Design, Modeling and Synthesis of 1,2,3-Triazole-Linked Nucleoside-Amino Acid Conjugates as Potential Antibacterial Agents.

Copper-catalyzed azide-alkyne cycloadditions (CuAAC or click chemistry) are convenient methods to easily couple various pharmacophores or bioactive molecules. A new series of 1,2,3-triazole-linked nucleoside-amino acid conjugates have been designed and synthesized in 57-76% yields using CuAAC. The azido group was introduced on the 5'-position of uridine or the acyclic analogue using the tosyl-azide exchange method and alkylated serine or proparylglycine was the alkyne. Modeling studies of the conjugates in the active site of LpxC indicate they have promise as antibacterial agents.


Introduction
Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions, also commonly known as click chemistry, are widely used in a variety of interdisciplinary fields such as organic synthesis, combinational chemistry, drug development, materials science, and bioorthogonal chemistry [1]. They require an azide and an alkyne to form a 1,2,3-triazole ring in the presence of Cu(I) ions. The use of CuAAC in bioorthogonal chemistry offers a multitude of advantages. The necessary alkyne and azide moieties share properties that are ideal under the conditions of bioorthogonal chemistry, such as their small size, stability, and absence within biological systems, preventing the disruption of native chemical reactions and structures within the studied system. The CuAAC reaction has fast kinetics and high yield [1]. The stability of the signature 1,2,3-triazole ring also protects the bioactive conjugates from metabolic degradation and allows for tolerance of a diverse range of reaction conditions, chemical environments, and reagents [1,2].
In addition to facilitating bioorthogonal chemical reactions, a broad assortment of triazole derivatives readily synthesized via CuAAC have shown promising results within the field of medicinal chemistry, with certain triazoles displaying antifungal activity against several Candida species [3]. Additionally, CuAAC has made it possible for researchers to prepare novel phospholipid-protein conjugates with high binding affinity for autoimmune antibodies [4]. Recent studies have described the use of 1,2,3-triazolyl nucleosides and nucleoside analogues as chitin synthase inhibitors [5], phosphoglycosyltransferase inhibitors [6] and anticancer [7][8][9], antiviral [10,11] and antimicrobial agents [12][13][14]. These developments, as well as the results described in this paper, highlight the practicality and versatility of the CuAAC reactions.
Although there have been many great technological advances in the health industry, the treatment of microbial diseases, including bacterial, viral, and parasitic infections, remains a challenge [15].
To further complicate problems, multidrug-resistant strains of Gram-negative bacteria present potential serious health issues [18]. The bacterial resistance and difficulty in treating Gram-negative bacterial infections have prompted researchers to develop novel and effective antibacterial therapies [17].
The outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS). An integral component of LPS is lipid A, which is a glucosamine-based phospholipid. Lipid A anchors LPS to the outer membrane and is essential for the growth and viability of the bacterium [17]. There are nine enzymes involved in the biosynthetic pathway of Lipid A [18]. Among these enzymes is UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC is responsible for catalyzing the first committed step in the synthesis of lipid A [18], and therefore, it is an attractive target for inhibiting the biosynthesis of lipid A, resulting in a compromised outer membrane.
Information from previous inhibitors and the structure of the natural substrate influenced rational design of the conjugates' structures. The natural substrate features three regions: a sugar with a hydrophobic tail (black), a zinc-binding motif (red), and a uracil-based nucleoside (blue) ( Figure 1A). The initial design of the conjugates featured a hydroxamic acid to bind to the zinc ion and a uracil-based nucleoside ( Figure 1B). The hydrophobic moiety was initially omitted for synthetic ease. The analogues are synthesized in two building blocks, the nucleoside block and the hydrophobic moiety with the hydroxamic acid, and then clicked together to form the triazole linkage ( Figure 1B). To further complicate problems, multidrug-resistant strains of Gram-negative bacteria present potential serious health issues [18]. The bacterial resistance and difficulty in treating Gram-negative bacterial infections have prompted researchers to develop novel and effective antibacterial therapies [17]. The outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS). An integral component of LPS is lipid A, which is a glucosamine-based phospholipid. Lipid A anchors LPS to the outer membrane and is essential for the growth and viability of the bacterium [17]. There are nine enzymes involved in the biosynthetic pathway of Lipid A [18]. Among these enzymes is UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC is responsible for catalyzing the first committed step in the synthesis of lipid A [18], and therefore, it is an attractive target for inhibiting the biosynthesis of lipid A, resulting in a compromised outer membrane.
