Synthesis and biological evaluation of γ-aminophosphonates as potent, sub-type selective sphingosine 1-phosphate receptor agonists and antagonists

Abstract

The synthesis of N-arylamide phosphonates and related arylether and arylamine analogues provided potent, subtype-selective agonists and antagonists of the five known sphingosine 1-phosphate (S1P) receptors (S1P_1–5). To this end, the syntheses of phosphoserine mimetics - selectively protected and optically active phosphonoserines - are described. In vitro binding assays showed that the implementation of phosphonates as phosphate mimetics provided compounds with similar receptor binding affinities as compared to their phosphate precursors. Meta-substituted arylamide phosphonates were discovered to be antagonists of the S1P₁ and S1P₃ receptors. When administered to mice, an antagonist blocked the lymphopenia evoked by a S1P receptor agonist and caused capillary leakage in both lung and kidney.

1. Introduction

Sphingosine 1-phosphate (S1P, Figure 1) receptors (S1P_1–5) are integral membrane G protein-coupled receptors that were initially referred to as endothelial differentiation gene (EDG) receptors, EDG-1, -5, -3, -6 and -8, respectively.¹ These receptors provide control over numerous aspects of cellular physiology when activated by endogenous S1P.^2,3 Particular attention has been accorded to the role of the S1P₁ receptor in modulating the immune system since the discovery of FTY720, an agonist at the S1P_1,3,4,5 receptors (Figure 1). S1P₁ receptor agonists have been shown to inhibit the egress of T-lymphocytes from secondary lymphoid tissues, and thus are thought to direct effector T-cells away from sites of inflammation.⁴

Figure 1.

Structures of endogenous sphingosine 1-phosphate (S1P) and the S1P receptor ligands, FTY720, VPC22173 and VPC23019.

FTY720 was developed from synthetic analog studies of myriocin (ISP-1).⁵ Eventually discovered to be a pro-drug, FTY720 is activated in vivo when phosphorylated by sphingosine kinase type 2 to form FTY720-P.⁶ The biological activity of FTY720 indicated that S1P receptors are valid targets for the treatment of autoimmune disorders and allograft rejection.⁷ It is hoped that S1P receptor agonists can modulate immune system function without the toxic liabilities attendant to existing immuno-therapeutics such as calcineurin inhibitors and corticosteroids.^7–9

The success of FTY720 in human investigation has prompted synthetic efforts to provide both pharmacological tools to study S1P signaling and therapeutics.^10–15 We reported previously the synthesis of aryl-amide containing phosphates (Figure 1, VPC22173 and VPC23019) and profiled their binding affinities at S1P receptors in vitro.^14,16 Among these compounds were the first S1P₁ receptor antagonists, e.g. VPC23019.¹⁶ In an effort to discover compounds with increased resistance to phosphatase-catalyzed hydrolysis (the deactivation pathway of S1P analogs), the synthesis of the corresponding phosphonates is reported herein. Further, the synthesis of related aryl-amine and aryl-ether containing phosphonates is discussed.

To initiate this work, strategies were pursued for the efficient synthesis of chiral phosphonoserines 4a and 4b, which are non-natural amino acids used to study protein phosphorylation.¹⁷ Previous syntheses of note include Barton and Vonder-Embse’s synthesis of the fully unprotected phosphonoserine from N-Cbz-glutamic acid in 4 steps and 58% yield, involving the use of white phosphorous (P₄).¹⁸ Perich and Johns published two syntheses, the most recent in 42% yield and seven steps, from properly protected glutamic acid and using a Barton – McCombie deoxygenation.¹⁹ Finally, other methods employed include enzymatic chiral resolutions of racemic materials, ^20–22 and the induction of chirality by chiral auxiliaries.^23–25 Methods reported in this paper lead to chiral phosphonoserines with protecting groups amenable to our synthetic approach, as well as peptide synthesis, in good yield from commercially available L- or D- serine and R- or S-glycidol.

Derived from protected R- and S- phosphonoserines 4a and 4b, N-aryl-amide phosphonates 12a–f provided similar binding affinities at S1P receptors as their phosphate precursors. Synthesis of the α-fluorophosphonate 13 showed similarly potent binding compared to its corresponding phosphonate 12a. Phosphonate analogues 12c, 12d (VPC44116) and 12f, of our previously described S1P_1,3 antagonists,¹⁶ proved to retain their activity as antagonists and provided pharmacological tools for in vivo studies. While a new class of arylether phosphonates 18a, 18b and 19 were relatively weak partial agonists or inactive, the aryl-amine 26 retained similar activity to its amide precursor.

