Synthesis of fluorinated carbocyclic pyrimidine nucleoside analogues

Abstract

Analogues of the canonical nucleosides have a longstanding presence and proven capability within medicinal chemistry and drug discovery research. The synthesis reported herein successfully replaces furanose oxygen with CF₂ and CHF in pyrimidine nucleosides, granting access to an alternative pharmacophore space. Key diastereoselective conjugate addition and fluorination methodologies are developed from chiral pool materials, establishing a robust gram-scale synthesis of 6′-(R)-monofluoro- and 6′-gem-difluorouridines. Vital intermediate stereochemistries are confirmed using X-ray crystallography and NMR analysis, providing an indicative conformational preference for these fluorinated carbanucleosides. Utilising these 6′-fluorocarbauridine scaffolds enables synthesis of related cytidine, ProTide and 2′-deoxy analogues alongside a preliminary exploration of their biological capabilities in cancer cell viability assays. This synthetic blueprint offers potential to explore fluorocarbanucleoside scaffolds, indicatively towards triphosphate analogues and as building blocks for oligonucleotide synthesis.

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Introduction

Nucleoside analogues possess a privileged and accomplished history within therapeutic intervention strategies, most notably in the fight against viruses and cancer.^1,2 They are also present within synthetic sequences of DNA and RNA, providing a cornerstone to nucleic acid therapies.³ Since its foundation in the 1950s, the field of nucleoside analogue chemistry has continually evolved, delivering generations of landscape defining therapeutics; notably this includes recent frontline treatments against COVID-19.^4,5 As a result of these successes, the requirement for synthetic capability, both chemical and enzymatic, to evolve new pharmacophore space and manufacture these materials efficiently is ever growing.^6–8

A commonly encountered structural change is modification of the ribose ring (Fig. 1a); examples here include cytarabine, remdesivir and gemcitabine. Within this subtext of ribose ring modification, replacement of furanose oxygen with other heteroatoms or functional groups is often explored (Fig. 1b). Examples here include forodosine, a 4′-azanucleoside, and 4′-thionucleosides.^9,10 The templating of additional ribose ring modifications, ontop of furanose oxygen replacement, enables further divergence to explore new modalities.^11,12

Fig. 1 (a) Examples of nucleoside analogue drugs with furanose ring modifications shown in blue (b) replacement of furanose oxygen with other heteroatoms and carbon, in blue (c) key recent examples of the bioisosteric O → CF₂ replacement for nucleoside analogues.

A third and perhaps more extensively interrogated class of ethereal replacement harnesses carbon;¹³ carbanucleosides (a cyclopentane core with oxygen replaced by CH₂) exist naturally as highly cytotoxic agents aristeromycin and neplanocin A. Carbocyclic nucleosides are resistant to enzymatic degradation, as the hemi-aminal linkage targeted by nucleoside phosphorylases is absent, and adopt alternative ring conformations compared to classical (C3′ or C2′ endo) systems.^14,15 The carbocylic structural motif has successfully underpinned development of the therapeutics abacavir and entecavir and has recently been included within synthetic RNA oligonucleotides.¹⁶

In addition to using CH₂ to replace the furanose ring oxygen, there are a limited number of reports that concomitantly build fluorine into this carbocylic modification, effectively introducing CF₂ (Fig. 1c). For example, 6′6′-difluorinated purines have been developed to target RNA virus activity.¹⁷ This suggested bioisosteric replacement (and the related CHF), originally proposed by Blackburn to replace ethereal oxygen in pyrophosphates,¹⁸ remains underexplored as a pharmacophore.^19–21 Synthetic methods to expedite accessing it,²² and further analogues therefrom, are therefore required to sustain the evolution of new generations of biorelevant nucleoside analogues.^17,23–25

Established benefits from introducing fluorine to nucleoside analogues are evidenced by the drugs sofosbuvir (2′-deoxy-2′-C-methyl-2′-fluoro) and gemcitabine (2′,2′-difluoro-2′-deoxycytidine), alongside the biological capabilities of including 2′-fluorinated monomer components within oligonucleotides for medicinal chemistry.^23,26 Notwithstanding this, the structural effect of replacing furanose oxygen with CF₂, both in free nucleosides and nucleotides, and related DNA/RNA sequences is an area primed for exploration.^27–29

In line with the achievements highlighted in Fig. 1c, we present herein our synthesis of a series of pyrimidine (uridine and cytidine) carbocyclic nucleosides, modified with β-mono- and di-fluorination at the 6′-position (Fig. 2). In addition, we access their ProTide prodrug forms and demonstrate the first examples of diversification to 2′-deoxygentated materials.

Fig. 2 Overview of synthetic targets: access to 6′-mono- and di-fluorinated carbocylic uridines as a point to diversify analogues series to ProTide, cytidine and 2′-deoxygenated forms.

Results and discussion

To access our fluorinated carbocylic nucleoside analogue series, we first required building blocks suitable for nucleobase installation and further diversification. Previous syntheses of bioisosteric CF₂ replacement involved starting from commercially available fragments, where the gem-difluorination is already in place,²⁵ alongside methods that add fluorine to the scaffold using electrophilic fluorination.²⁴ Jeong and colleagues have reported syntheses of fluorinated nucleoside analogues, where the core nucleoside is accessed from enone 1, most commonly effecting conjugate addition of a Gilman reagent, to diastereoselectively install a ^tBu ether protected C5′-hydroxymethyl unit, followed by electrophilic fluorination of an appropriate silyl enol ether.^17,30,31

Our synthesis began similarly, using commercial isopropylidine protected cyclopentenone 1, where we envisaged conjugate addition of a vinyl group (Scheme 1), effectively masking the C5′-primary alcohol until a later oxidative cleavage step. To achieve this, we adopted a report by Schneller accessing aristeromycin analogues, that used a combination of vinyl magnesium bromide, TMSCl, HMPA, and CuBr·Me₂S. However, in our hands we were unable to consistently replicate the yield of 80% reported for this transformation.³² Gratifyingly, when we modified the reagent combination to just vinyl magnesium bromide and CuI in THF, the successful isolation of 2 in 78% yield was enabled. This reaction proved reliable on scales up to 20 g and only one diastereoisomer of 2 was isolated, following chromatography. NMR data matched those reported by Schneller, indicating top face addition of the organocuprate, with the bottom face blocked by the 2,3-di-O-isopropylidine.

Scheme 1 Synthesis of fluorinated cyclopentanone building blocks. (a) VinylMgBr, CuI, THF, −78 °C; (b) TESCl, Li/HMDS, THF, −78 °C; (c) Selectfluor®, DMF, 0 °C–rt, 8/1, 4-(R)/4-(S); (d) TESCl, Li/HMDS, THF, −78 °C; (e) Selectfluor®, DMF, 0 °C–rt, observed as hydrate; box illustrates ¹⁹F NMR (377 MHz, CDCl₃) for 7, with splitting patterns for diastereotopic fluorines shown.

α-Ketofluorination of 2 was completed using a two-step procedure of silyl enol ether formation, followed by reaction with Selectfluor®. Formation of 3 proceeded in quantitative crude yield with a significant downfield chemical shift observed for the vinyl ether proton (δ_H = 4.65 ppm). Silyl enol ether 3 was used immediately for electrophilic fluorination, delivering 4 in an isolated yield of 73% and with an 8 : 1 preference for fluorination having occurred on the β-face of the system (i.e., giving the (R)-F diastereoisomer). Installation of fluorine α to the ketone in 2 was confirmed using ¹H and ¹³C NMR, with large ¹J and ²J couplings observed for the remaining α-proton in 4 (²J_H,F = 50.5 Hz and ¹J_C,F = 201.2 Hz). Electrophilic fluorination of 2 with a C4-vinyl group in place has not been reported previously and compares favourably to results obtained by Jeong using a ^tBu protected hydroxymethyl C4 substituent [5 : 1, (R)/(S)] and a C4-hydroxyethyl homologue [(R)-F selective].^17,31

The diastereomeric mixture 4 was inseparable by silica gel chromatography and the mixture was used in a second fluorination via TES-enol either 5, affording difluorinated 6 in 66% yield over the two steps and reliably delivering this material in >10 g quantities. Ketone 6 was observed by ¹H NMR to be in equilibrium with its hydrate form, 7. The presence of the diastereotopic CF₂ group within 7 was confirmed using ¹⁹F NMR; the expected doublet of doublets observed for each fluorine, with a ¹⁹F–¹⁹F geminal coupling constant of J = 237.5 Hz, alongside smaller ³J_F,H couplings to H₄ of 9.0 and 21.6 Hz respectively. In addition, for one fluorine a small coupling (J = 1.8 Hz, presumably to OH) could be observed, altering the multiplicity to a doublet of doublets of doublets (Scheme 1, box).

After a diastereoselective NaBH₄ reduction of monofluoroketone mixture 4 (Scheme 2), the resultant secondary alcohols 8–11 could be separated using chromatography with isolation of 10 as the major product in 70% yield. For this major (R)-F analogue, stereochemical assignment at C1 was confirmed through an X-Ray crystal structure of a derived C1-dinitrobenzoate (Scheme 2, boxes). The minor (S)-F diastereoisomer 8 was also obtained, in 3% yield, alongside the (R)-F C1 epimer 11, in 3% yield (compound 9 was not isolated). Both 8 and 11 were crystalline solids and X-ray crystallography also confirmed their stereochemical assignment at both C6 (fluorination site) and C1 (secondary alcohol, Scheme 2, boxes). Due to the small amount of 8 isolated, no further synthesis towards an (S)-F analogue was completed. Diastereoselective reduction of difluorinated 6/7 was completed in a similar manner giving alcohol 12 in 79% yield, with no evidence of a C1 epimer (Scheme 2).

Scheme 2 Diastereoselective reduction of fluorinated cyclopentanone building blocks. (a) NaBH₄, MeOH, rt.

Secondary alcohols 10 and 12 were next acetylated at C1 in excellent yield (95% and 94% for 13 and 14 respectively, Scheme 3). This delivered appropriate materials for oxidative alkene cleavage, first with AD-mix β, to give a vicinal diol mixture, followed by cleavage using NaIO₄. Subsequent reduction of the generated aldehyde with concomitant acetate removal furnished alcohols 15 and 16. gem-Difluoroalcohol 16 was crystalline and X-ray crystallography was again used to confirm the previous diastereoselective reduction had occurred from the top face (Scheme 3, box), to provide appropriate material for later installation of the nucleobase via stereochemical inversion at C1.

Scheme 3 Synthesis of mono- and gem-difluorinated uridines. (a) Ac₂O, pyridine, DCM, rt; (b) AD-mix β, ⁱPrOH, H₂O, rt; (c) for 15: NaIO₄ (1.7 equiv.), dioxane, H₂O, rt, 1 h; for 16: NaIO₄ (10 equiv.), dioxane, H₂O, 0 °C, 6 h; (d) NaBH₄, MeOH, rt; (e) TBDPSCl, imidazole, DMF; (f) Tf₂O, pyridine; (g) NaN₃, DMF, 100 °C; (h) H₂, Pd/C, MeOH, rt; (i) (3-ethoxyacryloyl)isocyanate, DMF, rt; (j) H₂SO₄, 1,4-dioxane, reflux; (k) TBAF, THF, rt.

This three-step cleavage proceeded efficiently in the case of the monofluorinated substrate, yielding 15 in 66% over three steps. A complication was however encountered with the difluorinated substrate 14, resulting in the desired material from NaIO₄-mediated diol cleavage isolable only as a minor component and an elimination product (the enal) observed as the major product from this transformation. This led to the formation of 17 (Scheme 3, box) as the major product, following redcution. By lowering the reaction temperature to 0 °C and increasing the equivalents of NaIO₄ five-fold, the unwanted elimination was suppressed and the desired material (16) was obtained. Following this optimisation, concomitant reduction and deprotection delivered 15 and 16, with 16 isolated in 60% yield over the three steps.

Alcohols 15 and 16 were next protected with TBDPS at the C5 position. Some additional TBDPS protection of the C1 secondary alcohol was observed in the case of 15. However, 15 was easily recoverable following global silyl-deprotection using TBAF in THF. Azidation of the remaining secondary alcohol was achieved via formation of an α-triflate, followed by stereoinversion using NaN₃ to give 18 and 19 in yields of 77% and 72% respectively over three steps. Direct S_N2 displacement of an α-mesylate with pyrimidine nucleobases or using Mitsunobu inversion were unsuccessful, in line with results reported for the related synthesis of fluorinated aristeromycin derivatives.³⁰

Acryloyl urea intermediates 20 and 21 were next obtained, first by reduction of 18 and 19 to the C1 amine, which was then treated with (3-ethoxy-acryloyl)isocyanate, freshly prepared by reaction of 3-ethoxy-acryloyl chloride with AgOCN in toluene,³³ to give 20 and 21 in yields of 84% and 87% over two steps. Finally, cyclisation to the desired pyrimidine and acetonide removal was achieved by treatment with H₂SO₄ in dioxane. A final TBAF deprotection yielded fluorinated uridines 22 and 23 in total yields of 9% and 6% respectively, and over 15 and 17 steps from 1.

With 22 and 23 in hand we were interested to compare elements of their NMR data to canonical uridine. We noted an upfield chemical shift for the pseudo-anomeric position in 22 (δ_H = 4.89 ppm, δ_C = 62.3 ppm) relative to uridine (δ_H = 5.83 ppm, δ_C = 91.7 ppm); this effect was less pronounced in gem-difluorinated 23 (δ_H = 5.37 ppm, δ_C = 63.6 ppm). Furthermore, Table 1 illustrates selected observed ³J and ¹J coupling constants for 22 and 23.

Table 1 Comparison of ³J and ¹J couplings for carbanucleosides 22 and 23 and uridine in D₂O at 400 MHz. Indicative 1′-exo envelope (₁E) conformation for 22 and highlighting observed ³J_H1–H2, ³J_H1–H6 and ³J_H1–F couplings. U = uridine

	X	^3 J (Hz)
		1′-2′	1′-6′	1′-F
	O	4.6	—	—
	CHF	10.5	3.4	30.6
	CF2	10.2	—	18.8, 10.0
		^1 J (Hz)
	X	1′	2′	3′	4′
	O	171.8	152.4	149.7	150.1
	CHF	140.9	145.9	151.4	128.1
	CF2	144.3	144.5	154.5	135.7

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First considering ³J_H–H couplings, we noted changes between H₁ and H₂ for both the mono- and difluorinated systems. The ³J_{H1′–H2′} coupling in uridine is 4.6 Hz, but this increased to >10 Hz for both 22 and 23, indicating a change to this dihedral angle. Carbanucleosides tend to adopt a 1′-exo envelope conformation (₁E),³⁴ due to lack of the anomeric effect and gauche interactions between the furanose oxygen and the 2′- and 3′-OH groups. This ₁E conformation is suggested to be energetically and sterically favourable, placing the bulky nucleobase in a pseudoequatorial position.^14,15,34 The sizable ³J_{H1′–H2′} coupling constants observed here for 22 and 23 are indicative of H₁–C₁–C₂–H₂ torsion angles tending to 150°, and tentatively support this ₁E conformational assignment in solution-phase. However, these couplings are larger than related values observed for non-fluorinated carbanucleosides (³J_{H1′–H2′} = 5.6 Hz for carbauridine in DMSO-d₆,¹⁶ and ³J_{H1′–H2′} = 8.5 Hz for aristeromycin and carbaguanosine in D₂O).³⁵

¹ J coupling constants can serve as convenient identifiers for the anomeric carbon within nucleoside rings, alongside supporting patterns of stereochemical identity (α and β anomers).³⁶ Recording ¹J C–H values for 22 and 23 (Table 1), we noted a significant decrease in this coupling constant at C1′ and C4′ when replacing furanosyl oxygen with CHF or CF₂. CHF showed the largest change relative to uridine (for C1′: ¹J_H,C = 171.8 Hz for uridine and 140.9 Hz for 22). This effect was not pronounced for C2′ or C3′, where the coupling constants remained similar to uridine. Since ¹J C–H coupling constants of carbon generally increase with the electronegativity of attached functional groups, these data exhibit the effect of the removal of oxygen from the ring, and indicate that replacing with fluorinated motifs produces coupling constants that resemble those for ring carbons bearing OH groups (i.e., ¹J_H,C = 149.7 Hz for C3′ in uridine and 140.9 Hz for C1′ in 22).

With established access to gram quantities of 22 and 23 we next sought to diversify these analogues, targeting modification of the pyrimidine base, ProTide forms and 2′-deoxygenation. Accordingly, cytidine analogues 26 and 27 were prepared in three steps from 22 and 23. Global hydroxyl group protection was performed to give acetate protected uridines 24 and 25 in excellent yields (Scheme 4). This was followed by treatment with 1,2,4-triazole and POCl₃ to provide an intermediate 4-triazole which was subsequently aminated alongside simultaneous hydroxyl group deprotection, using 35% NH₄OH, to deliver 26 and 27 in yields of 53% and 42% respectively over three steps.

Scheme 4 Synthesis of mono- and gem-di-fluorinated cytidines. (a) Ac₂O, pyridine, DMAP, DCM, rt; (b) 1,2,4-triazole, POCl₃, Et₃N, MeCN, rt; (c) 35% NH₄OH, H₂O, rt.

Uridine analogues 22 and 23 were next 3′,5′-O-protected with TIPDSiCl₂ in pyridine to give 28 and 29 in 76% and 43% yields respectively (Scheme 5). Some unwanted protection of the 2′-O-hydroxyl group was also observed, however, 22 or 23 could easily be regenerated using a global TBAF deprotection. A Barton-McCombie 2′-deoxygenation was next performed via the 2′-O-thiocarbamate. This intermediate was obtained from treatment of either 28 or 29 with 1,1′-thiocarbonyldiimidazole in MeCN, followed by radical mediated deoxygenation using Bu₃SnH and AIBN, delivering 3′,5′-O-protected uridines 30 and 31 in 52% and 68% yields respectively, over two steps. The target 2′-deoxy uridines 32 and 33 were subsequently obtained in 66% and 55% yields, following TIPDS-deprotection with TBAF. The 3′,5′-O-protected uridines 30 and 31 were also converted through to their cytidine derivatives using the procedures described above for 24 and 25, affording 34 and 35 in 11% and 9% yields respectively over three steps.

Scheme 5 Synthesis of 2′-deoxy mono- and gem-di-fluorinated uridine and cytidine. (a) TIPDSiCl₂, pyridine, rt; (b) TCDI, DMAP, MeCN, 45 °C; (c) Bu₃SnH, AIBN, toluene, reflux; (d) TBAF, THF, rt; (e) 1,2,4-triazole, POCl₃, Et₃N, MeCN, 0 °C–rt; (f) 35% NH₄OH, 1,4-dioxane, rt; (g) TBAF, THF, rt.

Finally, each of 22 and 23 were converted through to their ProTide phosphoramidate forms (Scheme 6).³⁷ Firstly, a 2′,3′-O-acetonide protecting group was installed, giving 36 and 37 in yields of 81% and 71% respectively. Deprotonation of the remaining 5′-OH with tert-butylmagnesium chloride, followed by reaction with commercial phosphoramidate reagent 38,³⁸ delivered uridine phosphoramidate analogues 39 and 40 in 24% and 27% yields respectively, following acetonide removal.

Scheme 6 Synthesis of uridine phosphoramidates. (a) H₂SO₄, Me₂CO, reflux; (b) 38, ^tBuMgCl, THF, −78 °C; (c) formic acid, H₂O, rt.

