Model Substrate/Inactivation Reactions for MoaA and Ribonucleotide Reductases: Loss of Bromo, Chloro, or Tosylate Groups from C2 of 1,5-Dideoxyhomoribofuranoses upon Generation of an α-Oxy Radical at C3 ^†

Abstract

We report studies on radical-initiated fragmentations of model 1,5-dideoxyhomoribofuranose derivatives with bromo, chloro, and tosyloxy substituents on C2. The effects of stereochemical inversion at C2 were probed with the corresponding arabino epimers. In all cases, the elimination of bromide, chloride, and tosylate anions occurred when the 3-hydroxyl group was unprotected. The isolation of deuterium-labeled furanone products established heterolytic cleavage followed by the transfer of deuterium from labeled tributylstannane. In contrast, 3-O-methyl derivatives underwent the elimination of bromine or chlorine radicals to give the 2,3-alkene with no incorporation of label in the methyl vinyl ether. More drastic fragmentation occurred with both of the 3-O-methyl-2-tosyloxy epimers to give an aromatized furan derivative with no deuterium label. Contrasting results observed with the present anhydroalditol models relative to our prior studies with analogously substituted nucleoside models have demonstrated that insights from biomimetic chemical reactions can provide illumination of mechanistic pathways employed by ribonucleotide reductases (RNRs) and the MoaA enzyme involved in the biosynthesis of molybdopterin.

1. Introduction

Ribonucleotide reductases (RNRs) effect conversion of nucleoside 5′-(di- or tri)phosphates into their 2′-deoxy counterparts and thereby provide the only de novo access to the monomeric precursors for DNA replication and repair [1]. Depletion of these crucial deoxynucleotide pools by inhibition of RNRs presents an inviting approach for rational drug design [2,3]. Mechanism-based inactivation of RNRs has been observed with 2′-chloro [4] and 2′-azido [5,6,7,8] analogues of pyrimidine nucleoside 5′-(di- or tri)phosphates, and the potent inactivator 2′-deoxy-2′,2′-difluorocytidine (gemcitabine) is a primary drug for the treatment of pancreatic and non-small cell lung cancers [2,3,9,10].

Abstraction of H3′ from the ribosyl moiety of the substrate in A (Figure 1, X = OH) by a thiyl radical (•SCys439) to give B (X = OH) is the postulated initial step of the RNR-catalyzed deoxygenation of ribonucleotides [1]. Enzyme-assisted loss of water from C2′ in B would produce the 3′-keto C2′ radical in C, and further electron and hydrogen-atom transfers via D and E would complete the conversion of substrate into the product 2′-deoxynucleotide in F [11,12].

Abstraction of H3′ from a 2′-chloro-2′-deoxynucleotide inactivator was proposed to convert A (X = Cl) to B (X = Cl). Spontaneous loss of chloride in B (X = Cl) followed by hydrogen transfer from the thiol to C2′ in C and electron transfer to the dithiol would produce the identical 3′-keto-2′-deoxynucleotide in D as with the substrate nucleotide [1,4,6]. Changes at the active site caused by the loss of Cl⁻ and H⁺ from the inactivator (rather than enzyme-assisted removal of H₂O from the substrate) were invoked to rationalize its dissociation from D of the enzyme. Successive β-eliminations of B(ase) and pyrophosphate would generate 2-methylene-3(2H)-furanone (G), a Michael acceptor, which could affect covalent alkylating of the RNR and cause mechanism-based inactivation. Incubation of gemcitabine 5′-diphosphate with RDPRs (ribonucleoside diphosphate reductases) caused inactivation of both of the R1 and R2 subunits [13,14,15,16,17].

The initial mechanistic steps are compatible with theoretical modeling studies [18,19], with the biomimetic chemical experiments reported by Giese [20] and Robins [21,22], and with Stubbe’s enzymatic studies with gemcitabine [13,14,15,16,17] and 2′-deoxy-2′-fluoromethylenecytidine [23,24]. However, the detection of ribosyl-based radicals during RNR-catalyzed deoxygenation of substrates remained elusive [25]. Two sets of EPR signals were observed during kinetic studies with the E441Q mutant of E. coli class I RDPR and cytidine 5′-diphosphate [26]. Signals attributed to the initially detected radical were consistent with those from a disulfide radical anion [27], and those from the second were compatible with appearance of a nucleotide-based radical of the semidione type [28]. The latter-type of signals was observed during the inactivation of RNR by gemcitabine nucleotides [16].

Giese [20] and Robins [21,22] designed chemical models to simulate such initiation/elimination cascades that begin with the generation of a C3′ radical during the reduction and mechanism-based inactivation mediated by RDPRs. Lenz and Giese employed photochemical fragmentation of adenosine 3′-selenoesters in aqueous methanol to generate C3′ radicals containing a 3′-hydroxyl group. The addition of an acetate buffer to the photolysis solution enhanced the rate of cleavage of water from C2′ as predicted for base-promoted assistance by an enzymatic carboxylate group [20]. Robins et al. designed 6′-O-nitro-2′-substituted homonucleoside derivatives that produced 6′-oxyl radicals upon treatment with tributylstannane/AIBN. The O6′ radicals were positioned to abstract H3′ with the generation of C3′ radicals containing a 3′-hydroxyl group. Fragmentation of a 2′-O-tosyl derivative occurred by anionic elimination [29], whereas a 2′-chloro analogue fragmented by a radical elimination pathway [30]. Nucleoside derivatives with a 2′-(azido, bromo, chloro, iodo, or methylthio) group also underwent elimination of a radical species upon generation of a radical center at C3′ [31].

Begley recently reported trapping of intermediates in the MoaA-catalyzed biosynthesis of molybdopterin using 2′-deoxyguanosine 5′-triphosphate and its 2′-chloro-2′-deoxy derivative as alternative substrate analogs [32]. A 5′-deoxyadenosyl radical cofactor abstracts H3′ from the GTP substrate (and also from the 2′-chloro ribo analog) with generation of C3′ radicals containing a 3′-hydroxyl group. The homolytic elimination of chlorine from C2′ would give the 2′,3′-enol, and β-eliminations of triphosphate and guanine would produce 2-methylene-3(2H)furanone (G, Figure 1) as described by us in 1996 [30].

Herein, we report that 1,5-dideoxyhomoribofuranose derivatives containing a 3-hydroxyl group with a bromo, chloro, or tosylate substituent at C2 fragment with loss of bromide, chloride, or tosylate anions upon generation of a radical center at C3. The ionic fragmentations of both ribo and arabino epimers were essentially equivalent. These model reactions provide additional experimental data that allow further illumination of mechanisms employed by ribonucleotide reductases [18,19] and the MoaA enzyme [32] involved in molybdopterin biosynthesis.

2. Results

2.1. Synthesis of 1,4-Anhydrohexitol Models

Methyl 5-deoxy-2,3-O-isopropylidene-β-D-ribo-hexofuranoside (1, Scheme 1) was prepared efficiently in 7 steps (~40% overall yield) from 1,2-O-isopropylidene-α-D-glucose [29]. Benzoylation of 1 and anomeric deoxygenation of 2 (BF₃•OEt₂/Et₃SiH) [33,34] gave a mixture of 4 and 3 (partial loss of the isopropylidene group). Reprotection of 3 gave the 1,4-anhydroalditol [35] 4 (44% overall from 1). Successive debenzoylation of 4, iodination of 5, and deprotection of 6 gave 6-iodo furanitol 7 (53% from 4). Nitrate displacement of iodide from 7 (AgNO₃/CH₃CN) gave 1,4-anhydro-5-deoxy-6-O-nitro-D-ribo-hexitol (8, 89%). Tin-mediated tosylation [36] of 8 gave a regioisomeric mixture from which the major 2-O-tosyl isomer 9 (48%) was isolated.

Tin-mediated benzylation of 8 with p-methoxybenzyl (PMB) chloride gave a mixture from which the 3-O-PMB regioisomer 10 (Scheme 2) was isolated. Tosylation of 10 followed by removal of the PMB group from 15 (ceric ammonium nitrate (CAN)) gave an alternative route to 9 (54% from 10). Mitsunobu chlorination of 10 (freshly prepared HCl•pyridine) gave the protected 2-chloro-arabino-hexofuranose 11 (62%). Analogous bromination of 10 with freshly prepared HBr•pyridine gave the 2-bromo product 12 (49%). Debenzylation of 11 and 12 (CAN) gave 13 (78%) and 14 (79%), respectively.

The 2-chloro and 2-bromo-D-ribo-hexofuranoses 20 and 22 (Scheme 2) were prepared from 10 by double inversion. Mitsunobu treatment of 10 (BzOH) produced the 2-O-benzoyl-D-arabino-hexofuranose 16a. Debenzoylation of 16a and treatment of 16b with TfCl/DMAP gave the reasonably stable triflate 17b. Displacement of triflate (LiCl or LiBr) and debenzylation (CAN) of the resulting 19 or 21 gave the 2-chloro or 2-bromo-D-ribo-hexofuranose 20 or 22. Tosylation of 16b followed by debenzylation of 17a gave the 2-O-tosyl-arabino product 18.

Tin-mediated methylation of 8 produced 23 and 24 (Scheme 3) and the major 3-O-methyl isomer 23 (38%) was isolated and tosylated to give the ribo tosylate 25. Mitsunobu treatment of 23 (BzOH), debenzoylation of 26, and tosylation of 27 gave arabino tosylate 30. Mitsunobu halogenations of 23 gave the 3-O-methyl-D-arabino 2-chloro 28 and 2-bromo 29 analogues. Triflation of 27 and displacement of triflate from 31 with LiCl or LiBr gave the ribo 2-chloro 32 and 2-bromo 33 compounds.

2.2. Radical Generation Studies

Treatment of the 6-O-nitro-2-O-tosyl ribo compound 9 with Bu₃SnH/AIBN/toluene/95 °C for 1 h gave an equilibrating mixture of the 3-oxo product 34a [(R)-2-(2-hydroxyethyl)-3(2H)-dihydrofuranone] and its cyclic hemiacetal 35a (66%, ~1:1; ¹H and ¹³C NMR) (Scheme 4). A minor amount (19%) of the 6-hydroxy-2-O-tosyl byproduct resulting from hydrogen transfer to the 6-oxyl radical without elimination of tosylate also was isolated (

Table 1. Products from 6-O-nitro-1,4-anhydrohexitols ^a.

Entry	Substrate			Reagent	Products	Ratio b	Yield c (%)
	No	X	Y
1	9	OTs	H	Bu3SnH	34a:35a	1:1	66 d
2	9	OTs	H	Bu3SnD	34b:35b	1:1	71 d
3	20	Cl	H	Bu3SnH	34a:35a	1:1	81
4	20	Cl	H	Bu3SnD	34b:35b	1:1	67
5	22	Br	H	Bu3SnH	34a:35a	1:1	64
6	22	Br	H	Bu3SnD	34b:35b	1:1	68
7	13	H	Cl	Bu3SnH	34a:35a	1:1	97
8	13	H	Cl	Bu3SnD	34b:35b	1:1	82
9	14	H	Br	Bu3SnH	34a:35a	1:1	67 e
10	14	H	Br	Bu3SnD	34b:35b	1:1	54 e
11	18	H	OTs	Bu3SnH	34a:35a	1:1	93
12	18	H	OTs	Bu3SnD	34b:35b	1:1	93

^a Reactions were performed on 0.057 mmol (9 or 18), 0.094 mmol (13 or 20), or 0.078 mmol (14 or 22) of substrates (0.029–0.047 M) with 5 equiv. of Bu₃SnH(D) and 2 equiv. of AIBN in toluene (95 °C, 1–2 h.). ^b Determined by ¹H NMR. ^c Isolated yields. ^d 1,5-Dideoxy-2-O-tosyl-D-ribohexofuranose also formed (19%). ^e Hydrodebrominated (2-deoxy) byproducts were also isolated (10–15%).

