Subscriber access provided by JUBILANT ORGANOSYS LTD Article High-Affinity Alkynyl Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT) Rocco L. Policarpo, Ludovic Decultot, Elizabeth May, Petr Kuzmic, Samuel Carlson, Danny Huang, Vincent Chu, Brandon A. Wright, Saravanakumar Dhakshinamoorthy, Aimo Kannt, Shilpa Rani, Sreekanth Dittakavi, Joseph D. Panarese, Rachelle Gaudet, and Matthew D. Shair J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01238 • Publication Date (Web): 07 Oct 2019 Downloaded from pubs.acs.org on October 8, 2019

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1 High-Affinity Alkynyl Bisubstrate Inhibitors of Nicotinamide 2 3 N-Methyltransferase (NNMT) 4 5 Rocco L. Policarpoa, Ludovic Decultota, Elizabeth Mayb, Petr Kuzmičc, Samuel Carlsonb, 6 7 Danny Huanga, Vincent Chua,1, Brandon A. Wrighta,2, Saravanakumar Dhakshinamoorthyd, 8 Aimo Kannte, Shilpa Ranid, Sreekanth Dittakavid, Joseph D. Panaresea,3, Rachelle Gaudetb, 9 10 Matthew D. Shaira* 11 12 13 aDepartment of Chemistry and Chemical Biology and bDepartment of Molecular and Cel- 14 15 lular Biology, Harvard University, Cambridge MA 02138, USA 16 cBioKin Ltd., Watertown MA 02472, USA 17 18 dJubilant Biosys Ltd., Yeshwantpur Bangalore - 560 022, Karnataka, India 19 eSanofi Research and Development, Industriepark Hoechst, H823, D-65926, Frankfurt am 20 21 Main, Germany 22 23 24 25 ABSTRACT 26 Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that methylates nic- 27 28 29 otinamide (NAM) using cofactor S-adenosylmethionine (SAM). NNMT overexpression 30 31 32 has been linked to diabetes, obesity, and various cancers. In this work, structure-based ra- 33 34 tional design led to the development of potent and selective alkynyl bisubstrate inhibitors 35 36 37 of NNMT. The reported nicotinamide-SAM conjugate (named NS1) features an alkyne as 38 39 40 a key design element that closely mimics the linear, 180° transition state geometry found 41 42 43 in the NNMT-catalyzed SAM → NAM methyl transfer reaction. NS1 was synthesized in 44 45 14 steps and found to be a high-affinity, subnanomolar NNMT inhibitor. An X-ray co- 46 47 48 crystal structure and SAR study revealed the ability of an alkynyl linker to span the methyl 49 50 51 transfer tunnel of NNMT with ideal shape complementarity. The compounds reported in 52 53 this work represent the most potent and selective NNMT inhibitors reported to date. The 54 55 1 56 57 58 59 60 ACS Paragon Plus Environment

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1 rational design principle described herein could potentially be extended to other methyl- 2 3 transferase enzymes. 4 5 6 7 8 9 INTRODUCTION 10 11 12 Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme responsible for the 13 14 methylation of nicotinamide (NAM) using the cofactor S-adenosylmethionine (SAM), re- 15 16 17 sulting in the production of 1-methylnicotinamide (MNAM) and S-adenosylhomocysteine 18 19 1-2 20 (SAH). While initially established as a vitamin B3 clearance enzyme, recent work has 21 22 shown NNMT to be an important regulator of several intracellular pathways in fat, liver, 23 24 25 and cancer cells.2 NNMT can regulate methyl donor metabolism by direct interaction with 26 27 3 28 proteins of the methionine cycle and can regulate hepatic nutrient metabolism through 29 30 Sirt1 stabilization4. By decreasing cellular SAM levels, NNMT can modulate the epige- 31 32 33 netic landscape of cancer cells5 and embryonic-stem cells6. 34 35 36 Accordingly, NNMT has emerged as a disease-relevant enzyme and a possible point of 37 38 39 therapeutic intervention. Abnormal NNMT expression is implicated in several diseases, 40 41 including diabetes7-8, obesity7, 9, and a variety of cancers2, 5, 10. Increased NNMT protein expres- 42 43 44 sion was observed in adipose and liver tissue of obese and diabetic mice7, while increased 45 46 47 NNMT mRNA expression was observed in humans11. NNMT is overexpressed in glioblas- 48 49 50 toma, leading to cellular SAM depletion, DNA hypomethylation, and accelerated tumor 51 52 53 54 55 2 56 57 58 59 60 ACS Paragon Plus Environment

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12 1 growth. Recently, NNMT was shown to be a master metabolic regulator of cancer-asso- 2 3 ciated fibroblasts, with NNMT expression supporting ovarian cancer migration, prolifera- 4 5 6 tion, and metastasis.13 The successful development of potent and selective NNMT inhibitors 7 8 9 would aid efforts to elucidate the role of NNMT in disease, potentially enabling new strat- 10 11 egies to treat a variety of metabolic disorders, cancers, and other pathologies. 12 13 14 Several NNMT inhibitors have been reported, including methylated quinolines14, nicotin- 15 16 17 amide analogues15-16, covalent inhibitors17-18, and amino-adenosine derived bisubstrate inhibi- 18 19 19-21 20 tors . Bisubstrate inhibitors are compounds that bind both the substrate and cofactor bind- 21 22 ing pockets within an enzyme active site. The design of bisubstrate inhibitors relates 23 24 25 closely to two well-established ligand design strategies: transition-state mimicry and frag- 26 27 22 28 ment-based design. Importantly, if two weak inhibitors that bind a protein at distinct sites 29 30 are linked in an optimal manner, the resulting bisubstrate inhibitors can obtain several or- 31 32 33 ders of magnitude increase in binding affinity.23-24 As a result, linker identity is crucial to 34 35 36 properly position the key interacting groups of the two component molecules and minimize 37 38 39 unfavorable interactions between the linker and the protein. 40 41 In this work, we used a combination of structure-based design and molecular docking to 42 43 44 develop high-affinity alkynyl bisubstrate inhibitors of NNMT. To begin, we examined the 45 46 47 substrate-bound NNMT co-crystal structure (PDB ID 3ROD) and noted that the NAM ni- 48 49 50 trogen and SAH sulfur atoms are positioned ~ 4 Å apart and reside in a small tunnel that 51 52 25 facilitates the SN2-type methyl transfer reaction (Figure 1a,b). We envisioned an alkyne as 53 54 55 3 56 57 58 59 60 ACS Paragon Plus Environment

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1 the optimal linker between a NAM-like and a SAM-like fragment, as an alkyne linker 2 3 resembles the linear, 180° transition state geometry found in the SAM → NAM methyl 4 5 6 transfer reaction (Figure 1c,d). 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figure 1. Overview of the alkynyl bisubstrate strategy. (a) Binding pose of ligands SAH 40 41 42 and NAM (teal) rendered from previously published co-crystal structure (PDB ID 3ROD). 43 44 45 (b) Graphical representation of the SAM → NAM methyl transfer reaction catalyzed by 46 47 48 NNMT. (c) Graphical representation of alkynyl bisubstrate inhibitor NS1 posed to mimic 49 50 the binding geometry of SAM/NAM. (d) Molecular docking output pose of alkynyl bisub- 51 52 53 strate inhibitor NS1 (black) overlaid with SAH and NAM from structure 3ROD (teal). 54 55 4 56 57 58 59 60 ACS Paragon Plus Environment

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1 Thus, we rationally designed alkynyl nicotinamide-SAM conjugate 1 (named NS1, 10). 2 3 Molecular docking with Glide26 predicted NS1 to be a better binder than SAM by > 3 4 5 6 kcal/mol (see Supporting Information Part 1). The docked pose of NS1 (Figure 1d) over- 7 8 9 lays well with NAM and SAH in the previously published substrate-bound crystal struc- 10 11 ture; the alkyne linker fits well into the methyl transfer tunnel. With molecular docking 12 13 14 suggesting NS1 as a potential NNMT inhibitor, we aimed to synthesize and test NS1 in an 15 16 17 NNMT inhibition assay. 18 19 20 6’-alkynyl nucleosides were first proposed and synthesized in 1990 as mixtures of al- 21 22 kynyl and amino acid diastereomers.27 Unfortunately, the designed alkynyl nucleosides 23 24 25 were poor inhibitors when tested against catechol O-methyltransferase (COMT).28 In this 26 27 28 work, we demonstrate that the alkynyl bisubstrate strategy toward SAM-dependent me- 29 30 thyltransferase inhibition is a valuable approach. We synthesized the rationally designed 31 32 33 alkynyl bisubstrate compound NS1 as a single enantiomer and diastereomer in 14 steps 34 35 36 and showed it to be a high-affinity, selective NNMT inhibitor with a Ki of 500 pM. 37 38 39 RESULTS AND DISCUSSION 40 41 42 NS1 Synthesis. The synthesis of NS1 (10, Scheme 1) began with commercially available 43 44 45 acetonide 1. Alkylation of the corresponding triflate with lithium TMS-acetylide followed 46 47 29 48 by silyl deprotection generated alkyne 2. Copper-catalyzed semi-reduction of 2 rendered 49 50 terminal alkene 3, which was converted to enal 4 by cross-metathesis with crotonaldehyde. 51 52 53 Solvent and catalyst screening (Table S1, Figure S1) revealed methyl tert-butyl ether 54 55 5 56 57 58 59 60 ACS Paragon Plus Environment

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30 1 (MTBE) and the commercially available Nitro-Grela olefin metathesis catalyst as an op- 2 3 timal solvent/catalyst system to effect this transformation. The key C6’ stereocenter was 4 5 6 installed by a rhodium-catalyzed asymmetric alkynylation31 to afford aldehyde 5 as a single 7 8 9 diastereomer (see Supporting Information Part 1, Section 3 for numbering scheme). A 10 11 Horner-Wadsworth-Emmons olefination with a phosphonoglycine derivative yielded en- 12 13 14 amide 6. The C17’ stereocenter was set via rhodium-catalyzed asymmetric hydrogenation 15 16 32 17 with a Burk-type 1,2-bis(phospholano)ethane (BPE) ligand, providing (S)-trifluoroa- 18 19 cetamide 7 as a single diastereomer. Subsequent acylation and Vorbrüggen glycosylation 20 21 22 promoted by a 2,6-di-tert-butyl-4-methylpyridinium triflate salt33 delivered nucleoside 8. 23 24 25 Introduction of the benzamide moiety by a Sonogashira cross-coupling and a global depro- 26 27 tection sequence completed the synthesis of NS1 (10) in a total of 14 steps with an overall 28 29 30 yield of 9.1%. The small-molecule X-ray crystal structure of NS1•TFA was obtained, con- 31 32 33 firming the regiochemistry of the adenine nucleobase (N9-linkage) as well as the configu- 34 35 36 ration of the C6’ and C17’ stereocenters. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 6 56 57 58 59 60 ACS Paragon Plus Environment

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1 H H H H H H H H O OMe 1) Tf O, 2,6-lutidine O OMe IPrCuCl, t-BuOK O OMe crotonaldehyde O O OMe 2 HO 2 CH2Cl2, - 78 °C PMHS, i-BuOH nitro-Grela (1 mol %) 3 H OO 2) TMS-CCH, n-BuLi, DMPU OO PhMe, RT, 93% OO MTBE, 40 °C, 71% OO 4 THF, - 78 °C then TBAF 5 1 68% (2 steps) 2 3 4 6 NHTFA TIPS-CCH H H OMe NHTFA NHTFA O 6’ O OMe MeO OMe H H H H 7 [Rh(OAc)(C2H4)2]2 P MeO O OMe H2 MeO O OMe (S)-DTBM-SegPhos O O , NaH (S,S)-MeBPE-Rh 17’ 8 H O O MeOH, 40 °C, 94% THF, 40 °C MeOH, RT, 92% OO OO OO 9 89%, 6:1 Z:E 10 5 TIPS 6 TIPS 7 TIPS 11 N N 12 1) TASF, DMF, 60 °C, 95% NHTFA NHBz 3-iodobenzamide NHTFA NHBz H H H H 13 2) TFA/H2O/CH2Cl2, RT MeO O N CuI, Pd(PPh3)4 MeO O N N N 14 3) Ac2O, Pyr, DMAP O N PhMe/DMF/i-Pr2NEt (5:1:1) O N CH2Cl2, RT, 52% (2 steps) 60 °C, 90% 15 4) N6-BzAd, BSA, DTBMP triflate AcO OAc AcO OAc EtCN, 95 °C, 67% 8 9 16 N NH2 17 NH3•TFA NH H H 2 HO 6’ O N O 18 17’ 1) LiOH aq., THF N 19 O N HO OH 20 2) NH3, MeOH then HPLC 21 88% (2 steps) 22 NH2

23 O NS1 (1 0) 24 25 Scheme 1. Synthesis and X-Ray Crystal Structure of NS1 (10) 26 27 28 Biochemical Evaluation of NS1 and Analogues. A fluorescence-based NNMT inhibi- 29 30 31 tion assay34 revealed NS1 to be a highly potent, subnanomolar (500 pM) NNMT inhibitor. 32 33 34 In the assay, NNMT catalyzes the SAM-dependent methylation of quinoline, forming 1- 35 36 methyquinolinium (1MQ) and SAH. Quinoline as opposed to NAM as methyl-group ac- 37 38 39 ceptor has been previously employed by Loring and Thompson35 who determined the de- 40 41 42 tailed kinetic mechanism of NNMT (Rapid Equilibrium Random Bi Bi in Cleland's no- 43 44 menclature36-38) and thus indirectly established that NAM and quinoline are mechanistically 45 46 47 equivalent as mutually interchangeable methyl group acceptors. 48 49 50 More importantly for the purposes of our inhibition study, quinoline enables continuous 51 52 53 real-time monitoring of the reaction progress, using fluorescence detection to monitor the 54 55 7 56 57 58 59 60 ACS Paragon Plus Environment

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1 appearance of 1MQ. In contrast, previously described NNMT assays using NAM as native 2 3 substrate were forced to rely on discontinuous (end-point) detection39 and are thus unsuita- 4 5 6 ble for real-time recording of continuous reaction progress, as was done in our study. Uti- 7 8 9 lization of a quinoline-based continuous real-time assay was especially important because 10 11 many enzymatic progress curves were prominently nonlinear, both in the presence and in 12 13 14 the absence of inhibitors (Figure 2). In the specific case of NS1, the reaction progress 15 16 17 curves were strongly nonlinear even within the first two minutes of the inhibition assay 18 19 and therefore no "initial linear region" could be identified. Thus, as a matter of principle, 20 21 22 the inhibitors reported in this study could not be studied by conventional initial rate kinetic 23 24 25 methods.40-41 26 27 28 Therefore we analyzed the full reaction time-course using the software package 29 30 DynaFit.42-43 The mathematical models for each of several postulated kinetic mechanisms 31 32 33 were systems of first-order Ordinary Differential Equations (ODEs) automatically derived 34 35 36 by the DynaFit software. Inhibition constants were computed as the ratio Ki = koff/kon, where 37 38 39 koff is the microscopic rate constant for the dissociation of the enzyme-inhibitor complex 40 41 and kon is the corresponding microscopic association rate constant. (for details, see Support- 42 43 44 ing Information Part 3, Section 2, pp. 13-15). Because the Ki values reported in this work 45 46 47 span six orders of magnitude, they are presented and discussed after logarithmic scaling, 48 49 as pKi (Figures 4, 5). 50 51 52 53 54 55 8 56 57 58 59 60 ACS Paragon Plus Environment

Page 9 of 188 Journal of Medicinal Chemistry

1 Note that the inhibition constants, Ki, reported in this study are true dissociation constants 2 3 app of the enzyme—inhibitor complex, as opposed to apparent inhibition constants, Ki , and 4 5 6 therefore are entirely independent of the nature of the particular substrate (quinoline vs. 7 8 44 9 NAM). This interpretation is based on the assumption, supported by the X-Ray structural 10 11 studies reported herein, that bisubstrate inhibitors in principle bind to the target enzyme in 12 13 14 such a way that the binding of a bisubstrate inhibitor precludes any simultaneous binding 15 16 17 of either of the two co-substrates. 18 19 20

21 0 nM 24 22 36 55

23 2 83 24 125 190 25 288 6 , rfu 436 26 0 661 1 27 F / 1 ∆ 28 29 30 0 31 0.1 32 0.05 33 0

residuals -0.05 34 35 0 200 400 600 800 36 t, sec 37 Figure 2. Kinetic studies of NNMT. A time course of the NNMT kinetic assay in the 38 39 40 absence (red circles) or in the presence (remaining symbols) of NS1. Inhibitor concentra- 41 42 Δ 43 tions are shown at top right. F are scaled fluorescence changes in relative fluorescence 44 45 units (“rfu”). The smooth curves represent the best least-squares fit to a system of first- 46 47 48 order differential equations automatically generated by the DynaFit software package42-43 49 50 51 according to the reaction mechanism shown in the Supporting Information (Part 3: Enzyme 52 53 Kinetics). 54 55 9 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 3 X-Ray Co-Crystal Structure of NS1 Bound to hNNMT. We determined the co-crystal 4 5 6 structure of NS1-bound NNMT (Figure 3, PDB ID 6ORR, Table S2) and observed an NS1- 7 8 9 shaped density in the NAM/SAM binding pocket in the electron density map (Figure 3a). 10 11 12 Fitting NS1 into the density decreased the Rfree value and improved the map quality, reveal- 13 14 ing that NS1 occupies both the NAM and SAM binding sites in the same orientation as the 15 16 17 native substrates, with the alkynyl linker occupying the methyl transfer tunnel. The NS1 18 19 20 alkyne is an optimal surrogate for the SAM-dependent methyl transfer reaction of NNMT 21 22 and effectively positions both structural components of NS1 properly in space, allowing 23 24 25 NS1 to capture similar binding interactions as the native substrates (Figure 3b). 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 10 56 57 58 59 60 ACS Paragon Plus Environment

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1 NS1 exploits the same amino acids that contact the native substrates to bind NNMT. To 2 3 stabilize the adenosine portion of NS1, D142 forms a hydrogen bond with the primary 4 5 6 amine on the adenine nucleobase, while the hydroxyl groups of the ribose hydrogen-bond 7 8 9 extensively with the backbone nitrogen atom of S87 and the side chains of D85 and N90 10 11 (Figure 3c). The carboxylic acid of the NS1 amino acid accepts four hydrogen bonds from 12 13 14 Y20, Y25, Y69, and T163, while the amino group hydrogen-bonds with the backbone car- 15 16 17 bonyl of G63 (Figure 3d). A hydrophobic clamp positions the aromatic ring of the ben- 18 19 zamide moiety between Y204 and L164, with S201 and S213 stabilizing the amide group 20 21 22 through hydrogen bonds (Figure 3e). 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 11 56 57 58 59 60 ACS Paragon Plus Environment

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1 Figure 3. Co-crystal structure of NS1-bound NNMT (PDB ID 6ORR). (a) A 2Fo-Fc omit 2 3 map contoured at 1σ reveals NS1-shaped electron density in the binding pocket of NNMT. 4 5 6 (b) NS1 (black) binds NNMT in an orientation that closely resembles previously published 7 8 9 co-crystal structure containing ligands SAH and NAM (cyan; PDB ID 3ROD). (c-e) NS1 10 11 also exploits the same protein interactions that NNMT uses to bind its native substrates. 12 13 14 Ligand-interacting residues from NNMT are shown in yellow; black dotted lines represent 15 16 17 hydrogen bonds. (f) MS2756-bound NNMT (brown; PDB ID 6B1A) superimposed on 18 19 NS1-bound NNMT. 20 21 22 23 24 25 Stucture-Activity Relationships (SAR). The synthesis of NS1 was designed to be 26 27 28 highly modular to facilitate a systematic structure-activity analysis of our bisubstrate ap- 29 30 31 proach (Figures 4,5). SAR analysis began with the minimal motif desthia-SAH (11) and 32 33 built up to the full NS1 structure incrementally (Figure 4a). Desthia-SAH, completely lack- 34 35 36 ing an alkynyl-benzamide side chain, was a poor NNMT inhibitor. Addition of an acetylene 37 38 39 (12) or a phenyl-acetylene (13) side chain yielded equally poor NNMT inhibitors. The 40 41 potency of the parent compound NS1 (10) highlights the importance of the benzamide 42 43 44 functionality in driving compound potency. 45 46 47 48 49 50 51 52 53 54 55 12 56 57 58 59 60 ACS Paragon Plus Environment

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1 A fundamental aspect of our design strategy relied on the C6’ stereocenter to properly 2 3 direct the NS1 alkyne toward the NAM binding pocket. Analysis of the NNMT crystal 4 5 6 structure (3ROD) suggested that the S stereochemistry would best capture the transition 7 8 9 state geometry modeled in Figure 1. Accordingly, NS1 was a better inhibitor than the NS1- 10 11 C6’ epimer (14) by > 200-fold in terms of the inhibition constant Ki. The linear vector 12 13 14 maintained by the alkynyl linker of NS1 proved optimal; NS1-alkane (15) was almost 10- 15 16 17 fold less potent than NS1 itself (in terms of Ki). The NS1-alkane 6’ epimer (16) and alkynyl 18 19 20 21 22

a N 23 NH2 NH H H 2 NH N N 2 NH2 HO O N NH2 NH H H 24 H H 2 HO O N N HO O N N N O 25 N O N HO OH O H H N HO OH HO OH 26 H 27 11 4.67 ± 0.01 12 4.32 ± 0.08 13 4.90 ± 0.03 28 NH N NH N N 2 NH2 2 NH2 NH2 NH NH N H H H H H H 2 2 NH2 29 HO O N O N H H HO HO O N HO O N N N N N O N N 30 O O N O N HO OH 31 HO OH HO OH HO OH NH 2 NH2 32 NH2 NH2 O 33 O O O 14 6.91 ± 0.07 NS1-alkane (15) 8.33 ± 0.03 16 6.97 ± 0.05 34 NS1 (10) pKi = 9.30 ± 0.07

NH2 35 O NH2 H H O NH2 HO O OMe

36 O N N NH HO OH NH H H 2 37 H H 2 O O N N N 38 N N N NH2 HO OH HO OH 39 O 40 17 6.58 ± 0.04 18 5.41 ± 0.05 19 4.11 ± 0.17 41

b N N N NH NH2 NH NH 42 H H 2 H H 2 H H 2 N O O O NH2 NH HO N N H2N N H H 2 43 HO O N N N N O N N O N N 44 O N HO OH HO OH HO OH 45 HO OH NH2 NH2 NH2 NH 46 2 O O O 47 O 20 4.65 ± 0.06 21 7.24 ± 0.02 22 5.98 ± 0.01 48 NH N N N N 2 NH2 NH2 NH NH O NH H H H H 2 H H H 2 H H 2 O N 49 MeO H2N O N H2N N O N O N HO N N N N O N N N N 50 O O NH2 HO OH 51 HO OH HO OH HO OH

NH 52 2 NH2 NH2 NH2 53 O O O O 54 23 6.36 ± 0.08 24 7.77 ± 0.02 25 7.12 ± 0.05 26 9.62 ± 0.05 55 13 56 57 58 59 60 ACS Paragon Plus Environment

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1 epimer 14 had similar pKi values (6.97 vs. 6.91, respectively). These results may be ex- 2 3 plained by the shape of the NNMT binding pocket; the narrow width of the methyl transfer 4 5 6 tunnel may accommodate linear alkynyl geometry better than a staggered aliphatic chain. 7 8 9 Furthermore, the rigidity of the alkynyl linker could lower the entropic cost of binding 10 11 relative to alkane containing analogues. 12 13 14 15 16 17 Figure 4. Structure-activity relationship (SAR) studies of bisubstrate NNMT inhibitors. 18 19 20 pKi values (-log10Ki) are shown in red and are used to facilitate comparison between inhib- 21 22 23 itors of widely varying potency. (a) Exploration of the alkynyl bisubstrate approach to 24 25 NNMT inhibition. (b) Amino acid modifications. 26 27 28 29 30 31 More drastic structural modifications to the NS1 scaffold included excision of the entire 32 33 34 amino-acid side chain (17) or the entire adenine nucleobase (19). Compound 17, a simpli- 35 36 37 fied bisubstrate inhibitor designed to target the adenosine and NAM binding pockets of 38 39 NNMT, was a weaker inhibitor than NS1 by ~ 500-fold in terms of Ki. Attempts to improve 40 41 42 the inhibitory activity of 17 by conformational restriction (via the installation of a cyclo- 43 44 45 propyl group; 18) proved detrimental. Removal of the adenine nucleobase (19) completely 46 47 abrogated NNMT inhibition. 48 49 50 SAR: Amino Acid Modification. Alteration of the NS1 amino acid (Figure 4b) revealed 51 52 53 the C17’ amino group of NS1 to be critical for inhibition, as compound 20 (lacking the 54 55 14 56 57 58 59 60 ACS Paragon Plus Environment

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1 C17’ amine) was a poor NNMT inhibitor. Removal of the carboxylic acid was better toler- 2 3 ated, as compound 21 (which retains the C17’ amine) had a pKi of 7.24 ± 0.02. Amide- 4 5 6 (22) and methyl ester-containing (23) analogues were poor NNMT inhibitors (5.98 ± 0.01 7 8 9 and 6.36 ± 0.08, respectively), while amino-amide- (24) or urea-containing (25) analogues 10 11 were slightly better (7.77 ± 0.02 and 7.12 ± 0.05, respectively). These results are consistent 12 13 14 with the structural features of 24 and 25, as both contain the same number of potential- 15 16 17 hydrogen bonding groups as NS1 and native substrate SAH (although differing slightly in 18 19 their donor/acceptor capacities and positions). Compound 22 has one fewer hydrogen- 20 21 22 bonding atom than NS1. In 23, the methyl ester likely adds too much bulk to fit in the 23 24 25 amino acid pocket while also reducing the hydrogen bonding ability of the amino acid 26 27 carboxylate. 28 29 30 Only one side chain modification yielded a more potent compound than NS1: the addition 31 32

33 of a single methylene unit in the NS1 backbone (26, homo-NS1). Homo-NS1 had a pKi of 34 35 36 9.62 ± 0.05 (compared to NS1 with pKi = 9.30 ± 0.07), indicating a ~ 2-fold increase in 37 38 39 inhibitory activity in terms of Ki. This finding is consistent with prior reports showing that 40 41 the addition of a methylene unit in amino-bisubstrate NNMT inhibitors can improve po- 42 43 44 tency.20 We also synthesized and tested previously reported compounds MS2734 and 45 46 47 MS2756 (Figure 5b). In our hands, MS2734—a one-carbon chain-extended homologue of 48 49 MS2756—was a 10-fold better inhibitor than MS2756 in terms of Ki (pKi of 7.05 ± 0.01 50 51 52 vs. 6.04 ± 0.06). The benefit of an extra methylene group in bisubstrate inhibitors could 53 54 55 15 56 57 58 59 60 ACS Paragon Plus Environment

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1 arise because an added methylene better mimics the longer C—S bonds of the native sub- 2 3 strates. The trimethylene linker of homo-NS1 would also be more flexible than the di- 4 5 6 methylene linker of NS1, which may allow the molecule more room to optimally satisfy 7 8 9 hydrogen bonds between the amino acid, benzamide, ribose and adenine moieties to the 10 11 NNMT binding pocket. 12 13 14 SAR: Aryl Modification. After examining the impact of side chain modifications on 15 16 17 inhibitor potency, we tested the effects of aryl group modification. We purposely designed 18 19 20 the NS1 synthesis to accommodate a late-stage Sonogashira cross-coupling reaction to al- 21 22 low for easy introduction of a desired aryl moiety late in our synthesis. Leveraging this 23 24 25 capability, we prepared gram quantities of intermediate 8 (Scheme 1) and synthesized a 26 27 28 variety of aryl substituted NS1 analogues (Figure 5a). 29 30 31 These studies revealed that proper positioning of an amide group on NS1 is essential for 32 33 NNMT binding, as para-benzamide (27), ortho-benzamide (28), and sulfonamide (29) 34 35 36 substituted NS1 analogues were much less potent than NS1. We designed fluorine (30), 37 38 39 methyl (31), trifluoromethyl (32), and chlorine (33) containing analogues to 1) bind a small 40 41 hydrophobic pocket in the nicotinamide binding site, 2) modulate the pKa of the NS1 ben- 42 43 44 zamide group through inductive effects, and 3) potentially alter the rotation of the NS1 45 46 47 amide relative to the NS1 benzamide phenyl ring. While 30, 31, and 32 were potent NNMT 48 49 50 inhibitors (pKi > 8.0), only chloro-substitution (33) yielded a more potent NNMT inhibitor 51 52 than NS1 (pKi = 9.72 ± 0.15). As shown in Figure 6a, there is a small hydrophobic region 53 54 55 16 56 57 58 59 60 ACS Paragon Plus Environment

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1 in the NAM binding pocket para to the alkyne of the NS1 scaffold. The small, hydrophobic 2 3 chlorine atom of 33 likely makes Van der Waals contacts with this region, providing addi- 4 5 6 tional binding affinity. 7 8 9 After interrogating ring substitution at the para-position, we attempted to restrict rotation 10 11 12 of the NS1 amide by converting the benzamide moiety to a benzolactam (Figure 5a, 34 and 13 14 35). Benzolactams 34 and 35 were moderate NNMT inhibitors, with a six-membered ring 15 16 17 (34) better tolerated than a five-membered ring (35). We also examined C13’/C14’ substi- 18 19 20 tution with methylenedioxy analogue 36 (pKi = 7.49 ± 0.05) or introduced a heterocyclic 21 22 nitrogen atom (to better mimic NAM, 37, 38, 39, 40). Compounds 36-40 all had pKi values 23 24 25 below that of NS1, demonstrating that C13’/C14’ substitution or inclusion of a nitrogen 26 27 28 atom are not beneficial in these cases. Lastly, we designed and synthesized amino naph- 29 30 thalene derivative 41 (a bisubstrate analogue of the previously reported NNMT inhibitor 31 32 45 33 5-amino-1-methylquinolinium ), but 41 was also a poor inhibitor (pKi = 5.43 ± 0.07). 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 17 56 57 58 59 60 ACS Paragon Plus Environment

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a 1 O

H2N NH2 NH2 NH 2 S 2 O O F O 3 O NH2 Me O 4 27 5.24 ± 0.02 28 4.71 ± 0.02 29 6.71 ± 0.05 30 8.73 ± 0.15 31 8.47 ± 0.09 N NH2 NH H H 2 5 O HO N O N 6 O N NH2 NH2 O NH O O 2 HO OH 7 CF3 O Cl O NH NH O 32 8.58 ± 0.08 33 9.72 ± 0.15 34 7.26 ± 0.05 35 5.73 ± 0.02 36 7.49 ± 0.05 8 NH2 9 O N N 10 N NH NH2 NH2 2 NH2 N O 11 O O O NH2 12 37 8.17 ± 0.05 38 8.05 ± 0.10 39 8.30 ± 0.02 40 7.45 ± 0.07 41 5.43 ± 0.07 13 NH N N b NH N 2 NH2 O NH 2 NH2 H H H H 2 14 H H O O HO O N HO N N N N HO N N N N N N 15 O N O NH2 16 HO OH HO OH HO OH 17 NH2 NH2 O NH2 18 O O VH45 5.21 ± 0.02 MS2756 6.04 ± 0.06 MS2734 7.05 ± 0.01 19 20 21 22

23 Figure 5. SAR studies of bisubstrate inhibitors. pKi values (-log10Ki) are shown in red and 24 25 26 are used to facilitate comparison between inhibitors of widely varying potency. (a) Modi- 27 28 fication of the benzamide moiety of NS1. (b) Previously published amino-bisubstrate 29 30 31 NNMT inhibitors were synthesized and tested alongside NS1 and analogues. 32 33 34 35 36 37 NS1-Alkane (15) vs. Amino-Bisubstrate Inhibitor MS2756. In the final stage of our 38 39 40 SAR analysis we compared our inhibitors with previously reported amino bisubstrate 41 42 NNMT inhibitors (Figure 5b). Previously reported compounds VH4519 , MS275620, and 43 44 45 MS273420 use a tertiary amine core and an alkyl tether to link a SAM-like fragment to a 46 47 48 NAM-like fragment. An alignment of NS1-bound NNMT with MS2756-bound NNMT20 49 50 51 shows the similar binding modes of the two compounds (Figure 3f). We synthesized and 52 53 tested all three of these compounds alongside NS1-alkane (15, Figure 4a) using the same 54 55 18 56 57 58 59 60 ACS Paragon Plus Environment

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1 assay protocols and kinetic analyses to determine Ki values. We compared MS2756 and 2 3 NS1-alkane directly because they are close structural analogues in which the tertiary amine 4 5 6 core of MS2756 has been replaced with an sp3 hybridized carbon stereocenter. These stud- 7 8 9 ies revealed that NS1-alkane was approximately 200-fold more potent than MS2756 in 10 11 terms of Ki. 12 13 14 The contrast between the observed activity of MS2756 and NS1-alkane is noteworthy; 15 16 17 changing a single non-hydrogen atom (tertiary nitrogen to sp3 carbon stereocenter) sub- 18 19 20 stantially alters compound potency. We hypothesize that a variety of factors could be re- 21 22 sponsible for this observation. It is evident that the stereochemical definition at the C6’ 23 24

25 stereocenter of NS1-alkane is beneficial as evidenced by the ~ 20-fold difference in Ki 26 27 28 between NS1-alkane and its C6’ epimer (16). A much larger difference (~ 250-fold) in Ki 29 30 between NS1 and its C6’ epimer (14) was also observed, demonstrating that as linker ri- 31 32 33 gidity increases, C6’ stereodefinition becomes more important. 34 35

36 While NS1-alkane and NS1 both show large differences in Ki values between their C6’ 37 38 39 stereoisomers, the tertiary nitrogen atom in MS2756 maintains sp3 tetrahedral geometry but 40 41 is free to invert rapidly at room temperature. In solution, we imagine that NS1-alkane and 42 43 44 NS1 assume the optimal tetrahedral geometry at C6’ necessary for binding, whereas 45 46 47 MS2756 is free to occupy conformations that are unproductive for binding because of ni- 48 49 50 trogen inversion. It is also likely that the hydrophobic, carbon-based scaffolds of NS1- 51 52 alkane and NS1 are preferred over the polar, tertiary amine core of MS2756. NS1-alkane 53 54 55 19 56 57 58 59 60 ACS Paragon Plus Environment

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1 and NS1 likely form favorable Van der Waals contacts with the NNMT active site, as the 2 3 closest residue to the NS1-alkane/NS1 C6’ carbon stereocenter is F15 (~ 4 Å away). This 4 5 6 phenylalanine residue presents a large hydrophobic46 surface in the NNMT ligand binding 7 8 9 pocket (Figure 6a), near the NS1-alkane/NS1 C6’ stereocenter. 10 11 12 Finally, the transition state structure of PNMT (phenylethanolamine N-methyltransfer- 13 14 ase47), the second closest homologue of NNMT, involves even distribution of positive 15 16 17 charge along the entire axis of methyl transfer.48 If NNMT has a transition state structure 18 19 20 similar to that of PNMT, analogues like MS2756, which localize positive charge on a single 21 22 nitrogen atom, do not accurately capture the transient electrostatics of the methyl transfer 23 24 25 transition state. While not positively charged, carbon-based linkers avoid the localized pos- 26 27 28 itive charge of MS2756 and other amino-bisubstrate inhibitors. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Figure 6. The chemical properties and geometry of NS1 closely match the hydrophobic 47 48 49 profile and shape of the NNMT binding pocket. (a) The binding pocket is buried in the 50 51 hydrophobic core of the protein (gray). (Inset) A solvent-accessible surface representation 52 53 54 55 20 56 57 58 59 60 ACS Paragon Plus Environment

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46 1 of the binding pocket colored by the Eisenberg hydrophobicity scale reveals a hydropho- 2 3 bic patch (dark red) that contacts the C6’ carbon atom of NS1. (b) A side-on view shows 4 5 6 that the binding pocket has shape complementarity specific to the S diastereomer of NS1 7 8 9 at the C6’ carbon. 10 11 12 13 14 Selectivity Evaluation. 15 While several previously reported NNMT inhibitors were de- 16 17 scribed as selective for NNMT, none of them have been tested against the closest homo- 18 19 20 logues25 of NNMT or other small-molecule methyltransferases49-50. We aimed to critically 21 22 23 evaluate the selectivity profile of NS1 by testing it against the methyltransferases most like 24 25 NNMT (Table 1). Sequence similarity analysis and generation of a sequence similarity 26 27 28 network51-52 (Figure S2, Table S3) showed that NNMT, INMT (indolethylamine N-methyl- 29 30 53 31 transferase ), and PNMT cluster tightly, with INMT and PNMT having 53% and 39% se- 32 33 quence identity to NNMT, respectively. A complementary approach based on structural 34 35 36 homology analysis via the DALI54 server demonstrated that INMT and PNMT are also the 37 38 39 closest structural homologues of NNMT (Figure S3, S4, Table S4).25 In addition to the se- 40 41 lectivity of NS1 amongst small-molecule methyltransferases, we tested NS1 against a few 42 43 44 representative DNA, histone, and protein-arginine methyltransferases (Table 1, Table S5). 45 46 47 These studies revealed thiopurine S-methyltransferase (TPMT) to be the closest off-target 48 49 50 of NS1, with an IC50 of ~ 1 µM. This result might be explained by the flexibility of the 51 52 TPMT active site55 and the ability of TPMT to bind a variety of small molecule substrates 53 54 55 21 56 57 58 59 60 ACS Paragon Plus Environment

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56 1 and inhibitors . INMT was the second closest off-target of NS1, with an IC50 of 3.4 µM 2 3 (Figure S5). 4 5 6 While in this case NS1 proved selective for NNMT, the introduction of different aryl 7 8 9 groups on the NS1 scaffold could likely be used to target other methyltransferases selec- 10 11 12 tively. In this work, cross coupling of compound 8 (Scheme 1) with 3-iodobenzamide gen- 13 14 erated NS1; cross coupling of 8 with indole or thiopurine fragments could provide inhibi- 15 16 17 tors of INMT and TPMT, respectively. In cases where off-target binding or inhibition is 18 19 20 observed, aryl groups could be modified until a desired selectivity profile is achieved. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 22 56 57 58 59 60 ACS Paragon Plus Environment

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1 Table 1. Methyltransferase Selectivity Profile of NS1 2 3 4 5 6 7 8 9 small-molecule inhibition inhibition athiopurine S-methyltransferase. bindolethyl- 10 methyltransferase at 10 µM (%) at 1 µM (%) 11 TPMTa 76 51 12 amine N-methyltransferase; *values interpo- 13 INMTb,* 53 14

14 COMTc 43 0 15 lated from the full IC50 curve shown in Figure 16 PNMTd 26 0 17 GAMTe 26 0 S5. ccatechol O-methyltransferase. 18 GNMTf 23 4 19 HNMTg 2 6 20 dphenylethanolamine N-methyltransferase. 21 protein or DNA IC (µM) 22 methyltransferase 50 23 eguanidinoacetate N-methyltransferase. fgly- hDNMT3ah 7.6 24 25 PRMT1i >10 cine N-methyltransferase. ghistamine N-me- 26 ASH1Lj >10 27 DOT1Lk >10 28 thyltransferase. hDNA (cytosine-5)-methyl- 29 EHMT1l >10 30 G9am >10 31 transferase 3A. iprotein arginine N-methyl- SETDB1 n >10 32 33 j k 34 transferase 1. histone-lysine N-methyltransferase ASH1L. histone-lysine N-methyltrans- 35 36 ferase, H3 lysine-79 specific. lhistone-lysine N-methyltransferase EHMT1. mhistone-lysine 37 38 39 N-methyltransferase EHMT2. nhistone-lysine N-methyltransferase SETDB1. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 23 56 57 58 59 60 ACS Paragon Plus Environment

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1 Thermal Stabilization and Cell-Based Evaluation. Along with biochemical profiling 2 3 of NS1 and analogues, we found that NS1 binds and stabilizes NNMT in lysate of K562 4 5 6 cells (a chronic myelogenous leukemia line known to express NNMT17). The cellular ther- 7 8 57 9 mal shift assay revealed that NS1 and 25 shift the melting curve of NNMT by 8 and 5 °C, 10 11 respectively (Figures S6, S8) and in a dose-dependent manner (Figures S7, S9). We also 12 13 14 evaluated several compounds for cytotoxicity via the CellTiter-Glo luminescent cell via- 15 16 15 17 bility assay in U2OS cells (a human bone osteosarcoma line known to express NNMT ). 18 19 None of the compounds tested (NS1, 21, 23, 24, 25) were toxic to U2OS cells after 48 h at 20 21 22 a 100 µM dose (Table S7). 23 24 25 We tested the ability of NS1, 21, 23, 24, and 25 to decrease MNAM levels in U2OS cells, 26 27 28 but the effects were modest (Table S8). NS1-methyl ester (23), designed to be more cell 29 30 permeable than NS1, performed best in this assay, decreasing MNAM levels by 30% at a 31 32 33 31.6 µM dose. A-B permeability testing (Caco-2, pH 6.5/7.4) showed that NS1 and ana- 34 35 36 logues have limited permeability (most permeable out of those tested was NS1-amine 21, 37 38 -6 39 1.3 x 10 cm/s, Table S9). Ongoing work is aimed at developing more cell-penetrant al- 40 41 kynyl NNMT inhibitors that would be useful probes in cell-based studies. 42 43 44 45 CONLUSIONS 46 47 48 In this work, we designed and synthesized the most potent and selective NNMT inhibitors 49 50 reported to date. A modular, scalable synthesis of alkynyl nucleosides enabled us to make 51 52 53 and test a variety of alkynyl bisubstrate inhibitors, leading to the discovery of NS1 as a 54 55 24 56 57 58 59 60 ACS Paragon Plus Environment

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1 selective, picomolar NNMT inhibitor. Alkynyl bisubstrate inhibition could be a general- 2 3 izable strategy; covalent tethering of alkynyl nucleosides to substrate mimics of other small 4 5 6 molecule methyltransferases could provide a method for selective inhibition of these en- 7 8 9 zymes. The stereodefined alkyne present in the NS1 scaffold could be used for click chem- 10 11 istry or other functionalization, enabling the development of bisubstrate probes or inhibi- 12 13 14 tors of protein arginine/lysine methyltransferases.58-59 15 16 17 Finally, this work demonstrates the value of alkynes in small-molecule ligand design in 18 19 20 the context of methyltransferase inhibition. Alkynes provide unique rigidity, geometry, and 21 22 spatial occupancy not captured by common fragment linkers. Current state-of-the-art bi- 23 24 25 substrate inhibitor designs often rely upon reductive amination, alkylation, and amide- 26 27 28 bond forming reactions to link fragments together. These reactions are generally fast and 29 30 easy to implement, but often produce highly flexible structures characterized by relatively 31 32 33 weak overall binding affinity. This report demonstrates that C6’ alkynyl nucleosides— 34 35 36 while more synthetically complex—can be used to generate potent bisubstrate methyl- 37 38 39 transferase inhibitors. 40 41 42 EXPERIMENTAL SECTION 43 44 45 General Chemistry Procedures 46 47 48 All reactions were performed under an atmosphere of nitrogen in flame- or oven-dried 49 50 51 glassware unless otherwise noted. Reactions were monitored by thin layer chromatography 52 53 (TLC) using Merck silica gel 60 covered glass backed plates (0.25 mm, F254). TLC plates 54 55 25 56 57 58 59 60 ACS Paragon Plus Environment

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1 were visualized under UV light and/or exposure to an acidic solution of p-anisaldehyde, 2 3 an aqueous solution of ceric ammonium molybdate (CAM), or an aqueous solution of po- 4 5 6 tassium permanganate, followed by heating. Reactions were also monitored by analytical 7 8 9 LCMS using an Agilent 6120 Quadrupole MS and an Agilent 1260 Infinity II LC system 10 11 equipped with (1) a Phenomenex Kinetex F5 column (2.6 µm, 100 Å, 100 × 3.0 mm), (2) 12 13 14 a Phenomenex Kinetex Biphenyl column (2.6 µm, 100 Å, 100 × 3.0 mm) or (3) a Thermo 15 16 × 17 Scientific Accucore aQ C18 column (2.6 µm, 100 2.1 mm). For analytical LCMS, sol- 18 19 vent A consisted of 0.1% (v/v) FA/water and solvent B consisted of 0.1% (v/v) FA/CH3CN. 20 21 22 Two methods were used: method A for columns 1, 2, and 3: 5% B for 1 min, 5% → 95% 23 24 25 B over 8 min, 95% B for 2 min, and reequilibration for 2 min; flow rate: 0.55 mL/min (1), 26 27 254 method B 28 0.95 mL/min (2), 0.70 mL/min (3), column temperature: 50 °C; UV , and for 29 30 columns 2 and 3: 0% B for 1 min, 0% → 40% B over 8 min, 40% → 95% B over 2 min, 31 32 33 95% B for 2 min, and reequilibration for 2 min; flow rate: 0.95 mL/min (2), 0.70 mL/min 34 35 36 (3), column temperature: 50 °C; UV254. Column 2 (method B) was used for the final analysis 37 38 of very polar compounds, including NS1 (10) and analogues. Flash column chromatog- 39 40 41 raphy was performed on an Interchim PuriFlash 215 system with prepacked silica gel car- 42 43 44 tridges (Büchi FlashPure (35 to 45 µm), Büchi Reveleris HP (16 to 24 µm), Teledyne Isco 45 46 RediSep Rf (35 to 70 µm) and Teledyne Isco RediSep Rf Gold (20 to 40 µm). Preparative 47 48 49 HPLC was performed on an Agilent 1260 Infinity II Preparative LC system equipped with 50 51 52 a Kromasil C18 column (10 µm, 100 Å, 21.2 × 250 mm) using solvents A (0.1% (v/v) 53 54 55 26 56 57 58 59 60 ACS Paragon Plus Environment

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1 FA/water) and B (0.1% (v/v) FA/CH 3CN) or A’ (0.1% (v/v) TFA/water) and B’ (0.1% (v/v) 2 3 TFA/CH3CN) at flow rate 10 mL/min delivering gradients specified in individual experi- 4 5 6 mental protocols. All compounds reported in this manuscript and Supporting Information 7 8 ≥ 1 13 9 have a purity of 95% as determined by analytical LCMS and NMR ( H and C) unless 10 11 otherwise noted. 12 13 14 15 16 17 Instrumentation 18 19 20 1H NMR spectra were recorded on Varian INOVA-600 or Varian INOVA-500 spectrom- 21 22 23 eters at ambient temperature. Chemical shifts are reported in ppm (δ scale) relative to re- 24 25 26 sidual undeuterated solvent (CDCl3: 7.26 (CHCl3), CD3OD: 3.31 (CD2HOD), CD3CN: 1.94 27 28 δ (CD2HCN), D2O: 4.79 (HOD)). Data are reported as follows: chemical shift ( ppm) (inte- 29 30 31 gration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = 32 33 34 broad, app = apparent, or a combination of these), coupling constant(s) J (Hz)). When a 35 36 mixture of deuterated solvents is employed, the residual undeuterated solvent used for ref- 37 38 39 erence is indicated in bold. 13C NMR spectra were recorded on Varian INOVA-500, Varian 40 41 42 MERCURY-400 and JEOL J400 spectrometers at ambient temperature. Data are reported 43 44 as follows: chemical shifts (δ scale, ppm) relative to the carbon resonances of the solvent 45 46

47 (CDCl3: 77.16, CD3OD: 49.00, CD3CN: 1.32). When a mixture of deuterated solvents is 48 49 50 employed, the solvent used for reference is indicated in bold. 19F NMR spectra were rec- 51 52 53 54 55 27 56 57 58 59 60 ACS Paragon Plus Environment

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1 orded on Varian INOVA-500 and Varian MERCURY-400 spectrometers at ambient tem- 2 3 δ perature. Data are reported as follows: chemical shifts ( scale, ppm) relative to CFCl3 and 4 5 6 referenced to benzotrifluoride (BTF), hexafluorobenzene (HFB), or trifluoroacetic acid 7 8 δ δ δ 9 (TFA) as internal standards (BTF IStd: –63.7, HFB IStd: –164.9, TFA IStd: –76.6). 10 11 31P NMR spectra were recorded on a Varian MERCURY-400 spectrometer at ambient tem- 12 13 14 perature. Data are reported as follows: chemical shifts (δ scale, ppm), calibrated to an ex- 15 16 δ δ 17 ternal standard of triphenyl phosphate in CDCl3 (0.0485 M, –17.6 relative to H3PO4 at 18 19 0). Infrared (FTIR) spectra were recorded on a Bruker Alpha FT-IR spectrophotometer. 20 21 22 Data is reported in frequency of absorption (cm-1). The IR spectrum of each compound 23 24 25 (solid or liquid) was acquired directly neat or on a thin layer at ambient temperature. High 26 27 resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II or an Agilent 28 29 30 6220 LC-TOF equipped with an electrospray ionization source (ESI+). 31 32 33 34 35 36 Materials 37 38 39 Commercial reagents and solvents were used as received unless otherwise noted. All sol- 40 41 42 vents were of commercially available anhydrous grade (DriSolv® or equivalent) unless 43 44 otherwise noted. The molarity of n-butyllithium solutions was determined by titration us- 45 46 47 ing N-benzylbenzamide60 or N-pivaloyl-o-toluidine61 with similar results (average of three 48 49 62 50 determinations). [Rh(OAc)(C2H4)2]2 was prepared according to a known procedure using 51 52 [RhCl(C2H4)2]2 (CAS: 12081-16-2) obtained from Strem Chemicals, Inc. (catalog #: 45- 53 54 55 28 56 57 58 59 60 ACS Paragon Plus Environment

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1 0270). The following catalysts were all obtained from Strem Chemicals, Inc.: nitro-Grela 2 3 catalyst ([1,3-bis(2,4,6-trimethylphenylimidazolidin-2-ylidene)]-(2-i-propoxy-5-nitroben- 4 5 6 zylidene)ruthenium(II) dichloride), CAS: 502964-52-5, catalog #: 44-0758; (S,S)-MeBPE- 7 8 63 9 Rh catalyst ((-)-1,2-bis((2S,5S)-2,5-dimethylphospholano)ethane(1,5-cyclooctadi- 10 11 ene)rhodium(I) tetrafluoroborate), CAS: 213343-65-8, catalog #: 45-0169; (S)-DTBM- 12 13 14 SegPhos ((S)-(+)-5,5'-bis[di(3,5-di-t-butyl-4-methoxyphenyl)phosphino]-4,4'-bi-1,3-ben- 15 16 17 zodioxole), CAS: 210169-40-7, catalog #: 15-0067; [Rh(nbd)2]BF4, CAS: 36620-11-8, cat- 18 19 alog #: 45-0230; (S,S)-MeDUPHOS ((+)-1,2-Bis((2S,5S)-2,5-dimethylphospholano)ben- 20 21 22 zene), CAS: 136735-95-0, catalog #: 15-0092. (±)-Z-α-phosphonoglycine trimethyl ester, 23 24 25 CAS: 88568-95-0, was obtained from Chem-Impex International, (Catalog #: 14125). 26 27 28 29 30 31 Synthesis of NS1 (10) 32 33 R R R R 34 (3a ,4 ,6 ,6a )-4-methoxy-2,2-dimethyl-6-(prop-2-yn-1-yl)tetrahydrofuro[3,4- 35 36 d][1,3]dioxole (2). The following is an adaption of a published procedure.64 To a stirred 37 38

39 solution of 2,6-lutidine (9.85 mL, 84.6 mmol, 1.1 equiv.) in CH2Cl2 (130 mL) at –78 °C 40 41 42 was added trifluoromethanesulfonic anhydride (13.7 mL, 81.5 mmol, 1.06 equiv.) over 5 43 44 min (flask A, 1 L rbf), followed by the addition of a solution of alcohol 1 (15.7 g, 76.9 45 46

47 mmol) in CH2Cl2 (33 mL) over 10 min. The mixture was stirred at –78 °C for 2 h and 48 49 50 warmed to 0 °C. Meanwhile, to a separate flask (flask B, 500 mL rbf) containing a solution 51 52 of TMS-acetylene (32.0 mL, 231 mmol, 3.00 equiv.) in dry THF (130 mL) at –78 °C was 53 54 55 29 56 57 58 59 60 ACS Paragon Plus Environment

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M 1 added n-butyllithium (2.56 in hexanes, 93.7 mL, 240 mmol, 3.12 equiv.) at a rate of 5 2 3 mL/min via syringe. The resulting mixture was stirred at –78 °C for 1.5 h and warmed to 4 5 6 0 °C. While the contents of flask B remained at 0 °C, flask A was removed from the ice/wa- 7 8 9 ter bath, volatiles were removed in vacuo (bath temperature at 20 °C) and a brief subjection 10 11 to high vacuum yielded a red-orange paste. The crude triflate in flask A was resuspended 12 13 14 in dry THF (65 mL) and DMPU (35 mL) was added. The contents were stirred vigorously 15 16 17 for 10 min until complete solubilization was noted and cooled to –78 °C. The contents of 18 19 flask B were cooled to –78 °C and transferred to flask A via cannula. The resulting mixture 20 21 22 was stirred at –78 °C for 1 h and at –12 °C for 3 h (NaCl/ice bath), before the slow addition 23 24 25 of TBAF (caution, gas evolution!, 1.0 M in THF, 250 mL, 250 mmol, 3.25 equiv.) via 26 27 cannula. The mixture was allowed to warm to RT and was stirred for another 30 min before 28 29 30 volatiles were removed in vacuo. The reddish-brown residue was resuspended in MTBE 31 32 33 (350 mL) and a sat. aq. NH4Cl solution (300 mL) was added. The layers were roughly 34 35 36 separated and the organic phase, largely emulsified, was filtered through a pad of 37 38 CaCO3/celite to give two clear layers in the filtrate. The layers were separated and the 39 40 41 combined aqueous phases were extracted with MTBE (2 × 200 mL) and EtOAc (200 mL). 42 43 44 The combined organic extracts were washed with a 5% aq. LiCl solution (800 mL) and 45 46 then with brine (800 mL), dried over MgSO4, filtered, and concentrated. The crude residue 47 48 49 was purified by dry column vacuum chromatography65 (cyclohexane/EtOAc, 0 → 30%) to 50 51 52 yield pure 2 as well as semi-pure 2 which was repurified by silica gel chromatography 53 54 55 30 56 57 58 59 60 ACS Paragon Plus Environment