2. Results and Discussion

2.1 Chemistry

2.1.1 Synthesis of aryl-amide-phosphonates 12a–f and 13

The production of phosphonate analogues containing an amide linker region was envisaged through the condensation of chiral phosphonoserines (L-or D-2-(N-tert-butoxycarbonyl amine)-4-phosphonyl butyric acids) 4a and 4b with various substituted anilines. The initial efforts (Scheme 1) towards this protected unnatural amino acid began with the synthesis of Garner’s Aldehyde, 1, from commercially available L-serine over five steps.²⁶ A Horner-Wadsworth-Emmons olefination performed from one of two bis-phosphonates installed the α-methylene or α-fluorophosphonates in 2a or 2b, respectively.²⁷ The fluorinated bisphosphonate used to synthesize 2b was derived, as previously described by Prestwich,²⁸ from the commercially available tetraethyl methylenebisphosphonate used to arrive at 2a.

Scheme 1.

Synthesis of (3R)-4a,b – Method A: a.) i. SOCl₂, MeOH, rt, 16h ii. Boc₂O, Et₃N, CH₂Cl₂, rt, 12h iii. 2,2-dimethoxypropane, p-TsOH, CH₂Cl₂, 0 °C to rt, 2h 62% (3 steps) iv. NaBH₄, LiCl, 3:2 EtOH/THF, 0 °C to rt, 4h 89% v. DMSO, (COCl)₂, CH₂Cl₂, −78 °C, then Et₃N −78 °C to rt, 2–4 h, 97% b.) tetraethyl methylenebisphosphonate, n-BuLi, THF, −78 °C, rt, overnight, 75% (2a) or tetraethyl 2-fluoromethynebisphosphonate, n-BuLi, THF, -78 °C, rt, overnight, 31% (2b) c.) H₂, Pd/C, rt., 12h, EtOH, 99% (3a) and 88% (3b) d.) Jones reagent, acetone, 0 °C to rt., 12h then, isopropyl alcohol, celite, rt, 15min., 59% (4a) and 48% (4b).

The resulting olefins 2a and 2b were reduced by hydrogenation over Pd/C to 3a and 3b. Selective acetonide deprotection proved to be low yielding in our initial efforts;²⁹ however, this led to the use of a convenient, simultaneous deprotection and oxidation with the Jones reagent in acetone.³⁰ This un-optimized method led to the protected amino-acids (R)-4a,b in 40% and 18% yields, respectively, in 3 steps from Garner’s Aldehyde.

A second avenue (Scheme 2) led to a shorter and more efficient synthesis of both (S)- and (R)-4a. For example, (R)-(+)-glycidol was benzyl protected,³¹ and the epoxide was opened by the resultant carbanion of diethyl methylphosphonate and n-BuLi, in the presence of BF₃·OEt₂, to yield alcohol 6.³² Installation of an azide at the 3-hydroxyl position proved to be the major impediment to this approach, as has been noted by others for similar substrates.³³ Mesylate formation followed by azide substitution suffered from a tendency for elimination over a range of temperatures. Therefore, a milder method of azide formation was pursued.

Scheme 2.

Synthesis of (3R)-[or (3S)-]4 – Method B: a.) BnBr, DMF, 60% NaH, 0 °C to rt, 3h, 78% b.) CH₃PO₃Et₂, n-BuLi, BF₃·OEt₂, THF, −78 °C to rt, 3h then, NH₄Cl, 1h, 96% c.) DPPA, DIAD, 3%-polymer-bound PhPPh₂, CH₂Cl₂, 0 °C to rt, 20h, 96%, d.) Boc₂O, H₂ (balloon), 20%^w/w Lindlar’s catalyst, MeOH, rt, 24h 77% e.) H₂ (balloon), Pd/C, EtOH, rt, 24h, 94% f.) TEMPO, bis(acetoxy)iodosobenzene, NaHCO₃, 1:1 CH₃CN/H₂O, rt, 3h., 38% or RuCl₃·hydrate, NaIO₄, 3:2:2 H₂O/CH₃CN/CCl₄, rt, 3h, 76%.

Diphenylphosphoryl azide (DPPA) under Mitsunobu conditions was successful in assembling the desired azides, 7, despite reported difficulties arising from congested secondary alcohols.³⁴ After extensive isolation efforts, near quantitative yields were found via this procedure. Purification issues led to the investigation of more convenient Mitsunobu reagents to preclude the difficult separation of polar products from the complex reaction mixture (polar phosphonate 7 was nearly inseparable from both triphenyl- and tributylphosphane oxides). The use of commercially available polymer-bound triphenylphosphine yielded equivalent conversions to azide 7. With the use of three equivalents of phosphine, the desired transformation was completed in fewer than 20 hours. The inclusion of a polymer-bound phosphine reagent allowed for standard purification of the crude reaction mixture, after filtration of the resultant phosphane oxide.

