bifunctional molecule. Each Z′ or Z is paired with and identifies a particular X′ or X, respectively. Optionally, reacted aqueous aliquot compositions containing approximately equal amounts of bifunctional molecules so formed are admixed to form a single composition containing an approximately equal mixture of the bifunctional molecules.

A contemplated masked ketone is a compound whose carbonyl group is protected from reaction during the arylation, but can be readily deprotected to again provide a carbonyl group. Illustrative masked carbonyl groups include oximes, hydrazones and ketals. When used as an oxime, the hydroxyl group is itself protected from reaction as by formation of an ether linkage as is illustrated hereinafter.

Useful Pd(II) catalysts are well known in the art. Exemplary catalysts include PdCl₂, Pd(TFA)₂, Pd (Piv)₂, [PdCl (C₃H₅)]₂, PdCl₂ (PFh₃)₂, Pd (PPh₃)₄, Pd₂ (dba)₃, [PdCl₂ (MeCN) 2], [Pd (OTf)₂·4MeCN], and [Pd(BF₄)₂ 4 MeCN]. Of these catalysts, Pd(TFA)₂, Pd(Piv)₂ and Pd(OAc)₂ are presently preferred. A contemplated catalyst is utilized in a catalytic amount. That amount is typically about 5 to about 40 mole percent based on the moles of reactive substrate, and more preferably about 10 to about 20 mole percent.

Illustrative basic salts include NaOAc, Na₂CO₃, NaHCO₃, Na_HPO₄, NazHPO₄, NaH=PO₄, Na₃PO₄, KHCO₃, KOAc, K₂CO₃, K₃PO₄, K₂HPO₄·3H₂O, Li₂CO₃ and Cs₂CO₃. Of those salts, NaOAc and Li₂CO₃ usually provided the highest yields and are preferred.

A scavenger such as sodium diethyldithio-carbamate trihydrate is typically added to the reaction mixture after the arylation reaction is completed to assist in recovery of the palladium catalyst. Use of about 70 to about 90 equivalents of the scavenger per equivalent of the tethered iodo-substituted aromatic ring moiety achieves recovery of the palladium and a maximal amount to the initial DNA present.

A contemplated reaction medium optionally, but preferably includes a ligand that can promote both C—H cleavage and the subsequent functionalization steps. Effective C—H functionalization often requires a synergistic relationship between ligand and substrate coordinated to the metal center.

Although not usually needed for some reaction to proceed, the presence of a ligand molecule usually boosts the yield of a desired product. A ligand is typically present in the reaction composition at about 10 to about 30 mole percent based on the moles of substrate. Preferably, the ligand is present at about 20 mole percent. Alternatively, a ligand is present in an amount that is about 1.5 to about 4 times the molar amount of said palladium (II) catalyst.

A contemplated palladium-catalyzed arylation reaction mixture is maintained at a temperature of about 70° to about 100° C. for a time period sufficient to carry out the electrophilic insertion and form an arylated reaction product.

More preferably, that temperature is about 75° to about 90° C. Reaction times are typically about 15 to about 30 hours, with times of about 18 to about 25 hours being usual.

The carboxylic acid, carboxamide or masked ketone functionality present in one or more aliquots of the one or more aqueous compositions that contain bifunctional molecules of Formula (I), X_nα, is reacted with one or more iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms, W′, that is the same or different from that present at terminus C′. One or more precursor nucleotide sequence identifiers, Y′ with Z_nα to form one or more aqueous compositions containing bifunctional molecules having the Formula (II), Y_nβ-Z_nα—A—B—C—X_nα—W_nβ. Reacted aqueous aliquot compositions so formed containing approximately equal amounts of bifunctional molecules are optionally admixed to form a single composition containing an approximately equal mixture of bifunctional molecules. Each Y′ or Y is paired with and identifies a particular W′ or W, respectively. “β” identifies one or more specific reacted chemical groups W and the corresponding one or more paired DNA sequence identifiers of Y in the bifunctional molecule of Formula (II).

The one or more iodo-substituted aromatic ring moieties, W_nβ, present in the aqueous composition or aliquots thereof containing a bifunctional molecule of Formula (II) is reacted by palladium-catalyzed arylation at the β C(sp³)—H or γ—C(sp³)—H position of one or more C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone unit that is the same or different from that reacted previously, V′. One or more precursor nucleotide sequence identifiers T′ is reacted with Y_nγ to form one or more compositions containing bifunctional molecules of Formula (III), T_nγ—Y_nβ—Z_nα—A—B—C—X_nα—W_nβ-V_nγ.

Each T′ or T is paired with and identifies a particular V′ or V, respectively. Additionally, γidentifies one or more specific C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units V and the corresponding one or more identifying paired DNA sequences of Y in the bifunctional molecule.

It is to be understood in regard to the above outlined reaction sequence, that at least one set of reaction steps includes a plurality of aliquots of an aqueous composition that contains a bifunctional molecule that individually reacted with a reactant different from that reacted in another aliquot at that step, followed by combining the aliquots produced to form an aqueous composition containing admixture of a plurality of different bifunctional molecules that constitute a library of said bifunctional molecules in an aqueous composition.

A linker is also referred to herein as a “head group”. An illustrative head group used herein is shown below along with two abbreviations often

used herein for it:

and DNA—NH₂. The depicted amine group, —NH₂, is a useful functionality to react with and tether the iodo-substituted aromatic moiety to the remainder of the head group, whereas the DNA sequences are readily extended by well-known methods of oligonucleotide synthesis.

The nucleotide sequence of the preferably double stranded oligonucleotide identifier Z′ is used to identify the specific X′ that was arylated at position subscript “n” in the “string” of organic residues added on to the linker. The subscripted Greek letter that accompanies each subscripted “n” stands for one up to about 40 different chemical structures and corresponding oligonucleotide sequences that identify each of those structures. In preferred practice, 1 to about 20 different chemical structures are utilized at each position “n” along with the same number of identifying DNA sequences at the same position “n” relative to B on the other side of the linear bifunctional molecule. More preferably, one to about 10 different chemical structures are utilized along with the same number of identifying DNA sequences present at each position in the bifunctional molecule.

In one preferred embodiment, a nucleotide sequence of a restriction endonuclease flanks both upstream and downstream of the X′-identifying sequence so that the positional identifier, as identified by endonuclease, can be readily removed for identification of its one or more reactant-identifying nucleotide sequences. It is preferred that the same endonuclease sequence is present on both sides of the identifying sequence so only one enzyme is needed to excise an identifier sequence at a given position in bifunctional molecule.

It is noted that each aqueous arylation is preferably carried out at a β—C(sp³)—H position relative to the carbonyl carbon of a carboxylic acid, carboxamide or masked ketone unit.

In a bifunctional molecule of Formula III, each T′ or T is paired with and identifies a particular V′ or V, respectively, and Y_ni, Z_n, A, B, C, X_n, and W_ni are as previously defined.

Two or more of the aliquots containing bifunctional molecules of Formula III are combined to form an aqueous composition containing an admixture of bifunctional molecules, thereby forming an aqueous composition containing the library. A library so formed can be isolated by separation from the aqueous composition as by lyophilization, precipitation with an organic solvent, and other means well known to skilled workers.

It is also to be noted that the synthesis of a contemplated library of bifunctional molecules need not stop after three cycles of reaction steps as illustrated above, but can continue for several further cycles of reaction that follow the pattern set out above. Two cycles to about 7 are preferred, with 3 to about 5 reaction cycles being more preferred.

When a plurality of different residues is desired at a given position in the sequence, the bifunctional molecule-containing aqueous composition is divided into at least as many aliquots as the number of different reactants desired to be added along with an identifying DNA sequence for each along with flanking positional identifiers if desired.

If it is desired that the next, adjacent, position be occupied by a mixture, each reacted aliquot is again split into at least the number of reactants to be used at this step. The members of the next set of reactants are individually reacted with the growing bifunctional molecule in each of the newly divided aliquots, as is the next DNA sequence that identifies each along with flanking restriction site sequences that identify the particular position, if desired.

The above variant and others of parallel synthesis whose products are pooled and then divided is illustrated using different syntheses in Brenner and Lerner, Proc Natl Acad Sci, USA 89:5381-5383 (June 1992), U.S. Pat. Nos. 5,573,905; 5,723,598 and 6,060,596, as well as in Houghten, Proc Natl Acad Sci, USA 82:5131-5135 (1985), Houghten et al., Biotechniques, 4(6):522-528 (1986), U.S. Pat. Nos. 4,631,211; 5,763,193, each of whose disclosures are incorporated by reference.

If it is not desired for that next possible position to be a mixture, all of the previously reacted aliquots are combined providing a single composition that contains the mixed bifunctional molecules. Those mixed bifunctional molecules can then be reacted with a single reactant, used without further reaction or recovered for later use.

Thus, T_nγ—Y_nβ—Z_nα—A is a polynucleotide sequence in which the specific identifiers identify the reacted aromatic ring moieties and C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone units that constitute the reacted building blocks of C—X_nα—W_nβ—V_nγ in a bifunctional molecule of the library. Preferably, linker B includes an oligonucleotide sequence that contains the recognition sequence of a predetermined restriction endonuclease that permits cleavage of the polynucleotide sequence of the T_nγ—Y_nβ—Z_nα—A identifiers.

It is further preferred that each identifier oligonucleotide sequence be double stranded DNA and that the specific identifier T_nγ—Y_nβ—Z_nα—A polynucleotide sequence also be double stranded DNA. It is also preferred that each of the specific identifier double stranded DNA oligonucleotides contains two restriction endonuclease recognition sites, one on either side of the specific identifier nucleotide sequence of the reactant used in synthesis. Yet another preference is that a C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone arylationally bonded to an aromatic ring moiety is bonded via a previously present β—C(sp³)—H position relative to the carbonyl group of the carboxylic acid, carboxamide or masked ketone.

It is further preferred in some embodiments that each aromatic ring moiety contain a six-membered ring, whereas it is preferred in other embodiments that each aromatic ring moiety contains a five-membered ring. In still other embodiments, both 5—and 6-membered aromatic ring moieties are present in a library of bifunctional molecules. In some preferred embodiments, each aromatic ring moiety is carbocyclic, whereas in other preferred embodiments each aromatic ring moiety is heterocyclic. In further preferred embodiments, at least one aromatic ring moiety is carbocyclic and at least one aromatic ring moiety is heterocyclic.

In still further preferences, at least one of the C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone contains a 3- or 4-membered ring bonded to the carbonyl carbon atom, and the reacting 3- or 4-membered ring contained a β—C(sp³)—H at the position of arylation bond formation.

A method of carrying out an aqueous arylation reaction of a C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone at a position β—or γ— to the position of the carboxylic acid, carboxamide or masked ketone carbonyl carbon atom is also disclosed below.

An iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms, a reactant that is a C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone containing a β—C(sp³)—H or γ—C(sp³)—H, a catalytic amount of a palladium (II) catalyst, a silver salt, a basic alkali metal salt, and an optionally present ligand that interacts with a Pd²⁺ ion in aqueous media are dissolved or dispersed in an aqueous medium to form an aqueous reaction medium. That aqueous reaction medium is maintained for a period of about 15 to about 30 hours at a temperature of about room temperature (about 20° C.) to about 100° C., and preferably at about 70° C. to about 100° C., to provide a product aromatic ring moiety free of secondary ring nitrogen atoms bonded between the ring position formally occupied by the iodo substituent to the former position occupied by the β—C(sp³)—H or γ—C(sp³)—H of the C₄-C₁6 aliphatic carboxylic acid, carboxamide or masked ketone.

In one preferred embodiment, the C₄-C₁₆ aliphatic carboxylic acid, carboxamide or masked ketone containing a β—C(sp³)—H or γ—C(sp³)—H is present in the aqueous reaction medium in a molar excess relative to the iodo-substituted aromatic moiety 10:1 to about 1200:1. In another preferred embodiment, the silver salt and basic alkali metal salt are present aqueous reaction medium at a molar ratio of about 2:1 to about 4:1, and the molar ratio of the silver salt to the iodo-substituted aromatic moiety is about 100:1 to about 400:1.

In another preferred embodiment, the ligand is present in an amount that is 1 to about about 1.5 to about 4 times the molar amount of the palladium (II) catalyst. A preferred ligand is selected from the group consisting of one or more of

In yet another preferred embodiment, the iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms contains a single 5- or 6-membered aromatic ring, is itself linked to a DNA—containing linker designated “DNA-” and is selected from the group consisting of one or more of

In some preferred embodiments, the masked ketone containing a β—C(sp³)—H or γ—C(sp³)—H is selected from the group consisting of one or more of

Results

Because carboxylic acids and (hetero)aryl iodides are ubiquitous building blocks for DNA—encoded libraries, the coupling of a DNA-tethered aryl iodide with a free carboxylic acid was first studied through Pd-catalyzed β—C—H arylation. This presented formidable challenges beyond the need to adapt the chemistry to aqueous conditions, notably the limited stability of the DNA towards low pH values and heat, the potential for interfering reactivity of the DNA bases, and the high dilution of the DNA-tethered component. Nonetheless, it was decided to develop this chemistry around free carboxylic acids.

Illustrative useful carboxylic acids having a β—C(sp³)—H or γ—C(sp³)—H are shown in the table below.

Guided by previous C—H arylation efforts¹⁴, palladium source, ligand, silver source, base, and co-solvent were screened as illustrated in Tables I and 2, below.

The optimized conditions gave product Compound 1 in 78% yield with no di- or tri-arylation of pivalic acid (entry 1, Table 1). Palladium, silver, and heat were mandatory (entries 2 to 4, Table 1). No ligand was required (entry 5, Table 1), but amino acid-derived ligands improved the yield, PGP-34,TRE with ligand L1 found to be optimal.

TABLE 1
Entry	Deviation from standard condition	1 (%)
1	none	78
2	without Pd(OAc)2	0
3	without Ag2CO3	0
4	r.t. instead of 80° C.	0
5	without L1	56
	instead of
6	S1b	S1a	0

The main side reaction, protodeiodination (e.g., Compound S1b), was always present to a small extent due to the use of excess silver salt and palladium. Compound S1b containing no iodide was subjected to the reaction conditions and recovered intact, confirming that coupling occurs at the aryl iodide and not at some undetermined location on the DNA tag (entry 6, Table 1). Dithiocarbamate releases DNA from DNA-palladium complex as a result of its stronger coordination with palladium leading to insoluble complex.

After each reaction, the mixture was incubated with sodium diethyldithiocarbamate trihydrate in order to scavenge palladium. The level of DNA recovery was significantly influenced by the loading of the dithiocarbamate scavenger.

Examining the results shown in FIG. 3A, a broad range of carboxylic acids adjacent to quaternary carbon atoms are suitable for this chemistry, including those containing hydroxyl, ethers or fluorine (Compounds 2 to 14). However, acids adjacent to a secondary or tertiary carbon react with <30% yield (Compounds 15 to 16). Importantly, cyclopropane—and cyclobutanecarboxylic acids (desirable as alkene isosteres) are competent coupling partners (Compounds 17 to 21).

The mechanism of the Pd-catalyzed β—C—H arylation is well known to give the cis stereoisomer exclusively. LCMS analysis was unable to determine whether chiral ligand L1 gave any absolute stereoinduction.

The results shown in FIG. 3B illustrate that the aryl iodide substitution pattern is flexible (Compounds 22 to 28, 39 to 44). Although previously-published reaction conditions in organic solvent could not couple heteroaryl iodides^24-25, it was believed that conditions that tolerate DNA—the bases that contain nitrogen heterocycles-should allow coupling of heteroaryl iodides (e.g., pyridines and pyrazoles). This was indeed the case; heteroaryl iodides successfully reacted with carboxylic acids under the same reaction conditions (Compounds 29 to 38, 42 to 44). Because aryl and heteroaryl iodides react under the same conditions, they can both be present in a contemplated split-pool synthesis of a DEL.

As shown as part of Table 1 above, the standard reaction conditions for the reactions of FIGS. 3A and 3B are based upon starting with 10 nanomoles (nmol) of the DNA-linked aromatic iodide that is reacted with 1000 equivalents (equiv) of the carboxylic acid containing a β—C(sp³)—H or γ—C(sp³)—H. That reaction takes place in the presence of 10 equivalents of Pd( )Ac)₂, 120 equivalents of the ligand, 300 equivalents of Ag₂CO₃, 150 equivalents of NaOAC that are dissolved or dispersed in a medium containing water, dimethylacetamide and hexafluoro-2-propanol present at a ratio of 8:1:1. The reaction was carried out at a temperature of 80° C. for a time period of 16 hours.

Unless otherwise noted, those conditions were used as the standard condition. For Compound 2: the carboxylic acid was present at 500 equiv. For Compounds 3-14, 18, 19, and 39-44: AgTFA, 300 equiv; carboxylic acid 300 equiv, 36 hours. For Compound 15: Ag₃PO₄, 200 equiv; carboxylic acid, 300 equiv; 36 hours. For Compound 21: carboxylic acid, 300 equiv; 36 hours. For Compound 15: AgOTf, 300 equiv; carboxylic acid, 300 equiv; 36 hours. For Compound 17: H₂O/DMA (6/1).

TABLE 2
Entry	Deviation from standard condition	45 (%)
1	none	69
2	without Pd(OAc)2	0
3	without AgOAc	24
4	without Li2CO3	35
5	AgTFA instead of AgOAc	46
6	Ag2CO3 instead of AgOAc	42
7	NaOAc instead of Li2CO3	46
8	Na2CO3 instead of Li2CO3	55
9	K2CO3 instead of Li2CO3	41
10	r.t. instead of 80° C.	60

Reevaluation of the silver source, base, and co-solvents in the absence of ligand led to optimized conditions that afforded Compound 45 in 69% yield (entry 1, Table 2). Palladium is essential for this transformation (entry 2, Table 2). Silver salts and bases are not strictly required but have a significant impact on yield (entries 3 to 9, Table 2). Surprisingly, this reaction was also found to work well at room temperature (entry 10, Table 2).

Amides derived from cyclopropane- or cyclobutanecarboxylic acid and a broad range of α-amino acids reacted smoothly both heated and at room temperatures (Compounds 45 to 56) as is also seen in the yields shown in FIG. 4A. Pd-catalyzed arylation proceeds only at the β—C—H bond from the amide; attempted coupling of Ac—L—Val—OH gave none of arylation product Compound 59 indicating that α-arylation did not occur. Two LC peaks having same mass were due to the generation of diastereomers after the C—H arylation. To demonstrate this, a representative off-DNA reaction was run to synthesize product Compound 51. As expected, a mixture of diastereomers were observed, although the ratio is slightly lower due to different reaction temperature.

Although amides derived from α-amino acids and containing cyclopropyl or cyclobutyl rings (desirable as alkene isosteres) were focused upon, the chemistry can be extended to other alkyl carboxylic acids (Compounds 57 and 58) and to β-amino acids (Compound 60). Diverse arene substitution patterns on the DNA-tethered aryl iodide were tolerated (Compounds 61 to 65) as seen in the yields shown in FIG. 4B.

As was the case for β—C—H arylation of carboxylic acids, the DNA-tolerant conditions for amide arylation can also be employed for coupling DNA-tethered heteroaryl iodides such as pyridines and pyrazoles (Compounds 66 to 72). If desired, the carboxylic acid of the product can be further modified (Compound 73) shown in FIG. 4C.

Having developed DNA-compatible C(sp³)—H arylations for carboxylic acids and amides, attention was turned to use of ketones in similar DNA—compatible C(sp³)—H arylations. Ketones are useful monomers for building DELs because they can be further elaborated via reductive amination. Guided by previous work using aminooxyacetic acids as removable directing groups to recruit palladium to activate the β—C—H bond of ketones¹⁶, a DNA-tolerant version of this reaction was created and optimized.

The optimized reaction illustrated in Table 3 and FIG. 5A through FIG. 5D gave arylation 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 the silver salt (entries 4-6, Table 3); ligand and base play a lesser role (entries 7 and 8, Table 3). Ligand L8 was also found to decrease the degradation of DNA and provide cleaner LC traces. Although a large excess of palladium is often associated with degradation of the DNA tag, this chemistry gives a higher yield at 20 mM Pd/L (entry 1, 40 equivalents) than at 30 equivalents (entry 9, Table 3).

TABLE 3
Entry	Deviation from standard condition	83 (%)
1	none	62
2	without Pd(OAc)2	0
3	r.t. instead of 80° C.	0
4	without AgTFA	4
5	Ag2CO3 (300 equiv) instead of AgTFA	25
6	AgOAc (500 equiv) instead of AgTFA	32
7	without L8	51
8	without NaOAc	55
9	Pd(OAc)2/L8 (30 equiv/30 equiv)	49
	instead of
10	S1b	S1a	0

The above conditions effect β—C—H arylation in diverse settings, including on acyclic ketone derivatives (Compounds 74 to 77, 84 to 89), at positions next to simple or complex rings (Compounds 78 to 82, 90, 91), and on simple or complex rings (Compounds 83, 92) as is seen by the yields shown in FIG. 5A. A broad range of functional groups are tolerated on the ketone to be arylated, including esters, ethers, acetals, and amides.

Ketone derivatives bearing β-quaternary centers can also be γ-arylated (Compound 93) as illustrated in FIG. 5A. The yields shown in FIG. 5B illustrate that DNA-tethered aryl iodide accepts different substitution patterns (Compounds 94 to 97) and heteroaryl iodides can react under the same conditions (Compounds 98 to 102).

The ability to convert the oxime ethers back into ketones is critical for implementing this chemistry in a DEL build. It was found that these oxime ethers readily hydrolyze in the presence of aniline and acetone (Compound 83 to 103), likely through equilibrium transimination with aniline and trapping of the free aminooxyacetic acid with acetone¹⁷. This reaction is illustrated in FIG. 5C.

Each of these C(sp³)—H activation reactions, as new disconnections for DEL synthesis, can incorporate unique structural motifs. The combination of multiple C(sp³)—H activations in DEL synthesis can further enhance the diversity. Hence, a multi-step synthesis on DNA consisting of β—C—H arylation of a carboxylic acid, amide formation, β—C—H arylation of a masked ketone, and ketone deprotection was embarked upon to demonstrate how these C—H activation chemistries can be combined to develop large DELs of diverse, drug-like compounds. A representative analog synthesis is shown in FIG. 5D.