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1 (hexanes/EtOAc, 0 → 30%). Compound 2 (11.1 g, 52.3 mmol, 68%) was obtained as a 2 3 ν −1 light-yellow oil. Rf = 0.60 (hexanes/EtOAc, 67:33); FTIR (neat), max (cm ): 3286, 2989, 4 5 1 6 2938, 2835, 1438, 1209, 1090, 1053, 1041; H NMR (600 MHz, CDCl3): δ 4.95 (s, 1H), 7 8 9 4.69 (d, J = 5.9 Hz, 1H), 4.60 (d, J = 5.9 Hz, 1H), 4.30 (dd, J = 9.3, 6.6 Hz, 1H), 3.32 (s, 10 11 3H), 2.52 (ddd, J = 16.7, 6.6, 2.7 Hz, 1H), 2.42 (ddd, J = 16.6, 9.4, 2.7 Hz, 1H), 2.04 (t, J 12 13 13 14 = 2.7 Hz, 1H), 1.46 (s, 3H), 1.31 (s, 3H); C NMR (151 MHz, CDCl3): δ 112.3, 109.6, 85.2, 15 16 + 17 85.1, 83.3, 80.2, 70.4, 54.7, 26.4, 24.9, 24.6; HRMS (ESI+): [C11H16O4Na] calc. 235.0941, 18 19 meas. 235.0938, Δ 1.0 ppm. 20 21 22 (3aR,4R,6R,6aR)-4-allyl-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole 23 24 25 (3). Alkyne 2 (10.6 g, 49.9 mmol) was reduced according to protocols adapted from Cox 26 27 28 et al.66 Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 à 29 30 30%) to afford alkene 3 (9.97 g, 46.5 mmol, 93%) as a clear, viscous oil. The flash column 31 32 33 chromatography removed ca. 98% of PHMS, yielding material that was suitable for use in 34 35 36 subsequent reactions. An analytically pure sample was prepared by subjecting 2.0 g of the 37 38 39 post-column material to short path distillation (Kugelrohr) at 130 °C and 200 mTorr. Rf = 40 41 ν −1 0.50 (hexanes/EtOAc, 80:20); FTIR (neat), max (cm ): 3078, 2987, 2938, 2833, 1643, 1381, 42 43 1 44 1372, 1209, 1193, 1105, 1090, 1059, 1031; H NMR (600 MHz, CDCl3): δ 5.82 (dddd, J = 45 46 47 16.7, 10.3, 7.4, 6.2 Hz, 1H), 5.15–5.09 (m, 2H), 4.96 (s, 1H), 4.61 (d, J = 5.9 Hz, 1H), 4.57 48 49 (dd, J = 6.0, 1.0 Hz, 1H), 4.23 (td, J = 7.8, 1.0 Hz, 1H), 3.34 (s, 3H), 2.46–2.38 (m, 1H), 50 51 13 52 2.32–2.24 (m, 1H), 1.48 (s, 3H), 1.31 (s, 3H); C NMR (126 MHz, CDCl3): δ 134.5, 117.7, 53 54 55 31 56 57 58 59 60 ACS Paragon Plus Environment

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+ 1 112.4, 109.6, 86.6, 85.7, 83.6, 55.0, 39.6, 26.6, 25.2; HRMS (ESI+): [C11H18O4Na] calc. 2 3 237.1095, meas. 237.1097, Δ 1.0 ppm. 4 5 6 (E)-4-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol- 7 8 9 4-yl)but-2-enal (4). To a solution of alkene 3 (2.0 g, 9.3 mmol) in MTBE (degassed prior 10 11 12 to addition by sparging with nitrogen for 15 min, 28 mL) at RT, were added E-crotonalde- 13 14 hyde (3.9 mL, 47 mmol, 5.0 equiv.) and nitro-Grela metathesis catalyst (63 mg, 93 µmol, 15 16 17 1 mol %) sequentially. The flask headspace was purged with nitrogen for 10 min and the 18 19 20 mixture was stirred at 40 °C for 48 h, at which point TLC analysis indicated ca. 50% con- 21 22 version. More nitro-Grela catalyst (63 mg, 93 µmol, 1 mol %) and E-crotonaldehyde (3.9 23 24 25 mL, 47 mmol, 5.0 equiv.) were added and the reaction mixture was stirred at 40 °C for 26 27 28 another 24 h, at which point complete conversion was indicated by TLC analysis. Volatiles 29 30 were removed in vacuo and the crude material was purified by silica gel chromatography 31 32

33 (hexanes/EtOAc, 2 → 50%) to afford enal 4 (1.60 g, 6.60 mmol, 71%) as a yellow oil. Rf 34 35 ν -1 36 = 0.41 (hexanes/EtOAc, 50:50); FTIR (neat), max (cm ): 2990, 2936, 2832, 2735, 1688, 37 38 39 1641, 1453, 1382, 1377, 1298, 1269, 1263, 1242, 1210, 1194, 1161, 1140, 1104, 1089, 40 41 1 1058, 1026; H NMR (600 MHz, CDCl3): δ 9.54 (d, J = 7.8 Hz, 1H), 6.84 (dt, J = 15.7, 42 43 44 6.8 Hz, 1H), 6.21 (ddt, J =15.7, 7.8, 1.5 Hz, 1H), 4.98 (s, 1H), 4.64 (d, J = 5.9 Hz, 1H), 45 46 47 4.56 (dd, J = 6.0, 1.1 Hz, 1H), 4.35 (ddd, J= 8.8, 6.4, 1.0 Hz, 1H), 3.34 (s, 3H), 2.68 (dddd, 48 49 J = 15.4, 8.5, 6.6, 1.6 Hz, 1H), 2.58 (dtd, J = 15.2, 6.7,1.5 Hz, 1H), 1.48 (s, 3H), 1.32 (s, 50 51 13 52 3H); C NMR (151 MHz, CDCl3): δ 193.6, 153.5, 134.8, 112.6, 109.8, 85.4, 85.3, 83.7, 53 54 55 32 56 57 58 59 60 ACS Paragon Plus Environment

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+ 1 54.7, 38.4, 26.5, 25.0; HRMS (ESI+): calcd. for [C12H18O5Na] 265.1046, meas. 265.1062, 2 3 Δ 6.0 ppm. 4 5 6 (R)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol- 7 8 9 4-yl)methyl)-5-(triisopropylsilyl)pent-4-ynal (5). Aldehyde 5 was prepared using proto- 10

11 31 12 cols adapted from Nishimura et al. A 250 mL flask was charged with [Rh(OAc)(C2H4)2]2 13 14 catalyst (126 mg, 0.289 mmol, 2.5 mol %), (S)-DTBM-SegPhos (818 mg, 0.693 mmol, 6.0 15 16 17 mol%), and a large stir bar. The flask headspace was evacuated and backfilled with nitro- 18 19 × 20 gen (3 ) and methanol was added (22 mL). The resulting orange suspension was stirred 21 22 vigorously at RT until a red-orange solution was obtained (30 min). At this time, a solution 23 24 25 of enal 4 (2.80 g, 11.6 mmol) in MeOH (10 mL) and TIPS-acetylene (5.19 mL, 23.1 mmol, 26 27 28 2.0 equiv.) were added sequentially to the reaction mixture. The resulting red mixture was 29 30 stirred at 40 °C for 15 h and volatiles were removed in vacuo. Crude material was purified 31 32 33 by silica gel chromatography (hexanes/EtOAc, 0 → 50%) to afford aldehyde 5 (4.60 g, 34 35 36 10.8 mmol, 94%) as a single diastereomer, as a light yellow oil. Rf = 0.42 (cyclohex- 37 38 ν -1 39 ane/EtOAc, 75:25); FTIR (neat), max (cm ): 2941, 2865, 2722, 2167, 1728, 1463, 1381, 40 41 1 1371, 1271, 1241, 1210, 1193, 1160, 1093, 1058, 1018; H NMR (600 MHz, CDCl3): δ 42 43 44 9.82 (t, J = 2.1 Hz, 1H), 4.95 (s, 1H), 4.61 (dd, J = 5.9, 1.2 Hz, 1H), 4.58 (d, J = 5.9 Hz, 45 46 47 1H), 4.49 (ddd, J = 10.6, 4.2, 1.2 Hz, 1H), 3.35 (s, 3H), 3.16 (dtd, J = 11.2, 6.9, 3.9 Hz, 48 49 1H), 2.60 (ddd, J = 16.5, 7.5, 2.2 Hz, 1H), 2.55 (ddd, J = 16.5, 6.4, 2.1 Hz, 1H), 1.74 (ddd, 50 51 52 J = 13.3, 10.6, 4.0 Hz, 1H), 1.64 (ddd, J = 13.2, 11.1, 4.2 Hz, 1H), 1.47 (s, 3H), 1.31 (s, 53 54 55 33 56 57 58 59 60 ACS Paragon Plus Environment

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13 1 3H), 1.11–0.97 (m, 21H); C NMR (151 MHz, CDCl3): δ 200.7, 112.6, 109.9, 108.6, 85.5, 2 3 85.3, 84.5, 84.2, 55.4, 49.0, 40.7, 26.7, 25.4, 25.1, 18.7, 11.3; HRMS (ESI+): calcd. for 4 5 + ∆ 6 [C23H40O5Si1Na] 447.2537, meas. 447.2527, 2.3 ppm. 7 8 9 Methyl (S,Z)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 10 11 d 12 ][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-2- 13 14 en-6-ynoate (6). A solution of methyl 2-(dimethoxyphosphoryl)-2-(2,2,2-trifluoroacetam- 15 16 17 ido)acetate (S2, 3.11 g, 10.6 mmol, 1.5 equiv.) in THF (21 mL) was added dropwise over 18 19 20 20 min to a stirred suspension of NaH (95%, 254 mg, 10.6 mmol, 1.5 equiv.) in THF (10 21 22 mL) at 0 °C. The resulting cloudy mixture was stirred at 0 °C for 30 min and a solution of 23 24 25 aldehyde 5 (3.00 g, 7.06 mmol) in THF (7.1 mL) was added over 5 min. The resulting 26 27 28 mixture was allowed to warm gradually to RT and was stirred for 2 h, at which point the 29 30 reaction media was heated to 40 °C and stirred for another 6 h. The mixture was allowed 31 32 33 to cool to RT and volatiles were removed in vacuo. The residue was partitioned between 34 35 36 MTBE (25 mL) and 1× PBS (15 mL). The mixture was stirred vigorously for 10 min and 37 38 × 39 the layers were separated. The aqueous phase was extracted with MTBE (3 25 mL). The 40 41 combined organic extracts were washed with brine (80 mL), dried over MgSO4, filtered, 42 43 44 and concentrated. Crude material was purified by silica gel chromatography (hex- 45 46 47 anes/EtOAc, 0 → 30%) to yield enamide 6 (3.74 g, 6.32 mmol, 89%) as a 6:1 mixture of 48 49 Z and E isomers as a light yellow oil. *Note: Z and E isomers are not separated for the 50 51

52 following step, as they both react to form the desired product. Rf = 0.48 (hexanes/EtOAc, 53 54 55 34 56 57 58 59 60 ACS Paragon Plus Environment

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ν -1 1 67:33); FTIR (neat), max (cm ): 3297, 2943, 2866, 1720, 1664, 1528, 1463, 1439, 1382, 2 3 1 1373, 1335, 1283, 1210, 1160, 1106, 1092, 1062, 1043, 1016; H NMR (600 MHz, CDCl3): 4 5 6 δ (Z-isomer) 7.73 (s, 1H), 7.07 (t, J = 7.2 Hz, 1H), 4.94 (s, 1H), 4.61 (dd, J = 6.0, 1.1 Hz, 7 8 9 1H), 4.58 (d, J = 5.9 Hz, 1H), 4.44 (ddd, J = 10.4, 4.4, 1.1 Hz, 1H), 3.81 (s, 3H), 3.32 (s, 10 11 3H), 2.87 (ddd, J = 8.3, 5.5, 3.7 Hz, 1H), 2.40 (ddd, J = 15.8, 7.2, 5.1 Hz, 1H), 2.26 (ddd, 12 13 14 J = 15.8, 8.7, 7.2 Hz, 1H), 1.71 (ddd, J = 13.3, 10.4, 4.2 Hz, 1H), 1.65 (ddd, J = 13.4, 10.8, 15 16 13 17 4.5 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06–1.02 (m, 21H); C NMR (126 MHz, CDCl3): 18

19 2 1 δ (Z-isomer) 163.3, 155.1 (q, JC-F = 38 Hz), 138.3, 133.3, 123.8, 115.6 (q, JC-F = 288 Hz), 20 21 22 112.0, 109.4, 109.1, 85.2, 84.2, 83.7, 54.5, 52.2, 40.6, 34.4, 29.2, 26.2, 24.8, 18.3, 11.0; 19F 23 24 25 NMR (471 MHz, CDCl3, BTF IStd): δ (Z-isomer) –76.4; HRMS (ESI+): calcd. for 26 27 + ∆ [C28H44F3N1O7Si1K] 630.2471, meas. 630.2462, 1.4 ppm. 28 29 30 Methyl (2S,5S)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 31 32 33 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-6- 34 35 36 ynoate (7). To a flask charged with olefin 6 (300 mg, 0.507 mmol), as a 6:1 mixture of Z 37 38 E 39 and isomers, was added MeOH (20 mL) at RT under a nitrogen atmosphere. The con- 40 41 tents were stirred until full dissolution was noted. Separately, (S,S)-MeBPE-Rh (28 mg, 42 43 44 0.051 mmol, 10 mol %) was weighed out into a vial in a glove box. The vial was removed 45 46 47 from the glove box, placed under a nitrogen atmosphere, and MeOH (10 mL) was added. 48 49 The methanolic solution of (S,S)-MeBPE-Rh was transferred via syringe to the methanolic 50 51 52 solution of olefin 6. The flask headspace was purged with hydrogen (balloon), the outlet 53 54 55 35 56 57 58 59 60 ACS Paragon Plus Environment

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1 needle was removed, and the balloon was refilled with hydrogen. The contents were stirred 2 3 vigorously at RT under a hydrogen atmosphere (balloon) for 4 h and volatiles were re- 4 5 6 moved in vacuo. Crude material was purified by silica gel chromatography (hex- 7 8 9 anes/EtOAc, 0 → 50%) to afford protected amino acid 7 (276 mg, 0.465 mmol, 92%) as a 10 11 single diastereomer, as a clear viscous oil. Rf = 0.45 (hexanes/EtOAc, 67:33); FTIR (neat), 12 13 ν -1 14 max (cm ): 3321, 2942, 2865, 2165, 1750, 1719, 1549, 1462, 1440, 1382, 1372, 1208, 1160, 15 16 1 17 1095, 1060, 1017; H NMR (600 MHz, CDCl3): δ 6.84 (d, J = 7.8 Hz, 1H), 4.93 (s, 1H), 18 19 4.66 (td, J = 7.7, 4.6 Hz, 1H), 4.61 (dd, J = 5.9, 1.2 Hz, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.43 20 21 22 (ddd, J = 10.1, 4.8, 1.2 Hz, 1H), 3.79 (s, 3H), 3.30 (s, 3H), 2.70–2.62 (m, 1H), 2.14 (dddd, 23 24 25 J = 14.0, 10.8, 5.8, 4.6 Hz, 1H), 2.08–1.99 (m, 1H), 1.68–1.57 (m, 2H), 1.54 (dddd, J = 26 27 J 28 13.1, 10.7, 5.9, 4.5 Hz, 1H), 1.46 (s, 3H), 1.36 (dddd, = 13.1, 11.2, 9.7, 4.4 Hz, 1H), (s, 29 30 13 2 3H), 1.08–1.03 (m, 21H); C NMR (126 MHz, CDCl3): δ 171.4, 156.9 (q, JC-F = 38 Hz), 31 32 1 33 115.7 (q, JC-F = 288 Hz), 112.5, 109.8, 109.5, 85.6, 85.5, 84.5, 83.7, 55.1, 53.0, 52.3, 41.1, 34 35 19 36 31.1, 30.1, 29.8, 26.7, 25.4, 18.7, 11.3; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.8; 37 38 + Δ HRMS (ESI+): calcd. for [C28H46F3N1O7Si1Na] 616.2888, meas. 616.2890, 0.4 ppm. 39 40 41 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-ethynyl-6-methoxy-6- 42 43 44 oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (8). To a 45 46 6 47 suspension of N -benzoyladenine (742 mg, 3.10 mmol, 2.15 equiv.) in propionitrile (dried 48 49 over 4 Å molecular sieves, 10 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide 50 51 52 (0.97 mL, 4.0 mmol, 2.75 equiv.). The resulting mixture was stirred at 80 °C for 10 min, 53 54 55 36 56 57 58 59 60 ACS Paragon Plus Environment

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6 1 upon which complete solubilization of N -benzoyladenine was observed. To a separate 2 3 flask charged with triacetate S4 (dried by azeotropic distillation with benzene (4 ×), 735 4 5 6 mg, 1.44 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (51 mg, 0.14 mmol, 10 7 8 9 mol %) was added propionitrile (10 mL). The resulting mixture was stirred at RT for 5 min 10 11 until a clear solution was obtained and the solution of silylated N6-benzoyladenine was 12 13 14 added. The reaction mixture was stirred at 95 °C for 3 h, cooled to RT and volatiles were 15 16 17 removed in vacuo. Crude material was purified by silica gel chromatography 18 19 (PhMe/MeCN, 0 → 50%) to afford nucleoside 8 (663 mg, 0.963 mmol, 67%) as a white 20 21 ν -1 22 amorphous solid. Rf = 0.48 (PhMe/MeCN, 50:50); FTIR (thin-film), max (cm ): 3273, 3086, 23 24 25 2928, 1748, 1720, 1611, 1582, 1511, 1487, 1456, 1374, 1329, 1243, 1217, 1182, 1160, 26

27 1 3 28 1099, 1074, 1047, 1029; H NMR (500 MHz, CDCl ): δ 9.09 (s, 1H), 8.77 (s, 1H), 8.09 (s, 29 30 1H), 8.03–7.99 (m, 2H), 7.62–7.57 (m, 1H), 7.53–7.48 (m, 2H), 7.06 (d, J = 7.9 Hz, 1H), 31 32 33 6.12 (d, J = 5.4 Hz, 1H), 6.07 (t, J = 5.4 Hz, 1H), 5.55 (dd, J = 5.5, 4.5 Hz, 1H), 4.61 (td, 34 35 36 J = 7.8, 5.0 Hz, 1H), 4.48 (ddd, J = 10.9, 4.5, 2.9 Hz, 1H), 3.77 (s, 3H), 2.59 (ddtd, J = 37 38 11.4, 9.1, 4.4, 2.5 Hz, 1H), 2.16 (d, J = 2.4 Hz, 1H), 2.15 (s, 3H), 2.11–2.00 (m, 2H), 2.06 39 40 41 (s, 3H), 1.95 (dddd, J = 13.9, 10.9, 7.7, 4.6 Hz, 1H), 1.86 (ddd, J = 13.8, 11.2, 2.9 Hz, 1H), 42 43 13 44 1.61–1.54 (m, 1H), 1.45 (dddd, J = 13.6, 10.9, 9.3, 4.6 Hz, 1H); C NMR (126 MHz, 45

46 2 CDCl3): δ 171.2, 169.8, 169.6, 165.2, 157.0 (q, JC-F = 37.6 Hz), 152.6, 151.6, 149.9, 142.4, 47 48 1 49 133.5, 132.8, 128.8, 128.1, 123.9, 115.7 (q, JC-F = 288 Hz), 87.0, 84.7, 80.7, 73.5, 72.9, 50 51 52 53 54 55 37 56 57 58 59 60 ACS Paragon Plus Environment

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19 1 71.7, 53.0, 52.3, 38.2, 30.8, 29.6, 27.9, 20.6, 20.4; F NMR (471 MHz, CDCl3, BTF IStd): 2 3 + ∆ δ –76.7; HRMS (ESI+): calcd. for [C31H31F3N6O9Na] 711.1988, meas. 711.1997, 1.2 ppm. 4 5 6 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-car- 7 8 9 bamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahy- 10 11 12 drofuran-3,4-diyl diacetate (9). To a vial charged with alkyne 8 (79 mg, 0.11 mmol) were 13 14 added 3-iodobenzamide (57 mg, 0.23 mmol, 2.0 equiv.), CuI (4 mg, 0.02 mmol, 20 mol 15 16

17 %), and Pd(PPh3)4 (7 mg, 6 µmol, 5 mol %). The vial headspace was purged with nitrogen 18 19 20 for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by sparging with 21 22 nitrogen for 15 min, 3.5 mL) was added at RT. The resulting mixture was stirred at 70 °C 23 24 25 for 3 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude 26 27 28 material was purified by silica gel chromatography (PhMe/MeCN, 0 → 100%) to afford 29 30 protected NS1 9 (83 mg, 0.10 mmol, 90%) as a white amorphous solid. Rf = 0.17 (EtOAc/i- 31 32 ν -1 33 PrOH, 50:50); FTIR (neat), max (cm ): 3255, 3064, 2929, 1744, 1717, 1666, 1610, 1578, 34 35 36 1511, 1486, 1455, 1374, 1329, 1240, 1212, 1180, 1158, 1096, 1073, 1046, 1029; 1H NMR 37 38 39 (600 MHz, CD2Cl2): δ 9.03 (br s, 1H), 8.75 (s, 1H), 8.14 (s, 1H), 8.00 (t, J = 1.3 Hz, 1H), 40 41 7.99 (d, J = 1.5 Hz, 1H), 7.87 (td, J = 1.8, 0.6 Hz, 1H), 7.77 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 42 43 44 7.64 (ddt, J = 8.0, 7.0, 1.3 Hz, 1H), 7.57–7.54 (m, 2H), 7.53 (dd, J = 1.6, 1.0 Hz, 1H), 7.40 45 46 47 (td, J = 7.8, 0.6 Hz, 1H), 7.15 (d, J = 7.7 Hz, 1H), 6.53 (br s, 1H), 6.18 (d, J = 5.8 Hz, 1H), 48 49 6.15 (t, J = 5.5 Hz, 1H), 5.70 (br s, 1H), 5.65 (dd, J = 5.3, 4.0 Hz, 1H), 4.65 (td, J = 7.5, 50 51 52 5.5 Hz, 1H), 4.54 (dt, J = 10.1, 3.9 Hz, 1H), 3.77 (s, 3H), 2.83 (ddt, J = 11.0, 9.0, 4.4 Hz, 53 54 55 38 56 57 58 59 60 ACS Paragon Plus Environment

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1 1H), 2.19 (ddd, J = 13.8, 10.1, 4.0 Hz, 1H), 2.15 (s, 3H), 2.11 (dt, J = 10.5, 5.1 Hz, 1H), 2 3 2.11–1.99 (m, 2H), 2.04 (s, 3H), 1.68 (ddt, J = 13.0, 10.7, 5.6 Hz, 1H), 1.57 (dddd, J = 4 5 13 6 13.5, 10.8, 9.3, 4.8 Hz, 1H); C NMR (101 MHz, CD2Cl2): δ 171.6, 170.4, 170.1, 168.8, 7 8 2 9 165.4, 157.4 (q, JC-F = 37 Hz), 152.8, 152.1, 150.3, 142.9, 134.8, 134.1, 134.0, 133.1, 131.1, 10

11 1 129.1, 128.9, 128.3, 127.4, 124.6, 123.9, 116.1 (q, JC-F = 287 Hz), 91.9, 87.3, 83.1, 81.5, 12 13 19 14 74.1, 73.0, 53.3, 53.0, 38.8, 31.4, 29.8, 29.1, 20.8, 20.6; F NMR (471 MHz, CDCl3, BTF 15 16 + ∆ 17 IStd): δ –76.7; HRMS (ESI+): calcd. for [C38H37F3N7O10] 808.2549, meas. 808.2538, 1.3 18 19 ppm. 20 21 22 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 23 24 25 hydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoic acid (NS1, 10). To a so- 26 27 28 lution of alkyne 9 (158 mg, 196 µmol) in THF (2.0 mL) at RT, was added a 0.5 M aq. LiOH 29 30 solution (2.0 mL). The resulting mixture was stirred for 2.5 h, cooled to 0 °C, and a 10% 31 32 33 aq. AcOH solution was added dropwise until pH 7 was reached. Volatiles were removed 34 35 36 in vacuo and the residue was dissolved in a solution of ammonia in methanol (7 N, 8 mL) 37 38 in vacuo 39 at RT. The resulting mixture was stirred for 18 h and volatiles were removed . 40 41 Crude material was purified by preparative HPLC (gradient elution: 5% → 55% B over 45 42 43 44 min) to afford NS1 10 (107 mg, 172 µmol, 88% over 2 steps) as the TFA salt. Single crys- 45 46 47 tals suitable for X-ray diffraction studies were grown from a 4:1:1 volumetric mixture of 48 49 10 mM aq. NS1-TFA:n-BuOH:i-PrOH via slow evaporation at 4 °C over two weeks. Full 50 51 52 small molecule X-ray crystallographic data for NS1 (10) are reported in the Supporting 53 54 55 39 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 Information. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.37 2 3 (s, 1H), 8.34 (s, 1H), 7.78 (td, J = 1.8, 0.6 Hz, 1H), 7.72 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 4 5 6 7.52 (dt, J = 7.8, 1.4 Hz, 1H), 7.40 (td, J = 7.8, 0.6 Hz, 1H), 6.01 (d, J = 5.1 Hz, 1H), 4.74 7 8 9 (t, J = 5.1 Hz, 1H), 4.38 (ddd, J = 10.3, 4.4, 3.2 Hz, 1H), 4.23 (t, J = 4.8 Hz, 1H), 4.02 (dd, 10 11 J = 6.6, 5.8 Hz, 1H), 2.81 (ddt, J = 10.6, 9.1, 4.5 Hz, 1H), 2.17 (dddd, J = 14.4, 11.5, 6.7, 12 13 14 4.5 Hz, 1H), 2.10–1.97 (m, 2H), 1.95–1.89 (m, 1H), 1.81–1.72 (m, 1H), 1.61–1.52 (m, 15 16 13 17 1H); C NMR (101 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 172.1, 171.3, 18 19 151.0, 149.4, 145.2, 144.1, 135.8, 134.4, 131.6, 129.9, 128.1, 124.5, 120.1, 92.8, 90.0, 20 21 + 22 83.9, 83.4, 74.8, 74.5, 53.5, 38.9, 30.9, 29.6, 28.7; HRMS (ESI+): calcd. for [C24H28N7O6] 23 24 25 510.2096, meas. 510.2082, ∆ 2.6 ppm. 26 27 28 29 30 31 Synthesis of Compounds 11-41 32 33 S R S R R H 34 ( )-2-amino-6-((2 ,3 ,4 ,5 )-5-(6-amino-9 -purin-9-yl)-3,4-dihydroxytetrahy- 35 36 drofuran-2-yl)hexanoic acid (11). To a solution of nucleoside S11 (73 mg, 0.10 mmol) in 37 38

39 a 4:2:1 mixture of MeOH, THF and water (35 mL) at RT was added CsOH•xH2O (100 mg). 40 41 42 The resulting mixture was stirred for 16 h and a 10% AcOH aq. solution was added drop- 43 44 wise until neutral pH was reached. Volatiles were removed in vacuo. Crude material was 45 46 47 purified by preparative HPLC (5% → 95% B’ over 80 min) to afford the partially depro- 48 49 50 tected carbamate. To a solution of the carbamate intermediate in a 10:1 mixture of i-PrOAc 51 52 t 53 and -BuOH (25 mL) at RT, was added Pd/C (10%, 65 mg, 61 µmol). The reaction mixture 54 55 40 56 57 58 59 60 ACS Paragon Plus Environment

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× 1 was purged with hydrogen (3 ) and stirred at RT under a hydrogen atmosphere (balloon) 2 3 for 2 h. The reaction rate appeared sluggish so Pd(OH)2/C (20%, 105 mg, 150 µmol) was 4 5 6 added as a suspension in a 1:1 mixture of i-PrOAc and AcOH (10 mL) and the resulting 7 8 9 mixture was stirred at 50 °C for 2 h. The reaction remained slow, so EtOH (20 mL) was 10 11 added. The temperature was lowered to 40 °C and the reaction media was stirred for 3 h, 12 13 14 upon which complete cleavage of the Cbz protecting group was observed.70 The flask head- 15 16 17 space was purged with nitrogen under vigorous stirring. The contents were filtered over a 18 19 pad of celite and the volatiles were removed in vacuo. Crude material was purified by 20 21 22 preparative HPLC (2.5% → 27.5% B’ over 80 min) to afford 11 (33 mg, 69 µmol, 66% 23 24 1 25 over 2 steps) as the TFA salt, as a fluffy white powder. H NMR (600 MHz, D2O): δ 8.48 26 27 J J J 28 (s, 1H), 8.45 (s, 1H), 6.12 (d, = 5.1 Hz, 1H), 4.82 (t, = 5.2 Hz, 1H), 4.29 (t, = 5.0 Hz, 29 30 1H), 4.21–4.16 (m, 1H), 4.00 (t, J = 6.2 Hz, 1H), 1.98 (app. ddt, J = 14.2, 10.9, 5.5 Hz, 31 32 13 33 1H), 1.94–1.86 (m, 1H), 1.88–1.77 (m, 2H), 1.59–1.39 (m, 4H); C NMR (101 MHz, D2O): 34 35 36 δ 173.2, 150.3, 148.6, 144.9, 143.0, 119.2, 88.4, 85.1, 74.1, 73.4, 53.6, 32.5, 30.0, 24.7, 37 38 + ∆ 24.2; HRMS (ESI+): calcd. for [C15H23N6O5] 367.1724, meas. 367.1728, 1.1 ppm. 39 40 41 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 42 43 44 hydrofuran-2-yl)methyl)hept-6-ynoic acid (12). To a solution of 8 (42 mg, 61 µmol) in 45 46 47 THF (1.0 mL) at RT, was added a 0.5 M LiOH aq. solution (1.0 mL). The resulting mixture 48 49 was stirred for 1 h, cooled to 0 °C, and a 10% AcOH aq. solution was added dropwise until 50 51 52 53 54 55 41 56 57 58 59 60 ACS Paragon Plus Environment

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1 pH 7 was reached. Volatiles were removed in vacuo. The residue was dissolved in a solu- 2 3 tion of ammonia in methanol (7 N, 3 mL) at RT. The resulting mixture was stirred for 16 h 4 5 6 and the volatiles were removed in vacuo. Crude material was purified by preparative HPLC 7 8 9 (2% → 42% B over 30 min followed by 42% → 95% B over 5 min) to afford 12 (22 mg, 10 11 44 µmol, 72% over 2 steps) as the TFA salt upon treatment with d-TFA as part of sample 12 13 1 14 preparation for NMR analysis. H NMR (500 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA 15 16 17 (40 µL)): δ 8.87 (s, 2H), 6.50 (d, J = 5.0 Hz, 1H), 5.19 (t, J = 5.1 Hz, 1H), 4.68 (t, J = 4.8 18 19 Hz, 1H), 4.49 (dd, J = 6.7, 5.8 Hz, 1H), 3.08 (dddd, J = 13.7, 7.2, 5.8, 4.3 Hz, 1H), 3.02 20 21 22 (d, J = 2.4 Hz, 1H), 2.59 (dddd, J = 14.2, 11.6, 6.8, 4.7 Hz, 1H), 2.53–2.45 (m, 1H), 2.41 23 24 25 (td, J = 10.0, 5.2 Hz, 1H), 2.33 (ddd, J = 14.0, 10.9, 3.4 Hz, 1H), 2.19 (tt, J = 12.3, 5.0 Hz, 26

27 13 J CD3CN 28 1H), 1.99 (dddd, = 13.4, 11.9, 9.1, 4.6 Hz, 1H); C NMR (101 MHz, (350 29 30 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 172.0, 150.9, 149.3, 145.2, 144.0, 120.0, 89.8, 86.1, 31 32 + 33 83.7, 74.8, 74.4, 73.1, 53.4, 38.8, 30.7, 28.8, 28.4; HRMS (ESI+): calcd. for [C17H23N6O5] 34 35 ∆ 36 391.1724, meas. 391.1722, 0.7 ppm. 37 38 S S R S R R H 39 (2 ,5 )-2-amino-5-(((2 ,3 ,4 ,5 )-5-(6-amino-9 -purin-9-yl)-3,4-dihydroxytetra- 40 41 hydrofuran-2-yl)methyl)-7-phenylhept-6-ynoic acid (13). To a solution of S12 (74 mg, 42 43 44 97 µmol) in THF (7.0 mL) at RT, was added a 0.5 M LiOH aq. solution (1.7 mL). The 45 46 47 resulting mixture was stirred for 3 h, cooled to 0 °C, and a 10% AcOH aq. solution was 48 49 added dropwise until pH 7 was reached. Volatiles were removed in vacuo. The residue was 50 51 52 dissolved in a solution of ammonia in methanol (7 N, 10 mL) at RT. The resulting mixture 53 54 55 42 56 57 58 59 60 ACS Paragon Plus Environment

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1 was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified 2 3 by preparative HPLC (5% → 45% B over 45 min followed by 45% → 95% B over 5 min) 4 5 6 to afford 13 (35 mg, 60 µmol, 62% over 2 steps) as the TFA salt upon treatment with d- 7 8 1 9 TFA as part of the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 10 11 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.84 (s, 1H), 8.82 (s, 1H), 7.84–7.80 (m, 2H), 7.78– 12 13 14 7.74 (m, 3H), 6.47 (d, J = 5.1 Hz, 1H), 5.19 (t, J = 5.1 Hz, 1H), 4.83 (ddd, J = 10.5, 4.5, 15 16 17 3.2 Hz, 1H), 4.68 (t, J = 4.8 Hz, 1H), 4.48 (dd, J = 6.7, 5.7 Hz, 1H), 3.27 (ddt, J = 10.7, 18 19 9.2, 4.5 Hz, 1H), 2.63 (dddd, J = 14.3, 11.5, 6.8, 4.5 Hz, 1H), 2.56–2.44 (m, 2H), 2.38 20 21 22 (ddd, J = 13.9, 10.8, 3.3 Hz, 1H), 2.23 (tt, J = 12.6, 5.0 Hz, 1H), 2.03 (dddd, J = 13.4, 11.9, 23 24 13 25 9.2, 4.5 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 26 27 28 172.0, 151.0, 149.4, 145.2, 144.0, 132.4, 129.5, 129.2, 123.9, 120.1, 91.7, 89.9, 84.2, 83.9, 29 30 + 74.8, 74.5, 53.5, 39.0, 30.9, 29.5, 28.7; HRMS (ESI+): calcd. for [C23H26N6O5Na] 489.1857, 31 32 33 meas. 489.1841, ∆ 3.2 ppm. 34 35 36 (2S,5R)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytet- 37 38 39 rahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoic acid (14). To a solu- 40 41 tion of S19 (50 mg, 62 µmol) in THF (1.2 mL) at RT, was added a 0.5 M LiOH aq. solution 42 43 44 (1.2 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. 45 46 47 solution (0.22 mL) was added until pH 7 was reached. Volatiles were removed in vacuo. 48 49 The residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The 50 51 52 resulting mixture was stirred for 18 h and the volatiles were removed in vacuo. Crude 53 54 55 43 56 57 58 59 60 ACS Paragon Plus Environment

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1 material was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% 2 3 → 95% B over 5 min) to afford 14 (38 mg, 62 µmol, quant. over 2 steps) as the TFA salt 4 5 6 upon treatment with d-TFA (50 µL), as part of the sample preparation for NMR analysis. 7 8 1 9 H NMR (600 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (50 µL)): δ 8.37 (s, 1H), 8.31 10 11 (s, 1H), 7.70 (ddd, J = 7.7, 1.7, 1.3 Hz, 1H), 7.67 (ddd, J = 1.7, 1.5, 0.4 Hz, 1H), 7.41 (ddd, 12 13 14 J = 7.7, 1.5, 1.3 Hz, 1H), 7.36 (app td, J = 7.7, 0.4 Hz, 1H), 6.00 (d, J = 5.2 Hz, 1H), 4.79 15 16 17 (app t, J = 5.2 Hz, 1H), 4.28 (ddd, J = 8.6, 5.1, 4.0 Hz, 1H), 4.24 (dd, J = 5.2, 4.0 Hz, 1H), 18 19 4.01 (app t, J = 6.4 Hz, 1H), 2.81 (app tt, J = 8.6, 5.3 Hz, 1H), 2.21 (dddd, J = 14.2, 11.5, 20 21 22 6.4, 5.4 Hz, 1H), 2.08 (app dt, J = 13.9, 8.6 Hz, 1H), 2.03–1.94 (m, 2H), 1.76–1.63 (m, 23 24 13 25 2H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (50 µL)): δ 172.2, 171.6, 26 27 28 150.9, 149.4, 145.2, 144.1, 135.7, 134.2, 131.3, 129.9, 128.1, 124.5, 120.0, 93.8, 90.0, 29 30 + 84.6, 82.9, 74.7, 74.7, 53.6, 38.9, 30.7, 29.8, 28.6; HRMS (ESI+): calcd. for [C24H28N7O6] 31 32 33 510.2096, meas. 510.2112, ∆ 3.2 ppm. 34 35 36 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 37 38 39 hydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)heptanoic acid (15). To a solution of 40 41 9 (20 mg, 25 µmol) in MeOH (1.3 mL) at RT was added Pd/C (10%, 8 mg, 7 µmol, 30 mol 42 43 44 %). The vial headspace was evacuated and refilled with hydrogen (3 ×) and the resulting 45 46 47 mixture was stirred vigorously at 50 °C under a hydrogen atmosphere (balloon) for 17 h 48 49 and filtered through a nylon syringe filter (13 mm, 0.22 µm). The solids were washed with 50 51 52 MeOH (5 × 1 mL) and the filtrates were concentrated. Crude alkane was used directly in 53 54 55 44 56 57 58 59 60 ACS Paragon Plus Environment

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1 the next step without further purification. To a solution of crude alkane in THF (0.5 mL) 2 3 at RT, was added a 0.5 M LiOH aq. solution (0.46 mL). The resulting mixture was stirred 4 5 6 for 1 h, cooled to 0 °C and a 1 M HCl aq. solution (0.11 mL) was added to reach pH 7. 7 8 9 Volatiles were removed in vacuo. The residue was dissolved in a solution of ammonia in 10 11 methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles 12 13 14 were removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B 15 16 17 over 30 min followed by 45% → 95% B over 5 min) to afford NS1-alkane (15) (16 mg, 18 19 25 µmol, quant. over 3 steps) as the TFA salt upon treatment with d-TFA as part of the 20 21 1 22 sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 23 24 25 µL)/d-TFA (40 µL)): δ 8.32 (s, 1H), 8.31 (s, 1H), 7.57– 7.56 (m, 1H), 7.54 (app dt, J = 7.3, 26 27 28 1.7 Hz, 1H), 7.32 (app dt, J = 7.7, 1.7 Hz, 1H), 7.30 (ddd, J = 7.7, 7.3, 0.5 Hz, 1H), 5.93 29 30 (d, J = 4.7 Hz, 1H), 4.64 (app t, J = 4.7 Hz, 1H), 4.13–4.10 (m, 1H), 4.10 (dd, J = 5.3, 4.7 31 32 33 Hz, 1H), 3.94 (app t, J = 6.1 Hz, 1H), 2.65–2.53 (m, 2H), 1.93–1.87 (m, 1H), 1.86–1.78 34 35 36 (m, 1H), 1.76–1.70 (m, 2H), 1.68–1.60 (m, 1H), 1.60–1.53 (m, 2H), 1.52–1.45 (m, 1H), 37

38 13 1.45–1.38 (m, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): 39 40 41 δ 172.5, 172.3, 151.0, 149.4, 145.3, 144.2, 144.0, 133.8, 133.4, 129.7, 128.4, 125.9, 120.0, 42 43 44 89.9, 83.4, 74.8, 74.7, 53.9, 37.4, 34.8, 34.3, 32.8, 29.0, 27.9; HRMS (ESI+): calcd. for 45

46 + ∆ [C24H32N7O6] 514.2414, meas. 514.2437, 4.5 ppm. 47 48 49 (2S,5R)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytet- 50 51 52 rahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)heptanoic acid (16). To a solution 53 54 55 45 56 57 58 59 60 ACS Paragon Plus Environment

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1 of 14 (10 mg, 16 µmol) in a 9:1 mixture of EtOH and water (2.0 mL) at RT was added 2 3 Pd/C (10%, 5 mg, 5 µmol, 30 mol %). The vial headspace was evacuated and refilled with 4 5 6 nitrogen (3 ×) followed by hydrogen (3 ×), and the resulting mixture was stirred vigorously 7 8 9 at 50 °C under a hydrogen atmosphere (balloon) for 2.5 h and filtered through a nylon 10 11 syringe filter (13 mm, 0.22 µm). The solids were washed with EtOH (3 × 1 mL) and the 12 13 14 filtrates were concentrated. Crude material was purified by preparative HPLC (5% → 45% 15 16 17 B’ over 30 min followed by 45% → 95% B’ over 5 min) to afford 16 (9 mg, 14 µmol, 89%) 18

19 1 as the TFA salt. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 µL)): δ 8.26 (s, 1H), 8.24 20 21 22 (s, 1H), 7.56–7.52 (m, 2H), 7.29–7.25 (m, 2H), 5.92 (d, J = 4.6 Hz, 1H), 4.64 (dd, J = 5.0, 23 24 25 4.6 Hz, 1H), 4.09 (app t, J = 5.0 Hz, 1H), 4.09–4.05 (m, 1H), 3.80 (app t, J = 6.1 Hz, 1H), 26 27 28 2.59–2.52 (m, 2H), 1.90–1.83 (m, 1H), 1.80–1.70 (m, 2H), 1.67 (ddd, J = 14.3, 7.6, 3.5 29 30 13 Hz, 1H), 1.64–1.51 (m, 3H), 1.48–1.37 (m, 2H); C NMR (126 MHz, CD3CN (350 µL)/D2O 31 32 33 (350 µL)): δ 173.1, 172.3, 152.4, 149.5, 147.5, 144.2, 143.0, 133.8, 133.2, 129.6, 128.3, 34 35 36 125.8, 119.9, 89.5, 83.6, 74.8, 74.6, 54.6, 37.7, 35.6, 34.3, 32.7, 28.1, 27.5; HRMS (ESI+): 37 38 + ∆ calcd. for [C24H32N7O6] 514.2409, meas. 514.2423, 2.9 ppm. 39 40 41 3-(4-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- 42 43 44 yl)but-1-yn-1-yl)benzamide (17). To a vial charged with S26 (58 mg, 97 µmol) was added 45 46 47 a solution of ammonia in methanol (7 N, 8.0 mL) at RT. The resulting mixture was stirred 48 49 for 17 h and volatiles were removed in vacuo. Crude material was purified by preparative 50 51 52 HPLC (10% → 45% B’ over 35 min followed by 45% → 95% B’ over 5 min) to afford 17 53 54 55 46 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 (35 mg, 86 µmol, 88%) as a white fluffy solid. H NMR (600 MHz, CD3CN (400 µL)/D2O 2 3 (400 µL)): δ 8.36 (s, 1H), 8.31 (s, 1H), 7.70–7.69 (m, 1H), 7.71–7.67 (m, 1H), 7.46–7.43 4 5 6 (m, 1H), 7.38–7.35 (m, 1H), 5.98 (d, J = 5.1 Hz, 1H), 4.70 (dd, J = 5.1, 4.7 Hz, 1H), 4.22– 7 8 9 4.18 (m, 2H), 2.53 (app dt, J = 17.3, 6.9 Hz, 1H), 2.49 (app dt, J = 17.3, 7.2 Hz, 1H), 2.03– 10

11 13 1.96 (m, 2H); C NMR (126 MHz, CD3CN (400 µL)/D2O (400 µL)): δ 171.3, 150.9, 149.4, 12 13 14 145.3, 143.8, 135.6, 134.2, 131.2, 129.9, 127.9, 124.6, 119.9, 91.6, 89.6, 84.8, 80.9, 74.8, 15 16 + ∆ 17 74.1, 32.6, 16.3; HRMS (ESI+): calcd. for [C20H20N6O4Na] 431.1438, meas. 431.1453, 3.4 18 19 ppm. 20 21 22 3-(((1R,2R)-2-((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydro- 23 24 25 furan-2-yl)cyclopropyl)ethynyl)benzamide (18). To a vial charged with S33 (50 mg, 82 26 27 28 µmol) was added a solution of ammonia in methanol (7 N, 6.0 mL) at RT. The resulting 29 30 mixture was stirred for 17 h and volatiles were removed in vacuo. Crude material was 31 32 33 purified by preparative HPLC (10% → 45% B’ over 30 min followed by 45% → 95% B’ 34 35 36 over 5 min) to afford 18 (32 mg, 76 µmol, 93%) as a white fluffy solid. 1H NMR (500 MHz, 37 38 39 CD3CN (350 µL)/D2O (350 µL)): δ 8.39 (s, 1H), 8.32 (s, 1H), 7.71 (td, J = 1.8, 0.6 Hz, 40 41 1H), 7.67 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.44 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.35 (td, J = 42 43 44 7.8, 0.6 Hz, 1H), 5.95 (d, J = 4.7 Hz, 1H), 4.73 (dd, J = 5.2, 4.7 Hz, 1H), 4.36 (dd, J = 5.2, 45 46 47 4.8 Hz, 1H), 3.45 (dd, J = 9.1, 4.8 Hz, 1H), 1.62 (dddd, J = 9.1, 8.8, 5.9, 4.4 Hz, 1H), 1.53 48 49 (ddd, J = 8.6, 5.4, 4.4 Hz, 1H), 1.05 (ddd, J = 8.8, 5.4, 4.8 Hz, 1H), 1.02 (ddd, J = 8.6, 5.9, 50 51 52 4.8 Hz, 1H); 13C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)): δ 171.2, 151.1, 149.3, 53 54 55 47 56 57 58 59 60 ACS Paragon Plus Environment

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1 145.4, 143.8, 135.6, 134.3, 131.3, 129.8, 127.8, 124.5, 120.0, 93.6, 89.7, 88.5, 76.7, 74.9, 2 3 + 74.4, 25.2, 12.9, 6.4; HRMS (ESI+): calcd. for [C21H21N6O4] 421.1619, meas. 421.1627, 4 5 6 ∆ 1.9 ppm. 7 8 9 (2S,5S)-2-amino-7-(3-carbamoylphenyl)-5-(((2R,3S,4R,5R)-3,4-dihydroxy-5-meth- 10 11 12 oxytetrahydrofuran-2-yl)methyl)hept-6-ynoic acid (19). To a solution of S35 (23 mg, 13 14 44 µmol) in THF (2.0 mL) at RT was added a 0.5 M LiOH aq. solution (2.0 mL). The 15 16 17 reaction mixture was stirred at RT for 3 h and a 10% AcOH aq. solution was added slowly 18 19 20 until pH 5 was obtained. Volatiles were removed in vacuo and the residue was purified by 21 22 preparative HPLC (5% → 55% B’ over 60 min followed by 55% → 95% B’ over 5 min) 23 24 1 25 to afford 19 (11 mg, 28 µmol, 63%) as the TFA salt. H NMR (600 MHz, D2O): δ 7.84 (t, 26 27 28 J = 1.8 Hz, 1H), 7.74 (ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.65 (ddt, J = 7.7, 1.6, 0.8 Hz, 1H), 29 30 7.47 (tt, J = 7.9, 0.6 Hz, 1H), 4.90 (d, J = 1.4 Hz, 1H), 4.31 (ddd, J = 10.2, 6.3, 3.0 Hz, 31 32 33 1H), 4.13 (dd, J = 6.3, 4.7 Hz, 1H), 4.11–4.06 (m, 2H), 3.40 (s, 3H), 2.97–2.89 (m, 1H), 34 35 36 2.25 (dddd, J = 14.2, 11.5, 6.7, 4.6 Hz, 1H), 2.16 (ddt, J = 14.4, 11.2, 5.5 Hz, 1H), 1.93 37 38 39 (ddd, J = 13.8, 10.9, 3.1 Hz, 1H), 1.85–1.80 (m, 1H), 1.77 (ddd, J = 13.7, 10.6, 4.5 Hz, 40 41 13 1H), 1.70–1.59 (m, 1H); C NMR (101 MHz, D2O): δ 172.6, 172.3, 135.3, 133.1, 130.5, 42 43 44 129.0, 127.2, 123.2, 108.2, 92.1, 82.7, 81.0, 74.6, 74.4, 55.2, 53.2, 39.5, 29.9, 29.1, 28.0; 45 46 + ∆ 47 HRMS (ESI+): calcd. for [C20H27N2O7] 407.1813, meas. 407.1809, 1.0 ppm. 48 49 S R S R R H 50 ( )-5-(((2 ,3 ,4 ,5 )-5-(6-amino-9 -purin-9-yl)-3,4-dihydroxytetrahydrofuran- 51 52 2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoic acid (20). To a solution of S41 (105 53 54 55 48 56 57 58 59 60 ACS Paragon Plus Environment

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M 1 mg, 0.151 mmol) in a 2:1 mixture of MeOH and THF (24 mL) at 0 °C was added a 0.2 2 3 LiOH aq. solution (7.5 mL, 3.01 mmol, 20 equiv.). The resulting mixture was stirred at RT 4 5 6 for 24 h and organics were removed in vacuo. A 1 M HCl aq. solution (1.7 mL) was added 7 8 9 and volatiles were removed in vacuo. Crude material was purified by preparative HPLC 10 11 (30% → 95% B over 35 min followed by 95% B over 5 min) to afford 20 (45 mg, 91 µmol, 12 13 1 14 60%) as a fluffy white solid. H NMR (600 MHz, CD3CN (400 µL)/D2O (400 µL)/d-TFA 15 16 17 (100 µL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.76 (dd, J = 1.8, 1.2 Hz, 1H), 7.70 (ddd, J = 7.8, 18 19 1.8, 1.2 Hz, 1H), 7.49 (app dt, J = 7.8, 1.2 Hz, 1H), 7.37 (app t, J = 7.8 Hz, 1H), 6.01 (d, J 20 21 22 = 5.3 Hz, 1H), 4.81–4.77 (dd, J = 5.3, 5.0 Hz, 1H), 4.41 (ddd, J = 10.6, 4.0, 3.1 Hz, 1H), 23 24 25 4.25–4.21 (dd, J = 5.0, 4.0 Hz, 1H), 2.78–2.71 (m, 1H), 2.36–2.26 (m, 2H), 2.01 (ddd, J = 26 27 J 28 13.9, 10.6, 4.1 Hz, 1H), 1.87 (ddd, = 13.9, 11.0, 3.1 Hz, 1H), 1.83–1.75 (m, 1H), 1.73– 29 30 13 1.64 (m, 1H), 1.61–1.49 (m, 2H); C NMR (126 MHz, CD3CN (400 µL)/D2O (400 µL)/d- 31 32 33 TFA (100 µL)): δ 178.3, 171.9, 151.1, 149.5, 145.3, 144.4, 136.0, 134.2, 131.5, 130.0, 34 35 36 128.1, 124.9, 120.2, 94.0, 90.3, 84.5, 82.9, 74.9, 74.8, 39.2, 35.2, 34.5, 29.8, 23.5; HRMS 37 38 + ∆ (ESI+): calcd. for [C24H27N6O6] 495.1987, meas. 495.1984, 0.4 ppm. 39 40 41 3-((S)-6-amino-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 42 43 44 hydrofuran-2-yl)methyl)hex-1-yn-1-yl)benzamide (21). To a vial charged with S47 (79 45 46 47 mg, 0.11 mmol) was added a solution of ammonia in methanol (7 N, 15 mL) at RT. The 48 49 resulting mixture was stirred for 24 h and volatiles were removed in vacuo. Crude material 50 51 52 was purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% 53 54 55 49 56 57 58 59 60 ACS Paragon Plus Environment

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1 B over 5 min) to afford 21 (33 mg, 57 µmol, 54%) as the TFA salt upon treatment with d- 2 3 1 TFA as part of the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 4 5

6 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.76 (ddd, J = 1.8, 1.5, 0.4 7 8 9 Hz, 1H), 7.71 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.50 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 7.39 10 11 (app td, J = 7.8, 0.4 Hz, 1H), 6.00 (d, J = 5.0 Hz, 1H), 4.71 (dd, J = 5.2, 5.0 Hz, 1H), 4.36 12 13 14 (ddd, J = 10.3, 4.5, 3.4 Hz, 1H), 4.21 (dd, J = 5.2, 4.5 Hz, 1H), 2.97–2.89 (m, 2H), 2.80– 15 16 17 2.74 (m, 1H), 1.98 (ddd, J = 14.1, 10.3, 4.3 Hz, 1H), 1.90 (ddd, J = 14.1, 10.7, 3.4 Hz, 1H), 18 19 1.89–1.82 (m, 1H), 1.78–1.70 (m, 1H), 1.61 (app ddt, J = 13.2, 10.7, 5.4 Hz, 1H), 1.54 20 21 13 22 (dddd, J = 13.2, 10.6, 9.1, 5.1 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 23 24 25 µL)/d-TFA (40 µL)): δ 171.4, 150.9, 149.4, 145.2, 144.0, 135.7, 134.2, 131.4, 129.9, 128.0, 26 27 124.5, 120.0, 93.3, 89.8, 83.9, 83.1, 74.8, 74.4, 40.2, 38.9, 32.3, 29.5, 25.8; HRMS (ESI+): 28 29 30 + ∆ calcd. for [C23H28N7O4] 466.2197, meas. 466.2219, 4.7 ppm. 31 32 33 3-((S)-7-amino-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 34 35 36 hydrofuran-2-yl)methyl)-7-oxohept-1-yn-1-yl)benzamide (22). To a vial charged with 37 38 S41 N 39 (105 mg, 0.151 mmol) was added a solution of ammonia in methanol (7 , 18 mL) at 40 41 RT. The resulting mixture was stirred at 40 °C for 5 days and volatiles were removed in 42 43 44 vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min fol- 45 46 47 lowed by 45% → 95% B over 5 min) to afford 22 (57 mg, 0.12 mmol, 77%) as a white 48

49 1 fluffy solid. H NMR (600 MHz, CD3CN (400 µL)/D2O (400 µL)/d-TFA (50 µL)): δ 8.35 50 51 52 (s, 1H), 8.32 (s, 1H), 7.75 (app t, J = 1.5 Hz, 1H), 7.69 (app dt, J = 7.8, 1.5 Hz, 1H), 7.48 53 54 55 50 56 57 58 59 60 ACS Paragon Plus Environment

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1 (app dt, J = 7.8, 1.5 Hz, 1H), 7.36 (app t, J = 7.8 Hz, 1H), 5.99 (d, J = 5.2 Hz, 1H), 4.75 2 3 (app t, J = 5.2 Hz, 1H), 4.38 (ddd, J = 10.6, 4.1, 3.1 Hz, 1H), 4.21 (dd, J = 5.2, 4.1 Hz, 4 5 6 1H), 2.73 (dddd, J = 10.9, 9.2, 5.4, 4.0 Hz, 1H), 2.23 (ddd, J = 14.6, 8.0, 7.2 Hz, 1H), 2.20 7 8 9 (ddd, J = 14.6, 7.8, 7.1 Hz, 1H), 1.99 (ddd, J = 13.8, 10.6, 4.1 Hz, 1H), 1.85 (ddd, J = 13.8, 10 11 11.0, 3.1 Hz, 1H), 1.82–1.73 (m, 1H), 1.71–1.62 (m, 1H), 1.59–1.47 (m, 2H); 13C NMR 12 13

14 (126 MHz, CD3CN (400 µL)/D2O (400 µL)/d-TFA (50 µL)): δ 179.5, 171.4, 150.9, 149.4, 15 16 17 145.2, 144.2, 135.8, 134.2, 131.4, 129.9, 128.0, 124.7, 120.1, 93.9, 90.1, 84.3, 82.8, 74.8, 18

19 + 74.6, 39.1, 35.6, 35.1, 29.7, 24.2; HRMS (ESI+): calcd. for [C24H27N7O5Na] 516.1966, meas. 20 21 22 516.1986, ∆ 3.9 ppm. 23 24 25 Methyl (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihy- 26 27 28 droxytetrahydrofuran-2-yl)methyl)-7-(3-carbamoylphenyl)hept-6-ynoate (23). To a 29 30 solution of S51 (32 mg, 34 µmol) in a 1:1 mixture of MeCN and CH2Cl2 (2.0 mL) at RT 31 32 33 was added piperidine (0.50 mL). The resulting mixture was stirred for 20 min and volatiles 34 35 36 were removed in vacuo. The residue was dissolved in a solution of ammonia in methanol 37 38 N 39 (7 , 4.5 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were 40 41 removed in vacuo. Crude material was purified by preparative HPLC (5% → 45% B’ over 42 43 44 35 min followed by 45% → 95% B’ over 5 min) to afford 23 (12 mg, 19 µmol, 27% over 45 46 1 47 2 steps) as the TFA salt. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 µL)): δ 8.33 (s, 48 49 1H), 8.30 (s, 1H), 7.77 (ddd, J = 1.9, 1.5, 0.5 Hz, 1H), 7.72 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 50 51 52 7.51 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 7.40 (app td, J = 7.8, 0.5 Hz, 1H), 5.98 (d, J = 5.0 Hz, 53 54 55 51 56 57 58 59 60 ACS Paragon Plus Environment