Compounds 22, 23, 26, 27, 32–35, 39 and 40 were evaluated in cellular viability assays against cancer cell lines U87-MG and PANC-1 (Table 2). 6-(R)-Monofluorocytidine 26 showed mild activity against U87-MG cells (Table 2, entry 5). Whilst the remaining compounds showed little activity below 100 μM, this may prove advantageous for exploring antiviral activity. In addition, whilst the ProTide strategy adopted here (compounds 39 and 40, Table 2, entries 11 & 12) did not translate to biological activity, it is possible that enzymatic release of the masked monophosphate was prevented in this assay and that alternative pronucleotide design could be considered; for example, the cycloSal approach obviates enzymatic activation of a prodrug unit.^39,40 Within a wider program of work, we will explore antiviral activity for the compounds reported herein, alongside targeting nucleotide triphosphate derivatives and associated in vitro polymerase activity, to further inform potential monophosphate prodrug candidate development.

Table 2 Biological evaluation of compounds 22, 23, 26, 27, 32–35, 39 and 40 against U87-MG and PANC-1 cell lines. Gemcitabine and cytarabine were included as positive controls

Entry	Compound	Cell viability (%)
		U87-MG		PANC-1
		100 μM	10 μM	100 μM	10 μM
a Gemcitabine evaluated at top concentration of 10 μM. b Cytarabine evaluated at top concentration of 100 μM.
1	Gemcitabine^a	—	32	—	37
2	Cytarabine^b	22	—	42	—
3	22	≥100	≥100	≥100	≥100
4	23	≥100	≥100	≥100	≥100
5	26	42	63	70	≥100
6	27	93	96	≥100	≥100
7	32	≥100	≥100	≥100	≥100
8	33	≥100	≥100	≥100	≥100
9	34	≥100	≥100	89	≥100
10	35	≥100	≥100	≥100	≥100
11	39	≥100	≥100	≥100	≥100
12	40	≥100	≥100	≥100	≥100

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Conclusion

In summary, we have developed a reliable and scalable synthesis of 6′-(R)-monofluoro and 6′-gem-difluorouridines as common building blocks for wider diversification to alternative pyrimidines, ProTide prodrug forms and 2′-deoxygentated nucleoside analogues. A robust conjugate addition using a vinyl organocuprate, followed by diastereoselective Selectfluor α-keto fluorination via the silyl enol ether delivers key scaffold materials. These are elaborated through 5-position alkene oxidation/cleavage and nucleobase installation, building up from a C1 amine using (3-ethoxyacryloyl)isocyanate. Importantly this alkene manipulation methodology lays a foundation for exploration of 4′-position modifications within this fluorocarbanucleoside scaffold. The synthesis is supported by key X-ray structures confirming cyclopentane ring stereochemistries. Additionally, key differences to canonical pyrimidine nucleoside ¹H NMR data are observed and work to further understand the conformational preferences for this analogues class, alongside their wider biological evaluation is currently underway.

Experimental section

General experimental methods

All chemicals were purchased from Acros Organics, Alfa Aesar, Biosynth Carbosynth, Fisher Scientific, Fluorochem, Sigma Aldrich or TCI Chemicals and were used without further purification unless otherwise stated. CuI was purified prior to use.⁴¹ The concentration of vinylmagnesium bromide was determined via iodometric titration⁴² prior to use. Anhydrous DMF, MeOH, pyridine and Et₃N were obtained from Sure/Seal™ bottles via chemical suppliers. Anhydrous THF, DCM and toluene were obtained by passing solvent through activated alumina columns and dispensed from a PureSolv MD ASNA solvent purification system and stored over 4 Å molecular sieves. Unless otherwise stated, all reactions were conducted using anhydrous solvents, under an atmosphere of N₂ which was passed through a Drierite® drying column. Thin layer chromatography (TLC) was performed using pre-coated 0.25 mm 60 F₂₅₄ silica gel plates (Merck). Visualisation was achieved using UV light (λ = 254 nm), and KMnO₄ staining followed by heating, or Ninhydrin staining followed by heating, or 5% H₂SO₄/EtOH staining followed by heating. Flash column chromatography was performed using silica gel, high purity grade, pore size 60 Å, 230–400 mesh particle size, 40–63 μm particle size (Sigma Aldrich). All final compounds were purified on an Agilent 1260 Infinity II preparative HPLC system equipped with a variable wavelength detector and a fraction collector, on a reverse phase column (Polaris 180 Å C18-A. 21.2 × 250 mm, 5 μm) to achieve a purity level >95%. Visualisation was achieved using UV detection at 254 nm. Optical rotations were recorded on a Bellingham + Stanley ADP430 (specific rotation, tube length: 50 mm, concentrations in g per 100 mL). All high-resolution mass spectra were measured at the EPSRC National Mass Spectrometry Facility at Swansea University, UK. All crystallographic data were collected on a Bruker D8 Quest ECO diffractometer using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) and a Photon II-C14 CPAD detector. The ADPs are rendered at 50% probability level. NMR spectra were recorded on a Bruker Avance 400 spectrometer. The chemical shift data for each signal are given as δ in units of parts per million (ppm) relative to tetramethylsilane, where δ = 0.00 ppm. The number of protons (n) for a given resonance is indicated by nH. The multiplicity of each signal is indicated by: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), p (pentet), sep (septet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dddd (doublet of doublet of doublet of doublets), dt (doublet of triplets), tt (triplet of triplets), dqd (doublet of quartets of doublets) or m (multiplet). Coupling constants (J) are quoted in Hz and calculated to the nearest 0.1 Hz. Cell culture: PANC-1 (ATCC, catalogue number: CRL-1469) cells were cultured at 37 °C with 5% CO₂ in DMEM (Corning, catalogue number: 10-013CV), supplemented with 10% heat-inactivated FBS (Corning, catalogue number: 35016CV) and 1X Non-essential amino acids [(0.1 mM per amino acid) (Corning, catalogue number: 25025CI)] and 1× Penicillin–Streptomycin Solution [Penicillin (100 IU) and Streptomycin (100 μg mL⁻¹) (Corning, catalogue number: 30002CI)]. U87-MG (ATCC, catalogue number: HTB-14) cells were cultured at 37 °C with 5% CO₂ in EMEM (Lonza, catalogue number: 12-611F), supplemented with 10% heat-inactivated FBS (Corning, catalogue number: 35016CV) and 1× non-essential amino acids (0.1 mM per amino acid) (Corning, catalogue number: 25025CI) and 1× Corning™ Penicillin–Streptomycin Solution [Penicillin (100 IU) and Streptomycin (100 μg mL⁻¹) (Corning, catalogue number 30002CI)].

(2R,3R,4R)-2,3-O-Isopropylidene-4-vinylcyclopentan-1-one 2. An oven dried multi-necked round bottom flask was charged with CuI (2.5 g, 13.0 mmol, 0.1 equiv.), evacuated and flushed with N₂ three times and heated to 100 °C under vacuum for 1 h with stirring. The reaction vessel was cooled to rt and THF (520 mL) was added. The mixture was stirred vigorously and cooled to −78 °C, at which point vinylmagnesium bromide (216 mL, 162 mmol, 1.3 equiv., 0.75 M in THF) was added dropwise. The reaction mixture was slowly warmed to −40 °C over 1 h, after which it was cooled to −78 °C again. (4R,5R)-4,5-O-Isopropylidene-2-cyclopentenone 1 (20.0 g, 130 mmol, 1.0 equiv.) in THF (130 mL) was added dropwise at 0.5 mL min⁻¹ using a syringe pump, and the reaction mixture was stirred at −78 °C for 2 h. TLC analysis (petroleum ether/Et₂O, 4 : 1) showed complete consumption of the starting material to a higher R_f. The reaction mixture was warmed to 0 °C and quenched with 5 : 1 sat. NH₄Cl/sat. Na₂S₂O₃ (v/v, 600 mL). The aqueous phase was extracted with EtOAc (3 × 500 mL), and the combined organic phases were washed with H₂O (500 mL), brine (200 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography on silica gel (0–20% Et₂O/petroleum-ether) to give 2 as a colourless oil (18.4 g, 101 mmol, 78%). R_f = 0.39 (petroleum-ether/Et₂O, 4 : 1); ¹H NMR (400 MHz, CDCl₃): δ 5.83 (1H, ddd, ³J_H5–H6b = 17.2 Hz, ³J_H5–H6a = 10.6 Hz, ³J_H5–H4 = 6.5 Hz, H₅), 5.15 (1H, d, ³J_H6a–H5 = 10.6 Hz, H_6a), 5.10 (1H, dd, ³J_H6b–H5 = 17.4 Hz, ²J_H6b–H6a = 1.6 Hz, H_6b), 4.64 (1H, d, ³J_H3–H2 = 5.2 Hz, H₃), 4.20 (1H, d, ³J_H2–H3 = 5.2 Hz, H₂), 3.11 (1H, dd, ³J_H4–H7a = 7.9 Hz, ³J_H4–H5 = 7.0 Hz, H₄), 2.84 (1H, dd, ²J_H7a–H7b = 18.3 Hz, ³J_H7a–H4 = 8.6 Hz, H_7a), 2.29 (1H, d, ²J_H7b–H7a = 18.2 Hz, H_7b), 1.45 (3H, s, CH₃), 1.35 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 213.2 (C₁, C=O), 137.2 (C₅), 116.5 (C₆), 112.5 [C(CH₃)₂], 81.4 (C₃), 77.9 (C₂), 39.8 (C₄), 38.6 (C₇), 26.9 [C(CH₃)₂], 24.9 [C(CH₃)₂]; HRMS (ESI): calculated for C₁₀H₁₄O₃Na [M + Na]⁺ 205.0835, found 205.0827. These data were in good agreement with literature values.³²

(2R,3R,4R)-2,3-O-Isopropylidene-4-vinyl-7-(R/S)-fluorocyclopentan-1-one 4. Ketone 2 (35.1 g, 193 mmol, 1.0 equiv.) was dissolved in THF (960 mL) and cooled to −78 °C. TESCl (64.0 mL, 386 mmol, 2.0 equiv.) was added followed by LiHMDS (64.5 g, 386 mmol, 2.0 equiv.) and the reaction was stirred at −78 °C for 2 h. TLC analysis (petroleum-ether/Et₂O, 4 : 1) showed complete conversion of the starting material to a higher R_f. The reaction was warmed to 0 °C and quenched with sat. NH₄Cl (400 mL). The aqueous phase was extracted with EtOAc (3 × 400 mL), and the combined organic phases were washed with H₂O (300 mL), brine (300 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The residue was dissolved in DMF (770 mL) and cooled to 0 °C, after which Selectfluor® (82.0 g, 231 mol, 1.2 equiv.) was added in three portions over 30 min. The reaction mixture was allowed to warm to rt and was stirred for 3 h, at which point TLC analysis (petroleum-ether/Et₂O, 4 : 1) showed complete conversion from a higher R_f to a lower R_f. The reaction mixture was cooled to 0 °C and quenched with sat. NH₄Cl (400 mL). The aqueous phase was extracted with EtOAc (4 × 300 mL), and the combined organic phases were washed with H₂O (3 × 200 mL), brine (200 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography on silica gel (0–50% Et₂O/petroleum-ether) to give 4, as an 8 : 1 inseparable mixture of diastereomers and a colourless oil (28.2 g, 140 mmol, 73%). R_f = 0.09 (petroleum-ether/Et₂O, 4 : 1); ¹H NMR (400 MHz, CDCl₃) (major): δ 5.58 (1H, ddd, ³J_H5–H6b = 17.3 Hz, ³J_H5–H6a = 10.8 Hz, ³J_H5–H4 = 7.8 Hz, H₅), 5.50 (1H, ddd, ²J_H7–F = 50.5 Hz, ³J_H7–H4 = 7.9 Hz, ⁴J_H7–H3 = 0.9 Hz, H₇), 5.31 (1H, dt, ³J_H6a–H5 = 10.8 Hz, ²J_H6a–H6b = 0.9 Hz, H_6a), 5.26 (1H, dt, ³J_H6b–H5 = 17.3 Hz, ²J_H6b–H6a = 1.1 Hz, H_6b), 4.74 (1H, ddd, ³J_H3–H2 = 5.7 Hz, ³J_H3–H4 = 4.7 Hz, ⁴J_H3–H7 = 0.9 Hz, H₃), 4.25 (1H, dd, ³J_H2–H3 = 6.0 Hz, ⁴J_H2–F = 2.6 Hz, H₂), 3.42–3.38 (1H, m, H₄), 1.48 (3H, s, CH₃), 1.35 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃) (major): δ 204.5 (d, ²J_C1–F = 13.6 Hz, C₁, C=O), 130.1 (d, ³J_C5–F = 5.4 Hz, C₅), 120.5 (d, ⁴J_C6–F = 1.5 Hz, C₆), 113.1 [C(CH₃)₂], 91.0 (d, ¹J_C7–F = 201.2 Hz, C₇), 78.1 (d, ³J_C3–F = 6.1 Hz, C₃), 74.0 (d, ³J_C2–F = 2.3 Hz, C₂), 43.5 (d, ²J_C4–F = 16.3 Hz, C₄), 26.3 [C(CH₃)₂], 24.0 [C(CH₃)₂]; ¹⁹F NMR (376 MHz, CDCl₃) (major): δ −216.6 (d, ²J_F–H7 = 50.5 Hz); HRMS (ESI): calculated for C₁₀H₁₄FO₃ [M + H]⁺ 201.0921, found 201.0919.

(2R,3R,4R)-2,3-O-Isopropylidene-4-vinyl-7-gem-difluorocyclopentan-1-one 6 (and hydrate 7). Monofluoroketone 4 (15.2 g, 76.0 mmol, 1.0 equiv.) was dissolved in THF (380 mL) and cooled to 78 °C. TESCl (25.5 mL, 152 mmol, 2.0 equiv.) was added dropwise, followed by LiHMDS (25.4 g, 152.0 mmol, 2.0 equiv.). The reaction was slowly warmed to rt, and stirred for a further 18 h, at which point TLC analysis (petroleum-ether/Et₂O, 4 : 1) showed complete conversion of the starting material to a higher R_f. The reaction was cooled to 0 °C and quenched with sat. NH₄Cl (200 mL). The aqueous phase was extracted with EtOAc (3 × 200 mL). The combined organic phases were washed with H₂O (150 mL), brine (150 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The residue was dissolved in DMF (380 mL) and cooled to 0 °C, after which Selectfluor® (32.3 g, 91.2 mmol, 1.2 equiv.) was added in two portions over 20 min. The reaction mixture was allowed to warm to rt, and stirred for 16 h, at which point TLC analysis (petroleum-ether/Et₂O, 4 : 1) showed complete conversion from a higher R_f to a lower R_f. The reaction mixture was cooled to 0 °C and quenched with sat. NH₄Cl (200 mL), and the aqueous phase was extracted with EtOAc (4 × 200 mL). The combined organic phases were washed with H₂O (3 × 100 mL), brine (150 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography on silica gel (0–50% Et₂O/petroleum-ether) to give 6, as a colourless oil (11.8 g, 50.1 mmol, 66%). R_f = 0.10 (petroleum-ether/Et₂O, 4 : 1); ¹H NMR (400 MHz, CDCl₃) (hydrate 7): δ 5.85 (1H, ddd, ³J_H5–H6a = 17.3 Hz, ³J_H5–H6b = 10.4 Hz, ³J_H5–H4 = 8.3 Hz, H₅), 5.38–5.34 (2H, m, H_6a, H_6b), 4.504.46 (1H, m, H₂), 4.44–4.42 (1H, m, H₃), 4.26 (1H, d, ⁴J_OH–F = 1.7 Hz, OH), 3.15–3.05 (1H, m, H₄), 3.02 (1H, s, OH), 1.57 (3H, s, CH₃), 1.37 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃) (hydrate 7): δ 129.7 (d, ³J_C5–F = 4.1 Hz, C₅), 124.1 (dd, ¹J_C7–F = 271.1 Hz, ¹J_C7–F = 253.5 Hz, C₇), 121.1 (C₆), 114.2 [C(CH₃)₂], 95.2 (dd, ²J_C1–F = 25.7 Hz, ²J_C1F = 22.0 Hz, C₁), 79.3 (d, ³J_C3–F = 3.2 Hz, C₃), 78.6 (dd, ³J_C2–F = 7.7 Hz, ³J_C2–F = 1.2 Hz, C₂), 52.5 (dd, ²J_C4–F = 20.6 Hz, ³J_C4–F 20.1 Hz, C₄), 26.2 [C(CH₃)₂], 24.8 [C(CH₃)₂]; ¹⁹F NMR (376 MHz, CDCl₃) (hydrate 7): δ −119.4 (ddd, ²J_F–F = 237.5, ³J_F–H4 = 9.0 Hz, ⁴J_F–OH = 1.8 Hz), −121.1 (dd, ²J_F–F = 237.5 Hz, ³J_F–H4 = 21.7 Hz); HRMS (ketone 6) (ESI): calculated for C₁₀H₁₃F₂O₃ [M + H]⁺ 219.0827, found 219.0826.

General procedure for the synthesis of 8–11. Ketone 4/6 (1.0 equiv.) was dissolved in MeOH (0.20 M) and cooled to 0 °C. NaBH₄ (2.0 equiv.) was added in three portions over 10 min, and the reaction mixture was allowed to warm to rt. The reaction mixture was stirred at rt for 16 h, at which point TLC analysis (petroleum-ether/Et₂O, 1 : 1) showed complete consumption of the starting material to a higher R_f. The reaction mixture was cooled to 0 °C and H₂O (200 mL) was added. The solvent was removed in vacuo, and the residue was partitioned between H₂O (200 mL) and EtOAc (200 mL). The aqueous phase was extracted with EtOAc (3 × 200 mL), and the combined organic phases were washed with brine (200 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography on silica gel (10–20% Et₂O/hexane).

(1R,2S,3R,4R,7S)-2,3-O-Isopropylidine-4-vinyl-7-fluorocyclopentan-1-ol 8. Alcohol 8 (403 mg, 2.0 mmol, 3%) a white crystalline solid, was obtained following the above procedure from a mixture of 4 (13.0 g, 65.0 mmol, 1.0 equiv.). R_f = 0.42 (petroleum-ether/Et₂O, 1 : 1); mp: 48–50 °C; [α]^24.9_D = −15.5 (c 2.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 5.62 (1H, ddd, ³J_H5–H6a = 17.6 Hz, ³J_H5–H6b = 10.7 Hz, ³J_H5–H4 = 7.0 Hz, H₅), 5.20–5.12 (2H, m, H_6a, H_6b), 4.79–4.65 (1H, m, H₇), 4.60–4.59 (2H, m, H₂, H₃), 4.11–4.00 (1H, m, H₁), 3.12 (1H, dd, ³J_H4–F = 18.6 Hz, ³J_H4–H3/H5 = 6.4 Hz, H₄), 2.76 (1H, d, ³J_OH–H1 = 10.5 Hz, OH), 1.51 (3H, s, CH₃), 1.34 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 133.6 (d, ³J_C5–F = 6.1 Hz, C₅), 117.9 (C₆), 112.0 [C(CH₃)₂], 97.1 (d, ¹J_C7–F = 189.6 Hz, C₇), 83.0 (C₃), 77.7 (C₂), 72.6 (d, ²J_C1–F = 17.0 Hz, C₁), 50.4 (d, ²J_C4–F = 19.1 Hz, C₄), 26.3 [C(CH₃)₂], 24.5 [C(CH₃)₂]; ¹⁹F NMR (376 MHz, CDCl₃): δ −197.9 (ddd, ²J_F–H = 52.0 Hz, ³J_F–H1 = 26.2 Hz, ³J_F–H4 = 18.4 Hz); HRMS (NSI): calculated for C₁₀H₁₅FO₃Na [M + Na]⁺ 225.0897, found 225.0898.