Thus, pyridyl iodide Compound 318 and pivalic acid were coupled to form intermediate Compound 31, and amide coupling with p-iodobenzyl amine then set up a second C—H arylation event. Coupling with a masked cyclobutyl ketone gave oxime ether Compound 105, whose hydrolysis revealed ketone Compound 106 with 6% overall yield over 4 steps as shown in FIG. 5E.

The above is only one of many sequences that a skilled worker can design by using sequential multiple C—H arylation steps in combination with common DNA-encoded library-building steps such as amide formation or reductive amination. Importantly, the ability to conduct the C—H arylations using DNA—tethered aryl iodides means that the full power split-pool combinatorial synthesis can be realized.

To evaluate DNA compatibility of the C—H activation chemistry with DEL synthesis, a select set of chemically modified on-DNA analogs (C—H arylation products 1, 45 and 83), along with their starting aryl iodide analog Sla were enzymatically ligated to a 65-mer dsDNA, so that the resulting oligomers were approximately equal in length to an encoding tag of a 3-cycle DEL build. All four ligation reactions proceeded smoothly, indicating that chemistry had no significant impact on encodability. In order to determine the amount of amplifiable DNA remaining after exposure to C—H activation conditions, the ligation products from 1, 45 and 83 were amplified by PCR and compared with those of Sla (untreated control). All three reactions showed satisfactory PCR viability (60-80% amplifiable DNA remaining). Moreover, Sanger sequencing reads also confirmed the integrity of their nucleobase sequence structures.

As purification is an inherently difficult process in split-pool synthesis, the reactivity required for DNA-compatible reactions must be devoid of unidentified byproducts that can complicate analysis. In this case, the main byproducts generated through our reaction platform consist only of starting material or its protodehalogenated derivative. Finally, we were able to obtain all products through our on-DNA C—H arylation platform in moderate and synthetically useful yields; higher than the threshold of 25% deemed practical in DEL synthesis.^10f Gratifyingly also, we were able to obtain 60-80% DNA recovery from qPCR experiments, greater than the acceptable 305 threshold deemed practical in these processes.^4c Altogether, these promising results further demonstrate the practicality of our DEL-compatible C(sp³)—H activation platform, enabling practitioners to rapidly generate structural complexity and diversity in a modular manner.

In summary, DEL-compatible C(sp³)—H activation reactions of carboxylic acid, amides, and ketones have been developed. Ligands were essential for the reactivity under DEL conditions. These protocols are compatible with C(sp³)—H bond of small rings and heterocyclic coupling partners that are desirable for improving drug-like properties.

Sequential C(sp³)—H activation for DEL synthesis provides a unique tool for constructing chemical diversity containing high C(sp³) character.

1. General Information

Equipment and Chemicals

VWR® modular heating block (64 wells) was used to heat PCR tubes to run the DNA reactions. 10K variable speed mini centrifuge (BT604) was purchased from BTLab Systems. VWR® 9 mm screw-thread polypropylene vials and screw caps were used to submit the samples to HPLC-MS. Hexafluoroisopropanol (HFIP) was purchased from Oakwood. N,N-Dimethyl-acetamide (DMA) was obtained from Honeywell. N,N-dimethylformamide (DMF) and acetonitrile (CH₃CN) were obtained by passing the previously degassed solvents through an activated alumina column. Deionized water was used in all the reactions. 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 used without further purification.

Ligands were prepared via previously published protocols except some of them are now commercially available. The iodo-substituted heteroaromatic acids were received from Pfizer. Carboxylic acids, amides and ketones bearing directing groups were synthesized via previously published protocols except some are also commercially available. DMTMM=4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

DNA Headpiece Materials

DNA headpiece (5′-/5Phos/GAGTCA/iSp9/-iAmM/-iSp9/-TGACTCCC-3′, below) was obtained from Biosearch Technologies, Petaluma, CA. The abbreviated DNA headpiece is shown below.

Analysis of DNA samples

DNA Concentration:

DNA samples subjected to HPLC-MS analysis were prepared as 0.1 mM in H₂O, assuming 100% of DNA total recovery after reaction.

Analysis:

One microliter of the DNA solution was analyzed on a Waters I-Class LC with a Waters BEH C18 of 114 mM HFIP and 14 mM Et₃N in water (A) and methanol (B) (0.3 mL/minute, 10-26% B over 10 minutes) at 60° C. The yield was determined by calculating the percentage of UV absorbance at 260 nm PGP-52,E corresponding to the product peak, ignoring potential UV absorption coefficient differences between DNA products and assuming 100% mass recovery. Peak identities were determined by ESI using the [M]³ion.

Deconvolution:

Data visualization and integration was performed with MassLynx™ V4.1 software.

Yield Calculation:

Ignoring UV coefficient difference for all DNA products and assuming 100% of DNA total recovery, the yield of DNA products was determined from UV absorbance trace (260 nm) peak area using the equation below:

$\mathrm{Yield}\left(\mathrm{product},\%\right)=\text{?}\times 100\%$ $\text{?}\text{indicates text missing or illegible when filed}$

MS Deconvolution:

Whereas multi-charged (negative) mass was observed, triply charged mass was determined to be base peak in all cases. Observed m/z could be calculated as m/z=[M]/z−1.00794.

2. Ligand Structures L1-L13

The Ligands L3, L5, L5-L13 were purchased PGP-53,C3 from Sigma-Aldrich, Nova Biochem, TCI and Combi-Blocks Inc. The Ligands L1^14b, L2¹⁸, L4¹⁹ were synthesized according to previous reports.

3. Preparation of DNA-Conjugated Aryl Iodides

3.1 General Procedure 1 for DNA-Conjugated Aryl Iodides

Materials

Headpiece: 20 mM in H₂O

Sodium carboxylate: 1.0 M in water [1 mmol acid was added into 1.0 mL aqueous NaOH (40 mg) solution]4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM): 1.0 M in water(294.7 mg DMTMM dissolved in 1 mL H₂O)Borate buffer: 100 mM in H₂O

General Procedure 1

1) To the headpiece solution (⁴00 nmol, 20 maintained at room temperature for 3 hours.

2) To the mixture was added 5 M NaCl stored at −20° C.). The mixture was then stored at −20° C. in a freezer for more than 30 minutes.

3) Centrifuge the sample for 7 minutes at 4° C. in a microcentrifuge at 10000 rpm. The supernatant was discarded and the precipitate was dried under vacuum. The pellet was then dissolved in deionized of N,N-heated at 70° C. for 12 hours.

4) Cooling to room temperature, 5 M NaCl sequentially added, and the resultant mixture was stored at −20° C. for 30 minutes. The mixture was centrifuged at 4° C. for 7 minutes at 10000 rpm before the resulting supernatant was removed, and the precipitate was dried under vacuum. The pellet was was used in next study without further purification.

5) HPLC-(10 mM) wa ₂O to prepare the testing sample at 0.1 mM concentration.

3.2 Structures of S1-S23

The iodo-substituted aromatic acids S1-S13 were purchased from Sigma-Aldrich, Oakwood, TCI and Combi-Blocks Inc. The heteroaromatic acids S14-323 were received from Pfizer.

3.3 LC Trace and Masa Characterization of S1-323

LC Trace and Masa of Sla

Following General Procedure 1

Yield: 85%

Exact mass: 5178.8211

Triply charged mass [M]/3-1.00794, calculated

1725.2658; observed 1725.2783.

Following General Procedure 1.

Yield: 83%

Exact mass: 5052.9244

Triply charged mass [M]/3-1.00794, calculated

1683.3002; observed 1683.3007.

Following General Procedure 1.

Yield: 83%

Exact mass: 5178.8211

Triply charged mass [M]/3-1.00794, calculated

1725.2658; observed 1725.2783.

Following General Procedure 1.

Yield: 78%

Exact mass: 5192.8367

Triply charged mass [M]/3-1.00794, calculated

1729.9376; observed 1729.9423.

Following General Procedure 1.

Yield: 74%

Exact mass: 5164.8054

Triply charged mass [M]/3-1.00794, calculated

1720.5939; observed 1720.6038.

Following General Procedure 1.

Yield: 69%

Exact mass: 5178.8211

Triply charged mass [M]/3-1.00794, calculated

1725.2658; observed 1725.2783.

Following General Procedure 1.

Yield: 67%

Exact mass: 5198.7664

Triply charged mass [M]/3-1.00794, calculated

1731.9142; observed 1731.9187.

Following General Procedure 1.

Yield: 83%

Exact mass: 5164.8054

Triply charged mass [M]/3-1.00794, calculated

1720.5939; observed 1720.6038.

Following General Procedure 1.

Yield: 76%

Exact mass: 5178.8211

Triply charged mass [M]/3-1.00794, calculated

1725.2658; observed 1725.2783.

Following General Procedure 1.

Yield: 71%

Exact mass: 5198.7664

Triply charged mass [M]/3-1.00794, calculated

1731.9142; observed 1731.9187.

Following General Procedure 1.

Yield: 85%

Exact mass: 5164.8054

Triply charged mass [M]/3-1.00794, calculated

1720.5939; observed 1720.6038.

Following General Procedure 1.

Yield: 75%

Exact mass: 5178.8211

Triply charged mass [M]/3-1.00794, calculated

1725.2658; observed 1725.2783.

Following General Procedure 1.

Yield: 71%

Exact mass: 5182.7960

Triply charged mass [M]/3-1.00794, calculated

1726.5907; observed 1726.6053.

Following General Procedure 1.

Yield: 55%

Exact mass: 5182.7960

Triply charged mass [M]/3-1.00794, calculated

Following General Procedure 1.

Yield: 57%

Exact mass: 5165.8007

Triply charged mass [M]/3-1.00794, calculated 1720.9256; observed 1720.9265.

Following General Procedure 1.

Yield: 65%

Exact mass: 5165.8007

Triply charged mass [M]/3-1.00794, calculated

1720.9256; observed 1720.9265.

Following General Procedure 1.

Yield: 79%

Exact mass: 5165.8007

Triply charged mass [M]/3-1.00794, calculated

1720.9256; observed 1720.9265.

Following General Procedure 1.

Yield: 59%

Exact mass: 5209.8269

Triply charged mass [M]/3-1.00794, calculated

1735.6010; observed 1735.6018.

Following General Procedure 1.

Yield: 56%

Exact mass: 5165.8007

Triply charged mass [M]/3-1.00794, calculated

1720.9256; observed 1720.9265.

Following General Procedure 1.

Yield: 77%

Exact mass: 5168.8116

Triply charged mass [M]/3-1.00794, calculated

1721.9293; observed 1721.9290.

Following General Procedure 1.

Yield: 76%

Exact mass: 5168.8116

Triply charged mass [M]/3-1.00794, calculated

1721.9293; observed 1721.9459.

Following General Procedure 1.

Yield: 69%

Exact mass: 5168.8116

Triply charged mass [M]/3-1.00794, calculated

1721.9293; observed 1721.9290.