Information from previous inhibitors and the structure of the natural substrate influenced rational design of the conjugates' structures. The natural substrate features three regions: a sugar with a hydrophobic tail (black), a zinc-binding motif (red), and a uracil-based nucleoside (blue) ( Figure 1A). The initial design of the conjugates featured a hydroxamic acid to bind to the zinc ion and a uracilbased nucleoside ( Figure 1B). The hydrophobic moiety was initially omitted for synthetic ease. The analogues are synthesized in two building blocks, the nucleoside block and the hydrophobic moiety with the hydroxamic acid, and then clicked together to form the triazole linkage ( Figure 1B). Herein, we report the design, computational analysis and synthesis of four novel nucleosideamino acid conjugates 1a, 2a, 3 and 4 ( Figure 2) conveniently coupled via CuAAC, positioning a 1,2,3triazole group in the center of the molecule, linking the nucleoside and amino acid. We chose to use amino acids because their properties can be easily modified and the carboxylic acid can be conveniently converted to a hydroxamic acid. Not only does the use of the click reaction provide the above-mentioned advantages, these compounds or their derivatives could be further functionalized to include additional amino acids or groups off the amino group of serine (Ser) or propargylglycine that would further modulate their Herein, we report the design, computational analysis and synthesis of four novel nucleosideamino acid conjugates 1a, 2a, 3 and 4 ( Figure 2) conveniently coupled via CuAAC, positioning a 1,2,3-triazole group in the center of the molecule, linking the nucleoside and amino acid. We chose to use amino acids because their properties can be easily modified and the carboxylic acid can be conveniently converted to a hydroxamic acid. To further complicate problems, multidrug-resistant strains of Gram-negative bacteria present potential serious health issues [18]. The bacterial resistance and difficulty in treating Gram-negative bacterial infections have prompted researchers to develop novel and effective antibacterial therapies [17]. The outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS). An integral component of LPS is lipid A, which is a glucosamine-based phospholipid. Lipid A anchors LPS to the outer membrane and is essential for the growth and viability of the bacterium [17]. There are nine enzymes involved in the biosynthetic pathway of Lipid A [18]. Among these enzymes is UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC is responsible for catalyzing the first committed step in the synthesis of lipid A [18], and therefore, it is an attractive target for inhibiting the biosynthesis of lipid A, resulting in a compromised outer membrane.
Information from previous inhibitors and the structure of the natural substrate influenced rational design of the conjugates' structures. The natural substrate features three regions: a sugar with a hydrophobic tail (black), a zinc-binding motif (red), and a uracil-based nucleoside (blue) ( Figure 1A). The initial design of the conjugates featured a hydroxamic acid to bind to the zinc ion and a uracilbased nucleoside ( Figure 1B). The hydrophobic moiety was initially omitted for synthetic ease. The analogues are synthesized in two building blocks, the nucleoside block and the hydrophobic moiety with the hydroxamic acid, and then clicked together to form the triazole linkage ( Figure 1B). Herein, we report the design, computational analysis and synthesis of four novel nucleosideamino acid conjugates 1a, 2a, 3 and 4 ( Figure 2) conveniently coupled via CuAAC, positioning a 1,2,3triazole group in the center of the molecule, linking the nucleoside and amino acid. We chose to use amino acids because their properties can be easily modified and the carboxylic acid can be conveniently converted to a hydroxamic acid. Not only does the use of the click reaction provide the above-mentioned advantages, these compounds or their derivatives could be further functionalized to include additional amino acids or groups off the amino group of serine (Ser) or propargylglycine that would further modulate their Not only does the use of the click reaction provide the above-mentioned advantages, these compounds or their derivatives could be further functionalized to include additional amino acids or groups off the amino group of serine (Ser) or propargylglycine that would further modulate their activity or properties. The modeling studies confirmed that these conjugates bind in the active site of LpxC and provided further insight into which conjugates show the most promise as inhibitors of LpxC.