, entry 1, footnote d). The ¹³C NMR spectrum of the mixture had a signal for the carbonyl carbon of 34a at 215.9 ppm and one for the hemiacetal carbon of 35a at 114.9 ppm. Analogous treatment of 9 with Bu₃SnD gave the 2-deuterio epimers (2R/S, ~1:1) of 34b/35b (71%, ~1:1; entry 2). The ¹H NMR spectrum of 34b/35b corresponded to that of 34a/35a with ~50% reduction of the integrated intensity for the H2/2′ signals and simplification of the H1/1′ signals. The ¹³C NMR spectrum of 34b/35b showed triplets from splitting with deuterium (1:1:1 intensity) at 36.7 and 38.9 ppm for C2. MS and HRMS spectra showed ~95% incorporation of deuterium. Benzoylation of the 34a/35a mixture provided a stable ketone benzoate 36a, and benzoylation of 34b/35b gave 36b (2-deuterio epimers).

The role of the proton on the 3-hydroxyl group was probed. Treatment of the 3-O-methyl-6-O-nitro-2-O-tosyl ribo substrate 25 produced 2-(2-hydroxyethyl)-3-methoxyfuran (37, 63%) plus the 6-hydroxy byproduct 38 (22%) (Scheme 5;

Table 2. Products from 3-O-methyl-6-O-nitro-1,4-anhydrohexitols ^a_.

Entry	Substrate			Reagent	Product	Yield b (%)
	No	X	Y
1	25	OTs	H	Bu3SnH	37	63 c
2	25	OTs	H	Bu3SnD	37	61 c
3	32	Cl	H	Bu3SnD	40	55
4	33	Br	H	Bu3SnD	40	37 d
5	28	H	Cl	Bu3SnD	40	63
6	29	H	Br	Bu3SnD	40	47 d
7	30	H	OTs	Bu3SnH	37	51 e
8	30	H	OTs	Bu3SnD	37	50 e

^a Reactions were performed with 0.055 mmol (25 or 30), 0.088 mmol (28 or 32), or 0.074 (29 or 33) of substrates (0.027–0.044 M), 5 equiv. of Bu₃SnH(D), and 2 equiv. of AIBN in toluene for 1.5 h at 95 °C. ^b Isolated yields. ^c Compound 38 (22%) also was isolated. ^d Hydrodebrominated 2-deoxy byproducts also were isolated (~15–25%). ^e Compound 39 (38%) also was isolated.

, entry 1). Treatment of 25 with Bu₃SnD gave 37 and 38 with no incorporation of deuterium (entry 2) and no epimerization at C3 [37]. Tosylate 25 was thermally stable in toluene at 95 °C. Treatment of the arabino 3-O-methyl-2-O-tosyl substrate 30 with Bu₃SnH gave 37 (51%) plus byproduct 39 (38%) (entry 7). Treatment of 30 with Bu₃SnD produced 37 and 39 without incorporation of deuterium or epimerization at C3 (entry 8).

Treatment of the 3-O-methyl-2-chloro (32) and 2-bromo (33) ribo substrates with Bu₃SnD resulted in formation of vinyl ether 40 with no incorporation of deuterium (entries 3 and 4). The ¹³C NMR spectrum of 40 had olefinic signals at 90.2 and 157.7 ppm, and the ¹H NMR spectrum had olefinic proton signals at 4.60–4.70 ppm. Treatment of the arabino 3-O-methyl-2-chloro (28) and bromo (29) compounds with Bu₃SnD also resulted in elimination of a halogen atom to give vinyl ether 40 with no incorporation of deuterium (entries 5 and 6).

Similar treatment of the 2-chloro (20) and 2-bromo (22) ribo analogs produced the same keto/hemiacetal mixture (34a/35a, ~1:1; 81%, entry 3; and 64%, entry 5). Treatment of 20 (and 22) with Bu₃SnD gave 34b/35b as 2(R/S)-deuterio epimers (entries 4 and 6). Toluene solutions of 9, 20, and 22 (without Bu₃SnH or AIBN) were heated independently at 95 °C for 4 h (also at 110 °C). Unchanged starting materials were recovered almost quantitatively, which confirmed their thermal stability under these conditions and excluded the possibility of initial dissociation of a substituent from C2. Compound 9 also was stable at 95 °C for 2.5 h in DMF.

The arabino 2-(chloro, bromo, and tosyloxy) compounds (13, 14, and 18) were subjected to the conditions used with the ribo epimers to probe the effect of stereochemical inversion at C2. The same equilibrating mixtures of 34a/35a were produced with Bu₃SnH (entries 7, 9, and 11) and the epimeric 2-deuterio derivatives 34b/35b were generated with Bu₃SnD (entries 8, 10, and 12). Incorporation of deuterium into the ketone/hemiacetal products demonstrated that elimination of tosylate, chloride, and bromide anions occurred with all three of the arabino compounds. Enhanced yields of the tautomeric product mixtures were isolated with the chloro (13, entries 7 and 8) and tosylate (18, entries 11 and 12) substrates. Byproduct formation was not observed with 18, whereas it was formed (~19%) with the ribo substrate 9 (entries 1 and 2, footnote d). Some hydrodebromination also was detected with the arabino 2-bromo epimer 14 (entries 9 and 10, footnote e).

Samples of 9, 13, 14, and 18 were treated with Bu₃SnH in deuterated toluene. Fragmentation of tosylate 9 in toluene-d₈ (Bu₃SnH/AIBN) at 75 °C was 90–95% complete in 2.5 h (TLC and ¹H NMR) and produced 34a/35a (80–85%) as sole products with no incorporation of deuterium. Analogous treatment of 9 at 55 °C for 2.5 h resulted in ~50% fragmentation. Treatment of the arabino tosylate 18 and chloride 13 substrates at 75 °C showed that the fragmentation of 18 was slightly faster than that of 13 (Figure 2; ¹H NMR spectra were used for substrate/products ratios, see the Experimental Section). Fragmentation of the ribo tosylate 9 to give 34a/35a proceeded at a rate similar to that determined with the arabino chloride 13. Treatment of the arabino bromide 14 at 75 °C produced 34a/35a plus 2-deoxy byproduct mixtures with ¹H NMR spectra that were too complex for quantitative analysis.

3. Discussion

The mechanism proposed [1] for conversion of ribonucleoside 5′-diphosphates (A, X = OH) to 2′-deoxynucleotides (F) by RDPRs (Figure 1, inside the boxes) is supported by biochemical, chemical, and theoretical modeling studies. However, the enzymatic processing of 2′-chloro analogs (A, X = Cl) could cause inactivation by different chemistry. Incubation of a 2′-chloro-2′-deoxynucleoside 5′-diphosphate with RDPR produced 2-methylene-3(2H)furanone (G), a Michael acceptor that could cause time-dependent enzyme inactivation. Stubbe rationalized [1] that spontaneous elimination of a chloride anion (and a proton) at the active site (rather than the enzyme-assisted removal of HOH with substrates) could cause active site changes resulting in dissociation of the 2′-deoxy-3′-oxo intermediate from D (Figure 1). Successive β-eliminations of pyrophosphate from C5′ and the base from C1′ could generate G in solution. However, the identical 3′-keto intermediate in D was postulated in the substrate to product sequence, which makes the presence of chloride and a proton the only difference for the inactivation sequence.

We reasoned that elimination of a chlorine atom from the initial C3′ radical was more likely. The electronegative character of C1′ would make the elimination of chloride (with generation of positive character at C2′) unfavorable, whereas loss of a chlorine atom with generation of a C2′ radical was well precedented [38], and generation of an enol would be energetically advantageous. Elimination of a chlorine atom (rather than chloride) at the active site could have serious consequences. The chlorine radical could attack a sulfhydryl group and the resulting sulfenyl chloride could react with nucleophilic groups in the enzyme or undergo hydrolysis to a sulfenic acid. Chlorine-atom abstraction of hydrogen from an amino acid residue (and resulting radical processes) and other chlorine-radical reactions would be possible, whereas such events would not occur with a ground state chloride anion. Radical-induced disruption of active-site architecture provides a more plausible explanation for dissociation of the 2′-deoxy-3′-oxo intermediate from D.

We have shown [31] that leaving-group radical stability is crucial for substituent elimination from C2′ upon generation of a radical at C3′. Treatment of 2′-(azido, bromo, chloro, iodo, or methylthio)-2′-deoxy-3′-O-phenoxythiocarbonyl nucleosides (Ha, Figure 3) with BuSnH resulted in generation of C3′ radicals Ia that underwent elimination of a radical from C2′ with formation of 2′,3′-unsaturated derivative J. In contrast, treatment of the 2′-(fluoro, mesyloxy, or tosyloxy)-3′-thionocarbonates Hb generated C3′ radicals Ib that abstracted hydrogen from the stannane to give 3′-deoxy-2′-(fluoro, mesyloxy, or tosyloxy) derivatives Kb. Elimination of a radical from C2′ of Ia gave J, whereas homolytic scission to release a high-energy fluorine atom or a mesyloxy or tosyloxy radical is energetically prohibitive—and did not occur. In those cases, abstraction of hydrogen from the stannane by the C3′ radical Ib gave the reduced products Kb.

Because the radicals I did not contain a 3′-hydroxyl group, we prepared model compounds that were more closely related to the natural substrates for RDPR-catalyzed 2′-deoxygenations. Treatment of the adenine 6′-O-nitro-2′-O-tosyl L and uracil 6′-O-nitro-2′-chloro M analogues with Bu₃SnD/AIBN/Δ generated the 6′-oxyl radicals N by attack of a stannyl radical at nitrate oxygen (Figure 4). Intramolecular abstraction of H3′ from N gave the C3′ radicals O or S. Loss of the proton from the 3′-hydroxyl group of O and elimination of the 2′-tosylate (with shift of the unpaired electron from C3′ to C2′ and generation of the O = C3′ double bond) would drive the overall elimination of toluenesulfonic acid from O to produce P. Deuterium transfer from the stannane to C2′ of P followed by elimination of the trans proton/deuteron and adenine from Q would give the observed 2-(2-hydroxyethyl)-3(2H)-furanone (R) containing deuterium at C4 (C2′ from the nucleoside). In contrast, loss of the chlorine atom from S would give enol T, which could undergo 1,4-elimination to U with no incorporation of deuterium (as observed). This demonstrated the distinct differences between TsO–C2′ bond cleavage by a two-electron heterolysis (in O) and cleavage of the chlorine–C2′ bond by a one-electron homolysis (in S) [21,29,30].

Barton’s nitrite ester [39] and Wagner’s δ-substituted aryl ketone [40] photolysis studies showed a strong preference for six-membered transition states for hydrogen abstraction by oxyl radicals. Fraser-Reid employed that [1,5]-hydrogen shift with oxyl radicals generated from carbohydrate nitrate esters and Bu₃SnH [41]. Radical-induced loss of ^–OTs and H⁺ (Figure 4, path a) is also analogous to an ionic LiEt₃BH-promoted rearrangement of 2′-O-tosyladenosine. Removal of the 3′-hydroxyl proton by Et₃BH^– initiated a [1,2]-hydride shift from C3′ to C2′ with displacement of tosylate from C2′ [42,43]. That rearrangement occurred also with 2′-chloro-2′-deoxyadenosine, but at a lower rate with the poorer leaving group (chloride).