Following General Procedure 1.

Yield: 67%

Exact mass: 5154.7847

Triply charged mass [M]/3-1.00794, calculated

1717.2536; observed 1717.2589.

Following General Procedure 1.

Yield: 69%

Exact mass: 5170.7618

Triply charged mass [M]/3-1.00794, calculated

1722.5793; observed 1722.5918.

4. Experimental Section for on-DNA C—H Arylation of Free Carboxylic Acids

4.1 Substrate Structures of Free Carboxylic Acids A1—A38

Carboxylic acids were obtained from the commercial sources or synthesized following the literature procedures^14b,20

TABLE 4
Evaluation of A1 Concentration
Entry	A1 (x mM)	Yield (%)
1	100 mM	51
2	200 mM	53
3	300 mM	60
4	500 mM	59
5	800 mM	66
6	1000 mM	78
7	2000 mM	65

TABLE 5
Evaluation of Base
Entry	Base (150 mM)	Yield (%)
1	NaOAc	78
2	KOAc	49
3	Na2CO3	47
4	K2CO3	65
5	NaHCO3	57
6	KHCO3	56
7	Na2HPO4	57
8	NaH2PO4	35
9	Na3PO4	48
10	K2HPO4	53
11	K3PO4	59

TABLE 6
Evaluation of Pd/L Concentration
Entry	Pd(OAc)2/L1 (x/y)	Yield (%)
1	10/10	67
2	10/15	72
3	10/20	78
4	10/30	49
5	10/40	52
6	5/20	42
7	15/20	60
8	20/20	65

TABLE 7
Evaluation of Standard Conditions
Entry	Deviation from above	Yield (%)
1	none	78
2	without Pd(OAc)2	0
3	without Ag2CO3	0
4	without NaOAc	77
5	without L1	56
6	H2O/DMA (9/1) instead of	76
	H2O/DMA/HFIP (8/1/1)
7	H2O instead of H2O/DMA/HFIP	76
	(8/1/1)
8	r.t. instead of 80° C.	0
9	L2 instead of L1	74
10	L3 instead of L1	66
11	L4 instead of L1	65
12	L5 instead of L1	54
13	S1b instead of S1a	0a
aOnly S1b was totally recovered.

4.3 DNA Recovery Investigations

TABLE NO. 8
Evaluation of DNA Recovery
	Scavenger
	(sodium	Total DNA
	diethyldithiocarbamate	Recovery
Entry	trihydrate)	(%)a
1	10 equiv	51
2	50 equiv	76
3	70 equiv	81
4	90 equiv	85
5	100 equiv	70
aThe total DNA recovery was calculated with S1a as the standard.

4.4 General Procedure 2 for on-DNA C—H Arylation of Free Carboxylic Acids

Condition A:

Materials

DNA-conjugated aryl iodide 8:10 mM in H₂O Carboxylic acid A: 3 M in DMA (Note: the high concentration may increase the total volume) L1:200 mM in hexafluoroisopropanol (HFIP) (4.4 mg in

NaOAc: 1.5 M in H₂

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

Procedure

• • 1) To prepared AgTFA (300 equiv, 0.66 mg) was added Pd(OAc):—drying, carboxylic acid A DNA-conjugated aryl iodide S (10 nmol, 1 NaOAc d. Finally, L1 solution (20 and the reaction mixture was heated at 80° C. for 36 hours.
  • 2) The reaction mixture was cooled to room resulting mixture was reheated at 80° C. for 30 minutes.
  • 3) The reaction mixture was cooled to room temperature, and 5 M NaCl solution (10% by volume, were added. The mixture was stored at a −20° C. freezer for more than 30 minutes.
  • 4) The sample was centrifuged for about 7 minutes in a microcentrifuge at 10000 rpm. The supernatant was discarded and the precipitate was dried under vacuum. The resulting DNA pellet was redissolved in H₂ 2 minutes in a microcentrifuge at 10000 rpm. An—MS.

Condition B:

Materials

S1a:10 mM in H₂O

Cyclopropanecarboxylic acid (A22): 3 M in H₂O

L1:200 mM in HFIP

Pd(OAc)₂: 100 mM in HFIP

NaOAc: 1.5 M in H₂O)

Sodium diethyldithiocarbamate trihydrate (scavenger):

1 M in H₂O

Procedure was added Pd(OAc)₂ L1 solution (20—drying, Ag₂CO₃ (300 equiv, 0.83 mg) was added, and then A22 (1000 equiv, 3.3 S1a (10 nmol, 1 NaOAc aqueous solution (150 vortexed. The reaction mixture was heated at 80° C. for 16 hours.

• • 2) The reaction mixture was cooled to room temperature, 9

resulting mixture was reheated at 80° C. for 30 minutes.

• • 3) The reaction mixture was cooled to room temperature, and 5 M NaCl solution (10% by volume, were added. The mixture was stored at −20=C in a freezer for more than 30 minutes.
  • 4) The sample was centrifuged for about 7 minutes in a microcentrifuge at 10000 rpm. The supernatant was discarded and the precipitate was dried under vacuum. The resulting DNA pellet was redissolved in H₂

2 minutes in a microcentrifuge at 10000 rpm. An—MS. 4.5 Scope and Limitations of Free Carboxylic Acids

TABLE 9
Scope and Limitations of Free Carboxylic Acids
	Free		Yield
Entry	Carboxylic Acid	Ag Salt (x mM)	(%)
1  2  3  4		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	31 60 N.D. 61
5  6  7  8		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	46 49 (62)a  5 22
9  10  11  12		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	72 24  8 39
13  14  15  16		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	56  9  8 20
17  18  19  20		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	48 11  7 35
21  22  23  24		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	32 11 18 20
25  26  27  28		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	19 12 N.D. 18
29  30  31  32		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	33 29 21 33
33  34  35  36		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	46 11  6 13
37  38  39  40		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	72 58  6 20
41  42  43  44		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	48 12  6 16
45  46  47  48		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	46  4  4 14
49  50  51  52		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	61 20 N.D. 33
53  54  55  56		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	64 39  7 35
57  58  59  60		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	49  4 trace  5
61  62  63  64		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	61  8 N.D. 11
65  66  67  68		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	5 21 28 22
69  70  71  72		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	16 28 16 14
73  74  75  76		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	trace 21 18 28
77  78  79  80		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	13  4 N.D. 24
81  82  83  84		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOTf (300 mM)	18 12 N.D. 26
85  86  87  88		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	12 22 (41)b 16 14
89  90  91  92		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	47  6 N.D. 12
93  94		AgTFA (300 mM) Ag2CO3 (300 mM)	40 N.D.
95  96  97  98		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	7 21  6  5
99 100 101 102		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	20 16 (39) 16 19
103 104 105 106		AgTFA (300 mM) Ag2CO3 (300 mM) Ag3PO4 (200 mM) AgOAc (300 mM)	34 40 24 25
107		AgTFA (300 mM)	N.D.
108 109		AgTFA (300 mM) Ag2CO3 (300 mM)	N.D. N.D.
110 111		AgTFA(300 mM) Ag2CO3 (300 mM)	N.D. Trace
112		AgTFA (300 mM)	N.D.
113		AgTFA (300 mM)	N.D.
114 115		AgTFA (300 mM) Ag2CO3 (300 mM)	N.D. N.D.
116 117		AgTFA (300 mM) Ag2CO3 (300 mM)	N.D. N.D.
118		AgTFA (300 mM)	N.D.
119		AgTFA (300 mM)	N.D.
120		AgTFA (300 mM)	N.D.
121		AgTFA (300 mM)	N.D.
aUsing A2 (500 mM);
bCondition B was followed;
cUsing A26 (1000 mM), 16 hours.
N.D. = not detected.

4.6 LC Trace and Mass Characterization of 1-51 LC Trace and Mass of 1

Following General Procedure 2 (Condition A) with A1 (1000 mM) except for employing Ag₂CO₃ instead of

AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 66/2/4

Exact mass: 5152.9766

Triply charged mass [M]/3-1.00794, calculated

1716.6509; observed 1716.6649.

LC Trace and Masa of 2

Following General Procedure 2 (Condition A) with A2 (500 mM) except for employing Ag:CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 53/6/24

Exact mass: 5166.9927

Triply charged mass (M)/3-1.00794, calculated

1721.3230; observed 1721.3342.

LC Trace and Mass of 3

Following General Procedure 2 (Condition A) with A3.

Yield:—

Ratio (product/deiodination/aryl iodide): 61/3/4

Exact mass: 5181.0081

Triply charged mass [M]/3-1.00794, calculated

1725.9948; observed 1725.9928.

LC Trace and Mass of 4

Following General Procedure 2 (Condition A) with A4.

Yield:—

Ratio (product/deiodination/aryl iodide): 48/5/6

Exact mass: 5195.0238

Triply charged mass [M]/3-1.00794, calculated

1730.6667; observed 1730.6748.

LC Trace and Mass of 5

Following General Procedure 2 (Condition A) with A5.

Yield:—

Ratio (product/deiodination/aryl iodide): 41/10/18

Exact mass: 5195.0238

Triply charged mass [M]/3-1.00794, calculated

1730.6667; observed 1730.6748.

LC Trace and Mass of 6

Following General Procedure 2 (Condition A) with A6.

Yield:—

Ratio (product/deiodination/aryl iodide): 27/12/10

Exact mass: 5214.9925

Triply charged mass [M]/3-1.00794, calculated

1737.3229; observed 1737.3254.

LC Trace and Mass of 7

Following General Procedure 2 (Condition A) with A7.

Yield:—

Ratio (product/deiodination/aryl iodide): 16/4/5

Exact mass: 5168.9718

Triply charged mass (M)/3-1.00794, calculated

1721.9827; observed 1721.9800.

LC Trace and Mass of 8

Following General Procedure 2 (Condition A) with A8.

Yield:—

Ratio (product/deiodination/aryl iodide): 28/3/11

Exact mass: 5193.0081

Triply charged mass (M)/3-1.00794, calculated

1729.9948; observed 1729.9934.

LC Trace and Mass of 7

Following General Procedure 2 (Condition A) with A9.

Yield:—

Ratio (product/deiodination/aryl iodide): 39/4/6

Exact mass: 5207.0238

Triply charged mass [M]/3-1.00794, calculated

1734.6667; observed 1734.6637.

LC Trace and Masa of 8

Following General Procedure 2 (Condition A) with A10.

Yield:—

Ratio (product/deiodination/aryl iodide): 61/5/0

Exact mass: 5237.0344

Triply charged mass [M]/3-1.00794, calculated

1744.6702; observed 1744.6731.

LC Trace and Masa of 9

Following General Procedure 2 (Condition A) with A11.

Yield:—

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

Exact mass: 5243.0238

Triply charged mass [M]/3-1.00794, calculated

1746.6667; observed 1746.6749.

LC Trace and Mass of 10

Following General Procedure 2 (Condition A) with A12.

Yield:—

Ratio (product/deiodination/aryl iodide): 39/9/4

Exact mass: 5287.0500

Triply charged mass [M]/3-1.00794, calculated

1761.3421; observed 1761.3392.

LC Trace and Mass of 11

Following General Procedure 2 (Condition A) with A13.