Computational
The triazole-linked conjugates were optimized in the LpxC active site using the M06-L [19] density functional theory methods with the 6-31G [20] basis set. The ligands were placed in a truncated active site (His265, Gly264, His58, Phe192, Phe194, Glu197, and Lys239) in an initial configuration similar to the natural substrate (from the crystal structure) [21]. The positions of the ligands and amino acid residue side chains were optimized in both a vacuum model and a solvated model. For the solvated model, we used implicit solvation with water via the PCM using default parameters [22]. Counterpoise-corrected [23] electronic interaction energies were calculated for the ligand and Zn 2+ and the ligand and all amino acid residues in both conditions using M06-L and the 6-311+G* basis set.
The results of the counterpoise-corrected electronic interaction energies of compounds 1b, 2b and 3 in the vacuum model are shown in Table 1. Negative numbers indicate attractive forces. Conjugates 1b and 2b were evaluated as hydroxamic acids to ensure the strongest interaction with the zinc ion. Nonetheless, in the vacuum model, conjugate 3 shows the most favorable interaction energy in the active site, with a large percentage of the total interaction energy coming from the interaction between the ligand and the zinc ion. Compounds 1b and 2b were designed to have added flexibility to adopt the optimal conformation and binding within the LpxC active site. However, there is a possibility the compounds had too much rotational freedom, resulting in lower interaction energies. Due to the less favorable computational results of compounds 1b and 2b, they were not investigated further. The results of the counterpoise-corrected interaction energies of the solvated model are summarized in the third and fourth columns of Table 1. The solvated model can be considered a more realistic model, as it is more representative of the conditions in vivo. Due to the favorability of compound 3 in the vacuum model, only 3 and 4 were investigated in the solvated model. It was concluded that 3 exhibits the strongest interaction energy because of the hydroxamic acid oxygen interacting with the backbone NH 2 of the histidine residue. These computational studies suggest that 3 and 4 may act as inhibitors of LpxC, where a nitrogen in the triazole ring provides an additional point of interaction with zinc.
The optimized complexes are shown in Figure 3. Since the Zn 2+ ion in the active site has a positive charge, it can bind with any portion of the substrate that is partially negative. The ligand binds Zn 2+ using the oxygens of the hydroxamic acid, producing a very strong attractive interaction. However, our results showed the triazole group also binds well to zinc, which may strengthen binding in the active site. All four conjugates bound Zn 2+ through the hydroxamic acid oxygens and with a nitrogen in the triazole ring. Lys239 and His58 have strong attractive interactions with conjugate 4. This is because the ligand has a −1 charge and both lysine and histidine are positively charged. The glutamate residues in the active site have strong repulsive interactions with 4 since glutamate is negatively charged, as is compound 4.