(1R,2S,3R,4R,7R)-2,3-O-Isopropylidine-4-vinyl-7-fluorocyclopentan-1-ol 10. Alcohol 10 (9.1 g, 45.1 mmol, 70%) a colourless oil, was obtained following the above procedure from a mixture of 4 (13.0 g, 65.0 mmol, 1.0 equiv.). R_f = 0.5 (petroleum-ether/Et₂O, 1 : 1); [α]^24.9_D = +8.6 (c 0.2, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 5.87 (1H, ddd, ³J_H5–H6a = 17.9 Hz, ³J_H5–H6b = 10.4 Hz, ³J_H5–H4 = 7.7 Hz, H₅), 5.30–5.24 (2H, m, H_6a, H_6b), 4.90 (1H, dt, ²J_H7–F = 50.7 Hz, ³J_H7–H1/H4 = 4.2 Hz, H₇), 4.73–4.69 (1H, m, H₂), 4.64–4.61 (1H, m, H₃), 4.17–4.11 (1H, m, H₁), 3.05–2.94 (1H, m, H₄), 2.76 (1H, dd, ³J_OH–H1 = 4.5 Hz, ⁴J_OH–F = 2.4 Hz, OH), 1.54 (3H, s, CH₃), 1.38 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 132.5 (d, ³J_C5–F = 6.1 Hz, C₅), 118.7 (C₆), 113.8 [C(CH₃)₂], 100.6 (d, ¹J_C7–F = 181.0 Hz, C₇), 82.8 (d, ³J_C3–F = 2.0 Hz, C₃), 77.7 (d, ³J_C2–F = 2.7 Hz, C₂), 72.0 (d, ²J_C1–F = 26.3 Hz, C₁), 50.0 (d, ²J_C4–F = 17.6 Hz, C₄), 26.3 [C(CH₃)₂], 24.5 [C(CH₃)₂]; ¹⁹F NMR (376 MHz, CDCl₃): δ −204.2 (ddd, ²J_F–H7 = 50.7 Hz, ³J_F–H4 = 26.2 Hz, ³J_F–H1 = 10.5 Hz); HRMS (NSI): calculated for C₁₀H₁₅FO₃Na [M + Na]⁺ 225.0897, found 225.0898.

(1S,2S,3R,4R,7R)-2,3-O-Isopropylidine-4-vinyl-7-fluorocyclopentan-1-ol 11. Alcohol 11 (391 mg, 1.9 mmol, 3%), a white crystalline solid, was obtained following the above procedure from 4 (13.0 g, 65.0 mmol, 1.0 equiv.). R_f = 0.38 (petroleum-ether/EtOAc, 1 : 1); mp: 86–87 °C; [α]^21.4_D = +33.4 (c 5, DCM); ¹H NMR (400 MHz, CDCl₃): δ 5.95 (1H, ddd, ³J_H5–H6a = 17.4 Hz, ³J_H5–H6b = 10.3 Hz, ³J_H5–H4 = 8.1 Hz, H₅), 5.31–5.22 (2H, m, H_6a, H_6b), 5.00 (1H, dt, ²J_H7–F = 52.4 Hz, ³J_H7–H1 = 3.8 Hz, H₇), 4.63 (1H, ddd, ³J_H3–H2 = 7.0 Hz, ³J_H3–H4 5.2 Hz, ⁴J_H3–F = 1.7 Hz, H₃), 4.57–4.53 (1H, m, H₂), 4.20 (1H, ddt, ³J_H1–F = 19.6 Hz, ³J_H1–OH = 6.8 Hz, ³J_H1–H2/H7 = 3.4 Hz, H₁), 2.81 (1H, ddt, ³J_H4–F = 28.4 Hz, ³J_H4–H5 = 8.5 Hz, ³J_H4–H3 = 4.5 Hz, H₄), 2.18 (1H, dd, ³J_OH–H1 = 7.3, ⁴J_OH–F = 2.5 Hz, OH), 1.50 (3H, s, CH₃), 1.31 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 133.0 (d, ³J_C5–F = 5.6 Hz, C₅), 118.6 (C₆), 113.0 [C(CH₃)₂], 98.4 (d, ¹J_C7–F = 181.8 Hz, C₇), 84.7 (d, ³J_C2–F = 1.0 Hz, C₂), 82.6 (d, ³J_C3–F = 1.7 Hz, C₃), 78.0 (d, ²J_C1–F = 16.7 Hz, C₁), 51.9 (d, ²J_C4–F = 17.5 Hz, C₄), 27.0 [C(CH₃)₂], 24.4 [C(CH₃)₂]; ¹⁹F NMR (377 MHz, CDCl₃): δ −210.9 (ddd, ²J_F–H7 = 49.9 Hz, ³J_F–H4 = 28.4 Hz, ³J_F–H1 = 19.6 Hz); HRMS (NSI): calculated for C₁₀H₁₅FO₃Na [M + Na]⁺ 225.0897, found 225.0898.

(1R,2S,3R,4R)-2,3-O-Isopropylidine-4-vinyl-7-gem-difluorocyclopentan-1-ol 12. Alcohol 12 (6.9 g, 31.3 mmol, 79%) a white waxy solid, was obtained following the above procedure from 6/7 (9.4 g, 40.0 mmol, 1.0 equiv.). R_f = 0.56 (petroleum ether/Et₂O, 1 : 1); [α]^24.3_D = −18.5 (c 4.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 5.80 (1H, ddd, ³J_H5–H6a = 17.8 Hz, ³J_H5–H6b = 10.2 Hz, 7.9 Hz, H₅), 5.17–5.32 (1H, m, H_6a, H_6b), 4.63–4.59 (1H, m, H₂), 4.55–4.52 (1H, m, H₃), 4.04 (1H, app. p, J = 6.0 Hz, H₁), 3.17–3.07 (1H, m, H₄), 2.90 (1H, dd, ³J_OH–H1 = 5.4 Hz, ⁴J_OH–F = 1.4 Hz, OH), 1.55 (3H, s, CH₃), 1.37 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 129.8 (dd, ³J_C5–F = 4.6 Hz, ³J_C5–F = 2.3 Hz, C₅), 126.9 (dd, ¹J_C7–F = 266.6 Hz, ¹J_C7–F = 249.7 Hz, C₇), 120.6 (C₆), 113.3 [C(CH₃)₂], 80.4 (dd, ³J_C3–F = 6.2 Hz, ³J_C3–F = 1.4 Hz, C₃), 75.1 (t, ³J_C2–F = 3.3 Hz, C₂), 70.8 (dd, ²J_C1–F = 31.8 Hz, ²J_C1–F = 20.0 Hz, C₁), 51.8 (dd, ²J_C4–F = 21.4 Hz, ²J_C4–F = 19.8 Hz, C₄), 26.0 [C(CH₃)₂], 24.5 [C(CH₃)₂]; ¹⁹F NMR (376 MHz, CDCl₃): δ −112.8 (dt, ²J_F–F = 245.0 Hz, ³J_F–H1/H4 = 6.6 Hz), −114.7 (ddd, ²J_F–F = 245.0 Hz, ³J_F–H1 = 19.6 Hz, ³J_F–H4 = 4.7 Hz); HRMS (ESI): calculated for C₁₀H₁₄F₂O₃Na [M + Na]⁺ 243.0803, found 243.0803.

General procedure for the synthesis of 13 & 14. Alcohol 10/12 (1.0 equiv.) was dissolved in DCM (0.5 M) and DMAP (0.1 equiv.), pyridine (2.0 equiv.) and Ac₂O (2.0 equiv.) were added. The reaction was stirred at rt for 2–4 h, at which point TLC analysis (hexane/EtOAc, 4 : 1) showed complete consumption of the starting material to a higher R_f. The reaction mixture was poured onto 1.0 M aqueous HCl (50 mL), and the aqueous phase was extracted with DCM (3 × 100 mL). The combined organic phases were washed with sat. NaHCO₃ (50 mL), H₂O (100 mL), brine (100 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. This material was used without further purification.

(1R,2R,3R,4R,7R)-1-O-Acetyl-2,3-O-isopropylidine-4-vinyl-7-fluorocyclopentane 13. Acetate 13 (6.6 g, 26.9 mmol, 95%) a colourless oil, was obtained following the above procedure from 10 (5.4 g, 26.9 mmol, 1.0 equiv.). R_f = 0.54 (hexane/EtOAc, 4 : 1); [α]^24.9_D = −62.5 (c 0.2, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 5.78 (1H, ddd, ³J_H5–H6a = 17.9 Hz, ³J_H5–H6b = 10.3 Hz, ³J_H5–H4 = 7.8 Hz, H₅), 5.30–5.25 (2H, m, H_6a, H_6b), 5.14 (1H, dt, ²J_H7–F = 52.5 Hz, ³J_H7–H4/H1 = 6.6 Hz, H₇), 4.96 (1H, dt, ³J_H1–F = 14.4 Hz, ³J_H1–H2/H7 = 6.4 Hz, H₁), 4.79 (1H, td, ³J_H1–H7 = 6.0 Hz, ³J_H2–H3 = 2.4 Hz, H₂), 4.58 (1H, dt, ³J_H3–H4 = 5.8 Hz, ³J_H3–H2 = 2.2 Hz, H₃), 3.11–3.05 (1H, m, H₄), 2.14 (3H, s, Ac–CH₃), 1.46 (3H, s, CH₃), 1.30 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 170.3 (C=O), 131.6 (d, ³J_C5–F = 6.1 Hz, C₅), 119.3 (C₆), 112.1 [C(CH₃)₂], 95.9 (d, ¹J_C7–F = 188.2 Hz, C₇), 81.7 (d, ³J_C3–F = 4.2 Hz, C₃), 75.1 (d, ²J_C1–F = 22.7 Hz, C₁), 74.9 (d, ³J_C2–F = 6.8 Hz, C₂), 47.5 (d, ²J_C4–F = 17.5 Hz, C₄), 26.1 [C(CH₃)₂], 24.5 [C(CH₃)₂], 20.7 (Ac-CH₃); ¹⁹F NMR (377 MHz, CDCl₃): δ −207.3 (dddt, ²J_F–H7 = 51.6 Hz, ³J_F–H1 = 14.5 Hz, ³J_F–H4 = 9.5 Hz, ⁴J_F–H2/H3 = 2.5 Hz); HRMS (ESI): calculated for C₁₂H₁₈FO₄ [M + H]⁺ 245.1184, found 254.1180.

(1R,2R,3R,4R)-1-O-Acetyl-2,3-O-isopropylidine-4-vinyl-7-gem-difluorocyclopentane 14. Acetate 14 (7.9 g, 30.4 mmol, 94%) a colourless oil, was obtained following the above procedure from 12 (6.1 g, 30.1 mmol, 1.0 equiv.). R_f = 0.39 (hexane/EtOAc, 4 : 1); [α]^24.5_D = −87.0 (c 2.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 5.82 (1H, ddd, ³J_H5–6a = 17.4 Hz, ³J_H5–H6b = 10.5 Hz, ³J_H5–H4 = 7.9 Hz, H₅), 5.45–5.33 (2H, m, H_6a, H_6b), 5.10 (1H, dt, ³J_H1–F = 8.1 Hz, ³J_H1–H2 = 5.4 Hz, H₁), 4.72 (1H, ddd, ³J_H2–H3 = 6.8 Hz, ³J_H2–H1 = 5.6 Hz, ⁴J_H2–F = 3.8 Hz, H₂), 4.53 (1H, ddd, ³J_H3–H2 = 6.3 Hz, ⁴J_H3–H4 = 3.8 Hz, ³J_H3–F = 1.7 Hz, H₃), 3.18–3.05 (1H, m, H₄), 2.19 (3H, s, Ac–CH₃), 1.50 (3H, s, CH₃), 1.33 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 169.3 (C=O), 129.4 (dd, ³J_C5–F = 4.9 Hz, ³J_C5–F = 1.5 Hz, C₅), 126.1 (dd, ¹J_C7–F = 269.7 Hz, ¹J_C7–F = 248.4 Hz, C₇), 121.0 (C₆), 113.6 [C(CH₃)₂], 80.6 (dd, ³J_C3–F = 6.8 Hz, ³J_C3–F = 0.8 Hz, C₃), 74.9 (dd, ³J_C2–F = 3.3 Hz, ³J_C2–F = 1.8 Hz, C₂), 71.8 (dd, ²J_C1–F = 33.5 Hz, ²J_C1–F = 18.7 Hz, C₁), 52.8 (dd, ²J_C4–F = 21.0 Hz, ²J_C4–F = 19.9 Hz, C₄), 26.1 [C(CH₃)₂], 24.7 [C(CH₃)₂], 20.6 (Ac–CH₃); ¹⁹F NMR (377 MHz, CDCl₃): δ −110.7 (dddd, ²J_F–F = 245.4 Hz, ³J_F–H1 = 9.2 Hz, ⁴J_F–H4 = 5.4 Hz, ⁴J_F–H3 = 1.5 Hz), −112.9 (ddddd, ²J_F–F = 245.5 Hz, ³J_F–H4 = 21.4 Hz, ³J_F–H1 = 8.1 Hz, ⁴J_F–H2 = 3.6 Hz, ⁴J_F–H3 = 1.8 Hz); HRMS (ESI): calculated for C₁₂H₁₆FO₄Na [M + Na]⁺ 285.0909, found 285.0914.

General procedures for the synthesis of 15 & 16.
Sharpless asymmetric dihydroxylation. AD-mix β (1.4 g per 1 mmol of substrate) was suspended in 1 : 1 ⁱPrOH : H₂O (v/v, 0.1 M), cooled to 0 °C and stirred vigorously for 30 min, before it was added to 13 or 14 (1.0 equiv.). The reaction mixture was allowed to warm to rt over 1 h and stirred for a further 6–16 h. TLC analysis (hexane/EtOAc, 1 : 1) showed complete consumption of the starting material to two spots of lower R_f. The reaction was quenched by the addition of Na₂SO₃ (1.5 g per 1 mmol of substrate), and the mixture was stirred for 30 min, at which point H₂O (200 mL) was added. The aqueous phase was extracted with EtOAc (3 × 300 mL), and the combined organic phases were washed with H₂O (400 mL), brine (200 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. This material was used without further purification.
Oxidative cleavage conditions A. Vicinal diol intermediate was dissolved in 1 : 5 H₂O/1,4-dioxane (v/v, 0.1 M) and NaIO₄ (1.7 equiv.) was added. The reaction mixture was stirred at rt for 1 h, at which point TLC analysis (hexane/EtOAc, 1 : 1) showed complete conversion from two spots to one with a higher R_f. The reaction mixture was diluted with H₂O (200 mL) and extracted with EtOAc (3 × 300 mL). The combined organic phases were washed with H₂O (500 mL), brine (200 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. This material was used without further purification.
Oxidative cleavage conditions B. Vicinal diol intermediate was dissolved in 1 : 5 H₂O/1,4-dioxane (v/v, 0.1 M) and cooled to 0 °C. NaIO₄ (10 equiv.) was added, and the reaction mixture was stirred at this temperature for 6 h, at which point TLC analysis (hexane/EtOAc, 1 : 1) showed complete conversion of two spots to two spots with a higher R_f. The reaction mixture was diluted with H₂O (200 mL) and extracted with EtOAc (3 × 300 mL). The combined organic phases were washed with H₂O (500 mL), brine (200 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. This material was used without further purification.
Reduction and acyl deprotection. Aldehyde intermediate was dissolved in MeOH (0.27 M), cooled to 0 °C and NaBH₄ (1.5 equiv.) was added in two portions over 10 min. The reaction mixture was allowed to warm to rt and stirred for a further 16 h, at which point TLC analysis (hexane/EtOAc, 1 : 1) showed complete conversion to a lower R_f. The reaction mixture was cooled to 0 °C and H₂O (100 mL) was added. The mixture was allowed to warm to rt and stirred for a further 2 h. The solvent was removed in vacuo and the residue was partitioned between H₂O (200 mL) and EtOAc (300 mL). The aqueous phase was extracted with EtOAc (3 × 300 mL) and the combined organic phases were washed with H₂O (200 mL), brine (100 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography on silica gel (10–50% EtOAc/hexane).

(1R,2S,3R,4R,6R)-2,3-O-Isopropylidine-4-hydroxymethyl-6-fluorocyclopentan-1-ol 15. Diol 15 (3.7 g, 18.0 mmol, 66%) a colourless oil, was obtained following the above procedures, using oxidative cleavage conditions A, from 13 (6.6 g, 26.9 mmol, 1.0 equiv.). R_f = 0.39 (petroleum-ether/EtOAc, 1 : 4); [α]^24.1_D = −13.8 (c 2.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 5.05 (1H, ddd, ²J_H6–F = 51.4 Hz, ³J_H6–H4 = 5.4 Hz, ³J_H6–H1 = 4.6 Hz, H₆), 4.67–4.65 (2H, m, H₂, H₃), 4.23 (1H, dd, ³J_H1–F = 13.3 Hz, ³J_{H1–H2/H6/OH} = 4.5 Hz, H₁), 3.90–3.82 (2H, m, H_5a, H_5b), 2.76 (1H, d, ³J_OH–H1 = 3.8 Hz, OH), 2.53 (1H, dddd, ³J_H4–F = 20.2 Hz, ³J_H4–H5a/H5b = 11.4 Hz, ³J_H4–H6 = 5.6 Hz, ³J_H4–H3 = 2.8 Hz, H₄), 1.51 (3H, s, CH₃), 137 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 113.0 [C(CH₃)₂], 100.1 (d, ¹J_C6–F = 180.7 Hz, C₇), 81.2 (d, ³J_C3–F = 2.2 Hz, C₃), 77.6 (d, ³J_C2–F = 4.8 Hz, C₂), 73.1 (d, ²J_C1–F = 25.7 Hz, C₁), 59.5 (d, ³J_C5–F = 8.4 Hz, C₅), 47.8 (d, ²J_C4–F = 17.7 Hz, C₄), 26.2 [C(CH₃)₂], 24.3 [C(CH₃)₂]; ¹⁹F NMR (376 MHz, CDCl₃): δ −206.5 (ddd, ²J_F–H6 = 51.5 Hz, ³J_F–H4 = 20.3 Hz, ³J_F–H1 = 13.7 Hz); HRMS (ESI): calculated for C₉H₁₅FO₄Na [M + Na]⁺ 229.0847, found 229.0848.

(1R,2S,3R,4R)-2,3-O-Isopropylidine-4-hydroxymethyl-6-gem-difluorocyclopentan-1-ol 16. Diol 16 (3.7 g, 16.7 mmol, 60%), a colourless crystalline solid, was obtained following the above procedures, oxidative cleavage conditions B, from 14 (7.2 g, 27.8 mmol, 1.0 equiv.). R_f = 0.21 (hexane/EtOAc, 1 : 1); mp: 92–95 °C; [α]^24.9_D = −36.3 (c 4.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 4.66 (1H, ddd, ³J_H2–H1 = 6.4 Hz, ³J_H2–H3 = 3.1 Hz, ⁴J_H2–OH = 1.3 Hz, H₂), 4.57 (1H, td, ³J_H3–H4 = 6.4 Hz, ³J_H3–H2 = 3.0 Hz, H₃), 4.19 (1H, dddd, ³J_H1–F = 14.0 Hz, ³J_H1–OH = 9.4 Hz, ³J_H1–F = 8.1 Hz, ³J_H1–H2 = 6.2 Hz, H₁), 3.96–3.81 (2H, m, H_5a, H_5b), 2.82 (1H, dd, ³J_OH–H1 = 9.4 Hz, ⁴J_OH–H2 = 1.1 Hz, OH), 2.56–2.48 (1H, m, H₄), 1.52 (3H, s, CH₃), 1.36 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 127.2 (dd, ¹J_C7–F = 258.8 Hz, ¹J_C7–F = 256.6 Hz, C₇), 111.9 [C(CH₃)₂], 79.0 (dd, ³J_C2–F = 4.6 Hz, ³J_C2–F = 2.5 Hz, C₂), 75.4 (app. d, ³J_C3–F = 8.8 Hz, C₃), 73.1 (dd, ²J_C1–F = 28.0 Hz, ²J_C1–F = 18.9 Hz, C₁), 58.9 (dd, ³J_C5–F = 6.2 Hz, ³J_C5–F = 5.8 Hz, C₅), 50.0 (dd, ²J_C4–F = 21.0 Hz, ²J_C4–F = 20.0 Hz, C₄), 26.0 [C(CH₃)₂], 24.3 [C(CH₃)₂]; ¹⁹F NMR (377 MHz, CDCl₃): δ −107.8 – −108.7 (m), −114.6 – −115.3 (m); HRMS (ESI): calculated for C₂₅H₃₂FO₄Si [M − H]⁻ 223.0787, found 223.0792.