Yield:—

Ratio (product/deiodination/aryl iodide): 52/4/5

Exact mass: 5211.0187

Triply charged mass [M]/3-1.00794, calculated

1735.9983; observed 1736.0114.

LC Trace and Mass of 12

Following General Procedure 2 (Condition A) with A14.

Yield:—

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

Exact mass: 5213.0144

Triply charged mass [M]/3-1.00794, calculated

1736.6635; observed 1736.6769.

LC Trace and Masa of 13

Following General Procedure 2 (Condition A) with A15.

Yield:—

Ratio (product/deiodination/aryl iodide): 42/5/6

Exact mass: 5263.0112

Triply charged mass [M]/3-1.00794, calculated

1753.3291; observed 1753.3391.

LC Trace and Mass of 14

Following General Procedure 2 (Condition A) with A16.

Yield:—

Ratio (product/deiodination/aryl iodide): 52/3/12

Exact mass: 5193.0081

Triply charged mass [M]/3-1.00794, calculated

1729.9948; observed 1730.0104.

LC Trace and Mass of 15

Following General Procedure 2 (Condition A) with A17

except for employing Ag₃PO₄ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 24/7/5

Exact mass: 5124.9455

Triply charged mass [M]/3-1.00794, calculated

1707.3072; observed 1707.3116.

LC Trace and Mass

Following General Procedure 2 (Condition A) with A18

except for employing Ag₂O₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 24/6/6

Exact mass: 5138.9612

Triply charged mass [M]/3-1.00794, calculated

1711.9791; observed 1711.9851.

LC Trace and Mass

Following General Procedure 2 (Condition A) with A19 except for employing AgOAc instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 24/17/3 Exact mass: 5152.9768

Triply charged mass [M]/3-1.00794, calculated

1716.6510; observed 1716.6649.

LC Trace and Mass

Following General Procedure 2 (Condition A) with A20 except for employing AgOAc instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 20/16/17

Exact mass: 5166.9925

Triply charged mass [M]/3-1.00794, calculated

1721.3229; observed 1721.3342.

LC Trace and Mass of 16

Following General Procedure 2 (Condition A) with A21 except for employing AgOTf instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 22/27/14

Exact mass: 5206.9486

Triply charged mass [M]/3-1.00794, calculated

1734.6416; observed 1734.6637.

LC Trace and Mass of 17

Following General Procedure 2 (Condition B) with A22.

Yield:—

Ratio (product/deiodination/aryl iodide): 35/9/7

Exact mass: 5136.9455

Triply charged mass [M]/3-1.00794, calculated

1711.3072; observed 1711.3074.

LC Trace and Mass of 18

Following General Procedure 2 (Condition A) with A23.

Yield:—

Ratio (product/deiodination/aryl iodide): 40/3/21

Exact mass: 5193.0081

Triply charged mass (M)/3-1.00794, calculated

1729.9948; observed 1729.9934.

LC Trace and Masa of 19

Following General Procedure 2 (Condition A) with A24.

Yield:—

Ratio (product/deiodination/aryl iodide): 34/0/29

Exact mass: 5255.0238

Triply charged mass (M)/3-1.00794, calculated

1750.6667; observed 1750.6652.

LC Trace and Mass

Following General Procedure 2 (Condition A) with A25 except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 18/15/6

Exact mass: 5164.9768

Triply charged mass [M]/3-1.00794, calculated

1720.6510; observed 1720.6547.

LC Trace and Moss of 20

Following General Procedure 2 (Condition A) with A26 (1000 mM) except for employing Ag₂CO₃ instead of

AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 33/23/5

Exact mass: 5150.9612

Triply charged mass [M]/3-1.00794, calculated

1715.9791; observed 1715.9694.

LC Trace and Ness of 21

Following General Procedure 2 (Condition A) with A27 except for employing Ag:CO₃ instead of AgTFA.

Yield:—

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

Exact mass: 5178.9925

Triply charged mass [M]/3-1.00794, calculated

1725.3229; observed 1725.3293.

LC Trace and Masa of 22

Following General Procedure 2 (Condition A) with S2 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 46/31/4

Exact mass: 5152.9768

Triply charged mass [M]/3-1.00794, calculated

1716.6510; observed 1716.6649.

LC Trace and Mass

Following General Procedure 2 (Condition A) with S3 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 57/3/15

Exact mass: 5166.9925

Triply charged mass [M]/3-1.00794, calculated

1721.3229; observed 1721.3342.

LC Trace and Mass

Following General Procedure 2 (Condition A) with S10 and A1 (1000 mM) except for employing Ag₂CO₃ instead

of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 57/8/23

Exact mass: 5138.9612

Triply charged mass [M]/3-1.00794, calculated

1711.9791; observed 1711.9851.

LC Trace and Mesa of 25

Following General Procedure 2 (Condition A) with S4 and A1 (1000 mM) except for employing Ag:CO₃ instead

of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 35/24/3

Exact mass: 5138.9612

Triply charged mass [M]/3-1.00794, calculated

1711.9791; observed 1711.9851.

LC Trace and Mass of 26

Following General Procedure 2 (Condition A) with S6 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 35/22/3

Exact mass: 5172.9222

Triply charged mass [M]/3-1.00794, calculated

1723.2995; observed 1723.3057.

LC Trace and Moss of 27

Following General Procedure 2 (Condition A) with S7 and A1 (1000 mM) except for employing Ag₂CO₃ instead

of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 50/5/22

Exact mass: 5138.9612

Triply charged mass [M]/3-1.00794, calculated

1711.9791; observed 1711.9851.

LC Trace and Ness of 28

Following General Procedure 2 (Condition A) with S9 and A1 (1000 mM) except for employing Ag:CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 41/11/18

Exact mass: 5172.9222

Triply charged mass [M]/3-1.00794, calculated

1723.2995; observed 1723.3057.

LC Trace and Masa of 29

Following General Procedure 2 (Condition A) with 814 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 42/10/2

Exact mass: 5139.9564

Triply charged mass [M]/3-1.00794, calculated

1712.3109; observed 1712.3240.

LC Trace and Mass of 30

Following General Procedure 2 (Condition A) with 815 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 53/7/4

Exact mass: 5139.9564

Triply charged mass [M]/3-1.00794, calculated

1712.3109; observed 1712.3240.

LC Trace and Mass of 31

Following General Procedure 2 (Condition A) with S18 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 41/7/2

Exact mass: 5139.9564

Triply charged mass [M]/3-1.00794, calculated

1712.3109; observed 1712.3070.

LC Trace and Mass of 32

Following General Procedure 2 (Condition A) with S16 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 30/18/28

Exact mass: 5139.9564

Triply charged mass [M]/3-1.00794, calculated

1712.3109; observed 1712.3240.

LC Trace and Mass of 33

Following General Procedure 2 (Condition A) with S17 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

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

Exact mass: 5183.9827

Triply charged mass [M]/3-1.00794, calculated

1726.9863; observed 1726.9968.

LC Trace and floss of 34

Following General Procedure 2 (Condition A) with 819 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 35/24/17

Exact mass: 5142.9673

Triply charged mass [M]/3-1.00794, calculated

1713.3145; observed 1713.3240.

LC Trace and Mass of 35

Following General Procedure 2 (Condition A) with 820 and A1 (1000 mM) except for employing Ag:CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 22/43/3

Exact mass: 5142.9673

Triply charged mass [M]/3-1.00794, calculated

1713.3145; observed 1713.3240.

LC Trace and Mass of 36

Following General Procedure 2 (Condition A) with 821 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 23/19/30

Exact mass: 5142.9673

Triply charged mass [M]/3-1.00794, calculated

1713.3145; observed 1713.3240.

LC Trace and Mass of 37

Following General Procedure 2 (Condition A) with 822 and A1 (1000 mM) except for employing Ag:CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 51/7/0

Exact mass: 5128.9405

Triply charged mass [M]/3-1.00794, calculated

1708.6389; observed 1708.6487.

LC Trace and Mass of 38

Following General Procedure 2 (Condition A) with 823 and A1 (1000 mM) except for employing Ag₂CO₃ instead of AgTFA.

Yield:—

Ratio (product/deiodination/aryl iodide): 46/12/0

Exact mass: 5144.9176

Triply charged mass [M]/3-1.00794, calculated

1713.9646; observed 1713.9681.

LC Trace and Mass of 39

Following General Procedure 2 (Condition A) with S2 and A23.

Yield:—

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

Exact mass: 5193.0081

Triply charged mass [M]/3-1.00794, calculated

1729.9948; observed 1729.9934.

LC Trace and Mass of 40

Following General Procedure 2 (Condition A) with S5 and A23.

Yield:—

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

Exact mass: 5193.0081

Triply charged mass [M]/3-1.00794, calculated

1729.9948; observed 1729.9934.

LC Trace and Masa of 41

Following General Procedure 2 (Condition A) with 87 and A23.

Yield:—

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

Exact mass: 5178.9925

Triply charged mass [M]/3-1.00794, calculated

1725.3229; observed 1725.3293.

LC Trace and Mass of 42

Following General Procedure 2 (Condition A) with S16 and A23.

Yield:—

Ratio (product/deiodination/aryl iodide): 21/3/25

Exact mass: 5179.9877

Triply charged mass [M]/3-1.00794, calculated

1725.6546; observed 1725.6526.

LC Trace and Mass of 43

Following General Procedure 2 (Condition A) with S20 and A23.

Yield:—

Ratio (product/deiodination/aryl iodide): 21/17/0

Exact mass: 5182.9986

Triply charged mass [M]/3-1.00794, calculated

1726.6583; observed 1726.6564.

LC Trace and Masa of 44

Following General Procedure 2 (Condition A) with S21 and A23.

Yield:—

Ratio (product/deiodination/aryl iodide): 20/12/11

Exact mass: 5182.9986

Triply charged mass [M]/3-1.00794, calculated

1726.6583; observed 1726.6564.

LC Trace and Mass

Following General Procedure 2 (Condition A) with S22 and A23.

Yield:—

Ratio (product/deiodination/aryl iodide): 18/7/0

Exact mass: 5168.9718

Triply charged mass [M]/3-1.00794, calculated

1721.9827; observed 1721.9800.

5. Experimental Section for on-DNA C-S Arylation of Amides

5.1 Substrate Structures of Amides B1-B16

Amides were obtained from the commercial sources or synthesized following the literature procedures.²¹

5.2 Condition Optimizations

TABLE 10
Evaluation of Ag salt
Entry	Ag salt (300 mM)	Yield (%)
1	AgOAc	46
2	AgNO3	12
3	AgTFA	46
4	Ag2CO3	42
5	AgOTs	35

TABLE 11
Evaluation of Base
Entry	Base (150 mM)	Yield (%)
1	NaOAC	46
2	Na2CO3	55
3	K2CO3	41
4	K3PO4	31
5	K2HPO4 • 3H2O	18
6	Li2CO3	69
7	CS2CO3	41

TABLE 12
Evaluation of Standard Conditions
Entry	Deviation from above	Yield (%)
1	none	69
2	without Pd(OAc)2	0
3	without AgOAc	24
4	without Li2CO3	35
5	r.t. instead of 80° C.	60

5.3 General Procedure 3 for on-DNA C—H Arylation of Amides

Materials

DNA-conjugated aryl iodide S: 10 mM in H₂O

Amide B: 2 M in DMA

Pd(OAc)₂: 200 mM in HFIP

Sodium diethyldithiocarbamate trihydrate (scavenger):

1 M in H₂O

Procedure

added Pd(OAc)₂-dry,

AgOAc (300 equiv, 0.5 mg), amide B

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

• • ) were added. The mixture was vortexed. The reaction mixture was heated at 80° C. for 20 hours.scavenger was added, and the mixture was reheated at 80° C. for 30 minutes.
  • 3) Cooling to room temperature, 5 M NaCl so stored in a freezer at −20° C. for more than 30 minutes.