Synthesis
To synthesize the acyclic nucleoside 5, uracil was reacted with 1,3-dioxolane according to the published procedure [24] to produce 5 in a 33% yield, (Scheme 1). The acyclic nucleoside 5 was tosylated in 6, followed by exchanging the tosylate for an azide 7 (Scheme 1) [25]. In our hands, tosylation of the acyclic nucleoside analogue proved to be difficult. Various attempts to synthesize 6 are depicted in Supplementary Table S1. A review of the literature for more successful methods was unproductive [25][26][27][28][29]. Both tosyl anhydride (Ts2O) or tosyl chloride (TsCl) have been used, as well as the presence or absence of an aqueous workup. Pure tosylate 6 was obtained with a yield of 23% and a clean 1 H-NMR. The azide reaction proceeded with success to about 70% yield. Similar reactions have been perfomed resulting in higher yields [29]. However, differences in solubility and polarity of their 5,6-substituted uracil analogue likely played a role in the inconsistency in their reported yield and what we obtained. Synthesis of the acyclic nucleoside-Ser conjugate is illustrated in Scheme 2. Commercially available Boc-L-Ser was alkylated with bromobutyne to produce 8 (Scheme 2). The alkylation reaction was first

Synthesis
To synthesize the acyclic nucleoside 5, uracil was reacted with 1,3-dioxolane according to the published procedure [24] to produce 5 in a 33% yield, (Scheme 1). The acyclic nucleoside 5 was tosylated in 6, followed by exchanging the tosylate for an azide 7 (Scheme 1) [25]. In our hands, tosylation of the acyclic nucleoside analogue proved to be difficult. Various attempts to synthesize 6 are depicted in Supplementary Table S1. A review of the literature for more successful methods was unproductive [25][26][27][28][29]. Both tosyl anhydride (Ts 2 O) or tosyl chloride (TsCl) have been used, as well as the presence or absence of an aqueous workup. Pure tosylate 6 was obtained with a yield of 23% and a clean 1 H-NMR. The azide reaction proceeded with success to about 70% yield. Similar reactions have been perfomed resulting in higher yields [29]. However, differences in solubility and polarity of their 5,6-substituted uracil analogue likely played a role in the inconsistency in their reported yield and what we obtained.

Synthesis
To synthesize the acyclic nucleoside 5, uracil was reacted with 1,3-dioxolane according to the published procedure [24] to produce 5 in a 33% yield, (Scheme 1). The acyclic nucleoside 5 was tosylated in 6, followed by exchanging the tosylate for an azide 7 (Scheme 1) [25]. In our hands, tosylation of the acyclic nucleoside analogue proved to be difficult. Various attempts to synthesize 6 are depicted in Supplementary Table S1. A review of the literature for more successful methods was unproductive [25][26][27][28][29]. Both tosyl anhydride (Ts2O) or tosyl chloride (TsCl) have been used, as well as the presence or absence of an aqueous workup. Pure tosylate 6 was obtained with a yield of 23% and a clean 1 H-NMR. The azide reaction proceeded with success to about 70% yield. Similar reactions have been perfomed resulting in higher yields [29]. However, differences in solubility and polarity of their 5,6-substituted uracil analogue likely played a role in the inconsistency in their reported yield and what we obtained. Synthesis of the acyclic nucleoside-Ser conjugate is illustrated in Scheme 2. Commercially available Boc-L-Ser was alkylated with bromobutyne to produce 8 (Scheme 2). The alkylation reaction was first Scheme 1. Synthesis of the azido acyclic nucleoside 7.
Synthesis of the acyclic nucleoside-Ser conjugate is illustrated in Scheme 2. Commercially available Boc-L-Ser was alkylated with bromobutyne to produce 8 (Scheme 2). The alkylation reaction was first attempted using NaH as the base, producing 8 in 10-23% yields [30]. Due to the poor yields and inconsistent purity of the compound, a different method was sought. Using K 2 CO 3 as a base, 8 was obtained in a yield of 38% [31]. attempted using NaH as the base, producing 8 in 10-23% yields [30]. Due to the poor yields and inconsistent purity of the compound, a different method was sought. Using K2CO3 as a base, 8 was obtained in a yield of 38% [31].
CuAAC was used to create the 1,2,3-triazole linkage to couple the nucleoside analogue and amino acid. Azide 7 and alkyne 8 were coupled together to form 1a [32]. Despite the difficulty in preparation of the azide and alkyne, the click reaction proceeded with ease, providing 1a in a 69% yield. Silica gel column chromatography eluted a pure compound, despite the impurities present in the azide.