Theoretical modeling [18,44] of RDPR-catalyzed 2′-deoxygenation invoked hydrogen bonding from the 3′-OH to a carboxylate group and from H-donors to 2′-OH. Analogous attraction between the cis 3-OH in 9 and a tosylate oxygen atom was considered for possible assistance of the heterolytic cleavage of the TsO–C2 linkage. However, treatment of the arabino tosylate 18 (no cis 3-OH) gave the same 34b/35b mixture in higher yield (93%) than with 9 (~70%). Byproduct with a hydroxyl group at C6 and tosylate at C2 was isolated (~19%) from treatment of 9, but no such arabino byproduct was observed with 18. These results are more consistent with a greater population of the C2-endo/C3-exo conformation range in 9 (reduction of strain with the 2-tosylate group) that would make a six-membered transition state for intramolecular abstraction of H3 by the 6-oxyl radical less favorable. Enhanced abstraction of deuterium from the stannane by the 6-oxyl radical would then occur to produce more of the byproduct. Greater C2-exo/C3-endo populations in 18 (reduction of strain between the arabino tosylate at C2 and the side chain at C4) would enhance the approach of the 6-oxyl radical for H3 abstraction and elimination of tosylate from the C3 radical to produce 34b/35b in higher yield (93%).

Thus, noteworthy mechanistic changes were observed within our model series. Anionic elimination of tosylate from C2′ of a nucleoside occurred (Figure 4, pathway a), whereas radical elimination of a chlorine atom (pathway b) was preferred. However, treatment of our ribo anhydroalditol tosylate 9, chloro 20, or bromo 22 substrates produced the same 2-deuterio-3-ketone 34b and hemiacetal 35b mixture. The arabino chloro 13 and bromo 14 epimers also gave 34b/35b (plus some hydrodebromination with 14). In every case, the loss of bromide or chloride anions from an intermediate C3 radical resulted in formation of deuterium-labeled materials rather than homolytic loss of a halogen atom to give the unlabeled alkene. The absence of an electronegative nucleobase on C1 (C1 is a –CH₂– moiety in the anhydroalditol models) allowed elimination of an anion from C2 when loss of the 3-hydroxyl proton could generate an O = C3 double bond.

Radical elimination occurred with the 2-chloro-3-O-methyl ribo 32 and arabino 28 substrates to give vinyl ether 40. Both 2-bromo-3-O-methyl ribo 33 and arabino 29 epimers also gave 40 (plus hydrodebrominated byproduct). Thus, when loss of the 3-hydroxyl proton and generation of a 3-ketone was prevented, elimination of a halogen atom occurred.

Ramos questioned our choice of toluene as solvent and lack of a basic residue in our prior nucleoside model studies and stated: “we concluded that the nature of the leaving substituent can be controlled; it will be anionic or radical depending on the presence or absence of a basic residue capable of deprotonating the 3′-HO group. In the enzyme, such functionality does exist, and so it can be concluded that the enzyme indeed eliminates anions, and not radicals” [19].

Clearly, the elimination of an anion is possible without a basic residue present in our current anhydroalditol models. Siegbahn [18] calculated a dielectric constant of ε ~ 4 in the region of the enzyme active site, which is approximated much more closely by that of toluene (ε ~ 2.4) than that of the aqueous methanol solutions (ε > 40) used by Lenz and Giese [20]. Thus, the intrinsic electronic status of the anomeric carbon C1′ (inductively negative in nucleosides) or C1 (inductively positive in anhydroalditols) as well as the nature of the leaving substituent (X• or A⁻) are major factors that control heterolytic elimination of an anion or homolytic elimination of a radical in biomimetic model reactions—and such factors also likely play a key role in enzyme-initiated inactivation processes.

Begley [32] also has invoked our elimination of a chlorine atom [30] upon abstraction of H3′ from 2′-chloro-2′-deoxyguanosine triphosphate by a 5′-deoxyadenosyl radical cofactor in MoaA. His 2′-deoxy-3′-ketone underwent the sequential β-eliminations [30] of triphosphate and guanine to give the same 2-methylene-3(2H)furanone (G, Figure 1) that could cause inactivation of RDPRs. Both of our ribo 32 and arabino 28 2-chloro epimers gave vinyl ether 40, whereas Begley’s arabino epimer remained unchanged during incubation with MoaA. The cis,cis configuration of H3′/Cl/guanine in his arabino isomer might restrict conformational ranges that would favor binding with MoaA, and/or hinder approach of the 5′-deoxyadenosyl cofactor toward H3′. As discussed above, the different product/byproduct ratios in our fragmentations of 9 and 18, and 25 and 30 might result from such conformational effects.

Treatment of the 3-O-methyl-2-O-tosyl ribo 25 or arabino 30 substrates produced the aromatized 2-(2-hydroxyethyl)-3-methoxyfuran (37) plus the respective 6-hydroxy-2-O-tosyl byproducts 38 or 39. Generation of a carbonyl group at C3 is precluded with 25 and 30, but a favorable six-membered transition state involving attraction between the α-proton on C1 and a tosylate oxygen with epimer 25 (Figure 5) might aid the loss of HOTs with generation of a 1,2-double bond. An analogous interaction involving the β-proton on C1 and a tosylate oxygen with arabino epimer 30 also is possible. Abstraction of hydrogen from C4 of the resulting resonance hybrid would produce furan 37. The observed similar yields of 37 plus the respective 2-O-tosyl byproducts 38 and 39 are consistent with parallel processes for the ribo 25 and arabino 30 epimers. Tosylate 25 was stable in toluene at 95 °C, which confirmed that generation of a C3 radical was necessary for the elimination of tosylate and production of 37.

4. Experimental Section

The ¹H (400 or 500 MHz) and ¹³C (100 or 125 MHz) NMR spectra were determined on Bruker Biospin spectrometers (Bruker, Billerica, MA, USA) with solutions in CDCl₃ unless otherwise noted. HRMS were obtained in TOF-ESI mode on Bruker Solarix FT-ICR instrument (Bruker, Billerica, MA, USA) unless otherwise noted. TLC was performed with Merck (Darmstadt, Germany) kieselgel 60-F₂₅₄ sheets and products were detected with 254 nm light or by visualization with Ce(SO₄)₂/(NH₄)₆Mo₇O₂₄∙4H₂O/H₂SO₄/H₂O reagent. Merck kieselgel 60 (230–400 mesh) was used for column chromatography. Reagent grade chemicals were used, and solvents were dried by reflux over and distillation from CaH₂ (except for THF/potassium) under argon or by passing the solvents through activated alumina cartridges using a solvent purification system.

Methyl 6-O-Benzoyl-1,5-dideoxy-2,3-O-isopropylidene-β-D-ribo-hexofuranoside (2). To a solution of methyl 1,5-dideoxy-2,3-isopropylidene-β-D-ribo-hexofuranoside [29] (1; 13.0 g, 58.8 mmol) and Et₃N (8.9 g, 12.3 mL, 88.2 mol) in CH₂Cl₂ (50 mL) was added dropwise BzCl (12.4 g, 10.2 mL, 88.2 mmol) at 0 °C. The cooling bath was removed and after 1 h the reaction mixture was quenched by addition of MeOH (2 mL). After additional 30 min the volatiles were evaporated and the residue was column chromatographed (hexanes → hexanes/EtOAc, 6:1) to give 2 as pale yellow oil (17.3 g, 92%): ¹H NMR δ 1.32, 1.49 (2 × s, 2 × 3H), 2.01–2.04 (m, 2H), 3.38 (s, 3H), 4.39–4.52 (m, 3H), 4.62–4.65 (m, 2H), 4.98 (s, 1H), 7.42–7.57 (m, 3H), 8.04–8.06 (m, 2H); ¹³C NMR δ 24.6, 26.2, 33.9, 54.8, 61.6, 83.6, 84.0, 85.2, 109.6, 112.0, 128.1, 129.3, 129.9, 132.6, 166.0; MS FAB m/z 345 (3, [M + Na]⁺), 291 (100, [M − OMe]⁺); HRMS ESI m/z calcd for C₁₇H₂₂O₆Na [M + Na]⁺ 345.1314, found 345.1317.

6-O-Benzoyl-1,5-dideoxy-D-ribo-hexofuranose (3) and 6-O-Benzoyl-1,5-dideoxy-2,3-O- isopropylidene-D-ribo-hexofuranose (4). To a stirred solution of 2 (18.0 g, 55.9 mmol) and Et₃SiH (19.4 g, 26.7 mL, 167.7 mmol) in CH₂Cl₂ (10 mL) was added BF₃•OEt₂ (23.8 g, 21.3 mL, 167.7 mmol). Mildly exothermic reaction ensued after approx. 15 min and the reaction mixture was allowed to stir for an additional 3 h. The reaction flask was placed in an ice slush and a saturated NaHCO₃/H₂O solution (200 mL) was added slowly with vigorous stirring. CH₂Cl₂ was added (50 mL), aqueous phase was separated and washed with CH₂Cl₂ (50 mL). Organic fractions were combined and evaporated to dryness. Column chromatography (hexanes/EtOAc, 6:1 → EtOAc) gave contaminated (~10%) 4 (1.5 g, 9%) and 3 (6.1 g, 43%) as syrup. Compound 3 had: ¹H NMR (CD₃OD) δ 1.87–1.94 (m, 1H), 2.12–2.19 (m, 1H), 3.70 (dd, J = 2.9, 9.8 Hz, 1H), 3.79 (dd, J = 5.4, 7.3 Hz, 1H), 3.85–3.89 (m, 1H), 4.07 (dd, J = 4.9, 9.8 Hz, 1H), 4.16 (td, J = 2.9, 4.9 Hz, 1H), 4.38–4.50 (m, 2H), 7.45–7.61 (m, 3H), 8.02–8.04 (m, 2H); ¹³C NMR (CD₃OD) δ 32.4, 61.9, 70.8, 72.2, 75.9, 78.9, 128.3, 129.5, 129.9, 133.0, 166.8; MS FAB m/z 275 (100, [M + Na]⁺); HRMS ESI m/z calcd for C₁₃H₁₆O₅Na [M + Na]⁺ 275.1531, found 275.0902.

Diol 3 (6.1 g, 24.2 mmol) and TsOH hydrate (0.5 g, 2.6 mmol) were dissolved in a mixture of acetone (40 mL) and 2,2-dimethoxypropane (10 mL) and left to stir at room temperature for 30 min. Neutralization with sat. NaHCO₃ (100 mL) followed by EtOAc extraction (2 × 50 mL) and evaporation to dryness provided oil that was filtered through silica to give 4 (6.4 g, 91%): ¹H NMR δ 1.33, 1.52 (2 × s, 2 × 3H), 1.86–1.92 (m, 2H), 3.86 (dd, J = 4.4, 10.7 Hz, 1H), 3.97 (dd, J = 1.5, 10.7 Hz, 1H), 4.23–4.26 (m, 1H), 4.36–4.49 (m, 2H), 4.52 (dd, J = 1.5, 6.3 Hz, 1H), 4.80–4.83 (m, 1H), 7.42–7.57 (m, 3H), 8.03–8.05 (m, 2H); ¹³C NMR δ 24.9, 26.5, 29.7, 61.5, 71.6, 80.8, 81.2, 85.0, 112.8, 128.3, 129.5, 130.1, 132.9, 166.4. MS FAB m/z 293 (100, [M + H]⁺), 315 (15, [M + Na]⁺); HRMS ESI m/z calcd for C₁₆H₂₀O₅Na [M + Na]⁺ 315.1208, found 315.1209.

1,5-Dideoxy-2,3-O-isopropylidene-D-ribo-hexofuranose (5). To a stirred solution of 4 (14.0 g, 47.9 mmol) in MeOH (50 mL) was added a solution of KOH (2.0 g, 35.7 mol) in MeOH (50 mL). After 1 h the volatiles were evaporated and the residue was column chromatographed (hexanes/EtOAc, 2:1 → EtOAc) to give 5 [45] (8.0 g, 87%): ¹H NMR δ 1.33, 1.51 (2 × s, 2 × 3H), 1.63–1.76 (m, 2H), 3.74–3.82 (m, 2H), 3.89 (dd, J = 4.4, 10.4 Hz, 1H), 3.94 (d, J = 10.3 Hz, 1H), 4.19–4.22 (m, 2H), 4.48 (dd, J = 1.5, 5.8 Hz, 1H), 4.81 (t, J = 5.1 Hz, 1H); ¹³C NMR δ 24.5, 26.2, 32.4, 59.2, 71.1, 80.4, 82.2, 84.6, 112.3. MS FAB m/z 189 (100, [M + H]⁺); HRMS ESI m/z calcd for C₉H₁₇O₄ [M + H]⁺ 189.1127, found 189.1123.