4) Centrifuge the sample for around 10 minutes in a microcentrifuge at 10000 rpm. The above supernatant was discarded and the precipitate was dried under vacuum. The DNA pellet was redissolved in H₂

a microcentrifuge at 10000 rpm. An al was taken and analyzed via HPLC-MS.

5.4 General Procedure 4 for Synthesis of Dipeptides

Materials

68: ca. 5 mM in H₂O

Glycine methyl ester hydrochloride: 1 M in H:0

DMTMM: 1 M in H:0

pH 5.5 phosphate buffer: 0.2 M NaH₂PO₄ in H₂O Procedure

68 solution was added 22

5.5 phosphate buffer, glycine methyl ester

temperature overnight (16-18 hours).

• • 2) Add 5 M NaCl solution (10% by volume) and cold ethanol (2.5 times by volume, ethanol stored at −20° C.). The mixture was stored at in a freezer at −20° C. for 1 hour.
  • 3) Centrifuge the sample for around 10 minutes in a microcentrifuge at 10000 rpm. The above

supernatant was discarded and the precipitate was dried under vacuum. The DNA pellet was redissolved in Hz

in a microcentrifuge at 10000 rpm. An aliquot (50 analyzed via HPLC-MS.

5.5 LC Trace and Mass Characterization of 52-80 LC Trace and Mass of 45

Following General Procedure 3 with Si.

Yield:—

Ratio (product/deiodination/aryl iodide): 59/0/3

Exact mass: 5221.9983

Triply charged mass [M]/3-1.00794, calculated

1739.6582; observed 1739.6648.

Following General Procedure 3 with B1 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 51/4/5

Exact mass: 5221.9983

Triply charged mass [M]/3-1.00794, calculated

1739.6582; observed 1739.6648.

LC Trace and Mass of 46

Following General Procedure 3 with B2.

Yield:—

Ratio (product/deiodination/aryl iodide): 56/3/0

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

Following General Procedure 3 with B2 except for running reaction and quenching with scavenger at room temperature.

Yield:—

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

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

LC Trace and Mass of 47

Following General Procedure 3 with B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 72/2/6

Exact mass: 5262.0296

Triply charged mass [M]/3-1.00794, calculated

1753.0019; observed 1752.9962.

Following General Procedure 3 with B3 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 55/6/28

Exact mass: 5262.0296

Triply charged mass [M]/3-1.00794, calculated

1753.0019; observed 1752.9962.

LC Trace and Mesa of 48

Following General Procedure 3 with B4.

Yield:—

Ratio (product/deiodination/aryl iodide): 29/10/0

Exact mass: 5219.9827

Triply charged mass [M]/3-1.00794, calculated

1738.9863; observed 1738.9816.

Following General Procedure 3 with B4 except for running reaction and quenching with scavenger at room temperature.

Yield:—

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

Exact mass: 5219.9827

Triply charged mass [M]/3-1.00794, calculated

1738.9863; observed 1738.9816.

LC Trace and Ness of 49

Following General Procedure 3 with B5.

Yield:—

Ratio (product/deiodination/aryl iodide): 56/3/4

Exact mass: 5248.0140

Triply charged mass (M)/3-1.00794, calculated

1748.3301; observed 1748.3356.

Following General Procedure 3 with B5 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 35/9/41

Exact mass: 5248.0140

Triply charged mass [M]/3-1.00794, calculated

1748.3301; observed 1748.3356.

LC Trace and Mass of 50

Following General Procedure 3 with 86.

Yield:—

Ratio (product/deiodination/aryl iodide): 48/6/5

Exact mass: 5193.9670

Triply charged mass [M]/3-1.00794, calculated

1730.3144; observed 1730.3170.

Following General Procedure 3 with B6 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 51/1/29

Exact mass: 5193.9670

Triply charged mass [M]/3-1.00794, calculated

1730.3144; observed 1730.3170.

LC Trace and Moss of 51

Following General Procedure 3 with 87.

Yield:—

Ratio (product/deiodination/aryl iodide): 63/3/0

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

Following General Procedure 3 with B7 except for running reaction and quenching with scavenger at room temperature.

Yield:—

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

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

LC Trace and Ness of 52

Following General Procedure 3 with 88.

Yield:—

Ratio (product/deiodination/aryl iodide):72/0/0

Exact mass: 5207.9827

Triply charged mass (M)/3-1.00794, calculated

1734.9863; observed 1734.9877.

Following General Procedure 3 with B8 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide):70/0/16

Exact mass: 5207.9827

Triply charged mass [M]/3-1.00794, calculated

1734.9863; observed 1734.9877.

LC Trace and Mass of 53

Following General Procedure 3 with B9.

Yield:—

Ratio (product/deiodination/aryl iodide): 62/2/0

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

Following General Procedure 3 with B9 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 70/5/9

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

LC Trace and Mass of 54

Following General Procedure 3 with B10.

Yield:—

Ratio (product/deiodination/aryl iodide): 72/2/3

Exact mass: 5250.0296

Triply charged mass [M]/3-1.00794, calculated

1749.0019; observed 1749.0034.

Following General Procedure 3 with B10 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 19/2/55

Exact mass: 5250.0296

Triply charged mass [M]/3-1.00794, calculated

1749.0019; observed 1749.0034.

LC Trace and Mass of 55

Following General Procedure 3 with B11.

Yield:—

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

Exact mass: 5248.0140

Triply charged mass (M)/3-1.00794, calculated

1748.3301; observed 1748.3356.

Following General Procedure 3 with B11 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 40/9/10

Exact mass: 5248.0140

Triply charged mass [M]/3-1.00794, calculated

1748.3301; observed 1748.3356.

LC Trace and Mass of 56

Following General Procedure 3 with B12.

Yield:—

Ratio (product/deiodination/aryl iodide): 63/3/2

Exact mass: 5248.0140

Triply charged mass [M]/3-1.00794, calculated

1748.3301; observed 1748.3356.

Following General Procedure 3 with B12 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 68/6/8

Exact mass: 5248.0140

Triply charged mass [M]/3-1.00794, calculated

1748.3301; observed 1748.3356.

LC Trace and Mass of 57

Following General Procedure 3 with B13.

Yield:—

Ratio (product/deiodination/aryl iodide): 43/6/22

Exact mass: 5272.0140

Triply charged mass [M]/3-1.00794, calculated

1756.3301; observed 1756.33413.

LC Trace and Mass of 58

Following General Procedure 3 with B14.

Yield:—

Ratio (product/deiodination/aryl iodide): 30/2/43

Exact mass: 5291.9593

Triply charged mass [M]/3-1.00794, calculated

1762.9785; observed 1762.9895.

Following General Procedure 3 with 814 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 13/6/58

Exact mass: 5291.9593

Triply charged mass [M]/3-1.00794, calculated

1762.9785; observed 1762.9895.

LC Trace and Moss of 60

Following General Procedure 3 with 816.

Yield:—

Ratio (product/deiodination/aryl iodide): 37/5/2

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

Following General Procedure 3 with B16 except for running reaction and quenching with scavenger at room temperature.

Yield:—

Ratio (product/deiodination/aryl iodide): 54/15/5

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

LC Trace and Ness of 61

Following General Procedure 3 with 87 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 50/2/0

Exact mass: 5262.0296

Triply charged mass [M]/3-1.00794, calculated

1753.0019; observed 1753.0133.

LC Trace and Masa of 62

Following General Procedure 3 with 89 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 55/5/0

Exact mass: 5295.9906

Triply charged mass [M]/3-1.00794, calculated

1764.3223; observed 1764.3309.

LC Trace and Masa of 63

Following General Procedure 3 with 813 and B3.

Yield:—

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

Exact mass: 5280.0202

Triply charged mass [M]/3-1.00794, calculated

1758.9988; observed 1759.0026.

LC Trace and Mass of 64

Following General Procedure 3 with 812 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 52/6/1

Exact mass: 5280.0202

Triply charged mass [M]/3-1.00794, calculated

1758.9988; observed 1759.0026.

LC Trace and Mesa of 65

Following General Procedure 3 with S3 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 50/I/O

Exact mass: 5290.0609

Triply charged mass (M)/3-1.00794, calculated

1762.3457; observed 1762.3534.

LC Trace and Mass of 66

Following General Procedure 3 with S18 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 45/2/0

Exact mass: 5263.0249

Triply charged mass [M]/3-1.00794, calculated

1753.3337; observed 1753.3391.

LC Trace and Masa of 67

Following General Procedure 3 with 815 and 83.

Yield:—

Ratio (product/deiodination/aryl iodide): 42/1/3

Exact mass: 5263.0249

Triply charged mass [M]/3-1.00794, calculated

1753.3337; observed 1753.3391.

LC Trace and Masa of 68

Following General Procedure 3 with 816 and 83.

Yield:—

Ratio (product/deiodination/aryl iodide): 53/2/3

Exact mass: 5263.0249

Triply charged mass [M]/3-1.00794, calculated

1753.3337; observed 1753.3391.

LC Trace and Mass of 69

Following General Procedure 3 with 817 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 56/9/0

Exact mass: 5307.0511

Triply charged mass [M]/3-1.00794, calculated

1768.0091; observed 1768.0140.

LC Trace and Mass of 70

Following General Procedure 3 with 821 and 83.

Yield:—

Ratio (product/deiodination/aryl iodide): 33/16/5

Exact mass: 5266.0358

Triply charged mass (M)/3-1.00794, calculated

1754.3373; observed 1754.3339.

LC Trace and Mass of 71

Following General Procedure 3 with 822 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 39/3/0

Exact mass: 5252.0089

Triply charged mass (M)/3-1.00794, calculated

1749.6617; observed 1749.6714.

LC Trace and Mass of 72

Following General Procedure 3 with S23 and B3.

Yield:—

Ratio (product/deiodination/aryl iodide): 52/3/0

Exact mass: 5267.9860

Triply charged mass [M]/3-1.00794, calculated

1754.9874; observed 1754.9857.

LC Trace and Mass of 73

Following General Procedure 4 with 68.

Yield:—

Exact mass: 5334.0620

Triply charged mass [M]/3-1.00794, calculated

1777.0127; observed 1777.0140.