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1 1H), 4.68 (dd, J = 5.2, 5.0 Hz, 1H), 4.34 (ddd, J = 10.0, 4.7, 3.4 Hz, 1H), 4.19 (dd, J = 5.2, 2 3 4.7 Hz, 1H), 4.04 (dd, J = 6.6, 6.1 Hz, 1H), 3.73 (s, 3H), 2.82–2.75 (m, 1H), 2.13 (dddd, J 4 5 6 = 14.3, 11.6, 6.6, 4.6 Hz, 1H), 2.06–1.95 (m, 2H), 1.91 (ddd, J = 13.9, 10.5, 3.4 Hz, 1H), 7 8 13 9 1.71 (app ddt, J = 12.8, 11.6, 5.1 Hz, 1H), 1.52 (dddd, J = 12.8, 11.7, 9.7, 4.6 Hz, 1H); C 10 11 NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)): δ 171.2, 170.9, 151.5, 149.4, 146.2, 12 13 14 143.6, 135.7, 134.3, 131.4, 129.9, 128.1, 124.3, 119.9, 92.7, 89.7, 83.6, 83.4, 74.7, 74.3, 15 16 + 17 54.3, 53.5, 38.7, 30.7, 29.5, 28.7; HRMS (ESI+): calcd. for [C25H30N7O6] 524.2252, meas. 18 19 524.2271, ∆ 3.6 ppm. 20 21 22 3-((3S,6S)-6,7-diamino-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydrox- 23 24 25 ytetrahydrofuran-2-yl)methyl)-7-oxohept-1-yn-1-yl)benzamide (24). To a vial charged 26 27 28 with 9 (44 mg, 54 µmol) was added a solution of ammonia in methanol (7 N, 15 mL) at 29 30 RT. The resulting mixture was stirred at 40 °C for 36 h and the volatiles were removed in 31 32 33 vacuo. Crude material was purified by preparative HPLC (5% → 45% B over 30 min fol- 34 35 36 lowed by 45% → 95% B over 5 min) to afford 24 (31 mg, 50 µmol, 91%) as the TFA salt 37

38 1 39 upon treatment with d-TFA as part of the sample preparation for NMR analysis. H NMR 40 41 (600 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (50 µL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 42 43 44 7.78–7.76 (m, 1H), 7.71 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.51 (ddd, J = 7.8, 1.5, 1.2 Hz, 45 46 47 1H), 7.39 (app td, J = 7.8, 0.6 Hz, 1H), 6.00 (d, J = 5.1 Hz, 1H), 4.73 (app t, J = 5.1 Hz, 48 49 1H), 4.37 (ddd, J = 10.3, 4.5, 3.4 Hz, 1H), 4.22 (dd, J = 5.1, 4.5 Hz, 1H), 3.96 (app t, J = 50 51 52 6.4 Hz, 1H), 2.79 (dddd, J = 10.6, 9.2, 4.5, 4.3 Hz, 1H), 2.13 (dddd, J = 14.2, 12.4, 6.4, 4.4 53 54 55 52 56 57 58 59 60 ACS Paragon Plus Environment

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1 Hz, 1H), 2.00 (ddd, J = 14.3, 10.3, 4.3 Hz, 1H), 1.98–1.89 (m, 2H), 1.67 (dddd, J = 13.1, 2 3 12.4, 4.8, 4.5 Hz, 1H), 1.56 (dddd, J = 13.1, 12.7, 9.2, 4.4 Hz, 1H); 13C NMR (126 MHz, 4 5

6 CD3CN (350 µL)/D2O (350 µL)/d-TFA (50 µL)): δ 172.4, 171.6, 151.0, 149.4, 145.2, 7 8 9 144.1, 135.9, 134.2, 131.6, 130.0, 128.1, 124.5, 120.1, 92.9, 90.0, 84.0, 83.4, 74.9, 74.5, 10

11 + 53.8, 38.8, 30.7, 29.8, 29.7; HRMS (ESI+): calcd. for [C24H28N8O5Na] 531.2075, meas. 12 13 14 531.2089, ∆ 2.7 ppm. 15 16 17 3-((S)-3-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofu- 18 19 20 ran-2-yl)methyl)-5-ureidopent-1-yn-1-yl)benzamide (25). To a vial charged with S58 21 22 (31 mg, 45 µmol) was added a solution of ammonia in methanol (7 N, 5.0 mL) at RT. The 23 24 25 resulting mixture was stirred for 17 h and the volatiles were removed in vacuo. Crude 26 27 28 material was purified by preparative HPLC (5% → 45% B over 35 min followed by 45% 29 30 → 95% B over 5 min) to afford 25 (18 mg, 36 µmol, 80%) as a fluffy white solid. 1H NMR 31 32 33 (600 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.54 (s, 1H), 8.51 (s, 1H), 34 35 36 7.96 (t, J = 1.7 Hz, 1H), 7.90 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 7.70 (dt, J = 7.7, 1.3 Hz, 1H), 37 38 39 7.58 (dd, J = 7.8, 0.6 Hz, 1H), 6.18 (d, J = 5.2 Hz, 1H), 4.93 (t, J = 5.2 Hz, 1H), 4.56 (dt, 40 41 J = 10.6, 3.5 Hz, 1H), 4.40 (t, J = 4.7 Hz, 1H), 3.50 (ddd, J = 13.3, 7.7, 5.4 Hz, 1H), 3.43 42 43 44 (dt, J = 13.7, 7.5 Hz, 1H), 2.98 (tt, J = 9.7, 4.5 Hz, 1H), 2.19 (ddd, J = 14.4, 10.5, 4.2 Hz, 45 46 47 1H), 2.08 (ddd, J = 13.9, 10.9, 3.2 Hz, 1H), 2.00–1.91 (m, 1H), 1.86 (dddd, J = 13.2, 9.7, 48

49 13 7.4, 5.4 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 50 51 52 171.6, 161.7, 151.0, 149.5, 145.2, 144.3, 135.9, 134.3, 131.5, 130.0, 128.2, 124.6, 120.1, 53 54 55 53 56 57 58 59 60 ACS Paragon Plus Environment

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1 93.0, 90.1, 84.1, 83.2, 74.8, 74.6, 39.5, 39.0, 35.3, 27.6; HRMS (ESI+): calcd. for 2 3 + ∆ [C23H26N8O5Na] 517.1918, meas. 517.1912, 1.2 ppm. 4 5 6 (2S,6S)-2-amino-6-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 7 8 9 hydrofuran-2-yl)methyl)-8-(3-carbamoylphenyl)oct-7-ynoic acid (26). To a solution of 10 11 M 12 S66 (69 mg, 84 µmol) in THF (1.5 mL) at RT was added a 0.5 LiOH aq. solution (1.5 13 14 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution 15 16 17 (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a 18 19 20 solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred 21 22 for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative 23 24 25 HPLC (5% → 25% B’ over 30 min followed by 25% → 95% B’ over 5 min) to afford 26 26 27 1 28 (9 mg, 14 µmol, 17% over 2 steps) as the TFA salt. H NMR (600 MHz, D2O (700 µL)): δ 29 30 8.42 (s, 1H), 8.30 (s, 1H), 7.70–7.65 (m, 1H), 7.51–7.48 (m, 1H), 7.37–7.33 (m, 2H), 6.08 31 32 33 (d, J = 5.0 Hz, 1H), 4.95 (dd, J = 5.1, 5.0 Hz, 1H), 4.47 (ddd, J = 9.2, 4.6, 3.2 Hz, 1H), 34 35 36 4.40 (dd, J = 5.1, 4.6 Hz, 1H), 3.95 (app t, J = 6.1 Hz, 1H), 2.89–2.84 (m, 1H), 2.08 (ddd, 37 38 39 J = 14.4, 9.2, 3.7 Hz, 1H), 2.00 (ddd, J = 14.4, 9.8, 3.2 Hz, 1H), 1.96–1.91 (m, 2H), 1.72– 40 41 13 1.60 (m, 3H), 1.56–1.49 (m, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)): δ 42 43 44 172.5, 171.3, 151.2, 149.4, 145.7, 143.8, 135.7, 134.2, 131.4, 129.9, 127.9, 124.5, 120.0, 45 46 47 93.7, 89.7, 83.9, 82.9, 74.7, 74.4, 53.7, 38.9, 34.9, 30.5, 29.5, 23.2; HRMS (ESI+): calcd. 48 49 + ∆ for [C25H30N7O6] 524.2252, meas. 524.2277, 4.7 ppm. 50 51 52 53 54 55 54 56 57 58 59 60 ACS Paragon Plus Environment

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S S R S R R H 1 (2 ,5 )-2-amino-5-(((2 ,3 ,4 ,5 )-5-(6-amino-9 -purin-9-yl)-3,4-dihydroxytetra- 2 3 hydrofuran-2-yl)methyl)-7-(4-carbamoylphenyl)hept-6-ynoic acid (27). To a solution 4 5 6 of S67 (18 mg, 22 µmol) in THF (0.41 mL) at RT was added a 0.5 M LiOH aq. solution 7 8 9 (0.41 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. 10 11 solution (0.10 mL) was added. Volatiles were removed in vacuo. The residue was dissolved 12 13 14 in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred 15 16 17 for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative 18 19 HPLC (5% → 45% B’ over 30 min followed by 45% → 95% B’ over 5 min) to afford 27 20 21 1 22 (7 mg, 11 µmol, 50% over 2 steps) as the TFA salt. H NMR (600 MHz, CD3CN (350 23 24 25 µL)/D2O (350 µL)): δ 8.32 (s, 1H), 8.30 (s, 1H), 7.73–7.70 (m, 2H), 7.43–7.39 (m, 26 27 J J J 28 2H), 5.97 (d, = 5.0 Hz, 1H), 4.71 (dd, = 5.3, 5.0 Hz, 1H), 4.33 (ddd, = 10.3, 4.5, 3.3 29 30 Hz, 1H), 4.19 (dd, J = 5.3, 4.5 Hz, 1H), 3.90 (dd, J = 6.8, 5.7 Hz, 1H), 2.83–2.76 (m, 1H), 31 32 33 2.10 (dddd, J = 14.2, 11.5, 6.8, 4.6 Hz, 1H), 2.03–1.96 (m, 2H), 1.94–1.88 (m, 1H), 1.73 34 35 13 36 (dddd, J = 12.8, 12.0, 5.4, 4.6 Hz, 1H), 1.55 (m, 1H); C NMR (126 MHz, CD3CN (350 37 38 µL)/D2O (350 µL)): δ 172.6, 171.4, 151.7, 149.4, 146.5, 143.6, 133.2, 132.5, 128.6, 127.7, 39 40 41 120.0, 94.7, 89.7, 83.7, 83.4, 74.7, 74.4, 53.9, 38.6, 30.9, 29.6, 28.8; HRMS (ESI+): calcd. 42 43 + ∆ 44 for [C24H28N7O6] 510.2096, meas. 510.2097, 0.2 ppm. 45 46 S S R S R R H 47 (2 ,5 )-2-amino-5-(((2 ,3 ,4 ,5 )-5-(6-amino-9 -purin-9-yl)-3,4-dihydroxytetra- 48 49 hydrofuran-2-yl)methyl)-7-(2-carbamoylphenyl)hept-6-ynoic acid (28). To a solution 50 51 52 of alkyne S68 (18 mg, 22 µmol) in THF (0.41 mL) at RT was added a 0.5 M LiOH aq. 53 54 55 55 56 57 58 59 60 ACS Paragon Plus Environment

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M 1 solution (0.41 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 2 3 HCl aq. solution (0.10 mL) was added. Volatiles were removed in vacuo. The residue was 4 5 6 dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture 7 8 9 was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified 10 11 by preparative HPLC (5% → 45% B’ over 30 min followed by 45% → 95% B’ over 5 min) 12 13 1 14 to afford 28 (8 mg, 13 µmol, 58% over 2 steps) as the TFA salt. H NMR (600 MHz, CD3CN 15 16 17 (350 µL)/D2O (350 µL)): δ 8.32 (s, 1H), 8.29 (s, 1H), 7.56–7.52 (m, 1H), 7.44–7.41 (m, 18 19 1H), 7.40 (app td, J = 7.6, 1.5 Hz, 1H), 7.37 (ddd, J = 7.6, 7.1, 2.0 Hz, 1H), 5.97 (d, J = 20 21 22 5.1 Hz, 1H), 4.71 (app t, J = 5.1 Hz, 1H), 4.34 (ddd, J = 10.1, 4.5, 3.4 Hz, 1H), 4.20–4.18 23 24 25 (m, 1H), 3.88 (dd, J = 6.7, 5.9 Hz, 1H), 2.84–2.76 (m, 1H), 2.09 (dddd, J = 14.2, 11.7, 6.7, 26 27 J 28 4.6 Hz, 1H), 2.01–1.95 (m, 2H), 1.95–1.89 (m, 1H), 1.72 (app ddt, = 13.4, 11.7, 4.9 Hz, 29 30 13 1H), 1.60–1.52 (m, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)): δ 172.8, 31 32 33 151.7, 149.4, 146.5, 143.6, 137.3, 134.1, 131.7, 129.4, 128.8, 121.3, 120.0, 97.1, 89.7, 34 35 + 36 83.7, 81.8, 74.6, 74.4, 54.1, 38.5, 30.9, 29.7, 28.9; HRMS (ESI+): calcd. for [C24H28N7O6] 37 38 510.2096, meas. 510.2095, ∆ 0.2 ppm. 39 40 41 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 42 43 44 hydrofuran-2-yl)methyl)-7-(3-sulfamoylphenyl)hept-6-ynoic acid (29). To a solution of 45 46 47 S69 (41 mg, 49 µmol) in THF (1.3 mL) at RT was added a 0.5 M LiOH aq. solution (1.1 48 49 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution 50 51 52 (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a 53 54 55 56 56 57 58 59 60 ACS Paragon Plus Environment

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N 1 solution of ammonia in methanol (7 , 5 mL) at RT. The resulting mixture was stirred for 2 3 18 h and volatiles were removed in vacuo. Crude material was purified by preparative 4 5 6 HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 29 7 8 9 (26 mg, 39 µmol, 81% over 2 steps) as the TFA salt upon treatment with d-TFA as part of 10

11 1 the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 12 13 14 µL)/d-TFA (40 µL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.82 (dd, J = 1.9, 1.5 Hz, 1H), 7.77 (ddd, 15 16 17 J = 7.8, 1.9, 1.2 Hz, 1H), 7.57 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 7.48 (app t, J = 7.8 Hz, 1H), 18 19 6.00 (d, J = 5.1 Hz, 1H), 4.73 (dd, J = 5.2, 5.1 Hz, 1H), 4.36 (ddd, J = 10.3, 4.5, 3.3 Hz, 20 21 22 1H), 4.22 (dd, J = 5.2, 4.5 Hz, 1H), 4.01 (dd, J = 6.7, 5.8 Hz, 1H), 2.86–2.78 (m, 1H), 2.16 23 24 25 (dddd, J = 14.4, 11.7, 6.7, 4.5 Hz, 1H), 2.10–2.02 (m, 1H), 2.01 (ddd, J = 14.3, 10.3, 4.1 26 27 J J 28 Hz, 1H), 1.96–1.90 (m, 1H), 1.77 (app ddt, = 13.0, 11.7, 5.1 Hz, 1H), 1.58 (dddd, = 29 30 13 13.0, 11.7, 9.6, 4.5 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA 31 32 33 (40 µL)): δ 172.1, 151.0, 149.4, 145.3, 144.1, 143.7, 136.4, 130.6, 129.6, 126.4, 125.2, 34 35 36 120.1, 93.8, 90.1, 83.9, 82.8, 74.9, 74.5, 53.6, 38.8, 30.9, 29.6, 28.7; HRMS (ESI+): calcd. 37 38 + ∆ for [C23H28N7O7S1] 546.1765, meas. 546.1764, 0.3 ppm. 39 40 41 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 42 43 44 hydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-fluorophenyl)hept-6-ynoic acid (30). To a 45 46 47 solution of S71 (44 mg, 53 µmol) in THF (1 mL) at RT, was added a 0.5 M LiOH aq. 48 49 solution (1 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl 50 51 52 53 54 55 57 56 57 58 59 60 ACS Paragon Plus Environment

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1 aq. solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dis- 2 3 solved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was 4 5 6 stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by 7 8 9 preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to 10 11 afford 30 (17 mg, 26 µmol, 50% over 2 steps) as the TFA salt upon treatment with d-TFA 12 13 1 14 as part of the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 15 16 17 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.76 (dd, J = 7.1, 2.3 Hz, 18 19 1H), 7.51 (ddd, J = 8.6, 4.8, 2.3 Hz, 1H), 7.15 (dd, J = 11.1, 8.6 Hz, 1H), 6.00 (d, J = 5.1 20 21 22 Hz, 1H), 4.72 (app t, J = 5.1 Hz, 1H), 4.35 (ddd, J = 10.3, 4.5, 3.3 Hz, 1H), 4.21 (dd, J = 23 24 25 5.1, 4.5 Hz, 1H), 4.01 (dd, J = 6.7, 5.8 Hz, 1H), 2.82–2.76 (m, 1H), 2.15 (dddd, J = 14.4, 26 27 J 28 11.7, 6.7, 4.5 Hz, 1H), 2.07–2.02 (m, 1H), 1.99 (ddd, = 13.9, 10.3, 4.5 Hz, 1H), 1.91 29 30 (ddd, J = 13.9, 10.8, 3.3 Hz, 1H), 1.75 (app ddt, J = 12.9, 11.7, 5.0 Hz, 1H), 1.56 (dddd, J 31 32 13 33 = 12.9, 11.9, 9.5, 4.5 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA 34 35 36 (40 µL)): δ 172.2, 167.5 (d, JC-F = 2.0 Hz), 160.7 (d, JC-F = 253 Hz), 151.0, 149.4, 145.3, 37 38 144.1, 137.7 (d, JC-F = 9.5 Hz), 134.8 (d, JC-F = 2.7 Hz), 122.4 (d, JC-F = 14 Hz), 120.9 (d, JC-F = 39 40

41 3.7 Hz), 120.1, 117.9 (d, JC-F = 25 Hz), 92.5, 90.0, 84.0, 82.4, 74.9, 74.5, 53.6, 38.9, 30.9, 42 43 19 44 29.6, 28.8; F NMR (471 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL), HFB IStd): 45 46 + ∆ δ –113.6; HRMS (ESI+): calcd. for [C24H26F1N7O6Na] 550.1821, meas. 550.1821, 0.0 ppm. 47 48 49 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 50 51 52 hydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-methylphenyl)hept-6-ynoic acid (31). To 53 54 55 58 56 57 58 59 60 ACS Paragon Plus Environment

Page 59 of 188 Journal of Medicinal Chemistry

M 1 a solution of S73 (26 mg, 32 µmol) in THF (1.5 mL) at RT was added a 0.5 LiOH aq. 2 3 solution (0.9 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl 4 5 6 aq. solution (0.17 mL) was added. Volatiles were removed in vacuo. The residue was dis- 7 8 9 solved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was 10 11 stirred for 18 h and volatiles were removed in vacuo. Crude material was purified by pre- 12 13 14 parative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to 15 16 17 afford 31 (11 mg, 17 µmol, 55% over 2 steps) as the TFA salt upon treatment with d-TFA 18

19 1 as part of the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 20 21

22 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.37 (br d, J = 1.8 Hz, 1H), 23 24 25 7.29 (dd, J = 7.9, 1.8 Hz, 1H), 7.17 (br d, J = 7.9 Hz, 1H), 5.99 (d, J = 5.1 Hz, 1H), 4.73 26 27 J J J 28 (app t, = 5.1 Hz, 1H), 4.35 (ddd, = 10.3, 4.5, 3.2 Hz, 1H), 4.21 (dd, = 5.1, 4.5 Hz, 29 30 1H), 4.00 (dd, J = 6.7, 5.8 Hz, 1H), 2.82–2.75 (m, 1H), 2.32 (s, 3H), 2.14 (dddd, J = 14.3, 31 32 33 11.7, 6.7, 4.5 Hz, 1H), 2.07–2.00 (m, 1H), 1.99 (ddd, J = 13.9, 10.3, 4.3 Hz, 1H), 1.90 34 35 36 (ddd, J = 13.9, 10.7, 3.2 Hz, 1H), 1.78–1.70 (app ddt, J = 13.0, 11.7, 5.1 Hz, 1H), 1.55 37

38 13 (dddd, J = 13.0, 11.8, 9.3, 4.5 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 39 40 41 µL)/d-TFA (40 µL)): δ 174.5, 172.2, 151.0, 149.4, 145.2, 144.1, 136.9, 136.5, 133.9, 132.1, 42 43 44 130.9, 121.4, 120.1, 92.1, 90.0, 83.9, 83.5, 74.8, 74.5, 53.6, 38.9, 30.9, 29.6, 28.8, 19.8; 45 46 + ∆ HRMS (ESI+): calcd. for [C25H29N7O6Na] 546.2072, meas. 546.2074, 0.5 ppm. 47 48 49 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 50 51 52 hydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-(trifluoromethyl)phenyl)hept-6-ynoic 53 54 55 59 56 57 58 59 60 ACS Paragon Plus Environment

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1 acid (32). To a solution of S75 (51 mg, 58 µmol) in THF (5.0 mL) at RT was added a 0.5 2 3 M LiOH aq. solution (1.5 mL). The resulting mixture was stirred for 3 h, cooled to 0 °C, 4 5 6 and a 10% AcOH aq. solution was added dropwise until pH 7 was reached. Volatiles were 7 8 9 removed in vacuo. The residue was dissolved in a solution of ammonia in methanol (7 N, 10 11 8 mL) at RT. The resulting mixture was stirred for 18 h and the volatiles were removed in 12 13 14 vacuo. Crude material was purified by preparative HPLC (5% → 35% B over 30 min fol- 15 16 17 lowed by 35% → 95% B over 5 min) to afford 32 (22 mg, 32 µmol, 55% over 2 steps) as 18 19 the TFA salt upon treatment with d-TFA as part of the sample preparation for NMR analy- 20 21 1 22 sis. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.82 (s, 1H), 23 24 25 8.80 (s, 1H), 8.12 (d, J = 8.2 Hz, 1H), 8.01 (ddt, J = 8.2, 1.8, 1.0 Hz, 1H), 8.00–7.97 (m, 26 27 28 1H), 6.45 (d, J = 5.0 Hz, 1H), 5.16 (t, J = 5.1 Hz, 1H), 4.82–4.79 (m, 1H), 4.65 (t, J = 4.8 29 30 Hz, 1H), 4.46 (dd, J = 6.7, 5.8 Hz, 1H), 3.32–3.25 (m, 1H), 2.60 (dddd, J = 14.3, 11.5, 6.7, 31 32 33 4.5 Hz, 1H), 2.54–2.43 (m, 2H), 2.41–2.35 (m, 1H), 2.27–2.18 (m, 1H), 2.03 (dddd, J = 34 35 13 36 13.4, 11.8, 9.3, 4.5 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA 37 38 (40 µL)): δ 172.0, 171.9, 151.0, 149.4, 145.2, 144.0, 136.0, 133.8, 132.0, 128.3, 127.8, 39 40 41 127.8, 127.7, 126.6, 126.3, 125.5, 123.4, 120.0, 117.9, 115.6, 113.3, 95.7, 89.9, 83.8, 82.3, 42 43 + 44 74.8, 74.4, 53.5, 38.7, 30.7, 29.6, 28.7; HRMS (ESI+): calcd. for [C25H26F3N7O6Na] 45 46 600.1789, meas. 600.1776, ∆ 2.2 ppm. 47 48 49 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 50 51 52 hydrofuran-2-yl)methyl)-7-(3-carbamoyl-4-chlorophenyl)hept-6-ynoic acid (33). To a 53 54 55 60 56 57 58 59 60 ACS Paragon Plus Environment

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M 1 solution of S77 (45 mg, 53 µmol) in THF (1 mL) at RT was added a 0.5 LiOH aq. 2 3 solution (1 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl 4 5 6 aq. solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dis- 7 8 9 solved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The resulting mixture was 10 11 stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified by 12 13 14 preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to 15 16 17 afford 33 (35 mg, 53 µmol, quant. over 2 steps) as the TFA salt upon treatment with d-TFA 18

19 1 as part of the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 20 21

22 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.36 (s, 1H), 8.34 (s, 1H), 7.48–7.47 (m, 1H), 7.38– 23 24 25 7.36 (m, 2H), 5.99 (d, J = 5.1 Hz, 1H), 4.72 (app t, J = 5.1 Hz, 1H), 4.34 (ddd, J = 10.4, 26 27 J J 28 4.5, 3.2 Hz, 1H), 4.20 (dd, = 5.1, 4.5 Hz, 1H), 4.00 (dd, = 6.7, 5.8 Hz, 1H), 2.79 (app 29 30 ddt, J = 10.7, 9.2, 4.5 Hz, 1H), 2.14 (dddd, J = 14.3, 11.6, 6.7, 4.5 Hz, 1H), 2.07–2.01 (m, 31 32 33 1H), 2.00 (ddd, J = 14.1, 10.4, 4.5 Hz, 1H), 1.91 (ddd, J = 14.1, 10.7, 3.2 Hz, 1H), 1.75 34 35 13 36 (dddd, J = 13.1, 11.6, 5.0, 4.5 Hz, 1H), 1.55 (dddd, J = 13.1, 11.8, 9.2, 4.5 Hz, 1H); C 37 38 NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 172.1, 171.1, 151.0, 39 40 41 149.4, 145.2, 144.1, 136.0, 135.1, 132.7, 131.3, 131.0, 123.1, 120.1, 93.9, 90.0, 83.9, 82.4, 42 43 + 44 74.8, 74.5, 53.5, 38.8, 30.8, 29.6, 28.7; HRMS (ESI+): calcd. for [C24H26Cl1N7O6Na] 45 46 566.1525, meas. 566.1528, ∆ 0.4 ppm. 47 48 49 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 50 51 52 hydrofuran-2-yl)methyl)-7-(1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)hept-6-ynoic 53 54 55 61 56 57 58 59 60 ACS Paragon Plus Environment

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1 acid (34). To a solution of S78 (29 mg, 35 µmol) in THF (0.65 mL) at RT was added a 0.5 2 3 M LiOH aq. solution (0.65 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, 4 5 6 and a 1 M HCl aq. solution (0.14 mL) was added. Volatiles were removed in vacuo. The 7 8 9 residue was dissolved in a solution of ammonia in methanol (7 N, 5 mL) at RT. The result- 10 11 ing mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was 12 13 14 purified by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B 15 16 17 over 5 min) to afford 34 (23 mg, 35 µmol, quant. over 2 steps) as the TFA salt upon treat- 18 19 ment with d-TFA as part of the sample preparation for NMR analysis. 1H NMR (600 MHz, 20 21

22 CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.08 (br d, J 23 24 25 = 7.7 Hz, 1H), 6.96 (dd, J = 7.7, 1.6 Hz, 1H), 6.82 (d, J = 1.6 Hz, 1H), 5.99 (d, J = 5.1 Hz, 26 27 J J J 28 1H), 4.73 (app t, = 5.1 Hz, 1H), 4.35 (ddd, = 10.4, 4.5, 3.2 Hz, 1H), 4.21 (dd, = 5.1, 29 30 4.5 Hz, 1H), 4.00 (dd, J = 6.7, 5.6 Hz, 1H), 2.89–2.85 (m, 2H), 2.78 (dddd, J = 10.8, 9.3, 31 32 33 4.7, 4.3 Hz, 1H), 2.53–2.48 (m, 2H), 2.13 (dddd, J = 14.3, 11.8, 6.7, 4.6 Hz, 1H), 2.03 34 35 36 (dddd, J = 14.3, 11.7, 5.6, 5.2 Hz, 1H), 1.99 (ddd, J = 14.0, 10.4, 4.3 Hz, 1H), 1.89 (ddd, J 37 38 = 14.0, 10.8, 3.2 Hz, 1H), 1.74 (dddd, J = 13.0, 11.8, 5.2, 4.7 Hz, 1H), 1.54 (dddd, J = 13.0, 39 40 13 41 11.7, 9.3, 4.6 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 42 43 44 µL)): δ 174.6, 172.2, 151.0, 149.4, 145.2, 144.1, 138.1, 129.2, 127.5, 125.6, 122.8, 120.1, 45 46 119.1, 91.7, 90.0, 83.9, 83.8, 74.8, 74.5, 53.5, 38.9, 30.9, 30.7, 29.5, 28.7, 25.4; HRMS 47 48 + ∆ 49 (ESI+): calcd. for [C26H30N7O6] 536.2252, meas. 536.2268, 3.0 ppm. 50 51 52 53 54 55 62 56 57 58 59 60 ACS Paragon Plus Environment

Page 63 of 188 Journal of Medicinal Chemistry

S S R S R R H 1 (2 ,5 )-2-amino-5-(((2 ,3 ,4 ,5 )-5-(6-amino-9 -purin-9-yl)-3,4-dihydroxytetra- 2 3 hydrofuran-2-yl)methyl)-7-(3-oxoisoindolin-5-yl)hept-6-ynoic acid (35). To a solution 4 5 6 of 8 (107 mg, 0.155 mmol) and 6-iodoisoindolin-1-one (101 mg, 0.388 mmol, 2.5 equiv.) 7 8 9 in a 5:3:1 mixture of toluene, DMF and i-Pr2NEt (degassed by sparging with nitrogen for 10 11 15 min, 4.7 mL) at RT were added CuI (7 mg, 0.04 mmol, 25 mol %) and Pd(PPh3)4 (9 mg, 12 13 14 8 µmol, 5 mol %) rapidly. The resulting mixture was stirred at 70 °C for 3 h. The solution 15 16 17 was allowed to cool to RT and volatiles were removed in vacuo. Crude material was filtered 18 19 through a short pad of silica gel (EtOAc/i-PrOH, 85:15) to afford the desired crude reaction 20 21 22 product which was used directly in the next step without further purification. To a solution 23 24 25 of the crude reaction product in THF (3 mL) at RT was added a 0.5 M LiOH aq. solution 26 27 (3 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. solution 28 29 30 (0.60 mL) was added. Volatiles were removed in vacuo. The residue was dissolved in a 31 32 33 solution of ammonia in methanol (7 N, 18 mL) at RT. The resulting mixture was stirred for 34 35 36 18 h and volatiles were removed in vacuo. Crude material was purified by preparative 37 38 HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) to afford 35 39 40 41 (35 mg, 55 µmol, 36% over 3 steps) as the TFA salt upon treatment with d-TFA as part of 42 43 1 44 the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 45 46 µL)/d-TFA (40 µL)): δ 8.36 (s, 1H), 8.33 (s, 1H), 7.65 (br s, 1H), 7.54 (dd, J = 7.9, 1.5 Hz, 47 48 49 1H), 7.48–7.45 (m, 1H), 6.01 (d, J = 5.1 Hz, 1H), 4.74 (app t, J = 5.1 Hz, 1H), 4.40 (br s, 50 51 52 2H), 4.38 (ddd, J = 10.2, 4.4, 3.3 Hz, 1H), 4.23 (app t, J = 4.8 Hz, 1H), 4.03 (app t, J = 6.2 53 54 55 63 56 57 58 59 60 ACS Paragon Plus Environment

Journal of Medicinal Chemistry Page 64 of 188

1 Hz, 1H), 2.82 (app ddt, J = 10.7, 9.2, 4.5 Hz, 1H), 2.18 (dddd, J = 14.4, 11.4, 6.7, 4.5 Hz, 2 3 1H), 2.06 (app ddt, J = 14.5, 11.5, 5.6 Hz, 1H), 2.01 (ddd, J = 14.1, 10.2, 4.2 Hz, 1H), 4 5 6 1.96–1.90 (m, 1H), 1.77 (app ddt, J = 12.9, 11.5, 5.0 Hz, 1H), 1.57 (dddd, J = 13.4, 11.7, 7 8 13 9 9.4, 4.4 Hz, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 10 11 172.8, 172.2, 150.9, 149.4, 145.2, 145.1, 144.1, 136.1, 132.7, 127.0, 124.9, 123.8, 120.0, 12 13 14 92.7, 90.0, 84.0, 83.4, 74.8, 74.5, 53.6, 47.0, 38.8, 30.9, 29.6, 28.8; HRMS (ESI+): calcd. 15 16 + ∆ 17 for [C25H28N7O6] 522.2096, meas. 522.2103, 1.4 ppm. 18 19 S S R S R R H 20 (2 ,5 )-2-amino-5-(((2 ,3 ,4 ,5 )-5-(6-amino-9 -purin-9-yl)-3,4-dihydroxytetra- 21 22 hydrofuran-2-yl)methyl)-7-(6-carbamoylbenzo[d][1,3]dioxol-4-yl)hept-6-ynoic acid 23 24 25 (36). To a solution of S81 (27 mg, 32 µmol) in THF (0.60 mL) at RT was added a 0.5 M 26 27 28 LiOH aq. solution (0.60 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and 29 30 a 1 M HCl aq. Solution (90 µL) was added. Volatiles were removed in vacuo. The residue 31 32 33 was dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting 34 35 36 mixture was stirred for 18 h and volatiles were removed in vacuo. Crude material was 37 38 39 purified by preparative HPLC (5% → 40% B’ over 40 min followed by 40% → 95% B’ 40 41 over 5 min) to afford 36 (16 mg, 24 µmol, 76% over 2 steps) as the TFA salt. 1H NMR (600 42 43

44 MHz, CD3CN (350 µL)/D2O (350 µL)): δ 8.31 (s, 1H), 8.29 (s, 1H), 7.32 (d, J = 1.8 Hz, 45 46 47 1H), 7.19 (d, J = 1.8 Hz, 1H), 6.05 (d, J = 1.0 Hz, 1H), 6.04 (d, J = 1.0 Hz, 1H), 5.96 (d, J 48 49 = 5.1 Hz, 1H), 4.71 (app t, J = 5.1 Hz, 1H), 4.32 (ddd, J = 10.4, 4.4, 3.2 Hz, 1H), 4.19 (dd, 50 51 52 J = 5.1, 4.4 Hz, 1H), 3.88 (dd, J = 6.7, 5.8 Hz, 1H), 2.84–2.78 (m, 1H), 2.08 (dddd, J = 53 54 55 64 56 57 58 59 60 ACS Paragon Plus Environment

Page 65 of 188 Journal of Medicinal Chemistry

1 14.2, 11.7, 6.7, 4.7 Hz, 1H), 2.03–1.97 (m, 2H), 1.90 (ddd, J = 13.9, 10.7, 3.2 Hz, 1H), 2 3 1.72 (app ddt, J = 13.1, 11.7, 5.0 Hz, 1H), 1.55 (dddd, J = 13.1, 11.7, 9.2, 4.7 Hz, 1H); 13C 4 5

6 NMR (101 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 172.4, 171.4, 152.8, 7 8 9 151.1, 149.5, 149.1, 145.4, 144.4, 127.8, 126.9, 120.1, 108.6, 105.4, 104.0, 97.2, 90.3, 10

11 + 84.1, 77.5, 74.9, 74.6, 53.8, 38.7, 30.9, 29.9, 28.9; HRMS (ESI+): calcd. for [C25H28N7O8] 12 13 14 554.1994, meas. 554.2004, ∆ 1.8 ppm. 15 16 17 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 18 19 20 hydrofuran-2-yl)methyl)-7-(6-carbamoylpyridin-2-yl)hept-6-ynoic acid (37). To a so- 21 22 lution of S82 (28 mg, 35 µmol) in THF (0.66 mL) at RT was added a 0.5 M LiOH aq. 23 24 25 solution (0.66 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M 26 27 28 HCl aq. solution (0.13 mL) was added. Volatiles were removed in vacuo. The residue was 29 30 dissolved in a solution of ammonia in methanol (7 N, 4 mL) at RT. The resulting mixture 31 32 33 was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified 34 35 36 by preparative (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to 37 38 39 afford 37 (19 mg, 30 µmol, 88% over 2 steps) as the TFA salt upon treatment with d-TFA 40 41 1 as part of the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 42 43

44 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.37 (s, 1H), 8.34 (s, 1H), 7.99 (dd, J = 7.9, 1.4 Hz, 45 46 47 1H), 7.96 (dd, J = 7.9, 7.5 Hz, 1H), 7.63 (dd, J = 7.5, 1.4 Hz, 1H), 6.00 (d, J = 5.0 Hz, 1H), 48 49 4.74 (dd, J = 5.2, 5.0 Hz, 1H), 4.37 (ddd, J = 10.1, 4.5, 3.4 Hz, 1H), 4.23 (dd, J = 5.2, 4.5 50 51 52 Hz, 1H), 4.02 (dd, J = 6.7, 5.8 Hz, 1H), 2.91–2.85 (m, 1H), 2.15 (dddd, J = 14.3, 11.6, 6.7, 53 54 55 65 56 57 58 59 60 ACS Paragon Plus Environment

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1 4.6 Hz, 1H), 2.10–2.02 (m, 2H), 1.98 (ddd, J = 13.9, 10.6, 3.4 Hz, 1H), 1.80 (app ddt, J = 2 3 13.1, 11.6, 5.0 Hz, 1H), 1.64 (dddd, J = 13.1, 11.8, 9.2, 4.6 Hz, 1H); 13C NMR (126 MHz, 4 5

6 CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 172.1, 167.9, 151.0, 149.6, 149.4, 7 8 9 145.2, 144.2, 142.3, 140.6, 131.6, 123.1, 120.1, 95.1, 90.0, 83.8, 82.2, 74.8, 74.4, 53.5, 10

11 + 38.3, 30.6, 29.6, 28.7; HRMS (ESI+): calcd. for [C23H27N8O6] 511.2048, meas. 511.2059, 12 13 14 ∆ 2.1 ppm. 15 16 17 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 18 19 20 hydrofuran-2-yl)methyl)-7-(2-carbamoylpyridin-4-yl)hept-6-ynoic acid (38). To a so- 21 22 lution of S83 (35 mg, 43 µmol) in THF (0.8 mL) at RT was added a 0.5 M LiOH aq. solution 23 24 25 (0.8 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. 26 27 28 solution (0.16 mL) was added. Volatiles were removed in vacuo. The residue was dissolved 29 30 in a solution of ammonia in methanol (7 N, 4 mL) at RT. The resulting mixture was stirred 31 32 33 for 18 h and volatiles were removed in vacuo. Crude material was purified by preparative 34 35 36 HPLC (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to afford 38 37 38 39 (19 mg, 30 µmol, 70% over 2 steps) as the TFA salt upon treatment with d-TFA as part of 40 41 1 the sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 µL)/D2O (350 42 43 44 µL)/d-TFA (40 µL)): δ 8.68 (dd, J = 5.8, 0.6 Hz, 1H), 8.37 (s, 1H), 8.35 (s, 1H), 8.27 (dd, 45 46 47 J = 1.6, 0.6 Hz, 1H), 7.90 (dd, J = 5.8, 1.6 Hz, 1H), 6.01 (d, J = 5.0 Hz, 1H), 4.73 (dd, J = 48 49 5.2, 5.0 Hz, 1H), 4.36 (ddd, J = 10.5, 4.4, 3.3 Hz, 1H), 4.22 (dd, J = 5.2, 4.4 Hz, 1H), 4.01 50 51 52 (dd, J = 6.6, 5.9 Hz, 1H), 2.95 (app ddt, J = 10.9, 9.3, 4.8 Hz, 1H), 2.14 (dddd, J = 14.3, 53 54 55 66 56 57 58 59 60 ACS Paragon Plus Environment

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1 11.6, 6.6, 4.6 Hz, 1H), 2.09–2.02 (m, 2H), 1.99 (ddd, J = 14.0, 10.9, 3.3 Hz, 1H), 1.82 (app 2 3 ddt, J = 13.3, 11.6, 4.8 Hz, 1H), 1.64 (dddd, J = 13.3, 11.9, 9.3, 4.6 Hz, 1H); 13C NMR 4 5 δ 6 (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): 172.1, 165.6, 151.0, 149.4, 7 8 9 145.5, 145.3, 145.2, 144.2, 141.1, 131.6, 127.6, 120.1, 105.9, 90.1, 83.6, 81.0, 74.8, 74.5, 10

11 + 53.5, 38.3, 30.5, 30.2, 28.7; HRMS (ESI+): calcd. for [C23H27N8O6] 511.2048, meas. 12 13 14 511.2050, ∆ 0.4 ppm. 15 16 17 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 18 19 20 hydrofuran-2-yl)methyl)-7-(5-carbamoylpyridin-3-yl)hept-6-ynoic acid (39). To a so- 21 22 lution of S84 (26 mg, 32 µmol) in THF (0.6 mL) at RT was added a 0.5 M LiOH aq. solution 23 24 25 (0.6 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. 26 27 28 solution (0.13 mL) was added. Volatiles were removed in vacuo. The residue was dissolved 29 30 in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture was stirred 31 32 33 for 18 h and the volatiles were removed in vacuo. Crude material was purified by prepar- 34 35 36 ative HPLC (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to afford 37 38 39 39 (16 mg, 26 µmol, 80% over 2 steps) as the TFA salt upon treatment with d-TFA as part 40 41 1 of the sample preparation for NMR analysis. H NMR (500 MHz, CD3CN (350 µL)/D2O 42 43 44 (350 µL)/d-TFA (40 µL)): δ 9.04 (d, J = 1.8 Hz, 1H), 8.89 (d, J = 1.8 Hz, 1H), 8.81 (app 45 46 47 t, J = 1.8 Hz, 1H), 8.37 (s, 1H), 8.36 (s, 1H), 6.01 (d, J = 4.9 Hz, 1H), 4.72 (app t, J = 4.9 48 49 Hz, 1H), 4.37 (ddd, J = 10.3, 4.9, 3.1 Hz, 1H), 4.22 (app t, J = 4.9 Hz, 1H), 4.01 (app t, J 50 51 52 = 6.3 Hz, 1H), 2.95–2.86 (m, 1H), 2.15 (dddd, J = 14.3, 11.6, 6.3, 4.8 Hz, 1H), 2.10–2.01 53 54 55 67 56 57 58 59 60 ACS Paragon Plus Environment

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13 1 (m, 2H), 1.98 (ddd, J = 13.7, 10.7, 3.1 Hz, 1H), 1.85–1.77 (m, 1H), 1.68–1.59 (m, 1H); C 2 3 NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 172.1, 165.8, 151.1, 4 5 6 149.4, 147.7, 147.0, 145.3, 144.1, 141.1, 133.9, 125.1, 120.1, 101.4, 90.1, 83.7, 77.4, 74.9, 7 8 + 9 74.5, 53.5, 38.4, 30.6, 30.0, 28.7; HRMS (ESI+): calcd. for [C23H27N8O6] 511.2048, meas. 10 11 511.2069, ∆ 4.0 ppm. 12 13 14 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 15 16 17 hydrofuran-2-yl)methyl)-7-(4-carbamoylpyridin-2-yl)hept-6-ynoic acid (40). To a so- 18 19 20 lution of S85 (22 mg, 27 µmol) in THF (1.0 mL) at RT was added a 0.5 M LiOH aq. solution 21 22 (1.0 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M HCl aq. 23 24 25 solution (0.20 mL) was added. Volatiles were removed in vacuo. The residue was dissolved 26 27 28 in a solution of ammonia in methanol (7 N, 4 mL) at RT. The resulting mixture was stirred 29 30 for 18 h and the volatiles were removed in vacuo. Crude material was purified by prepar- 31 32 33 ative HPLC (5% → 20% B over 30 min followed by 20% → 95% B over 5 min) to afford 34 35 36 40 (14 mg, 22 µmol, 82% over 2 steps) as the TFA salt upon treatment with d-TFA as part 37 38 1 39 of the sample preparation for NMR analysis. H NMR (500 MHz, CD3CN (350 µL)/D2O 40 41 (350 µL)/d-TFA (40 µL)): δ 8.73 (dd, J = 6.1, 0.7 Hz, 1H), 8.37 (s, 1H), 8.35 (s, 1H), 8.22 42 43 44 (dd, J = 1.9, 0.7 Hz, 1H), 8.14 (dd, J = 6.1, 1.9 Hz, 1H), 6.01 (d, J = 4.9 Hz, 1H), 4.73 (dd, 45 46 47 J = 5.1, 4.9 Hz, 1H), 4.35 (ddd, J = 10.2, 4.8, 3.4 Hz, 1H), 4.23 (dd, J = 5.1, 4.8 Hz, 1H), 48 49 4.01 (app t, J = 6.3 Hz, 1H), 3.03–2.96 (m, 1H), 2.18–1.99 (m, 4H), 1.89–1.80 (m, 1H), 50 51 13 52 1.74–1.64 (m, 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): 53 54 55 68 56 57 58 59 60 ACS Paragon Plus Environment

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1 δ 172.1, 166.8, 151.0, 150.0, 149.4, 145.3, 144.8, 144.2, 137.5, 129.7, 124.8, 120.1, 106.3, 2 3 90.1, 83.4, 76.1, 74.8, 74.4, 53.5, 38.0, 30.3, 30.2, 28.6; HRMS (ESI+): calcd. for 4 5 + ∆ 6 [C23H27N8O6] 511.2048, meas. 511.2055, 1.3 ppm. 7 8 9 (2S,5S)-2-amino-5-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetra- 10 11 12 hydrofuran-2-yl)methyl)-7-(5-aminonaphthalen-1-yl)hept-6-ynoic acid (41). To a so- 13 14 lution of S86 (34 mg, 41 µmol) in THF (0.76 mL) at RT was added a 0.5 M LiOH aq. 15 16 17 solution (0.76 mL). The resulting mixture was stirred for 1 h, cooled to 0 °C, and a 1 M 18 19 20 HCl aq. solution (0.17 mL) was added. Volatiles were removed in vacuo. The residue was 21 22 dissolved in a solution of ammonia in methanol (7 N, 4.5 mL) at RT. The resulting mixture 23 24 25 was stirred for 18 h and the volatiles were removed in vacuo. Crude material was purified 26 27 28 by preparative HPLC (5% → 45% B over 30 min followed by 45% → 95% B over 5 min) 29 30 to afford 41 (20 mg, 26 µmol, 64% over 2 steps) as the TFA salt upon treatment with d- 31 32 1 33 TFA as part of sample preparation for NMR analysis. H NMR (600 MHz, CD3CN (350 34 35 36 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 8.33 (s, 1H), 8.29 (app dt, J = 8.4, 1.0 Hz, 1H), 8.29 37 38 39 (s, 1H), 7.89 (app dt, J = 8.5, 1.0 Hz, 1H), 7.70 (dd, J = 7.2, 1.0 Hz, 1H), 7.63 (dd, J = 7.5, 40 41 1.0 Hz, 1H), 7.61 (dd, J = 8.5, 7.2 Hz, 1H), 7.57 (dd, J = 8.4, 7.5 Hz, 1H), 6.00 (d, J = 5.0 42 43 44 Hz, 1H), 4.77 (dd, J = 5.2, 5.0 Hz, 1H), 4.44 (ddd, J = 10.0, 4.6, 3.3 Hz, 1H), 4.28 (dd, J 45 46 47 = 5.2, 4.6 Hz, 1H), 4.06 (dd, J = 6.7, 5.6 Hz, 1H), 3.01–2.95 (m, 1H), 2.23 (dddd, J = 14.4, 48 49 11.6, 6.7, 4.6 Hz, 1H), 2.17–2.10 (m, 1H), 2.11 (ddd, J = 14.0, 10.0, 4.5 Hz, 1H), 2.05 50 51 52 (ddd, J = 14.0, 10.3, 3.3 Hz, 1H), 1.84 (app ddt, J = 13.1, 11.6, 5.1 Hz, 1H), 1.72–1.64 (m, 53 54 55 69 56 57 58 59 60 ACS Paragon Plus Environment

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13 1 1H); C NMR (126 MHz, CD3CN (350 µL)/D2O (350 µL)/d-TFA (40 µL)): δ 172.1, 150.8, 2 3 149.4, 145.1, 144.2, 134.6, 132.5, 128.5, 128.4, 127.5, 127.5, 127.4, 123.0, 122.7, 122.0, 4 5 6 119.9, 98.5, 90.1, 83.9, 81.3, 74.7, 74.4, 53.5, 38.6, 30.8, 29.7, 28.8; HRMS (ESI+): calcd. 7 8 + ∆ 9 for [C27H30N7O5] 532.2303, meas. 532.2325, 4.2 ppm. 10 11 12 13 14 Synthesis of Compounds S2-S86 15 16 17 Methyl 2-(dimethoxyphosphoryl)-2-(2,2,2-trifluoroacetamido)acetate (S2).67 To a so- 18 19 α 20 lution of (±)-Z- -phosphonoglycine trimethyl ester (S1) (3.31 g, 9.99 mmol) in CH2Cl2 (50 21 22 23 mL) at RT, was added Pd/C (10%, 0.53 g, 0.50 mmol, 5 mol %). The flask headspace was 24 25 × 26 evacuated and refilled with hydrogen (3 ) and the reaction mixture was stirred vigorously 27 28 under a hydrogen atmosphere (balloon) for 24 h. The flask headspace was evacuated and 29 30 31 refilled with nitrogen, and the contents were filtered through a pad of celite. The filtrate 32 33 34 was cooled to 0 °C and TFAA (3.47 mL, 25.0 mmol, 2.5 equiv.) was added dropwise. The 35 36 reaction mixture was stirred at RT for 12 h and the solution was concentrated to yield an 37 38 39 orange residue. Crude material was purified by silica gel chromatography (hex- 40 41 42 anes/EtOAc, 0 → 100%) to afford TFA protected phosphonate S2 (2.05 g, 6.99 mmol, 70% 43

44 -1 over 2 steps). Rf = 0.41 (EtOAc); FTIR (thin film), cm−1: νmax (cm ): 3197, 3046, 2964, 45 46 47 2861, 1757, 1722, 1559, 1433, 1360, 1307, 1257, 1212, 1180, 1150, 1035; 1H NMR (600 48 49 2 50 MHz, CDCl3): δ 7.23 (br s, 1H), 5.14 (dd, J H-P = 21.3, J = 8.7 Hz, 1H), 3.89 (s, 3H), 3.87 51

52 3 3 13 J H-P J J H-P J 3 53 (d, , = 11.2 Hz, 3H), 3.83 (d, , = 11.1 Hz, 3H); C NMR (126 MHz, CDCl ): δ 54 55 70 56 57 58 59 60 ACS Paragon Plus Environment

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2 3 1 2 1 165.5, 157.2 (qd, J C-F = 38 Hz, J C-P = 5.9 Hz), 115.7 (q, J C-F = 287 Hz), 54.5 (d, J C-P = 6.6 2 3 2 1 19 Hz), 54.1 (d, J C-P = 6.9 Hz), 53.3, 50.3 (d, J C-P = 152 Hz); F NMR (471 MHz, CDCl3, BTF 4 5 31 6 IStd): δ –76.5; P NMR (162 MHz, CDCl3): δ 16.1; HRMS (ESI+): calcd. for 7 8 + ∆ 9 [C7H12F3N1O6P1] 294.0360, meas. 294.0349, 3.7 ppm. 10 11 S S R R R R 12 Methyl (2 ,5 )-5-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 13 14 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept-6-ynoate (S3). To a vial 15 16 17 charged with 7 (182 mg, 0.307 mmol) was added DMF (3 mL) at RT under a nitrogen 18 19 20 atmosphere. A separate vial was charged with TASF (253 mg, 0.920 mmol, 3.0 equiv.) in 21 22 a glovebox, put under a nitrogen atmosphere, and DMF (2 mL) was added at RT. The 23 24 25 resulting solution was added to the vial containing alkyne 7 and the reaction mixture was 26 27 28 stirred at 60 °C for 2 h. Volatiles were removed in vacuo. The residue was partitioned 29 30 between Et2O (40 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (40 mL). The 31 32 × 33 layers were separated and the aqueous phase was extracted with Et2O (3 30 mL). The 34 35 36 combined organic extracts were dried over MgSO4, filtered and concentrated. Crude mate- 37 38 39 rial was purified by silica gel chromatography (hexanes/EtOAc, 5 → 50%) to yield S3 40 41 (128 mg, 0.293 mmol, 95%) as a colorless oil. Rf = 0.35 (hexanes/EtOAc, 60:40); FTIR 42 43 ν -1 44 (neat), max (cm ): 3292, 3089, 2990, 2954, 2938, 2836, 1747, 1719, 1553, 1455, 1440, 1383, 45 46 1 47 1275, 1209, 1160, 1104, 1060; H NMR (600 MHz, CDCl3): δ 6.94 (d, J = 7.9 Hz, 1H), 48 49 4.93 (s, 1H), 4.64 (td, J = 7.8, 4.9 Hz, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.55 (dd, J = 6.0, 1.0 50 51 52 Hz, 1H), 4.45 (ddd, J = 11.2, 3.9, 0.9 Hz, 1H), 3.80 (s, 3H), 3.31 (s, 3H), 2.66–2.58 (m, 53 54 55 71 56 57 58 59 60 ACS Paragon Plus Environment

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1 1H), 2.14 (d, J = 2.4 Hz, 1H), 2.10 (ddq, J = 14.0, 10.8, 5.2 Hz, 1H), 2.05–1.94 (m, 1H), 2 3 1.67 (ddd, J = 13.3, 11.2, 4.0 Hz, 1H), 1.55 (dddd, J = 21.7, 13.2, 10.9, 4.6 Hz, 2H), 1.47 4 5 6 (s, 3H), 1.40 (dddd, J = 16.5, 14.4, 8.7, 3.9 Hz, 1H), 1.30 (s, 3H); 13C NMR (126 MHz, 7 8 2 1 9 CDCl3): δ 171.3, 157.0 (q, J C-F = 38 Hz), 115.1 (q, J C-F = 287 Hz), 112.5, 110.1, 85.6, 85.1, 10 11 85.0, 84.5, 71.6, 55.3, 53.1, 52.6, 52.5, 40.4, 30.7, 29.8, 28.6, 26.6, 25.1; HRMS (ESI+): 12 13 + ∆ 14 calcd. for [C19H26F3N1O7Na] 460.1554, meas. 460.1569, 3.3 ppm. 15 16 17 (3R,4R,5R)-5-((2S,5S)-2-ethynyl-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetam- 18 19 20 ido)hexyl)tetrahydrofuran-2,3,4-triyl triacetate (S4). To a solution of acetonide S3 21 22 (1.21 g, 2.77 mmol) in CH2Cl2 (16.5 mL) at RT, was added a 2:1 mixture of TFA and water 23 24 25 (51 mL). The reaction mixture was stirred at RT for 21 h and volatiles were removed in 26 27 28 vacuo. The residue was resuspended in Ac2O (6.0 mL, 64 mmol, 23 equiv.) and pyridine 29 30 (12.0 mL, 149 mmol, 54 equiv.). The resulting mixture was stirred at RT for 22 h and 31 32 33 volatiles were removed in vacuo. Crude material was purified by silica gel chromatography 34 35 36 (hexanes/EtOAc, 0 → 40%) to afford triacetate S4 (735 mg, 1.44 mmol, 52% over 2 steps) 37 38 39 as a 1:1.7 mixture of α and β anomers, as a light yellow oil. Rf = 0.37 (α-anomer) / 0.46 40 41 ν -1 (β-anomer) (hexanes/EtOAc, 50:50); FTIR (thin-film), max (cm ): 3290, 2956, 1748, 1724, 42 43 1 44 1553, 1438, 1373, 1219, 1178, 1112, 1049, 1013; H NMR (600 MHz, CDCl3): δ α-anomer: 45 46 47 6.92 (d, J = 7.8 Hz, 1H), 6.36 (d, J = 4.6 Hz, 1H), 5.22 (dd, J = 6.8, 4.6 Hz, 1H), 5.05 (dd, 48 49 J = 6.8, 3.7 Hz, 1H), 4.63 (td, J = 7.6, 5.1 Hz, 1H), 4.43 (dt, J = 10.2, 3.6 Hz, 1H), 3.80 (s, 50 51 52 3H), 2.62 (dqd, J = 13.8, 4.6, 2.4 Hz, 1H), 2.13 (d, J = 2.4 Hz, 1H), 2.12 (s, 3H), 2.08 (s, 53 54 55 72 56 57 58 59 60 ACS Paragon Plus Environment