6-Iodo-1,5,6-trideoxy-2,3-O-isopropylidene-D-ribo-hexofuranose (6). To a stirred solution of 5 (9.0 g, 47.9 mmol), Ph₃P (15.1 g, 57.6 mmol) and imidazole (7.9 g, 116.2 mmol) in toluene (225 mL) was added iodine (14.6 g, 57.5 mmol) at 70 °C. The suspension was vigorously stirred another 2 h, then allowed to cool to ambient temperature and the solution decanted to another flask. Volatiles were evaporated and the residue was column chromatographed (hexanes → hexanes/EtOAc, 6:1) to give 6 (13.03 g, 85%): ¹H NMR δ 1.33, 1.52 (2 × s, 2 × 3H), 1.88–1.97 (m, 2H), 3.14–3.27 (m, 2H), 3.78 (dd, J = 4.4, 10.7 Hz, 1H), 3.95 (dd, J = 1.5, 10.7 Hz, 1H), 4.10 (ddd, J = 1.5, 4.9, 9.2 Hz, 1H), 4.23 (dd, J = 2.0, 6.3 Hz, 1H), 4.78 (m, 1H); ¹³C NMR δ 1.0, 24.7, 26.2, 34.0, 71.2, 80.4, 83.6, 84.1, 112.3; MS FAB m/z 321 (12, [M + Na]⁺), 319 (100); HRMS ESI m/z calcd for C₉H₁₅IO₃Na [M + Na]⁺ 320.9964, found 320.9970.

6-Iodo-1,5,6-trideoxy-D-ribo-hexofuranose (7). A solution of 6 (14.0 g, 47.0 mmol) in a mixture of MeOH (100 mL), H₂O (25 mL) and conc. HCl (25 mL) was stirred at room temperature for 3 h. Volatiles were evaporated and the residue was column chromatographed (hexanes/EtOAc, 2:1 → EtOAc) to give 7 (8.73 g, 72%): ¹H NMR δ 2.00–2.07 (m, 1H), 2.16–2.25 (m, 1H), 3.22–3.45 (m, 4H), 3.74–3.77 (m, 2H), 3.84 (br s, 1H), 4.10 (dd, J = 4.9, 10.3 Hz, 1H), 4.27 (br s, 1H); ¹³C NMR δ 1.7, 37.3, 70.9, 72.8, 75.6, 81.6; MS FAB m/z 258 (10, [M]⁺), 74 (100); HRMS ESI m/z calcd for C₆H₁₁IO₃, 257.9753, found 257.9740.

1,5-Dideoxy-6-O-nitro-D-ribo-hexofuranose (8). A suspension of 7 (12.0 g, 46.5 mmol) and AgNO₃ (15.8 g, 93.0 mmol) in CH₃CN (150 mL) was stirred at room temperature for 2 days. The yellow precipitate was filtered off, washed with EtOAc and the volatiles were evaporated. Column chromatography (hexanes/EtOAc, 1:1 → EtOAc) gave 8 (7.99 g, 89%): ¹H NMR δ 1.88–1.96 (m, 1H), 2.08–2.15 (m, 1H), 3.43 (br s, 2H), 3.73–3.83 (m, 3H), 4.12 (dd, J = 4.9, 10.3 Hz, 1H), 4.23–4.26 (m, 1H), 4.56–4.66 (m, 2H); ¹³C NMR δ 30.2, 70.1, 70.8, 72.6, 75.9, 77.8; MS FAB m/z 216 (100, [M + Na]⁺); HRMS ESI m/z calcd for C₆H₁₁NO₆Na [M + Na]⁺, 216.0484 found 216.0497.

1,5-Dideoxy-6-O-nitro-2-O-tosyl-D-ribo-hexofuranose (9). Method A. A solution of diol 8 (370 mg, 1.92 mmol) and Bu₂SnO (477 mg, 1.92 mmol) in anhydrous MeOH (40 mL) was heated in a sealed flask at 75 °C for 1 h. After the flask was cooled to 0 °C, Et₃N (1.15 g, 1.6 mL, 11.4 mmol) was added with stirring followed by TsCl (2.17 g, 11.4 mmol) dissolved in a minimum volume of acetone. The volatiles were evaporated, and the residue was suspended in acetone and deposited on silica. Column chromatography (hexanes/EtOAc, 6:1 → 3:1) gave a 5:2 mixture of two isomers (670 mg) from which the main product 9 (320 mg, 48%) was isolated after second column chromatography: ¹H NMR δ 1.85–1.92 (m, 1H), 2.09–2.17 (m, 1H), 2.46 (s, 3H), 2.76 (d, J = 7.8 Hz, 1H), 3.75 (dt, J = 3.9, 8.3 Hz, 1H), 3.80 (dd, J = 3.1, 11.2 Hz, 1H), 3.89 (dt, J = 5.4, 7.8 Hz, 1H), 4.06 (dd, J = 4.9, 11.2 Hz, 1H), 4.52–4.62 (m, 2H), 4.93 (dt, J = 3.0, 5.3 Hz, 1H), 7.38, 7.82 (2 × d, J = 8.3 Hz, 2 × 2H); ¹³C NMR δ 21.6, 30.2, 69.8, 70.1, 74.9, 77.8, 79.4, 127.8, 130.1, 132.8, 145.6; MS FAB m/z 370 (100, [M + Na]⁺); HRMS ESI m/z calcd for C₁₃H₁₇NO₈SNa [M + Na]⁺ 370.0573, found 370.0587.

Method B. Step a: TsCl (66 mg, 0.35 mmol) was added to a stirred solution of 10 (100 mg, 0.32 mmol) in anhydrous pyridine (1 mL) at ambient temperature. After 16 h, the volatiles were evaporated and residue was partitioned between ice-cold AcOH/H₂O (1:99, 30 mL) and CHCl₃ (30 mL). The aqueous layer was extracted with CHCl₃ and the combined organic phase was washed with ice-cold saturated NaHCO₃/H₂O (30 mL), brine (30 mL) and dried (Na₂SO₄). Column chromatography (EtOAc/hexane, 5:95 → 35:65) gave 1,5-dideoxy-3-O-(p-methoxybenzyl)- 6-O-nitro-2-O-tosyl-D-ribo-hexofuranose (15; 82 mg, 55%): ¹H NMR δ 1.78–1.81 (m, 1H), 2.00–2.02 (m, 1H), 2.47 (s, 3H), 3.59 (dd, J = 5.0, 8.6 Hz, 1H), 3.83 (s, 3H), 3.86 (dt, J = 2.2, 9.6 Hz, 2H), 4.04 (dd, J = 4.5, 11.2 Hz, 2H), 4.26 (d, J = 11.2 Hz, 1H), 4.47 (m, 2H), 4.58 (d, J = 11.2Hz, 1H), 5.13 (dt, J = 4.7, 7.0 Hz, 1H), 6.88 and 7.21 (2 × d, J = 9.5 Hz, 2 × 2H), 7.34 and 7.84 (2 × d, J = 8.3 Hz, 2 × 2H). In step b, A solution of CAN (176 mg, 0.32 mmol) and 15 (75 mg, 0.16 mmol) in MeCN (1.5 mL) and water (0.15 mL) was stirred at ambient temperature for 22 h. Volatiles were evaporated and the residue was column chromatographed (EtOAc/hexane, 3:97 → 30:70) to give 9 (54 mg, 98%) with data as above.

1,5-Dideoxy-3-O-(p-methoxybenzyl)-6-O-nitro-D-ribo-hexofuranose (10). A suspension of diol 8 (1.20 g, 6.22 mmol) and Bu₂SnO (1.70 g, 6.84 mmol) in anhydrous MeOH (10 mL) was heated in a sealed flask at 75 °C for 1 h. After the flask was cooled to ambient temperature, the volatiles were evaporated. DMF (5 mL) and PMBCl (1.95 g, 1.7 mL, 12.44) were added, and the reaction mixture was heated at 90 °C for 18 h. Volatiles were evaporated, and the residue was chromatographed (hexanes/EtOAc, 6:1 → 1:1) to give 1,5-dideoxy-2,3-di-O-(p-methoxybenzyl)-6-O-nitro-D-ribo- hexofuranose (1.55 g; 20% contaminated) followed by 1,5-dideoxy-2-O-(p-methoxybenzyl)-6-O-nitro-D-ribo-hexofuranose (0.47 g, 24%) and 10 (0.60 g; 31%) of sufficient purity for use in subsequent reaction. Sample of 10 was repurified on column chromatography for spectroscopic characterization: ¹H NMR δ 1.77–1.85 (m, 1H), 1.93–2.00 (m, 1H), 3.20 (br s, 1H), 3.60 (dd, J = 5.4, 7.3 Hz, 1H), 3.76 (dd, J = 2.9, 10.3 Hz, 1H), 3.81 (s, 3H), 3.85 (dt, J = 3.9, 7.3 Hz, 1H), 4.01 (dd, J = 4.9, 10.3 Hz, 1H), 4.18 (m, 1H), 4.47–4.61 (m, 4H), 6.88–6.92 (m, 2H), 7.24–7.30 (m, 2H); ¹³C NMR δ 30.5, 55.2, 69.1, 70.0, 72.6, 73.3, 76.1, 82.4, 114.0, 128.8, 129.7, 159.7; MS FAB m/z 336 (100, [M + Na]⁺); HRMS ESI m/z calcd for C₁₄H₁₉NO₇Na [M + Na]⁺ 336.1059, found 336.1072.

2-Chloro-3-O-(p-methoxybenzyl)-6-O-nitro-1,2,5-trideoxy-D-arabino-hexofuranose (11). To a solution of Ph₃P (2.18 g, 8.31 mmol) and DIAD (1.26 g, 1.22 mL, 6.23 mmol) in THF (14 mL) was added a solution of 10 (0.65 g, 2.08 mmol) in THF (6 mL) followed by freshly prepared HCl•pyridine (0.72 g, 6.23 mmol; prepared by slow addition of TMSCl (0.68 g, 0.8 mL, 6.31 mmol) to a solution of pyridine (0.98 g, 1.0 mL, 12.4 mmol) in MeOH (0.32 g, 0.4 mL, 9.9 mmol) and CH₂Cl₂ (10 mL) at 0 °C. Volatiles were evaporated and the crystalline residue was dried at 80 °C under vacuum overnight). The suspension was stirred overnight at room temperature. Volatiles were evaporated, and the residue was column chromatographed (hexanes/EtOAc, 10:1 → 6:1) to give 11 (0.43 g, 62%): ¹H NMR δ 2.05–2.10 (m, 2H), 3.82 (br s, 4H), 3.81–3.86 (m, 1H), 3.90 (br d, J = 3.4 Hz, 1H), 4.02 (d, J = 10.3 Hz, 1H), 4.13 (dd, J = 4.4, 10.7 Hz, 1H), 4.47–4.65 (m, 4H), 6.90–6.93 (m, 2H), 7.25–7.28 (m, 2H); ¹³C NMR δ 30.9, 55.2, 60.1, 70.0, 72.1, 74.2, 80.9, 89.8, 113.9, 128.8, 129.6, 159.5; HRMS ESI m/z Calcd for C₁₄H₁₈ClNO₆Na [M + Na]⁺ 354.0720, found 354.0706.