6. Experimental Section for on-DNA C—H Arylation of Ketones

6.1 Substrate Structures of Ketones Containing Directing Groups (DGs) C1-C30

Ketones containing directing groups C1-C30 were synthesized as previously reported.^16a

6.2 Condition Optimizations

TABLE 13
Ligand Optimization
Entry	Ligand (15 mM)	Yield (%)
1	L6	16
2	L7	32
3	L8	38
4	L9	36
5	L10	32
6	L11	33
7	L12	37
8	L13	27

TABLE 14
Co-solvent Optimization
Entry	H2O/Co-solvent (x/y)	Yield (%)
1	H2O/DMA (1 /1)	28
2	H2O/DMA (2/1)	39
3	H2O/DMF (2/1)	35
4	H2O/DMSO (2/1)	26

TABLE 15
Pd/L8 Concentration Optimization
Entry	Pd/L8 (x/y)	Yield (%)
1	20/10	33
2	20/15	48
3	20/20	48

TABLE 16
Solvent Ratio Optimization
Entry	H2O/DMA (x/y)	Yield (%)
1	4/1	51
2	9/1	62

TABLE 17
Evaluation of Standard Conditions
Entry	Deviation from above	Yield (%)
1	none	62
2	without Pd(OAc)2	0
3	without AgTFA	4
4	without NaOAc	55
5	Without L8	51
6	r.t instead of 80° C.	0
7	Ag2CO3 (150 mM) instead of AgTFA	25
	(250 mM)
8	AgOAC (250 mM) instead of AgTFA	32
	(250 mM)
9	C10 (100 mM) instead of 150 mM	61
10	C10 (200 mM) instead of 150 mM	53
11	C10 (250 mM) instead of 150 mM	54
12	L1 instead of L8	48
13	L5 instead of L8	45
14	Pd(OAC)2/L8 (15/15) instead of	49
	20/20
15	S1b instead of S1a	0a
aOnly S1b was totally recovered.

6.3 General Procedure 5 for on-DNA C—H Arylation of Ketones

Materials

DNA-conjugated aryl iodide S: 10 mM in H₂O

Ketone C: 3 M in DMA (Note: the high concentration may increase the total volume)

L8

Pd(OAc)₂

NaOAc: 1.5 M in H₂O)

Sodium diethyldithiocarbamate trihydrate (scavenger):

1 M in H:0

Procedure

• • 1) To prepared AgTFA (500 equiv, 1.1 mg) was added Pd(OAc): —dry, ketone C DNA-conjugated aryl iodide S (10 nmol, 1 LS (40), NaOAc

The mixture was vortexed.

The reaction mixture was heated at 80° C. for 20 hours. scavenger was added and reheating the mixture at 80° C. for 30 minutes.

• • 3) Cooling to room temperature, 5 M NaCl ored in a freezer at −20=C for more than 30 minutes.
  • 4) Centrifuge the sample for about 7 minutes in a microcentrifuge at 10000 rpm. The supernatant was discarded and the precipitate was dried under vacuum. The DNA pellet was redissolved in H₂) and centrifuged for about 2 minutes in was taken and analyzed via HPLC-MS.

6.4 Limitations of Ketones

6.5 General Procedure 6 for Removal of DG

solution of the DNA-tethered substrate 83 (ca. 10 reaction mixture was subsequently heated at 50° C. for 24 hours.

• • 2) Cooling to room temperature, 5 M NaCl mixture was stored in a freezer at −20° C. for more than 30 minutes.
  • 3) Centrifuge the sample for about 7 minutes in a microcentrifuge at 10000 rpm. The supernatant was discarded and the precipitate was dried under vacuum. The DNA pellet was redissolved in H₂

was taken and analyzed via HPLC-MS.

6.6 LC Trace and Mass Characterization of 81-119

LC Trace and Masa of 74

Following General Procedure 5 with C1.

Yield:—

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

Exact mass: 5252.0453

Triply charged mass [M]/3-1.00794, calculated

1749.6738; observed 1749.6714.

LC Trace and Mass of 75

Following General Procedure 5 with C2.

Yield:—

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

Exact mass: 5239.0296

Triply charged mass [M]/3-1.00794, calculated

1745.0019; observed 1745.0151.

LC Trace and Mass of 76

Following General Procedure 5 with C3.

Yield:—

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

Exact mass: 5252.0453

Triply charged mass [M]/3-1.00794, calculated

1749.6738; observed 1749.6886.

LC Trace and Mass of 77

Following General Procedure 5 with C₄.

Yield:—

Ratio (product/deiodination/aryl iodide): 47/3/0

Exact mass: 5224.0140

Triply charged mass (M)/3-1.00794, calculated

1740.3301; observed 1740.3309.

LC Trace and Masa of 78

Following General Procedure 5 with C5.

Yield:—

Ratio (product/deiodination/aryl iodide): 34/1/22

Exact mass: 5264.0453

Triply charged mass [M]/3-1.00794, calculated

1753.6738; observed 1753.6821.

LC Trace and Mass of 79

Following General Procedure 5 with C6.

Yield:—

Ratio (product/deiodination/aryl iodide): 17/7/39

Exact mass: 5336.0664

Triply charged mass [M]/3-1.00794, calculated

1777.6809; observed 1777.6874.

LC Trace and Mass of 80

Following General Procedure 5 with C7.

Yield:—

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

Exact mass: 5266.0245

Triply charged mass [M]/3-1.00794, calculated

1754.3336; observed 1754.3339.

LC Trace and Masa of 81

Following General Procedure 5 with C8.

Yield:—

Ratio (product/deiodination/aryl iodide): 29/2/8

Exact mass: 5321.0667

Triply charged mass [M]/3-1.00794, calculated

1772.6810; observed 1772.6836.

LC Trace and Masa of 82

Following General Procedure 5 with C9.

Yield:—

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

Exact mass: 5322.0507

Triply charged mass (M)/3-1.00794, calculated

1773.0090; observed 1773.0112.

LC Trace and Masa of 83

Following General Procedure 5 with C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 53/2/5

Exact mass: 5250.0296

Triply charged mass [M]/3-1.00794, calculated

1749.0019; observed 1749.0034.

LC Trace and Mesa

Following General Procedure 5 with C11.

Yield:—

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

Exact mass: 5278.0609

Triply charged mass [M]/3-1.00794, calculated

1758.3457; observed 1758.3500.

LC Trace and Mass

Following General Procedure 5 with C12.

Yield:—

Ratio (product/deiodination/aryl iodide): 17/4/33

Exact mass: 5308.0715

Triply charged mass [M]/3-1.00794, calculated

1768.3492; observed 1768.3584.

LC Trace and Mass of 84

Following General Procedure 5 with C13.

Yield:—

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

Exact mass: 5224.0140

Triply charged mass [M]/3-1.00794, calculated

1740.3301; observed 1740.3309.

LC Trace and Mass

Following General Procedure 5 with C14.

Yield:—

Ratio (product/deiodination/aryl iodide): 18/2/28

Exact mass: 5296.0351

Triply charged mass (M)/3-1.00794, calculated

1764.3371; observed 1764.3481.

LC Trace and Masa of 85

Following General Procedure 5 with C15.

Yield:—

Ratio (product/deiodination/aryl iodide): 33/1/9

Exact mass: 5312.0453

Triply charged mass [M]/3-1.00794, calculated

1769.6738; observed 1769.6847.

LC Trace and Mass of 86

Following General Procedure 5 with C₁₆.

Yield:—

Ratio (product/deiodination/aryl iodide): 38/0/6

Exact mass: 5238.0296

Triply charged mass [M]/3-1.00794, calculated

1745.0019; observed 1744.9980.

LC Trace and Mass of 87

Following General Procedure 5 with C17.

Yield:—

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

Exact mass: 5286.0296

Triply charged mass [M]/3-1.00794, calculated

1761.0019; observed 1761.0127.

LC Trace and Mass

Following General Procedure 5 with C18.

Yield:—

Ratio (product/deiodination/aryl iodide): 17/6/14

Exact mass: 5282.0194

Triply charged mass [M]/3-1.00794, calculated

1759.6652; observed 1759.6725.

LC Trace and Masa of 88

Following General Procedure 5 with C19.

Yield:—

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

Exact mass: 5372.1028

Triply charged mass (M)/3-1.00794, calculated

1789.6930; observed 1789.6906.

LC Trace and Mass of 89

Following General Procedure 5 with D20 except for

employing Pd(OAc): (15 mM) and L8 (15 mM) in H₂0/DMA (2/1).

Yield:—

Ratio (product/deiodination/aryl iodide): 29/2/10

Exact mass: 5328.0766

Triply charged mass [M]/3-1.00794, calculated

1775.0176; observed 1775.0293.

LC Trace and Moss of 90

Following General Procedure 5 with C21 except for employing Pd(OAc): (15 mM) and L8 (15 mM) in H₂O/DMA (2/1).

Yield:—

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

Exact mass: 5264.0453

Triply charged mass [M]/3-1.00794, calculated

1753.6738; observed 1753.6821.

LC Trace and Ness of 91

Following General Procedure 5 with C22.

Yield:—

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

Exact mass: 5276.0453

Triply charged mass (M)/3-1.00794, calculated

1757.6738; observed 1757.6803.

LC Trace and Moss of 92

Following General Procedure 5 with C23.

Yield:—

Ratio (product/deiodination/aryl iodide): 29/1/19

Exact mass: 5302.0609

Triply charged mass (M)/3-1.00794, calculated

1766.3457; observed 1766.3441.

LC Trace and Mesa

Following General Procedure 5 with C24.

Yield:—

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

Exact mass: 5266.0609

Triply charged mass (M)/3-1.00794, calculated

1754.3457; observed 1754.3511.

LC Trace and Mass

Following General Procedure 5 with C25.

Yield:—

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

Exact mass: 5252.0453

Triply charged mass [M]/3-1.00794, calculated

1749.6738; observed 1749.6886.

LC Trace and Mesa of 93

Following General Procedure 5 with C26.

Yield:—

Ratio (product/deiodination/aryl iodide): 19/2/30

Exact mass: 5306.0922

Triply charged mass [M]/3-1.00794, calculated

1767.6895; observed 1767.6869.

LC Trace and Mass

Following General Procedure 5 with 84 and C10.

Yield:—

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

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

LC Trace and Mass

Following General Procedure 5 with 35 and C10.

Yield:—

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

Exact mass: 5250.0296

Triply charged mass [M]/3-1.00794, calculated

1749.0019; observed 1749.0034.

LC Trace and Mass of 94

Following General Procedure 5 with 37 and C10.

Yield:—

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

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

LC Trace and Mesa of 95

Following General Procedure 5 with S9 and C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 26/3/16

Exact mass: 5269.9750

Triply charged mass [M]/3-1.00794, calculated

1755.6504; observed 1755.6549.

LC Trace and Mass of 96

Following General Procedure 5 with 810 and C10.

Yield:—

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

Exact mass: 5236.0140

Triply charged mass [M]/3-1.00794, calculated

1744.3301; observed 1744.3309.

LC Trace and Mass of 97

Following General Procedure 5 with 83 and C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 43/1/4

Exact mass: 5264.0454

Triply charged mass [M]/3-1.00794, calculated

1753.6739; observed 1753.6821.

LC Trace and Masa of 98

Following General Procedure 5 with 314 and C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 36/5/1

Exact mass: 5237.0092

Triply charged mass [M]/3-1.00794, calculated

1744.6618; observed 1744.6731.