A two-step reduction of 7, first in the presence of H₂, Boc₂O and Lindlar’s catalyst,³⁵ followed by H₂, Pd/C, yielded the protected amino alcohol 9 in 72% over two steps. Oxidation of the primary alcohol was performed by means of RuCl_3(cat.)/NaIO₄ conditions³⁶ (76%) to yield compound (R)-4a in 40% yield over 6 steps from optically active glycidol. (R)- and (S)-4a were further derived to the corresponding diastereomers, (R)- and (S)-4a-Phe-OMe, through a PyBOP mediated condensation with L-phenylalanine methyl ester.³⁸ This was done to ensure high enantiomeric excess of the desired carboxylic acids. These condensation reactions were high yielding and arrived at individual stereoisomers as determined by NMR.

Compounds 12a-f and 13 (Scheme 3) were synthesized by PyBOP initiated condensations³⁷ of (R)- or (S)-4a or (R)-4b with various aniline compounds 10a–d. Aqueous soluble carbodiimide (EDC methiodide) was also investigated as a condensation reagent; however, this method generally produced lower yields. The alkane hydrocarbons of unavailable alkylanilines were installed through Sonogashira couplings with 3-iodo-nitrobenzene,³⁸ followed by concomitant reduction of the nitro group and resulting triple bond. Pd couplings were also successfully performed following the amide formation with m- or p- iodoaniline to complete the desired phosphonates in a linear fashion. The compounds 11a–f were deprotected with bromotrimethylsilane followed by hydrolysis of the ensuing phosphonate silyl-oxy-esters. These conditions conveniently deprotected the N-Boc group, as well, to yield compounds 12a–f. α-Fluorophosphonate 13 was synthesized by the same protocol from 4b and 10a.

Scheme 3.

Synthesis of arylamide phosphonates 12 a–f & 13: a) PyBOP, 10 a–d, di-iso- propyl ethylamine (DIEA), CH₂Cl₂, 24–70% b.) TMSBr, CH₂Cl₂, rt, 4-6h then, 95:5 MeOH/H₂O, rt, 1–4h, 45-100%.

While the synthesis described from glycidol retained the reported efficiency up to half-gram scale, increasing material to greater than one gram proved detrimental to the formation of azide 7. Due to the demand for greater quantities of compounds 13b and 13d, further optimization of our synthesis from serine was undertaken (Scheme 4). Acetonide protected vinyl phosphonate 2 was converted to phosphonoserine 4a in two convenient steps. Compound 2, as displayed in Scheme 1, undergoes reduction within four to six hours under an atmosphere of H₂ and in the presence of 10% Pd/C (20^w/_w %). It was observed that a trace amount of the alcohol 9a was present at this time. The reaction was allowed to stir for one day at room temperature, and it was estimated by TLC that nearly half of the acetonide was hydrolyzed. Following further investigation, the use of an additional half equivalent of 10% Pd/C (10^w/_w%), added after one day of vigorous stirring, converted the remaining material to the reduced alcohol 9a in less than 72 hours.³⁹ This selective acetonide deprotection proved to be effective with greater than five grams of material and yielded more efficient conversions (>95%) than 1.5 equivalents of p-toluenesulfonic acid in ethanol (65–75 %). The alcohol was then converted to 4a with RuCl₃/NaIO₄ conditions, as previously described.

Scheme 4.

Efficient synthesis of 4a from 2a: a.) H₂, 10% Pd/C (20 ^w/_w% followed by 10 ^w/_w%), EtOH, rt, 3d, >95% b.) RuCl₃·H₂O_x, NaIO₄, 2:2:3 CCl₄/CH₃CN/H₂O, rt, 1–3h, 79%.

2.1.2 Synthesis of aryl ether phosphonates 18a, 18b, and 19

Using glycidol as an alternative starting material to serine led conveniently to phenolic ether compounds (Scheme 5). The synthesis began with a Mitsunobu condensation between p-octylphenol and glycidol. Chiral epoxides 14 were then opened, as previously described, to alcohols 15. For these substrates, DPPA/Mitsunobu conditions yielded products with sufficiently disparate polarities from tributylphosphane oxide. The azides, 16, were reduced and deprotected as described above to give enantiomers 18a and 18b. Compound 15a was deprotected to form 19 to confirm the overall effects of an amine at the 3-position of 18b.

Scheme 5.

Synthesis of arylether phosphonate analogues 18 a–b and 19: a.) DIAD, PPh₃, THF, 0 °C to rt, overnight, 80–84% b.) CH₃PO₃Et₂, n-BuLi, BF₃·OEt₂, THF, −78 °C to rt, 3h then, NH₄Cl, 1h, 72–87% c.) DPPA, DIAD, PPh₃, CH₂Cl₂, 0 °C to rt, 20h, 67–98% d.) H₂ (balloon), Pd/C, EtOH, formic acid cat., rt, 3.5h, quantitative e.) TMSBr, CH₂Cl₂, rt, 4–6h then, 95:5 MeOH/H₂O, rt, 2–4h, 79–100%.