(1R,2S,3R)-2,3-O-Isopropylidine-4-hydroxymethyl-6-fluoro-4,6-cyclopenten-1-ol 17. Diol 17 (200 mg, 0.98 mmol, 49%), a colourless syrup, was obtained following the above procedures, oxidative conditions A, from 14 (593 mg, 2.0 mmol, 1.0 equiv.). R_f = 0.11 (hexane/EtOAc, 1 : 1), [α]^21.6_D = +41.7 (c 2.0, MeOH); ¹H NMR (400 MHz, CDCl₃): δ 5.07 (1H, app t, ³J_H3–H2 = 6.6 Hz, H₃), 4.72 (1H, td, ³J_H2–H3 = 6.1 Hz, ³J_H2–H1 = 3.5 Hz, H₂), 4.48 (1H, app. br s, H₁), 4.41–4.28 (2H, m, H_5a, H_5b), 2.84 (1H, d, ³J_OH–H1 = 9.0 Hz, OH), 1.48 (3H, s, CH₃), 1.42 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 157.7 (d, ¹J_C6–F = 289.8 Hz, C₆), 116.6 (d, ²J_C4–F = 5.5 Hz, C₄), 112.7 [C(CH₃)₂], 78.8 (d, ³J_C3–F = 9.5 Hz, C₃), 73.8 (d, ³J_C2–F = 7.8 Hz, C₂), 69.0 (d, ²J_C1–F = 21.0 Hz, C₁), 55.0 (C₅), 27.5 [C(CH₃)₂], 26.2 [C(CH₃)₂]; ¹⁹F NMR (377 MHz, CDCl₃): δ −129.73 – −129.79 (m).

General procedure for the synthesis of 18 & 19. Diol (1.0 equiv.) was dissolved in DMF (0.5 M). Imidazole (2.0 equiv.) and TBDPSCl (1.0 equiv.) were added, and the reaction mixture was stirred at rt for 1–4 h, at which point TLC analysis (hexane/EtOAc, 4 : 1) showed complete consumption of the starting material to a higher R_f. H₂O (50 mL) was added and aqueous phase was extracted with EtOAc (3 × 100 mL). The combined organic phases were washed with H₂O (2 × 100 mL), brine (100 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography (0–10% EtOAc/hexane).

(1R,2S,3R,4S,6R)-2,3-O-Isopropylidine-4-O-(tert-butyldiphenylsilyl)methyl-6-fluorocyclopentan-1-ol. TBDPS alcohol (6.8 g, 15.3 mmol, 85%) a colourless oil, was obtained following the above procedure from 15 (3.7 g, 18.0 mmol, 1.0 equiv.). R_f = 0.16 (hexane/EtOAc, 9 : 1); [α]^25.0_D = −13.5 (c 0.6, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 7.70–7.67 (4H, m, ArH), 7.48–7.36 (6H, m, ArH), 5.06 (1H, dt, ²J_H6–F = 52.0 Hz, ³J_H6–H1/H4 = 6.0 Hz, H₆), 4.62 (1H, dt, ³J_H2–H1 = 6.3 Hz, ³J_H2–H3 = 2.1 Hz, H₂), 4.55 (1H, dt, ³J_H3–H4 = 6.3 Hz, ³J_H3–H2 = 2.2 Hz, H₃), 4.42 (1H, app. dq, ³J_H1–F = 14.6 Hz, ³J_H1–H2/H6 = 6.3 Hz, H₁), 3.89 (1H, dd, ²J_H5a–H5b = 10.3 Hz, ³J_H5a–H4 = 4.2 Hz, H_5a), 3.74 (1H, ddd, ²J_H5b–H5a = 10.4 Hz, ³J_H5b–H4 = 5.7 Hz, ⁴J_H5b–F = 1.9 Hz, H_5b), 2.81 (1H, dd, ³J_OH–H1 = 6.9 Hz, ⁴J_OH–F = 1.2 Hz, OH), 2.56–2.48 (1H, m, H₄), 1.51 (3H, s, CH₃), 1.35 (3H, s, CH₃), 1.08 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 135.68 (ArC), 135.66 (ArC), 133.0 (ArC), 132.8 (ArC), 129.89 (ArC), 129.85 (ArC), 127.83 (ArC), 127.81 (ArC), 112.0 [C(CH₃)₂], 99.3 (d, ¹J_C6–F = 185.0 Hz, C₆), 80.8 (d, ³J_C3–F = 3.5 Hz, C₃), 77.0 (d, ³J_C2–F = 7.1 Hz, C₂), 73.8 (d, ²J_C1–F = 24.4 Hz, C₁), 59.9 (d, ³J_C5–F = 8.3 Hz, C₅), 46.9 (d, ²J_C4–F = 18.0 Hz, C₄), 26.9 [C(CH₃)₃], 26.2 [C(CH₃)₂], 24.2 [C(CH₃)₂], 19.2 [C(CH₃)₃]; ¹⁹F NMR (376 MHz, CDCl₃): δ −208.5 (app. dt, ²J_F–H6 = 52.2 Hz, ³J_F–H1/H4 = 13.7 Hz); HRMS (NSI) calculated for C₂₅H₃₃FO₄SiNa [M + Na]⁺ 476.2024, found 467.2021.

(1R,2S,3R,4S)-2,3-O-Isopropylidine-4-O-(tert-butyldiphenylsilyl)methyl-6-gem-difluorocyclopentan-1-ol. TBDPS alcohol (7.5 g, 16.2 mmol, 97%) a colourless oil, was obtained following the above procedure from 16 (3.7 g, 16.7 mmol, 1.0 equiv.). R_f = 0.27 (hexane/EtOAc, 9 : 1); [α]^24.3_D = −4.6 (c 0.2, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 7.65–7.61 (4H, m, ArH), 7.49–7.37 (6H, m, ArH), 4.64–4.57 (2H, m, H₂, H₃), 4.46–4.34 (1H, m, H₁), 3.90 (1H, dd, ²J_H5a–H5b = 10.7 Hz, ³J_H5a–H4 = 2.5 Hz, H_5a), 3.68 (1H, dddd, ²J_H5b–H5a = 10.6 Hz, ³J_H5b–H4 = 3.3 Hz, ⁴J_H5b–F = 2.0 Hz, ⁴J_H5b–F = 1.3 Hz, H_5b), 2.84 (1H, dd, ³J_OH–H1 = 11.0 Hz, ³J_OH–F = 1.5 Hz, OH), 2.41 (1H, dt, ³J_H4–F = 17.0 Hz, ³J_H4–H5 = 3.0 Hz, H₄), 1.49 (3H, s, CH₃), 1.35 (3H, s, CH₃), 1.05 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 135.6 (ArC), 135.6 (ArC), 132.3 (ArC), 132.1 (ArC), 130.1 (ArC), 130.0 (ArC), 128.0 (ArC), 127.9 (ArC), 124.2 (dd, ¹J_C6–F = 275.3 Hz, ¹J_C6–F = 252.5 Hz, C₆), 111.4 [C(CH₃)₂], 79.2 (d, ³J_C2–F = 5.5 Hz, C₂), 75.5 (d, ³J_C3–F = 10.2 Hz, C₃), 74.0 (dd, ²J_C1–F = 26.3 Hz, ²J_C1–F = 19.0 Hz, C₁), 60.3 (dd, ³J_C5–F = 8.3 Hz, ³J_C5–F = 4.9 Hz, C₅), 50.2 (dd, ²J_C4–F = 21.0 Hz, ²J_C4–F = 20.2 Hz, C₄), 26.9 [C(CH₃)₃], 25.9 [C(CH₃)₂], 24.2 [C(CH₃)₂], 19.1 [C(CH₃)₃]; ¹⁹F NMR (377 MHz, CDCl₃): δ −107.4 (dt, ²J_F–F = 240.7 Hz, ³J_F–H1/H4 = 16.5 Hz), −114.0 – −114.9 (m); HRMS (NSI): calculated for C₂₅H₃₂F₂O₄SiNa [M + Na]⁺ 485.1930, found 485.1936.
Triflation. TBDPS alcohol (1.0 equiv.) was dissolved in pyridine (0.32 M) and cooled to −10 °C. Tf₂O (2.0 equiv.) was added dropwise. The reaction mixture was stirred at this temperature for 10 min, at which point TLC analysis (hexane/EtOAc, 9 : 1) showed complete consumption to a higher R_f. H₂O (50 mL) was added and the aqueous phase was extracted with Et₂O (3 × 200 mL). The combined organic phases were washed with sat. CuSO₄ (200 mL), followed by H₂O (200 mL), brine (200 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. This material was used without further purification.
Azidation. Triflate intermediate was dissolved in DMF (0.2 M), and NaN₃ (3.0 equiv.) was added. The reaction was stirred at 80–100 °C for 3–6 h, at which point TLC analysis (hexane/EtOAc 9 : 1) showed complete conversion from a lower R_f to higher R_f. The reaction was cooled to rt, and H₂O (100 mL) was added. The aqueous phase was extracted with Et₂O (3 × 200 mL), and the combined organic phases were washed with H₂O (2 × 100 mL), brine (100 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography (0–10% EtOAc/hexane).

(1S,2S,3R,4S,6R)-1-Azido-2,3-O-isopropylidine-4-O-(tert-butyldiphenylsilyl)methyl-6-fluorocyclopentan-1-ol 18. Azide 18 (6.5 g, 13.9 mmol, 91%) a colourless oil, was obtained following the above procedure from TBDPS alcohol (6.8 g, 15.3 mmol, 1.0 equiv.). R_f = 0.74 (hexane/EtOAc, 9 : 1); [α]^24.9_D = −8.0 (c 1.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 7.68–7.64 (4H, m, ArH), 7.46–7.35 (6H, m, ArH), 5.28 (1H, app. dt, ²J_H6–F = 52.5 Hz, ³J_H6–H1/H4 = 3.2 Hz, H₆), 4.66 (1H, ddd, ³J_H2–H3 = 7.3 Hz, ³J_H2–H1 = 6.0 Hz, ⁴J_H2–F = 1.3 Hz, H₂), 4.38–4.34 (1H, m, H₃), 3.87 (2H, d, ³J_H5a/H5b–H4 = 8.3 Hz, H_5a, H_5b), 3.71 (1H, ddd, ³J_H1–F = 28.0 Hz, ³J_H1–H2 = 5.8 Hz, ³J_H1–H6 = 3.0 Hz, H₁), 2.46 (1H, m, H₄), 1.50 (3H, s, CH₃), 1.28 (3H, s, CH₃), 1.06 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 135.6 (ArC), 135.6 (ArC), 133.3 (ArC), 133.1 (ArC), 129.8 (ArC), 127.8 (ArC), 127.7 (ArC), 114.26 [C(CH₃)₂], 97.0 (d, ¹J_C6–F = 184.0 Hz, C₆), 82.1 (C₂), 79.7 (C₃), 68.1 (d, ³J_C5–F = 16.0 Hz, C₅), 60.3 (d, ²J_C1–F = 7.4 Hz, C₁), 50.9 (d, ²J_C4–F = 17.7 Hz, C₄), 27.1 [C(CH₃)₃], 26.8 [C(CH₃)₃], 24.6 [C(CH₃)₂], 19.2 [C(CH₃)₂]; ¹⁹F NMR (377 MHz, CDCl₃): δ −206.64 (ddd, ²J_F–H6 = 52.5 Hz, ³J_F–H1 = 32.6 Hz, ³J_F–H4 = 28.1 Hz); HRMS (ASAP): calculated for C₂₅H₃₃FNO₃Si [M + H–N2]⁺ 442.2208, found 442.2203.

(1S,2S,3R,4S)-1-Azido-2,3-O-isopropylidine-4-O-(tert-butyldiphenylsilyl)methyl-6-gem-difluorocyclopentane 19. Azide 19 (5.8 g, 12.0 mmol, 74%) a colourless oil, was obtained following the above procedure from TBDPS alcohol (7.5 g, 16.2 mmol, 1.0 equiv.). R_f = 0.58 (hexane/EtOAc, 9 : 1); [α]^24.4_D = −28.0 (c 2.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 7.70–7.65 (4H, m, ArH), 7.47–7.37 (6H, m, ArH), 4.41–4.36 (1H, m, H₃), 4.36–4.30 (1H, m, H₂), 3.99–3.80 (3H, m, H₁, H_5a, H_5b), 2.75–2.62 (1H, m, H₄), 1.52 (3H, s, CH₃), 1.28 (3H, s, CH₃), 1.06 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 135.8 (ArC), 135.8 (ArC), 127.9 (ArC), 127.9 (ArC), 127.0 (dd, ¹J_C6–F = 264.0 Hz, ¹J_C6–F = 256.1 Hz, C₆), 113.5 [C(CH₃)₂], 79.7 (d, ³J_C2–F = 6.7 Hz, C₂), 77.5 (d, ³J_C3–F = 8.1 Hz, C₃) 69.2 (dd, ²J_C1–F = 23.9 Hz, ²J_C1–F = 18.7 Hz, C₁), 59.5 (d, ³J_C5–F = 6.8 Hz, C₅), 52.0 (t, ²J_C4–F = 19.7 Hz, C₄), 27.1 [C(CH₃)₃], 26.9 [C(CH₃)₂], 24.8 [C(CH₃)₂], 19.3 [C(CH₃)₃]; ¹⁹F NMR (377 MHz, CDCl₃): δ −99.7 (dt, ²J_F–F = 238.7 Hz, ³J_F–H1/H4 = 7.9 Hz), −118.8 – −119.6 (m); HRMS (ASAP): calculated for C₂₅H₃₂F₂N₃O₃Si [M + H]⁺ 488.2176, found 488.2182.

General procedure for the synthesis of 20 and 21.
Reduction. Azide 18 or 19 (1.0 equiv.) was dissolved in MeOH (0.2 M) and Pd/C (0.1 equiv.) was added. The reaction vessel was evacuated and refilled with H₂ seven times, and the reaction was stirred at rt for 4–18 h. TLC analysis (hexane/EtOAc, 9 : 1) showed complete consumption of the starting material to a lower R_f. The reaction mixture was filtered through a Celite® pad and eluted with MeOH. This material was used without further purification.

(1S,2S,3R,4S,6R)-1-Amino-2,3-O-isopropylidine-4-O-(tert-butyldiphenylsilyl)methyl-6-fluorocyclopentane. C1-Amine (6.1 g, 13.6 mmol, 98%) a colourless syrup, was obtained following the above procedure from 20 (6.5 g, 13.9 mmol, 1.0 equiv.). R_f = 0.12 (hexane/EtOAc, 1 : 1); [α]^24.7_D = +3.2 (c 0.4, CH₂Cl₂); ¹H NMR (400 MHz, CDCl_3:) δ 7.70–7.65 (4H, m, ArH), 7.45–7.34 (6H, m, ArH), 5.10 (1H, dt, ³J_H6–F = 53.0 Hz, ³J_H6–H1/H4 = 3.1 Hz, H₆), 4.35–4.27 (2H, m, H₂, H₃), 3.88–3.85 (2H, m, H_5a, H_5b), 3.36–3.25 (1H, m, H₁), 2.50–2.34 (1H, m, H₄), 1.49 (3H, s, CH₃), 1.26 (3H, s, CH₃), 1.05 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 135.6 (ArC), 135.6 (ArC), 133.5 (ArC), 133.6 (ArC), 129.7 (ArC), 129.7 (ArC), 127.7 (ArC), 127.7 (ArC), 113.5 [C(CH₃)₂], 99.2 (d, ¹J_C6–F = 176.3 Hz, C₆), 86.8 (C₂), 80.2 (C₃), 61.8 (d, ²J_C1–F = 17.3 Hz, C₁), 60.7 (d, ³J_C5–F = 7.4 Hz, C₅), 51.4 (d, ²J_C4–F = 18.0 Hz, C₄), 27.3 [C(CH₃)₃], 26.8 [C(CH₃)₃], 24.7 [C(CH₃)₂], 19.3 [C(CH₃)₂]; ¹⁹F NMR (377 MHz, CDCl₃): δ −210.5 (ddd, ²J_F–H6 = 53.0 Hz, ³J_F–H4 = 34.9 Hz, ³J_F–H1 = 30.5 Hz). HRMS (ESI): calculated for C₂₅H₃₅FNO₃Si [M + H]⁺ 444.2365, found 444.2365.

(1S,2S,3R,4S)-1-Amino-2,3-O-isopropylidine-4-O-(tert-butyldiphenylsilyl)methyl-6-gem-difluorocyclopentane. C1-Amine (5.1 g, 11.0 mmol, 99%) a colourless syrup, was obtained following the above procedure from 21 (5.4 g, 11.1 mmol, 1.0 equiv.). R_f = 0.51 (hexane/EtOAc, 1 : 4); [α]^22.3_D = −9.8 (c 0.5, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 7.70–7.66 (4H, m, ArH), 7.46–7.36 (6H, m, ArH), 4.35–4.31 (1H, m, H₃), 4.17–4.11 (1H, m, H₂), 3.96–3.86 (2H, m, H_5a, H_5b), 3.38 (1H, app. dt, ³J_H1–F = 17.7 Hz, ³J_H1–H2 = 5.9 Hz, H₁), 2.69–2.56 (1H, m, H₄), 1.51 (3H, s, CH₃), 1.28 (3H, s, CH₃), 1.06 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃) δ 135.7 (ArC), 135.6 (ArC), 133.2 (ArC), 133.1 (ArC), 129.8 (ArC), 129.8 (ArC), 112.7 (ArC), 82.8 (d, ³J_C2–F = 8.3 Hz, C₂), 77.2 (app. d, ³J_C3–F = 7.7 Hz, C₃), 62.7 (dd, ²J_C1–F = 23.2 Hz, ²J_C1–F = 21.0 Hz, C₁), 59.9 (d, ³J_C5–F = 6.8 Hz, C₅), 51.9 (t, ²J_C4–F = 20.5 Hz, C₄), 27.1 [C(CH₃)₂], 26.7 [C(CH₃)₃], 24.7 [C(CH₃)₂], 19.2 [C(CH₃)₃]; ¹⁹F NMR (377 MHz, CDCl₃): δ −104.4 (app. dt, ²J_F–F = 234.9 Hz, ³J_F–H1/H4 = 8.0 Hz), −123.2 (app. dt, ²J_F–F = 234.9 Hz, ³J_F–H1/H4 = 19.4 Hz); HRMS (ESI): calculated for C₂₅H₃₄F₂NO₃Si [M + H]⁺ 462.2271, found 462.2269.