The remaining conjugates 2-4 were synthesized from the protected 5′-azido uridine 11, which was synthesized in three steps (Scheme 3). Beginning with a ketal protection of the 2′-and 3′-hydroxyl groups on commercially available uridine to produce 9 (Scheme 3) [33]. The ketal production proceeded with ease, attaining a high yield of 91%. Following the protection, a tosylation reaction was performed to activate the 5′-hydroxyl group, producing 11 (Scheme 3) [25]. Once again, the tosylation reaction proceeded with some difficulty. There are literature procedures describing the use of either Ts2O or TsCl, both of which have been used with pyridine, as well as in the presence or absence of dichloromethane [25,34]. Various reaction conditions were attempted to determine the optimal conditions to produce a pure product in a high yield, including running the reaction for 2-4 h or overnight, at room temperature or slightly heated. Despite the literature [25] reporting a 98% yield on the formation of the tosylate, in our hands a maximum of 77% yield has been obtained with Ts2O and dichloromethane under a 2 h reflux. The tosyl group in 11 was replaced by an azide group in accordance with the literature procedure [25] (Scheme 3). The yields for this reaction are comparable to the literature.  CuAAC was used to create the 1,2,3-triazole linkage to couple the nucleoside analogue and amino acid. Azide 7 and alkyne 8 were coupled together to form 1a [32]. Despite the difficulty in preparation of the azide and alkyne, the click reaction proceeded with ease, providing 1a in a 69% yield. Silica gel column chromatography eluted a pure compound, despite the impurities present in the azide.
The remaining conjugates 2-4 were synthesized from the protected 5 -azido uridine 11, which was synthesized in three steps (Scheme 3). Beginning with a ketal protection of the 2 -and 3 -hydroxyl groups on commercially available uridine to produce 9 (Scheme 3) [33]. The ketal production proceeded with ease, attaining a high yield of 91%. Following the protection, a tosylation reaction was performed to activate the 5 -hydroxyl group, producing 11 (Scheme 3) [25]. Once again, the tosylation reaction proceeded with some difficulty. There are literature procedures describing the use of either Ts 2 O or TsCl, both of which have been used with pyridine, as well as in the presence or absence of dichloromethane [25,34]. Various reaction conditions were attempted to determine the optimal conditions to produce a pure product in a high yield, including running the reaction for 2-4 h or overnight, at room temperature or slightly heated. Despite the literature [25] reporting a 98% yield on the formation of the tosylate, in our hands a maximum of 77% yield has been obtained with Ts 2 O and dichloromethane under a 2 h reflux. The tosyl group in 11 was replaced by an azide group in accordance with the literature procedure [25] (Scheme 3). The yields for this reaction are comparable to the literature. attempted using NaH as the base, producing 8 in 10-23% yields [30]. Due to the poor yields and inconsistent purity of the compound, a different method was sought. Using K2CO3 as a base, 8 was obtained in a yield of 38% [31].
CuAAC was used to create the 1,2,3-triazole linkage to couple the nucleoside analogue and amino acid. Azide 7 and alkyne 8 were coupled together to form 1a [32]. Despite the difficulty in preparation of the azide and alkyne, the click reaction proceeded with ease, providing 1a in a 69% yield. Silica gel column chromatography eluted a pure compound, despite the impurities present in the azide.