2.1.3 Synthesis of aryl amine phosphonate 26

Secondary amine 26 was synthesized to view the effects of reducing the amide bond in 12d (Scheme 6) while retaining a functional group capable of donating a hydrogen bond. After activation of p-octylaniline by mono-tosyl protection, the amine was condensed under Mitsunobu conditions to form epoxide 21. Consecutive nucleophilic ring opening, azide formation with DPPA/Mitsunobu conditions, and reduction yielded amine 24. Deprotection of the N-tosyl group proved difficult. Sluggish reaction times and low yields were discovered with the planned Mg/MeOH deprotection,⁴¹ and dissolving metal conditions were used. Solid sodium in NH_3(l) unmasked the desired secondary amine in an un-optimized 26% yield. Compound 25 was deprotected with TMSBr to yield the ammonium bromide salt 26 .

Scheme 6.

Synthesis of arylamine phosphonate analogue 26: a.) DIAD, PPh₃, THF, 0 °C to rt, overnight, 69% b.) CH₃PO₃Et₂, n-BuLi, BF₃·OEt₂, THF, –78 °C to rt, 2h then, NH₄Cl, 2h, 97% c.) DPPA, DIAD, PPh₃, CH₂Cl₂, 0 °C to rt, 20h, 85% (containing 5% OPPh₃) d.) H₂ (balloon), 20%^w/^w Pd(OH)₂, 20:1 MeOH/conc. HCl, 1h, 100% e.)Na_(s), NH_3(l), –78 °C, 5 min.then EtOH, 25% (recovered 28% starting material) f.) TMSBr, CH₂Cl₂, rt, 4–6h then, 95:5 MeOH/H₂O, 4h, rt, 95%.

2.2 Biological Evaluation

2.2.1 [γ³⁵S]-GTP Binding Assay

γ³⁵S-GTP dependant receptor binding activity (Table 1) was determined in vitro for S1P, FTY720-P, and all final compounds, as previously communicated.¹⁶ Briefly, the expression of individual human S1P receptors and individual G protein subunits was forced in HEK293T cells. The membrane bound G protein α subunits yielded data by binding the labeled, non-hydrolyzable [γ³⁵S]-GTP when activated by an extracellular ligand. Following our discovery of the S1P_1,3 antagonist VPC23019 (B), the meta-substituted analogues were analyzed for their ability to antagonize S1P’s endogenous activity in the [γ³⁵S]-GTP assays. The effects on S1P’s endogenous binding constant were determined as previously discussed.¹⁶

Table 1.

[γ-³⁵S]-GTP binding assay in HEK293T cells over-expressed with subtype specific S1P receptors.^a

			Receptors
			S1P1		S1P2		S1P3		S1P4		S1P5
Compound	linker	Head Group	EC50a	Emax	EC50	Emax	EC50	Emax	EC50	Emax	EC50	Emax
FTY720-P	H2CCH2	phosphate	1.3	1.00	NA	0.00	0.1	0.50	4.0	0.90	4.0	0.56
VPC22173	amide	phosphate	58.0	1.10	NA	0.00	450.0	0.35	500.0	1.10	52.0	0.79
VPC2301S	amide	phosphate	NA	0.00	NA	0.00	NA	0.00	120.0	1.13	480.0	0.57
12 a	amide	phosphonate	3.6	0.96	270.0	0.50	43.0	0.88	230.0	0.67	30.0	0.77
b	amide	phosphonate	27.0	0.93	NA	0.00	270.0	0.38	2,300.0	0.80	76.0	0.64
c	amide	phosphonate	NA	0.00	n/a	n/a	NA	0.00	n/a	n/a	n/a	n/a
d	amide	phosphonate	NA	0.00	NA	0.00	NA	0.00	6,100.0	1.42	33.0	0.73
6	amide	phosphonate	1200.00	NA	NA	0.00	15000	0.30	n/a	n/a	n/a	n/a
f	amide	phosphonate	NA	0.00	NA	0.00	NA	0.00	n/a	n/a	n/a	n/a
13	amide	fluorophosphonate	2.1	0.99	490.0	0.47	23.0	0.96	170.0	0.68	19.0	0.77
18 a	ether	phosphonate	68.0	0.70	NA	0.00	NA	0.00	147.0	0.71	31.0	0.41
b	ether	phosphonate	140.0	0.64	530.0	0.50	100.0	0.53	150.0	0.90	47.0	0.50
19	ether	phosphonate	33.0	0.43	NA	0.00	NA	0.00	440,000.0	0.45	NA	0.00
26	amine	phosphonate	30.0	0.56	NA	0.00	NA	0.00	340.0	1.18	41.0	0.61

^aEC₅₀s are nM and determined by the mean of at least three experiments.