(3-Ethoxy-acryloyl)isocyanate. An oven dried multi-necked round bottom flask was charged with AgOCN (13.9 g, 92.9 mmol, 2.5 equiv.), heated to 100 °C for 1 h with stirring, and evacuated and flushed with N₂ three times. The reaction vessel was cooled to rt and toluene (106 mL) was added and the mixture stirred vigorously at reflux for 30 min. 3-Ethoxyacryloylchloride (5.0 g, 37.2 mmol, 1.0 equiv.) was added dropwise and the reaction mixture was stirred at reflux for a further 1 h. The reaction mixture was allowed to cool to rt, and the supernatant, a 0.35 M solution of (3-ethoxy-acryloyl)isocyanate, was transferred to an oven-dried round bottom flask via cannula and stored under an atmosphere of N₂. The product was stored at −25 °C prior to use.
Nucleobase synthesis. C1-Amine was dissolved in DMF (0.2 M) and cooled to 0 °C. (3-Ethoxy-acryloyl)isocyanate (2.0 equiv., 0.35 M in toluene,) was added dropwise, and the reaction was allowed to warm to rt and stirred for a further 16 h. TLC analysis (hexane/EtOAc, 1 : 1) showed complete consumption of the starting material to a higher R_f. The reaction mixture was cooled to 0 °C and quenched with sat. NaHCO₃ (100 mL) and the aqueous phase was extracted with EtOAc (3 × 100 mL). The combined organic phases were washed with H₂O (2 × 100 mL), brine (100 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography (10–50% EtOAc/hexane).

(1′S,2′S,3′R,4′S,6′R)-1′-(6-Ethoxyacryloylurea)-2′,3′-O-isopropylidine-4′-O-(tert-butyldiphenylsilyl)methyl-6′-fluorocyclopentane 20. Acryloyl urea 20 (5.3 g, 9.2 mmol, 68%) a white foam, was obtained following the above procedure from C1-amine (6.1 g, 13.6 mmol, 1.0 equiv.). R_f = 0.44 (hexane/EtOAc, 1 : 1); [α]^24.9_D = −9.0 (c 2.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 9.68 (1H, s, H₃), 9.24 (1H, d, ³J_H1–H1′ = 8.5 Hz, H₁), 7.69–7.65 (4H, m, ArH, H₆), 7.45–7.36 (6H, m, ArH), 5.40 (1H, d, ³J_H5–H6 = 12.2 Hz, H₅), 5.26 (1H, dt, ²J_H6′–F = 53.0 Hz, ³J_{H6′–H1′/H4′} = 3.0 Hz, H_6′), 4.58 (1H, dd, ³J_{H2′–H1′} = 6.2 Hz, ³J_{H2′–H3′} = 7.0 Hz, H_2′), 4.45 (1H, dddd, ³J_H1′–F = 31.2 Hz, ³J_H1′–H1 = 8.8 Hz, ³J_{H1′–H2′} = 6.1 Hz, ³J_{H1′–H6′} = 3.0 Hz, H_1′), 4.32 (1H, t, ³J_{H3′–H2′/H4′} = 6.8 Hz, H_3′), 3.98 (2H, q, J = 7.1 Hz, –OCH₂CH₃), 3.89 (2H, app. d, J = 8.1 Hz, H_5′a, H_5′b), 2.49 (1H, dddd, ²J_H4′–F = 35.8 Hz, ²J_{H4′–H5′a} = 10.3 Hz, ²J_{H4′–H5′b} = 8.0 Hz, ²J_{H4′–H6′} = 2.9 Hz, H_4′), 1.51 (3H, s, CH₃), 1.35 (3H, t, J = 7.1 Hz, –OCH₂CH₃), 1.26 (3H, s, CH₃), 1.05 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 168.3 (C₄, C=O), 163.1 (C₆), 155.2 (C₂, C=O), 135.6 (ArC), 133.4 (ArC), 133.2 (ArC), 129.7 (ArC), 127.8 (ArC), 127.7 (ArC), 114.0 [C(CH)₃], 98.1 (C₅), 84.5 (C_2′), 79.7 (C_3′), 67.6 (-OCH₂CH₃), 60.5 (d, ³J_C5′–F = 7.1 Hz, C_5′), 58.9 (d, ²J_C1′–F = 16.0 Hz, C_1′), 51.5 (d, ²J_C4′–F = 17.7 Hz, C_4′), 27.3 (CH₃), 26.8 (CH₃), 24.7 (CH₃), 14.6 (-OCH₂CH₃); ¹⁹F NMR (376 MHz, CDCl₃): δ −206.4 (ddd, ¹J_F–H6′ = 53.2 Hz, ²J_F–H4′ = 35.7 Hz, ²J_F–H1′ = 31.5 Hz); HRMS (ESI): calculated for C₃₁H₄₁FN₂O₆SiNa [M + Na]⁺ 607.2610, found 607.2604.

(1′S,2′S,3′R,4′S)-1′-(6-Ethoxyacryloylurea)-2′,3′-O-isopropylidine-4′-O-(tert-butyldiphenylsilyl)methyl-6′-gem-difluorocyclopentane 21. Acryloyl urea 21 (4.9 g, 8.1 mmol, 74%) a white foam, was obtained following the above procedure from C1-amine (5.1 g, 11 mmol, 1.0 equiv.). R_f = 0.76 (EtOAc/hexane, 4 : 1); [α]^24.5_D = −21.2 (c 1.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 9.96 (1H, br s, H₃), 9.29 (1H, d, ³J_H1–H1′ = 9.0 Hz, H₁, 7.74–7.63 (5H, m, ArH, H₆), 7.46–7.36 (6H, m, ArH), 5.39 (1H, d, ³J_H5–H6 = 12.3 Hz, H₅), 4.76–4.64 (1H, m, H_1′), 4.38–4.31 (2H, m, H_2′, H_3′), 4.04–3.93 (3H, m, H_5′a, –OCH₂CH₃), 3.89 (1H, dd, ²J_{H5′b–H5′a} = 10.6 Hz, ³J_{H5′b–H4′} = 6.3 Hz, H_5′b), 2.79–2.65 (1H, m, H_4′), 1.55 (1H, s, CH₃), 1.34 (3H, t, J = 7.1 Hz, –OCH₂CH₃), 1.29 (3H, s, CH₃), 1.07 (9H, s, 3 × CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 168.5 (C₄, C=O), 163.1 (C₆), 155.6 (C₂, C=O), 135.67 (ArC), 135.65 (ArC), 133.2 (ArC), 133.1 (ArC), 129.8 (ArC), 127.8 (ArC), 126.4 (dd, ¹J_C6′–F = 266.9 Hz, ¹J_C6′–F = 250.2 Hz, C_6′), 113.3 [C(CH₃)₂], 98.0 (C₅), 81.0 (d, ³J_C2′–F = 8.5 Hz, C_2′), 76.8 (d, ³J_C3′–F = 8.2 Hz, C_3′), 67.4 (–OCH₂CH₃), 59.6 (dd, ²J_C1′–F = 22.2 Hz, ²J_C1′–F = 18.3 Hz, C_1′), 59.4 (d, ³J_C5′–F = 6.7 Hz, C_5′), 51.6 (t, ³J_C4′–F = 19.6 Hz, C_4′), 27.2 [C(CH₃)₂], 26.7 [C(CH₃)₃], 24.9 [C(CH₃)₂], 19.2 [C(CH₃)₃], 14.5 (–OCH₂CH₃); ¹⁹F NMR (377 MHz, CDCl₃): δ −104.5 (dt, ²J_F–F = 237.8 Hz, ³J_{F–H1′/H4′} = 5.8 Hz), −120.1 (dt, ²J_F–F = 236.9 Hz, ³J_{F–H1′/H4′} = 23.0 Hz); HRMS (ESI): calculated for C₃₁H₄₀F₂N₂O₆SiNa [M + Na]⁺ 625.2516, found 625.2507.

General procedure for the synthesis of 22 & 23.
Cyclisation. Acryloyl urea 20 or 21 was dissolved in 1,4-dioxane (0.5 M), and H₂SO₄ (0.5 equiv., 2.0 M) was added. The reaction mixture was stirred at reflux for 3 h, at which point TLC analysis (EtOAc/hexane, 4 : 1) showed complete conversion of the starting material to a lower R_f. The reaction mixture was cooled to rt and solid Na₂CO₃ was added to adjust the pH to 7. The mixture was filtered, and the solvent of the filtrate removed in vacuo. This material was used without further purification.
Silyl deprotection. Uridine intermediate was dissolved in THF (0.2 M) and TBAF (1.0 equiv., 1.0 M in THF) was added. The reaction mixture was stirred at rt for 3 h, at which point TLC analysis (10% MeOH/DCM) showed complete conversion from a higher R_f to a lower R_f. The solvent was removed in vacuo, and the residue was purified via flash column chromatography on silica gel (0–10% MeOH/EtOAc).
HPLC purification. A sample of 22 or 23 was dissolved in H₂O (100 mg mL⁻¹), and injected onto a reverse phase column (see general experimental) and purified at a flow rate of 20.00 ml min⁻¹ using the following gradient system:

Time (min)	%A (H2O)	%B (MeOH)
0.0	95	5
5.0	95	5
12.0	0	100
15.0	0	100
15.1	95	5
20.0	95	5

(6′R)-6′-Fluorocarbauridine 22. Uridine derivative 22 (1.3 g, 4.7 mmol, 70%) a white solid after freeze drying, was obtained following the above procedure from 20 (3.9 g, 6.7 mmol, 1.0 equiv.). R_f = 0.12 (10% MeOH/DCM); ¹H NMR (400 MHz, D₂O): δ 7.69 (1H, dd, ³J_H6–H5 = 8.1 Hz, ⁵J_H6–F = 1.5 Hz, H₆), 5.79 (1H, d, ³J_H5–H6 = 7.6 Hz, H₅), 5.11 (1H, ddd, ²J_H6′–F = 54.9 Hz, ³J_{H6′–H4′} = 4.5 Hz, ³J_{H6′–H1′} = 3.7 Hz, H_6′), 4.86 (1H, ddd, ³J_H1′–F = 30.6 Hz, ³J_{H1′–H2′} = 10.5 Hz, ³J_{H1′–H6′} = 3.4 Hz, H_1′), 4.53 (1H, dd, ³J_{H2′–H1′} = 10.4 Hz, ³J_{H2′–H3′} = 6.6 Hz, H_2′), 3.99 (1H, dd, ³J_{H3′–H2′} = 6.4 Hz, ³J_{H3′–H4′} = 4.8 Hz, H_3′), 3.75 (2H, m, H_5′a, H_5′b) 2.37 (dddd, ³J_H4′–F = 31.3 Hz, ³J_{H4′–H5′a} = 12.2 Hz, ³J_{H4′–H5′b} = 7.7 Hz, ³J_{H4′–H3′/H6′} = 4.7 Hz, H_4′); ¹³C NMR (101 MHz, D₂O): δ 167.0 (C₄, C=O), 153.4 (C₂, C=O), 144.0 (d, ⁴J_C6–F = 4.8 Hz, C₆), 101.6 (C₅), 90.9 (d, ¹J_C6′–F = 180.8 Hz, C_6′), 70.2 (C_2′), 69.2 (C_3′), 61.6 (d, ²J_C1′–F = 16.4 Hz, C_1′), 58.3 (d, ³J_C5′–F = 11.3 Hz, C_5′), 50.6 (d, ²J_C4′–F = 18.1 Hz, C_4′); ¹⁹F NMR (377 MHz, D₂O): δ −207.8 (dt, ²J_F–H6′ = 55.0 Hz, ³J_{F–H1′/H4′} = 30.9 Hz); HRMS (ESI): calculated for C₁₀H₁₂FN₂O₅ [M − H]⁻ 259.0736, found 259.0738. These data were in good agreement with the literature.¹⁷

6′-gem-Difluorocarbauridine. Uridine derivative 23 (1.4 g, 5.1 mmol, 63%) a white solid after freeze drying, was obtained following the above procedure from 21 (4.9 g, 8.1 mmol, 1.0 equiv.). R_f = 0.13 (10% MeOH/DCM); ¹H NMR (400 MHz, MeOD): δ 7.68 (1H, dd, ³J_H6–H5 = 8.0 Hz, ⁵J_H6–F = 2.3 Hz, H₆), 5.73 (1H, d, ³J_H5–H6 = 8.1 Hz, H₆), 5.37 (1H, dt, ³J_H1′–F = 18.3 Hz, ³J_{H1′–H2′} = 10.2 Hz, H_1′), 4.43 (1H, dd, ³J_{H2′–H1′} = 10.7 Hz, ³J_{H2′–H3′} = 5.1 Hz, H_2′), 4.10–4.08 (1H, m, H_3′), 3.86–3.71 (2H, m, H_5′a, H_5′b), 2.63–2.46 (1H, m, H_4′); ¹³C NMR (101 MHz, MeOD): δ 165.9 (C₄, C=O), 153.3 (C₂, C=O), 144.5 (d, ⁴J_C6–F = 4.2 Hz, C6), 126.0 (dd, ¹J_C6′–F = 260.0 Hz, ¹J_C6′–F = 253.7 Hz, C_6′), 102.6 (C₅), 71.7 (d, ³J_C2′–F = 8.0 Hz, C_2′), 71.0 (dd, ³J_C3′–F = 5.3 Hz, ³J_C3′–F = 2.5 Hz, C_3′), 63.6 (dd, ²J_C1′–F = 24.3 Hz, ²J_C1′–F = 18.3 Hz, C_1′), 58.7 (d, ⁴J_C5′–F = 10.9 Hz, C_5′), 55.4 (t, ³J_C4′–F = 20.2 Hz, C_4′); ¹⁹F NMR (377 MHz, MeOD): δ −96.7 (ddd, ²J_F–F = 238.6 Hz, ³J_F–H4′ = 15.4 Hz, ³J_F–H1′ = 9.9 Hz), −117.0 (app. dt, ²J_F–F = 238.6 Hz, ³J_{F–H1′/H4′} = 15.6 Hz). HRMS (ESI): calculated for C₁₀H₁₁F₂N₂O₅ [M − H]⁻ 277.0642, found 277.0643. These data are in good agreement with literature values.¹⁷

General procedure for the synthesis of 24 & 25. Uridine 22 or 23 was suspended in DCM (0.2 M) and DMAP (0.1 equiv.), pyridine (6.0 equiv.) and Ac₂O (6.0 equiv.) were added. The reaction mixture was stirred at rt for 16 h, at which point TLC analysis (10% MeOH/DCM) showed complete consumption of the starting material to a higher R_f. The reaction mixture was poured onto 1.0 M aqueous HCl (2 mL), and the aqueous phase was extracted with DCM (3 × 10 mL). The combined organic phases were washed with sat. NaHCO₃ (5 mL), H₂O (5 mL), brine (5 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. The crude material was purified via flash column chromatography on silica gel (50–100%, EtOAc/Hexane).

(6′R)-2′,3′,5′-Tri-O-acetyl-6′-fluorocarbauridine 24. Triacetate 24 (68 mg, 0.18 mmol, 88%.) as a colourless syrup, was obtained following the above procedure from 22 (50.0 mg, 0.18 mmol, 1.0 equiv.). R_f = 0.53 (hexane/EtOAc, 1 : 4); [α]^22.1_D = −10.7 (c 0.5, MeOH); ¹H NMR (400 MHz, CDCl₃): δ 8.87 (1H, br s, NH), 7.32 (1H, dd, ³J_H6–H5 = 8.2 Hz, ⁵J_H6–F = 1.8 Hz, H₆), 5.77 (1H, dd, ³J_H5–H6 = 8.2 Hz, ⁶J_H5–F = 2.1 Hz, H₅), 5.64 (1H, dd, ³J_{H2′–H1′} = 9.9 Hz, ³J_{H2′–H3′} = 7.0 Hz, H_2′), 5.32–5.22 (2H, m, H_1′, H_3′), 5.13 (1H, dt, ²J_H6′–F = 50.7 Hz, H_6′), 4.34 (1H, ddd, ²J_{H5′a–H5′b} = 11.2 Hz, ³J_{H5′a–H4′} = 6.3 Hz, ⁴J_H5′a–F = 1.4 Hz, H_5′a), 4.26 (1H, dd, ²J_{H5′b–H5′a} = 11.3 Hz, ³J_{H5′b–H4′} = 8.8 Hz, H_5′b), 2.83–2.68 (1H, m, H_4′), 2.11 (3H, s, Ac–CH₃), 2.07 (3H, s, Ac–CH₃), 2.05 (3H, s, Ac–CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 170.5 (C=O), 169.8 (C=O), 169.7 (C=O), 162.4 (C₂, C=O), 151.2 (C₄, C=O), 141.2 (d, ⁴J_C6–F = 5.2 Hz, C₆), 102.8 (C₅), 90.3 (d, ¹J_C6′–F = 184.5 Hz, C_6′), 69.6 (C₂), 69.4 (C₃), 59.8 (d, ³J_C5′–F = 10.1 Hz, C_5′), 59.7 (d, ²J_C1′–F = 16.4 Hz, C_1′), 46.0 (d, ²J_C4′–F = 18.2 Hz, C_4′), 20.7 (Ac–CH₃), 20.5 (Ac–CH₃), 20.4 (Ac–CH₃); ¹⁹F NMR (377 MHz, CDCl₃) δ −206.2 (dt, ²J_F–H6′ = 54.5 Hz, ³J_{F–H1′/H4′} = 30.2 Hz); HRMS (NSI): calculated for C₁₆H₂₀FN₂O₈ [M + H]⁺ 387.1198, found 387.1200.

2′,3′,5′-Tri-O-acetyl-6′-gem-difluorocarbauridine 25. Triacetate 25 (48.3 mg, 0.12 mmol, 92%) a colourless syrup was obtained following the above procedure from 23 (36.1 mg, 0.13 mmol, 1.0 equiv.). R_f = 0.55 (hexane/EtOAc, 1 : 4); [α]^24.6_D = −33.2 (c 0.4, MeOH); ¹H NMR (400 MHz, CDCl₃): δ 9.58 (1H, br s, NH), 7.24 (1H, dd, ³J_H6–H5 = 8.2 Hz, ⁵J_H6–F = 2.5 Hz, H₆), 5.80 (1H, d, ³J_H5–H6 = 8.2 Hz, H₅), 5.59–5.51 (2H, m, H_1′, H_2′), 5.32–5.29 (1H, m, H_3′), 4.37–4.33 (2H, m, H_5′a, H_5′b), 3.03–2.90 (1H, m, H_4′), 2.14 (3H, s, Ac–CH₃), 2.09 (3H, s, Ac–CH₃), 2.05 (3H, s, Ac–CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 170.3 (C=O), 169.7 (C=O), 169.7 (C=O), 162.7 (C₂, C=O), 151.1 (C₄, C=O), 141.0 (d, ⁴J_C6–F = 4.8 Hz, C₆), 122.5 (dd, ¹J_C6′–F = 262.9 Hz, ¹J_C6′–F = 253.7 Hz, C_6′), 103.3 (C₅), 68.8 (d, ³J_C3′–F = 5.5 Hz, C_3′), 68.6 (d, ³J_C2′–F = 8.1 Hz, ³J_C2′–F), 60.4 (dd, ²J_C1′–F = 25.9 Hz, ²J_C1′–F = 18.6 Hz, C_1′), 58.6 (d, ³J_C5′–F = 9.0 Hz, C_5′), 48.3 (dd, ²J_C4′–F = 23.0 Hz, ²J_C4′–F 20.7 Hz, C_4′), 20.7 (Ac–CH₃), 20.6 (Ac–CH₃), 20.4 (Ac–CH₃); ¹⁹F NMR (377 MHz, CDCl₃): δ −100.8 (d, ²J_F–F = 240.8 Hz), −115.5 (app. dt, ²J_F–F = 240.5 Hz, ³J_{F–H1′/H4′} = 17.5 Hz); HRMS (NSI) calculated for C₁₆H₁₉F₂N₂O₈ [M + H]⁺ 405.1104, found 405.1104.