2-Chloro-1,2,5-trideoxy-6-O-nitro-D-arabino-hexofuranose (13). A solution of 11 (0.43 g, 1.30 mmol) and CAN (1.42 g, 2.59 mmol) in CH₃CN (7 mL) and H₂O (0.7 mL) was stirred for 1.5 h. The reaction mixture was concentrated, and the residue was deposited on silica gel and column chromatographed (hexanes/EtOAc, 10:1 → 3:1) to give 13 (0.21 g, 78%): ¹H NMR δ 2.10–2.22 (m, 2H), 2.69 (br s, 1H), 3.80–3.84 (m, 1H), 4.02 (br d, J = 10.8 Hz, 1H), 4.14–4.25 (m, 3H), 4.58–4.68 (m, 2H); ¹³C NMR δ 30.9, 62.3, 70.0, 73.3, 81.7, 83.2; MS FAB m/z 212 (5, [M + H]⁺), 120 (100); HRMS ESI m/z calcd for C₆H₁₁ClNO₅ [M + H]⁺ 212.0326, found 212.0327.

2-Bromo-1,2,5-trideoxy-6-O-nitro-D-arabino-hexofuranose (14). Step a: To a solution of Ph₃P (2.85 g, 10.09 mmol) and DIAD (1.65 g, 8.15 mmol) in THF (30 mL) was added a stirred solution of 10 (1.75 g, 5.43 mmol) in THF (10 mL) followed by freshly prepared HBr•pyridine (1.30 g, 8.15 mmol; prepared by slow addition of TMSBr (0.93 g, 0.8 mL, 6.06 mmol) to a solution of pyridine (0.98 g, 1.0 mL, 12.4 mmol) in MeOH (0.32 g, 0.4 mL, 9.9 mmol) and CH₂Cl₂ (10 mL) at 0 °C. Volatiles were evaporated and the crystalline residue was dried at 80 °C under vacuum overnight). The suspension was stirred at room temperature for 22 h. Volatiles were evaporated, and the residue was column chromatographed (hexanes/EtOAc, 10:1 → 6:1) to give crude 2-bromo-3-O-(p-methoxybenzyl)-6-O-nitro-1,2,5-trideoxy-D-arabino-hexofuranose (12; 1.0 g, 49%) which was directly used in next step. Step b. A solution of crude 12 (1.0 g, 2.66 mmol) and CAN (2.91 g, 5.32 mmol) in a mixture of CH₃CN (14 mL) and H₂O (1.4 mL) was stirred for 1.5 h. The resulting mixture was concentrated and the residue was deposited on silica and column chromatographed (hexanes/EtOAc, 10:1 → 4:1) to give 14 (0.54 g, 79%): ¹H NMR δ 2.14–2.26 (m, 2H), 3.80–3.86 (dt, J = 5.2, 8.8 Hz, 1H), 4.10 (dd, J = 4.0, 10.6 Hz, 1H), 4.16–4.22 (m, 1H), 4.26–4.33 (m, 2H), 4.58–4.72 (m, 2H); ¹³C NMR δ 31.2, 51.5, 69.9, 73.6, 81.5, 83.5. HRMS ESI/DART m/z calcd for C₆H₁₄⁷⁹BrN₂O₅ [M + NH₄]⁺ 273.0081, found 273.0084; calcd for C₆H₁₄⁸¹BrN₂O₅ [M + NH₄]⁺ 275.0061, found 275.0061.

1,5-Dideoxy-3-O-(p-methoxybenzyl)-6-O-nitro-D-arabino-hexofuranose (16b). Step a: A solution of compound 10 (2.70 g, 8.63 mmol) in THF (8 mL) followed by a solution of DIAD (2.09 g, 2.03 mL, 10.35 mmol) in THF (3 mL) were slowly (12 min) added to a stirred solution of Ph₃P (2.71 g, 10.35 mmol) and PhCO₂H (1.26 g, 10.35 mmol) in THF (40 mL) at –50 °C under nitrogen atmosphere. The resulting mixture was allowed to warm to ambient temperature within 1 h (it became colorless at –20 °C). Volatiles were evaporated, and the residue was column chromatographed (hexanes → hexanes/EtOAc, 3:1) to give 2-O-benzoyl-1,5-dideoxy-3-O-(p-methoxybenzyl)-6-O-nitro-D-arabino-hexofuranose (16a; 2.87 g, 80%): ¹H NMR δ 2.0–2.07 (m, 2H), 3.79 (s, 3H), 3.85 (td, J = 1.0, 4.6 Hz, 1H), 3.90 (td, J = 5.0, 7.6 Hz, 1H), 4.08–4.12 (m, 2H), 4.51–4.59 (m, 3H), 4.75 (d, J = 11.7 Hz, 1H), 5.43 (td, J = 1.4, 2.7 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.7, 2H), 7.47 (t, J = 7.7 Hz, 2H), 7.61 (tt, J = 1.5, 7.5 Hz, 1H), 8.03 (d, J = 7.6 Hz, 2H). Step b: A suspension of KOH (0.40 g, 7.14 mol) in MeOH (20 mL) was added to a stirred solution of 16a (2.82 g, 6.76 mmol) in MeOH (20 mL) at ambient temperature. After 1 h, the solution was neutralized with AcOH to pH 7. The volatiles were evaporated and the residue was column chromatographed (hexanes/EtOAc, 3:1 → 2:1) to give 16b (1.64 g, 77%): ¹H NMR δ 1.26 (d, J = 2.9 Hz, 1H), 2.00–2.04 (m, 2H), 3.64 (d, J = 2.9 Hz, 1H), 3.80 (br s, 4H), 3.83 (d, J = 10.3 Hz, 1H), 3.91 (dd, J = 3.9, 10.3 Hz, 1H), 4.27–4.29 (m, 1H), 4.48–4.58 (m, 4H), 6.88–6.91 (m, 2H), 7.24–7.27 (m, 2H); ¹³C NMR δ 30.9, 55.2, 70.2, 71.8, 74.1, 76.1 80.3, 89.2, 113.9, 128.4, 129.5, 159.4; MS FAB m/z 336 (100, [M + Na]⁺); HRMS ESI m/z calcd for C₁₄H₁₉NO₇Na [M + Na]⁺ 336.1059, found 336.1047.

1,5-Dideoxy-6-O-nitro-2-O-tosyl-D-arabino-hexofuranose (18). Step a: TsCl (134 mg, 0.70 mmol) was added to a stirred solution of 16b (100 mg, 0.32 mmol) in anhydrous pyridine (1mL) at ambient temperature. After 16 h, the volatiles were evaporated and residue was partitioned between ice-cold AcOH/H₂O (1:99, 30 mL) and CHCl₃ (30 mL). The organic layer was separated, and the aqueous layer was extracted with CHCl₃ (30 mL). Combined organic phase was washed with ice-cold saturated NaHCO₃/H₂O (30 mL), brine (30 mL) and dried (Na₂SO₄), concentrated in vacuo and column chromatographed (EtOAc/hexane, 5:95 → 35:65) to give 1,5-dideoxy-3-O-(p-methoxybenzyl)-6-O-nitro-2-O-tosyl-D-arabino-hexofuranose (17a; 128 mg, 86%) as a colorless oil: ¹H NMR δ 1.90–1.96 (m, 2H), 2.46 (s, 3H), 3.72–3.78 (m, 1H), 3.82 (s, 3H), 3.84 (dt, J = 1.2, 4.6 Hz, 1H), 3.87 (d, J = 4.0 Hz, 1H), 3.92 (d, J = 11.3 Hz, 1H), 4.33 (d, J = 11.3 Hz, 1H), 4.42–4.49 (m, 3H), 4.93 (dt, J = 1.2, 4.0 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H); ¹³C NMR δ 21.6, 30.3, 55.3, 69.8, 71.4, 71.8, 80.2, 83.9, 86.3, 114.0, 127.8, 128.8, 129.5, 130.1, 133.4, 145.4, 159.6. Step b: A solution of CAN (352 mg, 0.64 mmol) and 17a (100 mg, 0.21 mmol) in MeCN (1 mL) and H₂O (0.1 mL) was stirred at ambient temperature for 22 h. Volatiles were evaporated and the residue was column chromatographed (EtOAc/hexane, 3:97 → 30:70) to give 18 (73 mg, 98%) as a colorless oil: ¹H NMR δ 1.97–2.07 (m, 1H), 2.07–2.17 (m, 1H), 2.47 (s, 3H), 3.69–3.76 (m, 1H), 3.85–3.90 (m, 1H), 3.95 (dd, J = 5.5, 11.3 Hz, 1H), 4.17 (dd, J = 2.5, 6.0 Hz, 1H), 4.51–4.63 (m, 2H), 4.76 (dt, J = 2.5, 5.5 Hz, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.5 Hz, 2H); ¹³C NMR δ 21.6, 30.2, 69.7, 70.3, 80.5, 80.9, 86.5, 127.9, 130.1, 132.8, 145.6. HRMS ESI/DART m/z calcd for C₁₃H₂₁N₂O₈S [M + NH₄]⁺ 365.1013, found 365.1031.

2-Chloro-1,2,5-trideoxy-3-O-(p-methoxybenzyl)-6-O-nitro-D-ribo-hexofuranose (19). Step a: TfCl (160 mg, 0.95 mmol) was added to a stirred solution of 16b (250 mg, 0.80 mmol) and DMAP (295 mg, 2.4 mmol) in anhydrous CH₂Cl₂ (2 mL) at 0 °C (ice-bath). After 1 h, the reaction mixture was partitioned between ice-cold AcOH/H₂O (1:99, 30 mL) and CH₂Cl₂ (30 mL). The aqueous layer was extracted with CH₂Cl₂ (30 mL) and the combined organic phase was washed with ice-cold saturated NaHCO₃/H₂O (30 mL), brine (30 mL) and dried (Na₂SO₄) to give 1,5-dideoxy-3-O-(p-methoxybenzyl)-2-O-(trifluoromethanesulfonyl)-6-O-nitro-D-arabino-hexofuraose as a colorless oil (17b; 313 mg, 88%) of sufficient purity to be used in next step. Column chromatography (EtOAc/hexane, 5:95 → 30:70) gave pure sample of 17b: ¹H NMR δ 1.93–2.02 (m, 2H), 3.80–3.84 (m, 1H), 3.82 (s, 3H), 3.92 (dt, J = 1.2, 4.6 Hz, 1H), 4.02 (dd, J = 3.5, 12.0 Hz, 1H), 4.18 (br d, J = 12.1 Hz, 1H), 4.47–4.52 (m, 3H), 4.66 (d, J = 11.5 Hz, 1H), 5.32 (d, J = 3.5 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H). Step b: A solution of crude 17b (50 mg, 0.11 mmol; from step a) and dried LiCl (23 mg, 0.54 mmol) in DMF (1 mL) was stirred for 5 h at ambient temperature under N₂. Volatiles were evaporated and residue was partitioned between ice-cold saturated NaHCO₃/H₂O (15 mL) and EtOAc (15 mL). The separated organic phase was washed with brine (30 mL), dried (Na₂SO₄) and column chromatographed (EtOAc/hexane, 5:95 → 15:85) to give 19 (30 mg, 80.6%) as a colorless oil: ¹H NMR δ 1.82–1.91 (m, 1H), 2.01–2.1 (m, 1H), 3.72 (dd, J = 5.2, 8.2 Hz, 1H), 3.82 (s, 3H), 3.99 (dt, J = 4.0, 8.2 Hz, 1H), 4.06 (dd, J = 2.8, 10.7 Hz, 1H), 4.30 (dd, J = 5.07,10.7 Hz, 1H), 4.40 (d, J = 11.2 Hz, 1H), 4.44 (dt, J = 2.2, 5.2 Hz, 2H), 4.48–4.59 (m, 2H), 4.69 (d, 11.3 Hz, 1H), 6.90 (d, J = 8.7 Hz, 2H), 7.30 (d, J = 8.7 Hz, 2H); ¹³C NMR δ 30.4, 55.2, 57.8, 69.9, 72.1, 74.0, 76.3, 81.5, 114.0, 128.8, 129.8, 159.7. HRMS ESI m/z calcd for C₁₄H₁₈³⁵ClNO₆Na [M + Na]⁺ 354.0715, found 354.0718; calcd for C₁₄H₁₈³⁷ClNO₆Na [M + Na]⁺ 356.0691, found 356.0694.