Emax values are normalized to the maximal activation of endogenous S1P, at each receptor.

NA = no activation

n/a = not available

Para substituted phosphonates 12a, 12b (VPC44152), 12e, 18a, 18b, and 19 showed various activities as agonists. Phosphonate 12b (VPC44152) was twice as potent as corresponding phosphate A (VPC22173) at S1P₁ and S1P₃, while less potent at S1P₄ and S1P₅. Phosphonate 12a gained activity across all receptors with comparison to A and displayed similar potency to FTY720-P and S1P at S1P₁. The replacement of the amide linkage with an ether resulted in the loss of activity, at S1P₁ and S1P₃, for 18a compared to 12b, implicating the importance of available hydrogen-bond donation alpha to the phenyl ring. Interestingly, epimer 18b was considerably less potent than 18a at S1P₁ but displayed modest activity at all five S1P receptors. γ-Hydroxyphosphonate 19 was less potent than 18a at S1P₁ and functionally inactive at S1P_2–5, which is consistent with the two point binding model for S1P receptor interaction.⁴² Compared with our previously described phosphate agonist A, phosphonates 12a, 12b (VPC44152), and 12e retained similar potency and efficacy. Meta-substituted compounds 12c, 12d (VPC44116), and 12f showed no agonist activity at S1P₁ and S1P₃ receptors; rather, meta substituted compounds displayed antagonists activity against S1P binding to the S1P₁ and S1P₃ receptors.

To characterize these compounds, Schild regressions were performed as described in earlier work.¹⁶ These experiments revealed arylamides 12d and 12f as potent antagonists at the S1P₁ and S1P₃ receptors. Arylamine 26 displayed antagonist activity at both receptors with a preference for S1P₃. The most promising antagonist, 12d (VPC44116), was compared with its phosphate precursor VPC23019. VPC23019 and VPC44116 were nearly indistinguishable in their affinity for the S1P₁ and S1P₃ receptors (K_i values of about 30nM and 300nM, respectively). This was described in more detail by radioligand displacement experiments, described previously,¹⁶ revealing IC₅₀s for the phosphate and phosphonate to be 31 nM and 72 nM, respectively (not shown).

We have demonstrated previously that VPC44116 opposes the protective effect of FTY720 in a mouse model of acute renal injury.⁴³ To characterize this compound further, we injected mice with doses up to 45 mg/kg body weight and measured blood lymphocytes. Lymphopenia (a decrease in circulating lymphocytes below the normal range) is a convenient index of S1P₁ receptor agonist action. The quintessential S1P agonist, FTY720, has been proposed to operate as a functional antagonist through receptor desensitization mechanisms⁴⁴ – a hypothesis that suggests a direct receptor antagonist would behave likewise. Nevertheless, no significant change in circulating lymphocyte numbers was observed at any dose of VPC44116 tested (not shown). However, 12d (VPC44116 (meta)) blocked the lymphopenia evoked by its positional isomer, the agonist 12b (VPC44152 (para)) (Figure 2). A S1P receptor antagonist similar to VPC44116 but containing a hexyl (vs. octyl in VPC44116) group caused vascular leakage in lung when administered to mice.⁴⁵ To learn whether VPC44116 behaved similarly, we injected VPC44116 into mice followed by Evans blue dye as described previously.⁴³ After sacrifice, we found extravasation of the dye into lung and kidney, but not into heart, liver, testes or intestine (Figure 3).

Figure 2.

The lymphopenia evoked by the S1P agonist, 12b (VPC44152) is blocked by co-administration of the S1P receptor antagonist 12d (VPC44116). Groups of 3 C57BL/6 x sv129/J mice injected with vehicle (2% hydroxypropyl β-cyclodextrin), VPC44116 (22 mg/kg) and/or VPC44152 (18 mg/kg). After 16 hours, blood was drawn from the orbital sinuses and lymphocytes were measure with a Hemavet blood analyzer. Data are presented as mean ± S.E.

Figure 3.

Effect of VPC44116 on vascular permeability. C57BL/6 mice were treated with vehicle (2% hydroxypropyl β-cyclodextrin) or VPC44116 (25 mg/kg) two hours prior to injection 2% Evans Blue dye (EBD) (20 mg/kg) into the jugular vein 30 min. before harvesting tissues. EBD was extracted into formamide, measured in a spectrometer and the amount of extravasated EBD in tissues was calculated from a standard curve. Values are means ± SE; n = 4 for each group. **P < 0.05, **P < 0.01 compared with vehicle treatment.