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1 3H), 2.08–2.04 (m, 1H), 2.07 (s, 3H), 2.01 (dddd, J = 18.5, 9.1, 5.3, 2.9 Hz, 1H), 1.84 (ddd, 2 3 J = 14.1, 10.8, 3.5 Hz, 1H), 1.65–1.59 (m, 1H), 1.59–1.53 (m, 1H), 1.40 (dddd, J = 13.1, 4 5 6 11.0, 9.5, 4.8 Hz, 1H); β-anomer: 7.02 (d, J = 8.1 Hz, 1H), 6.10 (s, 1H), 5.31 (dd, J = 4.9, 7 8 9 0.9 Hz, 1H), 5.16 (dd, J = 7.2, 4.7 Hz, 1H), 4.64 (td, J = 8.2, 4.6 Hz, 1H), 4.33 (ddd, J = 10 11 10.1, 7.1, 2.7 Hz, 1H), 3.81 (s, 3H), 2.66 (ttd, J = 9.6, 4.9, 2.4 Hz, 1H), 2.13 (d, J = 2.4 Hz, 12 13 14 1H), 2.12 (s, 3H), 2.10 (s, 3H), 2.08–2.04 (m, 1H), 2.06 (s, 3H), 2.05–1.97 (m, 1H), 1.84 15 16 17 (ddd, J = 13.1, 9.9, 2.8 Hz, 1H), 1.69–1.61 (m, 1H), 1.60–1.55 (m, 1H), 1.47 (dddd, J = 18

19 13 13.2, 11.3, 8.9, 4.5 Hz, 1H); C NMR (126 MHz, CDCl3): δ α-anomer: 171.1, 169.9, 169.5, 20 21 2 1 22 169.3, 156.9 (q, J C-F = 38 Hz), 115.7 (q, J C-F = 288 Hz), 93.7, 84.6, 81.2, 72.5, 71.5, 69.7, 23 24 25 52.9, 52.3, 38.9, 30.4, 29.1, 28.4, 21.0, 20.5, 20.2; β-anomer: 171.1, 170.1, 169.7, 169.4, 26

27 2 1 156.9 (q, J C-F = 38 Hz), 115.6 (q, J C-F = 288 Hz), 98.3, 84.9, 79.8, 74.3, 73.8, 71.3, 52.9, 28 29 30 19 52.3, 39.6, 30.6, 29.5, 28.0, 21.0, 20.6, 20.2; F NMR (471 MHz, CDCl3, BTF IStd): δ – 31 32 + ∆ 33 75.8; HRMS (ESI+): calcd. for [C21H26F3N1O10Na] 532.1401, meas. 532.1407, 1.2 ppm. 34 35 36 Methyl 6-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]di- 37 38 oxol-4-yl)hexanoate (S6).68 39 To a 3-necked flask charged with pyridine (10 mL) were added 40 41 NaI (1.49 g, 10.0 mmol, 1.0 equiv.) and FeCl3•6H2O (1.35 g, 5.00 mmol, 50 mol %) se- 42 43 44 quentially, both in one portion. Zinc powder (3.92 g, 60.0 mmol, 6.0 equiv.) was added 45 46 47 portionwise to the reaction mixture (Caution, exothermic), monitoring the temperature so 48 49 that it remains below 40 °C. Upon complete addition of the zinc powder, the resulting 50 51 52 mixture was allowed to cool gradually to RT and a solution of riboside S569 (3.14 g, 10.0 53 54 55 73 56 57 58 59 60 ACS Paragon Plus Environment

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1 mmol) in methyl acrylate (6.0 mL) was added over 10 min via syringe. The reaction mix- 2 3 ture was stirred at RT for 2 h, before the addition of toluene (100 mL) and filtration over 4 5 6 celite. Volatiles were removed in vacuo to give methyl ester S6 (2.52 g, 9.25 mmol, 93%) 7 8 9 which was of suitable purity to be used in the next step without further purification. Rf = 10 11 ν -1 0.42 (cyclohexane/EtOAc, 67:33); FTIR (thin film), max (cm ): 2989, 2940, 1735, 1437, 12 13 1 δ 14 1372, 1270, 1239, 1208, 1194, 1159, 1090, 1057, 1011; H NMR (600 MHz, CDCl3): 15 16 17 4.94 (s, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.52 (dd, J = 5.9, 1.1 Hz, 1H), 4.13 (ddd, J = 8.9, 18 19 6.4, 1.1 Hz, 1H), 3.67 (s, 3H), 3.34 (s, 2H), 2.35 (td, J = 7.4, 1.4 Hz, 2H), 1.86 – 1.76 (m, 20 21 22 1H), 1.71 (ddtd, J = 13.2, 10.1, 7.3, 5.7 Hz, 1H), 1.62 (dddd, J = 14.1, 10.1, 8.9, 5.1 Hz, 23 24 25 1H), 1.53 (ddt, J = 13.4, 10.4, 6.0 Hz, 1H), 1.47 (s, 3H), 1.31 (s, 3H); 13C NMR (151 MHz, 26 27 δ CDCl3): 173.5, 112.1, 109.5, 86.7, 85.5, 84.1, 54.9, 51.4, 34.4, 33.5, 26.4, 24.9, 21.7; 28 29 30 + ∆ HRMS (ESI+): calcd. for [C13H22O6Na] 297.1309, meas. 297.1307, 0.7 ppm. 31 32 33 4-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4- 34 35 36 yl)butanal (S7). To an oversized flask (250 mL) containing a solution of methyl ester S6 37 38 i 39 (2.15 g, 7.83 mmol) in toluene (20 mL) at –85 °C ( -PrOH/liquid nitrogen bath) was added 40 41 i-Bu2AlH (1.0 M in hexanes, 8.0 mL, 8.0 mmol, 1.02 equiv.) over 40 min via syringe pump, 42 43 44 down the side-wall of the flask. Upon complete addition of i-Bu2AlH, the reaction mixture 45 46 47 was stirred at –85 °C for 1 h, before the slow addition of MeOH (4 mL) over 20 min down 48 49 the side-wall of the flask, followed by a potassium sodium tartrate sat. aq. solution (40 mL) 50 51 52 and EtOAc (50 mL). The reaction mixture was removed from the cooling bath and allowed 53 54 55 74 56 57 58 59 60 ACS Paragon Plus Environment

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1 to warm gradually to RT. Water (10 mL) and brine (10 mL) were added, and the biphasic 2 3 mixture was stirred for another 10 min. The layers were separated and the aqueous phase 4 5 6 was extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with 7 8 9 brine (200 mL), dried over MgSO4, filtered and concentrated to give aldehyde S7 (1.80 g, 10 11 7.37 mmol, 94%) as a light-yellow oil which was of suitable purity to be used in the next 12 13

14 step without further purification. Rf = 0.31 (cyclohexane/EtOAc, 67:33); FTIR (thin film), 15 16 ν -1 17 max (cm ): 2988, 2938, 2832, 2723, 1724, 1457, 1412, 1381, 1372, 1273, 1240, 1209, 1193, 18

19 1 1160, 1087, 1058, 1018; H NMR (600 MHz, CDCl3): δ 9.73 (t, J = 1.6 Hz, 1H), 4.89 (s, 20 21 22 1H), 4.55 (d, J = 6.0 Hz, 1H), 4.48 (dd, J = 6.0, 1.1 Hz, 1H), 4.09 (ddd, J = 9.0, 6.3, 1.1 23 24 25 Hz, 1H), 3.30 (s, 3H), 2.45 (dddd, J = 8.7, 7.1, 3.4, 1.6 Hz, 2H), 1.81–1.71 (m, 1H), 1.72– 26 27 1.63 (m, 1H), 1.59 (dtd, J = 13.6, 9.2, 5.2 Hz, 1H), 1.49 (ddt, J = 13.5, 10.1, 6.1 Hz, 1H), 28 29 30 13 1.43 (s, 3H), 1.27 (s, 3H); C NMR (151 MHz, CDCl3): δ 202.0, 112.3, 109.6, 86.8, 85.5, 31 32 33 84.1, 55.1, 43.4, 34.4, 26.6, 25.0, 18.9. 34 35 36 Methyl (Z)-2-(((benzyloxy)carbonyl)amino)-6-((3aR,4R,6R,6aR)-6-methoxy-2,2-di- 37 38 methyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)hex-2-enoate (S8) 39 . To a stirred suspension 40 41 of (±)-Cbz-α-phosphonoglycine trimethyl ester (S1) (343 mg, 1.04 mmol, 1.2 equiv.) in 42 43 44 THF (3.0 mL) at –78 °C was added 1,1,3,3-tetramethylguanidine (0.12 mL, 0.95 mmol, 45 46 47 1.1 equiv.). The resulting mixture was stirred at –78 °C for 15 min, before the addition of 48 49 a solution of aldehyde S7 (211 mg, 0.864 mmol) in THF (2.0 mL). The reaction mixture 50 51 52 was stirred at –78 °C for 30 min and then placed in an ice/water bath and stirred for 1 h. 53 54 55 75 56 57 58 59 60 ACS Paragon Plus Environment

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1 The reaction was quenched by the addition of a 10% AcOH aq. solution (2 mL) and water 2 3 (10 mL) was added. The layers were separated and the aqueous phase was extracted with 4 5 × 6 CH2Cl2 (3 15 mL). The combined organic extracts were washed with water (10 mL) and 7 8 1 9 brine (10 mL), dried over MgSO4, filtered and concentrated. H NMR analysis of the crude 10 11 revealed a 15:1 mixture of Z and E isomers. Crude material was purified by silica gel chro- 12 13 14 matography (2,2,4-trimethylpentane/EtOAc, 0 → 50%). Z and E isomers separated and 15 16 17 only fractions containing Z isomers were collected to afford enamide S8 (353 mg, 0.785 18 19 mmol, 91 %) as a clear viscous oil. Rf = 0.44 (cyclohexane/EtOAc, 50:50); FTIR (thin film), 20 21 ν -1 22 max (cm ): 3316, 2988, 2942, 1716, 1657, 1500, 1455, 1437, 1381, 1373, 1270, 1213, 1160, 23 24 1 25 1105, 1089, 1047; H NMR (600 MHz, CDCl3): δ 7.41–7.30 (m, 5H), 6.61 (t, J = 7.2 Hz, 26 27 J J 28 1H), 5.14 (s, 2H), 4.93 (s, 1H), 4.58 (d, = 5.9 Hz, 1H), 4.49 (d, = 5.9 Hz, 1H), 4.12 (dd, 29 30 J = 8.3, 6.3 Hz, 1H), 3.75 (s, 3H), 3.33 (s, 3H), 2.31–2.18 (m, 2H), 1.68–1.48 (m, 4H), 31 32 13 33 1.47 (s, 3H), 1.30 (s, 3H); C NMR (151 MHz, CDCl3): δ 165.1, 137.5, 136.1, 128.6, 128.3, 34 35 36 128.2, 112.3, 109.6, 86.9, 85.6, 84.2, 67.4, 55.0, 52.5, 34.7, 28.1, 26.6, 25.1; HRMS 37 38 + ∆ (ESI+): calcd. for [C23H32NO8] 450.2122, meas. 450.2124, 0.3 ppm. 39 40 41 Methyl (S)-2-(((benzyloxy)carbonyl)amino)-6-((3aR,4R,6R,6aR)-6-methoxy-2,2-di- 42 43 44 methyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)hexanoate (S9). To a flask charged with 45 46 47 enamide S8 (179 mg, 0.398 mmol, exclusively Z isomer) was added MeOH (5 mL) at RT 48 49 under a nitrogen atmosphere. The contents were stirred until full dissolution was noted. 50 51

52 Separately, [Rh(nbd)2]BF4 (49 mg, 0.12 mmol, 30 mol %) and (S,S)-Me-DUPHOS(43 mg, 53 54 55 76 56 57 58 59 60 ACS Paragon Plus Environment

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1 0.14 mmol, 35 mol %) were weighed out into the same vial in a glove box. The vial was 2 3 removed from the glove box, placed under a nitrogen atmosphere and MeOH (10 mL) was 4 5 6 added. The flask headspace was evacuated and backfilled with hydrogen (3 ×). The meth- 7 8 9 anolic solution of enamide S8 was transferred to the vial containing the catalyst (2 mL of 10 11 MeOH used for vial rinse) and the contents were stirred vigorously at RT under a hydrogen 12 13 14 atmosphere (balloon) for 1 h. Volatiles were removed in vacuo and crude material was 15 16 17 purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 50%) to afford 18 19 protected amino acid S9 (172 mg, 0.381 mmol, 96%) as a clear viscous oil and as a single 20 21 ν -1 22 diastereomer. Rf = 0.32 (cyclohexane/EtOAc, 67:33); FTIR (thin film), max (cm ): 3338, 23 24 25 2988, 2939, 2862, 1720, 1523, 1455, 1439, 1381, 1373, 1348, 1259, 1239, 1209, 1161, 26

27 1 3 J 28 1106, 1087, 1056, 1026; H NMR (600 MHz, CDCl ): δ 7.38–7.28 (m, 5H), 5.31 (d, = 29 30 8.4 Hz, 1H), 5.09 (s, 2H), 4.92 (s, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.48 (d, J = 6.0 Hz, 1H), 31 32 33 4.37 (td, J = 8.0, 5.1 Hz, 1H), 4.10 (dd, J = 8.9, 5.8 Hz, 1H), 3.73 (s, 3H), 3.31 (s, 3H), 34 35 36 1.88–1.79 (m, 1H), 1.64 (app. h, J = 8.1 Hz, 1H), 1.58 (app. q, J = 8.8 Hz, 1H), 1.49–1.43 37

38 13 (m, 2H), 1.46 (s, 3H), 1.40–1.32 (m, 3H), 1.30 (s, 3H); C NMR (151 MHz, CDCl3): δ 39 40 41 173.0, 155.9, 136.3, 128.5, 128.2, 128.1, 112.2, 109.4, 87.0, 85.6, 84.1, 67.0, 54.9, 53.8, 42 43 + 44 52.3, 34.8, 32.6, 26.5, 25.8, 25.0; HRMS (ESI+): calcd. for [C23H34NO8] 452.2280, meas. 45 46 452.2285, ∆ 1.1 ppm. 47 48 49 (3R,4R,5R)-5-((S)-5-(((benzyloxy)carbonyl)amino)-6-methoxy-6-oxohexyl)tetrahy- 50 51 52 drofuran-2,3,4-triyl triacetate (S10). To a vial charged with acetonide S9 (171 mg, 0.378 53 54 55 77 56 57 58 59 60 ACS Paragon Plus Environment

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1 mmol) was added a 4:1 mixture of acetic acid and water (7.5 mL) at RT. The resulting 2 3 mixture was stirred at 85 °C for 8 h. Volatiles were removed in vacuo and the residue was 4 5 6 dried by azeotropic distillation with benzene (1 × 10 mL). Crude material was used directly 7 8 9 in the next step without further purification. To a solution of the crude triol in CH2Cl2 (4.0 10 11 mL) at RT, were added pyridine (0.50 mL, 6.2 mmol, 16 equiv.), Ac2O (0.50 mL, 5.3 mmol, 12 13 14 14 equiv.) and DMAP (5 mg, 0.04 mmol, 0.11 equiv.) sequentially. The resulting mixture 15 16 17 was stirred at RT for 2 h and the volatiles were removed in vacuo. Crude material was 18 19 purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to af- 20 21 22 ford triacetate S10 (152 mg, 0.290 mmol, 77% over 2 steps) as a ca. 1:1 mixture of α and 23 24 25 β anomers as a colorless oil. Rf = 0.28 (α-anomer)/0.37 (β-anomer) (EtOAc); FTIR (thin 26 27 ν -1 28 film), max (cm ): 3359, 2950, 1743, 1721, 1523, 1455, 1437, 1371, 1216, 1110, 1047, 1010; 29 30 1 H NMR (600 MHz, CDCl3): δ α-anomer: 7.38–7.27 (m, 5H), 6.34 (d, J = 4.6 Hz, 1H), 31 32 33 5.29 (d, J = 8.2 Hz, 1H), 5.18 (dd, J = 6.8, 4.6 Hz, 1H), 5.09 (s, 2H), 5.01 (dd, J = 6.8, 3.6 34 35 36 Hz, 1H), 4.35 (td, J = 7.8, 5.1 Hz, 1H), 4.20–4.15 (m, 1H), 3.73 (s, 3H), 2.10 (s, 3H), 2.09 37 38 (s, 3H), 2.05 (s, 3H), 1.83–1.77 (m, 1H), 1.69–1.54 (m, 3H), 1.47–1.42 (m, 1H), 1.39–1.28 39 40 41 (m, 3H); β-anomer: 7.38–7.28 (m, 5H), 6.10 (d, J = 1.1 Hz, 1H), 5.31 (d, J = 8.1 Hz, 1H), 42 43 44 5.29 (dd, J = 4.8, 1.2 Hz, 1H), 5.14 (dd, J = 6.9, 4.8 Hz, 1H), 5.10 (s, 2H), 4.36 (td, J = 45 46 7.8, 5.0 Hz, 1H), 4.12 (td, J = 7.4, 5.2 Hz, 1H), 3.73 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 47 48 49 2.05 (s, 3H), 1.85–1.78 (m, 1H), 1.68–1.55 (m, 2H), 1.51–1.42 (m, 1H), 1.40–1.29 (m, 50 51 13 52 4H); C NMR (126 MHz, CDCl3): δ mixture of α and β-anomers: 172.9, 170.2, 169.8, 53 54 55 78 56 57 58 59 60 ACS Paragon Plus Environment

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1 169.4, 155.9, 136.3, 128.6, 128.2, 128.2, 98.3, 94.0, 83.3, 81.4, 74.6, 73.6, 72.3, 69.9, 67.0, 2 3 53.8, 52.4, 33.8, 33.1, 32.5, 25.0, 25.0, 24.9, 24.8, 21.1, 21.1, 20.7, 20.6, 20.6, 20.4; HRMS 4 5 + ∆ 6 (ESI+): calcd. for [C25H34NO11] 524.2126, meas. 524.2134, 1.5 ppm. 7 8 9 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-5-(((benzyloxy)car- 10 11 12 bonyl)amino)-6-methoxy-6-oxohexyl)tetrahydrofuran-3,4-diyl diacetate (S11). To a 13 14 suspension of N6-benzoyladenine (149 mg, 0.624 mmol, 2.15 equiv.) in propionitrile (dried 15 16 17 over 4 Å M.S., 6.0 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.21 mL, 0.87 18 19 20 mmol, 3.00 equiv.). The resulting mixture was stirred at 95 °C for 10 min, upon which 21 22 complete solubilization of N6-benzoyladenine was observed. To a separate flask charged 23 24 25 with triacetate S10 (152 mg, 0.290 mmol) and 2,6-di-tert-butyl-4-methylpyridinium tri- 26 27 28 flate (21 mg, 58 µmol, 20 mol %) was added propionitrile (6.0 mL). The resulting mixture 29 30 was stirred at RT for 5 min, until a clear solution was obtained. This clear solution was 31 32 33 added to the solution of silylated N6-benzoyladenine. The reaction mixture was stirred at 34 35 36 95 °C for 2.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was 37 38 39 purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100% then 40 41 EtOAc/i-PrOH, 0 → 15%) to afford nucleoside S11 (148 mg, 0.211 mmol, 73%) as a white 42 43 ν -1 44 amorphous solid. Rf = 0.28 (EtOAc); FTIR (thin film), max (cm ): 3330, 2950, 1744, 1703, 45 46 1 47 1609, 1581, 1510, 1487, 1454, 1408, 1373, 1330, 1239, 1214, 1177, 1095, 1047, 1028; H 48 49 NMR (600 MHz, CDCl3): δ 9.28 (br s, 1H), 8.74 (s, 1H), 8.11 (s, 1H), 8.01–7.97 (m, 2H), 50 51 52 7.59–7.53 (m, 1H), 7.49–7.44 (m, 2H), 7.33–7.24 (m, 5H), 6.12 (d, J = 5.3 Hz, 1H), 5.94 53 54 55 79 56 57 58 59 60 ACS Paragon Plus Environment

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1 (t, J = 5.5 Hz, 1H), 5.45 (d, J = 3.5 Hz, 1H), 5.44 (t, J = 5.3 Hz, 1H), 5.07 (s, 2H), 4.34 (td, 2 3 J = 8.0, 5.1 Hz, 1H), 4.15 (dt, J = 8.8, 4.9 Hz, 1H), 3.69 (s, 3H), 2.11 (s, 3H), 2.04 (s, 3H), 4 5 6 1.85–1.70 (m, 3H), 1.65–1.57 (m, 1H), 1.51–1.42 (m, 1H), 1.41–1.30 (m, 3H); 13C NMR 7 8 9 (126 MHz, CDCl3): δ 172.9, 169.7, 169.5, 165.0, 155.9, 152.7, 151.8, 149.9, 141.9, 136.3, 10 11 133.6, 132.8, 128.8, 128.5, 128.2, 128.1, 128.0, 123.9, 86.5, 82.4, 73.3, 73.1, 67.0, 53.7, 12 13 + 14 52.4, 32.9, 32.5, 24.9, 24.9, 20.6, 20.4; HRMS (ESI+): calcd. for [C35H38N6O10Na] 725.2542, 15 16 ∆ 17 meas. 725.2573, 4.3 ppm. 18 19 R R R R H S S 20 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-((2 ,5 )-6-methoxy-6-oxo-2-(phe- 21 22 nylethynyl)-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate 23 24 25 (S12). To a vial charged with nucleoside 8 (82 mg, 0.12 mmol) were added iodobenzamide 26 27 28 (49 mg, 0.24 mmol, 2.0 equiv.), CuI (5 mg, 0.02 mmol, 20 mol %) and Pd(PPh3)4 (7 mg, 6 29 30 µmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mix- 31 32

33 ture of toluene, DMF and i-Pr2NEt (degassed by sparging with nitrogen for 15 min, 3.5 34 35 36 mL) was added at RT. The resulting mixture was stirred at 70 °C for 3 h. The solution was 37 38 in vacuo 39 allowed to cool to RT and volatiles were removed . Crude material was purified 40 41 by silica gel chromatography (EtOAc/i-PrOH, 0 → 5%) to afford alkyne S12 (76 mg, 99 42 43 ν -1 44 µmol, 83%) as a white amorphous solid. Rf = 0.48 (EtOAc); FTIR (thin-film), max (cm ): 45 46 47 3297, 3082, 2954, 1745, 1717, 1610, 1582, 1509, 1489, 1455, 1373, 1328, 1239, 1212, 48

49 1 1176, 1159, 1096, 1072, 1047, 1029; H NMR (500 MHz, CDCl3): δ 8.95 (s, 1H), 8.81 (s, 50 51 52 1H), 8.13 (s, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.61 (t, J = 7.2 Hz, 1H), 7.52 (t, J = 7.5 Hz, 53 54 55 80 56 57 58 59 60 ACS Paragon Plus Environment

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1 2H), 7.40 (ddt, J = 5.3, 2.7, 1.4 Hz, 2H), 7.31 (tt, J = 3.8, 2.2 Hz, 3H), 6.92 (d, J = 5.8 Hz, 2 3 1H), 6.14 (d, J = 5.4 Hz, 1H), 6.09 (t, J = 5.4 Hz, 1H), 5.59 (t, J = 4.9 Hz, 1H) 4.66 (td, J 4 5 6 = 7.7, 5.0 Hz, 1H), 4.53 (ddd, J = 10.8, 4.5, 2.9 Hz, 1H), 3.78 (s, 3H), 2.82 (ddt, J = 10.9, 7 8 9 9.1, 4.5 Hz, 1H), 2.16 (s, 3H), 2.19–2.09 (m, 1H), 2.07 (s, 3H), 2.06–1.92 (m, 2H), 1.70– 10

11 13 1.60 (m, 2H), 1.58–1.46 (m, 1H); C NMR (126 MHz, CDCl3): δ 171.2, 169.8, 169.6, 12 13 2 14 164.8, 157.0 (q, JC-F = 38 Hz), 152.9, 151.7, 149.8, 142.4, 133.5, 132.9, 131.8, 128.4, 128.2, 15 16 1 17 128.0, 124.0, 123.2, 115.7 (q, JC-F = 288 Hz), 90.0, 87.1, 83.8, 81.0, 77.3, 73.7, 73.0, 53.1, 18

19 19 52.4, 38.8, 31.1, 30.0, 28.8, 20.7, 20.5; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.7; 20 21 + ∆ 22 HRMS (ESI+): calcd. for [C37H36F3N6O9] 765.2490, meas. 765.2504, 1.8 ppm. 23 24 25 (S)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol- 26 27 28 4-yl)methyl)-5-(triisopropylsilyl)pent-4-ynal (S13). A 50 mL flask was charged with 29 30 [Rh(OAc)(C2H4)2]2 (60 mg, 0.14 mmol, 2.5 mol %) and (R)-DTBMSegPhos (389 mg, 0.329 31 32 33 mmol, 6.0 mol %). The flask headspace was purged with nitrogen for 10 min followed by 34 35 36 the addition of MeOH (14 mL). The resulting orange suspension was stirred vigorously at 37 38 39 RT, until a clear solution was obtained (15 min), followed by the sequential addition of 40 41 enal 4 (1.33 g, 5.49 mmol) in MeOH (4 mL) and TIPS-acetylene (2.46 mL, 11.0 mmol, 2.0 42 43 44 equiv.). The resulting red mixture was stirred at 40 °C for 15 h and volatiles were removed 45 46 47 in vacuo. Crude material was purified by silica gel chromatography (hexanes/acetone, 0 48 49 → 30%) to afford aldehyde S13 (2.00 g, 4.71 mmol, 86 %) as a single diastereomer, as a 50 51 ν -1 52 light yellow oil. Rf = 0.31 (hexanes/acetone, 85:15); FTIR (thin film), max (cm ): 2942, 2866, 53 54 55 81 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 2169, 1729, 1464, 1382, 1211, 1160, 1108; H NMR (600 MHz, CDCl3): δ 9.83 (dd, J = 2 3 2.1, 1.7 Hz, 1H), 4.95 (s, 1H), 4.60 (s, 2H), 4.39 (app t, J = 7.7 Hz, 1H), 3.34 (s, 3H), 3.09 4 5 6 (app tdd, J = 7.7, 6.5, 6.0 Hz, 1H), 2.65 (ddd, J = 16.6, 6.0, 1.7 Hz, 1H), 2.60 (ddd, J = 7 8 9 16.6, 7.7, 2.1 Hz, 1H), 1.89 (app dt, J = 13.6, 7.7 Hz, 1H), 1.73 (ddd, J = 13.6, 7.7, 6.5 Hz, 10

11 13 1H), 1.47 (s, 3H), 1.31 (s, 3H), 1.06–1.03 (m, 21H); C NMR (126 MHz, CDCl3): δ 200.8, 12 13 14 112.6, 109.6, 108.8, 85.6, 84.8, 83.9, 83.8, 55.2, 47.8, 39.9, 26.7, 25.3, 24.2, 18.7, 11.3; 15 16 + ∆ 17 HRMS (ESI+): calcd. for [C23H40O5Si1Na] 447.2537, meas. 447.2523, 3.2 ppm. 18 19 R Z R R R R 20 Methyl ( , )-5-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 21 22 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-2- 23 24 25 en-6-ynoate (S14). To a suspension of NaH (95%, 45 mg, 1.8 mmol, 1.5 equiv.) in THF 26 27 28 (2.5 mL) at 0 °C, was added a solution of phosphonate S2 (517 mg, 1.76 mmol, 1.5 equiv.) 29 30 in THF (2.5 mL) dropwise. The resulting cloudy mixture was stirred at 0 °C for 30 min 31 32 33 and a solution of aldehyde S13 (499 mg, 1.18 mmol) in THF (2.5 mL) was added in one 34 35 36 portion. The yellow reaction mixture was stirred at 40 °C for 1.5 h and cooled to 0 °C, 37 38 39 before the addition of an aqueous pH 7 buffer (6 mL) and Et2O (6 mL). The layers were 40 41 × separated and the aqueous phase was extracted with Et2O (3 6 mL). The combined or- 42 43 44 ganic extracts were dried over MgSO4, filtered and concentrated. Crude material was puri- 45 46 47 fied by silica gel chromatography (hexanes/EtOAc, 1 → 35%) to afford olefin S14 (651 48 49 mg, 1.10 mmol, 94%) as a 6:1 mixture of Z and E isomers as a colorless oil. Rf = 0.28 50 51 ν -1 52 (hexanes/EtOAc, 80:20); FTIR (thin film), max (cm ): 3305, 2943, 2865, 2168, 1721, 1665, 53 54 55 82 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 1536, 1463, 1439, 1382, 1274, 1210, 1160, 1107, 1061; H NMR (600 MHz, CDCl3): δ 2 3 7.78 (s, 1H), 7.09 (dd, J = 7.5, 7.2 Hz, 1H), 4.95 (s, 1H), 4.60 (d, J = 6.0 Hz, 1H), 4.55 (dd, 4 5 6 J = 6.0, 0.9 Hz, 1H), 4.37 (app td, J = 7.5, 0.9 Hz, 1H), 3.81 (s, 3H), 3.34 (s, 3H), 2.81 7 8 9 (dddd, J = 9.0, 7.5, 7.0, 4.6 Hz, 1H), 2.45 (ddd, J = 15.7, 7.2, 4.6 Hz, 1H), 2.29 (ddd, J = 10 11 15.7, 9.0, 7.5 Hz, 1H), 1.89 (app dt, J = 13.7, 7.5 Hz, 1H), 1.70 (ddd, J = 13.7, 7.5, 7.0 Hz, 12 13 13 14 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06–1.04 (m, 21H); C NMR (101 MHz, CDCl3): δ 163.8, 15 16 2 1 17 155.2 (q, JC-F = 38 Hz), 137.7, 123.7, 115.8 (q, JC-F = 288 Hz), 112.6, 109.5, 109.3, 85.6, 84.8, 18

19 19 83.9, 55.1, 52.9, 40.0, 33.8, 28.8, 26.6, 25.2, 18.7, 11.3; F NMR (471 MHz, CDCl3, BTF 20 21 + 22 IStd): δ –76.3, –77.2; HRMS (ESI+): calcd. for [C28H44F3N1O7Si1Na] 614.2731, meas. 23 24 25 614.2735, ∆ 0.7 ppm. 26 27 S R R R R R 28 Methyl (2 ,5 )-5-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 29 30 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-7-(triisopropylsilyl)hept-6- 31 32 33 ynoate (S15). To a flask charged with enamide S14 (6:1 mixture of Z and E isomers, 651 34 35 36 mg, 1.10 mmol) was added MeOH (32 mL) under a nitrogen atmosphere. The contents 37 38 39 were stirred until full solubilization was observed. In a glove box, a separate vial was 40 41 charged with (S,S)-MeBPE-Rh (31 mg, 55 µmol, 5 mol %). The vial was removed from 42 43 44 the glove box, placed under a nitrogen atmosphere and MeOH (4 mL) was added. The 45 46 47 resulting orange solution was transferred to the flask containing the methanolic solution of 48 49 enamide S14 via syringe. The flask headspace was evacuated and refilled with hydrogen 50 51 52 53 54 55 83 56 57 58 59 60 ACS Paragon Plus Environment

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× 1 (3 ), and the reaction mixture was stirred vigorously under a hydrogen atmosphere (bal- 2 3 loon) for 1 h. Volatiles were removed in vacuo. Crude material was purified by silica gel 4 5 6 chromatography (2,2,4-trimethylpentane/EtOAc, 1 → 50%) to afford protected amino acid 7 8 9 S15 (627 mg, 1.06 mmol, 96%) as a single diastereomer, as a colorless oil. Rf = 0.30 (hex- 10 11 ν -1 anes/EtOAc, 85:15); FTIR (thin film), max (cm ): 3319, 2942, 2865, 2166, 1749, 1720, 12 13 1 14 1549, 1461, 1382, 1208, 1159, 1106, 1092, 1061; H NMR (500 MHz, CDCl3): δ 6.90 (d, 15 16 17 J = 7.5 Hz, 1H), 4.93 (s, 1H), 4.64 (app td, J = 7.5, 5.2 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 18 19 4.53 (dd, J = 5.9, 0.8 Hz, 1H), 4.36 (app td, J = 7.8, 0.8 Hz, 1H), 3.79 (s, 3H), 3.33 (s, 3H), 20 21 22 2.58–2.51 (m, 1H), 2.29 (dddd, J = 13.9, 11.4, 5.2, 4.7 Hz, 1H), 1.90–1.81 (m, 2H), 1.63 23 24 25 (ddd, J = 13.6, 7.8, 6.7 Hz, 1H), 1.59–1.44 (m, 2H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06–1.02 26

27 13 2 1 3 JC-F JC-F 28 (m, 21H); C NMR (126 MHz, CDCl ): δ 171.4, 156.9 (q, = 38 Hz), 115.7 (q, = 288 29 30 Hz), 112.6, 109.6, 109.5, 85.6, 84.9, 83.9, 83.5, 55.1, 53.1, 52.7, 40.3, 30.4, 30.1, 29.3, 31 32 19 33 26.6, 25.2, 18.7, 11.3; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.8; HRMS (ESI+): 34 35 + ∆ 36 calcd. for [C28H46F3N1O7Si1Na] 616.2888, meas. 616.2890, 0.3 ppm. 37 38 S R R R R R 39 Methyl (2 ,5 )-5-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 40 41 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept-6-ynoate (S16). To a solu- 42 43 44 tion of TIPS alkyne S15 (108 mg, 0.182 mmol) in DMF (4 mL) at RT, was added a solution 45 46 47 of TASF (0.15 g, 0.55 mmol, 3.0 equiv.) in DMF (3.3 mL). The resulting mixture was 48 49 stirred at 60 °C for 2.5 h and volatiles were removed in vacuo. The residue was partitioned 50 51

52 between Et2O (7 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (7 mL). The 53 54 55 84 56 57 58 59 60 ACS Paragon Plus Environment

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× 1 layers were separated and the aqueous phase was extracted with Et2O (3 7 mL). The 2 3 combined organic extracts were dried over MgSO4, filtered and concentrated. Crude mate- 4 5 6 rial was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 5 → 50%) 7 8 9 to afford alkyne S16 (80 mg, 0.18 mmol, quant.) as a colorless oil. Rf = 0.26 (hex- 10 11 ν -1 anes/EtOAc, 70:30); FTIR (thin film), max (cm ): 3288, 2936, 1747, 1719, 1552, 1456, 12 13 1 14 1440, 1383, 1374, 1209, 1160, 1106, 1091, 1059; H NMR (500 MHz, CDCl3): δ 6.96 (d, 15 16 17 J = 7.5 Hz, 1H), 4.93 (s, 1H), 4.63 (ddd, J = 7.5, 7.0, 5.8 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 18 19 4.51 (dd, J = 5.9, 1.0 Hz, 1H), 4.33 (app td, J = 7.5, 1.0 Hz, 1H), 3.80 (s, 3H), 3.33 (s, 3H), 20 21 22 2.50 (ddddd, J = 9.5, 7.3, 6.8, 4.6, 2.4 Hz, 1H), 2.22 (dddd, J = 14.0, 11.2, 5.8, 4.8 Hz, 1H), 23 24 25 2.13 (d, J = 2.4 Hz, 1H), 1.90–1.82 (m, 2H), 1.64 (ddd, J = 13.7, 7.5, 6.8 Hz, 1H), 1.60– 26

27 13 3 28 1.44 (m, 2H), 1.47 (s, 3H), 1.30 (s, 3H); C NMR (126 MHz, CDCl ): δ 171.3, 156.9 (q, 29 30 2 1 JC-F = 38 Hz), 115.7 (q, JC-F = 288 Hz), 112.6, 109.8, 85.6, 85.4, 84.6, 84.0, 71.2, 55.2, 53.1, 31 32 19 33 52.6, 39.8, 29.8, 29.6, 28.2, 26.6, 25.1; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.8; 34 35 + ∆ 36 HRMS (ESI+): calcd. for [C19H26F3N1O7Na] 460.1554, meas. 460.1562, 1.9 ppm. 37 38 R R R R S 39 (3 ,4 ,5 )-5-((2 ,5 )-2-ethynyl-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetam- 40 41 ido)hexyl)tetrahydrofuran-2,3,4-triyl triacetate (S17). To a solution of acetonide S16 42 43

44 (80 mg, 0.18 mmol) in CH2Cl2 (0.8 mL) at 0 °C, was added a 4:1 mixture of TFA and water 45 46 47 (3.6 mL). The resulting mixture was stirred at RT for 5.5 h. Volatiles were removed in 48 49 vacuo and the residue was dried by azeotropic distillation with benzene (2 × 0.8 mL). 50 51 52 Crude material was used directly in the next step without further purification. To a solution 53 54 55 85 56 57 58 59 60 ACS Paragon Plus Environment

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1 of the crude triol in CH2Cl2 (3.6 mL) at RT, were added pyridine (0.15 mL, 1.8 mmol, 10 2 3 equiv.), Ac2O (0.17 mL, 1.8 mmol, 10 equiv.) and DMAP (16 mg, 0.13 mmol, 0.70 equiv.) 4 5 6 sequentially. The resulting mixture was stirred at RT for 15 h and volatiles were removed 7 8 9 in vacuo. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 5 → 10 11 70%) to afford triacetate S17 (60 mg, 0.12 mmol, 64% over 2 steps) as a 2:1 mixture of 12 13

14 diastereomers, as a colorless oil. Rf = 0.35/0.43 (hexanes/EtOAc, 50:50); FTIR (thin film), 15 16 ν -1 1 17 max (cm ): 3290, 2930, 1746, 1722, 1554, 1438, 1372, 1214, 1111, 1011; H NMR (500 18 19 MHz, CDCl3): δ 7.12 (d, J = 8.1 Hz, 2H), 6.98 (d, J = 8.0 Hz, 1H), 6.35 (d, J = 4.5 Hz, 20 21 22 1H), 6.06 (d, J = 1.0 Hz, 2H), 5.30 (dd, J = 4.8, 1.0 Hz, 2H), 5.25–5.20 (m, 3H), 5.17 (dd, 23 24 25 J = 6.9, 3.5 Hz, 1H), 4.66–4.59 (m, 3H), 4.38–4.32 (m, 3H), 3.80 (s, 3H), 3.79 (s, 6H), 26 27 J J 28 2.60–2.50 (m, 3H), 2.25–2.15 (m, 2H), 2.14 (d, = 2.5 Hz, 1H), 2.13 (d, = 2.5 Hz, 2H), 29 30 2.12 (s, 6H), 2.11 (s, 3H), 2.11 (s, 3H), 2.09 (s, 6H), 2.07 (s, 6H), 2.06 (s, 3H), 1.93–1.83 31 32 13 33 (m, 4H), 1.83–1.74 (m, 6H), 1.61–1.47 (m, 6H); C NMR (126 MHz, CDCl3): δ 171.2, 34 35 2 2 36 170.2, 170.0, 169.9, 169.7, 169.6, 169.5, 157.1 (q, JC-F = 38 Hz), 156.9 (q, JC-F = 37 Hz), 37

38 1 1 115.8 (q, JC-F = 287 Hz), 115.7 (q, JC-F = 288 Hz), 98.5, 93.9, 85.2, 85.0, 81.3, 79.2, 74.4, 39 40 41 73.7, 72.0, 71.6, 71.2, 69.9, 53.1, 53.1, 52.6, 52.5, 38.4, 37.9, 29.7, 29.6, 29.5, 27.8, 27.3, 42 43 19 44 21.2, 21.1, 20.8, 20.6, 20.6, 20.4; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.75, –76.76; 45 46 + ∆ HRMS (ESI+): calcd. for [C21H26F3N1O10Na] 532.1401, meas. 532.1416, 2.8 ppm. 47 48 49 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2R,5S)-2-ethynyl-6-methoxy-6- 50 51 52 oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S18). To a 53 54 55 86 56 57 58 59 60 ACS Paragon Plus Environment

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6 1 suspension of N -benzoyladenine (113 mg, 0.471 mmol, 4.0 equiv.) in propionitrile (dried 2 3 over 4 Å M.S., 2.4 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.14 mL, 0.59 4 5 6 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which com- 7 8 6 9 plete solubilization of N -benzoyladenine was observed. To a separate flask charged with 10 11 triacetate S17 (dried by azeotropic distillation with benzene (4 ×), 60 mg, 0.12 mmol) and 12 13 14 2,6-di-tert-butyl-4-methylpyridinium triflate (21 mg, 59 µmol, 50 mol %) was added pro- 15 16 17 pionitrile (2.4 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution 18 19 was obtained, and the solution of silylated N6-benzoyladenine was added. The reaction 20 21 22 mixture was stirred at 95 °C for 5.5 h, cooled to RT and the volatiles were removed in 23 24 25 vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpen- 26 27 tane/EtOAc, 30 → 100%) to afford nucleoside S18 (53 mg, 77 µmol, 65%) as a colorless 28 29 30 ν -1 oil. Rf = 0.33 (hexanes/EtOAc, 20:80); FTIR (thin film), max (cm ): 3283, 3084, 2929, 1745, 31 32 33 1720, 1612, 1583, 1511, 1487, 1456, 1374, 1244, 1218, 1075, 1029; 1H NMR (500 MHz, 34 35 36 CDCl3): δ 9.07 (s, 1H), 8.79 (s, 1H), 8.11 (s, 1H), 8.04–7.98 (m, 2H), 7.63–7.57 (m, 1H), 37 38 7.54–7.49 (m, 2H), 7.04 (br d, J = 7.6 Hz, 1H), 6.12 (d, J = 5.5 Hz, 1H), 6.04 (app t, J = 39 40 41 5.5 Hz, 1H), 5.58 (dd, J = 5.5, 4.5 Hz, 1H), 4.60 (ddd, J = 7.6, 7.0, 5.4 Hz, 1H), 4.38 (ddd, 42 43 44 J = 7.5, 5.8, 4.5 Hz, 1H), 3.76 (s, 3H), 2.57 (dddd, J = 14.0, 8.2, 6.4, 2.4 Hz, 1H), 2.23– 45 46 2.12 (m, 2H), 2.15 (s, 3H), 2.15 (d, J = 2.4 Hz, 1H), 2.06 (s, 3H), 2.00–1.93 (m, 1H), 1.86– 47 48 13 49 1.77 (m, 1H), 1.54–1.48 (m, 2H); C NMR (100 MHz, CDCl3): δ 171.2, 169.8, 169.6, 50 51 2 52 164.8, 156.9 (q, JC-F = 38 Hz), 153.0, 151.8, 149.9, 142.0, 133.5, 133.0, 129.0, 128.0, 124.0, 53 54 55 87 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 115.7 (q, JC-F = 288 Hz), 86.7, 85.1, 80.6, 73.3, 72.8, 71.5, 53.2, 52.5, 37.7, 29.6, 29.6, 27.5, 2 3 19 20.7, 20.5; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.7; HRMS (ESI+): calcd. for 4 5 + ∆ 6 [C31H32F3N6O9] 689.2177, meas. 689.2178, 0.1 ppm. 7 8 9 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2R,5S)-2-((3-car- 10 11 12 bamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahy- 13 14 drofuran-3,4-diyl diacetate (S19). To a solution of nucleoside S18 (53 mg, 77 µmol) and 15 16 17 3-iodobenzamide (48 mg, 0.19 mmol, 2.5 equiv.) in a 5:1:1 mixture of toluene, DMF and 18 19 20 i-Pr2NEt (degassed by sparging with nitrogen for 15 min, 3.9 mL) at RT, were introduced 21 22 CuI (4 mg, 0.02 mmol, 25 mol %) and Pd(PPh3)4 (4 mg, 4 µmol, 5 mol %) rapidly. The 23 24 25 resulting mixture was stirred at 60 °C for 2 h. The solution was allowed to cool to RT and 26 27 28 volatiles were removed in vacuo. Crude material was purified by silica gel chromatography 29 30 (EtOAc/i-PrOH, 0 → 5%) to afford alkyne S19 (50 mg, 62 µmol, 80%) as a colorless oil. 31 32 ν -1 33 Rf = 0.43 (EtOAc/i-PrOH, 95:5); FTIR (thin film), max (cm ): 3284, 2932, 1746, 1716, 1668, 34 35 1 36 1611, 1580, 1455, 1375, 1242, 1215, 1183, 1160, 1075; H NMR (600 MHz, CDCl3): δ 37 38 39 9.23 (s, 1H), 8.75 (s, 1H), 8.18 (s, 1H), 8.04–7.98 (m, 2H), 7.84–7.82 (m, 1H), 7.80 (ddd, 40 41 J = 7.7, 1.9, 1.3 Hz, 1H), 7.61–7.56 (m, 1H), 7.52–7.47 (m, 2H), 7.46 (app dt, J = 7.7, 1.3 42 43 44 Hz, 1H), 7.36 (br d, J = 7.3 Hz, 1H), 7.34 (app t, J = 7.7 Hz, 1H), 6.91 (br s, 1H), 6.16 (d, 45 46 47 J = 5.7 Hz, 1H), 6.13 (dd, J = 5.7, 5.4 Hz, 1H), 5.99 (br s, 1H), 5.76 (dd, J = 5.4, 4.3 Hz, 48 49 1H), 4.68–4.63 (m, 1H), 4.43 (ddd, J = 7.0, 5.5, 4.3 Hz, 1H), 3.76 (s, 3H), 2.87–2.81 (m, 50 51 52 1H), 2.33–2.26 (m, 1H), 2.26 (ddd, J = 14.2, 8.4, 7.0 Hz, 1H), 2.14 (s, 3H), 2.06 (s, 3H), 53 54 55 88 56 57 58 59 60 ACS Paragon Plus Environment

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13 1 2.05–2.01 (m, 1H), 1.97–1.89 (m, 1H), 1.61–1.55 (m, 2H); C NMR (126 MHz, CDCl3): δ 2 3 2 171.3, 170.0, 169.6, 168.8, 165.0, 157.1 (q, JC-F = 38 Hz), 152.9, 151.9, 149.9, 142.2, 134.5, 4 5 1 6 133.6, 133.5, 133.0, 130.6, 129.0, 128.8, 128.1, 127.7, 124.1, 123.3, 115.7 (q, JC-F = 288 7 8 19 9 Hz), 91.7, 86.7, 82.9, 81.1, 73.2, 72.7, 532, 52.7, 37.6, 30.2, 29.6, 28.0, 20.8, 20.5; F NMR 10

11 + (471 MHz, CDCl3, BTF IStd): δ –76.7; HRMS (ESI+): calcd. for [C38H37F3N7O10] 808.2549, 12 13 14 meas. 808.2541, ∆ 0.9 ppm. 15 16 17 3-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4- 18 19 71 72 20 yl)propan-1-ol (S21). To a solution of known α,β-unsaturated methyl ester S20 (1.22 g, 21 22 4.72 mmol) in a 10:1 mixture of Et2O and MeOH (22 mL) at 0 °C, was added a solution of 23 24

25 LiBH4 (2.0 M in THF, 9.45 mL, 18.9 mmol, 4.0 equiv.) dropwise. The resulting mixture was 26 27 28 allowed to warm gradually to RT and stirred for 17 h, before the addition of a 1:1 mixture 29 30 of a NH4Cl sat. aq. solution and water (35 mL). The layers were separated and the aqueous 31 32 × 33 phase was extracted with Et2O (3 35 mL). The combined organic extracts were washed 34 35 36 with brine (100 mL), dried over MgSO4, filtered and concentrated to give alcohol S21 (1.10 37 38 39 g, 4.72 mmol, quant.) as a colorless oil. Rf = 0.24 (hexanes/EtOAc, 50:50); FTIR (thin film), 40 41 ν -1 1 max (cm ): 3422, 2989, 2937, 1449, 1373, 1210, 1160, 1088, 1054, 1015; H NMR (600 42 43 44 MHz, CDCl3): δ 4.95 (s, 1H), 4.61 (d, J = 5.9 Hz, 1H), 4.54 (dd, J = 5.9, 1.0 Hz, 1H), 4.20– 45 46 47 4.16 (m, 1H), 3.70–3.67 (m, 2H), 3.36 (s, 3H), 1.76–1.59 (m, 4H), 1.48 (s, 3H), 1.32 (s, 48

49 13 3H); C NMR (151 MHz, CDCl3): δ 112.4, 109.7, 87.2, 85.5, 84.3, 62.3, 55.1, 31.6, 29.6, 50 51 + ∆ 52 26.6, 25.1; HRMS (ESI+): calcd. for [C11H20O5Na] 255.1203, meas. 255.1196, 2.6 ppm. 53 54 55 89 56 57 58 59 60 ACS Paragon Plus Environment

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R R R R d 1 3-((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- ][1,3]dioxol-4- 2 3 73 yl)propanal (S22). To a solution of alcohol S21 (400 mg, 1.72 mmol) in CH2Cl2 (11.5 mL) 4 5 6 at RT, was added DMP (876 mg, 2.07 mmol, 1.2 equiv.) in one portion. The resulting mix- 7 8 9 ture was stirred at RT for 1 h, before the addition of a 1:1 mixture of a Na2S2O3 sat. aq. 10 11 solution and a NaHCO3 sat. aq. solution (10 mL). The biphasic mixture was stirred vigor- 12 13 14 ously at RT for 10 min and the layers were separated. The aqueous phase was extracted 15 16 × 17 with Et2O (3 10 mL) and the combined organic extracts were washed with brine (40 mL), 18 19 dried over MgSO4, filtered and concentrated. Crude material was purified by silica gel 20 21 22 chromatography (pentane/EtOAc, 2 → 50%) to afford aldehyde S22 (324 mg, 1.41 mmol, 23 24 ν -1 25 82%) as a colorless oil. Rf = 0.32 (hexanes/EtOAc, 75:25); FTIR (thin film), max (cm ): 2988, 26

27 1 3 28 2936, 2834, 2724, 1721, 1373, 1209, 1160, 1090, 1058; H NMR (500 MHz, CDCl ): δ 29 30 9.79 (t, J = 1.3 Hz, 1H), 4.93 (s, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.52 (dd, J = 5.9, 1.1 Hz, 31 32 33 1H), 4.14 (ddd, J = 9.2, 6.5, 1.1 Hz, 1H), 3.32 (s, 3H), 2.62 (dddd, J = 18.1, 7.9, 6.6, 1.3 34 35 36 Hz, 1H), 2.57 (dddd, J = 18.1, 8.2, 6.9, 1.3 Hz, 1H), 1.89–1.82 (m, 2H), 1.45 (s, 3H), 1.30 37

38 13 (s, 3H); C NMR (151 MHz, CDCl3): δ 201.4, 112.5, 109.9, 86.3, 85.6, 84.1, 55.3, 40.8, 39 40 + ∆ 41 27.5, 26.6, 25.1; HRMS (ESI+): calcd. for [C11H18O5Na] 253.1046, meas. 253.1036, 4.0 42 43 44 ppm. 45 46 R R R R 47 (3a ,4 ,6 ,6a )-4-(but-3-yn-1-yl)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 48 49 d][1,3]dioxole (S23).74 To a solution of aldehyde S22 (324 mg, 1.41 mmol) in MeOH (6.0 50 51

52 mL) at RT, was added K2CO3 (583 mg, 4.22 mmol, 3.0 equiv.), followed by a solution of 53 54 55 90 56 57 58 59 60 ACS Paragon Plus Environment

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1 dimethyl-1-diazo-2-oxopropyl phosphonate (338 mg, 1.76 mmol, 1.25 equiv.) in MeOH 2 3 (3.0 mL) under vigorous stirring. The resulting yellow suspension was stirred at RT for 3 4 5

6 h. The reaction was quenched by the addition of a NH4Cl sat. aq. solution (15 mL) and Et2O 7 8 9 (25 mL) was added. The layers were separated and the aqueous phase was extracted with 10 11 × Et2O (3 25 mL). The combined organic extracts were dried over MgSO4, filtered and 12 13

14 concentrated. Crude material was purified by silica gel chromatography (hexane/Et2O, 2 15 16 17 → 50%) to afford alkyne S23 (254 mg, 1.12 mmol, 80%) as a colorless oil. Rf = 0.50 (hex- 18

19 1 anes/EtOAc, 75:25); H NMR (600 MHz, CDCl3): δ 4.95 (s, 1H), 4.60 (d, J = 5.9 Hz, 1H), 20 21 22 4.56 (dd, J = 5.9, 1.0 Hz, 1H), 4.30 (ddd, J = 9.8, 5.8, 1.0 Hz, 1H), 3.34 (s, 3H), 2.36 (dddd, 23 24 25 J = 16.8, 7.8, 5.9, 2.6 Hz, 1H), 2.31 (dddd, J = 16.8, 7.8, 7.3, 2.6 Hz, 1H), 1.98 (t, J = 2.6 26 27 J J 28 Hz, 1H), 1.80 (dddd, = 13.4, 9.8, 7.3, 5.9 Hz, 1H), 1.74 (app dtd, = 13.4, 7.8, 5.8 Hz, 29 30 13 1H), 1.48 (s, 3H), 1.31 (s, 3H); C NMR (151 MHz, CDCl3): δ 112.5, 109.8, 85.9, 85.7, 31 32 + 33 84.1, 83.3, 69.1, 55.3, 33.9, 26.6, 25.1, 15.6; HRMS (ESI+): calcd. for [C12H18O4Na] 34 35 ∆ 36 249.1097, meas. 249.1118, 8.4 ppm. 37 38 R R R 39 (3 ,4 ,5 )-5-(but-3-yn-1-yl)tetrahydrofuran-2,3,4-triyl triacetate (S24). To a vial 40 41 charged with acetonide S23 (50 mg, 0.22 mmol) was added a 4:1 mixture of acetic acid 42 43 44 and water (2.5 mL) at RT. The resulting mixture was stirred at 80 °C for 5 h. Volatiles were 45 46 × 47 removed in vacuo and the residue was dried by azeotropic distillation with benzene (2 48 49 1.0 mL). Crude material was used directly in the next step without further purification. To 50 51

52 a solution of the crude triol in CH2Cl2 (2.5 mL) at RT, were added pyridine (0.18 mL, 2.2 53 54 55 91 56 57 58 59 60 ACS Paragon Plus Environment