2-Chloro-1,2,5-trideoxy-6-O-nitro-D-ribo-hexofuranose (20). A solution of CAN (74 mg, 0.13 mmol) and 19 (15 mg, 0.04 mmol) in MeCN (1mL) and H₂O (0.1 mL) was stirred at ambient temperature for 22 h. Volatiles were evaporated and the residue was column chromatographed (EtOAc/hexane, 3:97 → 30:70) to give 20 (6.2 mg, 66%) as a colorless oil: ¹H NMR δ 1.87–1.98 (m, 1H), 2.11–2.20 (m, 1H), 2.25 (d, J = 8.7 Hz, 1H), 3.82–3.88 (m, 1H), 3.93–4.00 (m, 1H), 4.02 (dd, J = 4.0, 10.7 Hz, 1H), 4.33 (dd, J = 5.4, 10.5 Hz, 1H), 4.44–4.48 (m, J = 4.2, 5.3 Hz, 1H), 4.55–4.67 (m, 2H); ¹³C NMR δ 30.5, 61.1, 69.8, 72.9, 75.7, 78.3; HRMS ESI/DART m/z calcd for C₆H₁₄³⁵ClN₂O₅ [M + NH₄]⁺ 229.0586, found 229.0588; calcd for C₆H₁₄³⁷ClN₂O₅ [M + NH₄]⁺ 231.0562, found 231.0561.

2-Bromo-1,2,5-trideoxy-3-O-(p-methoxybenzyl)-6-O-nitro-D-ribo-hexofuranose (21). A solution of 17b (30 mg, 0.06 mmol; prepared as described for 19, step a) and dried LiBr (29 mg, 0.33 mmol) in DMF (1 mL) was stirred for 7 h at ambient temperature under N₂. Volatiles were evaporated and the resulting residue was partitioned between ice-cold saturated NaHCO₃/H₂O (15 mL) and EtOAc (15 mL). The separated organic phase was washed with brine (30 mL), dried (Na₂SO₄) and column chromatographed (EtOAc/hexane, 5:95 → 15:85) to give 21 (18 mg, 71%) as a colorless oil: ¹H NMR δ 1.83–1.94 (m, 1H), 2.02–2.12 (m, 1H), 3.6 (dd, J = 5.0, 7.6 Hz, 1H), 3.84 (s, 3H), 4.03 (dt, J = 4.0, 8.0 Hz, 1H), 4.22 (dd, J = 2.8, 10.6 Hz, 1H), 4.44 (d, J = 11.1 Hz, 1H), 4.43–4.51 (m, 2 H), 4.51–4.61 (m, 2H), 4.71 (d, J = 11.1 Hz, 1H) 6.92 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H); ¹³C NMR δ 30.4, 49.7, 55.3, 69.9, 72.1, 74.1, 76.6, 81.2, 114.0, 128.81, 129.8, 159.71. HRMS ESI m/z calcd for C₁₄H₁₈⁷⁹BrNO₆Na [M + Na]⁺ 398.0210, found 398.0203; calcd for C₁₄H₁₈⁸¹BrNO₆Na [M + Na]⁺ 400.0191, found 400.0183.

2-Bromo-1,2,5-trideoxy-6-O-nitro-D-ribo-hexofuranose (22). A solution of CAN (78 mg, 0.14 mmol) and 21 (18 mg, 0.05 mmol) in MeCN (1 mL) and water (0.1 mL) was stirred at ambient temperature for 22 h. Volatiles were evaporated and the residue was column chromatographed (EtOAc/hexane, 3:97 → 30:70) to give 22 (12 mg, 96%) as a colorless oil: ¹H NMR δ 1.89–1.98 (m, 1H), 2.11–2.20 (m, 2H), 3.78–3.84 (m, 1H), 3.85–3.92 (m, 1H), 4.13 (dd, J = 4.6 Hz, 1H), 4.43 (dd, J = 5.4 Hz, 1H), 4.48 (dd, J = 5.0 Hz, 1H), 4.55–4.70 (m, 2H); ¹³C NMR δ 31.6, 53.7, 69.8, 72.9, 75.3, 78.8. HRMS ESI/DART m/z calcd for C₆H₁₄⁷⁹BrN₂O₅ [M + NH₄]⁺ 273.0081, found 273.0077; calcd for C₆H₁₄⁸¹BrN₂O₅ [M + NH₄]⁺ 275.0066, found 275.0058.

1,5-Dideoxy-3-O-methyl-6-O-nitro-D-ribo-hexofuranose (23) and 1,5-Dideoxy-2-O-methyl- 6-O-nitro-D-ribo-hexofuranose (24) and: A suspension of 8 (0.34 g, 1.76 mmol) and Bu₂SnO (0.44 g, 1.76 mmol) in anhydrous MeOH (8 mL) was refluxed for 30 min. Volatiles were evaporated after the flask was cooled to ambient temperature. DMF (1 mL) and MeI (1.14 g, 0.5 mL, 8.03 mmol) were added, the flask was sealed and the solution was stirred at 40 °C for 12 h. Volatiles were evaporated and the residue was column chromatographed (hexanes/EtOAc, 4:1 → 1:1) to give 23 (145 mg, 38%) and 24 (134 mg, 35%). Compound 23 had: ¹H NMR δ 1.85–1.92 (m, 1H), 2.09–2.16 (m, 1H), 2.79 (d, J = 8.8 Hz, 1H), 3.44 (s, 3H), 3.67 (ddd, J = 3.9, 7.8, 8.8 Hz, 1H), 3.74–3.84 (m, 3H), 4.06 (dd, J = 4.4, 5.4 Hz, 1H), 4.56–4.65 (m, 2H); ¹³C NMR δ 30.5, 57.7, 70.0, 70.1, 75.4, 78.6, 79.4; MS FAB m/z 230 (100, [M + Na]⁺); HRMS ESI m/z calcd for C₇H₁₃NO₆Na [M + Na]⁺ 230.0641, found 230.0651. Compound 24 had: ¹H NMR δ 1.89–1.96 (m, 1H), 2.04–2.11 (m, 1H), 2.70 (d, J = 3.9 Hz, 1H), 3.45 (dd, J = 4.9, 6.8 Hz, 1H), 3.47 (s, 3H), 3.79 (dd, J = 2.9, 9.8 Hz, 1H), 3.86 (ddd, J = 3.9, 6.8, 8.3 Hz, 1H), 3.79 (dd, J = 4.4, 10.2 Hz, 1H), 4.31 (m, 1H), 4.55–4.65 (m, 2H); ¹³C NMR δ 30.8, 58.3, 68.9, 70.0, 73.2, 75.9, 85.2; MS FAB m/z 208 (100, [M + H]⁺); HRMS ESI m/z calcd for C₇H₁₄NO₆ [M + H]⁺ 208.0821, found 208.0819.

1,5-Dideoxy-2-O-tosyl-3-O-methyl-6-O-nitro-D-ribo-hexofuranose (25). TsCl (101 mg, 0.53 mmol) was added to a stirred solution of 23 (100 mg, 0.48 mmol) in anhydrous pyridine (1 mL) at ambient temperature. After 16 h, the volatiles were evaporated and residue was partitioned between ice-cold AcOH/H₂O (1:99, 30 mL) and CHCl₃ (30 mL). The organic layer was separated, and the aqueous layer was extracted with CHCl₃ (30 mL). Combined organic phase was washed with ice-cold NaHCO₃/H₂O (30 mL), brine (30 mL), dried (Na₂SO₄), concentrated in vacuo and column chromatographed (EtOAc/hexane, 5:95 → 35:65) to give 25 (110 mg, 63%) as a colorless oil: ¹H NMR δ 1.86–1.96 (m, 1H), 2.05–2.15 (m, 1H), 2.48 (s, 3H), 3.31 (s, 3H), 3.47 (dd, J = 5.0, 8.6 Hz, 1H), 3.85 (dd, J = 4.2, 8.6 Hz, 1H), 3.89 (dd, J = 2.44, 11.5 Hz, 1H), 4.07 (dd, J = 4.44, 11.5 Hz, 1H), 4.51–4.63 (m, 2H), 5.12 (dt, J = 2.2, 4.7 Hz, 1H), 7.39 (d, J = 8.15 Hz, 2H), 7.85 (d, J = 8.15 Hz, 2H); ¹³C NMR δ 21.6, 30.5, 58.3, 69.8, 70.9, 75.9, 76.7, 83.7, 127.8, 129.9, 133.7, 145.2; HRMS ESI/DART m/z calcd for C₁₄H₂₃N₂O₈S [M + NH₄]⁺ 379.1170, found 379.1172.

2-O-Benzoyl-1,5-dideoxy-3-O-methyl-6-O-nitro-D-arabino-hexofuranose (26). Compound 23 (0.20 g, 0.97 mmol) in THF (6 mL) was added to a stirred solution of Ph₃P (0.31 g, 1.16 mmol) and PhCO₂H (0.14 g, 1.16 mmol) in THF (5 mL) at −50 °C. After 5 min. DIAD (0.23 g, 0.22 mL, 1.16 mmol) in THF (2 mL) was added slowly over 12 min. The reaction mixture was allowed to warm to room temperature within 1 h (it became colorless at −20 °C). Volatiles were evaporated, and the residue was column chromatographed (hexanes → hexanes/EtOAc, 10:1) to give 26 (0.23 g, 76%): ¹H NMR δ 2.07–2.17 (m, 2H), 3.50 (s, 3H), 3.71 (d, J = 3.4 Hz, 1H), 3.86–3.90 (m, 1H), 4.03–4.07 (m, 2H), 4.57–4.66 (m, 2H), 5.38–5.40 (m, 1H), 7.44–7.62 (m, 3H), 8.02–8.14 (m, 2H); ¹³C NMR δ 30.8, 58.0, 70.0, 71.7, 77.9, 80.4, 89.6, 128.4, 129.4, 130.1, 133.4, 165.7; MS FAB m/z 334 (15, [M + Na]⁺), 312 (100, [M + H]⁺); HRMS ESI m/z calcd for C₁₄H₁₇O₇NNa [M + Na]⁺ 334.0903, found 334.0904.

1,5-Dideoxy-3-O-methyl-6-O-nitro-D-arabino-hexofuranose (27). KOH (1.62 g, 28.9 mmol) in MeOH (80 mL) was added to a stirred solution of 26 (1.82 g, 5.85 mmol) in MeOH (80 mL). The reaction mixture was left to stir at room temperature for 1 h, then was neutralized with 5% HCl/H₂O. Volatiles were evaporated, and the residue was column chromatographed (hexanes/EtOAc, 3:1 → 1:1) to give 27 (0.87 g, 72%): ¹H NMR (300 MHz) δ 2.07–2.14 (m, 2H), 2.80 (br s, 1H), 3.42 (s, 3H), 3.48 (td, J = 1.1, 3.9 Hz, 1H), 3.79 (ddd, J = 3.9, 6.1, 7.3 Hz, 1H), 3.85 (br d, J = 10.3 Hz, 1H), 3.90 (dd, J = 3.7, 10.3 Hz, 1H), 4.27–4.29 (m, 1H), 4.54–4.67 (m, 2H); ¹³C NMR δ 30.9, 57.5, 70.2, 73.9, 75.2, 80.2, 91.6; MS FAB m/z 208 (5, [M + H]⁺), 71 (100); HRMS ESI m/z calcd for C₇H₁₄NO₆ [M + H]⁺ 208.0821, found 208.0807.