3. Conclusion

The synthetic methods described provide entry to multiple oxidation states and/or orthogonal protecting groups of phosphonoserines, including an isoelectric α-fluorophosphonate. This is accomplished from two convenient materials of the current chiral pool, L- or D- serine (eight steps and 39% yield) and R- or S- glycidol (six steps and 40% yield). Optically active agonists and antagonists of the S1P receptors are described. Replacing the phosphorous-oxygen bond with a phosphorous-carbon bond provides bioactive agents with putative resistance to degradative phosphatase activity. The phosphonate-containing antagonist, 12d (VPC44116), opposed the lymphopenia evoked by its agonist isomer, 12b (VPC44152), but did not affect numbers of circulating lymphocytes when injected alone. However, injection of VPC44116 alone caused capillary leakage in lung and kidney.

4. Experimental

4.1. General Experimental

All reactions were performed under an inert atmosphere using flame dried glassware. Reaction solvents methylene chloride, diethyl ether, tetrahydrofuran, and toluene were obtained from OptiDry canisters (<50 ppm H₂O, Fisher Scientific) and passed through an activated alumina (activity I) column, directly into the reaction flask when possible. Dimethylformamide was obtained from an OptiDry canister without further drying prior to use. All other solvents were used as obtained. All commercially available reagents were purchased from either Aldrich (Milwaukee, WI), Sigma (St. Louis, MO), Acros (Pittsburg, PA), or Advanced Chem Tech (Louisville, KY) and used as obtained unless otherwise stated. The reactions were monitored by analytical thin layered chromatography using Merck silica gel F-254 pre-coated aluminum-backed plates. R_f values refer to column chromatography eluent, unless otherwise noted. When not reported, R_f value ≈ 0.00. Silicycle Ultra Pure Silica Gel (230–400 mesh) or Fisher Scientific Silica Gel 60 Sorbent (230–400 mesh) was used for all normal phase chromatography. All yields refer to chromatographically and spectroscopically pure compounds, unless otherwise indicated.

Optical rotations were measured on a Perkin-Elmer model 343 polarimeter with a sodium lamp at 23 ± 2 °C in the stated solvent; [α]_D values are given in 10⁻¹ deg cm² g⁻¹. ¹H and ¹³C NMR spectra were recorded on UnityInova 300 (75) and 500 (125) MHz spectrometers (Varian). Chemical shifts are reported in δ (ppm) units using ¹H (residual) and ¹³C signals from CDCl₃ as an internal standard (7.26 and 77.23 ppm, respectively) unless otherwise specified. Elemental analysis was performed by Atlantic Microlab, Inc. (Norcross, GA) for C, H, and N. Elemental analyses were run in duplicate after thorough drying, before submission and when obtained by the vendor. Low Resolution Electrospray Ionization (ESI) was performed at the University of Virginia Mass Spectometry Laboratory. High-Resolution Mass Spectrometry (HRMS) was performed at the Mass Spectrometry Laboratory at University of Illinois Urbana-Champaign (Micromass Q-T of Ultima).

4.1.1. 4-formyl-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (1)

L-serine (5.00 g, 0.048 mol) was dissolved in 100 mL of methanol and cooled to <0 °C (brine/ice). Thionyl chloride (20.8 mL, 0.286 mol) was added slowly by syringe. The mixture was stirred overnight and then concentrated and co-evaporated with ether multiple times to eliminate excess thionyl chloride and provide the desired methyl ester that was shown to be >95% pure by ¹H NMR. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 4.07 (t, J = 4.0 Hz, 1H), 3.98 (d, J = 4.0 Hz, 2H), 3.83 (s, 3H) ppm.

The amino ester was reconstituted in CH₂Cl₂ (100 mL) and triethylamine (16.6 mL, 0.119 mol) was added dropwise at 0 °C. To this stirring solution was added di-tert-butyl dicarbonate (11.420 g, 0.052 mol) in one portion. The reaction was stirred until the starting material was consumed, as determined by TLC (1:1 EtOAc/Hex). The reaction mixture was concentrated and dissolved in EtOAc (50 mL) then washed with saturated NaHCO_3(aq) (3×25 mL) followed by brine (3×25 mL). The organic layer was dried over Na₂SO_4(s), filtered and concentrated. ¹H NMR (300 MHz, CDCl₃, 23°, δ): 5.54 (brd, 1H), 4.35 (m, 1H), 3.89 (ddd, J = 13.8, 11.2, and 3.7 Hz, 2H), 3.76 (s, 3H), 2.76 (bs, 1H), 1.43 (s, 9H) ppm.