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1 mmol, 10 equiv.), Ac2O (0.21 mL, 2.2 mmol, 10 equiv.) and DMAP (19 mg, 0.15 mmol, 2 3 0.70 equiv.) sequentially. The resulting mixture was stirred at RT for 2 h and volatiles were 4 5 6 removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trime- 7 8 9 thylpentane/EtOAc, 2 → 60%) to afford triacetate S24 (58 mg, 0.19 mmol, 88% over 2 10 11 steps) as a 1:1 mixture of diastereomers, as a colorless oil; Rf = 0.42/0.50 (hexanes/EtOAc, 12 13 ν -1 14 50:50); FTIR (thin film), max (cm ): 3285, 2958, 2920, 2850, 2117, 1742, 1433, 1371, 1214, 15 16 1 17 1107, 1010; H NMR (600 MHz, CDCl3): δ 6.36 (d, J = 4.6 Hz, 1H), 6.12 (d, J = 1.0 Hz, 18 19 1H), 5.30 (dd, J = 4.8, 1.0 Hz, 1H), 5.21 (dd, J = 6.8, 4.6 Hz, 1H), 5.21 (dd, J = 7.0, 4.8 20 21 22 Hz, 1H), 5.10 (dd, J = 6.8, 3.6 Hz, 1H), 4.33 (ddd, J = 8.5, 4.9, 3.6 Hz, 1H), 4.27 (ddd, J 23 24 25 = 8.5, 7.0, 4.6 Hz, 1H), 2.39–2.25 (m, 4H), 2.11 (s, 6H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 26 27 J J 28 3H), 2.05 (s, 3H), 1.97 (t, = 2.8 Hz, 1H), 1.96 (t, = 2.6 Hz, 1H), 1.95–1.86 (m, 2H), 29 30 13 1.86–1.78 (m, 2H); C NMR (126 MHz, CDCl3): δ 170.2, 170.0, 169.8, 169.6, 169.5, 169.2, 31 32 33 98.3, 93.9, 83.0, 82.8, 82.1, 80.3, 74.7, 73.5, 72.1, 70.0, 69.4, 69.2, 33.2, 32.3, 21.2, 21.2, 34 35 + 36 20.8, 20.7, 20.6, 20.4, 14.9, 14.8; HRMS (ESI+): calcd. for [C14H18O7Na] 321.0945, meas. 37 38 321.0952, ∆ 2.3 ppm. 39 40 41 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-(but-3-yn-1-yl)tetrahydrofuran- 42 43 44 3,4-diyl diacetate (S25). To a suspension of N6-benzoyladenine (160 mg, 0.670 mmol, 4.0 45 46 47 equiv.) in propionitrile (dried over 4 Å M.S., 5.0 mL) at RT, was added N,O-bis(trime- 48 49 thylsilyl)acetamide (0.20 mL, 0.84 mmol, 5.0 equiv.). The resulting mixture was stirred at 50 51 52 80 °C for 10 min, upon which complete solubilization of N6-benzoyladenine was observed. 53 54 55 92 56 57 58 59 60 ACS Paragon Plus Environment

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1 To a separate flask charged with triacetate S24 (dried by azeotropic distillation with ben- 2 3 zene (4 ×), 50 mg, 0.17 mmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (30 mg, 4 5 6 84 µmol, 50 mol %) was added propionitrile (5.0 mL). The resulting mixture was stirred 7 8 6 9 at RT for 5 min, until a clear solution was obtained, and the solution of silylated N -ben- 10 11 zoyladenine was added. The reaction mixture was stirred at 95 °C for 17 h, cooled to RT 12 13 14 and the volatiles were removed in vacuo. Crude material was purified by silica gel chro- 15 16 17 matography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford nucleoside S25 (73 mg, 18

19 ν -1 0.15 mmol, 91%) as a colorless oil. Rf = 0.31 (EtOAc); FTIR (thin film), max (cm ): 3291, 20 21 22 3091, 2934, 1747, 1699, 1609, 1582, 1510, 1486, 1455, 1373, 1239, 1217, 1099, 1073, 23 24 1 25 1053; H NMR (600 MHz, CDCl3): δ 9.12 (s, 1H), 8.78 (s, 1H), 8.09 (s, 1H), 8.04–7.98 (m, 26 27 J J 28 2H), 7.62–7.57 (m, 1H), 7.54–7.48 (m, 2H), 6.13 (d, = 5.4 Hz, 1H), 6.04 (dd, = 5.6, 5.4 29 30 Hz, 1H), 5.57 (dd, J = 5.6, 4.7 Hz, 1H), 4.37 (ddd, J = 9.2, 4.7, 4.3 Hz, 1H), 2.38 (dddd, J 31 32 33 = 17.0, 7.0, 5.6, 2.6 Hz, 1H), 2.31 (dddd, J = 17.0, 8.5, 6.8, 2.6 Hz, 1H), 2.15 (s, 3H), 2.16– 34 35 13 36 2.09 (m, 1H), 2.06 (s, 3H), 2.06–2.00 (m, 1H), 1.99 (t, J = 2.6 Hz, 1H); C NMR (151 37 38 MHz, CDCl3): δ 169.8, 169.6, 164.8, 153.0, 151.7, 149.9, 142.1, 133.6, 133.0, 129.0, 39 40 41 128.0, 124.0, 86.9, 82.8, 81.3, 73.3, 73.1, 69.6, 31.8, 20.7, 20.5, 14.8; HRMS (ESI+): calcd. 42 43 + ∆ 44 for [C24H24N5O6] 478.1721, meas. 478.1728, 1.5 ppm. 45 46 R R R R H 47 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-(4-(3-carbamoylphenyl)but-3-yn- 48 49 1-yl)tetrahydrofuran-3,4-diyl diacetate (S26). To a vial charged with alkyne S25 (72 mg, 50 51 52 0.15 mmol) were added 3-iodobenzamide (75 mg, 0.30 mmol, 2.0 equiv.), CuI (7 mg, 38 53 54 55 93 56 57 58 59 60 ACS Paragon Plus Environment

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1 µmol, 25 mol %) and Pd(PPh3)4 (9 mg, 8 µmol, 5 mol %). The vial headspace was purged 2 3 with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by 4 5 6 sparging with nitrogen for 15 min, 6.0 mL) was added at RT. The resulting mixture was 7 8 9 stirred at 80 °C for 1 h. The solution was allowed to cool to RT and volatiles were removed 10 11 in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 12 13

14 20%) to afford alkyne S26 (58 mg, 97 µmol, 64%) as a colorless oil. Rf = 0.24 (EtOAc/i- 15 16 ν -1 17 PrOH, 90:10); FTIR (thin film), max (cm ): 3187, 2931, 1747, 1664, 1609, 1578, 1511, 1486, 18

19 1 1454, 1373, 1239, 1215, 1097, 1049; H NMR (600 MHz, CDCl3): δ 9.30 (s, 1H), 8.76 (s, 20 21 22 1H), 8.17 (s, 1H), 8.04–7.97 (m, 2H), 7.83 (app t, J = 1.5 Hz, 1H), 7.74 (app dt, J = 7.8, 23 24 25 1.5 Hz, 1H), 7.59–7.55 (m, 1H), 7.50–7.45 (m, 3H), 7.33 (app t, J = 7.8 Hz, 1H), 6.64 (br 26 27 J J J 28 s, 1H), 6.17 (d, = 5.6 Hz, 1H), 6.13 (br s, 1H), 6.11 (app t, = 5.6 Hz, 1H), 5.71 (dd, = 29 30 5.6, 4.3 Hz, 1H), 4.40 (ddd, J = 8.1, 4.9, 4.3 Hz, 1H), 2.61 (app dt, J = 17.2, 6.3 Hz, 1H), 31 32 33 2.53 (ddd, J = 17.2, 8.4, 6.1 Hz, 1H), 2.23–2.17 (m, 1H), 2.14 (s, 3H), 2.16–2.09 (m, 1H), 34 35 13 36 2.05 (s, 3H); C NMR (101 MHz, CDCl3): δ 170.0, 169.7, 168.9, 165.0, 152.9, 151.9, 37 38 149.9, 142.2, 134.7, 133.7, 133.5, 133.0, 130.6, 128.9, 128.7, 128.1, 127.1, 124.1, 124.0, 39 40 41 89.7, 86.7, 81.8, 80.8, 73.3, 73.0, 31.7, 20.8, 20.5, 15.6; HRMS (ESI+): calcd. for 42 43 + ∆ 44 [C31H29N6O7] 597.2092, meas. 597.2097, 0.8 ppm. 45 46 R R R R R R d 47 ((1 ,2 )-2-((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- ][1,3]di- 48 49 oxol-4-yl)cyclopropyl)methanol (S28). The following protocol was adapted from an early 50 51 52 53 54 55 94 56 57 58 59 60 ACS Paragon Plus Environment

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1 publication reporting the asymmetric cyclopropanation of allylic alcohols with dioxaboro- 2 3 lane ligands.75 To a stirred solution of diethylzinc in hexanes (1.0 M in hexanes, 4.46 mL, 4 5

6 4.46 mmol, 2.23 equiv.) was added CH2Cl2 (8.0 mL). The mixture was cooled to 0 °C and 7 8 9 diiodomethane (0.72 mL, 8.9 mmol, 4.45 equiv.) was added dropwise over 5 min. The 10 11 reaction mixture was stirred at 0 °C for 15 min (white precipitate was formed), and a pre- 12 13 14 cooled (0 °C) solution of butylboronic acid N,N,N’,N’-tetramethyl-D-tartaric acid diamide 15 16 72 17 ester (360 mg, 2.28 mmol, 1.14 equiv.) and allylic alcohol S27 (457 mg, 2.00 mmol) in 18 19 CH2Cl2 (12 mL) was rapidly added via syringe. The resulting mixture was stirred at RT for 20 21

22 1 h, cooled to 0 °C and a NH4Cl sat. aq. solution (15 mL) was added slowly. The layers 23 24 × 25 were separated and the aqueous phase was extracted with CH2Cl2 (3 30 mL). The com- 26 27 bined organic extracts were passed through a phase separator (Biotage Isolute), dried over 28 29 30 MgSO4, filtered and concentrated. Crude material was purified by silica gel chromatog- 31 32 33 raphy (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford cyclopropyl alcohol S28 (382 34 35 36 mg, 1.56 mmol, 78%) as a 12:1 mixture of cyclopropyl diastereomers (major=desired), as 37 38 ν -1 a colorless oil. Rf = 0.24 (cyclohexane/EtOAc, 50:50); FTIR (thin film), max (cm ): 3446, 39 40 41 2991, 2935, 2251, 2041, 1458, 1382, 1373, 1274, 1210, 1194, 1159, 1105, 1089, 1053, 42 43 1 44 1030; H NMR (600 MHz, CDCl3): δ 4.95 (s, 1H), 4.74 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 45 46 5.9 Hz, 1H), 3.51–3.43 (m, 3H), 3.38 (s, 3H), 1.45 (s, 3H), 1.31 (s, 3H), 1.21 (dddd, J = 47 48 49 12.0, 8.4, 6.9, 5.1 Hz, 1H), 0.94 (ddt, J = 10.4, 9.0, 4.7 Hz, 1H), 0.53 (ddt, J = 17.2, 8.4, 50 51 13 52 5.1 Hz, 2H); C NMR (151 MHz, CDCl3): δ 112.1, 109.1, 91.1, 85.3, 84.0, 65.5, 54.4, 26.3, 53 54 55 95 56 57 58 59 60 ACS Paragon Plus Environment

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+ ∆ 1 24.8, 20.8, 20.4, 8.1; HRMS (ESI+): calcd. for [C12H20O5Na] 267.1203, meas. 267.1202, 2 3 0.2 ppm. 4 5 6 (1R,2R)-2-((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]di- 7 8 9 oxol-4-yl)cyclopropane-1-carbaldehyde (S29). To a solution of cyclopropyl alcohol S28 10 11 12 (245 mg, 1.00 mmol) in CH2Cl2 (20 mL) open to atmosphere at RT was added DMP (510 13 14 mg, 1.20 mmol, 1.2 equiv.) portionwise over 5 min. The reaction mixture was stirred for 2 15 16

17 h and was diluted with Et2O (10 mL). The suspension was filtered and the volatiles were 18 19 20 removed in vacuo. The crude residue was purified by silica gel chromatography (2,2,4- 21 22 trimethylpentane/EtOAc, 0 → 50%) to yield aldehyde S29 (182 mg, 0.751 mmol, 75%) as 23 24 25 a clear oil. In our hands, aldehyde S29 was best prepared, purified and used immediately 26 27 1 28 in the next reaction. Rf = 0.39 (cyclohexane/EtOAc, 67:33); H NMR (600 MHz, CDCl3): δ 29 30 9.16 (d, J = 4.9 Hz, 1H), 4.96 (s, 1H), 4.75 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 5.8 Hz, 1H), 31 32 33 3.55 (d, J = 10.0 Hz, 1H), 3.28 (s, 3H), 2.02 (app dtd, J = 7.8, 4.9, 3.9 Hz, 1H), 1.79 (app 34 35 36 tdd, J = 10.1, 8.9, 6.4, 4.0 Hz, 1H), 1.45 (s, 3H), 1.33 (dd, J = 8.9, 4.9 Hz, 1H), 1.31 (s, 37

38 13 39 3H), 1.07 (ddd, J = 8.3, 6.4, 4.9 Hz, 1H); C NMR (151 MHz, CDCl3): δ 199.9, 112.5, 40 41 109.2, 89.3, 85.4, 84.0, 54.6, 28.9, 26.4, 26.1, 25.0, 12.7. 42 43 44 (3aR,4R,6R,6aR)-4-((1R,2R)-2-ethynylcyclopropyl)-6-methoxy-2,2-dimethyltetra- 45 46 47 hydrofuro[3,4-d][1,3]dioxole (S30). To a solution of aldehyde S29 (182 mg, 0.751 mmol) 48 49 50 in MeOH (4.0 mL) at RT was added K2CO3 (312 mg, 2.26 mmol, 3.0 equiv.) followed by a 51 52 solution of dimethyl-1-diazo-2-oxopropyl phosphonate (180 mg, 0.939 mmol, 1.25 equiv.) 53 54 55 96 56 57 58 59 60 ACS Paragon Plus Environment

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1 in MeOH (3.0 mL) under vigorous stirring. The resulting yellow suspension was stirred at 2 3 RT for 3 h. The reaction was quenched by the addition of a NH4Cl sat. aq. solution (10 mL) 4 5

6 and CH2Cl2 (15 mL) was added. The layers were separated and the aqueous phase was ex- 7 8 × 9 tracted with CH2Cl2 (3 15 mL). The combined organic extracts were dried over MgSO4, 10 11 filtered and concentrated. Crude material was purified by silica gel chromatography (cy- 12 13 14 clohexane/EtOAc, 0 → 50%) to afford alkyne S30 (98 mg, 0.41 mmol, 55 %) as a colorless 15 16 17 oil. Alkyne S30 crystallized when stored as a neat oil at –20 °C for 1 week. A small mole- 18 19 cule X-ray structure of S30 was obtained, confirming its absolute configuration as shown 20 21 ν 22 in the Supporting Information. Rf = 0.68 (cyclohexane/EtOAc, 67:33); FTIR (thin film), max 23 24 25 (cm-1): 3283, 2989, 2935, 2834, 2116, 1458, 1407, 1382, 1373, 1307, 1273, 1233, 1209, 26

27 1 3 28 1194, 1159, 1104, 1090, 1056, 1034, 1023; H NMR (600 MHz, CDCl ): δ 4.99 (s, 1H), 29 30 4.73 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 5.9 Hz, 1H), 3.42 (s, 3H), 3.39–3.34 (m, 1H), 1.80 31 32 33 (d, J = 2.0 Hz, 1H), 1.45 (s, 3H), 1.44–1.38 (m, 1H), 1.38–1.32 (m, 1H), 1.3 (s, 3H), 0.94 34 35 13 36 (app dt, J = 8.5, 5.0 Hz, 1H), 0.79–0.74 (m, 1H); C NMR (151 MHz, CDCl3): δ 112.4, 37 38 109.1, 90.5, 85.9, 85.6, 84.1, 64.8, 54.6, 26.5, 26.1, 25.0, 13.2, 6.1; HRMS (ESI+): calcd. 39 40 + ∆ 41 for [C13H19O4] 239.1278, meas. 239.1288, 4.4 ppm. 42 43 44 (3R,4R,5R)-5-((1R,2R)-2-ethynylcyclopropyl)tetrahydrofuran-2,3,4-triyl triacetate 45 46 47 (S31). To a vial charged with acetonide S30 (97 mg, 0.41 mmol) was added a 4:1 mixture 48 49 of acetic acid and water (5.0 mL) at RT. The resulting mixture was stirred at 80 °C for 6 h. 50 51 52 Volatiles were removed in vacuo and the residue was dried by azeotropic distillation with 53 54 55 97 56 57 58 59 60 ACS Paragon Plus Environment

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× 1 benzene (2 1.0 mL). Crude material was used directly in the next step without further 2 3 purification. To a solution of the crude triol in CH2Cl2 (5.0 mL) at RT, were added pyridine 4 5

6 (0.99 mL, 12 mmol, 30 equiv.), Ac2O (0.50 mL, 5.3 mmol, 13 equiv.) and DMAP (10 mg, 7 8 9 0.082 mmol, 0.20 equiv.) sequentially. The resulting mixture was stirred at RT for 2 h and 10 11 volatiles were removed in vacuo. Crude material was purified by silica gel chromatography 12 13 14 (2,2,4-trimethylpentane/EtOAc, 0 → 100%) to afford triacetate S31 (63 mg, 0.20 mmol, 15 16 17 50% over 2 steps) as a 4:5 mixture of α and β anomers, as a colorless oil. Rf = 0.60 (α- 18

19 ν -1 anomer) / 0.74 (β-anomer) (cyclohexane/EtOAc, 50:50); FTIR (thin film), max (cm ): 3284, 20 21 1 22 2580, 1742, 1431, 1370, 1210, 1103, 1044, 1009; H NMR (400 MHz, CDCl3): δ α-anomer: 23 24 25 6.38 (dd, J = 3.9, 0.8 Hz, 1H), 5.26–5.19 (m, 2H), 3.85 (dd, J = 6.8, 2.9 Hz, 1H), 2.12 (s, 26 27 J J 28 3H), 2.09 (s, 3H), 2.07 (s, 3H), 1.82 (d, = 2.1 Hz, 1H), 1.42 (dddd, = 8.8, 6.7, 6.0, 4.5 29 30 Hz, 1H), 1.31 (app. dtd, J = 9.5, 5.0, 2.1 Hz, 1H), 0.97 (app. dt, J = 8.7, 5.1 Hz, 1H), 0.91 31 32 33 (ddd, J = 8.9, 6.1, 4.8 Hz, 1H); β-anomer: 6.12 (d, J = 1.1 Hz, 1H), 5.33 (dd, J = 4.8, 1.1 34 35 36 Hz, 1H), 5.30 (dd, J = 6.6, 4.8 Hz, 1H), 3.75 (t, J = 6.8 Hz, 1H), 2.12 (s, 3H), 2.10 (s, 3H), 37 38 2.07 (s, 3H), 1.82 (d, J = 2.1 Hz, 1H), 1.39 (dddd, J = 8.8, 7.0, 6.0, 4.4 Hz, 1H), 1.37–1.26 39 40 41 (m, 1H), 0.95 (app. dt, J = 8.7, 5.0 Hz, 1H), 0.85 (ddd, J = 8.8, 6.0, 4.8 Hz, 1H); 13C NMR 42 43 44 (151 MHz, CDCl3): δ α-anomer: 170.1, 169.7, 169.4, 93.9, 85.2, 84.5, 72.6, 70.0, 65.3, 45 46 23.7, 21.1, 20.8, 20.4, 11.7, 4.6; β-anomer: 169.8, 169.5, 169.3, 98.1, 82.8, 74.6, 73.8, 65.1, 47 48 + 49 55.5, 24.0, 21.2, 20.7, 20.6, 11.7, 4.4; HRMS (ESI+): calcd. for [C15H18O7Na] 333.0945, 50 51 52 meas. 333.0970, ∆ 7.5 ppm. 53 54 55 98 56 57 58 59 60 ACS Paragon Plus Environment

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R R R R H R R 1 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-((1 ,2 )-2-ethynylcyclopro- 2 3 pyl)tetrahydrofuran-3,4-diyl diacetate (S32). To a suspension of N6-benzoyladenine 4 5 6 (114 mg, 0.478 mmol, 2.15 equiv.) in propionitrile (dried over 4 Å M.S., 6.0 mL) at RT, 7 8 9 was added N,O-bis(trimethylsilyl)acetamide (0.16 mL, 0.67 mmol, 3.00 equiv.). The re- 10 11 sulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N6- 12 13 14 benzoyladenine was observed. To a separate flask charged with triacetate S31 (dried by 15 16 × 17 azeotropic distillation with benzene (4 ), 69 mg, 0.22 mmol) and 2,6-di-tert-butyl-4- 18 19 methylpyridinium triflate (16 mg, 44 µmol, 20 mol %) was added propionitrile (6.0 mL). 20 21 22 The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and 23 24 25 the solution of silylated N6-benzoyladenine was added. The reaction mixture was stirred at 26 27 95 °C for 2.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was 28 29 30 filtered through a short pad of silica gel (EtOAc/i-PrOH, 90:10) to afford alkyne S32, 31 32 33 which was further purified by preparative HPLC (5% → 95% B’ over 40 min) to afford 34 35 36 alkyne S32 (63 mg, 0.13 mmol, 58%) as a white amorphous solid. Rf = 0.19 (EtOAc); FTIR 37 38 ν -1 (thin film), max (cm ): 3295, 3020, 2931, 1748, 1696, 1650, 1612, 1583, 1512, 1488, 1458, 39 40 1 41 1375, 1240, 1215, 1072, 1050; H NMR (500 MHz, CDCl3): δ 8.80 (s, 1H), 8.24 (s, 1H), 42 43 44 8.07–8.02 (m, 2H), 7.65–7.59 (m, 1H), 7.56–7.50 (m, 2H), 6.13 (d, J = 5.4 Hz, 1H), 6.07 45 46 (app t, J = 5.4 Hz, 1H), 5.66 (dd, J = 5.4, 4.3 Hz, 1H), 3.68 (dd, J = 8.3, 4.3 Hz, 1H), 2.15 47 48 49 (s, 3H), 2.08 (s, 3H), 1.83 (d, J = 2.1 Hz, 1H), 1.78–1.71 (m, 1H), 1.45–1.39 (m, 1H), 1.06 50 51 52 (app dt, J = 8.8, 5.2 Hz, 1H), 0.92 (ddd, J = 8.8, 5.8, 5.0 Hz, 1H); 13C NMR (126 MHz, 53 54 55 99 56 57 58 59 60 ACS Paragon Plus Environment

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1 CDCl3): δ 169.7, 169.5, 165.1, 152.0, 151.9, 149.7, 142.5, 133.2, 133.1, 129.0, 128.3, 2 3 123.7, 87.1, 85.2, 84.9, 73.6, 73.2, 65.5, 23.6, 20.7, 20.5, 12.1, 5.3; HRMS (ESI+): calcd. 4 5 + ∆ 6 for [C25H24N5O6] 490.1721, meas. 490.1725, 0.8 ppm. 7 8 9 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((1R,2R)-2-((3-car- 10 11 12 bamoylphenyl)ethynyl)cyclopropyl)tetrahydrofuran-3,4-diyl diacetate (S33). To a 13 14 vial charged with alkyne S32 (48 mg, 98 µmol) were added 3-iodobenzamide (61 mg, 0.25 15 16

17 mmol, 2.5 equiv.), CuI (5 mg, 0.03 mmol, 25 mol %) and Pd(PPh3)4 (6 mg, 5 µmol, 5 mol 18 19 20 %). The vial headspace was purged with nitrogen for 10 min and a 5:1:1 mixture of toluene, 21 22 DMF and i-Pr2NEt (degassed by sparging with nitrogen for 15 min, 5.0 mL) was added at 23 24 25 RT. The resulting mixture was stirred at 60 °C for 1.5 h. The solution was allowed to cool 26 27 28 to RT and volatiles were removed in vacuo. Crude material was filtered through a short 29 30 pad of silica gel (EtOAc/i-PrOH, 75:25) to afford benzamide S33, which was further puri- 31 32 33 fied by preparative HPLC (15% → 95% B’ over 30 min) to afford benzamide S33 (50 mg, 34 35 36 82 µmol, 84%) as a white amorphous solid. Rf = 0.35 (EtOAc/i-PrOH, 95:5); FTIR (thin 37 38 ν -1 39 film), max (cm ): 3351, 3199, 3067, 2934, 1748, 1664, 1611, 1583, 1514, 1457, 1375, 1241, 40 41 1 1215, 1072; H NMR (500 MHz, CDCl3): δ 8.80 (s, 1H), 8.35 (s, 1H), 8.03–7.99 (m, 2H), 42 43 44 7.76–7.74 (m, 1H), 7.74–7.71 (m, 1H), 7.62–7.57 (m, 1H), 7.52–7.46 (m, 3H), 7.33 (t, J = 45 46 47 7.7 Hz, 1H), 6.88 (br s, 1H), 6.80 (br s, 1H), 6.28 (dd, J = 5.8, 5.3 Hz, 1H), 6.17 (d, J = 5.8 48 49 Hz, 1H), 5.70 (dd, J = 5.3, 3.5 Hz, 1H), 3.69 (dd, J = 9.0, 3.5 Hz, 1H), 2.17 (s, 3H), 2.08 50 51 52 (s, 3H), 2.00–1.94 (m, 1H), 1.61–1.56 (m, 1H), 1.17 (app dt, J = 8.7, 5.2 Hz, 1H), 1.02 53 54 55 100 56 57 58 59 60 ACS Paragon Plus Environment

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13 1 (app dt, J = 8.7, 5.4 Hz, 1H); C NMR (126 MHz, CDCl3): δ 170.2, 169.8, 169.6, 165.4, 2 3 151.9, 151.8, 149.6, 143.2, 135.4, 133.4, 132.8, 132.7, 130.7, 129.0, 128.9, 128.3, 127.2, 4 5 6 124.1, 123.6, 92.1, 87.5, 86.3, 76.3, 74.1, 72.9, 24.3, 20.8, 20.5, 13.0, 6.4; HRMS (ESI+): 7 8 + ∆ 9 calcd. for [C32H29N6O7] 609.2092, meas. 609.2114, 3.6 ppm. 10 11 S S R R R R 12 Methyl (2 ,5 )-7-(3-carbamoylphenyl)-5-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dime- 13 14 thyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept- 15 16 17 6-ynoate (S34). To a vial charged with alkyne S3 (140 mg, 0.320 mmol) were added 3- 18 19 20 iodobenzamide (197 mg, 0.799 mmol, 2.5 equiv.), CuI (15 mg, 80 µmol, 25 mol %) and 21 22 Pd(PPh3)4 (19 mg, 16 µmol, 5 mol %). The vial headspace was purged with nitrogen for 5 23 24

25 min and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by sparging with nitrogen 26 27 28 for 15 min, 7.1 mL) was added at RT. The resulting mixture was stirred at 60 °C for 1 h. 29 30 The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude mate- 31 32 33 rial was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 0 → 100%) 34 35 36 to afford alkyne S34 (72 mg, 0.13 mmol, 40%) as a colorless solid. Rf = 0.43 (EtOAc); 37 38 ν -1 39 FTIR (thin-film), max (cm ): 3307, 3066, 2935, 2253, 1719, 1665, 1612, 1575, 1438, 1382, 40 41 1 1274, 1209, 1158; H NMR (500 MHz, CDCl3): δ 7.83 (td, J = 1.8, 0.6 Hz, 1H), 7.75 (ddd, 42 43 44 J = 7.8, 1.9, 1.2 Hz, 1H), 7.51 (dt, J = 7.7, 1.4 Hz, 1H), 7.41–7.30 (m, 2H), 6.47 (br. s, 1H), 45 46 47 5.97 (br. s, 1H), 4.95 (s, 1H), 4.69 (td, J = 7.6, 5.2 Hz, 1H), 4.61 (s, 2H), 4.50 (dd, J = 10.8, 48 49 4.2 Hz, 1H), 3.80 (s, 3H), 3.34 (s, 3H), 2.87 (ddt, J = 10.6, 9.0, 4.4 Hz, 1H), 2.26–2.04 (m, 50 51 13 52 2H), 1.81–1.50 (m, 4H), 1.48 (s, 3H), 1.31 (s, 3H); C NMR (126 MHz, CDCl3): δ 171.5, 53 54 55 101 56 57 58 59 60 ACS Paragon Plus Environment

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2 1 1 168.8, 157.2 (q, JC-F = 38 Hz), 134.9, 133.5, 130.8, 128.7, 127.2, 123.9, 115.8 (q, JC-F = 288 2 3 Hz), 112.5, 110.2, 91.7, 85.6, 85.3, 84.5, 83.0, 55.4, 53.2, 52.7, 40.5, 30.9, 29.7, 29.4, 26.6, 4 5 19 6 25.0; F NMR (376 MHz, CDCl3, BTF IStd): δ –76.8; HRMS (ESI+): calcd. for 7 8 + ∆ 9 [C26H31F3N2O8Na] 579.1925, meas. 579.1928, 0.5 ppm. 10 11 S S R S R R 12 Methyl (2 ,5 )-7-(3-carbamoylphenyl)-5-(((2 ,3 ,4 ,5 )-3,4-dihydroxy-5-methox- 13 14 ytetrahydrofuran-2-yl)methyl)-2-(2,2,2-trifluoroacetamido)hept-6-ynoate (S35). To a 15 16 17 vial charged with acetonide S34 (38 mg, 68 µmol) was added a 2:1 mixture of MeOH and 18 19 20 TFA (6.0 mL) at RT. The vial was sealed tightly and the reaction mixture was stirred at 85 21 22 °C for 6 h. Volatiles were removed in vacuo and the residue was purified by silica gel 23 24 25 chromatography (EtOAc/i-PrOH, 0 → 15%) to afford, in order of elution, β-anomer S35 26 27 28 (15 mg, 29 µmol, 42%) and the α-anomer counterpart (14 mg, 27 µmol, 40%) as off-white 29 30 solids. Only β-anomer S35 was used in the subsequent step. Rf = 0.37 (β-anomer) 31 32 ν -1 33 (EtOAc/MeOH, 91:9); FTIR (thin-film), max (cm ): 3349, 3076, 2921, 1719, 1665, 1613, 34 35 36 1598, 1573, 1438, 1383, 1250, 1211, 1180, 1161, 1127, 1103, 1063, 1029; 1H NMR (600 37 38 39 MHz, (CD3)2CO): δ β-anomer: 8.82 (d, J = 8.1 Hz, 1H), 7.96 (t, J = 1.8 Hz, 1H), 7.88 (dt, 40 41 J = 7.9, 1.4 Hz, 1H), 7.56 (dt, J = 7.7, 1.4 Hz, 1H), 7.54 (br. s, 1H), 7.44 (t, J = 7.7 Hz, 42 43 44 1H), 6.70 (br. s, 1H), 4.74 (s, 1H), 4.62 (ddd, J = 9.6, 8.0, 4.9 Hz, 1H), 4.23 (d, J = 4.1 Hz, 45 46 47 1H), 4.18 (ddd, J = 10.5, 6.5, 2.8 Hz, 1H), 4.12 (d, J = 7.2 Hz, 1H), 3.98 (td, J = 6.7, 4.8 48 49 Hz, 1H), 3.91 (t, J = 4.4 Hz, 1H), 3.74 (s, 3H), 3.29 (s, 3H), 2.98 (ddd, J = 15.3, 9.5, 4.7 50 51 52 Hz, 1H), 2.23 (dddd, J = 13.7, 10.9, 6.2, 4.8 Hz, 1H), 2.15 (ddt, J = 13.8, 9.6, 5.0 Hz, 1H), 53 54 55 102 56 57 58 59 60 ACS Paragon Plus Environment

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1 1.91 (ddd, J = 13.4, 10.6, 2.9 Hz, 1H), 1.80 (dddd, J = 13.1, 10.0, 6.2, 4.9 Hz, 1H), 1.75– 2 3 13 1.67 (m, 1H), 1.65 (ddd, J = 13.3, 10.6, 4.4 Hz, 1H); C NMR (101 MHz, (CD3)2CO): δ β- 4 5 6 anomer: 171.9, 168.3, 135.7, 135.1, 131.5, 129.4, 127.9, 124.9, 109.6, 93.3, 83.1, 82.0, 7 8 19 9 76.4, 76.2, 54.9, 53.6, 52.9, 42.1, 32.3; F NMR (471 MHz, CDCl3 (500 µL)/(CD3)2CO (50 10

11 + µL), BTF IStd): δ β-anomer: –76.6; HRMS (ESI+): calcd. for [C23H28F3N2O8] 517.1792, 12 13 14 meas. 517.1788, ∆ 0.8 ppm. 15 16 17 Methyl (S,E)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 18 19 d 20 ][1,3]dioxol-4-yl)methyl)-7-(triisopropylsilyl)hept-2-en-6-ynoate (S36). To a solution 21 22 of 5 (728 mg, 1.71 mmol) in PhMe (16 mL) at RT, was added methyl (triphenylphospho- 23 24 25 ranylidene)acetate (1.06 g, 3.18 mmol, 1.85 equiv.) in one portion. The resulting mixture 26 27 28 was stirred at 50 °C for 2 h and volatiles were removed in vacuo. Crude material was 29 30 purified by silica gel chromatography (hexanes/EtOAc, 2 → 40%) to afford S36 (747 mg, 31 32

33 1.55 mmol, 91%) as a 13:1 mixture of E and Z isomers, as a colorless oil. Rf = 0.35 (hex- 34 35 ν -1 36 anes/Et2O, 70:30); FTIR (thin film), max (cm ): 2942, 2865, 2169, 1726, 1660, 1463, 1436, 37

38 1 39 1381, 1371, 1270, 1210, 1159, 1093, 1060; H NMR (600 MHz, CDCl3): δ 7.00 (app dt, J 40 41 = 15.6, 7.3 Hz, 1H), 5.88 (app dt, J = 15.6, 1.4 Hz, 1H), 4.93 (s, 1H), 4.61 (dd, J = 5.9, 1.1 42 43 44 Hz, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.45 (ddd, J = 10.6, 4.3, 1.1 Hz, 1H), 3.71 (s, 3H), 3.31 45 46 47 (s, 3H), 2.80 (dddd, J = 11.2, 7.5, 6.0, 3.7 Hz, 1H), 2.43–2.34 (m, 2H), 1.69 (ddd, J = 13.3, 48 49 10.6, 3.7 Hz, 1H), 1.60 (ddd, J = 13.3, 11.2, 4.3 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H), 1.06– 50 51 13 52 1.02 (m, 21H); C NMR (126 MHz, CDCl3): δ 166.7, 145.9, 123.3, 112.5, 109.8, 109.3, 53 54 55 103 56 57 58 59 60 ACS Paragon Plus Environment

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1 85.6, 85.5, 84.5, 83.7, 55.2, 51.5, 40.6, 38.4, 29.7, 26.7, 25.4, 18.8, 11.3; HRMS (ESI+): 2 3 + ∆ calcd. for [C26H44O6Si1Na] 503.2799, meas. 503.2787, 2.5 ppm. 4 5 6 Methyl (S)-5-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 7 8 9 d][1,3]dioxol-4-yl)methyl)-7-(triisopropylsilyl)hept-6-ynoate (S37). To a vial charged 10 11 12 with S36 (747 mg, 1.55 mmol) were added PhMe (degassed by sparging with nitrogen for 13 14 15 min, 16 mL) and t-BuOH (degassed by sparging with nitrogen for 15 min, 0.45 mL, 4.7 15 16 17 mmol, 3.0 equiv.) at RT, followed by the addition of (BDP)CuH (1.0 M in PhMe, 3.1 mL, 18 19 20 3.1 mmol, 2.0 equiv.). The resulting mixture was stirred for 4.5 h and volatiles were re- 21 22 moved in vacuo. Crude material was purified by silica gel chromatography (hex- 23 24

25 anes/EtOAc, 1 → 25%) to afford S37 (750 mg, 1.55 mmol, quant.) as a colorless oil. Rf = 26 27 ν -1 28 0.37 (hexanes/EtOAc, 80:20); FTIR (thin film), max (cm ): 2942, 2865, 2165, 1741, 1462, 29

30 1 1381, 1371, 1209, 1160, 1092, 1059; H NMR (600 MHz, CDCl3): δ 4.93 (s, 1H), 4.63 (dd, 31 32 33 J = 5.9, 1.1 Hz, 1H), 4.57 (d, J = 5.9 Hz, 1H), 4.46 (ddd, J = 10.4, 4.6, 1.1 Hz, 1H), 3.65 34 35 36 (s, 3H), 3.31 (s, 3H), 2.63 (app dtd, J = 11.0, 7.1, 3.7 Hz, 1H), 2.38–2.29 (m, 2H), 1.90– 37 38 39 1.82 (m, 1H), 1.82–1.73 (m, 1H), 1.67 (ddd, J = 13.3, 10.4, 3.7 Hz, 1H), 1.59 (ddd, J = 40 41 13.3, 11.0, 4.6 Hz, 1H), 1.51–1.46 (m, 2H), 1.46 (s, 3H), 1.30 (s, 3H), 1.07–1.03 (m, 21H); 42 43 13 44 C NMR (126 MHz, CDCl3): δ 173.9, 112.4, 110.5, 109.8, 85.8, 85.6, 84.6, 82.8, 55.2, 45 46 47 51.6, 41.1, 35.2, 33.9, 30.0, 26.7, 25.4, 22.8, 18.8, 11.4; HRMS (ESI+): calcd. for 48 49 + ∆ [C26H46O6Si1Na] 505.2956, meas. 505.2961, 0.9 ppm. 50 51 52 53 54 55 104 56 57 58 59 60 ACS Paragon Plus Environment

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S R R R R 1 Methyl ( )-5-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 2 3 d][1,3]dioxol-4-yl)methyl)hept-6-ynoate (S38). To a solution of TIPS alkyne S37 (750 4 5 6 mg, 1.55 mmol) in THF (16 mL) at 0 °C, was added TBAF (1.0 M in THF, 1.94 mL, 1.94 7 8 9 mmol, 1.25 equiv.) slowly. The resulting mixture was stirred at RT for 1 h, followed by the 10 11 addition of water (15 mL). The layers were separated and the aqueous phase was extracted 12 13 × 14 with Et2O (3 15 mL). The combined organic extracts were dried over MgSO4, filtered and 15 16 17 concentrated. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 18 19 1 → 30%) to afford alkyne S38 (468 mg, 1.43 mmol, 92%) as a colorless oil. Rf = 0.35 20 21 ν -1 22 (hexanes/EtOAc, 75:25); FTIR (thin film), max (cm ): 3273, 2959, 2926, 1738, 1438, 1373, 23 24 1 25 1258, 1089, 1014; H NMR (600 MHz, CDCl3): δ 4.93 (s, 1H), 4.59 (d, J = 6.0 Hz, 1H), 26 27 J J 28 4.57 (dd, = 6.0, 0.8 Hz, 1H), 4.48 (ddd, = 11.1, 3.9, 0.8 Hz, 1H), 3.66 (s, 3H), 3.32 (s, 29 30 3H), 2.64–2.57 (m, 1H), 2.37–2.29 (m, 2H), 2.11 (d, J = 2.4 Hz, 1H), 1.88–1.80 (m, 1H), 31 32 33 1.79–1.72 (m, 1H), 1.69 (ddd, J = 13.3, 11.2, 3.9 Hz, 1H), 1.56 (ddd, J = 13.3, 11.1, 4.0 34 35 13 36 Hz, 1H), 1.52–1.48 (m, 2H), 1.48 (s, 3H), 1.30 (s, 3H); C NMR (126 MHz, CDCl3): δ 37 38 173.9, 112.4, 110.1, 85.9, 85.7, 85.2, 84.6, 70.9, 55.3, 51.6, 40.4, 34.8, 33.8, 28.7, 26.6, 39 40 + ∆ 41 25.2, 22.6; HRMS (ESI+): calcd. for [C17H26O6Na] 349.1622, meas. 349.1621, 0.3 ppm. 42 43 44 (3R,4R,5R)-5-((S)-2-ethynyl-6-methoxy-6-oxohexyl)tetrahydrofuran-2,3,4-triyl tri- 45 46 47 acetate (S39). To a vial charged with acetonide S38 (468 mg, 1.43 mmol) at 0 °C, was 48 49 added a 4:1 mixture of TFA and water (10 mL). The resulting mixture was stirred at RT for 50 51 52 5.5 h. Volatiles were removed in vacuo and the residue was dried by azeotropic distillation 53 54 55 105 56 57 58 59 60 ACS Paragon Plus Environment

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× 1 with benzene (2 2 mL). Crude material was used directly in the next step without further 2 3 purification. To a solution of the crude triol in CH2Cl2 (14 mL) at RT, were added pyridine 4 5

6 (1.16 mL, 14.3 mmol, 10 equiv.), Ac2O (1.36 mL, 14.3 mmol, 10 equiv.) and DMAP (123 7 8 9 mg, 1.00 mmol, 0.70 equiv.) sequentially. The resulting mixture was stirred at RT for 1 h 10 11 and volatiles were removed in vacuo. Crude material was purified by silica gel chromatog- 12 13 14 raphy (hexanes/EtOAc, 2 → 60%) to afford triacetate S39 (200 mg, 0.500 mmol, 35% over 15 16 17 2 steps) as a 2:1 mixture of diastereomers, as a colorless oil; Rf = 0.27/0.32 (hex- 18

19 ν -1 anes/EtOAc, 60:40); FTIR (thin film), max (cm ): 3280, 2952, 2923, 1739, 1436, 1371, 20 21 1 22 1216, 1110, 1011; H NMR (600 MHz, CDCl3): δ 6.35 (d, J = 4.6 Hz, 1H), 6.13 (d, J = 1.1 23 24 25 Hz, 2H), 5.31 (dd, J = 4.8, 1.1 Hz, 2H), 5.22 (dd, J = 6.8, 4.6 Hz, 1H), 5.18 (dd, J = 6.9, 26 27 J J 28 4.8 Hz, 2H), 5.06 (dd, = 6.8, 3.7 Hz, 1H), 4.46 (app dt, = 10.3, 3.7 Hz, 1H), 4.40 (ddd, 29 30 J = 10.3, 6.9, 3.1 Hz, 2H), 3.66 (s, 9H), 2.63–2.57 (m, 3H), 2.37–2.28 (m, 6H), 2.11 (s, 31 32 33 6H), 2.11 (s, 3H), 2.10 (s, 3H), 2.07 (s, 6H), 2.06 (s, 6H), 2.05 (s, 3H), 1.89–1.80 (m, 3H), 34 35 36 1.80– 1.69 (m, 6H), 1.65 (ddd, J = 13.9, 10.3, 4.2 Hz, 2H), 1.62 (ddd, J = 13.4, 10.3, 4.0 37

38 13 Hz, 1H), 1.55–1.45 (m, 6H); C NMR (126 MHz, CDCl3): δ 173.9, 173.9, 170.2, 170.0, 39 40 41 169.8, 169.6, 169.5, 169.2, 98.5, 93.9, 85.7, 85.4, 81.5, 79.9, 74.7, 74.0, 72.7, 71.1, 70.9, 42 43 44 69.9, 51.7, 40.2, 39.2, 34.7, 34.7, 33.8, 33.8, 28.5, 28.2, 22.5, 22.5, 21.2, 21.2, 20.8, 20.7, 45 46 + ∆ 20.7, 20.4; HRMS (ESI+): calcd. for [C19H26O9Na] 421.1469, meas. 421.1490, 4.9 ppm. 47 48 49 50 51 52 53 54 55 106 56 57 58 59 60 ACS Paragon Plus Environment

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R R R R H S 1 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-(( )-2-ethynyl-6-methoxy-6- 2 3 oxohexyl)tetrahydrofuran-3,4-diyl diacetate (S40). To a suspension of N6-benzoylade- 4 5 6 nine (240 mg, 1.00 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 4.2 mL) at RT, 7 8 9 was added N,O-bis(trimethylsilyl)acetamide (0.31 mL, 1.3 mmol, 5.0 equiv.). The result- 10 11 ing mixture was stirred at 80 °C for 10 min, upon which complete solubilization of N6- 12 13 14 benzoyladenine was observed. To a separate flask charged with triacetate S39 (dried by 15 16 × 17 azeotropic distillation with benzene (4 ), 100 mg, 0.251 mmol) and 2,6-di-tert-butyl-4- 18 19 methylpyridinium triflate (45 mg, 0.13 mmol, 50 mol %) was added propionitrile (4.2 mL). 20 21 22 The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and 23 24 25 the solution of silylated N6-benzoyladenine was added. The reaction mixture was stirred at 26 27 95 °C for 2.5 h, cooled to RT and the volatiles were removed in vacuo. Crude material was 28 29 30 purified by silica gel chromatography (PhMe/MeCN, 2 → 60%) to afford nucleoside S40 31 32 ν - 33 (102 mg, 0.177 mmol, 70%) as a colorless oil. Rf = 0.44 (EtOAc); FTIR (thin film), max (cm 34 35 1 36 ): 3285, 2952, 2925, 2855, 1744, 1703, 1610, 1582, 1510, 1486, 1455, 1241, 1219, 1096, 37

38 1 1074, 1047; H NMR (600 MHz, CDCl3): δ 8.98 (br s, 1H), 8.80 (s, 1H), 8.07 (s, 1H), 8.04– 39 40 41 8.00 (m, 2H), 7.63–7.59 (m, 1H), 7.55–7.50 (m, 2H), 6.14–6.11 (m, 2H), 5.59–5.54 (m, 42 43 44 1H), 4.53 (ddd, J = 11.0, 4.4, 2.8 Hz, 1H), 3.66 (s, 3H), 2.59–2.51 (m, 1H), 2.35–2.25 (m, 45 46 2H), 2.16 (s, 3H), 2.16–2.10 (m, 1H), 2.13 (d, J = 2.4 Hz, 1H), 2.07 (s, 3H), 1.88–1.80 (m, 47 48 13 49 2H), 1.75–1.67 (m, 1H), 1.55–1.48 (m, 2H); C NMR (126 MHz, CDCl3): δ 173.8, 169.8, 50 51 52 169.5, 164.7, 152.9, 151.7, 149.9, 142.4, 133.6, 133.0, 129.0, 128.0, 124.1, 87.2, 85.5, 53 54 55 107 56 57 58 59 60 ACS Paragon Plus Environment

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1 81.0, 73.8, 72.9, 71.2, 51.7, 38.4, 34.7, 33.8, 28.1, 22.5, 20.8, 20.5; HRMS (ESI+): calcd. 2 3 + ∆ for [C29H32N5O8] 578.2245, meas. 578.2242, 0.5 ppm. 4 5 6 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-((3-car- 7 8 9 bamoylphenyl)ethynyl)-6-methoxy-6-oxohexyl)tetrahydrofuran-3,4-diyl diacetate 10 11 12 (S41). To a solution of alkyne S40 (102 mg, 0.177 mmol) and 3-iodobenzamide (131 mg, 13 14 0.530 mmol, 3.0 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by 15 16 17 sparging with nitrogen for 15 min, 9.0 mL) at RT, were introduced CuI (8 mg, 0.04 mmol, 18 19 20 25 mol %) and Pd(PPh3)4 (10 mg, 8.8 µmol, 5 mol %) rapidly. The resulting mixture was 21 22 stirred at 60 °C for 2.5 h. The solution was allowed to cool to RT and volatiles were re- 23 24 25 moved in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i- 26 27 28 PrOH, 0 → 10%) to afford benzamide S41 (105 mg, 0.151 mmol, 85%) as a white amor- 29

30 ν -1 phous solid. Rf = 0.39 (EtOAc/i-PrOH, 95:5); FTIR (thin film), max (cm ): 3340, 3198, 2925, 31 32 33 2855, 1747, 1669, 1611, 1581, 1513, 1487, 1456, 1375, 1244, 1221, 1097, 1047; 1H NMR 34 35 36 (600 MHz, CDCl3): δ 9.12 (s, 1H), 8.79 (s, 1H), 8.11 (s, 1H), 8.03–8.00 (m, 2H), 7.87– 37 38 39 7.86 (m, 1H), 7.75 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.62–7.57 (m, 1H), 7.54–7.47 (m, 3H), 40 41 7.36 (app t, J = 7.8 Hz, 1H), 6.51 (br s, 1H), 6.18 (dd, J = 5.9, 5.2 Hz, 1H), 6.15 (d, J = 5.9 42 43 44 Hz, 1H), 5.98 (br s, 1H), 5.66 (dd, J = 5.2, 3.8 Hz, 1H), 4.53 (app dt, J = 10.0, 3.8 Hz, 1H), 45 46 47 3.65 (s, 3H), 2.76 (app dtd, J = 11.1, 7.0, 3.7 Hz, 1H), 2.39–2.29 (m, 2H), 2.20 (ddd, J = 48 49 13.6, 10.0, 3.7 Hz, 1H), 2.15 (s, 3H), 2.05 (s, 3H), 1.98 (ddd, J = 13.6, 11.1, 3.8 Hz, 1H), 50 51 13 52 1.92–1.84 (m, 1H), 1.81–1.73 (m, 1H), 1.63–1.57 (m, 2H); C NMR (126 MHz, CDCl3): δ 53 54 55 108 56 57 58 59 60 ACS Paragon Plus Environment

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1 173.9, 170.0, 169.7, 168.8, 164.8, 152.9, 151.8, 149.9, 142.4, 134.9, 133.6, 133.6, 133.0, 2 3 130.8, 129.0, 128.7, 128.0, 127.0, 124.3, 124.0, 92.3, 86.9, 82.5, 81.5, 74.0, 72.8, 51.7, 4 5 + 6 38.7, 35.0, 33.8, 29.0, 22.7, 20.8, 20.6; HRMS (ESI+): calcd. for [C36H36N6O9Na] 719.2436, 7 8 ∆ 9 meas. 719.2452, 2.3 ppm. 10 11 S E R R R R 12 Triisopropyl(( , )-3-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydro- 13 14 furo[3,4-d][1,3]dioxol-4-yl)methyl)-6-nitrohex-5-en-1-yn-1-yl)silane (S42). To a solu- 15 16

17 tion of aldehyde 5 (385 mg, 0.907 mmol) in MeNO2 (9 mL) at 0 °C, was added Et3N (0.13 18 19 20 mL, 0.91 mmol, 1.00 equiv.). The resulting mixture was stirred at RT for 17 h and volatiles 21 22 were removed in vacuo. To a solution of the residue in CH2Cl2 (9 mL) at –78 °C were added 23 24

25 i-Pr2NEt (0.40 mL, 2.3 mmol, 2.50 equiv.) and MsCl (90 µL, 1.1 mmol, 1.25 equiv.) se- 26 27 28 quentially. The resulting mixture was stirred at –78 °C for 2 h, before the addition of a 29 30 NH4Cl sat. aq. solution (8 mL). The biphasic mixture was allowed to warm to RT and the 31 32 × 33 layers were separated. The aqueous phase was extracted with CH2Cl2 (3 8 mL). The com- 34 35 36 bined organic extracts were dried over Na2SO4, filtered and concentrated. Crude material 37 38 39 was purified by silica gel chromatography (hexanes/EtOAc, 2 → 25%) to afford S42 (319 40 41 mg, 0.682 mmol, 75% over 2 steps) as a colorless oil. Rf = 0.46 (hexanes/EtOAc, 75:25); 42 43 ν -1 1 44 FTIR (thin film), max (cm ): 2941, 2865, 2168, 1655, 1528, 1463, 1351, 1210, 1092; H 45 46 47 NMR (600 MHz, CDCl3): δ 7.33 (ddd, J = 13.4, 7.9, 7.5 Hz, 1H), 7.06 (app dt, J = 13.4, 48 49 1.4 Hz, 1H), 4.95 (s, 1H), 4.62 (dd, J = 5.9, 1.1 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.46 50 51 52 (ddd, J = 10.6, 4.3, 1.1 Hz, 1H), 3.34 (s, 3H), 2.89 (dddd, J = 10.9, 7.9, 5.5, 4.0 Hz, 1H), 53 54 55 109 56 57 58 59 60 ACS Paragon Plus Environment

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1 2.49 (dddd, J = 14.6, 7.5, 5.5, 1.4 Hz, 1H), 2.41 (app dtd, J = 14.6, 7.9, 1.4 Hz, 1H), 1.72 2 3 (ddd, J = 13.3, 10.6, 4.0 Hz, 1H), 1.66 (ddd, J = 13.3, 10.9, 4.3 Hz, 1H), 1.47 (s, 3H), 1.31 4 5 13 6 (s, 3H), 1.07–1.03 (m, 21H); C NMR (126 MHz, CDCl3): δ 141.0, 139.2, 112.7, 110.0, 7 8 9 108.0, 85.5, 85.3, 85.1, 84.4, 55.4, 40.8, 34.4, 29.6, 26.7, 25.4, 18.7, 11.3; HRMS (ESI+): 10 11 + ∆ calcd. for [C24H41N1O6Si1Na] 490.2595, meas. 490.2580, 3.2 ppm. 12 13 14 2,2,2-trifluoro-N-((S)-4-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydro- 15 16 17 furo[3,4-d][1,3]dioxol-4-yl)methyl)-6-(triisopropylsilyl)hex-5-yn-1-yl)acetamide 18 19 20 (S43). To a solution of S42 (319 mg, 0.682 mmol) in THF (6.8 mL) at 0 °C, was added 21 22 LiAlH4 (1.0 M in Et2O, 4.8 mL, 4.8 mmol, 7.0 equiv.) dropwise. The resulting mixture was 23 24 25 stirred at RT for 4 h. The reaction was quenched by the dropwise addition of water (0.18 26 27 28 mL), a 15% NaOH aq. solution (0.18 mL) and water (0.54 mL), sequentially, at 0 °C and 29 30 Na2SO4 was added. The mixture was stirred vigorously for 20 min and filtered through a 31 32 33 cotton plug to remove the white solids. The filtrate was concentrated and the crude material 34 35 36 was used directly in the next step without further purification. To a solution of the crude 37 38 39 amine in THF (6.8 mL) at 0 °C, were added Et3N (5.7 mL, 41 mmol, 60 equiv.) and ethyl 40 41 trifluoroacetate (2.4 mL, 21 mmol, 30 equiv.) sequentially. The resulting mixture was 42 43 44 stirred at RT for 17 h and volatiles were removed in vacuo. Crude material was purified by 45 46 47 silica gel chromatography (hexanes/EtOAc, 2 → 50%) to afford trifluoroacetamide S43 48 49 (252 mg, 0.470 mmol, 69% over 2 steps) as a colorless oil. Rf = 0.47 (hexanes/EtOAc, 50 51 ν -1 52 65:35); FTIR (thin film), max (cm ): 3306, 2941, 2865, 2166, 1705, 1559, 1463, 1381, 1209, 53 54 55 110 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 1161, 1106, 1061; H NMR (600 MHz, CDCl3): δ 6.27 (br s, 1H), 4.94 (s, 1H), 4.62 (dd, J 2 3 = 5.9, 0.7 Hz, 1H), 4.58 (d, J = 5.9 Hz, 1H), 4.45 (ddd, J = 10.3, 4.7, 0.7 Hz, 1H), 3.44– 4 5 6 3.39 (m, 2H), 3.32 (s, 3H), 2.69–2.63 (m, 1H), 1.88–1.80 (m, 1H), 1.76 (app ddq, J = 13.4, 7 8 9 9.9, 6.7 Hz, 1H), 1.68 (ddd, J = 13.4, 10.3, 4.1 Hz, 1H), 1.62 (ddd, J = 13.4, 10.7, 4.7 Hz, 10 11 1H), 1.56–1.48 (m, 2H), 1.47 (s, 3H), 1.31 (s, 3H), 1.08–1.03 (m, 21H); 13C NMR (126 12 13 2 1 14 MHz, CDCl3): δ 157.3 (q, JC-F = 37 Hz), 116.0 (q, JC-F = 288 Hz), 112.5, 110.1, 109.8, 85.7, 15 16 19 17 85.6, 84.5, 83.4, 55.2, 41.2, 39.8, 32.8, 30.0, 26.9, 26.7, 25.4, 18.8, 11.3; F NMR (471 18