General procedure for the synthesis of 26 & 27.
Installation of 1,2,4-triazole. Triacetate 24 or 25 (1.0 equiv.) was dissolved in MeCN (0.1 M) and cooled to 0 °C. 1,2,4-Triazole (23 equiv.), POCl₃ (2.4 equiv.) and Et₃N (23 equiv.) were added and the reaction mixture was allowed to slowly warm to rt and stirred for 18 h. TLC analysis (EtOAc/hexane, 4 : 1) showed majority conversion of the starting material to a lower R_f. The reaction mixture was cooled to 0 °C and quenched with sat. NaHCO₃ (5 mL). The aqueous phase was extracted with EtOAc (3 × 10 mL), and the combined organic phases were washed with H₂O (10 mL), brine (10 mL), dried over MgSO₄, filtered and the solvent removed in vacuo.
Amination and deprotection. 1,2,4-Triazole derivative was dissolved in 35% NH₄OH/1,4-dioxane (1 : 1, v/v, 0.1 M) and the reaction vessel was sealed. The reaction mixture was stirred at rt for 18 h, at which point TLC analysis (5% MeOH/DCM) showed complete conversion from a higher R_f to a lower R_f. The solvent was removed in vacuo, and the residue was purified via flash column chromatography on silica gel (10% MeOH/EtOAc).
HPLC purification. A sample of 26 or 27 was dissolved in H₂O (100 mg mL⁻¹), and injected onto a reverse phase column (see general experimental) and purified at a flow rate of 20.00 ml min⁻¹ using the following gradient system:

Time (min)	%A (H2O)	%B (MeOH)
0.0	96	4
10.0	96	4
12.0	0	100
16.0	0	100
16.5	96	4
20.0	96	4

(6′R)-6′-Fluorocarbacytidine 26. Cytidine derivative 26 (23.7 mg, 90 μmol, 50%) a white solid after freeze drying, was obtained following the above procedure from 24 (68 mg, 0.18 mmol, 1.0 equiv.). R_f = 0.06 (10% MeOH/DCM); ¹H NMR (400 MHz, D₂O): δ 7.65 (1H, dd, ³J_H6–H5 = 7.6 Hz, ⁵J_H6–F = 1.6 Hz, H₆), 5.97 (1H, d, ³J_H5–H6 = 7.5 Hz, H₅), 5.10 (1H, ddd, ²J_H6′–F = 54.9 Hz, ³J_{H6′–H4′} = 4.6 Hz, ³J_{H6′–H1′} = 3.6 Hz, H_6′), 4.89 (1H, ddd, ³J_H1′–F = 30.7 Hz, ³J_{H1′–H2′} = 10.6 Hz, ³J_{H1′–H6′} = 3.3 Hz, H_H1′), 4.52 (1H, dd, ³J_{H2′–H1′} = 10.6 Hz, ³J_{H2′–H3′} = 6.6 Hz, H_2′), 3.99 (1H, dd, ³J_{H3′–H2′} = 6.5 Hz, ³J_{H3′–H4′} = 4.5 Hz, H_3′), 3.77–3.73 (1H, m, H_5′a, H_5′b), 2.38 (1H, dddd, ³J_H4′–F = 31.2 Hz, ³J_{H4′–H5′a} = 12.0 Hz, ³J_{H4′–H5′b} = 7.5 Hz, ³J_{H4′–H3′/H6′} = 4.6 Hz, H_4′); ¹³C NMR (101 MHz, D₂O): δ 166.0 (C₂, C=O), 158.7 (C₄), 143.8 (d, ⁴J_C6–F = 4.2 Hz, C₆), 95.7 (C₅), 91.0 (d, ¹J_C6′–F = 180.6 Hz, C_6′), 70.3 (C₂), 69.3 (C₃), 62.3 (d, ²J_C1′–F = 16.5 Hz, C_1′), 58.3 (d, ³J_C5′–F = 11.3 Hz, C_5′), 50.6 (d, ²J_C4′–F = 18.1 Hz, C_4′); ¹⁹F NMR (377 MHz, D₂O): δ −208.2 (dt, ²J_F–H6′ = 55.0 Hz, ³J_{F–H1′/H4′} = 31.0 Hz); HRMS (NSI): calculated for C₁₀H₁₃FN₃O₄ [M − H]⁻ 258.0896, found 258.0895. These data are in good agreement with literature.¹⁷

6′-gem-Difluorocarbacytidine 27. Cytidine derivative 27 (19.3 mg, 70 μmol, 58%) a white solid after freeze drying, was obtained following the above procedure from 25 (48.3 mg, 0.12 mmol, 1.0 equiv.). R_f = 0.07 (10% MeOH/DCM); ¹H NMR (400 MHz, D₂O): δ 7.59 (1H, dd, ³J_H6–H5 = 7.6 Hz, ⁵J_H6–F = 2.5 Hz, H₆), 6.00 (1H, d, ³J_H5–H6 = 7.5 Hz, H₅), 5.44–5.24 (1H, m, H_1′), 4.43 (1H, dd, ³J_{H2′–H1′} = 10.7 Hz, ³J_{H2′–H3′} = 5.6 Hz, H_2′), 4.11–4.00 (1H, m, H_3′), 3.90–3.70 (2H, m, H_5′a, H_5′b), 2.61 (1H, m, H_4′); ¹³C NMR (101 MHz, D₂O): δ 166.0 (C₂, C=O), 158.7 (C₄), 143.5 (d, ⁴J_C6–F = 4.0 Hz, C₆), 124.2 (dd, ¹J_C6′–F = 259.8 Hz, ¹J_C6′–F 253.5 Hz, C_6′), 96.4 (C₅), 69.9 (d, ³J_C2′–F = 8.0 Hz, C_2′), 68.9 (dd, ³J_C3′–F = 6.2 Hz, ³J_C3′–F = 2.1 Hz, C_3′), 62.7 (dd, ²J_C1′–F = 23.8 Hz, ²J_C1′–F = 18.7 Hz, C_1′), 57.3 (d, ⁴J_C5′–F = 10.2 Hz, C_5′), 52.9 (t, ³J_C4′–F = 20.3 Hz, C_4′); ¹⁹F NMR (377 MHz, D₂O): δ −97.9 (ddd, ²J_F–F = 236.5 Hz, ³J_F–H4′ = 14.0 Hz, ³J_F–H1′ = 8.7 Hz), −117.2 (app. dt, ²J_F–F = 236.7 Hz, ³J_{F–H1′/H4′} = 16.9 Hz); HRMS (NSI): calculated for C₁₀H₁₂F₂N₃O₄ [M − H]⁻ 276.0801, found 276.0797. These data were in good agreement with literature.¹⁷

General procedure for the synthesis of 28 & 29. Uridine derivative 22 or 23 (1.0 equiv.) was dissolved in pyridine (0.2 M), cooled to 0 °C and TIPDSiCl₂ (1.1 equiv.) was added. The reaction was allowed to warm to rt, and stirred for a further 2 h, at which point TLC analysis (10% MeOH/DCM) showed complete conversion of the starting material to a higher R_f. The reaction was quenched with MeOH (5 mL) and the solvent was removed in vacuo. The crude material was purified via flash column chromatography on silica gel (20–100% EtOAc/Hexane).

(6′R)-3′,5′-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-6′-fluorocarbauridine. Uridine derivative 28 (1.0 g, 2.1 mmol, 76%) a white foam, was obtained following the above procedure from 22 (720 mg, 2.8 mmol, 1.0 equiv.). R_f = 0.38 (hexane/EtOAc, 1 : 1); [α]^23.4_D = −73.4 (c 1.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 9.32 (1H, br s, NH), 7.41 (1H, d, ³J_H6–H5 = 7.6 Hz, H₆), 5.75 (1H, d, ³J_H5–H6 = 8.1 Hz, H₅), 5.09 (1H, ddd, ²J_H6′–F = 55.2 Hz, ³J_{H6′–H4′} = 4.1 Hz, ³J_{H6′–H1′} = 3.1 Hz, H_6′), 4.74 (1H, ddd, ³J_H1′–F = 30.4 Hz, ³J_{H1′–H2′} = 9.2 Hz, ³J_{H1′–H6′} = 2.7 Hz, H_1′), 4.55–4.45 (2H, m, H_2′, H_3′), 4.14 (1H, dd, ²J_{H5′a–H5′b} = 12.2 Hz, ³J_{H5′a–H4′} = 4.0 Hz, H_5′a), 3.93 (1H, dd, ²J_{H5′b–H5′a} = 12.0 Hz, ³J_{H5′b–H4′} = 10.4 Hz, H_5′b), 3.03 (1H, d, ³J_OH–H1′ = 8.6 Hz, OH), 2.46 (1H, m, H_4′), 1.11–1.01 (28H, m, [–OSi(CH)₂(CH₃)₄]₂); ¹³C NMR (101 MHz, CDCl₃): δ 163.1 (C₂, C=O), 151.8 (C₄, C=O), 141.7 (d, ⁴J_C6–F = 4.1 Hz, C₆), 102.4 (C₅), 92.2 (d, ¹J_C6′–F = 180.6 Hz, C_6′), 71.3 (d, ³J_C3′–F = 0.6 Hz, C_3′), 70.4 (d, ³J_C2′–F = 1.7 Hz, C_2′), 63.0 (d, ²J_C1′–F = 16.4 Hz, C_1′), 61.9 (d, ³J_C5′–F = 11.8 Hz, C_5′), 53.3 (d, ²J_C4′–F = 17.6 Hz, C_4′), 17.7 {[–OSi(CH)₂(CH₃)₄]₂}, 17.5 {[–OSi(CH)₂(CH₃)₄]₂}, 17.4 {[–OSi(CH)₂(CH₃)₄]₂}, 17.24 {[–OSi(CH)₂(CH₃)₄]₂}, 17.22 {[–OSi(CH)₂(CH₃)₄]₂}, 17.19 {[–OSi(CH)₂(CH₃)₄]₂}, 17.15 {[–OSi(CH)₂(CH₃)₄]₂}, 17.11 {[–OSi(CH)₂(CH₃)₄]₂}, 17.05 {[–OSi(CH)₂(CH₃)₄]₂}, 13.50 {[–OSi(CH)₂(CH₃)₄]₂}, 13.47 {[–OSi(CH)₂(CH₃)₄]₂}, 13.19 {[–OSi(CH)₂(CH₃)₄]₂}, 13.18 {[–OSi(CH)₂(CH₃)₄]₂}, 12.6 {[–OSi(CH)₂(CH₃)₄]₂}; ¹⁹F NMR (377 MHz, CDCl₃) δ −207.0 (ddd, ²J_F–H6′ = 55.3 Hz, ³J_F–H4′ = 35.2 Hz, ³J_F–H1′ = 30.6 Hz); HRMS (NSI): calculated for C₂₂H₄₀FN₂O₆Si₂ [M + H]⁺ 503.2403, found 503.2402.

3′,5′-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-6′-gem-difluorocarbauridine 29. Uridine derivative 29 (310 mg, 0.60 mmol, 43%) a cream foam, was obtained following the above procedure from 23 (400 mg, 1.4 mmol, 1.0 equiv.). R_f = 0.46 (hexane/EtOAc, 1 : 1); [α]^20.3_D = −65.9 (c 0.5, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 9.22 (1H, s, NH), 7.17 (1H, dd, ³J_H6–H5 = 8.2 Hz, ⁵J_H6–F = 1.5 Hz, H₆), 5.76 (1H, d, ³J_H5–H6 = 8.1 Hz, H₅), 5.08 (1H, dt, ³J_H1′–F = 19.4 Hz, ³J_{H1′–H2′} = 7.2 Hz, H_1′), 4.57 (1H, app. t, ³J_{H3′–H2′/H4′} = 6.5 Hz, H_3′), 4.37 (1H, app. q, ³J_{H2′–H1′/H3′/OH} = 6.0 Hz, H_2′), 4.15 (1H, dd, ²J_{H5′a–H5′b} = 12.3 Hz, ³J_{H5′a–H4′} = 4.2 Hz, H_5′a), 4.02 (1H, dd, ²J_{H5′b–H5′a} = 12.3 Hz, ³J_{H5′b–H4′} = 7.5 Hz, H_5′b), 3.20 (1H, d, ³J_OH–H2′ = 6.0 Hz, OH), 2.72–2.58 (1H, m, H_4′), 1.10–1.04 (28H, m, [–OSi(CH)₂(CH₃)₄]₂); ¹³C NMR (101 MHz, CDCl₃): δ 162.8 (C₂, C=O), 151.0 (C₄, C=O), 141.9 (d, ⁴J_C6–F = 4.3 Hz, C₆), 123.5 (dd, ¹J_C6′–F = 262.3 Hz, ¹J_C6′–F = 251.3 Hz, C_6′), 103.0 (C₅), 70.1 (d, ³J_C3′–F = 6.8 Hz, C_3′), 69.6 (d, ³J_C2′–F = 8.7 Hz, C₂), 64.4 (dd, ²J_C1′–F = 22.3 Hz, ²J_C1′–F = 17.7 Hz, C_1′), 58.1 (d, ³J_C5′–F = 8.1 Hz, C_5′), 52.9 (t, ²J_C4′–F = 19.5 Hz, C_4′), 17.4 {[–OSi(CH)₂(CH₃)₄]₂}, 17.3 {[–OSi(CH)₂(CH₃)₄]₂}, 17.2 {[–OSi(CH)₂(CH₃)₄]₂}, 17.1 {[–OSi(CH)₂(CH₃)₄]₂}, 17.00 {[–OSi(CH)₂(CH₃)₄]₂}, 16.99 {[–OSi(CH)₂(CH₃)₄]₂}, 16.9 {[–OSi(CH)₂(CH₃)₄]₂}, 13.3 {[–OSi(CH)₂(CH₃)₄]₂}, 13.2 {[–OSi(CH)₂(CH₃)₄]₂}, 12.9 {[–OSi(CH)₂(CH₃)₄]₂}, 12.5 {[–OSi(CH)₂(CH₃)₄]₂}; ¹⁹F NMR (377 MHz, CDCl₃): δ −106.5 (d, ²J_F–F = 237.5 Hz), −118.7 (dt, ²J_F–F = 237.2 Hz, ³J_{F–H1′/H4′} = 21.5 Hz); HRMS (NSI): calculated for C₂₂H₃₈F₂N₂O₆Si₂Na [M + Na]⁺ 543.2129, found 543.2121.

General procedure for the synthesis of 30 & 31.
Synthesis of thiocarbamate. Uridine derivative 28 or 29 (1.0 equiv.) was dissolved in MeCN (0.2 M). TCDI (1.1 equiv.) was added followed by DMAP (0.1 equiv.) and the reaction mixture was heated to 45 °C and stirred for 4 h. TLC analysis (hexane/EtOAc, 1 : 1) showed complete consumption of the starting material to a lower R_f. The reaction mixture was cooled to rt, and the solvent was removed in vacuo. The residue was partitioned between H₂O (10 mL) and EtOAc (10 mL), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The combined organic phases were washed with brine, dried over MgSO₄, filtered and the solvent removed in vacuo. This material was used without further purification.
Barton McCombie deoxygenation. Thiocarbamate intermediate was charged to an oven dried multinecked RB flask equipped with a magnetic stirrer, and the reaction vessel was evacuated and refilled with N₂ × 3. The crude material was dissolved in degassed toluene (0.2 M), and Bu₃SnH (1.5 equiv.) and AIBN (0.16 equiv.) were added. The reaction mixture was stirred at reflux for 30 min, at which point TLC analysis (1 : 1, hexane/EtOAc) showed complete conversion from a lower R_f to a higher R_f. The reaction mixture was cooled to rt, and the solvent removed in vacuo. The residue was purified via flash column chromatography on silica gel (20–50% EtOAc/hexane).

(6′R)-2′-Deoxy-3′,5′-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-6′-fluorocarbauridine 30. 2′-Deoxyuridine derivative 30 (80 mg, 0.16 mmol, 75%) a colourless syrup, was obtained following the above procedure from 28 (100 mg, 0.21 mmol, 1.0 equiv.). R_f = 0.57 (hexane/EtOAc, 1 : 1); [α]^24.3_D = −52.3 (c 0.5, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃): δ 9.70 (1H, br s, NH), 7.23 (1H, dd, ³J_H6–H5 = 8.1 Hz, ⁵J_H6–F = 0.9 Hz, H₆), 5.66 (1H, d, ³J_H5–F = 8.1 Hz, H₅), 5.22 (1H, dddd, ³J_H1′–F = 30.6 Hz, ³J_{H1′–H2′a} = 11.3 Hz, ³J_{H1′–H2′b} = 9.2 Hz, ³J_{H1′–H6′} = 2.4 Hz, H_1′), 5.01 (1H, dt, ²J_H6′–F = 56.0 Hz, ³J_{H6′–H1′/H4′} = 3.0 Hz, H_6′), 4.60 (1H, ddd, ³J_{H3′–H2′a} = 7.7 Hz, ³J_{H3′–H4′} = 5.2 Hz, ³J_{H3′–H2′b} = 2.3 Hz, H_3′), 4.08 (1H, dd, ²J_{H5′a–H5′b} = 12.0 Hz, ³J_{H5′a–H4′} = 3.7 Hz, H_5′a), 3.88 (1H, dd, ²J_{H5′b–5′a} = 11.9 Hz, ³J_{H5′b–H4′} = 9.4 Hz, H_5′b), 2.38 (1H, dt, ²J_{H2′a–H2′b} = 12.8 Hz, ³J_{H2′a–H1′} = 12.8 Hz, ³J_{H2′a–H3′} = 8.3 Hz, H_2′a), 2.36–2.20 (1H, m, H_4′), 2.11 (1H, ddd, ³J_H2′b = 11.8 Hz, ³J_{H2′b–H1′} = 8.7 Hz, ³J_{H2′b–H3′} = 2.0 Hz, H_2′b), 1.05–0.93 (28H, m, [–OSi(CH)₂(CH₃)₄]₂); ¹³C NMR (101 MHz, CDCl₃): δ 162.3 (C₂, C=O), 150.4 (C₄, C=O), 140.7 (d, ⁴J_C6–F = 5.5 Hz, C₆), 101.0 (C₅), 95.3 (d, ¹J_C6′–F = 181.5 Hz, C_6′), 71.4 (C_3′), 60.8 (d, ³J_C5′–F = 10.8 Hz, C_5′), 54.3 (d, ²J_{C1′/C4′–F} = 17.1 Hz, C_1′, C_4′), 35.0 (C_2′), 16.6 {[–OSi(CH)₂(CH₃)₄]₂}, 16.4 {[–OSi(CH)₂(CH₃)₄]₂}, 16.4 {[–OSi(CH)₂(CH₃)₄]₂}, 16.4 {[–OSi(CH)₂(CH₃)₄]₂}, 16.4 {[–OSi(CH)₂(CH₃)₄]₂}, 16.2 {[–OSi(CH)₂(CH₃)₄]₂}, 15.98 {[–OSi(CH)₂(CH₃)₄]₂}, 15.95 {[–OSi(CH)₂(CH₃)₄]₂}, 12.33 {[–OSi(CH)₂(CH₃)₄]₂}, 12.30 {[–OSi(CH)₂(CH₃)₄]₂}, 11.9 {[–OSi(CH)₂(CH₃)₄]₂}, 11.5 {[–OSi(CH)₂(CH₃)₄]₂}; ¹⁹F NMR (377 MHz, CDCl₃) δ −209.2 (ddd, ²J_F–H6′ = 55.9 Hz, ³J_F–H4′ = 36.6 Hz, ³J_F–H1′ = 30.7 Hz).