The remaining conjugates 2-4 were synthesized from the protected 5′-azido uridine 11, which was synthesized in three steps (Scheme 3). Beginning with a ketal protection of the 2′-and 3′-hydroxyl groups on commercially available uridine to produce 9 (Scheme 3) [33]. The ketal production proceeded with ease, attaining a high yield of 91%. Following the protection, a tosylation reaction was performed to activate the 5′-hydroxyl group, producing 11 (Scheme 3) [25]. Once again, the tosylation reaction proceeded with some difficulty. There are literature procedures describing the use of either Ts2O or TsCl, both of which have been used with pyridine, as well as in the presence or absence of dichloromethane [25,34]. Various reaction conditions were attempted to determine the optimal conditions to produce a pure product in a high yield, including running the reaction for 2-4 h or overnight, at room temperature or slightly heated. Despite the literature [25] reporting a 98% yield on the formation of the tosylate, in our hands a maximum of 77% yield has been obtained with Ts2O and dichloromethane under a 2 h reflux. The tosyl group in 11 was replaced by an azide group in accordance with the literature procedure [25] (Scheme 3). The yields for this reaction are comparable to the literature. Alkyne 8 and azide 11 were clicked together to form the uridine-Ser conjugate 2a as shown in Scheme 4. Initially the reaction ran 48 h with a copper wire-wrapped stir bar to provide product 2a in 45% yield [32]. However, when Cu powder was used, the reaction yield improved significantly to 76%. Due to the synthetic challenges observed in the preparation of 1a and 2a, further deprotection and conversion to the hydroxamic acid was not pursued. Alkyne 8 and azide 11 were clicked together to form the uridine-Ser conjugate 2a as shown in Scheme 4. Initially the reaction ran 48 h with a copper wire-wrapped stir bar to provide product 2a in 45% yield [32]. However, when Cu powder was used, the reaction yield improved significantly to 76%. Due to the synthetic challenges observed in the preparation of 1a and 2a, further deprotection and conversion to the hydroxamic acid was not pursued. Following the challenges with alkylating Boc-L-Ser, we envisioned the use of propargylglycine to bypass the need for the alkylation reaction. Therefore, commercially available L-propargylglycine was used as the amino acid to click to the nucleoside. The use of propargylglycine provides significant advantages in that it allows further functionalization of the amino acid, including the convenient coupling of another amino acid or any other pharmacophore on either the nitrogen or carboxylic acid and the conversion to a hydroxamic acid or ester.
The synthesis of the uridine-propargylglycine conjugates began with the conversion of the carboxylic acid of L-propargylglycine to the protected hydroxamic acid intermediates 12 and 13. Compound 12 was successfully synthesized with an 82% yield using commercially available Boc-Lpropargylglycine and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (THP-hydroxylamine), 2-chloro-4,6dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) (Scheme 5) [35]. Commercially available Fmoc-L-propargylglycine was initially converted to the protected hydroxamic acid 13 using CDMT and NMM [35]. However, when standard dicyclohexylcarbodiimide (DCC) coupling was used, purification was easier and higher yields were obtained. Next, the Fmoc protecting group was removed using a 20% solution of piperidine in dimethylformamide (DMF), and immediately coupled with biphenyl-4-carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce 14 in a 35% yield over the two steps (Scheme 5) [36]. Not surprisingly, the use of diethylamine and longer reaction times during the Fmoc deprotection resulted in lower yields and racemization.  Following the challenges with alkylating Boc-L-Ser, we envisioned the use of propargylglycine to bypass the need for the alkylation reaction. Therefore, commercially available L-propargylglycine was used as the amino acid to click to the nucleoside. The use of propargylglycine provides significant advantages in that it allows further functionalization of the amino acid, including the convenient coupling of another amino acid or any other pharmacophore on either the nitrogen or carboxylic acid and the conversion to a hydroxamic acid or ester.
The synthesis of the uridine-propargylglycine conjugates began with the conversion of the carboxylic acid of L-propargylglycine to the protected hydroxamic acid intermediates 12 and 13. Compound 12 was successfully synthesized with an 82% yield using commercially available Boc-L-propargylglycine and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (THP-hydroxylamine), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) (Scheme 5) [35]. Commercially available Fmoc-L-propargylglycine was initially converted to the protected hydroxamic acid 13 using CDMT and NMM [35]. However, when standard dicyclohexylcarbodiimide (DCC) coupling was used, purification was easier and higher yields were obtained. Next, the Fmoc protecting group was removed using a 20% solution of piperidine in dimethylformamide (DMF), and immediately coupled with biphenyl-4-carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce 14 in a 35% yield over the two steps (Scheme 5) [36]. Not surprisingly, the use of diethylamine and longer reaction times during the Fmoc deprotection resulted in lower yields and racemization. Alkyne 8 and azide 11 were clicked together to form the uridine-Ser conjugate 2a as shown in Scheme 4. Initially the reaction ran 48 h with a copper wire-wrapped stir bar to provide product 2a in 45% yield [32]. However, when Cu powder was used, the reaction yield improved significantly to 76%. Due to the synthetic challenges observed in the preparation of 1a and 2a, further deprotection and conversion to the hydroxamic acid was not pursued. Following the challenges with alkylating Boc-L-Ser, we envisioned the use of propargylglycine to bypass the need for the alkylation reaction. Therefore, commercially available L-propargylglycine was used as the amino acid to click to the nucleoside. The use of propargylglycine provides significant advantages in that it allows further functionalization of the amino acid, including the convenient coupling of another amino acid or any other pharmacophore on either the nitrogen or carboxylic acid and the conversion to a hydroxamic acid or ester.