The crude oil was dissolved in acetone (120 mL) and 2,2-dimethoxypropane (87 mL, 15 eq.). The solution was stirred at room temperature and BF₃·OEt₂ (1.2 mL, 9.52 mmol) was added. The reaction mixture turned a yellow-orange hue and was stirred for 2.5 hours. When the reaction was complete, by TLC analysis, the solution was treated with 99% Et₃N (1.2 mL) and the solvent was removed. The brown oil was then partitioned between diethyl ether and saturated NaHCO_{3 (aq)}. The aqueous layer was extracted with diethyl ether (4×25 mL) and the organic layers were combined, dried (Na₂SO₄), and concentrated to a yellow oil (> 90% pure by ¹H NMR). R_{f(1:1 EtOAc/Hexanes)} = 0.73. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): Major rotamer = 4.37 (dd, J = 7.0, 3.1 Hz, 1H), 4.13 (dt, J = 9.23, 7.0Hz, 2H), 3.74 (S, 3H), 1.52 (s, 3H), 1.48 (s, 3H), 1.40 (s, 9H) ppm; Minor rotamer = 4.48 (dd, J = 6.6, 2.6 Hz, 1H), 4.04 (dt, J = 7.3, 2.9 Hz, 2H), 3.74 (s, 3H), 1.66 (s, 3H), 1.62 (s, 3H) 1.48 (s, 9H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): Major rotamer = 151.38, 80.51, 66.45, 59.46, 52.49, 28.46, 27.60, 25.15, 24.57 ppm; Minor rotamer = 151.38, 80.51, 66.20, 59.38, 52.61, 28.54, 27.60, 26.21, 25.35 ppm.

A mixture of NaBH₄ (2.247 g, 59.08 mmol) and LiCl (2.505 g, 59.08 mmol) was prepared in EtOH (42 mL), at 0 °C. A solution of the purified acetonide (7.659 g, 29.54 mmol) dissolved in THF (30 mL) was then added dropwise to the reaction mixture. The mixture was warmed to room temperature and stirred for four hours. After four hours the precipitate was filtered over celite and washed with EtOH. The filtrate was then concentrated and reconstituted in H₂O (50 mL) and EtOAc (50 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3×25 mL). The organic layers were combined and washed with brine (2×25 mL), dried (Na₂SO_{4 (s)}), concentrated, and the crude oil was purified by column chromatography (25–50% EtOAc/Hexanes) to give 6.101 g (89%) of the alcohol as a white solid. Rf_{(1:1 EtOAc/Hexanes)} = 0.86. ¹H NMR (300 MHz, CDCl₃, 35 °C, δ) = 4.07 (m, 2H), 3.75 (s, 1H), 3.68 (m, 2H), 1.52 (s, 6H), 1.49 (s, 9H) ppm.

To a stirring solution of 2.0 M oxalyl chloride, (COCl)₂, (11.78 mL, 23.56 mmol) at −78 °C was added drop-wise a solution of DMSO (2.1 mL, 23.56 mmol) in CH₂Cl₂ (33 mL). This solution was allowed to stir at −78 °C for 15 minutes before a solution of the primary alcohol in CH₂Cl₂ was added drop-wise. The solution was stirred for 35 minutes before Et₃N was added in a drop-wise manner at −78 °C. Following the addition of base, the reaction mixture was allowed to warm to 0 °C and the reaction was quenched with saturated NH₄Cl_(aq). The mixture was separated and the organic layer was washed with sat. NaHCO_3(aq) (2×25 mL) followed by brine (2×25 mL). The organic layer was dried (MgSO₄) and concentrated to a clear oil which was used immediately in the following reaction without further purification. R_{f(1:3 EtOAc/Hexanes)} = 0.35. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ) = Major rotamer: 9.35 (d, 1H, J = 2.4 Hz), 4.02 (m, 1H), 3.88 (m, 2H), 1.43 (s, 3H), 1.34 (s, 3H), 1.22 (9H) ppm; and Minor rotamer: 9.38 (m, 1H), 4.13 (m, 1H), 3.88 (m, 2H), 1.39 (s, 3H), 1.33–1.27 (m, 12H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ) = Major rotamer: 198.84, 151.07, 94.72, 80.55, 64.53, 63.58, 27.99, 25.51, 23.54 ppm; and Minor rotamer: 199.02, 152.29, 94.05, 80.92, 64.60, 63.16, 28.03, 26.45, 24.47 ppm.