2′-Deoxy-3′,5′-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-6′-gem-difluorocarbauridine 31. 2′-Deoxyuridine derivative 31 (170 mg, 0.34 mmol, 68%) a colourless syrup, was obtained following the above procedure from 29 (250 mg, 0.50 mmol, 1.0 equiv.). R_f = 0.54 (hexane/EtOAc, 1 : 1); [α]^25.9_D = −46.5 (c 2.0, CH₂Cl₂); ¹H NMR (400 MHz, CDCl3): δ 8.29 (1H, br s, NH), 7.17 (1H, dd, ³J_H6–H5 = 8.2 Hz, ⁵J_H6–F = 2.5 Hz, H₆), 5.76 (1H, dd, ³J_H5–H6 = 8.2 Hz, ⁶J_H5–F = 1.9 Hz, H₅), 5.58 (1H, dtd, ³J_H1′–F = 16.7 Hz, ³J_{H1′–H2′a} = 9.9 Hz, ³J_{H1′–H2′b} = 6.5 Hz, H_1′), 4.58 (1H, dd, ³J_H3′–H4 = 13.4 Hz, ³J_{H3′–H2′a/H2′b} = 6.3 Hz, H_3′), 4.17–3.98 (2H, m, H_5′a, H_5′b), 2.51–2.39 (1H, m, H_4′), 2.38–2.24 (2H, m, H_2′a, H_2′b), 1.11–1.02 (28 H, m, [–OSi(CH)₂(CH₃)₄]₂); ¹³C NMR (101 MHz, CDCl₃): δ 162.0 (C₂, C=O), 150.7 (C₄, C=O), 141.1 (d, ⁴J_C6–F = 5.9 Hz, C₆), 102.8 (C₅), 68.8 (C_3′), 58.1 (d, ³J_C5′–F = 6.8 Hz, C_5′), 55.0 (C_1′), 54.7 (C_4′), 34.7 (d, ³J_C2′–F = 5.2 Hz, C_2′), 17.44 {[–OSi(CH)₂(CH₃)₄]₂}, 17.36 {[–OSi(CH)₂(CH₃)₄]₂}, 17.32 {[–OSi(CH)₂(CH₃)₄]₂}, 17.29 {[–OSi(CH)₂(CH₃)₄]₂}, 17.1 {[–OSi(CH)₂(CH₃)₄]₂}, 17.0 {[–OSi(CH)₂(CH₃)₄]₂}, 16.9 {[–OSi(CH)₂(CH₃)₄]₂}, 13.3 {[–OSi(CH)₂(CH₃)₄]₂}, 13.1 {[–OSi(CH)₂(CH₃)₄]₂}, 12.8 {[–OSi(CH)₂(CH₃)₄]₂}, 12.5 {[–OSi(CH)₂(CH₃)₄]₂}; ¹⁹F NMR (377 MHz, CDCl3): δ −108.8 (dt, ²J_F–F = 232.4 Hz, ³J_F–H4′ = 7.3 Hz), −121.6 (ddd, ²J_F–F = 232.2 Hz, ³J_F–H4′ = 26.0 Hz, ³J_F–H1′ = 20.3 Hz); HRMS (NSI): calculated for C₂₂H₃₉F₂N₂O₅Si₂ [M + H]⁺ 505.2360, found 505.2360.

General procedure for the synthesis of 32 & 33. 2′-Deoxyuridine derivative 30 or 31 (1.0 equiv.) was dissolved in THF (0.2 M) and TBAF (2.0 equiv., 1.0 M in THF) was added. The reaction was stirred at rt for 16 h, at which point TLC analysis (10% MeOH/DCM) showed complete consumption of the starting material to a lower R_f. DOWEX 50WX8 (H⁺) resin (500 mg), CaCO₃ (180 mg) and MeOH (1.0 mL) were added, and the reaction mixture was stirred for a further 1 h. The reaction mixture was filtered through a Celite® plug and eluted with MeOH. The material was purified via flash column chromatography on silica gel (0–10% MeOH/EtOAc).
HPLC purification. A sample of 32 or 33 was dissolved in 9 : 1 H₂O/MeOH (v/v, 100 mg mL⁻¹), and injected onto a reverse phase column (see general experimental) and purified at a flow rate of 20.00 ml min⁻¹ using the following gradient system:

Time (min)	%A (H2O)	%B (MeOH)
0.0	90	10
5.0	90	10
12.0	0	100
15.0	0	100
15.1	90	10
20.0	90	10

(6′R)-2′-Deoxy-6′-fluorocarbauridine 32. 2′-Deoxyuridine analogue 32 (17 mg, 73 μmol, 66%) a white solid after freeze drying, was obtained following the above procedure from 30 (54 mg, 0.11 mmol, 1.0 equiv.). R_f = 0.27 (10% MeOH/DCM); [α]^23.6_D = −68.9 (c 1.0, MeOH); ¹H NMR (400 MHz, D₂O): δ 7.64 (1H, dd, ³J_H6–H5 = 8.1 Hz, ⁵J_H6–F = 1.4 Hz, H₆), 5.74 (1H, d, ³J_H5–H6 = 8.0 Hz, H₅), 5.27–4.98 (2H, m, H_1′, H_6′), 4.28–4.16 (1H, m, H_3′), 3.78–3.70 (2H, m, H_5a′, H_5′b), 2.47 (1H, ddd, ²J_{H2′a–H2′b} = 14.2 Hz, ³J_{H2′a–H1′} = 10.8 Hz, ³J_{H2′a–H3′} = 8.3 Hz, H_2′a), 2.41–2.11 (1H, m, H_4′), 2.11–1.96 (1H, m, H_2′b); ¹³C NMR (101 MHz, D₂O): δ 166.3 (C₂, C=O), 152.5 (C₄, C=O), 144.6 (d, ⁴J_C6–F = 5.3 Hz, C₆), 101.1 (C₅), 94.4 (d, ¹J_C6′–F = 181.3 Hz, C_6′), 70.2 (C_3′), 58.1 (d, ³J_C5′–F = 9.7 Hz, C_5′), 55.7 (d, ²J_C1′–F = 16.4 Hz, C_1′), 53.2 (d, ²J_C4′–F = 17.7 Hz, C_4′), 34.4 (C_2′); ¹⁹F NMR (377 MHz, D₂O) δ −209.7 (app. dt, ²J_F–H6′ = 54.5 Hz, ³J_{F–H1′/H4′} = 32.2 Hz); HRMS (NSI): calculated for C₁₀H₁₂FN₂O₄ [M − H]⁻ 243.0787, found 243.0783.

2′-Deoxy-6′-gem-difluorocarbauridine 33. 2′-Deoxyuridine analogue 33 (0.12 mmol, 55%) as a white solid, was obtained following the above procedure from 31 (112 mg, 0.22 mmol, 1.0 equiv.). R_f = 0.30 (10% MeOH/DCM); [α]^25.4_D = −82.1 (c 0.4, MeOH); ¹H NMR (400 MHz, D₂O) δ 7.57 (1H, dd, ³J_H6–H5 = 8.1 Hz, ⁵J_H6–F = 2.5 Hz, H₆), 5.78 (1H, d, ³J_H5–H6 = 8.0 Hz, H₅), 5.48 (1H, td, ³J_H1′–F = 18.5 Hz, ³J_{H1′–H2′a/2′b} = 10.0 Hz, H_1′), 4.21–4.14 (1H, m, H_3′), 3.88–3.74 (2H, m, H_5′a/5′b), 2.51 (1H, tdd, ³J_H4′–F = 10.3 Hz, 9.8, ³J_{H4′–H3′} = 5.7 Hz, H_4′), 2.41 (1H, ddd, ²J_{H2′a–H2′b} = 14.4 Hz, ³J_{H2′a–H1′} = 10.5 Hz, ³J_{H2′a–H3′} = 7.5 Hz, H_2′a), 2.15 (1H, ddd, ²J_{H2′b–H2′a} = 14.0 Hz, ³J_{H2′b–H1′} = 9.8 Hz, ³J_{H2′b–H3′} = 3.9 Hz, H_2′b); ¹³C NMR (101 MHz, D₂O): δ 167.0 (C2, C=O), 153.2 (C₄, C=O), 143.9 (d, ⁴J_C6–F = 4.5 Hz, C₆), 127.1 (dd, ¹J_C6′–F = 260.7 Hz, ¹J_C6′–F = 254.0 Hz, C_6′), 102.0 (C₅), 67.7 (d, ³J_C3′–F = 8.4 Hz, C_3′), 57.1 (d, ³J_C5′–F = 8.8 Hz, C_5′), 56.3 (dd, ²J_C1′–F = 25.2 Hz, ²J_C1′–F = 18.4 Hz, C_1′), 53.6 (t, ²J_C4′–F = 19.5 Hz, C_4′), 33.5 (d, ³J_C2′–F = 6.3 Hz, C_2′); ¹⁹F NMR (377 MHz, D₂O): δ −102.8 (app. dt, ²J_F–F = 232.4 Hz, ³J_{F–H4′/H1′} = 9.2 Hz), −120.5 (app. dt, ²J_F–F = 232.4 Hz, ³J_{F–H1′/H4′} = 19.6 Hz); HRMS (NSI): calculated for C₁₀H₁₁F₂N₂O₄ [M-H]⁻ 261.0692, found 261.0692

General procedure for the synthesis of 34 & 35.
Installation of 1,2,4-triazole. Uridine 30 or 31 (1.0 equiv.) was dissolved in MeCN (0.1 M) and cooled to 0 °C. 1,2,4-Triazole (23 equiv.), POCl₃ (2.4 equiv.) and Et₃N (23 equiv.) were added and the reaction mixture was allowed to slowly warm to rt and stirred for 18 h. TLC analysis (4 : 1 EtOAc/hexane) showed majority conversion of the starting material to a lower R_f. The reaction mixture was cooled to 0 °C and quenched with sat. NaHCO₃ (5 mL). The aqueous phase was extracted with EtOAc (3 × 10 mL), and the combined organic phases were washed with H₂O (10 mL), brine (10 mL), dried over MgSO₄, filtered and the solvent removed in vacuo.
Amination. 1,2,4-Triazole derivative was dissolved in 35% NH₄OH/1,4-dioxane (1 : 1, v/v, 0.1 M) and the reaction vessel was sealed. The reaction mixture was stirred at rt for 18 h, at which point TLC analysis (5% MeOH/DCM) showed complete conversion from a higher R_f to a lower R_f. The solvent was removed in vacuo, and the residue was passed through a silica plug (eluting first with 1 : 1 hexane/EtOAc, then 5% MeOH/EtOAc).

(6′R)-2′-Deoxy-3′,5′-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-6′-fluorocarbacytidine. 2′-Deoxycytidine derivative (24 mg, 50 μmol, 51%) a colourless syrup, was obtained following the above procedure from 30 (47 mg, 98 μmol, 1.0 equiv.). R_f = 0.49 (EtOAc/hexane, 4 : 1); [α]^24.5_D = −55.8 (c 1.0, MeOH); ¹H NMR (400 MHz, MeOD): δ 8.30 (2H, br s, NH₂), 7.62 (1H, dd, ³J_H6–H5 = 7.5 Hz, ⁵J_H6–F = 1.3 Hz, H₆), 5.87 (1H, d, ³J_H5–H6 = 7.5 Hz, H₅), 5.27 (1H, dddd, ³J_H1′–F = 30.5 Hz, ³J_{H1′–H2′a} = 11.6 Hz, ³J_{H1′–H2′b} = 8.2 Hz, ³J_{H1′–H6′} = 2.5 Hz, H_1′), 5.21 (1H, dt, ²J_H6′–F = 56.1 Hz, ³J_{H6′–H1′/H4′} = 3.1 Hz, H_6′), 4.71 (1H, ddd, ³J_{H3′–H2′a} = 7.3 Hz, ³J_{H3′–H4′} = 4.9 Hz, ³J_{H3′–H2′b} = 2.1 Hz, H_3′), 4.17 (1H, dd, ²J_{H5′a–H5′b} = 11.9 Hz, ³J_{H5′a–H4′} = 3.8 Hz, H_H5′a), 3.97 (1H, dd, ²J_{H5′b–H5′a} = 11.8 Hz, ³J_{H5′b–H4′} = 9.8 Hz, H_5′b), 2.64 (1H, td, ²J_{H2′a–2′b} = 12.8 Hz, ³J_{H2′a–H1′} = 12.8 Hz, ³J_{H2′a–H3′} = 7.9 Hz, H_2′a), 2.40 (1H, dtd, ³J_H4′–F = 35.8 Hz, ³J_{H4′–H5′b} = 8.6 Hz, ³J_{H4′–H3′/H5′a/H6′} = 4.0 Hz, H_4′), 2.10 (ddd, ²J_{H2′b–H2′a} = 13.1 Hz, ³J_{H2′b–H1′} = 8.3 Hz, ³J_{H2′b–H3′} = 1.9 Hz, H_2′b), 1.14–1.02 (28H, m, [–OSi(CH)₂(CH₃)₄]₂); ¹³C NMR (101 MHz, MeOD): δ 167.3 (C₂, C=O), 159.0 (C₄), 144.6 (d, ⁴J_C6–F = 4.3 Hz, C₆), 97.1 (d, ¹J_C6′–F = 180.7 Hz, C_6′), 95.5 (C₅), 74.1 (C_3′), 63.3 (d, ³J_C5′–F = 11.5 Hz, C_5′), 58.0 (d, ²J_C1′–F = 16.7 Hz, C_1′), 57.0 (d, ²J_C4′–F = 17.1 Hz, C_4′), 36.8 (C_2′), 18.1 {[–OSi(CH)₂(CH₃)₄]₂}, 18.0 {[–OSi(CH)₂(CH₃)₄]₂}, 17.94 {[–OSi(CH)₂(CH₃)₄]₂}, 17.92 {[–OSi(CH)₂(CH₃)₄]₂}, 17.85 {[–OSi(CH)₂(CH₃)₄]₂}, 17.7 {[–OSi(CH)₂(CH₃)₄]₂}, 17.54 {[–OSi(CH)₂(CH₃)₄]₂}, 17.51 {[–OSi(CH)₂(CH₃)₄]₂}, 14.6 {[–OSi(CH)₂(CH₃)₄]₂}, 14.5 {[–OSi(CH)₂(CH₃)₄]₂}, 14.2 {[–OSi(CH)₂(CH₃)₄]₂}, 13.8 {[–OSi(CH)₂(CH₃)₄]₂}; ¹⁹F NMR (377 MHz, CDCl₃): δ −207.68 (ddd, ²J_F–H6′ = 56.0 Hz, ³J_F–H4′ = 35.6 Hz, ³J_F–H1′ = 30.6 Hz); HRMS (NSI): calculated for C₂₂H₄₀FN₃O₄Si₂ [M + H]⁺ 486.2614, found 486.2614.

2′-Deoxy-3′,5′-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-6′-gem-difluorocarbacytidine. 2′-Deoxycytidine derivative (38 mg, 76 μmol, 59%) a colourless syrup, was obtained following the above procedure from 31 (65 mg, 0.13 mol, 1.0 equiv.). R_f = 0.68 (5% MeOH/DCM); [α]^24.6_D = −36.7 (c 2.0, MeOH); ¹H NMR (400 MHz, MeOD): δ 8.31 (2H, br s, NH₂), 7.55 (1H, dd, ³J_H6–H5 = 7.5 Hz, ⁵J_H6–F = 2.5 Hz, H₆), 5.93 (1H, d, ³J_H5–H6 = 7.5 Hz, H₅), 5.71 (1H, ddd, ³J_H1′–F = 19.9 Hz, ³J_H1′–F = 17.0 Hz, ³J_{H1′–H2′a/H2′b} = 9.8 Hz, H_1′), 4.66 (1H, dd, ³J_{H3′–H4′} = 12.9 Hz, ³J_{H3′–H2′} 6.8 Hz, 1H), 4.17–4.02 (2H, m, H_5′a, H_5′b), 2.58–2.44 (2H, m, H_2′a, H_4′), 2.27 (1H, ddd, ²J_{H2′b–H2′a} = 14.8 Hz, ³J_{H2′b–H1′} = 10.5 Hz, ³J_{H2′b–H3′} = 4.9 Hz, H_2′b), 1.14–1.06 (28H, m, [–OSi(CH)₂(CH₃)₄]₂); ¹³C NMR (101 MHz, MeOD) δ 167.1 (C₂, C=O), 158.6 (C₄), 144.6 (d, ⁴J_C6–F = 5.0 Hz, C₆), 127.9 (dd, ¹J_C6′–F = 261.5 Hz, ¹J_C6′–F = 252.9 Hz, C_6′), 96.3 (C₅), 70.8 (d, ³J_C3′–F = 9.2 Hz, C_3′), 59.6 (d, ³J_C5′–F = 7.8 Hz, C_5′), 57.2 (dd, ²J_C1′–F = 24.2 Hz, ²J_C1′–F = 17.3 Hz, C_1′), 56.6 (t, ²J_C4′–F = 19.0 Hz, C_4′), 35.7 (d, ³J_C2′–F = 5.7 Hz, C_2′), 18.0 {[–OSi(CH)₂(CH₃)₄]₂}, 17.9 {[–OSi(CH)₂(CH₃)₄]₂}, 17.83 {[-OSi(CH)₂(CH₃)₄]₂}, 17.80 {[–OSi(CH)₂(CH₃)₄]₂}, 17.7 {[–OSi(CH)₂(CH₃)₄]₂}, 17.52 {[-OSi(CH)₂(CH₃)₄]₂}, 17.49 {[–OSi(CH)₂(CH₃)₄]₂}, 14.54 {[-OSi(CH)₂(CH₃)₄]₂}, 14.47 {[–OSi(CH)₂(CH₃)₄]₂}, 14.4 {[–OSi(CH)₂(CH₃)₄]₂}, 13.8 {[–OSi(CH)₂(CH₃)₄]₂}; ¹⁹F NMR (377 MHz, MeOD) δ −109.35 (dt, ²J_F–F = 231.6 Hz, ³J_F–H4′ = 6.8 Hz), −122.6 (dt, ²J_F–F = 231.0 Hz, ³J_{F–H1′/H4′} = 23.1 Hz); HRMS (NSI): calculated for C₂₂H₃₉F₂N₃O₄Si₂ [M + H]⁺ 504.2520, found 504.2519.
Desilylation. 2′-Deoxycytidine derivative (1.0 equiv.) was dissolved in THF (0.2 M), and TBAF (2.0 equiv., 1.0 M in THF) was added. The reaction was stirred at rt for 16 h, at which point TLC analysis (10% MeOH/DCM) showed complete consumption of the starting material to a lower R_f. DOWEX 50WX8 (H⁺) resin (400 mg), CaCO₃ (160 mg) and MeOH (1.0 mL) were added, and the reaction mixture was stirred for a further 1 h. The reaction mixture was filtered through a Celite® plug and eluted with MeOH. The solvent as removed in vacuo, and the residue was purified via flash column chromatography (0–10% MeOH/EtOAc).
HPLC purification. A sample of 34 or 35 was dissolved in H₂O (100 mg mL⁻¹), and injected onto a reverse phase column (see general experimental) and purified at a flow rate of 20.00 ml min⁻¹ using the following gradient system:

Time (min)	%A (H2O)	%B (MeOH)
0.0	96	4
12.0	96	4
15.0	0	100
18.0	0	100
18.5	96	4
22.0	96	4

(6′R)-2′-Deoxy-6′-fluorocarbacytidine 34. 2′-Deoxycyridine analogue 34 (4.3 mg, 18 μmmol, 22%) a white solid after freeze drying, was obtained following the above procedure (40 mg, 82 μmol, 1.0 equiv.). R_f = 0.05 (10% MeOH/DCM); ¹H NMR (400 MHz, MeOD): δ 7.64 (1H, dd, ³J_H6–H5 = 7.5 Hz, ⁵J_H6–F = 1.6 Hz, H₆), 5.87 (1H, d, ³J_H5–H6 = 7.5 Hz, H₅), 5.30 (1H, dddd, ³J_H1′–F = 30.7 Hz, ³J_{H1′–H2′a} = 11.4 Hz, ³J_{H1′–H2′b} = 8.6 Hz, ³J_{H1′–H6′} = 2.8 Hz, H_1′), 5.15 (1H, app. dt, ²J_H6′–F = 55.7 Hz, ³J_{H6′–H1′/H4′} = 3.2 Hz, H_6′), 4.16 (1H, ddd, ³J_{H3′–H2′a} = 8.5 Hz, ³J_{H3′–H4′} = 6.2 Hz, ³J_{H3′–H2′b} = 2.7 Hz, H_3′), 3.76 (2H, app. d, ²J_{H5′a–H5′b} = 7.9 Hz, ³J_{H5′a/H5′b–H4′} = 7.9 Hz, H_5′a, H_5′b), 2.49 (1H, ddd, ²J_{H2′a–H2′b} = 13.3 Hz, ³J_{H2′a–H1′} = 11.8 Hz, ³J_{H2′a–H3′} = 8.1 Hz, H_2′a), 2.34–2.17 (1H, m, H_4′), 2.00 (1H, ddd, ²J_{H2′b–H2′a} = 12.4 Hz, ³J_{H2′b–H1′} = 8.8 Hz, ³J_{H2′b–H3′} = 2.8 Hz, H_2′b); ¹⁹F NMR (377 MHz, MeOD): δ −212.0 (app. dt, ²J_F–H6′ = 54.7 Hz, ³J_{F–H1′/H4′} = 31.7 Hz); HRMS (NSI): calculated for C₁₀H₁₃FN₃O₃ [M − H]⁻ 242.0946, found 242.0942.