LC Trace and Masa of 99

Following General Procedure 5 with S15 and C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 38/3/0

Exact mass: 5237.0092

Triply charged mass [M]/3-1.00794, calculated

1744.6618; observed 1744.6731.

LC Trace and Masa of 100

Following General Procedure 5 with 818 and C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 29/6/2

Exact mass: 5237.0092

Triply charged mass [M]/3-1.00794, calculated

1744.6618; observed 1744.6731.

LC Trace and Mass of 101

Following General Procedure 5 with 816 and C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 29/6/26

Exact mass: 5237.0092

Triply charged mass [M]/3-1.00794, calculated

1744.6618; observed 1744.6731.

LC Trace and Mass

Following General Procedure 5 with 819 and C10 except for employing H₂O/DMR (2/1).

Yield:—

Ratio (product/deiodination/aryl iodide): 13/10/29

Exact mass: 5240.0201

Triply charged mass [M]/3-1.00794, calculated

1745.6654; observed 1745.6653.

LC Trace and lass of 102

Following General Procedure 5 with 323 and C10.

Yield:—

Ratio (product/deiodination/aryl iodide): 28/3/14

Exact mass: 5241.9704

Triply charged mass [M]/3-1.00794, calculated

1746.3155; observed 1746.3156.

LC Trace and lass of 103

Following General Procedure 6 with 83.

Yield:—

Exact mass: 5148.9819

Triply charged mass [M]/3-1.00794, calculated

1715.3194; observed 1715.3248.

7. Representative Synthesis of Compound 106 Step 1: 1^st C (sp³)—H activation

LC Trace and Mass of 31, see above.

• • Step 2: Amidation

LC Trace and Mass of 104

Following General Procedure 4 with Compound 31 and 4-iodobenzyl-amine.

Yield:—

Exact mass: 5354.9160

Triply charged mass [M]/3-1.00794, calculated

1783.9641; observed 1783.9607.

• • Step 3: 2^nd C(sp³)—H activation

LC Trace and Mass of 105

Following General Procedure 5 with 104 and C10.

Yield:—

Exact mass: 5426.1246

Triply charged mass [M]/3-1.00794, calculated

1807.7003; observed 1807.7014.

Step 4: DG removal PGP-371,C3

LC Trace and Mass of 106

Following General Procedure 6 with 105.

Yield:—

Exact mass: 5325.0769

Triply charged mass [M]/3-1.00794, calculated

1774.0177; observed 1774.0288.

CITATIONS

• • 1. Brenner& Lerner, Proc. Natl. Acad. Sci. U.S.A. 89, 5381-5383 (1992).
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Each of the patents, patent applications and articles cited herein is incorporated by reference. The articles “α” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.

Claims

1. A method for preparing an aqueous composition containing a library having a plurality of different bifunctional molecules comprising the steps of: a) reacting in one or more aliquots of an aqueous composition of a bifunctional linker molecule B having termini A′ and C′ according to the formula A′-B-C′, wherein terminus C′ contains a bonded iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms and terminus A′ has an identifier nucleotide precursor, Z′, by palladium-catalyzed arylation via the linker terminal C′ iodo-substituted aromatic ring moiety at a β—C(sp3)—H or γ—C(sp3)—H position of one or more reactant C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone units, X′, wherein a different X′ is present in each aliquot, and adding one or more nucleotide precursors Z‘ to terminus A’ of said linker, a different Z′ being added in each aliquot, to form an aqueous composition containing bifunctional molecules having the formula Znα—A—B—C—Xnα, where n is a position identifier for X and Z and is an integer from 1 to 10, such that when n is 1, X and Z are located most proximal to the linker B, andαidentifies one or more specific reactant C4-C16 aliphatic carboxylic acid, carboxamide or ketone units X, and the corresponding one or more identifying paired DNA sequences of Z in the bifunctional molecule reactant, where each Z′ or Z is paired with and identifies a particular X′ or X, respectively;a′) optionally admixing reacted aqueous aliquot compositions containing approximately equal amounts of bifunctional molecules so formed to form a single composition containing a mixture of said bifunctional molecules;b) reacting the carboxylic acid, carboxamide or ketone functionality present in one or more aliquots of said aqueous composition that contains bifunctional molecules of step a) or a′), Xnα, with one or more iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms, W′, and reacting one or more nucleotide precursor Y′ with Znα to form one or more aqueous compositions containing bifunctional molecules having the formula Ynβ—Znα—A—B—C—Xnα—Wnβ, andb′) optionally admixing reacted aqueous aliquot compositions containing approximately equal amounts of bifunctional molecules so formed to form a single composition containing a mixture of said bifunctional molecules; where each Y′ or Y is paired with and identifies a particular W′ or W, respectively;where Z, A, C, X, Y and W are reacted forms of the corresponding Z′, A′, C′, X′, Y′ and W′;β identifies one or more specific chemical groups W and the corresponding one or more identifying paired DNA sequences of Y in the bifunctional molecule reactant;c) palladium-catalyzed arylating the iodo-substituted aromatic ring moiety, Wnβ, present in the aqueous composition or aliquots thereof formed in step b) to the β—C(sp3)—H or γ—C(sp3)—H position of a C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone unit, V′, and reacting nucleotide precursor T′ with Ynγ to form one or more compositions containing bifunctional molecules having the formula Tnγ—Ynβ—Znα—A—B—C—Xnu—Wnβ—Vnγ; where each T′ or T is paired with and identifies a particular V′ or V, respectively;γidentifies one or more specific C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone units V and the corresponding one or more identifying paired DNA sequences of Y in the bifunctional molecule reactant;wherein at least one of reaction steps a), b) and c) includes a plurality of aliquots of an aqueous composition that contains a bifunctional molecule that individually reacted with a reactant different from that reacted in another aliquot at that step, followed by combining the aliquots produced to form an aqueous composition containing admixture of a plurality of different bifunctional molecules that constitute a library of said bifunctional molecules in an aqueous composition.

2. A method for preparing a library having a plurality of different bifunctional molecules comprising the step d) of recovering said library from said aqueous composition.

3. A library having a plurality of different bifunctional molecules having the formula Tnγ—Ynβ—Znα—A—B—C—Xnα—Wnβ—Vnγwherein B is a linker having reacted termini A and C wherein terminus C contains a bonded iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms and reacted terminus A is a reacted precursor identifier nucleotide, Z,where n is a position identifier for each reacted pair in the bifunctional molecule and is an integer from 1 to 10, such that when n is 1, reacted residues Xnα and Znα are located most proximal to the linker B, αidentifies one or more specific reacted C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone residues, X, and the corresponding one or more paired identifying DNA sequences of Z in the bifunctional molecule,Wnβ is the residue of one or more iodo-substituted aromatic moieties that is bonded to the Xnα residue via said reacted carboxylic acid, carboxamide or masked ketone functionality, and Ynβ is a nucleotide identifier sequence for the one or more specific iodo-substituted aromatic compounds utilized in synthesis, andVnγ is the residue of one or more specific C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone unit residues arylatedly linked at a previously present β—C(sp3)—H or γ—C(sp3)—H position of the carboxylic acid, carboxamide or masked ketone to the Wnp aromatic ring at the position previously occupied iodo group, and Tn7 is a nucleotide identifier sequence of the specific C4-C16 aliphatic carboxylic acid, carboxamide or ketone unit utilized in synthesis,wherein at least one of paired positions (Xnα and Znα), (Wnβ and Ynβ) and (Vnγ and Tnγ) contains a plurality of specific C4-C16 aliphatic carboxylic acid, carboxamide or ketone unit residues and corresponding nucleotide identifier sequence therefor.

4. The library according to claim 3, wherein linker B includes an oligonucleotide that contains the recognition sequence of a predetermined restriction endonuclease.

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

6. The library according to claim 5, wherein each of Tnγ, Ynβ, and Znα is a double stranded DNA oligonucleotide.

7. The library according to claim 6, wherein each of said double stranded DNA oligonucleotides contains two restriction endonuclease recognition sites, one on either side of the specific identifier nucleotide sequence of the reactant used in synthesis.

8. The library according to claim 3, wherein a carboxylic acid, carboxamide or masked ketone arylationally bonded to an aromatic moiety is bonded via a previously present β—C(sp3)—H position relative to the carbonyl group of the or masked ketone.

9. The library according to claim 3, wherein each said aromatic moiety contains a six-membered ring.

10. The library according to claim 3, wherein each said aromatic moiety contains a five-membered ring.

11. The library according to claim 3, wherein each said aromatic moiety is carbocyclic.

12. The library according to claim 3, wherein one of said aromatic moieties is heterocyclic.

13. The library according to claim 3, wherein each said aromatic moiety is carbocyclic.

14. The library according to claim 3, wherein at least one of said C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone contains a 3- or 4-membered ring bonded to the carbonyl group, said 3- or 4-membered ring containing the β—C(sp3)—H at the position of arylation bond formation.

15. A method of carrying out an arylation reaction of a C4-C16 aliphatic carboxylic acid, carboxamide or ketone at a position β— or γ— to the position of the carboxylic acid, carboxamide or masked ketone carbonyl carbon that comprises the steps of dissolving or dispersing in an aqueous medium an iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms, a C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone containing a β—C(sp3)—H or γ—C(sp3)—H, a catalytic amount of a palladium (II) catalyst, a silver salt, a basic alkali metal salt, and an optionally present ligand that interacts with a Pd2 ion in aqueous media to form an aqueous reaction medium, and maintaining said aqueous reaction medium for a period of about 15 to about 30 hours at a temperature of about 70° to about 100° C. to provide an aromatic ring moiety that is free of secondary ring nitrogen atoms C—C-bonded between said ring position formally occupied by said iodo substituent to said former position occupied by said β—C(sp3)—H or γ—C(sp3)—H of said C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone.

16. The method according to claim 15, wherein said C4-C16 aliphatic carboxylic acid, carboxamide or masked ketone containing a β—C(sp3)—H or γ—C(sp3)—H is present in said aqueous reaction medium in a molar excess relative to said iodo-substituted aromatic moiety 10:1 to about 1200:1.

17. The method according to claim 15, wherein said silver salt and said basic alkali metal salt are present aqueous reaction medium at a molar ratio of about 2:1 to about 4:1, and the molar ratio of said silver salt to said iodo-substituted aromatic moiety is about 100:1 to about 400:1.

18. The method according to claim 15, wherein said ligand is present in an amount that is about 1.5 to about 4 times the molar amount of said palladium (II) catalyst.

19. The method according to claim 15, wherein said ligand is selected from the group consisting of one or more of

20. The method according to claim 15, wherein said iodo-substituted aromatic ring moiety that is free of secondary ring nitrogen atoms contains a single 5- or 6-membered aromatic ring, is itself linked to a DNA-containing linker designated “DNA-” and is selected from the group consisting of one or more of

21. The method according to claim 15, wherein said masked ketone containing a β—C(sp3)—H or γ—C(sp3)—H is selected from the group consisting of one or more of

22. The method according to claim 15, wherein said carboxylic acid containing a β—C(sp3)—H or γ—C(sp:3)—H is selected from the group consisting of one or more of