19 + MHz, CDCl3, BTF IStd): δ –76.9; HRMS (ESI+): calcd. for [C26H44F3N1O5Si1Na] 558.2833, 20 21 22 meas. 558.2858, ∆ 4.4 ppm. 23 24 25 2,2,2-trifluoro-N-((S)-4-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydro- 26 27 d 28 furo[3,4- ][1,3]dioxol-4-yl)methyl)hex-5-yn-1-yl)acetamide (S44). To a solution of 29 30 TIPS alkyne S43 (196 mg, 0.366 mmol) in DMF (3 mL) at RT, was added a solution of 31 32 33 TASF (0.30 g, 1.2 mmol, 3.0 equiv.) in DMF (4 mL). The resulting mixture was stirred at 34 35 36 60 °C for 2.5 h and volatiles were removed in vacuo. The residue was partitioned between 37 38 39 Et2O (7 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (7 mL). The layers were 40 41 × separated and the aqueous phase was extracted with Et2O (3 7 mL). The combined or- 42 43 44 ganic extracts were dried over MgSO4, filtered and concentrated. Crude material was puri- 45 46 47 fied by silica gel chromatography (hexanes/EtOAc, 5 → 50%) to afford alkyne S44 (117 48 49 g, 0.308 mmol, 84%) as an amorphous white solid. Rf = 0.32 (hexanes/EtOAc, 65:35); FTIR 50 51 ν -1 1 52 (thin film), max (cm ): 3298, 2937, 1706, 1558, 1454, 1382, 1209, 1159, 1104, 1059; H 53 54 55 111 56 57 58 59 60 ACS Paragon Plus Environment

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1 NMR (600 MHz, CDCl3): δ 6.43 (br s, 1H), 4.94 (s, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.57 2 3 (dd, J = 6.0, 0.9 Hz, 1H), 4.47 (ddd, J = 11.1, 4.0, 0.9 Hz, 1H), 3.42–3.37 (m, 2H), 3.32 (s, 4 5 6 3H), 2.66–2.60 (m, 1H), 2.14 (d, J = 2.4 Hz, 1H), 1.86–1.78 (m, 1H), 1.74 (app ddt, J = 7 8 9 14.0, 10.3, 7.0 Hz, 1H), 1.69 (ddd, J = 13.3, 11.1, 4.0 Hz, 1H), 1.59 (ddd, J = 13.3, 10.9, 10 11 4.0 Hz, 1H), 1.53–1.49 (m, 2H), 1.48 (s, 3H), 1.32–1.30 (s, 3H); 13C NMR (126 MHz, 12 13 2 1 14 CDCl3): δ 157.4 (q, JC-F = 37 Hz), 116.0 (q, JC-F = 288 Hz), 112.5, 110.1, 85.6, 85.6, 85.1, 15 16 19 17 84.5, 71.3, 55.3, 40.5, 39.8, 32.3, 28.6, 26.8, 26.6, 25.1; F NMR (471 MHz, CDCl3, BTF 18

19 + ∆ IStd): δ –76.9; HRMS (ESI+): calcd. for [C17H24F3N1O5Na] 402.1499, meas. 402.1510, 2.7 20 21 22 ppm. 23 24 25 (3R,4R,5R)-5-((S)-2-ethynyl-5-(2,2,2-trifluoroacetamido)pentyl)tetrahydrofuran- 26 27 28 2,3,4-triyl triacetate (S45). To a solution of S44 (117 mg, 0.308 mmol) in CH2Cl2 (1.5 mL) 29 30 at 0 °C, was added a 4:1 mixture of TFA and water (6.0 mL). The resulting mixture was 31 32 33 stirred at RT for 15 h. Volatiles were removed in vacuo and the residue was dried by azeo- 34 35 36 tropic distillation with benzene (2 × 2 mL). Crude material was used directly in the next 37 38 39 step without further purification. To a solution of the crude triol in CH2Cl2 (6.0 mL) at RT, 40 41 were added pyridine (0.37 mL, 4.6 mmol, 15.0 equiv.), Ac2O (0.44 mL, 4.6 mmol, 15.0 42 43 44 equiv.) and DMAP (39 mg, 0.32 mmol, 1.05 equiv.) sequentially. The resulting mixture 45 46 47 was stirred at RT for 2.5 h, before the addition of water (6 mL). The layers were separated 48 49 × and the aqueous phase was extracted with CH2Cl2 (3 6 mL). The combined organic ex- 50 51

52 tracts were dried over MgSO4, filtered and concentrated. Crude material was purified by 53 54 55 112 56 57 58 59 60 ACS Paragon Plus Environment

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1 silica gel chromatography (hexanes/EtOAc, 5 → 70%) to afford S45 (74 mg, 0.16 mmol, 2 3 53% over 2 steps) as a 1:1 mixture of diastereomers, as a colorless oil. Rf = 0.30/0.41 (hex- 4 5 ν -1 6 anes/EtOAc, 50:50); FTIR (thin film), max (cm ): 3288, 2933, 1749, 1720, 1558, 1435, 7 8 1 9 1373, 1218, 1180, 1158, 1012; H NMR (600 MHz, CDCl3): δ 6.70–6.62 (m, 2H), 6.34 (d, 10 11 J = 4.5 Hz, 1H), 6.10 (d, J = 0.9 Hz, 1H), 5.30 (dd, J = 4.8, 0.9 Hz, 1H), 5.21 (dd, J = 6.7, 12 13 14 4.5 Hz, 1H), 5.16 (dd, J = 7.0, 4.8 Hz, 1H), 5.04 (dd, J = 6.7, 3.7 Hz, 1H), 4.44 (app dt, J 15 16 17 = 10.1, 3.7 Hz, 1H), 4.36 (ddd, J = 10.1, 7.0, 3.0 Hz, 1H), 3.42– 3.33 (m, 4H), 2.66–2.59 18 19 (m, 2H), 2.13 (d, J = 2.4 Hz, 2H), 2.11 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 20 21 22 (s, 3H), 2.05 (s, 3H), 1.87–1.76 (m, 4H), 1.75–1.68 (m, 2H), 1.68–1.60 (m, 2H), 1.57–1.43 23 24 13 25 (m, 4H); C NMR (126 MHz, CDCl3): δ 170.3, 170.1, 169.9, 169.7, 169.5, 169.4, 157.4 (q, 26

27 2 2 1 1 JC-F JC-F JC-F JC-F 28 = 37 Hz), 157.4 (q, = 37 Hz), 116.0 (q, = 288 Hz), 116.0 (q, = 288 Hz), 98.5, 29 30 93.8, 85.5, 85.2, 81.5, 79.9, 74.5, 73.9, 72.7, 71.4, 71.2, 69.9, 40.1, 39.7, 39.6, 39.3, 32.1, 31 32 19 33 32.0, 28.5, 28.1, 26.6, 26.3, 21.2, 21.2, 20.8, 20.7, 20.6, 20.4; F NMR (471 MHz, CDCl3, 34 35 + 36 BTF IStd): δ –76.88, –76.90; HRMS (ESI+): calcd. for [C19H24F3N1O8Na] 474.1346, meas. 37 38 474.1358, ∆ 2.5 ppm. 39 40 41 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-ethynyl-5-(2,2,2-trifluoroa- 42 43 44 cetamido)pentyl)tetrahydrofuran-3,4-diyl diacetate (S46). To a suspension of N6-ben- 45 46 47 zoyladenine (72 mg, 0.30 mmol, 4.0 equiv.) in propionitrile (dried over 4 Å M.S., 1.5 mL) 48 49 at RT, was added N,O-bis(trimethylsilyl)acetamide (92 µL, 0.38 mmol, 5.0 equiv.). The 50 51 52 resulting mixture was stirred at 80 °C for 10 min, upon which complete solubilization of 53 54 55 113 56 57 58 59 60 ACS Paragon Plus Environment

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6 1 N -benzoyladenine was observed. To a separate flask charged with triacetate S45 (dried by 2 3 azeotropic distillation with benzene (4 ×), 34 mg, 75 µmol) and 2,6-di-tert-butyl-4- 4 5 6 methylpyridinium triflate (13 mg, 38 µmol, 50 mol %) was added propionitrile (1.5 mL). 7 8 9 The resulting mixture was stirred at RT for 5 min, until a clear solution was obtained, and 10 11 the solution of silylated N6-benzoyladenine was added. The reaction mixture was stirred at 12 13 14 95 °C for 4 h, cooled to RT and the volatiles were removed in vacuo. Crude material was 15 16 17 purified by silica gel chromatography (PhMe/MeCN, 0 → 50%) to afford nucleoside S46 18 19 (35 mg, 56 µmol, 74%) as a colorless oil. Rf = 0.31 (hexanes/EtOAc, 25:75); FTIR (thin 20 21 ν -1 22 film), max (cm ): 3289, 2928, 1749, 1708, 1612, 1583, 1457, 1245, 1218, 1159, 1076, 1049; 23 24 1 25 H NMR (600 MHz, CDCl3): δ 9.15 (s, 1H), 8.75 (s, 1H), 8.07 (s, 1H), 8.04–7.98 (m, 2H), 26 27 28 7.62–7.57 (m, 1H), 7.53–7.48 (m, 2H), 6.71–6.62 (m, 1H), 6.13–6.09 (m, 2H), 5.55 (app 29 30 t, J = 4.3 Hz, 1H), 4.49 (ddd, J = 10.8, 4.3, 3.1 Hz, 1H), 3.34 (m, 2H), 2.59–2.52 (m, 1H), 31 32 33 2.16 (s, 3H), 2.15 (d, J = 2.4 Hz, 1H), 2.12–2.06 (ddd, J = 13.5, 10.8, 4.2 Hz, 1H), 2.06 (s, 34 35 36 3H), 1.87 (ddd, J = 13.5, 11.2, 3.1 Hz, 1H), 1.82–1.74 (m, 1H), 1.67 (app tdd, J = 13.4, 37

38 13 8.5, 6.5 Hz, 1H), 1.56–1.45 (m, 2H); C NMR (126 MHz, CDCl3): δ 169.9, 169.6, 164.9, 39 40 2 41 157.4 (q, JC-F = 37 Hz), 152.8, 151.7, 149.9, 142.4, 133.5, 133.0, 129.0, 128.0, 124.1, 116.0 42 43 1 44 (q, JC-F = 288 Hz), 87.2, 85.2, 80.9, 73.7, 72.9, 71.5, 39.6, 38.5, 32.2, 28.0, 26.7, 20.8, 20.5; 45

46 19 + F NMR (471 MHz, CDCl3, BTF IStd): δ –76.9; HRMS (ESI+): calcd. for [C29H30F3N6O7] 47 48 49 631.2123, meas. 631.2142, ∆ 3.1 ppm. 50 51 52 53 54 55 114 56 57 58 59 60 ACS Paragon Plus Environment

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R R R R H S 1 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-(( )-2-((3-car- 2 3 bamoylphenyl)ethynyl)-5-(2,2,2-trifluoroacetamido)pentyl)tetrahydrofuran-3,4-diyl 4 5 6 diacetate (S47). To a solution of S46 (33 mg, 52 µmol) and 3-iodobenzamide (39 mg, 0.16 7 8 9 mmol, 3.0 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by sparging 10 11 with nitrogen for 15 min, 2.6 mL) at RT, were introduced CuI (3 mg, 0.01 mmol, 25 mol 12 13

14 %) and Pd(PPh3)4 (3 mg, 3 µmol, 5 mol %) rapidly. The resulting mixture was stirred at 60 15 16 17 °C for 2 h. The solution was allowed to cool to RT and volatiles were removed in vacuo. 18 19 Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 10%) to 20 21

22 afford alkyne S47 (32 mg, 43 µmol, 82%) as a white amorphous solid. Rf = 0.28 (EtOAc); 23 24 ν -1 25 FTIR (thin film), max (cm ): 3306, 2928, 1749, 1709, 1667, 1611, 1581, 1456, 1376, 1245, 26

27 1 3 28 1217, 1157, 1098, 1049; H NMR (500 MHz, CDCl ): δ 9.32 (s, 1H), 8.75 (s, 1H), 8.12 (s, 29 30 1H), 8.04–7.99 (m, 2H), 7.89–7.87 (m, 1H), 7.76 (ddd, J = 7.7, 1.9, 1.2 Hz, 1H), 7.61–7.56 31 32 33 (m, 1H), 7.52–7.47 (m, 3H), 7.34 (app td, J = 7.7, 0.5 Hz, 1H), 7.19–7.16 (m, 1H), 6.80 34 35 36 (br s, 1H), 6.18 (dd, J = 5.8, 5.2 Hz, 1H), 6.14 (d, J = 5.8 Hz, 1H), 6.08 (br s, 1H), 5.63 37 38 (dd, J = 5.2, 3.8 Hz, 1H), 4.51 (ddd, J = 10.2, 3.8, 3.6 Hz, 1H), 3.43–3.31 (m, 2H), 2.76 39 40 41 (m, 1H), 2.15 (s, 3H), 2.19–2.12 (m, 1H), 2.06 (s, 3H), 1.96 (ddd, J = 13.3, 11.0, 3.6 Hz, 42 43 13 44 1H), 1.88–1.79 (m, 1H), 1.79–1.69 (m, 1H), 1.64–1.52 (m, 2H); C NMR (126 MHz, 45

46 2 CDCl3): δ 170.1, 169.7, 168.9, 165.1, 157.6 (q, JC-F = 37 Hz), 152.7, 151.8, 150.0, 142.6, 47 48 1 49 134.7, 133.6, 133.5, 133.0, 131.0, 129.0, 128.7, 128.1, 127.3, 124.3, 123.7, 116.0 (q, JC-F = 50 51 52 288 Hz), 91.8, 87.0, 82.9, 81.3, 73.9, 72.7, 39.9, 38.7, 32.5, 28.8, 26.7, 20.8, 20.6; 19F NMR 53 54 55 115 56 57 58 59 60 ACS Paragon Plus Environment

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+ 1 (471 MHz, CDCl3, BTF IStd): δ –76.8; HRMS (ESI+): calcd. for [C36H34F3N7O8Na] 2 3 772.2313, meas. 772.2306, ∆ 0.9 ppm. 4 5 6 Methyl (2S,5S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- 7 8 9 (((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)me- 10 11 12 thyl)hept-6-ynoate (S48). To a solution of acetonide S3 (136 mg, 311 µmol) in THF (3.0 13 14 mL) was added a 0.5 M LiOH aq. solution (1.3 mL) at 0 °C. After 75 min, more 0.5 M 15 16 17 LiOH aq. solution (1.5 mL) was added. The reaction mixture was allowed to warm gradu- 18 19 20 ally to RT and was stirred for 8 h. More 0.5 M LiOH aq. solution (1.5 mL) was added and 21 22 the resulting mixture was stirred for 1 h. The reaction mixture was cooled to 0 °C and a 1 23 24

25 M HCl aq. solution was added dropwise until pH 6 was obtained. A NaHCO3 sat. aq. solu- 26 27 28 tion (5.0 mL) was added slowly followed by 1,4-dioxane (3.0 mL). A solution of Fmoc-Cl 29 30 in 1,4-dioxane (0.39 M, 1.0 mL, 0.39 mmol, 1.25 equiv.) was added dropwise over 5 min 31 32 33 under vigorous stirring. After 1 h, more Fmoc-Cl in 1,4-dioxane (0.39 M, 2.0 mL, 0.78 34 35 36 mmol, 2.50 equiv.) was added and the reaction mixture was stirred for 2 h. The reaction 37 38 M 39 was quenched by the slow addition of a 1 HCl aq. solution (8 mL) at 0 °C followed by 40 41 CH2Cl2 (8 mL). The layers were separated using a phase separator (Biotage Isolute) and the 42 43 × 44 aqueous phase was extracted with CH2Cl2 (2 8 mL). The combined organic extracts were 45 46 47 concentrated and the residue was dissolved in MeOH (20 mL). (Trimethylsilyl)diazome- 48 49 thane (2.0 M in hexanes, 5.0 mL) was added to the methanolic solution over 2 min and the 50 51 52 reaction mixture was stirred for 1 h. More (trimethylsilyl)diazomethane (2.0 M in hexanes, 53 54 55 116 56 57 58 59 60 ACS Paragon Plus Environment

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1 3.0 mL) was added and the resulting mixture was stirred for 1 h. The reaction was quenched 2 3 by the dropwise addition of glacial AcOH (4 mL) over 10 min and the volatiles were re- 4 5 6 moved in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trime- 7 8 9 thylpentane/EtOAc, 0 → 100%) to afford carbamate S48 (83 mg, 0.15 mmol, 47% over 3 10 11 steps) as an amorphous white solid. Rf = 0.56 (cyclohexane/EtOAc, 50:50); FTIR (thin 12 13 ν -1 14 film), max (cm ): 3300, 2987, 2947, 1723, 1526, 1447, 1377, 1341, 1212, 1163, 1099, 1061; 15 16 1 17 H NMR (500 MHz, CDCl3): δ 7.77 (dq, J = 7.5, 1.0 Hz, 2H), 7.60 (t, J = 6.8 Hz, 2H), 7.40 18 19 (tq, J = 7.4, 1.0 Hz, 2H), 7.32 (tt, J = 7.5, 1.4 Hz, 2H), 5.31 (d, J = 8.5 Hz, 1H), 4.94 (s, 20 21 22 1H), 4.58 (q, J = 6.0 Hz, 2H), 4.48 (dd, J = 11.1, 4.0 Hz, 1H), 4.46–4.33 (m, 3H), 4.23 (t, 23 24 25 J = 7.1 Hz, 1H), 3.77 (s, 3H), 3.31 (s, 3H), 2.66–2.60 (m, 1H), 2.14 (d, J = 2.3 Hz, 1H), 26 27 J J 28 2.06–1.95 (m, 1H), 1.95–1.85 (m, 1H), 1.69 (ddd, = 14.8, 11.2, 4.0 Hz, 1H), 1.58 (ddd, 29 30 = 16.3, 12.3, 4.7 Hz, 2H), 1.52fz (m, 1H), 1.49 (s, 3H), 1.31 (s, 3H); 13C NMR (126 MHz, 31 32 33 CDCl3): δ 172.9, 156.1, 144.0, 143.8, 141.4, 127.8, 127.2, 125.2, 125.1, 120.1, 112.4, 34 35 36 110.1, 110.1, 85.6, 85.1, 84.5, 71.4, 67.2, 55.3, 53.8, 52.6, 47.3, 40.4, 31.1, 30.5, 28.7, 26.6, 37 38 + ∆ 25.1; HRMS (ESI+): calcd. for [C32H37N1O8Na] 586.2411, meas. 586.2407, 0.8 ppm. 39 40 41 (3R,4R,5R)-5-((2S,5S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-ethynyl- 42 43 44 6-methoxy-6-oxohexyl)tetrahydrofuran-2,3,4-triyl triacetate (S49). To a vial charged 45 46 47 with acetonide S48 (43 mg, 76 µmol) was added a 4:1 mixture of acetic acid and water 48 49 (3.5 mL) at RT. The resulting mixture was stirred at 80 °C for 10 h. Volatiles were removed 50 51 52 in vacuo and the residue was dried by azeotropic distillation with benzene (2 × 1.0 mL). 53 54 55 117 56 57 58 59 60 ACS Paragon Plus Environment

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1 Crude material was used directly in the next step without further purification. To a solution 2 3 of the crude triol in CH2Cl2 (3.5 mL) at RT, were added pyridine (60 µL, 0.76 mmol, 10 4 5

6 equiv.), Ac2O (70 µL, 0.76 mmol, 10 equiv.) and DMAP (7 mg, 53 µmol, 0.70 equiv.) 7 8 9 sequentially. The resulting mixture was stirred at RT for 2 h and volatiles were removed in 10 11 vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpen- 12 13 14 tane/EtOAc, 5 → 60%) to afford S49 (42 mg, 66 µmol, 87% over 2 steps) as a 1:1 mixture 15 16 17 of diastereomers, as a colorless oil. Rf = 0.30/0.38 (hexanes/EtOAc, 50:50); FTIR (thin 18

19 ν -1 film), max (cm ): 3355, 3294, 2954, 2924, 2853, 1744, 1722, 1526, 1451, 1372, 1218, 1107, 20 21 1 22 1050, 1012; H NMR (500 MHz, CDCl3): δ 7.78–7.75 (m, 4H), 7.63–7.58 (m, 4H), 7.42– 23 24 25 7.38 (m, 4H), 7.34–7.29 (m, 4H), 6.36 (d, J = 4.6 Hz, 1H), 6.14 (d, J = 1.0 Hz, 1H), 5.38 26 27 J J J 28 (br d, = 8.5 Hz, 1H), 5.34 (br d, = 8.4 Hz, 1H), 5.32 (dd, = 4.8, 1.0 Hz, 1H), 5.23 (dd, 29 30 J = 6.8, 4.6 Hz, 1H), 5.18 (dd, J = 7.1, 4.8 Hz, 1H), 5.07 (dd, J = 6.8, 3.6 Hz, 1H), 4.46 31 32 33 (app dt, J = 10.2, 3.6 Hz, 1H), 4.42–4.36 (m, 7H), 4.23 (app t, J = 7.0 Hz, 2H), 3.76 (s, 34 35 36 6H), 2.69–2.57 (m, 2H), 2.13 (d, J = 2.4 Hz, 1H), 2.12 (d, J = 2.4 Hz, 1H), 2.12 (s, 3H), 37 38 2.11 (s, 3H), 2.10 (s, 3H), 2.07 (s, 6H), 2.06 (s, 3H), 2.00–1.75 (m, 6H), 1.70–1.43 (m, 39 40 13 41 6H); C NMR (126 MHz, CDCl3): δ 172.9, 172.8, 170.2, 170.0, 169.8, 169.6, 169.5, 169.3, 42 43 44 156.1, 156.0, 144.0, 144.0, 143.9, 143.8, 141.4, 141.4, 127.8, 127.2, 125.2, 120.1, 120.1, 45 46 98.5, 93.9, 85.3, 85.1, 81.4, 79.9, 74.6, 73.9, 72.6, 71.5, 71.3, 69.9, 67.1, 53.8, 53.7, 52.6, 47 48 49 47.3, 40.1, 39.2, 31.0, 30.4, 30.3, 28.6, 28.2, 21.2, 21.2, 20.8, 20.7, 20.7, 20.4; HRMS 50 51 + ∆ 52 (ESI+): calcd. for [C34H37N1O11Na] 658.2259, meas. 658.2248, 1.7 ppm. 53 54 55 118 56 57 58 59 60 ACS Paragon Plus Environment

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R R R R S S H 1 (2 ,3 ,4 ,5 )-2-((2 ,5 )-5-((((9 -fluoren-9-yl)methoxy)carbonyl)amino)-2- 2 3 ethynyl-6-methoxy-6-oxohexyl)-5-(6-benzamido-9H-purin-9-yl)tetrahydrofuran-3,4- 4 5 6 diyl diacetate (S50). To a suspension of N6-benzoyladenine (63 mg, 0.26 mmol, 4.0 equiv.) 7 8 9 in propionitrile (dried over 4 Å M.S., 1.5 mL) at RT, was added N,O-bis(trimethylsilyl)ac- 10 11 etamide (80 µL, 0.33 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 12 13 14 min, upon which complete solubilization of N6-benzoyladenine was observed. To a sepa- 15 16 × 17 rate flask charged with triacetate S49 (dried by azeotropic distillation with benzene (4 ), 18 19 42 mg, 66 µmol) and 2,6-di-tert-butyl-4-methylpyridinium triflate (12 mg, 33 µmol, 50 20 21 22 mol %) was added propionitrile (1.5 mL). The resulting mixture was stirred at RT for 5 23 24 25 min, until a clear solution was obtained, and the solution of silylated N6-benzoyladenine 26 27 was added. The reaction mixture was stirred at 90 °C for 4 h, cooled to RT and the volatiles 28 29 30 were removed in vacuo. Crude material was purified by silica gel chromatography (2,2,4- 31 32 33 trimethylpentane/EtOAc, 30 → 100%) to afford nucleoside S50 (44 mg, 54 µmol, 82%) as 34 35 ν -1 36 a colorless oil; Rf = 0.49 (EtOAc); FTIR (thin film), max (cm ): 3300, 2953, 2926, 2854, 37 38 1745, 1704, 1610, 1582, 1510, 1452, 1241, 1217, 1098, 1073, 1047; 1H NMR (500 MHz, 39 40

41 CDCl3): δ 8.79 (s, 1H), 8.13 (s, 1H), 8.02–7.97 (m, 2H), 7.76–7.72 (m, 2H), 7.62–7.57 (m, 42 43 44 3H), 7.53–7.49 (m, 2H), 7.40–7.35 (m, 2H), 7.31–7.27 (m, 2H), 6.13 (br d, J = 5.5 Hz, 45 46 1H), 6.05 (br t, J = 5.5 Hz, 1H), 5.55 (br t, J = 5.0 Hz, 1H), 5.35 (br d, J = 8.4 Hz, 1H), 47 48 49 4.54–4.48 (m, 1H), 4.46–4.35 (m, 3H), 4.23 (br t, J = 7.1 Hz, 1H), 3.74 (s, 3H), 2.67–2.58 50 51 52 (m, 1H), 2.16 (s, 3H), 2.15 (d, J = 2.4 Hz, 1H), 2.12–2.05 (m, 1H), 2.07 (s, 3H), 2.00–1.92 53 54 55 119 56 57 58 59 60 ACS Paragon Plus Environment

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13 1 (m, 1H), 1.92–1.80 (m, 2H), 1.64–1.45 (m, 2H); C NMR (126 MHz, CDCl3): δ 172.8, 2 3 169.8, 169.6, 164.8, 156.1, 152.8, 151.7, 149.8, 144.0, 143.8, 142.3, 141.4, 133.5, 133.0, 4 5 6 129.0, 128.0, 127.8, 127.2, 125.2, 123.9, 120.1, 87.1, 85.1, 80.9, 73.7, 73.0, 71.6, 67.1, 7 8 + 9 53.6, 52.6, 47.3, 38.5, 31.0, 30.5, 28.0, 20.8, 20.5; HRMS (ESI+): calcd. for [C44H43N6O10] 10 11 815.3035, meas. 815.3028, ∆ 0.9 ppm. 12 13 14 (2R,3R,4R,5R)-2-((2S,5S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((3- 15 16 17 carbamoylphenyl)ethynyl)-6-methoxy-6-oxohexyl)-5-(6-benzamido-9H-purin-9- 18 19 20 yl)tetrahydrofuran-3,4-diyl diacetate (S51). To a vial charged with S50 (61 mg, 75 21 22 µmol) were added 2-iodobenzamide (46 mg, 0.19 mmol, 2.5 equiv.), CuI (4 mg, 0.02 23 24

25 mmol, 25 mol %) and Pd(PPh3)4 (4 mg, 4 µmol, 5 mol %). The vial headspace was purged 26 27 28 with nitrogen for 10 min and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by 29 30 sparging with nitrogen for 15 min, 2.5 mL) was added at RT. The resulting mixture was 31 32 33 stirred at 60 °C for 2 h. The solution was allowed to cool to RT and volatiles were removed 34 35 36 in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i-PrOH, 0 → 37 38 ν - 39 25%) to afford S51 (68 mg, 73 µmol, 97%) as a light yellow oil. FTIR (thin film), max (cm 40 41 1): 3357, 3070, 2951, 2928, 1746, 1703, 1669, 1613, 1582, 1520, 1452, 1376, 1245, 1219, 42 43 1 44 1103, 1080, 1050; H NMR (500 MHz, CDCl3): δ 8.78 (s, 1H), 8.36 (s, 1H), 8.01 (br d, J = 45 46 47 7.7 Hz, 2H), 7.90–7.86 (m, 1H), 7.77 (dt, J = 7.9, 1.5 Hz, 1H), 7.74–7.69 (m, 2H), 7.63– 48 49 7.58 (m, 1H), 7.57–7.52 (m, 3H), 7.52–7.48 (m, 2H), 7.39–7.33 (m, 3H), 7.28–7.24 (m, 50 51 52 2H), 6.92 (br s, 1H), 6.82 (br s, 1H), 6.17 (br d, J = 5.8 Hz, 1H), 6.10 (br t, J = 5.8 Hz, 1H), 53 54 55 120 56 57 58 59 60 ACS Paragon Plus Environment

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1 5.71–5.65 (m, 1H), 5.51 (br d, J = 8.3 Hz, 1H), 4.56–4.50 (m, 1H), 4.49–4.41 (m, 1H), 2 3 4.41–4.32 (m, 2H), 4.18 (br t, J = 7.0 Hz, 1H), 3.75 (s, 3H), 2.89–2.80 (m, 1H), 2.16 (s, 4 5 6 3H), 2.19–2.12 (m, 1H), 2.06 (s, 3H), 2.12–2.02 (m, 2H), 1.99–1.89 (m, 1H), 1.74–1.64 7 8 13 9 (m, 1H), 1.64–1.54 (m, 1H); C NMR (126 MHz, CDCl3): δ 172.8, 170.3, 170.2, 169.8, 10 11 165.6, 156.2, 152.0, 151.8, 149.4, 143.9, 143.7, 143.3, 141.4, 135.2, 133.5, 132.6, 131.1, 12 13 14 129.0, 128.9, 128.4, 127.9, 127.9, 127.4, 127.2, 127.2, 125.1, 123.9, 123.3, 120.1, 92.0, 15 16 17 87.1, 82.9, 81.7, 73.8, 72.9, 67.2, 53.6, 52.8, 47.2, 38.6, 31.1, 30.5, 28.8, 20.8, 20.5; HRMS 18

19 + ∆ (ESI+): calcd. for [C51H47N7O11Na] 956.3226, meas. 956.3214, 1.2 ppm. 20 21 22 (S)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol- 23 24 25 4-yl)methyl)-5-(triisopropylsilyl)pent-4-yn-1-ol (S52). To a solution of 5 (5.50 g, 12.9 26 27 28 mmol) in MeOH (100 mL) at –78 °C was added NaBH4 (588 mg, 15.5 mmol, 1.2 equiv.) 29 30 portionwise. The reaction mixture was allowed to warm to 0 °C over 1 h, then was warmed 31 32 33 to RT and stirred for 1 h. Volatiles were removed in vacuo. The residue was partitioned 34 35 36 between CH2Cl2 (30 mL) and a NH4Cl sat. aq. solution (25 mL). The layers were separated 37 38 × 39 and the aqueous phase was extracted with CH2Cl2 (3 25 mL). The combined organic ex- 40 41 tracts were dried over MgSO4 and filtered over a silica plug to yield S52 as a yellow oil 42 43 44 (5.29 g, 12.4 mmol, 95%) which was of suitable purity to be used in the next step without 45 46 47 further purification. While the protocol reported here was performed on purified starting 48 49 material, it was found that direct treatment of the rhodium-catalyzed conjugate alkynyla- 50 51

52 tion reaction mixture (5) with NaBH4 at 0 °C reliably generated alcohol S52 in > 90% yield 53 54 55 121 56 57 58 59 60 ACS Paragon Plus Environment

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1 over two steps (alkynylation/reduction). Rf = 0.28 (hexanes/EtOAc, 67:33); FTIR (thin- 2 3 ν -1 film), max (cm ): 3424, 2936, 2891, 2864, 2163, 1463, 1377, 1372, 1347, 1272, 1242, 1211, 4 5 1 6 1193, 1161, 1104, 1092, 1057, 1019; H NMR (600 MHz, CDCl3): δ 4.95 (s, 1H), 4.63 (dd, 7 8 9 J = 6.0, 1.2 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.48 (ddd, J = 10.3, 4.6, 1.2 Hz, 1H), 3.88– 10 11 3.80 (m, 2H), 3.33 (s, 3H), 2.83 (dddd, J = 10.9, 9.3, 5.2, 4.0 Hz, 1H), 1.80–1.62 (m, 5H), 12 13 13 14 1.47 (s, 3H), 1.31 (s, 3H), 1.10–1.00 (m, 21H); C NMR (151 MHz, CDCl3): δ 112.5, 110.6, 15 16 17 109.7, 85.6, 84.5, 83.3, 61.2, 55.2, 41.2, 38.5, 27.4, 26.7, 25.4, 18.8, 11.3; HRMS (ESI+): 18

19 + ∆ calcd. for [C23H43O5Si1] 427.2874, meas. 427.2870, 0.9 ppm. 20 21 22 (S)-3-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol- 23 24 25 4-yl)methyl)pent-4-yn-1-ol (S53). To a solution of TIPS-alkyne S52 (3.81 g, 8.92 mmol) 26 27 28 in THF (15 mL) was added TBAF (1.0 M in THF, 15.0 mL, 15.0 mmol, 1.7 equiv.) at RT. 29 30 The reaction mixture was stirred for 3 h and the volatiles were removed in vacuo. The 31 32

33 residue was partitioned between Et2O (40 mL) and a NH4Cl sat. aq. solution (40 mL). The 34 35 × 36 layers were separated and the aqueous phase was extracted with Et2O (4 40 mL). The 37 38 39 combined organic extracts were washed with brine (120 mL), dried over MgSO4, filtered 40 41 and concentrated. Crude material was purified by silica gel chromatography (hex- 42 43 44 anes/EtOAc, 0 → 100%) to afford alkyne S53 (2.22 g, 8.21 mmol, 92%) as a colorless oil. 45 46 47 The pure material crystallized upon storage at –20 °C over three weeks. The crystals were 48 49 of suitable quality to obtain X-ray crystallographic data which is reported in the Supporting 50 51 ν -1 52 Information. Rf = 0.31 (cyclohexane/EtOAc, 50:50); FTIR (thin-film), max (cm ): 3446, 53 54 55 122 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 3281, 2988, 2937, 2836, 1142, 1382, 1374, 1274, 1241, 1210, 1161, 1090, 1056; H NMR 2 3 (500 MHz, CDCl3): δ 4.94 (s, 1H), 4.60 (d, J = 6.0 Hz, 1H), 4.58 (dd, J = 6.0, 0.8 Hz, 1H), 4 5 6 4.50 (ddd, J = 11.2, 4.0, 0.8 Hz, 1H), 3.87–3.76 (m, 2H), 3.33 (s, 3H), 2.86–2.76 (m, 1H), 7 8 9 2.15 (d, J = 2.4 Hz, 1H), 1.79–1.66 (m, 3H), 1.62 (ddd, J = 13.3, 11.1, 4.0 Hz, 1H), 1.48 10

11 13 (s, 3H), 1.31 (s, 3H); C NMR (126 MHz, CDCl3): δ 112.4, 110.1, 85.9, 85.7, 85.1, 84.5, 12 13 + 14 71.2, 60.8, 55.4, 40.5, 38.0, 26.6, 25.8, 25.1; HRMS (ESI+): calcd. for [C14H23O5] 271.1540, 15 16 ∆ 17 meas. 271.1547, 2.6 ppm. 18 19 N S R R R R d 20 -(( )-3-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- ][1,3]di- 21 22 oxol-4-yl)methyl)pent-4-yn-1-yl)-2-nitrobenzenesulfonamide (S54). To a solution of al- 23 24 76 25 cohol S53 (550 mg, 2.04 mmol), DMEAD (1.19 g, 5.09 mmol, 2.5 equiv.) and PPh3 (1.39 26 27 28 g, 5.29 mmol, 2.6 equiv.) in a 4:1 mixture of toluene and THF (110 mL) stirred at 0 °C 29 30 over activated 4 Å M.S. (1.65 g), was added a solution of 2-NsNH2 (1.44 g, 7.12 mmol, 3.5 31 32 33 equiv.) in THF (22 mL, prepared over 1.65 g of activated 4 Å M.S.) over 5 min. The reac- 34 35 36 tion mixture was allowed to warm gradually to RT and was stirred for 72 h. The media was 37 38 in vacuo 39 filtered and the volatiles were removed . Crude material was purified by silica gel 40 41 chromatography (hexanes/EtOAc, 0 → 100%) to afford S54 (496 mg, 1.09 mmol, 54%) as 42 43

44 a white amorphous solid and unreacted S53 (81 mg, 0.30 mmol, 15%). Rf = 0.27 (hex- 45 46 ν -1 47 anes/EtOAc, 50:50); FTIR (thin-film), max (cm ): 3289, 2989, 2936, 2836, 1540, 1442, 48 49 1415, 1363, 1343, 1300, 1274, 1241, 1210, 1192, 1162, 1087, 1056; 1H NMR (600 MHz, 50 51

52 CDCl3): δ 8.16–8.11 (m, 1H), 7.89–7.83 (m, 1H), 7.78–7.70 (m, 2H), 5.50 (t, J = 6.1 Hz, 53 54 55 123 56 57 58 59 60 ACS Paragon Plus Environment

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1 1H), 4.93 (s, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.54 (dd, J = 6.0, 0.9 Hz, 1H), 4.43 (ddd, J = 2 3 11.1, 3.9, 0.9 Hz, 1H), 3.32 (s, 3H), 3.30–3.26 (m, 2H), 2.71 (dddd, J = 14.6, 8.9, 4.5, 2.9 4 5 6 Hz, 1H), 2.13 (d, J = 2.4 Hz, 1H), 1.77 (dtd, J = 13.2, 7.5, 4.8 Hz, 1H), 1.71–1.63 (m, 2H), 7 8 13 9 1.58 (ddd, J = 13.3, 11.0, 4.0 Hz, 1H), 1.47 (s, 3H), 1.30 (s, 3H); C NMR (126 MHz, 10 11 CDCl3): δ 148.1, 133.7, 133.6, 132.9, 131.1, 125.5, 112.4, 110.1, 85.5, 84.8, 84.7, 84.4, 12 13 + 14 71.9, 55.4, 41.9, 40.3, 35.2, 26.5, 26.5, 25.0; HRMS (ESI+): calcd. for [C20H26N2O8S1Na] 15 16 ∆ 17 477.1302, meas. 477.1287, 3.2 ppm. 18 19 R R R S 20 (3 ,4 ,5 )-5-(( )-2-(2-((4-nitrophenyl)sulfonamido)ethyl)but-3-yn-1-yl)tetrahy- 21 22 drofuran-2,3,4-triyl triacetate (S55). To a solution of acetonide S54 (210 mg, 0.462 23 24

25 mmol) in CH2Cl2 (0.80 mL) at 0 °C, was added a 4:1 mixture of TFA and water (4.0 mL). 26 27 28 The resulting mixture was stirred at RT for 15 h. Volatiles were removed in vacuo and the 29 30 residue was dried by azeotropic distillation with benzene (2 × 1.0 mL). Crude material was 31 32 33 used directly in the next step without further purification. To a solution of the crude triol 34 35 36 in CH2Cl2 (4.8 mL) at RT, were added pyridine (0.37 mL, 4.6 mmol, 10 equiv.), Ac2O (0.44 37 38 39 mL, 4.6 mmol, 10 equiv.) and DMAP (11 mg, 92 µmol, 20 mol %) sequentially. The re- 40 41 sulting mixture was stirred at RT for 1 h and volatiles were removed in vacuo. Crude ma- 42 43 44 terial was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 15 → 45 46 47 75%) to afford S55 (70 mg, 0.13 mmol, 29% over 2 steps) as a 1.8:1 mixture of diastere- 48 49 ν - omers, as a colorless oil. Rf = 0.31/0.40 (hexanes/EtOAc, 40:60); FTIR (thin film), max (cm 50 51 52 1): 3288, 2925, 2853, 1747, 1541, 1424, 1368, 1222, 1167, 1124, 1082, 1058, 1012; 1H 53 54 55 124 56 57 58 59 60 ACS Paragon Plus Environment

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1 NMR (600 MHz, CDCl3): δ 8.16–8.11 (m, 2.8H), 7.89–7.84 (m, 2.8H), 7.79–7.71 (m, 2 3 5.6H), 6.32 (d, J = 4.6 Hz, 1.0H), 6.11 (d, J = 1.0 Hz, 1.8H), 5.55 (app t, J = 6.1 Hz, 1.0H), 4 5 6 5.52 (app t, J = 6.1 Hz, 1.8H), 5.29 (dd, J = 4.9, 1.0 Hz, 1.8H), 5.19 (dd, J = 6.7, 4.6 Hz, 7 8 9 1.0H), 5.16 (dd, J = 6.9, 4.9 Hz, 1.8H), 5.02 (dd, J = 6.7, 3.7 Hz, 1.0H), 4.40 (ddd, J = 10 11 10.3, 3.7, 3.3 Hz, 1.0H), 4.34 (ddd, J = 10.2, 6.9, 3.1 Hz, 1.8H), 3.33–3.22 (m, 5.6H), 2.70– 12 13 14 2.61 (m, 2.8H), 2.14 (d, J = 2.4 Hz, 2.8H), 2.12 (s, 5.4H), 2.11 (s, 3.0H), 2.10 (s, 3.0H), 15 16 17 2.08 (s, 5.4H), 2.06 (s, 5.4H), 2.06 (s, 3.0H), 1.86–1.73 (m, 5.6H), 1.71–1.60 (m, 4.6H), 18

19 13 1.56 (ddd, J = 14.0, 10.3, 4.2 Hz, 1.0H); C NMR (126 MHz, CDCl3): δ 170.2, 170.0, 20 21 22 169.8, 169.6, 169.5, 169.2, 148.2, 133.8, 133.7, 133.7, 132.9, 132.9, 131.2, 131.2, 125.6, 23 24 25 125.6, 98.4, 93.8, 84.5, 84.3, 81.2, 79.6, 74.6, 73.8, 72.5, 72.1, 72.0, 69.9, 41.9, 40.1, 39.0, 26 27 35.2, 35.0, 26.6, 26.2, 21.2, 21.2, 20.8, 20.7, 20.7, 20.4; HRMS (ESI+): calcd. for 28 29 30 + ∆ [C22H26N2O11S1Na] 549.1150, meas. 549.1138, 2.1 ppm. 31 32 33 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-2-(2-((4-nitrophenyl)sulfon- 34 35 36 amido)ethyl)but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate (S56). To a suspension 37

38 6 39 of N -benzoyladenine (93 mg, 0.39 mmol, 2.15 equiv.) in propionitrile (dried over 4 Å 40 41 M.S., 4.5 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (121 µL, 0.497 mmol, 42 43 44 2.75 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which complete 45 46 6 47 solubilization of N -benzoyladenine was observed. To a separate flask charged with tri- 48 49 acetate S55 (dried by azeotropic distillation with benzene (4 ×), 95 mg, 0.18 mmol) and 50 51 52 53 54 55 125 56 57 58 59 60 ACS Paragon Plus Environment

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1 2,6-di-tert-butyl-4-methylpyridinium triflate (13 mg, 36 µmol, 20 mol %) was added pro- 2 3 pionitrile (4.5 mL). The resulting mixture was stirred at RT for 5 min, until a clear solution 4 5 6 was obtained, and the solution of silylated N6-benzoyladenine was added. The reaction 7 8 9 mixture was stirred at 95 °C for 30 h, cooled to RT and the volatiles were removed in 10 11 vacuo. Crude material was purified by silica gel chromatography (PhMe/MeCN, 0 → 50%) 12 13 14 and lyophilized to afford nucleoside S56 (120 mg, 0.170 mmol, 94%) as a white fluffy 15 16 ν -1 17 solid. Rf = 0.36 (EtOAc); FTIR (thin-film), max (cm ): 3295, 3097, 2945, 1746, 1699, 1610, 18 19 1582, 1540, 1509, 1485, 1455, 1423, 1365, 1338, 1298, 1240, 1218, 1164, 1074, 1047; 1H 20 21

22 NMR (600 MHz, CDCl3): δ 9.09 (s, 1H), 8.76 (s, 1H), 8.10 (dd, J = 7.7, 1.6 Hz, 1H), 8.07 23 24 25 (s, 1H), 8.02 (dt, J = 7.1, 1.4 Hz, 2H), 7.80 (dd, J = 7.6, 1.6 Hz, 1H), 7.72 (td, J = 7.6, 1.6 26 27 J J 28 Hz, 1H), 7.68 (td, = 7.6, 1.6 Hz, 1H), 7.63–7.59 (m, 1H), 7.55–7.49 (m, 2H), 6.10 (d, 29 30 = 4.7 Hz, 2H), 5.55 (q, J = 6.2, 5.4 Hz, 2H), 4.46 (ddd, J = 11.0, 4.5, 2.8 Hz, 1H), 3.25 (dt, 31 32 33 J = 7.5, 6.2 Hz, 2H), 2.62 (dddq, J = 11.2, 9.0, 4.6, 2.5 Hz, 1H), 2.17 (d, J = 2.4 Hz, 1H), 34 35 36 2.16 (s, 3H), 2.07–2.12 (m, 1H), 2.07 (s, 3H), 1.85 (ddd, J = 13.8, 11.2, 2.9 Hz, 1H), 1.82– 37

38 13 1.73 (m, 2H), 1.69 (ddt, J = 13.5, 9.9, 6.3 Hz, 1H); C NMR (126 MHz, CDCl3): δ 169.9, 39 40 41 169.6, 164.8, 152.9, 151.6, 150.0, 148.1, 142.5, 133.8, 133.7, 133.6, 133.0, 133.0, 131.2, 42 43 44 129.0, 128.1, 125.5, 124.1, 87.2, 84.4, 80.8, 77.4, 73.7, 72.9, 72.2, 41.9, 38.3, 35.1, 26.1, 45 46 + ∆ 20.8, 20.6; HRMS (ESI+): calcd. for [C32H32N7O10S1] 706.1926, meas. 706.1922, 0.5 ppm. 47 48 49 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-4-(3-carbamoylphenyl)-2-(2- 50 51 52 ((4-nitrophenyl)sulfonamido)ethyl)but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate 53 54 55 126 56 57 58 59 60 ACS Paragon Plus Environment

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1 (S57). To a vial charged with S56 (54 mg, 77 µmol) were added 3-iodobenzamide (29 mg, 2 3 0.12 mmol, 1.5 equiv.), CuI (3 mg, 0.02 mmol, 20 mol %) and Pd(PPh3)4 (4 mg, 4 µmol, 5 4 5 6 mol %). The vial headspace was purged with nitrogen for 10 min and a 5:1:1 mixture of 7 8 9 toluene, DMF and i-Pr2NEt (degassed by sparging with nitrogen for 15 min, 3.0 mL) was 10 11 added at RT. The resulting mixture was stirred at 70 °C for 4 h. The solution was allowed 12 13 14 to cool to RT and volatiles were removed in vacuo. Crude material was purified by silica 15 16 17 gel chromatography (EtOAc/i-PrOH, 0 → 15%) to afford benzamide S57 (50 mg, 60 µmol, 18 19 ν 78%) as a white amorphous solid. Rf = 0.16 (EtOAc/i-PrOH, 91:9); FTIR (thin-film), max 20 21 22 (cm-1): 2926, 1746, 1667, 1610, 1579, 1540, 1511, 1484, 1455, 1366, 1339, 1298, 1241, 23 24 1 25 1215, 1163, 1073, 1047; H NMR (500 MHz, CDCl3): δ 9.13 (s, 1H), 8.76 (s, 1H), 8.11 (s, 26 27 J 28 1H), 8.10–8.07 (m, 1H), 8.05–8.00 (m, 2H), 7.87–7.86 (m, 1H), 7.80 (ddd, = 7.8, 1.9, 1.2 29 30 Hz, 1H), 7.79–7.76 (m, 1H), 7.71–7.64 (m, 2H), 7.63–7.58 (m, 1H), 7.55–7.47 (m, 3H), 31 32 33 7.38 (td, J = 7.7, 0.6 Hz, 1H), 6.56 (br s, 1H), 6.17 (t, J = 5.5 Hz, 1H), 6.13 (d, J = 5.8 Hz, 34 35 36 1H), 5.84 (br s, 1H), 5.79 (t, J = 6.1 Hz, 1H), 5.64 (dd, J = 5.3, 4.0 Hz, 1H), 4.50 (dt, J = 37 38 10.3, 3.7 Hz, 1H), 3.38–3.29 (m, 1H), 3.32–3.23 (m, 1H), 2.87 (tt, J = 10.2, 4.2 Hz, 1H), 39 40 41 2.23–2.10 (m, 1H), 2.16 (s, 3H), 2.06 (s, 3H), 1.98 (ddd, J = 13.5, 11.1, 3.6 Hz, 1H), 1.92– 42 43 13 44 1.73 (m, 2H); C NMR (101 MHz, CDCl3): δ 170.1, 169.7, 168.6, 164.9, 152.8, 151.7, 45 46 150.0, 148.1, 142.6, 134.7, 133.8, 133.6, 133.6, 133.5, 133.0, 133.0, 131.2, 130.9, 129.0, 47 48 49 128.8, 128.1, 127.5, 125.5, 124.3, 123.4, 91.1, 87.1, 83.5, 81.2, 77.4, 77.2, 76.9, 73.8, 72.7, 50 51 52 53 54 55 127 56 57 58 59 60 ACS Paragon Plus Environment

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+ 1 42.1, 38.5, 35.3, 27.0, 20.8, 20.6; HRMS (ESI+): calcd. for [C39H37N8O11S1] 825.2297, meas. 2 3 825.2300, ∆ 0.4 ppm. 4 5 6 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((S)-4-(3-carbamoylphenyl)-2-(2- 7 8 9 ureidoethyl)but-3-yn-1-yl)tetrahydrofuran-3,4-diyl diacetate (S58). To a solution of 10 11 12 S57 (112 mg, 0.136 mmol) in a 2:1 mixture of MeCN and DMF (10.5 mL) at 0 °C, were 13 14 added thiophenol (56 µL, 0.54 mmol, 4.0 equiv.) and Cs2CO3 (354 mg, 1.09 mmol, 8.0 15 16 17 equiv.) sequentially. The resulting mixture was stirred at RT for 1 h and was filtered twice 18 19 20 through a nylon syringe filter (13 mm, 0.22 µm). Volatiles were removed in vacuo. The 21 22 crude amine was used directly in the next step without further purification. To a solution 23 24

25 of the crude amine in a 1:1 mixture of CH2Cl2 and i-PrOH (13.5 mL) at RT, was added 26 27 28 (trimethylsilyl)isocyanate (1.29 mL, 9.51 mmol, 70 equiv.). The resulting mixture was 29 30 stirred at RT for 17 h and the volatiles were removed in vacuo. Crude material was purified 31 32 33 by preparative HPLC (5% → 95% B over 40 min) to afford S58 (24 mg, 35 µmol, 26% 34 35 1 36 over two steps) as a white fluffy solid. H NMR (600 MHz, CD3OD): δ 8.78 (s, 1H), 8.58 37 38 39 (s, 1H), 8.13–8.08 (m, 2H), 7.94 (t, J = 1.8 Hz, 1H), 7.83 (dt, J = 7.9, 1.6 Hz, 1H), 7.71– 40 41 7.64 (m, 1H), 7.61–7.54 (m, 3H), 7.43 (t, J = 7.8 Hz, 1H), 6.35 (d, J = 5.3 Hz, 1H), 6.20 42 43 44 (t, J = 5.5 Hz, 1H), 5.68 (t, J = 5.2 Hz, 1H), 4.60 (ddd, J = 10.5, 4.8, 3.1 Hz, 1H), 3.40 45 46 47 (ddd, J = 13.1, 7.4, 5.3 Hz, 1H), 3.28 (dt, J = 13.5, 7.4 Hz, 1H), 2.91 (ddt, J = 11.0, 9.3, 48 49 4.5 Hz, 1H), 2.28 (ddd, J = 14.1, 10.5, 4.0 Hz, 1H), 2.18 (s, 3H), 2.07 (s, 3H), 2.03 (ddd, J 50 51 52 = 14.0, 11.2, 3.2 Hz, 1H), 1.82 (dtd, J = 12.7, 7.6, 5.1 Hz, 1H), 1.74 (dddd, J = 13.0, 9.5, 53 54 55 128 56 57 58 59 60 ACS Paragon Plus Environment

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13 1 7.3, 5.4 Hz, 1H); C NMR (126 MHz, CD3OD): δ 171.6, 171.4, 171.3, 168.2, 162.1, 153.5, 2 3 153.1, 151.4, 145.2, 135.7, 135.3, 134.9, 133.9, 131.8, 129.8, 129.7, 129.5, 128.1, 125.7, 4 5 6 125.2, 92.8, 88.5, 83.5, 82.1, 75.2, 74.2, 39.6, 36.9, 28.0, 20.5, 20.3; HRMS (ESI+): calcd. 7 8 + ∆ 9 for [C34H35N8O8] 683.2572, meas. 683.2598, 3.8 ppm. 10 11 S R R R R d 12 ( )-4-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- ][1,3]dioxol- 13 14 4-yl)methyl)-6-(triisopropylsilyl)hex-5-ynenitrile (S59). To a solution of S52 (0.54 g, 15 16 17 1.3 mmol) in benzene (44 mL) at RT, were added acetone cyanohydrin (0.17 mL, 1.9 mmol, 18 19 20 1.5 equiv.), Pn-Bu3 (0.47 mL, 1.9 mmol, 1.5 equiv.) and a solution of TMAD (0.33 g, 1.9 21 22 mmol, 1.5 equiv.) in benzene (20 mL). The resulting mixture, which quickly became thick 23 24 25 and cloudy, was stirred at RT for 2 h and volatiles were removed in vacuo. Crude material 26 27 28 was purified by silica gel chromatography (hexanes/EtOAc, 2 → 40%) to afford S59 (0.49 29 30 g, 1.1 mmol, 89%) as a colorless oil. Rf = 0.44 (hexanes/EtOAc, 70:30); FTIR (thin film), 31 32 ν -1 33 max (cm ): 2941, 2865, 2247, 2166, 1463, 1381, 1371, 1241, 1210, 1192, 1160, 1092, 1057, 34 35 1 36 1018; H NMR (500 MHz, CDCl3): δ 4.94 (s, 1H), 4.61 (dd, J = 6.0, 1.0 Hz, 1H), 4.58 (d, 37 38 39 J = 6.0 Hz, 1H), 4.45 (ddd, J = 10.6, 4.5, 1.0 Hz, 1H), 3.34 (s, 3H), 2.81 (dddd, J = 10.6, 40 41 10.2, 4.6, 4.2 Hz, 1H), 2.59 (ddd, J = 16.9, 8.0, 5.6 Hz, 1H), 2.54 (ddd, J = 16.9, 8.0, 7.8 42 43 44 Hz, 1H), 1.86 (app dtd, J = 13.2, 8.0, 4.6 Hz, 1H), 1.74 (dddd, J = 13.2, 10.2, 7.8, 5.6 Hz, 45 46 47 1H), 1.69 (ddd, J = 13.2, 10.6, 4.2 Hz, 1H), 1.64 (ddd, J = 13.2, 10.6, 4.5 Hz, 1H), 1.46 (s, 48

49 13 3H), 1.30 (s, 3H), 1.07–1.03 (m, 21H); C NMR (100 MHz, CDCl3): δ 119.4, 112.6, 109.9, 50 51 52 53 54 55 129 56 57 58 59 60 ACS Paragon Plus Environment

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1 108.0, 85.5, 85.3, 84.8, 84.4, 55.4, 40.8, 31.6, 29.8, 26.7, 25.4, 18.8, 15.4, 11.3; HRMS 2 3 + ∆ (ESI+): calcd. for [C24H41N1O4Si1Na] 458.2697, meas. 458.2691, 1.2 ppm. 4 5 6 Methyl (S)-6-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 7 8 9 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-8-(triisopropylsilyl)oct-2-en- 10 11 . 12 7-ynoate (S61) To a solution of nitrile S59 (0.49 g, 1.1 mmol) in PhMe (22 mL) at –78 13 14 °C, was added DIBALH (1.0 M in hexanes, 1.68 mL, 1.68 mmol, 1.5 equiv.) dropwise. The 15 16 17 resulting mixture was stirred at –78 °C for 1.3 h. The reaction was quenched by the addition 18 19 20 of a potassium sodium tartrate sat. aq. solution (22 mL), followed by the addition of Et2O 21 22 (15 mL) and brine (15 mL). The biphasic mixture was stirred vigorously at RT for 45 min. 23 24 × 25 The layers were separated and the aqueous phase was extracted with Et2O (3 25 mL). 26 27 28 The combined organic extracts were dried over Na2SO4, filtered and concentrated. Crude 29 30 material was filtered through a short pad of silica gel (hexanes/EtOAc, 75:25) to afford 31 32 33 crude aldehyde S60 which was used directly in the next step without further purification. 34 35 36 To a suspension of NaH (95%, 33 mg, 1.3 mmol, 1.5 equiv.) in THF (1.7 mL) at 0 °C, was 37 38 S2 39 added a solution of phosphonate (0.39 g, 1.3 mmol, 1.5 equiv.) in THF (2.0 mL) drop- 40 41 wise. The resulting cloudy mixture was stirred at 0 °C for 30 min and a solution of aldehyde 42 43 44 S60 (0.38 g, 0.88 mmol) in THF (3.7 mL) was added in one portion. The yellow reaction 45 46 47 mixture was stirred at 45 °C for 20 h and cooled to 0 °C, before the addition of an aqueous 48 49 pH 7 buffer (5 mL) and Et2O (5 mL). The layers were separated and the aqueous phase was 50 51 × 52 extracted with Et2O (3 10 mL). The combined organic extracts were dried over MgSO4, 53 54 55 130 56 57 58 59 60 ACS Paragon Plus Environment