2-Chloro-3-O-methyl-6-O-nitro-1,2,5-trideoxy-D-arabino-hexofuranose (28). Solution of 23 (0.60 g, 2.90 mmol) in THF (15 mL) was added to a stirred solution of Ph₃P (1.52 g, 5.78 mmol) and DIAD (0.89 g, 0.86 mL, 4.38 mmol) in THF (15 mL) followed by addition of freshly prepared HCl•pyridine (0.50 g, 4.33 mmol). The suspension was stirred overnight at room temperature. Volatiles were evaporated, and the residue was chromatographed (hexanes → hexanes/EtOAc, 6:1) to give 28 (0.38 g, 61%): ¹H NMR δ 2.14–2.18 (m, 2H), 3.44 (s, 3H), 3.73 (d, J = 3.9 Hz, 1H), 3.78–3.82 (m, 1H), 4.02 (d, J = 10.7 Hz, 1H), 4.10 (dd, J = 4.4, 10.7 Hz, 1H), 4.24–4.27 (m, 1H), 4.56–4.67 (m, 2H); ¹³C NMR δ 31.3, 58.1, 59.4, 70.0, 74.2, 80.9, 92.8; HRMS ESI/DART m/z calcd for C₇H₁₆³⁵ClN₂O₅ [M + NH₄]⁺ 243.0742, found 243.0752; calcd for C₇H₁₆³⁷ClN₂O₅ [M + NH₄]⁺ 245.0716, found 245.0715.

2-Bromo-3-O-methyl-6-O-nitro-1,2,5-trideoxy-D-arabino-hexofuranose (29). Solution of 23 (0.65 g, 3.13 mmol) in THF (15 mL) was added to a stirred solution of Ph₃P (1.64 g, 6.25 mmol) and DIAD (0.95 g, 0.92 mL, 4.69 mmol) in THF (15 mL) followed by addition of freshly prepared HBr•pyridine (0.75 g, 4.69 mmol). The suspension was stirred overnight at room temperature. Volatiles were evaporated, and the residue was column chromatographed (hexanes/EtOAc, 10:1 → 6:1) to give 29 (0.39 g, 46%): ¹H NMR δ 2.19–2.27 (m, 2H), 3.49 (s, 3H), 3.82 (dt, J = 4.2, 6.8 Hz, 1H), 3.92 (d, J = 3.8 Hz, 1H), 4.14 (dd, J = 1.86, 11.0 Hz, 1H), 4.21 (dd, J = 4.47, 11.0 Hz, 1H), 4.27–4.31 (m, 1H), 4.58–4.70 (m, 2H); ¹³C NMR δ 31.5, 48.5, 58.0, 70.0, 74.6, 81.2, 93.0; HRMS ESI/DART m/z calcd for C₇H₁₆⁷⁹BrN₂O₅ [M + NH₄]⁺ 287.0237, found 287.0252; calcd for C₇H₁₆⁸¹BrN₂O₅ [M + NH₄]⁺ 289.0218, found 289.0233.

1,5-Dideoxy-2-O-tosyl-3-O-methyl-6-O-nitro-D-arabino-hexofuranose (30). TsCl (116 mg, 1.057 mmol) was added to a stirred solution of 27 (200 mg, 0.965 mmol) in anhydrous pyridine (1 mL) at ambient temperature. After 16 h, the volatiles were evaporated and residue was partitioned between ice-cold AcOH/H₂O (1:99, 30 mL) and CHCl₃ (30 mL). The organic layer was separated, and the aqueous layer was extracted with CHCl₃ (30 mL). Combined organic phase was washed with ice-cold saturated NaHCO₃/H₂O (30 mL), brine (30 mL) and dried (Na₂SO₄), concentrated in vacuo and column chromatographed (EtOAc/hexane, 5:95 → 35:65) to give 30 (240 mg, 69%) as a colorless oil: ¹H NMR δ 1.96–2.11 (m, 2H), 2.47 (s, 3H), 3.29 (s, 3H), 3.65 (td, J = 1.2, 4.4 Hz, 1H), 3.69–3.75 (m, 1H), 3.81 (dd, J = 4.1, 11.4 Hz, 1H), 3.92 (d, J = 11.4 Hz, 1H), 4.48–4.60 (m, 2H), 4.86 (td, J = 1.2, 4.1 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H). ¹³C NMR δ 21.6, 30.5, 58.0, 69.8, 71.3, 80.1, 83.1, 89.4, 127.8, 130.0, 133.4, 145.4; HRMS ESI/DART m/z calcd for C₁₄H₂₃N₂O₈S [M + NH₄]⁺ 379.1170, found 379.1169.

2-Chloro-1,2,5-trideoxy-3-O-methyl-6-O-nitro-D-ribo-hexofuranose (32). Step a: TfCl (123 μL, 195 mg, 1.16 mmol) was added to a stirred solution of 27 (200 mg, 0.96 mmol) and DMAP (354 mg, 2.9 mmol) in anhydrous CH₂Cl₂ (2 mL) at 0 °C (ice-bath). After 1 h, the reaction mixture was partitioned between ice-cold AcOH/H₂O (1:99, 30 mL) and CH₂Cl₂ (30 mL). The aqueous layer was extracted with CH₂Cl₂ (30 mL) and the combined organic phase was washed with ice-cold saturated NaHCO₃/H₂O (30 mL), brine (30 mL) and dried (Na₂SO₄) to give 31 as a colorless oil (243 mg, 74%) of sufficient purity to be used in next step. Column chromatography (EtOAc/hexane, 5:95 → 30:70) gave pure sample of 31: ¹H NMR δ 2.01–2.09 (m, 1H), 2.09–2.19 (m, 1H), 3.46 (s, 3H), 3.75–3.82 (m, 2H), 3.98 (dd, J = 3.6, 12.1 Hz, 1H), 4.17 (d, J = 12.1 Hz, 1H), 4.53–4.65 (m, 2H), 5.26 (d, J = 3.5 Hz, 1H); Step b. A solution of crude 31 (100 mg, 0.29 mmol; from step a) and dried LiCl (62.5 mg, 1.47 mmol) in DMF (1 mL) was stirred for 5 h at ambient temperature under N₂. Volatiles were evaporated and residue was partitioned between ice-cold saturated NaHCO₃/H₂O (15 mL) and EtOAc (15 mL). The separated organic phase was washed with brine (15 mL), dried (Na₂SO₄) and column chromatographed (EtOAc/hexane, 5:95 → 15:85) to give 32 (49 mg, 74%) as a colorless oil: ¹H NMR δ 1.89–2.02 (m, 1H), 2.06–2.18 (m, 1H), 3.44 (s, 3H), 3.58 (dd, J = 5.1, 8.0 Hz, 1H), 3.95 (td, J = 4.1, 8.3 Hz, 1H), 4.05 (dd, J = 2.7, 10.8 Hz, 1H), 4.32 (dd, J = 4.8, 10.8 Hz, 1H), 4.49 (td, J = 2.8, 5.0 Hz, 1H), 4.53–4.64 (m, 2H); ¹³C NMR δ 30.7, 57.6, 58.1, 69.9, 73.9, 76.1, 84.7; HRMS ESI/DART m/z calcd for C₇H₁₆³⁵ClN₂O₅ [M + NH₄]⁺ 243.0742, found 243.0747; calcd for C₇H₁₆³⁷ClN₂O₅ [M + NH₄]⁺ 245.0718, found 245.0716.

2-Bromo-1,2,5-trideoxy-3-O-methyl-6-O-nitro-D-ribo-hexofuranose (33). A solution of crude 31 (100 mg, 0.29 mmol; prepared as described for 32, step a) and dried LiBr (77 mg, 0.88 mmol) in DMF (1 mL) was stirred for 5 h at ambient temperature under N₂. Volatiles were evaporated and residue was partitioned between ice-cold saturated NaHCO₃/H₂O (15 mL) and EtOAc (15 mL). The separated organic phase was washed with brine (15 mL), dried (Na₂SO₄) and column chromatographed (EtOAc/hexane, 5:95 → 15:85) to give 33 (41 mg, 52%) as a colorless oil: ¹H NMR δ 1.90–2.0 (m, 1H), 2.06–2.16 (m, 1H), 3.39–3.45 (m, 4H), 3.97 (td, J = 4.2, 8.0 Hz, 1H), 4.20 (dd, J = 3.0, 11 Hz, 1H), 4.45 (dd, J = 4.8, 11.0 Hz, 1H), 4.50 (td, J = 2.9, 5.0 Hz, 1H), 4.53–4.64 (m, 2H); ¹³C NMR δ 30.7, 49.5, 58.2, 69.9, 74.0, 76.5, 84.4; HRMS ESI/DART m/z calcd for C₇H₁₆⁷⁹BrN₂O₅ [M + NH₄]⁺ 287.0237, found 287.0239; calcd for C₇H₁₆⁸¹BrN₂O₅ [M + NH₄]⁺ 289.0222, found 289.0218.

Biomimetic studies with 6-O-nitro-1,4-anhydrohexitols. Typical Procedure. A solution of 9 (20 mg, 0.06 mmol), Bu₃SnH (77 μL, 83 mg, 0.28 mmol), and AIBN (18 mg, 0.12 mmol) in dried toluene (2 mL) was deoxygenated (Ar) for 20 min and then heated at 95 °C for 1 h. Volatiles were evaporated carefully (at 25 °C and diminished pressure ~40 mmHg) and the residue was purified by column chromatography (EtOAc/hexane, 10:90 → 70:30) to give 1,2,5-trideoxy-D-glycero-hexofuranose-3-ulose 34a in equilibrium mixture (~1:1) with cyclic hemiacetal 35a (5 mg, 67%) as a colorless oil: HRMS ESI/DART m/z calcd for C₆H₁₁O₃ [M + H]⁺ 131.0703, found 131.0707. Ketone 34a had: ¹H NMR δ 1.84–1.93 (m, 1H), 1.95–2.06 (m, 2H), 2.15–2.27 (m, 2H), 3.74–3.85 (m, 2H), 3.88 (dd, J = 4.9, 7.4 Hz, 1H), 4.04–4.16 (m, 2H); ¹³C NMR δ 32.9, 38.9, 59.8, 68.9, 78.6, 215.9. Hemiacetal 35a had: 1.95–2.06 (m, 1H), 2.15–2.27 (m, 1H), 2.46–2.64 (m, 3H), 3.93–4.03 (m, 2H), 4.04–4.16 (m, 1H) 4.28 (dd, J = 2.27, 5.6 Hz, 1H), 4.37 (dt, J = 4.1, 9.2 Hz, 1H); ¹³C NMR δ 32.1, 36.7, 64.6, 68.3, 85.7, 114.9.

Also isolated from the reaction mixture was 1,5-dideoxy-2-O-tosyl-D-ribo-hexofuranose (3.3 mg, 19%): ¹H NMR δ 1.81–1.94 (m, 2H), 2.46 (s, 3H), 3.74–3.85 (m, 4H), 3.86–3.93 (m, 1H), 4.12 (dd, J = 4.9, 11.2 Hz, 1H), 4.95 (dt, J = 3.0, 5.3 Hz, 1H), 7.38 & 7.82 (2 × d, J = 8.3 Hz, 2 × 2H).