2′-Deoxy-6′-gem-difluorocarbacytidine 35. 2′-Deoxycytidine analogue 35 (4.0 mg, 15 μmmol, 15%) a white solid after freeze drying, was obtained following the above procedure (50 mg, 0.1 mmol, 1.0 equiv.). R_f = 0.4 (10% MeOH/DCM); ¹H NMR (400 MHz, D₂O): δ 7.54 (1H, dd, ³J_H6–H5 = 7.6 Hz, ⁵J_H6–F = 2.6 Hz, H₆), 5.96 (1H, d, ³J_H5–H6 = 7.6 Hz, H₅), 5.56 (1H, td, ³J_H1′–F = 18.5 Hz, ³J_{H1′–H2′a/H2′b/F} = 9.9 Hz, H_1′), 4.34–4.04 (1H, m, H_3′), 3.92–3.72 (2H, m, H_5′a, H_5′b), 2.58–2.44 (1H, m, H_4′), 2.39 (1H, ddd, ²J_{H2′a–H2′b} = 14.3 Hz, ³J_{H2′a–H1′} = 10.6 Hz, ³J_{H2′a–H3′} = 7.5 Hz, H_2′a), 2.14 (1H, ddd, ²J_{H2′b–H2′a} = 14.1 Hz, ³J_{H2′b–H1′} = 9.9 Hz, ³J_{H2′b–H3′} = 3.9 Hz, H_3′); ¹³C NMR (101 MHz, D₂O): δ 165.9 (C₂, C=O), 158.5 (C₄), 143.8 (d, ⁴J_C6–F = 4.6 Hz, C₆), 126.2 (m, C_6′), 96.0 (C₅), 67.8 (d, ⁴J_C3′–F = 8.5 Hz, C_3′), 57.1 (d, ⁴J_C5′–F = 8.5 Hz, C_5′), 56.8 (dd, ³J_C1′–F = 25.3 Hz, ³J_C1′–F = 18.3 Hz, C_1′), 53.8 (t, ³J_C4′–F = 19.6 Hz, C_4′), 33.9 (d, ⁴J_C2′–F = 6.5 Hz, C_2′); ¹⁹F NMR (377 MHz, D₂O): δ −102.8 (app. dt, ²J_F–F = 231.5 Hz, ³J_{F–H1′/H4′} = 9.2 Hz), −120.7 (app. dt, ²J_F–F = 231.5 Hz, ³J_{F–H1′/H4′} = 19.6 Hz); HRMS (NSI): calculated for C₁₀H₁₂F₂N₃O₃ [M − H]⁻ 260.0852, found 260.0848.

General procedure for the synthesis of 36 & 37. Uridine analogue 22 or 23 (1.0 equiv.) was suspended in acetone (0.2 M) and conc. H₂SO₄ (1 drop) was added. The reaction mixture was stirred at reflux for 2 h, at which point TLC analysis (10% MeOH/DCM) showed majority conversion of the starting material to a higher R_f. The reaction mixture was cooled to rt and the pH adjusted to 7 with solid Na₂CO₃. The mixture was filtered, and the solvent removed in vacuo. The crude material was purified via flash column chromatography on silica gel (0–10%, MeOH/EtOAc).

(6′R)-2′,3′-O-Isopropylidine-6′-fluorocarbauridine 36. Uridine derivative 36 (41 mg, 0.14 mmol, 81%) a colourless syrup, was obtained following the above procedure from 22 (43.3 mg, 0.17 mmol, 1.0 equiv.). R_f = 0.37 (5% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃): δ 9.42 (1H, br s, NH), 7.40 (1H, d, ³J_H6–H5 = 7.6 Hz, H₆), 5.76 (1H, d, ³J_H5–H6 = 8.1 Hz, H₅), 5.27 (1H, dt, ²J_H6′–F = 54.3 Hz, ³J_{H6′–H1′/H4′} = 3.0 Hz, H_6′), 5.12 (1H, ddd, ³J_H1′–F = 33.9 Hz, ³J_{H1′–H2′} = 7.2 Hz, ³J_{H1′–H6′} = 2.5 Hz, H_1′), 4.92 (1H, app. t, ³J_{H2′–H1′/H3′} = 7.3 Hz, H_2′), 4.69–4.62 (1H, app. t, ³J_{H3′–H2′/H4′} = 6.2 Hz, H_3′), 3.97–3.86 (2H, m, H_5′a, H_5′b), 2.66–2.49 (1H, m, H_4′), 1.55 (3H, s, CH₃), 1.34 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 162.8 (C₂, C=O), 151.1 (C₄, C=O), 141.6 (C₆), 115.2 [C(CH₃)₂], 102.6 (C₅), 97.8 (d, ¹J_C6′–F = 181.0 Hz, C_6′), 80.2 (C_2′), 79.7 (C_3′), 62.7 (d, ²J_C1′–F = 15.7 Hz, C_1′), 59.6 (d, ³J_C5′–F = 8.4 Hz, C₅), 50.6 (d, ²J_C4′–F = 17.5 Hz, C_4′), 27.3 [C(CH₃)₂], 24.9 [C(CH₃)₂]; ¹⁹F NMR (377 MHz, CDCl₃): δ −205.2 (app. dt, ²J_F–H6′ = 54.3 Hz, ³J_{F–H1′/H4′} = 34.8 Hz); HRMS (NSI) calculated for C₁₃H₁₇FN₂O₅Na [M + Na]⁺ 323.1014, found 232.1014. These data were in good agreement with literature.¹⁷

2′,3′-O-Isopropylidine-6′-gem-difluorocarbauridine 37. Uridine derivative 37 (34 mg, 0.11 mmol, 71%) a colourless syrup, was obtained following the above procedure from 23 (41.6 mg, 0.15 mmol, 1.0 equiv.). R_f = 0.38 (5% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃): δ 9.45 (1H, br s, –NH), 7.29 (1H, dd, ³J_H6–H5 = 8.1 Hz, ⁵J_H6–F = 1.7 Hz, H₆), 5.79 (1H, d, ³J_H5–H6 = 8.1 Hz, H₅), 5.29 (1H, dt, ³J_H1′–F = 20.7 Hz, ³J_{H1′–H2′} = 6.7 Hz, H_1′), 4.79 (1H, app. t, ³J_{H2′–H1′/H3′} = 6.7 Hz, H_2′), 4.70 (1H, dd, ³J_{H3′–H2′} = 6.6 Hz, ³J_{H3′–H4′} = 5.3 Hz, H_3′), 4.05–3.93 (1H, m, H_5′a, H_5′b), 2.85–2.71 (1H, m, H_4′), 2.32 (1H, br s, OH), 1.57 (3H, s, CH₃), 1.34 (3H, s, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 162.9 (C₂, C=O), 151.1 (C₄, C=O), 141.8 (app. d, ⁴J_C6–F = 5.2 Hz, C₆), 127.1 (dd, ¹J_C6′–F = 266.1 Hz, ¹J_C6′–F = 253.3 Hz, C_6′), 114.2 [C(CH₃)₂], 103.2 (C₅), 77.9 (d, ³J_C2′–F = 7.8 Hz, C_2′), 77.2 (app. d, ³J_C3′–F = 8.1 Hz, C_3′), 64.2 (dd, ²J_H1′–F = 24.2 Hz, ²J_H1′–F = 17.8 Hz, C_1′), 58.1 (app. d, ³J_C5′–F = 7.9 Hz, C_5′), 52.1 (app. t, ²J_C4′–F = 19.7 Hz, C_4′), 27.2 [C(CH₃)₂], 25.0 [C(CH₃)₂]; ¹⁹F NMR (377 MHz, CDCl₃): δ −100.0 (app. d, ²J_F–F = 237.2 Hz), −116.4 (app. dt, ²J_F–F = 237.4, ³J_{F–H1′/H4′} = 21.8 Hz); HRMS (NSI) calculated for C₁₃H₁₆F₂N₂O₅Na [M + Na]⁺ 341.0919, found 341.0921. These data were in good agreement with literature.¹⁷

General procedure for the synthesis of 39 & 40.
Synthesis of phosphoramidate intermediate. Uridine derivative 36 or 37 (1.0 equiv.) was suspended in THF (0.2 M) and cooled to 0 °C. ^tBuMgCl (2.0 equiv., 1.0 M in THF) was added dropwise and the reaction mixture was stirred for 1 h. Isopropyl [(S)-(perfluorophenoxy)(phenoxy)phosphoryl]-L-alaninate 38 (1.1 equiv.) was added in one portion and the reaction mixture was allowed to warm to rt and stirred for 4 h. TLC analysis showed complete consumption of the starting material to a higher R_f. The reaction was quenched with MeOH (1 mL), and the solvent removed in vacuo. The residue was partitioned between H₂O (5 mL) and EtOAc (5 mL), and the aqueous phase was extracted with EtOAc (2 × 5 mL), washed with brine (5 mL), dried over MgSO₄, filtered and the solvent removed in vacuo. This material was used without further purification.
Deprotection. Phosphoramidate intermediate was dissolved in 1 : 1 formic acid/H₂O (v/v, 0.1 M) and stirred at rt for 16 h. The solvent was removed in vacuo and the crude material was purified via flash column chromatography on silica gel (0–5% MeOH/EtOAc).
HPLC purification. A sample of 39 or 40 was dissolved in 1 : 1 H₂O/MeOH (v/v, 100 mg mL⁻¹), and injected onto a reverse phase column (see general experimental) and purified at a flow rate of 20.00 ml min⁻¹ using the following gradient system:

Time (min)	%A (H2O)	%B (MeOH)
0.0	50	50
5.0	50	50
12.0	0	100
15.0	0	100
15.1	50	50
20.0	50	50

[P(S),6′R]-5′-[Phenoxy(isopropyl-L-alaninate)]phosphate-6′-fluorocarbauridine 39. Phosphoramidate 39 (8.9 mg, 20 μmol, 24%) a white solid after freeze drying, was obtained following the above procedure from 36 (20.9 mg, 0.07 mmol, 1.0 equiv.). R_f = 0.09 (5% MeOH/DCM); ¹H NMR (400 MHz, MeOD): δ 7.65 (1H, dd, ³J_H6–H5 = 8.2 Hz, ⁵J_H6′–F = 1.5 Hz, H₆), 7.39–7.34 (2H, m, ArH), 7.27–7.16 (3H, m, ArH), 5.69 (1H, d, ³J_H5–H6 = 8.1 Hz, H₅), 5.06 (1H, dt, ²J_H6′–F = 55.2 Hz, ³J_{H6′–H1′/H4′} = 4.1 Hz, H_6′), 4.97 (1H, sep, ³J_{[CH(CH3)2]–[CH(CH3)2]} = 6.3 Hz, [CH(CH₃)₂]), 4.89 (1H, ddd, ³J_H1′–F = 30.3 Hz, ³J_{H1′–H2′} = 10.0 Hz, ³J_{H1′–H6′} = 3.5 Hz, H_1′), 4.46 (1H, dd, ³J_{H2′–H1′} = 9.9 Hz, ³J_{H2′–H3′} = 6.6 Hz, H_2′), 4.27 (2H, m, H_5′a, H_5′b), 4.00 (1H, dd, ³J_{H3′–H2′} = 6.1 Hz, ³J_{H3′–H4′} = 5.1 Hz, H_3′), 3.90 (1H, dq, ³J_AlaCH–P = 9.7 Hz, ³J_{AlaCH–AlaCH3} = 7.1 Hz, AlaCH), 2.63–2.48 (1H, m, H_4′), 1.34 (3H, dd, ³J_{AlaCH3–AlaCH} = 7.1 Hz, ⁴J_AlaCH3–P = 0.9 Hz, Ala–CH₃), 1.22 (6H, d, ³J_{[CH(CH3)2]–[CH(CH3)2]} = 6.3 Hz, [CH(CH₃)₂]); ¹³C NMR (101 MHz, MeOD): δ 174.5 (d, ³J_{Ala C=O–P} = 5.4 Hz, Ala C=O), 166.3 (C₄, C=O), 153.4 (C₂, C=O), 152.3 (d, ²J_ArC–P = 7.0 Hz, ArC), 144.6 (d, ⁴J_C6–F = 3.9 Hz, C₆), 130.8 (d, ⁴J_ArC–P = 0.6 Hz, ArC), 126.1 (d, ⁵J_ArC–P = 1.2 Hz, ArC), 121.5 (d, ³J_ArC–P = 4.7 Hz, ArC), 102.0 (C₅), 91.8 (d, ¹J_C6′–F = 182.3 Hz, C_6′), 71.6 (C_2′), 70.4 (C_3′), 70.1 [CH(CH₃)₂], 64.6 (dd, ²J_C5′–P = 11.3 Hz, ³J_C5′–F = 5.6 Hz, C_5′), 63.5 (d, ²J_C1′–F = 16.3 Hz, C_1′), 51.7 (Ala–CH), 51.0 (dd, ²J_C4′–F = 17.5 Hz, ³J_C4′–P = 7.5 Hz, C_4′), 22.0 [CH(CH₃)₂], 21.9 [CH(CH₃)₂], 20.5 (d, ³J_AlaCH3–P = 6.5 Hz, Ala–CH₃); ¹⁹F NMR (377 MHz, MeOD): δ −208.4 (dt, ²J_F–H6′ = 55.4 Hz, ³J_{F–H1′/H4′} = 30.3 Hz); ³¹P {¹H} NMR (162 MHz, MeOD): δ 3.46 (s); HRMS (NSI): calculated for C₂₂H₂₈FN₃O₉P [M − H]⁻ 528.1553, found 528.1547. These data are in good agreement with literature.¹⁷

[P(S)]-5′-[Phenoxy(isopropyl-L-alaninate)]phosphate-6′-gem-difluorocarbauridine 40. Phosphoramidate 40 (14.9 mg, 30 μmol, 27%) a white solid after freeze drying, was obtained following the above procedure from 37 (33.6 mg, 0.11 mmol, 1.0 equiv.). R_f = 0.11 (5% MeOH/DCM); ¹H NMR (400 MHz, MeOD): δ 7.54 (dd, ³J_H6–C5 = 8.1 Hz, ⁵J_H6–F = 2.4 Hz, H₆), 7.42–7.34 (2H, m, ArH), 7.28–7.18 (3H, m, ArH), 5.71 (d, ³J_H5–H6 = 8.1 Hz, H₅), 5.35 (app. dt, ³J_H1′–F = 18.9 Hz, ³J_{H1′–H2′} = 9.6 Hz, H_1′), 4.98 (1H, sep, ³J_{[CH(CH3)2]–[CH(CH3)2]} = 6.7 Hz, [CH(CH₃)₂]), 4.39–4.24 (3H, m, H_2′, H_5′a, H_5′b), 4.12–4.08 (1H, m, H_3′), 3.90 (1H, dq, ³J_AlaCH–P = 9.8 Hz, ³J_{AlaCH–AlaCH3} = 7.1 Hz, AlaCH), 2.83–2.70 (1H, m, H_4′), 1.35 (3H, dd, ³J_{AlaCH3–AlaCH} = 7.1 Hz, ⁴J_AlaCH3–P = 0.8 Hz, AlaCH₃), 1.23 (6H, d, ³J_{[CH(CH3)2]– [CH(CH3)2]} = 6.3 Hz, [CH(CH₃)₂]); ¹³C NMR (101 MHz, MeOD): δ 173.0 (d, ³J_C=O–P = 5.3 Hz, C=O Ala), 164.4 (C=O, C₂), 151.8 (C=O, C₄), 150.8 (d, ²J_ArC–P = 7.0 Hz, ArC), 142.9 (d, ⁴J_C6–F = 4.1 Hz, C₆), 129.4 (d, ⁴J_ArC–P = 0.6 Hz, ArC), 124.8 (d, ⁵J_ArC–P = 1.0 Hz, ArC), 123.8 (dd, ¹J_C6′–F = 261.1, ¹J_C6′–F = 253.7 Hz, C = 6′), 120.0 (d, ³J_ArC–P = 4.8 Hz, ArC), 101.3 (C₅), 69.8 (d, ³J_C2′–F = 7.8 Hz, C_2′), 68.9 (dd, ³J_C3′–F = 5.6 Hz, ³J_C3′–F = 1.7 Hz, C₃), 68.8 [CH(CH₃)₂], 62.2 (dd, ²J_C1′–F = 24.2 Hz, ²J_C1′–F = 18.2 Hz, C_1′), 61.9 (dd, ³J_C5′–P = 11.0 Hz, ³J_C5′–F = 5.4 Hz, C_5′), 51.9 (ddd, ³J_C4′–F = 21.6 Hz, ³J_C4′–F = 19.0 Hz, ³J_C4′–P = 8.3 Hz, C_4′) 50.2 (Ala–CH), 20.6 [CH(CH₃)₂], 20.5 [CH(CH₃)₂], 19.1 (d, ³J_CH3–P = 6.5 Hz, Ala–CH₃); ¹⁹F NMR (377 MHz, MeOD): δ −98.6 (ddd, ²J_F–F = 238.7 Hz, ³J_F–H4′ = 13.0 Hz, ³J_F–H1′ = 9.7 Hz), −117.0 (app. dt, ²J_F–F = 238.6 Hz, ³J_{F–H1′/H4′} = 16.7 Hz); ³¹P {¹H} NMR (162 MHz, MeOD): δ 3.29 (s); HRMS (NSI): calculated for C₂₂H₂₇F₂N₄O₉P [M − H]⁻ 546.1458, found 541.1460. These data were in good agreement with literature.¹⁷

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Keele University, Riboscience LLC and the Royal Society of Chemistry (Research Enablement Grant E21-7055928079) are thanked for funding to C. M. M. B. UK Research and Innovation (UKRI, Future Leaders Fellowship, MR/T019522/1) are thanked for project grant funding to G. J. M. We also thank the EPSRC UK National Mass Spectrometry Facility (NMSF) at Swansea University.

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Footnote

† Electronic supplementary information (ESI) available: Relevant spectral NMR, X-ray crystallography and HPLC data for compounds 2–40 alongside cell viability assay data. CCDC 2193041–2193043 and 2218823. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d2ob01761j

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