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1 filtered and concentrated. Crude material was purified by silica gel chromatography (hex- 2 3 anes/EtOAc, 1 → 40%) to afford olefin S61 (0.44 g, 0.72 mmol, 82%) as a 1:1 mixture of 4 5

6 Z and E isomers, as a colorless oil. Rf = 0.17/0.31 (hexanes/EtOAc, 80:20); FTIR (thin 7 8 ν -1 9 film), max (cm ): 3305, 2942, 2865, 2164, 1729, 1661, 1541, 1463, 1439, 1382, 1372, 1209, 10

11 1 1159, 1104, 1092, 1060; H NMR (500 MHz, CDCl3): δ 8.30 (s, 1H), 7.60 (s, 1H), 7.37 12 13 14 (app t, J = 7.6 Hz, 1H), 6.86 (app t, J = 7.3 Hz, 1H), 4.94 (s, 1H), 4.94 (s, 1H), 4.63 (dd, J 15 16 17 = 6.0, 1.2 Hz, 1H), 4.62 (dd, J = 6.0, 1.2 Hz, 1H), 4.58 (d, J = 6.0 Hz, 1H), 4.58 (d, J = 6.0 18 19 Hz, 1H), 4.49–4.43 (m, 2H), 3.87 (s, 3H), 3.80 (s, 3H), 3.32 (s, 3H), 3.32 (s, 3H), 2.89– 20 21 22 2.75 (m, 2H), 2.74–2.61 (m, 2H), 2.45–2.36 (m, 1H), 2.36–2.26 (m, 1H), 1.74–1.57 (m, 23 24 13 25 8H), 1.46 (s, 6H), 1.31 (s, 6H), 1.08–1.00 (m, 42H); C NMR (126 MHz, CDCl3): δ 163.9, 26

27 2 2 JC-F JC-F 28 163.8, 155.1 (q, = 38 Hz), 155.0 (q, = 38 Hz), 140.7, 135.5, 123.6, 122.9, 115.8 (q, 29 30 1 1 JC-F = 288 Hz), 115.6 (q, JC-F = 288 Hz), 112.5, 112.5, 110.1, 109.8, 109.8, 109.8, 85.7, 85.6, 31 32 33 85.6, 84.5, 84.5, 83.4, 83.2, 55.2, 55.2, 53.1, 53.0, 41.2, 41.1, 35.6, 34.1, 30.3, 30.2, 27.2, 34 35 19 36 26.7, 25.4, 25.4, 18.8, 18.8, 11.4, 11.3; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.4, – 37 38 + ∆ 77.2; HRMS (ESI+): calcd. for [C29H46F3N1O7Si1Na] 628.2888, meas. 628.2894, 1.0 ppm. 39 40 41 Methyl (2S,6S)-6-(((3aR,4R,6R,6aR)-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 42 43 44 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)-8-(triisopropylsilyl)oct-7- 45 46 47 ynoate (S62). In a glove box, [Rh(nbd)2]BF4 (47 mg, 0.13 mmol, 20 mol %) and (S,S)- 48 49 MeBPE (36 mg, 0.14 mmol, 22 mol %) were placed in two separate vials, capped with a 50 51 52 septum and placed under an atmosphere of nitrogen out of the glovebox. MeOH (3 mL) 53 54 55 131 56 57 58 59 60 ACS Paragon Plus Environment

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1 was added to the vial containing (S,S)-MeBPE and the resulting colorless solution was 2 3 transferred to the vial charged with [Rh(nbd)2]BF4. More MeOH (2 mL) was used for rins- 4 5 6 ing. The red solution of (S,S)-MeBPE-Rh was added to a solution of olefin S61 (0.38 g, 7 8 9 0.63 mmol) in MeOH (16 mL). The flask headspace was evacuated and refilled with hy- 10 11 drogen (3 ×), and the reaction mixture was stirred vigorously at RT under an atmosphere 12 13 14 of hydrogen (balloon) for 2 h. Volatiles were removed in vacuo. Crude material was puri- 15 16 17 fied by silica gel chromatography (hexanes/EtOAc, 1 → 60%) to afford protected amino 18 19 acid S62 (0.35 g, 0.57 mmol, 90%) as a colorless oil. Rf = 0.40 (hexanes/EtOAc, 70:30); 20 21 ν -1 22 FTIR (thin film), max (cm ): 3317, 2941, 2865, 2165, 1749, 1724, 1549, 1462, 1382, 1208, 23 24 1 25 1160, 1105, 1094, 1060; H NMR (500 MHz, CDCl3): δ 6.84 (d, J = 7.8 Hz, 1H), 4.94 (s, 26 27 J J 28 1H), 4.65–4.60 (m, 2H), 4.58 (d, = 5.9 Hz, 1H), 4.44 (ddd, = 10.1, 4.7, 1.1 Hz, 1H), 29 30 3.79 (s, 3H), 3.31 (s, 3H), 2.65–2.58 (m, 1H), 1.98–1.89 (m, 1H), 1.88–1.78 (m, 1H), 1.69– 31 32 33 1.54 (m, 3H), 1.51–1.42 (m, 3H), 1.46 (s, 3H), 1.31 (s, 3H), 1.07–1.01 (m, 21H); 13C NMR 34 35 2 1 36 (126 MHz, CDCl3): δ 171.4, 156.9 (q, JC-F = 38 Hz), 115.7 (q, JC-F = 288 Hz), 112.5, 110.3, 37 38 109.8, 85.7, 85.6, 84.5, 82.9, 55.2, 53.1, 52.7, 41.2, 35.3, 31.9, 30.1, 26.7, 25.4, 22.7, 18.8, 39 40 19 41 11.4; F NMR (376 MHz, CDCl3, BTF IStd): δ –76.8; HRMS (ESI+): calcd. for 42 43 + ∆ 44 [C29H48F3N1O7Si1Na] 630.3044, meas. 630.3046, 0.3 ppm. 45 46 S S R R R R 47 Methyl (2 ,6 )-6-(((3a ,4 ,6 ,6a )-6-methoxy-2,2-dimethyltetrahydrofuro[3,4- 48 49 d][1,3]dioxol-4-yl)methyl)-2-(2,2,2-trifluoroacetamido)oct-7-ynoate (S63). To a solu- 50 51 52 tion of TIPS alkyne S62 (381 mg, 0.627 mmol) in DMF (3.2 mL) at RT, was added a 53 54 55 132 56 57 58 59 60 ACS Paragon Plus Environment

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1 solution of TASF (518 mg, 1.88 mmol, 3.0 equiv.) in DMF (3.2 mL). The resulting mixture 2 3 was stirred at 60 °C for 2.5 h and volatiles were removed in vacuo. The residue was parti- 4 5

6 tioned between Et2O (15 mL) and a 1:1 mixture of brine and 5% LiCl aq. solution (15 mL). 7 8 × 9 The layers were separated and the aqueous phase was extracted with Et2O (3 15 mL). 10 11 The combined organic extracts were dried over MgSO4, filtered and concentrated. Crude 12 13 14 material was purified by silica gel chromatography (hexanes/EtOAc, 2 → 50%) to afford 15 16 17 alkyne S63 (250 mg, 0.554 mmol, 88%) as a colorless oil. Rf = 0.48 (hexanes/EtOAc, 18

19 ν -1 60:40); FTIR (thin film), max (cm ): 3309, 2937, 2867, 1747, 1717, 1550, 1440, 1374, 1208, 20 21 1 22 1158, 1089, 1058; H NMR (500 MHz, CDCl3): δ 6.90 (d, J = 7.5 Hz, 1H), 4.93 (s, 1H), 23 24 25 4.63 (ddd, J = 7.5, 7.1, 5.6 Hz, 1H), 4.59 (d, J = 6.0 Hz, 1H), 4.56 (dd, J = 6.0, 0.9 Hz, 26 27 J 28 1H), 4.46 (ddd, = 11.0, 4.1, 0.9 Hz, 1H), 3.79 (s, 3H), 3.31 (s, 3H), 2.63–2.55 (m, 1H), 29 30 2.10 (d, J = 2.4 Hz, 1H), 1.97–1.89 (m, 1H), 1.80 (dddd, J = 13.8, 10.4, 7.1, 4.6 Hz, 1H), 31 32 33 1.66 (ddd, J = 13.4, 11.0, 4.1 Hz, 1H), 1.60–1.52 (m, 2H), 1.47 (s, 3H), 1.51–1.39 (m, 3H), 34 35 13 2 1 36 1.30 (s, 3H); C NMR (126 MHz, CDCl3): δ 171.4, 156.9 (q, JC-F = 38 Hz), 115.7 (q, JC-F = 37 38 288 Hz), 112.4, 110.1, 85.6, 85.6, 85.2, 84.5, 71.1, 55.3, 53.1, 52.6, 40.5, 34.4, 31.6, 28.5, 39 40 19 41 26.6, 25.1, 22.5; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.8; HRMS (ESI+): calcd. for 42 43 + ∆ 44 [C20H28F3N1O7Na] 474.1710, meas. 474.1721, 2.3 ppm. 45 46 R R R S S 47 (3 ,4 ,5 )-5-((2 ,6 )-2-ethynyl-7-methoxy-7-oxo-6-(2,2,2-trifluoroacetamido)hep- 48 49 tyl)tetrahydrofuran-2,3,4-triyl triacetate (S64). To a solution of acetonide S63 (125 mg, 50 51

52 0.277 mmol) in CH2Cl2 (1.1 mL) at 0 °C, was added a 4:1 mixture of TFA and water (5.5 53 54 55 133 56 57 58 59 60 ACS Paragon Plus Environment

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1 mL). The resulting mixture was stirred at RT for 5.5 h. Volatiles were removed in vacuo 2 3 and the residue was dried by azeotropic distillation with benzene (2 × 1.5 mL). Crude 4 5 6 material was used directly in the next step without further purification. To a solution of the 7 8 9 crude triol in CH2Cl2 (5.5 mL) at RT, were added pyridine (0.22 mL, 2.8 mmol, 10 equiv.), 10 11 Ac2O (0.26 mL, 2.8 mmol, 10 equiv.) and DMAP (24 mg, 0.19 mmol, 0.70 equiv.) sequen- 12 13 14 tially. The resulting mixture was stirred at RT for 18 h and volatiles were removed in vacuo. 15 16 17 Crude material was purified by silica gel chromatography (2,2,4-trimethylpentane/EtOAc, 18 19 5 → 70%) to afford triacetate S64 (93 mg, 0.18 mmol, 64% over 2 steps) as a 2:1 mixture 20 21

22 of diastereomers, as a colorless oil. Rf = 0.23/0.29 (hexanes/EtOAc, 50:50); FTIR (thin 23 24 ν -1 25 film), max (cm ): 3293, 2954, 2867, 1747, 1723, 1553, 1438, 1372, 1216, 1180, 1162, 1113, 26

27 1 3 J 28 1051, 1012; H NMR (500 MHz, CDCl ): δ 6.96–6.89 (m, 3H), 6.35 (d, = 4.6 Hz, 1H), 29 30 6.12 (d, J = 1.0 Hz, 2H), 5.30 (dd, J = 4.8, 1.0 Hz, 2H), 5.22 (dd, J = 6.8, 4.6 Hz, 1H), 5.17 31 32 33 (dd, J = 7.1, 4.8 Hz, 2H), 5.05 (dd, J = 6.8, 3.8 Hz, 1H), 4.65–4.60 (m, 3H), 4.45 (ddd, J = 34 35 36 10.3, 3.8, 3.3 Hz, 1H), 4.38 (ddd, J = 10.2, 7.1, 3.0 Hz, 2H), 3.79 (s, 6H), 3.79 (s, 3H), 37 38 2.63–2.55 (m, 3H), 2.12 (s, 6H), 2.11 (s, 3H), 2.10 (s, 3H), 2.09 (d, J = 2.4 Hz, 2H), 2.09 39 40 41 (d, J = 2.4 Hz, 1H), 2.07 (s, 6H), 2.07 (s, 6H), 2.05 (s, 3H), 1.99–1.89 (m, 3H), 1.85–1.73 42 43 13 44 (m, 6H), 1.66–1.53 (m, 6H), 1.53–1.37 (m, 9H); C NMR (126 MHz, CDCl3): δ 171.4, 45

46 2 2 170.2, 170.1, 169.9, 169.7, 169.5, 169.3, 156.9 (q, JC-F = 38 Hz), 156.9 (q, JC-F = 38 Hz), 47 48 1 49 115.7 (q, JC-F = 288 Hz), 98.5, 93.8, 85.3, 85.1, 81.4, 79.8, 74.6, 73.9, 72.6, 71.2, 71.1, 69.9, 50 51 52 53.1, 52.6, 40.3, 39.3, 34.4, 34.4, 31.6, 31.6, 28.5, 28.1, 22.5, 22.4, 21.2, 21.2, 20.8, 20.7, 53 54 55 134 56 57 58 59 60 ACS Paragon Plus Environment

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19 1 20.7, 20.4; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.8; HRMS (ESI+): calcd. for 2 3 + ∆ [C22H28F3N1O10Na] 546.1558, meas. 546.1565, 1.3 ppm. 4 5 6 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,6S)-2-ethynyl-7-methoxy-7- 7 8 9 oxo-6-(2,2,2-trifluoroacetamido)heptyl)tetrahydrofuran-3,4-diyl diacetate (S65). To a 10

11 6 12 suspension of N -benzoyladenine (302 mg, 1.26 mmol, 4.0 equiv.) in propionitrile (dried 13 14 over 4 Å M.S., 6.0 mL) at RT, was added N,O-bis(trimethylsilyl)acetamide (0.39 mL, 1.6 15 16 17 mmol, 5.0 equiv.). The resulting mixture was stirred at 80 °C for 10 min, upon which com- 18 19 6 20 plete solubilization of N -benzoyladenine was observed. To a separate flask charged with 21 22 triacetate S64 (dried by azeotropic distillation with benzene (4 ×), 165 mg, 0.315 mmol) 23 24 25 and 2,6-di-tert-butyl-4-methylpyridinium triflate (56 mg, 0.16 mmol, 50 mol %) was added 26 27 28 propionitrile (6.0 mL). The resulting mixture was stirred at RT for 5 min, until a clear 29 30 solution was obtained, and the solution of silylated N6-benzoyladenine was added. The 31 32 33 reaction mixture was stirred at 95 °C for 5.5 h, cooled to RT and the volatiles were removed 34 35 36 in vacuo. Crude material was purified by silica gel chromatography (2,2,4-trimethylpen- 37 38 39 tane/EtOAc, 30 → 100%) to afford nucleoside S65 (209 mg, 0.297 mmol, 94%) as a col- 40 41 ν -1 orless oil. Rf = 0.43 (hexanes/EtOAc, 20:80); FTIR (thin film), max (cm ): 3287, 2955, 2925, 42 43 44 2868, 1748, 1721, 1612, 1583, 1511, 1488, 1457, 1245, 1219, 1187, 1161, 1049; 1H NMR 45 46 47 (600 MHz, CDCl3): δ 9.13 (br s, 1H), 8.76 (s, 1H), 8.07 (s, 1H), 8.04–7.98 (m, 2H), 7.61– 48 49 7.57 (m, 1H), 7.53–7.48 (m, 2H), 7.05 (br d, J = 7.9 Hz, 1H), 6.12–6.10 (m, 2H), 5.57– 50 51 52 5.54 (m, 1H), 4.60 (ddd, J = 7.9, 7.4, 5.4 Hz, 1H), 4.50 (ddd, J = 11.0, 4.5, 2.8 Hz, 1H), 53 54 55 135 56 57 58 59 60 ACS Paragon Plus Environment

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1 3.75 (s, 3H), 2.55–2.48 (m, 1H), 2.15 (s, 3H), 2.12 (d, J = 2.4 Hz, 1H), 2.10–2.03 (m, 1H), 2 3 2.06 (s, 3H), 1.89 (app ddt, J = 13.9, 10.8, 5.4 Hz, 1H), 1.83 (ddd, J = 13.8, 11.4, 2.8 Hz, 4 5 6 1H), 1.74 (dddd, J = 13.9, 10.7, 7.4, 4.8 Hz, 1H), 1.61–1.52 (m, 1H), 1.52–1.42 (m, 2H), 7 8 13 2 9 1.42–1.33 (m, 1H); C NMR (126 MHz, CDCl3): δ 171.4, 169.8, 169.6, 164.8, 156.9 (q, JC- 10

11 1 F = 38 Hz), 152.8, 151.7, 149.9, 142.4, 133.6, 133.0, 129.0, 128.0, 124.2, 115.7 (q, JC-F = 288 12 13 14 Hz), 87.2, 85.1, 80.9, 73.7, 72.9, 71.4, 53.0, 52.5, 38.6, 34.4, 31.6, 28.1, 22.6, 20.7, 20.5; 15 16 19 + 17 F NMR (376 MHz, CDCl3, BTF IStd): δ –76.8; HRMS (ESI+): calcd. for [C32H34F3N6O9] 18 19 703.2334, meas. 703.2326, ∆ 1.1 ppm. 20 21 22 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,6S)-2-((3-car- 23 24 25 bamoylphenyl)ethynyl)-7-methoxy-7-oxo-6-(2,2,2-trifluoroacetamido)heptyl)tetra- 26 27 28 hydrofuran-3,4-diyl diacetate (S66). To a vial charged with nucleoside S65 (89 mg, 0.13 29 30 mmol) were added 3-iodobenzamide (78 mg, 0.32 mmol, 2.5 equiv.), CuI (6 mg, 0.03 31 32

33 mmol, 25 mol %) and Pd(PPh3)4 (7 mg, 6 µmol, 5 mol %). The vial headspace was purged 34 35 36 with nitrogen for 10 min and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by 37 38 39 sparging with nitrogen for 15 min, 4.2 mL) was added at RT. The resulting mixture was 40 41 stirred at 60 ◦C for 2 h. The solution was allowed to cool to RT and volatiles were removed 42 43 44 in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 45 46 47 95:5) to afford benzamide S66, which was further purified by preparative HPLC (15% → 48 49 95% B’ over 30 min) to afford benzamide S66 (69 mg, 84 µmol, 66%) as a white amor- 50 51 ν -1 52 phous solid. Rf = 0.51 (EtOAc/i-PrOH, 95:5); FTIR (thin film), max (cm ): 3279, 3071, 2928, 53 54 55 136 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 1748, 1719, 1667, 1612, 1580, 1512, 1487, 1457, 1438, 1377, 1245, 1219, 1159, 1097; H 2 3 NMR (500 MHz, CDCl3): δ 8.79 (s, 1H), 8.46 (s, 1H), 8.07–8.01 (m, 2H), 7.87 (app t, J = 4 5 6 1.8 Hz, 1H), 7.80 (ddd, J = 7.8, 1.8, 1.3 Hz, 1H), 7.64–7.60 (m, 1H), 7.55–7.49 (m, 3H), 7 8 9 7.39 (app t, J = 7.8 Hz, 1H), 7.18 (br d, J = 7.8 Hz, 1H), 7.12 (br s, 2H), 6.20 (d, J = 5.8 10 11 Hz, 1H), 6.12 (dd, J = 5.8, 5.4 Hz, 1H), 5.65 (dd, J = 5.4, 4.0 Hz, 1H), 4.63 (app td, J = 12 13 14 7.8, 5.1 Hz, 1H), 4.54 (app dt, J = 9.6, 4.0 Hz, 1H), 3.74 (s, 3H), 2.80–2.72 (m, 1H), 2.17 15 16 17 (s, 3H), 2.13 (ddd, J = 13.8, 9.6, 3.6 Hz, 1H), 2.06 (s, 3H), 2.05–1.92 (m, 2H), 1.85–1.76 18

19 13 (m, 1H), 1.71–1.45 (m, 4H); C NMR (126 MHz, CDCl3): δ 171.5, 170.7, 170.2, 169.9, 20 21 2 22 165.9, 157.2 (q, JC-F = 38 Hz), 152.0, 151.2, 149.2, 143.5, 135.3, 133.7, 132.3, 132.2, 130.9, 23 24 1 25 129.1, 128.9, 128.5, 127.5, 123.9, 122.8, 115.7 (q, JC-F = 288 Hz), 92.0, 87.4, 82.7, 81.8, 26

27 19 73.8, 72.9, 53.1, 52.7, 38.8, 34.6, 31.8, 29.0, 23.1, 20.8, 20.5; F NMR (471 MHz, CDCl3, 28 29 30 + BTF IStd): δ –76.8; HRMS (ESI+): calcd. for [C39H38F3N7O10Na] 844.2524, meas. 844.2532, 31 32 33 ∆ 0.8 ppm. 34 35 36 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((4-car- 37 38 bamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahy- 39 40 41 drofuran-3,4-diyl diacetate (S67). To a vial charged with alkyne 8 (20 mg, 29 µmol) were 42 43 44 added 4-iodobenzamide (18 mg, 73 µmol, 2.5 equiv.), CuI (1 mg, 7 µmol, 25 mol %) and 45 46 47 Pd(PPh3)4 (2 mg, 2 µmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min 48 49 and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by sparging with nitrogen for 50 51 52 15 min, 1.5 mL) was added at RT. The resulting mixture was stirred at 60 °C for 3 h. The 53 54 55 137 56 57 58 59 60 ACS Paragon Plus Environment

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1 solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material 2 3 was filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne S67, 4 5 6 which was further purified by preparative HPLC (15% → 95% B’ over 30 min) to afford 7 8 1 9 alkyne S67 (19 mg, 24 µmol, 81%) as a white amorphous solid. H NMR (500 MHz, 10 11 CD3OD): δ 8.79 (s, 1H), 8.63 (s, 1H), 8.11–8.07 (m, 2H), 7.86–7.80 (m, 2H), 7.70–7.65 12 13 14 (m, 1H), 7.60–7.55 (m, 2H), 7.48–7.44 (m, 2H), 6.34 (d, J = 5.0 Hz, 1H), 6.16 (dd, J = 5.7, 15 16 17 5.0 Hz, 1H), 5.66 (dd, J = 5.7, 5.0 Hz, 1H), 4.56 (ddd, J = 10.3, 5.0, 3.6 Hz, 1H), 4.53 (dd, 18 19 J = 9.6, 4.9 Hz, 1H), 3.73 (s, 3H), 2.92–2.84 (m, 1H), 2.23 (ddd, J = 14.0, 10.3, 3.9 Hz, 20 21 22 1H), 2.15 (s, 3H), 2.18–2.10 (m, 1H), 2.06 (s, 3H), 2.05–2.00 (m, 2H), 1.74–1.60 (m, 2H); 23 24 13 2 25 C NMR (126 MHz, CD3OD): δ 172.5, 171.6, 171.5, 171.3, 168.7, 159.1 (q, JC-F = 37 Hz), 26 27 28 153.1, 153.0, 151.0, 145.5, 134.6, 134.2, 134.2, 132.6, 129.8, 129.6, 128.7, 128.2, 125.0, 29 30 1 117.4 (q, JC-F = 287 Hz), 94.3, 88.8, 83.9, 82.2, 75.0, 74.2, 53.8, 53.1, 39.4, 32.6, 29.9, 29.7, 31 32 19 33 20.5, 20.3; F NMR (376 MHz, CD3OD, BTF IStd): δ –76.5; HRMS (ESI+): calcd. for 34 35 + ∆ 36 [C38H37F3N7O10] 808.2549, meas. 808.2556, 0.9 ppm. 37 38 R R R R H S S 39 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-((2 ,5 )-2-((2-car- 40 41 bamoylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahy- 42 43 44 drofuran-3,4-diyl diacetate (S68). To a vial charged with alkyne 8 (20 mg, 29 µmol) were 45 46 47 added 2-iodobenzamide (18 mg, 73 µmol, 2.5 equiv.), CuI (1 mg, 7 µmol, 25 mol %) and 48 49 Pd(PPh3)4 (2 mg, 2 µmol, 5 mol %). The vial headspace was purged with nitrogen for 5 min 50 51

52 and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by sparging with nitrogen for 53 54 55 138 56 57 58 59 60 ACS Paragon Plus Environment

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1 15 min, 1.5 mL) was added at RT. The resulting mixture was stirred at 60 °C for 3 h. The 2 3 solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material 4 5 6 was filtered through a short pad of silica gel (EtOAc/i-PrOH, 75:25) to afford alkyne S68, 7 8 9 which was further purified by preparative HPLC (15% → 95% B’ over 30 min) to afford 10 11 alkyne S68 (18 mg, 22 µmol, 77%) as a white amorphous solid. 1H NMR (600 MHz, 12 13

14 CD3OD): δ 8.80 (s, 1H), 8.68 (s, 1H), 8.13–8.07 (m, 2H), 7.70–7.67 (m, 1H), 7.61–7.57 15 16 17 (m, 3H), 7.48–7.45 (m, 1H), 7.42 (app td, J = 7.5, 1.6 Hz, 1H), 7.39 (app td, J = 7.5, 1.6 18 19 Hz, 1H), 6.36 (d, J = 5.2 Hz, 1H), 6.15 (dd, J = 5.7, 5.2 Hz, 1H), 5.62 (dd, J = 5.7, 5.0 Hz, 20 21 22 1H), 4.62 (ddd, J = 10.4, 5.0, 3.0 Hz, 1H), 4.52 (dd, J = 9.8, 4.9 Hz, 1H), 3.73 (s, 3H), 23 24 25 2.92–2.86 (m, 1H), 2.22 (ddd, J = 13.7, 10.4, 4.2 Hz, 1H), 2.16 (s, 3H), 2.16–2.09 (m, 1H), 26

27 13 3 28 2.07 (s, 3H), 2.08–2.01 (m, 2H), 1.72–1.61 (m, 2H); C NMR (126 MHz, CD OD): δ 173.2, 29 30 2 172.6, 171.7, 171.3, 168.6, 159.1 (q, JC-F = 38 Hz), 153.1, 152.4, 150.6, 145.5, 139.2, 134.6, 31 32 1 33 134.2, 134.1, 131.2, 129.9, 129.8, 129.6, 129.2, 128.8, 122.1, 117.4 (q, JC-F = 287 Hz), 96.7, 34 35 19 36 88.8, 82.3, 82.2, 74.9, 74.2, 53.8, 53.1, 39.2, 32.5, 30.0, 29.7, 20.6, 20.3; F NMR (471 37

38 + MHz, CD3OD, BTF IStd): δ –76.5; HRMS (ESI+): calcd. for [C38H37F3N7O10] 808.2549, 39 40 41 meas. 808.2547, ∆ 0.1 ppm. 42 43 44 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-6-methoxy-6-oxo-2-((3- 45 46 47 sulfamoylphenyl)ethynyl)-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran-3,4- 48 49 diyl diacetate (S69). To a solution of alkyne 8 (41 mg, 59 µmol) and 3-iodobenzenesul- 50 51

52 fonamide (25 mg, 89 µmol, 1.5 equiv.) in a 5:1:1 mixture of toluene, DMF and i-Pr2NEt 53 54 55 139 56 57 58 59 60 ACS Paragon Plus Environment

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1 (degassed by sparging with nitrogen for 15 min, 2.0 mL) at RT, were introduced CuI (3 2 3 mg, 0.01 mmol, 25 mol %) and Pd(PPh3)4 (3 mg, 3 µmol, 5 mol %) rapidly. The resulting 4 5 6 mixture was stirred at 50 °C for 17 h. The solution was allowed to cool to RT and volatiles 7 8 9 were removed in vacuo. Crude material was purified by silica gel chromatography 10 11 (EtOAc/i-PrOH, 0 → 10%) to afford alkyne S69 (41 mg, 49 µmol, 82%) as a colorless oil. 12 13 ν -1 14 Rf = 0.29 (EtOAc/i-PrOH, 95:5); FTIR (thin film), max (cm ): 3301, 3074, 2924, 1748, 1721, 15 16 1 17 1612, 1583, 1456, 1336, 1245, 1219, 1162, 1097; H NMR (600 MHz, CDCl3): δ 9.11 (s, 18 19 1H), 8.75 (s, 1H), 8.16 (s, 1H), 8.02–7.96 (m, 2H), 7.85 (app t, J = 1.2 Hz, 1H), 7.77 (app 20 21 22 dt, J = 7.8, 1.2 Hz, 1H), 7.62–7.57 (m, 1H), 7.53–7.48 (m, 2H), 7.47 (app dt, J = 7.8, 1.2 23 24 25 Hz, 1H), 7.37 (app t, J = 7.8 Hz, 1H), 7.27 (br d, J = 7.7 Hz, 1H), 6.15 (d, J = 5.7 Hz, 1H), 26 27 J J 28 6.13 (dd, = 5.7, 5.3 Hz, 1H), 5.63 (br s, 2H), 5.62 (dd, = 5.3, 4.2 Hz, 1H), 4.65 (app td, 29 30 J = 7.7, 5.5 Hz, 1H), 4.47 (ddd, J = 9.7, 4.2, 3.5 Hz, 1H), 3.77 (s, 3H), 2.86–2.81 (m, 1H), 31 32 33 2.21–2.14 (m, 1H), 2.16 (s, 3H), 2.10 (app ddt, J = 14.0, 11.1, 5.5 Hz, 1H), 2.06 (s, 3H), 34 35 13 36 2.05–1.98 (m, 2H), 1.69–1.60 (m, 1H), 1.60–1.52 (m, 1H); C NMR (126 MHz, CDCl3): δ 37

38 2 171.3, 170.1, 169.8, 165.0, 157.2 (q, JC-F = 38 Hz), 152.8, 151.8, 149.8, 142.7, 142.6, 135.2, 39 40 1 41 133.4, 133.1, 129.8, 129.1, 129.0, 128.1, 125.5, 124.4, 124.1, 115.7 (q, JC-F = 288 Hz), 92.7, 42 43 19 44 87.0, 82.2, 81.1, 73.6, 72.8, 53.3, 52.5, 38.1, 30.8, 29.8, 28.6, 20.8, 20.6; F NMR (471 45

46 + MHz, CDCl3, BTF IStd): δ –76.6; HRMS (ESI+): calcd. for [C37H37F3N7O11S1] 844.2218, 47 48 49 meas. 844.2198, ∆ 2.4 ppm. 50 51 52 53 54 55 140 56 57 58 59 60 ACS Paragon Plus Environment

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1 2-fluoro-5-iodobenzamide (S70). To a vial charged with 2-fluoro-5-iodobenzonitrile 2 3 (0.99 g, 4.0 mmol) was added H2SO4 (98%, 4.0 mL). The resulting mixture was stirred at 4 5 6 70 °C for 2 h, cooled to 0 °C, before the addition of a 1 N NaOH aq. solution (15 mL) and 7 8 9 a NaHCO3 sat. aq. solution (5 mL). The resulting white precipitate was filtered to yield 2- 10 11 fluoro-5-iodobenzamide (S70) as a pale orange, off-white powder (1.1 g, 4.0 mmol, 12 13 ν -1 14 quant.). Rf = 0.43 (hexanes/EtOAc, 50:50); FTIR (thin film), max (cm ): 3361, 3305, 3165, 15 16 1 17 1710, 1656, 1625, 1598, 1564, 1475, 1427, 1363, 1254, 1228; H NMR (500 MHz, 18 19 CD3OD): δ 8.09 (1H, dd, J = 6.8, 2.4 Hz), 7.85 (1H, ddd, J = 8.7, 4.7, 2.4 Hz), 7.03 (1H, 20 21 13 22 dd, J = 10.7, 8.7 Hz); C NMR (101 MHz, CD3OD): δ 166.9, 161.5 (d, JC-F = 251 Hz), 143.3 23 24 25 (d, JC-F = 8.7 Hz), 140.5 (d, JC-F = 2.6 Hz), 125.7 (d, JC-F = 15 Hz), 119.6 (d, JC-F = 25 Hz), 88.0 26

27 19 (d, JC-F = 3.7 Hz); F NMR (471 MHz, CD3OD, BTF IStd): δ –116.3; HRMS (ESI+): calcd. 28 29 30 + ∆ for [C7H5F1I1N1O1Na] 287.9292, meas. 287.9283, 3.1 ppm. 31 32 33 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4-fluor- 34 35 36 ophenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofu- 37 38 ran-3,4-diyl diacetate (S71). 8 39 To a vial charged with nucleoside (31 mg, 46 µmol) were 40 41 added 2-fluoro-5-iodobenzamide (S70) (20 mg, 75 µmol, 1.65 equiv.), CuI (2 mg, 9 µmol, 42 43 44 20 mol %) and Pd(PPh3)4 (3 mg, 2 µmol, 5 mol %). The vial headspace was purged with 45 46 47 nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed by sparging 48 49 with nitrogen for 15 min, 3.5 mL) was added at RT. The resulting mixture was stirred at 50 51 52 60 °C for 1 h. More 2-fluoro-5-iodobenzamide (10 mg, 38 µmol, 0.83 equiv.) in a 5:1:1 53 54 55 141 56 57 58 59 60 ACS Paragon Plus Environment

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1 mixture of toluene, DMF and i-Pr2NEt (2.0 mL) was added and the reaction mixture was 2 3 stirred at 60 °C for another 3 h. The solution was allowed to cool to RT and volatiles were 4 5 6 removed in vacuo. Crude material was purified by silica gel chromatography (EtOAc/i- 7 8 9 PrOH, 0 → 10%) to afford alkyne S71 (34 mg, 41 µmol, 91%) as a white amorphous solid. 10 11 ν -1 FTIR (thin-film), max (cm ): 3256, 3082, 3060, 3026, 2924, 2853, 1748, 1719, 1672, 1611, 12 13 14 1583, 1513, 1491, 1453, 1366, 1245, 1216, 1183, 1159, 1110, 1074, 1048, 1029; 1H NMR 15 16 17 (600 MHz, CDCl3): δ 9.02 (s, 1H), 8.80 (s, 1H), 8.14 (dd, J = 7.5, 2.3 Hz, 1H), 8.12 (s, 18 19 1H), 8.03 (d, J = 7.4 Hz, 2H), 7.69–7.59 (m, 1H), 7.55–7.44 (m, 3H), 7.09 (dd, J = 11.5, 20 21 22 8.5 Hz, 1H), 7.05 (br d, J = 7.8 Hz, 1H), 6.68 (br d, J = 10.9 Hz, 1H), 6.14 (d, J = 5.4 Hz, 23 24 25 1H), 6.11 (t, J = 5.4 Hz, 1H), 5.95 (br s, 1H), 5.60 (t, J = 5.0 Hz, 1H), 4.65 (td, J = 7.7, 5.0 26 27 J J 28 Hz, 1H), 4.50 (ddd, = 10.7, 4.6, 3.0 Hz, 1H), 3.79 (s, 3H), 2.82 (ddt, = 10.9, 9.1, 4.5 29 30 Hz, 1H), 2.17 (s, 3H), 2.18–2.08 (m, 2H), 2.08 (s, 3H), 2.04–1.92 (m, 2H), 1.64 (ddd, J = 31 32 33 18.6, 9.3, 5.4 Hz, 1H), 1.52 (dddd, J = 13.5, 11.0, 9.4, 4.6 Hz, 1H); 13C NMR (101 MHz, 34 35 36 CDCl3): δ 171.3, 169.9, 169.7, 164.7, 164.1, 164.1, 161.6, 159.1, 157.3, 156.9, 156.5, 37 38 152.9, 151.7, 149.9, 142.4, 137.1, 137.0, 135.8, 135.8, 133.6, 133.0, 132.3, 132.2, 132.1, 39 40 41 132.1, 129.0, 128.7, 128.6, 128.0, 124.1, 120.5, 120.5, 120.4, 120.3, 117.2, 116.7, 116.4, 42 43 44 114.3, 91.1, 91.0, 87.2, 81.9, 80.9, 77.4, 73.7, 73.0, 53.3, 52.5, 38.6, 31.0, 30.0, 28.7, 23.6, 45

46 19 20.8, 20.6; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.7, –113.0; HRMS (ESI+): calcd. 47 48 + ∆ 49 for [C38H35F4N7O10Na] 848.2272, meas. 848.2274, 0.2 ppm. 50 51 52 53 54 55 142 56 57 58 59 60 ACS Paragon Plus Environment

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77 1 5-iodo-2-methylbenzamide (S72). To a vial charged with 5-iodo-2-methylbenzonitrile 2 3 (0.12 g, 0.50 mmol) and t-BuOK (0.17 g, 1.5 mmol, 3.0 equiv.) was added t-BuOH (4.5 4 5 6 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 60 °C for 24 h, 7 8 9 before the addition of water (10 mL) and EtOAc (20 mL). The layers were separated and 10 11 the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic extracts 12 13

14 were washed with brine (80 mL), dried over MgSO4, filtered and concentrated. Crude ma- 15 16 17 terial was dissolved in hot toluene, recrystallized and washed with hexanes to yield 5-iodo- 18 19 2-methylbenzamide (S72) (85 mg, 0.33 mmol, 65% yield) as pale yellow crystals. Rf = 0.20 20 21 ν -1 22 (hexanes/EtOAc, 50:50); FTIR (thin film), max (cm ): 3371, 3187, 1649, 1582, 1559, 1401, 23 24 1 25 1373; H NMR (600 MHz, CDCl3): δ 7.77 (d, J = 1.8 Hz, 1H), 7.65 (dd, J = 8.2, 1.8 Hz, 26 27 1H), 6.99 (d, J = 8.2 Hz, 1H), 5.68 (br s, 1H), 5.62 (br s, 1H), 2.43 (s, 3H); 13C NMR (126 28 29 30 MHz, CDCl3): δ 170.2, 139.3, 137.3, 136.2, 135.7, 133.2, 90.2, 19.7; HRMS (ESI+): calcd. 31 32 + ∆ 33 for [C8H9I1N1O1] 261.9723, meas. 261.9721, 0.9 ppm. 34 35 36 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4- 37 38 methylphenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahy- 39 40 41 drofuran-3,4-diyl diacetate (S73). To a solution of alkyne 8 (50 mg, 73 µmol) and 5- 42 43 44 iodo-2-methylbenzamide (S72) (47 mg, 0.18 mmol, 2.5 equiv.) in a 5:1:1 mixture of tolu- 45 46 47 ene, DMF and i-Pr2NEt (degassed by sparging with nitrogen for 15 min, 3.6 mL) at RT, 48 49 were introduced CuI (3 mg, 0.02 mmol, 25 mol %) and Pd(PPh3)4 (4 mg, 4 µmol, 5 mol %) 50 51 52 rapidly. The resulting mixture was stirred at 60 °C for 2 h. The solution was allowed to 53 54 55 143 56 57 58 59 60 ACS Paragon Plus Environment

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1 cool to RT and volatiles were removed in vacuo. Crude material was filtered through a 2 3 short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne S73, which was further pu- 4 5 6 rified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne S73 (40 mg, 49 7 8 ν -1 9 µmol, 67%) as a white amorphous solid. Rf = 0.34 (EtOAc); FTIR (thin film), max (cm ): 10 11 3341, 1747, 1721, 1668, 1612, 1584, 1457, 1376, 1246, 1220, 1099, 1075, 1052; 1H NMR 12 13

14 (500 MHz, CDCl3): δ 9.28 (s, 1H), 8.74 (s, 1H), 8.13 (s, 1H), 8.04–7.98 (m, 2H), 7.61– 15 16 17 7.56 (m, 1H), 7.52–7.47 (m, 2H), 7.46 (d, J = 1.8 Hz, 1H), 7.42 (br d, J = 7.7 Hz, 1H), 7.28 18 19 (dd, J = 7.8, 1.8 Hz, 1H), 7.11 (br d, J = 7.8 Hz, 1H), 6.32 (br s, 1H), 6.16 (br s, 1H), 6.13 20 21 22 (d, J = 5.6 Hz, 1H), 6.11 (dd, J = 5.6, 5.1 Hz, 1H), 5.61 (dd, J = 5.1, 4.2 Hz, 1H), 4.64 (app 23 24 25 td, J = 7.7, 5.2 Hz, 1H), 4.48 (ddd, J = 10.0, 4.2, 3.6 Hz, 1H), 3.75 (s, 3H), 2.79 (dddd, J 26 27 28 = 10.9, 9.3, 4.8, 4.0 Hz, 1H), 2.42 (s, 3H), 2.13 (s, 3H), 2.17–2.07 (m, 2H), 2.05 (s, 3H), 29 30 2.07–1.99 (m, 1H), 1.96 (ddd, J = 13.9, 10.9, 3.6 Hz, 1H), 1.63 (dddd, J = 13.4, 10.8, 5.8, 31 32 13 33 4.8 Hz, 1H), 1.53 (dddd, J = 13.4, 10.6, 9.3, 4.8 Hz, 1H); C NMR (126 MHz, CDCl3): δ 34 35 2 36 171.4, 171.4, 170.0, 169.6, 165.0, 157.1 (q, JC-F = 38 Hz), 152.8, 151.8, 149.9, 142.4, 136.8, 37

38 1 135.2, 133.5, 133.1, 133.0, 131.3, 130.6, 128.9, 128.1, 124.2, 120.6, 115.7 (q, JC-F = 288 39 40 41 Hz), 90.5, 86.9, 83.0, 81.2, 73.7, 72.8, 53.2, 52.5, 38.5, 31.0, 29.8, 28.7, 20.8, 20.5, 20.1; 42 43 19 + 44 F NMR (471 MHz, CDCl3, BTF IStd): δ –76.6; HRMS (ESI+): calcd. for [C39H39F3N7O10] 45 46 822.2705, meas. 822.2718, ∆ 1.6 ppm. 47 48 49 5-iodo-2-(trifluoromethyl)benzamide (S74). To a vial charged with 5-iodo-2-(trifluo- 50 51 52 romethyl)benzonitrile (0.30 g, 1.0 mmol) and t-BuOK (0.67 g, 6.0 mmol, 6.0 equiv.) was 53 54 55 144 56 57 58 59 60 ACS Paragon Plus Environment

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1 added t-BuOH (5.0 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 2 3 60 °C for 5 min, cooled to RT and stirred for 48 h. The reaction was quenched by the 4 5 6 addition of water (10 mL). The resulting suspension was filtered to produce an orange 7 8 9 solid. Crude material was purified by silica gel chromatography (hexanes/EtOAc, 0 → 10 11 100%) to yield 5-iodo-2-(trifluoromethyl)benzamide (S74) as a white solid (0.13 g, 0.41 12 13 ν -1 14 mmol, 41%). Rf = 0.46 (hexanes/EtOAc, 50:50); FTIR (thin film), max (cm ): 3369, 3183, 15 16 1 17 1649, 1588, 1569, 1400, 1378, 1311, 1287; H NMR (500 MHz, CD3OD): δ 8.01 (1H, ddq, 18 19 J = 8.3, 1.7, 0.9 Hz), 7.95–7.92 (1H, m), 7.50 (1H, br d, J = 8.3 Hz); 13C NMR (126 MHz, 20 21

22 CD3OD): δ 171.3, 140.3, 138.6 (q, JC-F = 2.1 Hz), 138.3, 129.0 (q, JC-F = 4.8 Hz), 127.7 (q, JC- 23 24 19 25 F = 32.6 Hz), 125.1 (q, JC-F = 273 Hz), 99.4 (q, JC-F = 1.4 Hz); F NMR (471 MHz, CD3OD, 26

27 + 8 5 3 1 1 1 28 BTF IStd): δ –60.3; HRMS (ESI+): calcd. for [C H F I N O Na] 337.9260, meas. 337.9260, 29 30 ∆ 0.0 ppm. 31 32 33 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4-(tri- 34 35 36 fluoromethyl)phenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetam- 37 38 39 ido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S75). To a vial charged with alkyne 8 (82 40 41 mg, 0.12 mmol) were added 5-iodo-2-(trifluoromethyl)benzamide (S74) (62 mg, 0.20 42 43

44 mmol, 1.65 equiv.), CuI (5 mg, 24 µmol, 20 mol %) and Pd(PPh3)4 (7 mg, 6 µmol, 5 mol 45 46 47 %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, 48 49 DMF and i-Pr2NEt (degassed by sparging with nitrogen for 15 min, 3.5 mL) was added at 50 51 52 RT. The resulting mixture was stirred at 70 °C for 3 h. The solution was allowed to cool to 53 54 55 145 56 57 58 59 60 ACS Paragon Plus Environment

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1 RT and volatiles were removed in vacuo. Crude material was purified by silica gel chro- 2 3 matography (EtOAc/i-PrOH, 0 → 5%) to afford alkyne S75 (90 mg, 0.10 mmol, 86%) as 4 5 ν -1 6 a white amorphous solid. Rf = 0.48 (PhMe/MeCN, 50:50); FTIR (thin-film), max (cm ): 7 8 9 3306, 2926, 2165, 1747, 1719, 1672, 1609, 1582, 1511, 1487, 1456, 1410, 1373, 1313, 10

11 1 1289, 1242, 1216, 1175, 1133, 1114, 1074, 1039; H NMR (500 MHz, CDCl3): δ 8.98 (s, 12 13 14 1H), 8.78 (s, 1H), 8.11 (s, 1H), 8.04–7.98 (m, 2H), 7.63–7.59 (m, 2H), 7.58 (dd, J = 1.6, 15 16 17 0.8 Hz, 1H), 7.57–7.49 (m, 2H), 7.48 (ddt, J = 8.2, 1.8, 0.9 Hz, 1H), 7.13 (d, J = 7.7 Hz, 18 19 1H), 6.20 (br s, 1H), 6.16–6.12 (m, 2H), 6.05 (br s, J = 2.7 Hz, 1H), 5.62 (td, J = 4.2, 0.8 20 21 22 Hz, 1H), 4.65 (td, J = 7.6, 5.2 Hz, 1H), 4.46 (dt, J = 10.3, 3.5 Hz, 1H), 2.86 (ddt, J = 10.6, 23 24 25 9.0, 4.5 Hz, 1H), 2.23–2.17 (m, 1H), 2.16 (s, 3H), 2.12 (ddd, J = 10.7, 8.4, 5.4 Hz, 1H), 26 27 J J 28 2.07 (s, 3H), 2.04–1.96 (m, 2H), 1.66 (ddt, = 13.1, 10.7, 5.4 Hz, 1H), 1.56 (ddd, = 8.9, 29 30 13 6.3, 4.6 Hz, 1H); C NMR (101 MHz, CDCl3): δ 171.3, 170.0, 169.6, 169.3, 165.0, 157.2 31 32 2 33 (q, JC-F = 38 Hz), 152.7, 151.7, 149.9, 142.5, 135.2, 133.4, 133.0, 132.8, 132.2, 132.1, 131.7, 34 35 2 1 36 128.9, 128.6, 128.5, 128.1, 127.4, 126.6, 126.5, 126.3 (q, JC-F = 32 Hz), 124.7, 123.4 (q, JC- 37

38 1 F = 274 Hz), 115.7 (q, JC-F = 287 Hz), 94.4, 87.0, 81.7, 81.0, 77.3, 73.6, 72.7, 53.1, 52.4, 38.1, 39 40 19 41 30.8, 29.7, 28.6, 20.7, 20.5; F NMR (471 MHz, CDCl3, BTF IStd): δ –60.0, –76.7; HRMS 42 43 + ∆ 44 (ESI+): calcd. for [C39H36F6N7O10] 876.2422, meas. 876.2426, 0.4 ppm. 45 46 47 2-chloro-5-iodobenzamide (S76). To a vial charged with 2-chloro-5-iodobenzonitrile 48 49 (1.32 g, 5.01 mmol) and t-BuOK (1.69 g, 15.0 mmol, 3.0 equiv.) was added t-BuOH (10.0 50 51 52 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 60 °C for 5 min, 53 54 55 146 56 57 58 59 60 ACS Paragon Plus Environment

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1 cooled to RT and stirred for 1 h. The reaction was quenched by the addition of water (10 2 3 mL). The solid amide was filtered and recrystallized from absolute ethanol to give 2- 4 5

6 chloro-5-iodobenzamide (S76) as a white solid (896 mg, 3.18 mmol, 64%). Rf = 0.35 (hex- 7 8 ν -1 9 anes/EtOAc, 50:50); FTIR (thin film), max (cm ): 3373, 3180, 1655, 1618, 1462, 1397, 10

11 1 1362; H NMR (500 MHz, CD3OD): δ 7.82 (1H, d, J = 2.2 Hz), 7.76 (1H, dd, J = 8.5, 2.2 12 13 13 14 Hz), 7.24 (1H, d, J = 8.5 Hz); C NMR (151 MHz, CD3OD): δ 170.5, 141.1, 139.2, 138.6, 15 16 + ∆ 17 132.9, 131.8, 92.0; HRMS (ESI+): calcd. for [C7H6Cl1I1N1O1] 281.9177, meas. 281.9188, 18 19 3.9 ppm. 20 21 22 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((3-carbamoyl-4-chlo- 23 24 25 rophenyl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofu- 26 27 28 ran-3,4-diyl diacetate (S77). To a solution of alkyne 8 (50 mg, 73 µmol) and 2-chloro-5- 29 30 iodobenzamide (S76) (51 mg, 0.18 mmol, 2.5 equiv.) in a 5:1:1 mixture of toluene, DMF 31 32

33 and i-Pr2NEt (degassed by sparging with nitrogen for 15 min, 3.6 mL) at RT, were intro- 34 35 36 duced CuI (3 mg, 0.02 mmol, 25 mol %) and Pd(PPh3)4 (4 mg, 4 µmol, 5 mol %) rapidly. 37 38 39 The resulting mixture was stirred at 70 °C for 3 h. The solution was allowed to cool to RT 40 41 and volatiles were removed in vacuo. Crude material was filtered through a short pad of 42 43 44 silica gel (EtOAc/i-PrOH, 90:10) to afford alkyne S77, which was further purified by pre- 45 46 47 parative HPLC (15% → 95% B over 30 min) to afford alkyne S77 (45 mg, 53 µmol, 74%) 48 49 ν -1 as a white amorphous solid. Rf = 0.48 (EtOAc/i-PrOH, 95:5); FTIR (thin film), max (cm ): 50 51 52 3320, 3085, 1748, 1719, 1673, 1613, 1583, 1456, 1374, 1246, 1219, 1047; 1H NMR (600 53 54 55 147 56 57 58 59 60 ACS Paragon Plus Environment

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1 MHz, CDCl3): δ 9.29 (s, 1H), 8.74 (s, 1H), 8.12 (s, 1H), 8.04–7.97 (m, 2H), 7.69 (d, J = 2 3 2.0 Hz, 1H), 7.60–7.56 (m, 1H), 7.50–7.46 (m, 3H), 7.31 (dd, J = 8.3, 2.0 Hz, 1H), 7.28 4 5 6 (d, J = 8.3 Hz, 1H), 6.65 (s, 1H), 6.48 (s, 1H), 6.16–6.08 (m, 2H), 5.58 (app t, J = 4.5 Hz, 7 8 9 1H), 4.63 (app td, J = 7.8, 5.1 Hz, 1H), 4.46 (ddd, J = 10.5, 4.5, 3.1 Hz, 1H), 3.75 (s, 3H), 10 11 2.80 (dddd, J = 11.1, 9.1, 5.3, 4.6 Hz, 1H), 2.14 (s, 3H), 2.19–2.11 (m, 1H), 2.12–2.06 (m, 12 13 14 1H), 2.05 (s, 3H), 2.03–1.96 (m, 1H), 1.94 (ddd, J = 13.8, 11.1, 3.1 Hz, 1H), 1.63 (app ddt, 15 16 13 17 J = 13.0, 10.8, 5.3 Hz, 1H), 1.58–1.49 (m, 1H); C NMR (126 MHz, CDCl3): δ 171.4, 18

19 2 170.0, 169.7, 167.7, 165.0, 157.2 (q, JC-F = 38 Hz), 152.8, 151.7, 149.9, 142.5, 134.4, 134.1, 20 21 1 22 133.6, 133.5, 133.0, 130.5, 130.5, 128.9, 128.1, 124.1, 122.5, 115.7 (q, JC-F = 288 Hz), 92.5, 23 24 25 87.1, 81.9, 80.9, 73.6, 72.8, 53.2, 52.5, 38.3, 30.9, 29.8, 28.7, 20.8, 20.5; 19F NMR (471 26

27 + MHz, CDCl3, BTF IStd): δ –76.6; HRMS (ESI+): calcd. for [C38H36Cl1F3N7O10] 842.2159, 28 29 30 meas. 842.2168, ∆ 1.0 ppm. 31 32 33 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-6-methoxy-6-oxo-2-((1- 34 35 36 oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)ethynyl)-5-(2,2,2-trifluoroacetam- 37 38 ido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S78) 8 39 . A vial was charged with alkyne 40 41 (40 mg, 58 µmol), 7-bromo-3,4-dihydroisoquinolin-1(2H)-one (33 mg, 0.15 mmol, 2.5 42 43 44 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol %). The vial headspace was purged with nitro- 45 46 47 gen for 5 min and a 5:1:1.5 mixture of toluene, DMF and i-Pr2NH (degassed by sparging 48 49 with nitrogen for 15 min, 1.5 mL) was added at RT. The resulting mixture was further 50 51

52 degassed by sparging with nitrogen for 5 min and a solution of Pd(Pt-Bu3)2 (3 mg, 6 µmol, 53 54 55 148 56 57 58 59 60 ACS Paragon Plus Environment

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1 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and i-Pr2NH (1.5 mL) was added. The 2 3 resulting mixture was stirred at 55 °C for 6 h. The solution was allowed to cool to RT and 4 5 6 volatiles were removed in vacuo. Crude material was filtered through a short pad of silica 7 8 9 gel (EtOAc/i-PrOH, 70:30) to afford alkyne S78, which was further purified by preparative 10 11 HPLC (15% → 95% B over 30 min) to afford alkyne S78 (29 mg, 35 µmol, 60%) as a 12 13 ν -1 14 white amorphous solid. FTIR (thin film), max (cm ): 3262, 3082, 2938, 1749, 1720, 1681, 15 16 1 17 1612, 1579, 1513, 1482, 1456, 1373, 1243, 1218, 1097, 1048; H NMR (600 MHz, CDCl3): 18 19 δ 9.16 (s, 1H), 8.78 (s, 1H), 8.78 (s, 1H), 8.13 (s, 1H), 8.05–8.00 (m, 2H), 7.62–7.58 (m, 20 21 22 1H), 7.54–7.49 (m, 2H), 7.24 (br d, J = 7.7 Hz, 1H), 7.08 (d, J = 7.8 Hz, 1H), 7.01 (dd, J 23 24 25 = 7.8, 1.2 Hz, 1H), 6.83 (d, J = 1.2 Hz, 1H), 6.14 (d, J = 5.6 Hz, 1H), 6.11 (dd, J = 5.6, 5.3 26 27 J J J 28 Hz, 1H), 5.59 (dd, = 5.3, 4.3 Hz, 1H), 4.64 (app td, = 7.7, 5.6 Hz, 1H), 4.49 (ddd, = 29 30 10.4, 4.3, 3.1 Hz, 1H), 3.76 (s, 3H), 2.93 (app dd, J = 8.5, 6.6 Hz, 2H), 2.82–2.76 (m, 1H), 31 32 33 2.61 (app dd, J = 8.5, 6.6 Hz, 2H), 2.17–2.10 (m, 1H), 2.15 (s, 3H), 2.10–2.05 (m, 1H), 34 35 36 2.06 (s, 3H), 2.04–1.97 (m, 1H), 1.96 (ddd, J = 13.8, 11.0, 3.1 Hz, 1H), 1.64 (app ddt, J = 37