Analogous treatment of 9 (20 mg, 0.06 mmol) with Bu₃SnD (77 μL, 83 mg, 0.28 mmol), instead of Bu₃SnH gave 2-deuterio epimers (2R/S, ~1:1) of 34b in equilibrium mixture (~1:1) with 35b (5.2 mg, 71%) as a colorless oil: ¹H NMR spectrum of 34b/35b corresponded to this of the above 34a/35a with reduction of the integrated intensity for the H2/2′ signal at δ 2.15–2.27 and 2.46–2.64 to approximately half and simplification of the H1/1′ signals at δ 4.04–4.16 (m, 1H) and 4.33–4.40 (m, 1H). ¹³C NMR spectrum of 34b/35b showed triplets at δ 36.7 and 38.9 (J = 20.1 Hz) for C2 carbons because of splitting to deuterium and two close peaks of equal intensity for each hemiacetal carbons. HRMS ESI/DART m/z calcd for C₆H₁₀DO₃ [M + H]⁺ 132.0765, found 132.0768. Isotopic incorporation (MS) was calculated to be 85–95% for [²H] isotopomers of 34b/35b depends on the experiments. The ¹³C NMR spectrum for the sample of 34b/35b (2R/S, ~1:1, [²H] incorporation ~85%) showed residual peaks at 38.9 ppm and 36.7 for the unlabeled 34a and 35a, respectively and isotopically upfield shifted carbon signals for 34b (two sets of triplets of equal intensity at 38.60 and 38.63 ppm with J_C2–D = 20.1 Hz) and 35b two sets of triplets of equal intensity at 36.35 and 3.36 with J_C2–D = 20.1 Hz), respectively.

6-O-Benzoyl-1,2,5-trideoxy-D-glycero-hexofuranose-3-ulose (36a). BzCl (23 µL, 28 mg, 0.2 mmol), pyridine (44 µL, 43 mg, 0.54 mmol), and DMAP (4 mg, 0.032 mmol) were added to a stirred solution of 34a/35a (30 mg, 0.23 mmol), in CH₂Cl₂ (2 mL). Stirring was continued at ambient temperature for 3 h and MeOH (0.3 mL) was added. Volatiles were evaporated and the residue was chromatographed (EtOAc/hexane, 5:95 → 15:85) to give 36a (22 mg, 81%) as an colorless oil: ¹H NMR δ 2.06–2.16 (m, 1H), 2.18–2.28 (m, 1H), 2.51 (dd, J = 6.50, 8.26 Hz, 2H), 3.90 (dd, J = 4.71, 7.0 Hz, 1H), 4.05–4.13 (m, 1H), 4.28–4.36 (m, 1H), 4.39–4.46 (m, 1H), 4.47–4.54 (m, 1H), 7.41–7.47(m, 2H), 7.56 (tt, J = 1.5, 7.4 Hz, 1H), 8.0 (d, J = 8.57 Hz, 2H); ¹³C NMR δ 29.9, 36.8, 60.9, 64.7, 76.8, 128.5, 129.7, 130.2, 133.1, 166.5, 215.5; HRMS TOF/DART m/z calcd for C₁₃H₁₈NO₄ [M + NH₄]⁺ 252.1230, found 252.1234.

6-O-Benzoyl-2-deuterio-1,2,5-trideoxy-D-glycero-hexofuranose-3-ulose (36b). Treatment of 34b/35b (30 mg, 0.23 mmol) with BzCl, as described for 36a, gave 36b (12 mg, 67%) as a colorless oil: ¹H NMR spectrum of 36b corresponded to this of the above 36a with reduction of the integrated intensity for the H2/2′ signal at δ 2.51 to half and simplification of the H1/1′ signals at 4.05–4.13 and 4.28–4.36. ¹³C NMR spectrum showed triplet at δ 36.4 (J = 20.1 Hz) for C2 because of splitting to deuterium. HRMS ESI/DART m/z calcd for C₁₃H₁₃DNaO₄ [M + Na]⁺ 258.0847, found 258.0836.

Biomimetic studies with 3-O-methyl-6-O-nitro-1,4-anhydrohexitols. 2-(2-Hydroxyethyl)-3-methoxyfuran (37). A solution of 25 (25 mg, 0.069 mmol), Bu₃SnH (92 μL, 100 mg, 0.34 mmol), and AIBN (22.7 mg, 0.14 mmol) in dried toluene (2 mL), was deoxygenated (Ar) for 20 min and then heated at 95 °C for 1.5 h. Volatiles were evaporated and the residue was purified by column chromatography (EtOAc/hexane, 10:90 → 75:25) to give 37 (6.2 mg, 63%) followed by 1,5-dideoxy-2-O-tosyl-3-O-methyl-D-ribo-hexofuranose (38, 4.8 mg, 22%) as a colorless oils. Compound 37 had: ¹H NMR δ 2.89 (t, J = 6.0 Hz, 2H), 3.76 (s, 3H), 3.86 (t, J = 6.0 Hz, 2H), 6.31 (d, J = 2.1 Hz, 1H), 7.16 (d, J = 2.1 Hz, 1H); ¹³C NMR δ 29.3, 59.4, 60.9, 102.9, 136.7, 139.7, 144.3; HRMS ESI/FT-ICR m/z calcd for C₇H₁₁O₃ [M + H]⁺ 143.0702, found 143.0701. Compound 38 had: ¹H NMR δ 1.74–1.84 (m, 1H), 1.85–1.94 (m, 1H), 2.29 (t, J = 5.9 Hz, 1H), 2.46 (s, 3H), 3.32 (s, 3H), 3.49 (dd, J = 4.9, 8.3 Hz, 1H), 3.72–3.79 (m, 2H), 3.85–3.94 (m, 2H), 4.07 (dd, J = 4.7, 11.3 Hz, 1H), 5.11 (dt, J = 2.4, 4.8 Hz, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H); ¹³C NMR δ 21.6, 35.6, 58.3, 60.6, 70.9, 76.5, 79.1, 83.8, 127.8, 129.9, 133.8, 145.1; HRMS ESI/DART m/z calcd for C₁₄H₂₁O₆S [M + H]⁺ 317.1053, found 317.1055.

Analogous treatment of 25 with Bu₃SnD gave 37 (6.0 mg, 61%) and (38, 4.8 mg, 22%) with spectroscopic data as above.

Treatment of 30 (25 mg, 0.069 mmol) with Bu₃SnH, as described for 37, gave 37 (5.0 mg, 51%) with data as above and 1,5-dideoxy-2-O-tosyl-3-O-methyl-D-arabino-hexofuranose 39 (8.3 mg, 38%) as a colorless oil: ¹H NMR δ 1.86–1.94 (m, 2H), 2.04–2.11 (m, 1H), 2.47 (s, 3H), 3.31 (s, 3H), 3.70 (dt, J = 1.2, 5.0 Hz, 1H), 3.73–3.82 (m, 3H), 3.83 (d, J = 4.3 Hz, 1H), 3.93 (d, J = 11.7 Hz, 1H), 4.88 (dt, J = 1.3, 4.2 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H); ¹³C NMR δ 21.6, 35.3, 58.0, 60.5, 71.3, 82.7, 83.4, 89.7, 127.8, 130.0, 133.5, 145.3; HRMS ESI/DART m/z calcd for C₁₄H₂₁O₆S [M + H]⁺ 317.1053, found 317.1050.

Analogous treatment of 30 with Bu₃SnD gave 37 (4.9 mg, 50%) and 39 (8.2 mg, 38%) with spectroscopic data as above.

1,2,5-Trideoxy-3-O-methyl-D-glycero-hex-2-enofuranose (40). Typical Procedure. A solution of 29 (20 mg, 0.074 mmol), Bu₃SnD (99 μL, 108 mg, 0.369 mmol), and AIBN (24 mg, 0.146 mmol) in dried toluene (2 mL), was deoxygenated (Ar) for 20 min and then heated at 95 °C for 2 h. Volatiles were evaporated and the residue was purified by column chromatography (EtOAc/hexane, 10:90 → 70:30) to give 40 [22] (5.0 mg, 47%) as a colorless oil: ¹H NMR δ 1.73–1.87 (m, 1H), 1.94–2.09 (m, 1H), 2.58 (t, J = 5.6 Hz, 1H), 3.68 (s, 3H), 3.76–3.82 (m, 2H), 4.60–4.70 (m, 3H), 4.73–4.79 (m, 1H); ¹³C NMR δ 35.6, 57.6, 60.6, 72.8, 81.2, 90.2, 157.7; HRMS ESI/DART m/z calcd for C₇H₁₆NO₃ [M + NH₄]⁺ 162.1125, found 162.1131.

Comparison of Reaction Rates of 9, 13 or 18 with Bu₃SnH. Independent solutions of 0.057 mmol samples of 9, 13 and 18 in toluene–d₈ (2.0 mL) were treated with 5 molar equiv of Bu₃SnH and 2 molar equiv of AIBN at 75 °C. Aliquots of the individual reaction mixtures (0.3 mL) were diluted in toluene–d₈ (0.2 mL) and directly analyzed by ¹H NMR. The 34a/35a (1:1)/starting material ratios were obtained by integrating disappearance of the peak at 4.55 ppm for H2 of 9 or 18 or at 3.96 ppm for H6 of 13 and appearance of the peak at 4.10 ppm for the H4 of 34a. The determinations were conducted under the pseudo-first-order conditions:

k₁t = −2.303 log(C/C_o) + a

where C/C_o is the ratio of the concentration of starting material 9, 18, or 13 in the mixture at time t to the initial concentration of starting material. Values of the term [−log(C/C_o)] were plotted against [t(min)k(s⁻¹) = k₁ (min⁻¹⁾/3600].

5. Summary and Conclusions

Remarkable changes between anionic and radical elimination mechanisms occurred with our 1,4-anhydrofuranitols. All 2-(bromo, chloro, and tosylate) epimers that contained a 3-hydroxyl group underwent two-electron elimination of bromide, chloride, or tosylate ions to give the same furanone 34a or deuterated furanone 34b. The inductively donating C1 of the furanitols allows for the elimination of an anion from C2 upon generation of a radical center at C3. Loss of the 3-hydroxyl proton produces a 3-oxo-C2 radical, which abstracts hydrogen from HSnBu₃ or deuterium from DSnBu₃.

All of the 3-O-methyl-2-bromo- and 2-chloro analogues underwent one-electron elimination of a bromine or chlorine atom to produce 2-(2-hydroxyethyl)-3-methoxy-2,5-dihydrofuran 40. No deuterium incorporation occurred in these cases. The 3-O-methyl-2-O-tosyl epimers underwent more drastic fragmentation upon generation of a C3 radical. Elimination of toluenesulfonic acid from C2/C1 and abstraction of hydrogen from C4 produced the aromatized 2-(2-hydroxyethyl)-3-methoxyfuran 37.

The inductively negative anomeric carbon (C1′) in nucleos(t)ides disfavors elimination of a chloride anion from C2′. Loss of Cl• followed by 1,4-elimination of the 3′-hydroxyl proton and base gives the unlabeled 2-(2-hydroxyethyl)-3(2H)-furanone (path b, Figure 4). The abstraction of deuterium from DSnBu₃ by Cl• provides a chain propagation step and so no label is incorporated into the furanone product.

By contrast, dissociation of a prohibitively high-energy tosyloxy radical is precluded so that departure of a tosylate anion from C2′ occurs. Loss of the 3′-hydroxyl proton gives a 3′-oxo-C2′ radical, which abstracts deuterium from DSnBu₃ and undergoes elimination of H/D and nucleobase to produce 4-deuterio-2-(2-hydroxyethyl)-3(2H)-furanone (path a, Figure 4).

Our combined results with nucleoside and anhydroalditol models provide data for plausible mechanistic rationalization of the two-electron elimination of hydrogen-bonded water from substrate nucleoside di(or tri)phosphates, and for one-electron dissociation of a chlorine atom from 2′-chloro-2′-deoxynucleoside di(or tri)phosphate inactivators of ribonucleotide reductases and the MoaA enzyme. Such Cl• radicals could react with active site components and contribute to enzyme inactivation.

Theoretical studies [18,19,44] employ major assumptions and calculation simplifications relative to actual biological systems. Mechanistic hypotheses based on reactions executed by modified enzymes [26,46] also involve unnatural “biomimetic” models—whether prepared by chemical synthesis or molecular biology. Combination of theoretical, biochemical, and biomimetic modeling provides more insightful approximations that any one of the individual models. Results of our present studies add clarity to hypotheses postulated for the radical chemistry-based inactivation of RNRs [30] and MoaA [32].