The synthesis of the uridine-propargylglycine conjugates began with the conversion of the carboxylic acid of L-propargylglycine to the protected hydroxamic acid intermediates 12 and 13. Compound 12 was successfully synthesized with an 82% yield using commercially available Boc-Lpropargylglycine and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (THP-hydroxylamine), 2-chloro-4,6dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) (Scheme 5) [35]. Commercially available Fmoc-L-propargylglycine was initially converted to the protected hydroxamic acid 13 using CDMT and NMM [35]. However, when standard dicyclohexylcarbodiimide (DCC) coupling was used, purification was easier and higher yields were obtained. Next, the Fmoc protecting group was removed using a 20% solution of piperidine in dimethylformamide (DMF), and immediately coupled with biphenyl-4-carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce 14 in a 35% yield over the two steps (Scheme 5) [36]. Not surprisingly, the use of diethylamine and longer reaction times during the Fmoc deprotection resulted in lower yields and racemization. Following preparation of the appropriate alkynes 12 and 14, they were coupled to azide 11 via a Cu-catalyzed cycloaddition [32] (Scheme 6). The click reaction was first sonicated for 10-15 min before stirring overnight and proceeded in 76 and 57% yields. Finally, the Boc group was removed (3) and the 2 -and 3 -hydroxyl groups were deprotected with trifluoroacetic acid (TFA) to yield 3 and 4 in an 83% and 70% yield, respectively (Scheme 6). The final products were precipitated from a methanolic solution using diethyl ether. The final conjugates 3 and 4 were characterized by 1 H-and 13 C-NMR and HRMS. The purity was confirmed by HPLC.
Following preparation of the appropriate alkynes 12 and 14, they were coupled to azide 11 via a Cu-catalyzed cycloaddition [32] (Scheme 6). The click reaction was first sonicated for 10-15 min before stirring overnight and proceeded in 76 and 57% yields. Finally, the Boc group was removed (3) and the 2′-and 3′-hydroxyl groups were deprotected with trifluoroacetic acid (TFA) to yield 3 and 4 in an 83% and 70% yield, respectively (Scheme 6). The final products were precipitated from a methanolic solution using diethyl ether. The final conjugates 3 and 4 were characterized by 1 H-and 13 C-NMR and HRMS. The purity was confirmed by HPLC.

General Methods
Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-Seal bottles under argon. All other reagents and solvents were purchased as the highest grade available from Acros (Fisher Scientific, Pittsburgh, PA, USA) or Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification. Boc-L-Propargylglycine was purchased from Aurum Pharmatech (Franklin Park, NJ, USA) and Fmoc-L-propargylglycine was purchased from AK Scientific (Union City, CA, USA). All moisture-sensitive reactions were carried out using dry solvents Following preparation of the appropriate alkynes 12 and 14, they were coupled to azide 11 via a Cu-catalyzed cycloaddition [32] (Scheme 6). The click reaction was first sonicated for 10-15 min before stirring overnight and proceeded in 76 and 57% yields. Finally, the Boc group was removed (3) and the 2′-and 3′-hydroxyl groups were deprotected with trifluoroacetic acid (TFA) to yield 3 and 4 in an 83% and 70% yield, respectively (Scheme 6). The final products were precipitated from a methanolic solution using diethyl ether. The final conjugates 3 and 4 were characterized by 1 H-and 13 C-NMR and HRMS. The purity was confirmed by HPLC.