38 13 12.9, 10.6, 5.3 Hz, 1H), 1.57–1.49 (m, 1H); C NMR (126 MHz, CDCl3): δ 171.8, 171.4, 39 40 2 41 169.9, 169.6, 164.9, 157.1 (q, JC-F = 38 Hz), 152.8, 151.8, 150.0, 142.4, 137.4, 133.5, 133.0, 42 43 1 44 129.0, 128.1, 128.0, 126.7, 124.2, 124.0, 122.5, 118.4, 115.7 (q, JC-F = 288 Hz), 90.4, 87.0, 45 46 83.1, 81.1, 73.8, 72.9, 53.2, 52.5, 38.7, 31.1, 30.6, 30.0, 28.8, 25.4, 20.8, 20.6; 19F NMR 47 48 + 49 (471 MHz, CDCl3, BTF IStd): δ –76.6; HRMS (ESI+): calcd. for [C40H39F3N7O10] 834.2705, 50 51 52 meas. 834.2702, ∆ 0.3 ppm. 53 54 55 149 56 57 58 59 60 ACS Paragon Plus Environment

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d 1 7-iodobenzo[ ][1,3]dioxole-5-carboxamide (S80). To a solution of known aldehyde 7- 2 3 iodobenzo[d][1,3]dioxole-5-carbaldehyde (S79)78-79 (500 mg, 1.81 mmol) in t-BuOH (20 4 5

6 mL) at RT, was added a solution of NaClO2 (328 mg, 3.62 mmol, 2.0 equiv.) and NaH2PO4 7 8 9 (0.11 g, 0.91 mmol, 0.50 equiv.) in water (4 mL). The resulting pale yellow solution was 10 11 cooled to 0 °C and 2-methyl-2-butene (4.0 mL, 38 mmol, 21 equiv.) was added dropwise. 12 13

14 The reaction mixture was stirred at RT for 20 h, upon which more NaClO2 (164 mg, 1.81 15 16 17 mmol, 1.0 equiv.) and NaH2PO4 (54 mg, 0.45 mmol, 0.25 equiv.) were added as a solution 18 19 in water (2 mL). The resulting mixture was stirred at RT for 72 h and diluted with a 10% 20 21

22 H3PO4 aq. solution (10 mL). Water (20 mL) and EtOAc (40 mL) were added. The layers 23 24 25 were separated and the aqueous phase was extracted with EtOAc (2 × 40 mL). The com- 26 27 bined organic extracts were washed with brine (80 mL), dried over Na2SO4, filtered and 28 29 30 concentrated to give a white solid. Crude material was used directly in the next step without 31 32 33 further purification. To a flask charged with the crude carboxylic acid and HATU (758 mg, 34 35 36 1.99 mmol, 1.1 equiv.) were added DMF (20 mL) and i-Pr2NEt (0.63 mL, 3.6 mmol, 2.0 37 38 equiv.) at RT. The resulting brown mixture was stirred for 30 min and NH4Cl (116 mg, 2.18 39 40 41 mmol, 1.2 equiv.) was added. The flask headspace was flushed with nitrogen and the reac- 42 43 44 tion mixture was stirred for 24 h, before the addition of EtOAc (30 mL) and water (20 mL). 45 46 The layers were separated and the aqueous phase was extracted with EtOAc (3 × 30 mL). 47 48 49 The combined organic extracts were washed with a 5% LiCl aq. solution (100 mL) and 50 51 52 53 54 55 150 56 57 58 59 60 ACS Paragon Plus Environment

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× 1 brine (2 100 mL), dried over Na2SO4, filtered and concentrated. Crude material was pu- 2 3 rified by silica gel chromatography (hexanes/EtOAc, 5 → 100%) to afford iodobenzamide 4 5

6 S80 (348 mg, 1.20 mmol, 66% over 2 steps) as a white solid. Rf = 0.15 (hexanes/EtOAc, 7 8 ν -1 9 50:50); FTIR (thin film), max (cm ): 3355, 3187, 2924, 2854, 1658, 1592, 1479, 1424, 1254, 10

11 1 1044; H NMR (500 MHz, CD3OD): δ 7.77 (d, J = 1.6 Hz, 1H), 7.31 (d, J = 1.6 Hz, 1H), 12 13 13 14 6.11 (s, 2H); C NMR (101 MHz, CD3OD): δ 170.1, 153.9, 148.1, 132.3, 130.7, 108.7, 15 16 + ∆ 17 103.0, 70.6; HRMS (ESI+): calcd. for [C8H7I1N1O3] 291.9465, meas. 291.9461, 1.4 ppm. 18 19 R R R R H S S 20 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-((2 ,5 )-2-((6-car- 21 22 bamoylbenzo[d][1,3]dioxol-4-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetam- 23 24 25 ido)hexyl)tetrahydrofuran-3,4-diyl diacetate (S81). To a vial charged with alkyne 8 (30 26 27 28 mg, 44 µmol) were added iodobenzamide S80 (32 mg, 0.11 mmol, 2.5 equiv.), CuI (2 mg, 29 30 0.01 mmol, 25 mol %) and Pd(PPh3)4 (3 mg, 2 µmol, 5 mol %). The vial headspace was 31 32

33 purged with nitrogen for 5 min and a 5:1:1 mixture of toluene, DMF and i-Pr2NEt (degassed 34 35 36 by sparging with nitrogen for 15 min, 2.3 mL) was added at RT. The resulting mixture was 37 38 39 stirred at 60 °C for 3 h. The solution was allowed to cool to RT and volatiles were removed 40 41 in vacuo. Crude material was filtered through a short pad of silica gel (EtOAc/i-PrOH, 42 43 44 80:20) to afford alkyne S81, which was further purified by preparative HPLC (15% → 45 46 47 95% B’ over 30 min) to afford alkyne S81 (27 mg, 32 µmol, 73%) as a white amorphous 48 49 ν -1 solid. FTIR (thin film), max (cm ): 3351, 3222, 3077, 2937, 1746, 1717, 1659, 1593, 1424, 50 51 1 52 1243, 1203, 1045; H NMR (500 MHz, CD3OD): δ 8.80 (s, 1H), 8.70 (s, 1H), 8.12–8.06 53 54 55 151 56 57 58 59 60 ACS Paragon Plus Environment

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1 (m, 2H), 7.70–7.65 (m, 1H), 7.60–7.55 (m, 2H), 7.45 (d, J = 1.7 Hz, 1H), 7.28 (d, J = 1.7 2 3 Hz, 1H), 6.34 (d, J = 5.0 Hz, 1H), 6.14 (dd, J = 5.7, 5.0 Hz, 1H), 6.10 (d, J = 1.1 Hz, 1H), 4 5 6 6.10 (d, J = 1.1 Hz, 1H), 5.65 (dd, J = 5.7, 5.1 Hz, 1H), 4.58 (ddd, J = 10.2, 5.1, 3.2 Hz, 7 8 9 1H), 4.52 (dd, J = 9.6, 5.0 Hz, 1H), 3.73 (s, 3H), 2.93–2.86 (m, 1H), 2.22 (ddd, J = 14.1, 10 11 10.2, 4.1 Hz, 1H), 2.15 (s, 3H), 2.17–2.10 (m, 1H), 2.06 (s, 3H), 2.07–1.98 (m, 2H), 1.72– 12 13 13 14 1.58 (m, 2H); C NMR (126 MHz, CD3OD): δ 172.5, 171.6, 171.3, 170.7, 168.7, 159.1 (q, 15 16 2 17 JC-F = 38 Hz), 153.0, 152.9, 152.2, 150.4, 149.4, 145.6, 134.4, 134.3, 129.8, 129.6, 128.9, 18

19 1 126.7, 124.3, 117.0 (q, JC-F = 288 Hz), 108.4, 105.6, 103.9, 96.7, 88.9, 82.2, 77.8, 74.9, 74.3, 20 21 19 22 53.7, 53.1, 39.3, 32.5, 29.9, 29.6, 20.5, 20.3; F NMR (471 MHz, CDCl3, BTF IStd): δ – 23 24 + ∆ 25 76.7; HRMS (ESI+): calcd. for [C39H37F3N7O12] 852.2447, meas. 852.2483, 4.3 ppm. 26 27 R R R R H S S 28 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-((2 ,5 )-2-((6-carbamoylpyridin- 29 30 2-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran- 31 32 33 3,4-diyl diacetate (S82). A vial was charged with alkyne 8 (35 mg, 51 µmol), 6-bro- 34 35 36 mopyridine-2-carboxamide (26 mg, 0.13 mmol, 2.5 equiv.) and CuI (2 mg, 0.01 mmol, 25 37 38 39 mol %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of 40 41 toluene, DMF and i-Pr2NH (degassed by sparging with nitrogen for 15 min, 1.3 mL) was 42 43 44 added at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 45 46 47 min and a solution of Pd(Pt-Bu3)2 (3 mg, 5 µmol, 10 mol %) in a 5:1:1.5 mixture of toluene, 48 49 DMF and i-Pr2NH (1.3 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. 50 51 52 53 54 55 152 56 57 58 59 60 ACS Paragon Plus Environment

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1 The solution was allowed to cool to RT and volatiles were removed in vacuo. Crude mate- 2 3 rial was filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne 4 5 6 S82, which was further purified by preparative HPLC (15% → 95% B over 30 min) to 7 8 9 afford alkyne S82 (28 mg, 35 µmol, 68%) as a white amorphous solid. Rf = 0.43 (EtOAc/i- 10 11 ν -1 PrOH, 95:5); FTIR (thin film), max (cm ): 3292, 2928, 1748, 1718, 1690, 1611, 1584, 1511, 12 13 1 14 1487, 1457, 1377, 1245, 1220, 1161, 1075; H NMR (600 MHz, CDCl3): δ 9.16 (s, 1H), 15 16 17 8.78 (s, 1H), 8.14 (s, 1H), 8.10 (dd, J = 7.8, 1.1 Hz, 1H), 8.05–7.99 (m, 2H), 7.95 (d, J = 18 19 4.0 Hz, 1H), 7.78 (app t, J = 7.8 Hz, 1H), 7.62–7.57 (m, 1H), 7.54–7.48 (m, 3H), 7.33 (br 20 21 22 d, J = 7.7 Hz, 1H), 6.15 (d, J = 5.9 Hz, 1H), 6.14 (dd, J = 5.9, 4.8 Hz, 1H), 5.92 (d, J = 4.0 23 24 25 Hz, 1H), 5.60 (dd, J = 4.8, 3.9 Hz, 1H), 4.66 (app td, J = 7.7, 5.1 Hz, 1H), 4.51 (ddd, J = 26 27 J 28 10.2, 3.9, 3.4 Hz, 1H), 3.77 (s, 3H), 2.90–2.83 (m, 1H), 2.22 (ddd, = 14.0, 10.2, 4.1 Hz, 29 30 1H), 2.15 (s, 3H), 2.18–2.11 (m, 1H), 2.09–2.02 (m, 2H), 2.06 (s, 3H), 1.71 (app ddt, J = 31 32 13 33 13.1, 10.7, 5.4 Hz, 1H), 1.65–1.57 (m, 1H); C NMR (126 MHz, CDCl3): δ 171.3, 169.9, 34 35 2 36 169.6, 166.3, 164.9, 157.1 (q, JC-F = 38 Hz), 152.8, 151.8, 150.0, 149.9, 142.4, 141.9, 137.6, 37

38 1 133.5, 133.0, 129.8, 129.0, 128.1, 124.2, 121.7, 115.7 (q, JC-F = 288 Hz), 91.3, 87.0, 82.9, 39 40 19 41 81.2, 73.8, 72.7, 53.2, 52.5, 38.3, 30.8, 29.8, 28.7, 20.8, 20.5; F NMR (471 MHz, CDCl3, 42 43 + ∆ 44 BTF IStd): δ –76.6; HRMS (ESI+): calcd. for [C37H36F3N8O10] 809.2501, meas. 809.2504, 45 46 0.4 ppm. 47 48 49 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((2-carbamoylpyridin- 50 51 52 4-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran- 53 54 55 153 56 57 58 59 60 ACS Paragon Plus Environment

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1 3,4-diyl diacetate (S83). A vial was charged with alkyne 8 (40 mg, 58 µmol), 4-bromo- 2 3 picolinamide (29 mg, 0.15 mmol, 2.5 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol %). The 4 5 6 vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF 7 8 9 and i-Pr2NH (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. 10 11 The resulting mixture was further degassed by sparging with nitrogen for 5 min and a 12 13

14 solution of Pd(Pt-Bu3)2 (3 mg, 6 µmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and 15 16 17 i-Pr2NH (1.5 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solu- 18 19 tion was allowed to cool to RT and volatiles were removed in vacuo. Crude material was 20 21 22 filtered through a short pad of silica gel (EtOAc/i-PrOH, 70:30) to afford alkyne S83, 23 24 25 which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford 26 27 ν - S83 max 28 alkyne (35 mg, 43 µmol, 75%) as a white amorphous solid. FTIR (thin film), (cm 29 30 1): 3283, 3079, 2931, 1748, 1721, 1690, 1611, 1599, 1583, 1456, 1356, 1243, 1218, 1074; 31 32 1 33 H NMR (600 MHz, CDCl3): δ 9.07 (s, 1H), 8.78 (s, 1H), 8.48 (dd, J = 5.0, 0.8 Hz, 1H), 34 35 36 8.12 (dd, J = 1.6, 0.8 Hz, 1H), 8.11 (s, 1H), 8.04–8.00 (m, 2H), 7.82 (d, J = 4.2 Hz, 1H), 37 38 7.62–7.58 (m, 1H), 7.54–7.49 (m, 2H), 7.38 (dd, J = 5.0, 1.6 Hz, 1H), 7.31 (d, J = 7.8 Hz, 39 40 41 1H), 6.15–6.11 (m, 2H), 5.93 (d, J = 4.2 Hz, 1H), 5.62–5.58 (m, 1H), 4.65 (app td, J = 7.8, 42 43 44 5.4 Hz, 1H), 4.47 (ddd, J = 10.8, 4.3, 3.0 Hz, 1H), 3.78 (s, 3H), 2.88 (app ddt, J = 10.8, 45 46 9.2, 4.6 Hz, 1H), 2.22–2.15 (m, 1H), 2.17 (s, 3H), 2.11 (ddt, J = 14.2, 11.0, 5.4 Hz, 1H), 47 48 49 2.08 (s, 3H), 2.03–1.95 (m, 2H), 1.67 (app ddt, J = 13.4, 10.8, 5.4 Hz, 1H), 1.57 (dddd, J 50 51 13 52 = 13.4, 11.0, 9.2, 4.6 Hz, 1H); C NMR (126 MHz, CDCl3): δ 171.3, 169.9, 169.7, 166.4, 53 54 55 154 56 57 58 59 60 ACS Paragon Plus Environment

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2 1 164.8, 157.1 (q, JC-F = 38 Hz), 152.9, 151.7, 149.9, 149.6, 148.5, 142.5, 133.6, 133.0, 133.0, 2 3 1 129.0, 128.4, 128.1, 124.8, 124.2, 115.7 (q, JC-F = 288 Hz), 96.7, 87.3, 81.1, 80.8, 73.7, 72.9, 4 5 19 6 53.3, 52.4, 38.3, 30.8, 29.9, 28.9, 20.8, 20.6; F NMR (471 MHz, CDCl3, BTF IStd): δ – 7 8 + ∆ 9 76.6; HRMS (ESI+): calcd. for [C37H36F3N8O10] 809.2501, meas. 809.2491, 1.2 ppm. 10 11 R R R R H S S 12 (2 ,3 ,4 ,5 )-2-(6-benzamido-9 -purin-9-yl)-5-((2 ,5 )-2-((5-carbamoylpyridin- 13 14 3-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran- 15 16 17 3,4-diyl diacetate (S84). A vial was charged with alkyne 8 (40 mg, 58 µmol), 5-bromon- 18 19 20 icotinamide (29 mg, 0.15 mmol, 2.5 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol %). The 21 22 vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, DMF 23 24

25 and i-Pr2NH (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added at RT. 26 27 28 The resulting mixture was further degassed by sparging with nitrogen for 5 min and a 29 30 solution of Pd(Pt-Bu3)2 (3 mg, 6 µmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and 31 32

33 i-Pr2NH (1.5 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solu- 34 35 36 tion was allowed to cool to RT and volatiles were removed in vacuo. Crude material was 37 38 i S84 39 filtered through a short pad of silica gel (EtOAc/ -PrOH, 70:30) to afford alkyne , 40 41 which was further purified by preparative HPLC (15% → 95% B over 30 min) to afford 42 43 ν - 44 alkyne S84 (26 mg, 32 µmol, 55%) as a white amorphous solid. FTIR (thin film), max (cm 45 46 1 47 ): 3263, 3085, 2954, 2928, 1748, 1720, 1677, 1612, 1583, 1456, 1376, 1245, 1219, 1075; 48

49 1 H NMR (600 MHz, CDCl3): δ 9.11 (s, 1H), 8.94 (d, J = 1.4 Hz, 1H), 8.77 (s, 1H), 8.67 (d, 50 51 52 J = 2.0 Hz, 1H), 8.16–8.11 (m, 2H), 8.01 (m, 2H), 7.63–7.58 (m, 1H), 7.55–7.48 (m, 2H), 53 54 55 155 56 57 58 59 60 ACS Paragon Plus Environment

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1 7.46 (br d, J = 7.5 Hz, 1H), 6.83 (br s, 1H), 6.17 (dd, J = 5.7, 5.1 Hz, 1H), 6.15 (d, J = 5.7 2 3 Hz, 1H), 6.05 (br s, 1H), 5.66 (dd, J = 5.1, 4.0 Hz, 1H), 4.65 (app td, J = 7.5, 5.4 Hz, 1H), 4 5 6 4.48 (ddd, J = 9.8, 4.0, 3.8 Hz, 1H), 3.79 (s, 3H), 2.88–2.82 (m, 1H), 2.21–2.15 (m, 1H), 7 8 9 2.16 (s, 3H), 2.15–2.08 (m, 1H), 2.07 (s, 3H), 2.08–1.99 (m, 2H), 1.72–1.64 (m, 1H), 1.63– 10

11 13 1.54 (m, 1H); C NMR (126 MHz, CDCl3): δ 171.3, 170.1, 169.8, 166.9, 165.0, 157.3 (q, 12 13 2 14 JC-F = 38 Hz), 154.5, 152.8, 151.8, 149.9, 147.6, 142.6, 138.1, 133.5, 133.1, 129.0, 128.5, 15 16 1 17 128.1, 124.3, 120.2, 115.7 (q, JC-F = 288 Hz), 95.3, 87.1, 81.2, 79.9, 73.7, 72.7, 53.3, 52.6, 18

19 19 38.3, 30.9, 29.7, 28.9, 20.8, 20.6; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.6; HRMS 20 21 + ∆ 22 (ESI+): calcd. for [C37H36F3N8O10] 809.2501, meas. 809.2511, 1.2 ppm. 23 24 25 (2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((2S,5S)-2-((4-carbamoylpyridin- 26 27 28 2-yl)ethynyl)-6-methoxy-6-oxo-5-(2,2,2-trifluoroacetamido)hexyl)tetrahydrofuran- 29 30 3,4-diyl diacetate (S85). A vial was charged with alkyne 8 (35 mg, 51 µmol), 2-bromo- 31 32 33 isonicotinamide (26 mg, 0.13 mmol, 2.5 equiv.) and CuI (2 mg, 0.01 mmol, 25 mol %). 34 35 36 The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of toluene, 37 38 i 2 39 DMF and -Pr NH (degassed by sparging with nitrogen for 15 min, 1.3 mL) was added at 40 41 RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and a 42 43 44 solution of Pd(Pt-Bu3)2 (3 mg, 5 µmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF and 45 46 47 i-Pr2NH (1.3 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The solu- 48 49 tion was allowed to cool to RT and volatiles were removed in vacuo. Crude material was 50 51 52 filtered through a short pad of silica gel (EtOAc/i-PrOH, 95:5) to afford alkyne S85, which 53 54 55 156 56 57 58 59 60 ACS Paragon Plus Environment

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1 was further purified by preparative HPLC (15% → 95% B over 30 min) to afford alkyne 2 3 S85 (22 mg, 27 µmol, 54%) as a light yellow amorphous solid. Rf = 0.31 (EtOAc/i-PrOH, 4 5 ν -1 6 95:5); FTIR (thin film), max (cm ): 3289, 2925, 1747, 1719, 1681, 1612, 1583, 1549, 1457, 7 8 1 9 1414, 1376, 1245, 1220, 1162, 1047; H NMR (600 MHz, CDCl3): δ 9.18 (s, 1H), 8.77 (s, 10 11 1H), 8.64 (dd, J = 5.2, 0.9 Hz, 1H), 8.13 (s, 1H), 8.05–7.98 (m, 2H), 7.79 (dd, J = 1.7, 0.9 12 13 14 Hz, 1H), 7.65 (dd, J = 5.2, 1.7 Hz, 1H), 7.62–7.58 (m, 1H), 7.59–7.56 (m, 1H), 7.55–7.48 15 16 17 (m, 2H), 7.11 (br s, 1H), 6.14 (br s, 1H), 6.14 (dd, J = 5.4, 5.0 Hz, 1H), 6.12 (d, J = 5.4 Hz, 18 19 1H), 5.66 (dd, J = 5.0, 4.4 Hz, 1H), 4.62 (app q, J = 6.9 Hz, 1H), 4.53 (ddd, J = 10.1, 4.4, 20 21 22 3.6 Hz, 1H), 3.77 (s, 3H), 2.85 (app ddt, J = 10.9, 9.0, 4.3 Hz, 1H), 2.15 (s, 3H), 2.19–2.09 23 24 25 (m, 3H), 2.06 (s, 3H), 2.07–2.01 (m, 1H), 1.73–1.66 (m, 1H), 1.65–1.57 (m, 1H); 13C NMR 26

27 2 3 JC-F 28 (126 MHz, CDCl ): δ 171.2, 170.1, 169.8, 166.8, 165.0, 157.4 (q, = 38 Hz), 152.8, 29 30 151.7, 150.7, 149.9, 143.9, 142.6, 141.0, 133.5, 133.1, 129.0, 128.1, 125.2, 124.2, 120.9, 31 32 1 33 115.7 (q, JC-F = 288 Hz), 92.3, 87.2, 83.1, 80.9, 73.7, 72.9, 53.3, 52.8, 38.2, 31.0, 29.6, 28.8, 34 35 19 36 20.8, 20.6; F NMR (471 MHz, CDCl3, BTF IStd): δ –76.6; HRMS (ESI+): calcd. for 37 38 + ∆ [C37H36F3N8O10] 809.2501, meas. 809.2502, 0.1 ppm. 39 40 41 (2R,3R,4R,5R)-2-((2S,5S)-2-((5-aminonaphthalen-1-yl)ethynyl)-6-methoxy-6-oxo-5- 42 43 44 (2,2,2-trifluoroacetamido)hexyl)-5-(6-benzamido-9H-purin-9-yl)tetrahydrofuran- 45 46 47 3,4-diyl diacetate (S86). A vial was charged with alkyne 8 (42 mg, 61 µmol), 5-bromo- 48 49 naphthalen-1-ylamine (34 mg, 0.15 mmol, 2.5 equiv.) and CuI (3 mg, 0.02 mmol, 25 mol 50 51 52 53 54 55 157 56 57 58 59 60 ACS Paragon Plus Environment

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1 %). The vial headspace was purged with nitrogen for 5 min and a 5:1:1.5 mixture of tolu- 2 3 ene, DMF and i-Pr2NH (degassed by sparging with nitrogen for 15 min, 1.5 mL) was added 4 5 6 at RT. The resulting mixture was further degassed by sparging with nitrogen for 5 min and 7 8 9 a solution of Pd(Pt-Bu3)2 (3 mg, 6 µmol, 10 mol %) in a 5:1:1.5 mixture of toluene, DMF 10 11 and i-Pr2NH (1.5 mL) was added. The resulting mixture was stirred at 50 °C for 5 h. The 12 13 14 solution was allowed to cool to RT and volatiles were removed in vacuo. Crude material 15 16 17 was filtered through a short pad of silica gel (EtOAc/iPrOH, 70:30) to afford alkyne S86, 18 19 which was further purified by preparative HPLC (20% → 95% B over 30 min) to afford 20 21

22 alkyne S86 (34 mg, 41 µmol, 67%) as a light yellow amorphous solid. Rf = 0.42 (EtOAc); 23 24 ν -1 25 FTIR (thin film), max (cm ): 3315, 3066, 2924, 1748, 1721, 1611, 1582, 1514, 1456, 1369, 26

27 1 3 28 1243, 1218, 1179, 1074; H NMR (600 MHz, CDCl ): δ 9.02 (s, 1H), 8.80 (s, 1H), 8.12 (s, 29 30 1H), 8.03–7.99 (m, 2H), 7.80 (app dt, J = 8.5, 1.0 Hz, 1H), 7.70 (app dt, J = 8.3, 1.0 Hz, 31 32 33 1H), 7.62–7.58 (m, 2H), 7.53–7.50 (m, 2H), 7.37 (dd, J = 8.5, 7.1 Hz, 1H), 7.34 (dd, J = 34 35 36 8.3, 7.4 Hz, 1H), 7.04 (br d, J = 7.7 Hz, 1H), 6.80 (dd, J = 7.4, 1.0 Hz, 1H), 6.15 (d, J = 37 38 5.3 Hz, 1H), 6.09 (dd, J = 5.5, 5.3 Hz, 1H), 5.61 (dd, J = 5.5, 4.8 Hz, 1H), 4.69 (app td, J 39 40 41 = 7.7, 4.9 Hz, 1H), 4.65 (ddd, J = 10.8, 4.8, 2.8 Hz, 1H), 4.32 (br s, 2H), 3.74 (s, 3H), 2.97 42 43 44 (app ddt, J = 10.8, 9.1, 4.5 Hz, 1H), 2.24–2.16 (m, 2H), 2.13 (s, 3H), 2.14–2.04 (m, 2H), 45 46 2.06 (s, 3H), 1.72 (dddd, J = 13.3, 10.8, 6.0, 4.8 Hz, 1H), 1.61 (dddd, J = 13.3, 10.9, 9.4, 47 48 13 2 49 4.5 Hz, 1H); C NMR (126 MHz, CDCl3): δ 171.3, 169.9, 169.6, 164.7, 157.0 (q, JC-F = 38 50 51 52 Hz), 152.9, 151.7, 149.9, 142.7, 142.3, 134.5, 133.6, 133.0, 130.7, 129.0, 128.0, 127.3, 53 54 55 158 56 57 58 59 60 ACS Paragon Plus Environment

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1 1 124.1, 124.1, 123.5, 121.5, 121.3, 117.0, 115.7 (q, JC-F = 288 Hz), 110.4, 94.9, 87.2, 82.4, 2 3 19 81.0, 73.7, 73.1, 53.2, 52.5, 38.9, 31.2, 30.3, 29.3, 20.8, 20.6; F NMR (471 MHz, CDCl3, 4 5 + ∆ 6 BTF IStd): δ –76.7; HRMS (ESI+): calcd. for [C41H39F3N7O9] 830.2756, meas. 830.2772, 7 8 9 1.9 ppm. 10 11 12 13 14 Cellular Thermal Shift Assay (CETSA) 15 16 17 K562 Cell Culture. The human chronic myeloid leukemia cell line K562 (ATCC CCL- 18 19 20 243) was cultured in RPMI 1640 with Glutamax (Gibco) supplemented with 10% fetal 21 22 23 bovine serum (Atlanta Biologicals), and 100 IU penicillin and 100 mg/mL streptomycin 24 25 26 (Corning). Cells were cultured in T175 flasks with 50 mL of media per flask (Corning) in 27 28 × a 37 °C incubator with 5% CO2 until the cells reached a density of 2 106 cells/mL. 29 30 31 Preparation of K562 Cell Lysate. K562 cells were cultured as described above. Cells were 32 33 34 washed with PBS and then resuspended in an appropriate volume of PBS supplemented 35 36 × 7 37 with cOmplete protease inhibitor cocktail (Roche) to obtain a final cell count of 3.0 10 38 39 to 3.6 × 107 cells/mL. The suspension was transferred to microcentrifuge tubes and sub- 40 41 42 jected to three rounds of freeze-thaw using liquid nitrogen and a room temperature water 43 44 45 bath. The cell lysate was clarified by centrifuging at 17,000g for 20 min at 4 °C, and the 46 47 supernatant was transferred to a new microcentrifuge tube. Lysate protein concentration 48 49 50 was determined by BCA assay and the lysate was diluted to a protein concentration of 3 51 52 53 54 55 159 56 57 58 59 60 ACS Paragon Plus Environment

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1 mg/mL with PBS supplemented with protease inhibitor cocktail. Aliquots were snap frozen 2 3 in liquid nitrogen and stored at –80 °C. 4 5 6 CETSA with K562 Cell Lysate. The CETSA assay was adapted from previously published 7 8 9 protocols.80-81 K562 lysate prepared as described above was incubated at room temperature 10 11 12 with DMSO, 100 µM of NS1 (10), or 100 µM of compound 25 for 30 min. A Bio-Rad 13 14 C1000 Touch Thermal Cycler was preheated to the following temperatures using the gra- 15 16 17 dient setting: 37 °C, 38.8 °C, 41.6 °C, 45.2 °C, 50.1 °C, 53.7 °C, 56 °C, 57.9 °C, 60.8 °C, 18 19 20 64.3 °C, 69.1 °C, 72.9 °C. DMSO-treated and compound-treated lysate were aliquotted 21 22 into twelve PCR tubes, and each tube was heated at one of the temperatures listed above 23 24 25 for 3 min. The samples were cooled at room temperature for 3 min, then snap frozen in 26 27 28 liquid nitrogen. When thawed, the samples were centrifuged at 17,000g for 20 min at 4 °C. 29 30 The supernatant was transferred to a new tube, mixed with NuPAGE LDS sample buffer 31 32 33 (Novex) and β-mercaptoethanol (2.5%). The samples were heated at 70 °C for 10 min and 34 35 36 analyzed by SDS-PAGE and western blot using an anti-NNMT mouse antibody (Abcam, 37 38 39 ab119758, 1:1000 dilution), an anti-actin HRP-conjugated mouse antibody (Abcam, 40 41 ab49900, 1:250000 dilution), and an anti-mouse HRP-conjugated IgG (Promega, W402B, 42 43 44 1:5000 dilution). 45 46 47 Isothermal Dose Response (IDTR) CETSA with K562 Cell Lysate. K562 lysate prepared 48 49 50 as described above was incubated at room temperature with DMSO or various concentra- 51 52 tions of NS1 (10) or compound 25 for 30 min. A Bio-Rad C1000 Touch thermal cycler was 53 54 55 160 56 57 58 59 60 ACS Paragon Plus Environment

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1 preheated to 57 °C. Samples were aliquotted into PCR tubes, and each tube was heated at 2 3 57 °C in the thermal cycler for 3 min. The samples were cooled at room temperature for 3 4 5 6 min and then snap frozen in liquid nitrogen. When thawed, the samples were centrifuged 7 8 9 at 17,000g for 20 min at 4 °C. The supernatant was prepared for western blot analysis as 10 11 described in the CETSA procedure above. 12 13 14 15 16 17 Cytotoxicity Assay in U2OS Cells. Cytotoxicity evaluation was performed with the 18 19 20 Promega CellTiter-Glo Luminescent Cell Viability Assay. U2OS cells were maintained in 21 22 23 DMEM-F12 glutamax complete growth media supplemented with 10% heat-inactivated 24 25 fetal bovine serum (HI FBS). CellTiter-Glo Buffer & CellTiter-Glo Substrate were thawed 26 27 28 at room temperature prior to use. An appropriate volume (10 mL) of CellTiter-Glo Buffer 29 30 31 was transferred into the amber bottle containing CellTiter-Glo Substrate to reconstitute the 32 33 lyophilized enzyme/substrate mixture. The contents were gently vortexed to obtain a ho- 34 35 36 mogeneous solution. U2OS cells were seeded at 10000 cells/150 µL/well in 96 well cell 37 38 39 culture plate and incubated overnight at 37 °C and 5% CO2. The next day, media was 40 41 changed (150 µL), various concentrations of compounds (50 µL from 4× stock) were tested 42 43 44 in duplicates, at eight concentrations, in semi log dilution pattern, with starting concentra- 45 46 47 tion of 100 µM. Final concentration of DMSO in the assay was 1 %. 10 µM, 5 µM and 3 48 49 50 µM of doxorubicin hydrochloride was used as positive control in the assay. The plates were 51 52 incubated for 24 h and 48 h at 37 °C with 5% CO2. After 24 h and 48 h of incubation, 53 54 55 161 56 57 58 59 60 ACS Paragon Plus Environment

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1 CellTiter-Glo Reagent (Promega) preparation was added to the cell culture medium present 2 3 in each well in a 1:1 ratio by volume (e.g., add 50 µL of reagent to 50 µL of medium 4 5 6 containing cells). The plate was gently vortexed for 10 min on an orbital shaker to induce 7 8 9 cell lysis. The contents of the plate were transferred to an opaque half area 96 well plate. 10 11 The plate was incubated at RT for 10 min to stabilize the luminescence signal. Lumines- 12 13 14 cence output was read on a Tecan Spark plate reader. The % cell viability was calculated 15 16 17 relative to DMSO control. 18 19 20 21 22 23 MNAM Measurement in U2OS Cells by LC-MS/MS 24 25 26 Materials. Acetonitrile (Millipore Sigma 1000292500), D4-MNAM (BioOrganics SRK(I)- 27 28 257-3226), MNAM (BioOrganics BST(I)-256-3271), DPBS (Invitrogen 14190-136), Top 29 30 31 Seal-A Plus (Perkin Elmer 6050185), 96-well plate sterile TC treated (Eppendorf 32 33 34 0030730119), 96 well plate transfer plate (Costar 3364), reference compound 6-methoxy 35 36 nicotinamide/(alternate name: JBSNF-0088) (Arbor chemical corporation Limited, 37 38 39 P180810). U2OS cells were obtained from ATCC and maintained in DMEM-F12 growth 40 41 42 media containing 10% HI FBS and 1% Pen-Strep (filter sterilized) in 37 °C incubator with 43 44 5% CO2. Extraction buffer was prepared by adding internal standard (D4 MNAM, 20 45 46 47 ng/mL) to 100% acetonitrile. 48 49 50 Protocol. Cells were maintained in cell culture flasks until seeding into microwell plates. 51 52 53 Media was removed, cells were washed with DPBS (-/-) and then detached with TrypLE 54 55 162 56 57 58 59 60 ACS Paragon Plus Environment

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2 1 Express (4mL) into a 150 cm flask (incubate at 37 °C for 5 min). The reaction was stopped 2 3 by the addition of 5 mL media and cells were counted with a Vi-CELL cell counter. Cells 4 5 6 were seeded into a 96-well microplate (100 µL/well = 10000 cells/well) and incubated for 7 8 9 24 h at 37 °C, 5% CO2, 95% humidity with the lid on. Media was replaced by addition of 10 11 100 µL of media/compound mixture and cells were incubated for 24 h at 37 °C, 5% CO2, 12 13 14 95% humidity with the lid on. The media/compound mixture was removed and cells were 15 16 17 washed two times with 150 µL of DPBS(-/-). The DPBS was removed and 100 µL of 18 19 extraction buffer was added. The plate was incubated for 20 min at RT with mild shaking. 20 21 22 100 µL autoclaved water was added and the plate was mixed gently. The plate was centri- 23 24 25 fuged at 5000g for 10 min and then 150 µL of the extraction buffer/water mixture (final 26 27 D4 MNAM concentration of 10 ng/mL) was transferred into a 96-well microplate. MNAM 28 29 30 levels were quantified by LC-MS/MS according to methods previously reported by Kannt 31 32 33 et al.15 34 35 36 37 38 39 ASSOCIATED CONTENT 40 41 42 43 Supporting Information 44 45 46 The Supporting Information (SI) is available free of charge on the ACS Publications web- 47 48 site. 49 50 51 52 53 54 55 163 56 57 58 59 60 ACS Paragon Plus Environment

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Supplementary figures and tables, synthetic schemes, and experimental protocols not listed 1 2 3 in the main manuscript, including: molecular docking, protein expression and purification, 4 5 6 biochemical assays, bioinformatic analyses, and protein crystallography (SI: Part 1); NMR 7 8 9 spectra (SI: Part 2); Enzyme Kinetics (SI: Part 3); Small molecule X-ray crystallography 10 11 data for NS1 (10), S30, S53 (CIF files); Molecular formula strings for compounds 10-41 12 13 14 (CSV). 15 16 17 18 19 20 AUTHOR INFORMATION 21 22 23 Corresponding Author 24 25 26 Email for M.D.S.: [email protected] 27 28 29 ORCID 30 31 32 Rocco L. Policarpo: 0000-0001-9819-2394 33 34 35 Ludovic Decultot: 0000-0002-2607-2016 36 37 38 Elizabeth May: 0000-0002-5804-9574 39 40 Petr Kuzmič: 0000-0001-5250-8381 41 42 43 Samuel Carlson: 0000-0001-9881-4210 44 45 46 Danny Huang: 0000-0001-9542-5092 47 48 Vincent Chu: 0000-0003-4499-2059 49 50 51 Brandon Wright: 0000-0003-4149-5292 52 53 54 Saravanakumar Dhakshinamoorthy: 0000-0001-6309-5041 55 164 56 57 58 59 60 ACS Paragon Plus Environment

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Aimo Kannt: 0000-0002-5197-2286 1 2 3 Shilpa Rani: 0000-0001-7774-1542 4 5 6 Sreekanth Dittakavi: 0000-0002-5638-327X 7 8 9 Rachelle Gaudet: 0000-0002-9177-054X 10 11 12 13 14 Notes 15 16 17 The authors declare no competing financial interests. All authors have given approval to 18 19 20 the final version of the manuscript. 21 22 23 24 Current Author Addresses: 25 26 27 Vincent Chu: Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., 28 29 Boston, MA 02115, USA 30 31 32 Brandon Wright: Department of Chemistry, University of California, Berkeley, CA 94720, 33 34 35 USA 36 37 Joseph Panarese: Enanta Pharmaceuticals, 500 Arsenal St., Watertown, MA 02472, USA 38 39 40 41 ACKNOWLEDGMENTS 42 43 44 Thank you to Dr. Shao-Liang Zheng for assistance with small molecule X-ray crystallog- 45 46 raphy and to Cheryl Arrowsmith for supplying the NNMT and INMT plasmids used in this 47 48 49 study (via Addgene). We thank the National Science Foundation for funding Rocco L. 50 51 52 Policarpo, as the material presented in this manuscript is based upon work supported by 53 54 55 165 56 57 58 59 60 ACS Paragon Plus Environment

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the NSF Graduate Research Fellowship under Grant No. DGE1144152. We thank the Har- 1 2 3 vard Blavatnik Biomedical Accelerator for funding portions of this project and the Harvard 4 5 6 College Research Program for providing funding to Danny Huang and Brandon A. Wright. 7 8 9 The authors thank Grace Kenney for assistance with sequence similarity network genera- 10 11 tion, James Tucker for helpful discussion and manuscript feedback, and Kevin Dalton for 12 13 14 assistance with crystallographic data processing and refinement. We thank Liz Hedstrom 15 16 17 (Brandeis University) for helpful comments on the manuscript and Paul Thompson 18 19 (UMass Medical School) for providing raw kinetic data from a previously published report. 20 21 22 We gratefully acknowledge the beamline staff of NE-CAT at the Advanced Photon Source 23 24 25 (Argonne, IL, USA) for help with data collection. NE-CAT is funded by NIH (P41 26 27 GM103403 and S10 RR029205) and the Advanced Photon Source by the US Department 28 29 30 of Energy (DE-AC02-06CH11357). 31 32 33 34 ABBREVIATIONS 35 36 37 NNMT, nicotinamide N-methyltransferase; SAM, S-adenosylmethionine; NAM, nicotin- 38 39 amide; MNAM, 1-methylnicotinamide; SAH, S-adenosylhomocysteine; COMT, catechol 40 41 42 O-methyltransferase; BPE, 1,2-bis(phospholano)ethane; INMT, indolethylamine N-me- 43 44 45 thyltransferase; PNMT, phenylethanolamine N-methyltransferase; TPMT, thiopurine S- 46 47 methyltransferase; IStd, internal standard; rbf, round-bottom flask; PMHS, polymethylhy- 48 49 50 drosiloxane. 51 52 53 54 Accession Codes 55 166 56 57 58 59 60 ACS Paragon Plus Environment

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1 The coordinates and structure factors of the hNNMT-NS1 (10) co-crystal structure have 2 3 been deposited with the RCSB Protein Data Bank under accession code 6ORR. The au- 4 5 6 thors will release the atomic coordinates upon article publication. 7 8 9 REFERENCES 10 11 12 1. Currently, there are many abbreviations used for nicotinamide and 1- 13 14 15 methylnicotinamide in the literature, leading to inconsistency and confusion. We use the 16 17 18 standardized nomenclature proposed by Pissios in a 2017 review article on NNMT (see 19 20 following citation). 21 22 23 2. Pissios, P., Nicotinamide N-methyltransferase: more than a vitamin B3 clearance 24 25 26 enzyme. Trends Endocrinol. Metab. 2017, 28 (5), 340-353. 27 28 3. Hong, S.; Zhai, B.; Pissios, P., Nicotinamide N-methyltransferase interacts with 29 30 31 enzymes of the methionine cycle and regulates methyl donor metabolism. Biochemistry 32 33 34 2018, 57 (40), 5775-5779. 35 36 37 4. Hong, S.; Moreno-Navarrete, J. M.; Wei, X.; Kikukawa, Y.; Tzameli, I.; Prasad, D.; 38 39 Lee, Y.; Asara, J. M.; Fernandez-Real, J. M.; Maratos-Flier, E.; Pissios, P., Nicotinamide 40 41 42 N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein 43 44 45 stabilization. Nat. Med. 2015, 21 (8), 887-894. 46 47 5. Ulanovskaya, O. A.; Zuhl, A. M.; Cravatt, B. F., NNMT promotes epigenetic 48 49 50 remodeling in cancer by creating a metabolic methylation sink. Nat. Chem. Biol. 2013, 9 51 52 53 (5), 300-306. 54 55 167 56 57 58 59 60 ACS Paragon Plus Environment

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22 hydrogenation catalyst was prepared freshly before use from [Rh(nbd)2]BF4 (CAS: 36620- 23 24 25 11-8, obtained from Strem Chemicals, Inc. (catalog #: 45-0230)) and (S,S)-MeBPE ligand 26 27 (CAS: 136779-26-5, obtained from Strem Chemicals, Inc. (catalog #: 15-0105)). 28 29 30 64. Ellis, F.; Fulton, H. E.; Hall, A.; Jaxa-Chamiec, A. A.; Swanson, S.; Vile, S. 31 32 33 Acetylene Compounds. WO2004026890 (A1), 2004. 34 35 36 65. Pedersen, D. S.; Rosenbohm, C., Dry column vacuum chromatography. Synthesis 37 38 2001, 2001 (16), 2431-2434. 39 40 41 66. Cox, N.; Dang, H.; Whittaker, A. M.; Lalic, G., Copper-catalyzed semi-reduction of 42 43 44 alkynes. Org. Synth. 2016, 93, 385-398. 45 46 67. Becerra-Figueroa, L.; Movilla, S.; Prunet, J.; Miscione, G. P.; Gamba-Sánchez, D., 47 48 49 An intramolecular oxa-Michael reaction on α,β-unsaturated α-amino-δ- 50 51 52 53 54 55 177 56 57 58 59 60 ACS Paragon Plus Environment

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1 hydroxycarboxylic acid esters. synthesis of functionalized 1,3-dioxanes. Org. Biomol. 2 3 Chem. 2018, 16 (8), 1277-1286. 4 5 6 68. Blanchard, P.; Da Silva, A. D.; El Kortbi, M. S.; Fourrey, J. L.; Robert-Gero, M., 7 8 9 Zinc-iron couple induced conjugate addition of alkyl halide derived radicals to activated 10 11 olefins. J. Org. Chem. 1993, 58 (23), 6517-6519. 12 13 14 69. Li, W.; Niu, Y.; Xiong, D.-C.; Cao, X.; Ye, X.-S., Highly substituted cyclopentane– 15 16 17 CMP conjugates as potent sialyltransferase inhibitors. J. Med. Chem. 2015, 58 (20), 7972- 18 19 7990. 20 21 22 70. The authors note that the N-Cbz cleavage reaction rate increased markedly upon 23 24 25 addition of EtOH. 26 27 71. Blanchard, P.; El Kortbi, M. S.; Fourrey, J. L.; Lawrence, F.; Robert-Gero, M., 28 29 30 Relationship between chemical structure and antileishmanial effect of sinefungine 31 32 33 analogues. Nucleosides Nucleotides 1996, 15 (6), 1121-1135. 34 35 36 72. Sarabia, F.; Martín-Ortiz, L.; López-Herrera, F. J., A convergent synthetic approach 37 38 to the nucleoside-type liposidomycin antibiotics. Org. Lett. 2003, 5 (21), 3927-3930. 39 40 41 73. Petakamsetty, R.; Ansari, A.; Ramapanicker, R., Diastereoselective synthesis of 42 43 44 furanose and pyranose substituted glycine and alanine derivatives via proline-catalyzed 45 46 asymmetric α-amination of aldehydes. Carbohydr. Res. 2016, 435, 37-49. 47 48 49 50 51 52 53 54 55 178 56 57 58 59 60 ACS Paragon Plus Environment

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1 74. Herrera, A. J.; Rondón, M.; Suárez, E., Stereocontrolled photocyclization of 1,2- 2 3 diketones: application of a 1,3-acetyl group transfer methodology to carbohydrates. J. Org. 4 5 6 Chem. 2008, 73 (9), 3384-3391. 7 8 9 75. Charette, A. B.; Juteau, H.; Lebel, H.; Molinaro, C., Enantioselective 10 11 cyclopropanation of allylic alcohols with dioxaborolane ligands: scope and synthetic 12 13 14 applications. J. Am. Chem. Soc. 1998, 120 (46), 11943-11952. 15 16 17 76. Hagiya, K.; Muramoto, N.; Misaki, T.; Sugimura, T., DMEAD: a new dialkyl 18 19 azodicarboxylate for the Mitsunobu reaction. Tetrahedron 2009, 65 (31), 6109-6114. 20 21 22 77. Bergström, M.; Suresh, G.; Naidu, V. R.; Unelius, C. R., N-iodosuccinimide (NIS) 23 24 25 in direct aromatic iodination. Eur. J. Org. Chem. 2017, 2017 (22), 3234-3239. 26 27 28 78. Nammalwar, B.; Bunce, R. A.; Berlin, K. D.; Bourne, C. R.; Bourne, P. C.; Barrow, 29 30 E. W.; Barrow, W. W., Approaches to iodinated derivatives of vanillin and isovanillin. 31 32 33 Org. Prep. Proced. Int. 2012, 44 (2), 146-152. 34 35 36 79. Kim, Y.; Park, H.; Lee, J.; Tae, J.; Kim, H. J.; Min, S.-J.; Rhim, H.; Choo, H., 5- 37 38 HT7 receptor modulators: amino groups attached to biphenyl scaffold determine functional 39 40 41 activity. Eur. J. Med. Chem. 2016, 123, 180-190. 42 43 44 80. Martinez Molina, D.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E. A.; Dan, 45 46 C.; Sreekumar, L.; Cao, Y.; Nordlund, P., Monitoring drug target engagement in cells and 47 48 49 tissues using the cellular thermal shift assay. Science 2013, 341 (6141), 84-87. 50 51 52 53 54 55 179 56 57 58 59 60 ACS Paragon Plus Environment

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1 81. Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; 2 3 Nordlund, P.; Martinez Molina, D., The cellular thermal shift assay for evaluating drug 4 5 6 target interactions in cells. Nat. Protoc. 2014, 9 (9), 2100-2122. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 TABLE OF CONTENTS GRAPHIC 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 180 56 57 58 59 60 ACS Paragon Plus Environment a b Page 181 of 188 Journal of Medicinal ChemistryO NH2 NH2 N N 1 NAM N 2 N N 3 O 4 Me 5 ~4 Å OH 180° ~4 Å S 6 180° OH 7 (S) 8 SAM H2N 9 10 CO2H c11 O d NH2 12 NH2 13 N 14 N 15 N 16 N 17 O 18 ~4 Å OH ~4 Å H 19 180° OH 180° 20 (S) 21 H N NS1 ACS Paragon Plus Environment 22 2 23 CO2H 24 H H H H H H H H O OMe 1) Tf O, 2,6-lutidine O OMe IPrCuCl, t-BuOK O OMe crotonaldehyde O O OMe HO 2 CH2Cl2, - 78 °C Journal of MedicinalPMHS, i-BuOH Chemistry nitro-Grela (1 mol %) Page 182 of 188 H OO 2) TMS-CCH, n-BuLi, DMPU OO PhMe, RT, 93% OO MTBE, 40 °C, 71% OO THF, - 78 °C then TBAF 1 2 3 4 1 68% (2 steps) 2 NHTFA TIPS-CCH H H OMe NHTFA NHTFA O 6’ O OMe MeO OMe H H H H [Rh(OAc)(C3 2H4)2]2 P MeO O OMe H2 MeO O OMe (S)-DTBM-SegPhos O O , NaH (S,S)-MeBPE-Rh 17’ 4 H O O MeOH, 40 °C, 94% THF, 40 °C MeOH, RT, 92% OO OO OO 5 89%, 6:1 Z:E 6 5 TIPS 6 TIPS 7 TIPS 7 N N 1) 8TASF, DMF, 60 °C, 95% NHTFA NHBz 3-iodobenzamide NHTFA NHBz H H H H 2) 9TFA/H2O/CH2Cl2, RT MeO O N CuI, Pd(PPh3)4 MeO O N N N 3) 10Ac2O, Pyr, DMAP O N PhMe/DMF/i-Pr2NEt (5:1:1) O N CH2Cl2, RT, 52% (2 steps) 60 °C, 90% 116 AcO OAc AcO OAc 4) N -BzAd, BSA, DTBMP triflate 8 9 12EtCN, 95 °C, 67% 13 N NH2 NH3TFA NH 14 H H 2 HO 6’ O N O 17’ 1) 15LiOH aq., THF N 16 O N 2) NH3, MeOH HO OH 17then HPLC 88%18 (2 steps) ACS Paragon Plus Environment 19 NH2 NS1 20 O (10) 21 0 nM PageJournal 183 of 188of Medicinal Chemistry 24 36 55 2 83 125 190 1 288 6 , rfu 436 20 661 3 1 F / 1 4∆ 5 6

7 0 0.1 80.05 9 0 residuals 10-0.05 ACS Paragon Plus Environment 11 0 200 400 600 800 12 t, sec 13 a b c Journal of Medicinal Chemistry Page 184 of 188

1 2 3 D142 4 5 V143 S87 6 N90 7 D85 8 9 10 11 d12 e f 13 14 15 Y204 16 17 G63 S201 18 Y20 T163 19 S213 20 Y69 Y25 L164 21 22 ACS Paragon Plus Environment 23 24 25 a N NH2 NH H H 2 NH N N 2 NH2 HO O N Page 185 of 188 NH2 JournalNH of Medicinal ChemistryH H H H 2 HO O N N HO O N N N O N O N HO OH O H H N HO OH HO OH H 1 11 4.67 ± 0.01 12 4.32 ± 0.08 13 4.90 ± 0.03 2

NH N NH N N 2 NH2 2 NH2 NH2 NH NH N 3 H H H H H H 2 2 NH2 HO O N O N H H HO HO O N HO O N N N N N 4 O N N O O N O N HO OH 5 HO OH HO OH HO OH

NH 6 2 NH2 NH2 NH2 O 7 O O O 14 6.91 ± 0.07 NS1-alkane (15)8.33 ± 0.03 16 6.97 ± 0.05 8 NS1 (10) pKi = 9.30 ± 0.07

9 NH2 O NH2 H H 10 O NH2 HO O OMe O N N NH HO OH 11 NH H H 2 H H 2 O O N N 12 N N N N NH2 13 HO OH HO OH O 14 17 6.58 ± 0.04 18 5.41 ± 0.05 19 4.11 ± 0.17 15 b16 N N N NH NH2 NH NH H H 2 H H 2 H H 2 N O O O NH2 NH HO N N H2N N 17 H H 2 HO O N N N N O N N O N N 18 O N HO OH HO OH HO OH 19 HO OH 20 NH2 NH2 NH2 NH2 21 O O O O 20 4.65 ± 0.06 21 7.24 ± 0.02 22 5.98 ± 0.01 22 NH N N N N 2 NH2 NH2 NH NH O NH 23 H H H H 2 H H H 2 H H 2 O N MeO H2N O N H2N N O N O N HO N N N N 24 O N N N N O O NH2 25 HO OH HO OH HO OH HO OH

26 NH ACS Paragon Plus Environment 2 NH2 NH2 NH2

27 O O O O 28 23 6.36 ± 0.08 24 7.77 ± 0.02 25 7.12 ± 0.05 26 9.62 ± 0.05 29 a O Journal of Medicinal Chemistry Page 186 of 188 H2N NH2 NH2 NH S 2 O O F O O NH2 Me O 27 5.24 ± 0.02 28 4.71 ± 0.02 29 6.71 ± 0.05 30 8.73 ± 0.15 31 8.47 ± 0.09 1 N NH2 NH H H 2 O 2 HO N O N O N NH2 NH2 O NH O O 2 3 HO OH CF3 O Cl O NH NH O 4 32 8.58 ± 0.08 33 9.72 ± 0.15 34 7.26 ± 0.05 35 5.73 ± 0.02 36 7.49 ± 0.05 5 NH2 O 6 N N N NH NH2 NH2 2 NH2 7 N O 8 O O O NH2 37 8.17 ± 0.05 38 8.05 ± 0.10 39 8.30 ± 0.02 40 7.45 ± 0.07 41 5.43 ± 0.07 9 10 NH N N b NH N 2 NH2 O NH 2 NH2 H H H H 2 H H O O 11 HO O N HO N N N N HO N N N N N N 12 O N O NH2 HO OH HO OH HO OH 13 ACS Paragon Plus Environment NH2 NH2 14 O NH2 O O 15 VH45 5.21 ± 0.02 MS2756 6.04 ± 0.06 MS2734 7.05 ± 0.01 16 a b Page 187 of 188 Journal of Medicinal Chemistry

1 2 3 90° 4 5 C6’ 6 C6’ 7 ACS Paragon Plus Environment 8 9 10 small-molecule inhibition inhibition methyltransferaseJournal of Medicinalat 10 µM Chemistry (%) Pageat 1188 µM of (%) 188

TPMTa 76 51

1 INMTb,* 53 14 2 COMTc 43 0 3 4 PNMTd 26 0 5 GAMTe 26 0

6 GNMTf 23 4 7 8 HNMTg 2 6 9protein or DNA IC (µM) 10methyltransferase 50 11 hDNMT3ah 7.6 12 13 PRMT1i >10

14 ASH1Lj >10 15 DOT1Lk >10 16 17 EHMT1l >10 ACS Paragon Plus Environment 18 G9am >10 19

20 SETDB1n >10