General Methods
Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-Seal bottles under argon. All other reagents and solvents were purchased as the highest grade available from Acros (Fisher Scientific, Pittsburgh, PA, USA) or Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification. Boc-L-Propargylglycine was purchased from Aurum Pharmatech (Franklin Park, NJ, USA) and Fmoc-L-propargylglycine was purchased from AK Scientific (Union City, CA, USA). All moisture-sensitive reactions were carried out using dry solvents

General Methods
Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-Seal bottles under argon. All other reagents and solvents were purchased as the highest grade available from Acros (Fisher Scientific, Pittsburgh, PA, USA) or Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification. Boc-L-Propargylglycine was purchased from Aurum Pharmatech (Franklin Park, NJ, USA) and Fmoc-L-propargylglycine was purchased from AK Scientific (Union City, CA, USA). All moisture-sensitive reactions were carried out using dry solvents and under slight pressure of ultra-pure argon. Commercially available disposable syringes were used for transferring reagents and solvents. All single syntheses were conducted in conventional flasks under an atmosphere of dry argon. Proton ( 1 H) and carbon ( 13 C) NMR spectra were recorded on a 400 MHz spectrometer (Varian, Palo Alto, CA USA). NMR data are reported as follows: chemical shifts (δ) are reported in parts per million (ppm) referenced to 1 H (CDCl 3 at 7.27, CD 3 OD at 3.31, DMSO-d 6 at 2.50), 13 C (CDCl 3 at 77.16, CD 3 OD at 49.00, DMSO-d 6 at 39.52), multiplicity (s = singlet, br s = broad singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, quin = quintet, m = multiplet) and coupling constants (J) are reported in Hz. Column chromatography was conducted using silica gel (Silicycle 55-65 Å). Purity of the compounds was confirmed to be greater than 95% via HPLC analysis (LC-6AD pumps, detection with a SPD-M20A PDA and CBM-20A communication, Shimadzu, Columbia, MD, USA) and a Hypersil Gold C18 column, (250 × 4.6 mm, particle size = 5 µm, Thermo Fisher Scientific, Waltham, MA, USA). High resolution mass spectra (HRMS) were obtained in the University of California Riverside High Resolution Mass Spectrometry facility using +ESI.

Computational Methods
Starting from the LpxC crystal structure with the natural substrate bound in the active site (PDB ID:2IER) [21], proposed analogues were placed in the active site in a similar configuration to the natural substrate. Optimizations were completed using M06-L/6-31G. M06-L was used since it is effective for intermolecular forces and interactions with metals [19]. A relaxed active site where the heavy atoms on the amino acid side chains and all protons were allowed to adjust their positions was used. Optimizations with implicit solvation were done via the PCM model using default parameters. The solvent in these cases was water. Interaction energies were also calculated from the solvent optimized structures. In all cases, counterpoise corrected interaction energies were calculated for the ligand/amino acid pairs and ligand/Zn 2+ using M06-L/6-311+G*. Calculations were performed using Gaussian09 [37].  . The alkyne container was rinsed with CH 3 CN (0.5 mL) and added to the reaction flask. To the flask was added DI water (0.4 mL, 1.74 mmol/mL) and Cu powder (9 mg, 0.14 mmol). The reaction was ultra-sonicated for 10 min before being stirred at 35 • C overnight. At 21 h, a second addition of Cu powder (9 mg, 0.14 mmol) was added. The reaction flask was ultra-sonicated for 10 min and stirred at 35 • C. At 24 h, the reaction was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (CH 2 Cl 2 :MeOH, 5%) to afford the desired product 1 as a white solid (112 mg, 69%). R f (9:1 CH 2 Cl 2 -MeOH) = 0. 38