4.1.2.1 4-[2-(diethoxyphosphoryl)vinyl]-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester ((3R)-2a)

To tetraethyl methylenebisphosphonate (4.28 mL, 17.278 mmol) in THF (40 mL) at −78 °C was added 2.5 M n-BuLi in Hexanes (6.28 mL, 15.707 mmol). After 15 minutes of stirring at low temperature, aldehyde 1 (3.601 g, 15.707 mmol) was added in THF (40 mL) and the reaction was allowed to warm to room temperature with continued stirring overnight. The reaction mixture was concentrated to 1 to 2 mLs and purified by column chromatography (500 mL SiO₂, 5% MeOH in CHCl₃) to yield 5.440 g (95%, two steps) of clear oil. R_f = 0.65. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ) = 6.63 (dt, 1H, J = 5.9, 17.8 Hz), 5.72 (dt, 1H, J = 15.8, 17.8 Hz), 4.40 (dt, 1H), 4.04 (m, 5H), 3.77 (m, 1H), 1.59 (m, 3H), 1.42 (m, 12H), 1.25 (dt, 6H, J = 1.5, 7.0 Hz) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 157.4, 150.3, 127.3, 119.7, 117.2, 92.6, 67.5, 62.1, 59.6 (d, J = 22.7 Hz), 28.5, 26.7, 25.7, and 16.6 (d, J = 8.1 Hz) ppm. [α]²³_D = −64.1° (c = 1.00, MeOH). HRMS (ES+) calculated m/z = 364.1889, experimental m/z = 364.1893.

4.1.2.2 4-[2-(diethoxyphosphoryl)vinyl]-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester ((3S)-2a)

The above procedure for the synthesis of (3R)-2a was employed using the epimer of 1 (1.146 g, 5.00 mmol) in THF (15 mL), 2.5M n-BuLi in Hexanes (2.00 mL, 5.00 mmol) and tetraethyl methylenebisphosphonate (1.36 mL, 5.5 mmol) yielded 1.728 g (>95%, two steps) of clear oil. Rf, ¹H and ¹³C NMR were consistent with data reported for (3R)-2a. [α]²³_D = +65.9° (c = 1.03, MeOH). HRMS (ES+) calculated m/z = 364.1889, experimental m/z = 364.1893.

4.1.3 4-[2-(diethoxyphosphoryl)-2-fluorovinyl]-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester (2b)

The above procedure for synthesis of 2a was employed using 1 (229 mg, 1.00 mmol) in THF (2.5 mL), tetraethyl 2-fluoromethylene-bisphosphonate (337 mg, 1.10 mmol) in 2.5 mL of THF, and 2.5M n-BuLi in Hexanes (0.4 mL, 1.00 mmol). The reaction yielded 119 mg (31%) of 2b as a clear liquid. Rf_(EtOAc) = 0.36. ¹H NMR (300 MHz, CDCl₃, 23°C, δ): 5.92 (dt, J = 37.6, 7.9 Hz, 1H), 3.71 (m, 1H), 4.10 (m, 5H), 3.72 (m, 1H), 1.41 (m, 6H), 1.39 (m, 9H), 1.29 (m, 6H) ppm.

4.1.4. General Procedure I: Hydrogenation /or Hydrogenolysis (3a)

To a solution of phosphonate 2a (845 mg, 2.33 mmol) dissolved in 25 mL of anhydrous EtOH was added 10% Pd/C (20% ^w/_w). The reaction flask was repeatedly filled with H₂ (balloon) and evacuated. Following three to five repetitions, the reaction was allowed to stir under an H₂ atmosphere for 4 h. The mixture was filtered over celite and washed with EtOH. The filtrate, which required no further purification, was concentrated to 844 mg (99%) of clear oil. Rf_(EtOAc) = 0.29. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 4.09 (dq, J = 3.3, 7.0 Hz, 4H), 3.94 (m, 1H), 3.68 (m, 2H), 1.80 (m, 4H), 1.59 (s, 3H) 1.54 (s, 3H), and 1.32 (t, J = 7.0 Hz, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 156.4, 92.5, 89.8, 65.0, 61.9 (d, J = 6.6Hz), 31.2, 28.6, 23.4 (d, J = 20.1Hz), 21.6 (d, J = 20.1 Hz), 16.7 (d, J = 6.0Hz) ppm.

4.1.5. 4-[2-(diethoxyphosphoryl)-2-fluoroethyl]-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (3b)

General Procedure I was performed on 2b (141 mg, 0.37 mmol) to give 125 mg (88%) of 3b as a clear oil. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 5.24 (d, J = 14.3 Hz, 1H), 4.76 (m, 1H), 4.14 (m, 4H), 4.03 (m, 1H), 3.84 (dd, J = 8.1 Hz, 2H), 3.60 (m, 1H), 2.17 (m, 2H), 1.50 (m, 3H), 1.40 (m, 12H), 1.28 (m, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 1.52.96, 68.24, 66.42, 63.34, 55.68, 28.44, 23.20, 16.55 ppm.

4.1.6. 2-tert-butoxycarbonylamino-4-(diethoxyphosphoryl)butyric acid ((2R)-4a)

In a stirring solution of 3a (844 mg, 2.31 mmol) and 5 mL of acetone, at 0 °C was added Jones’ reagent (1.73 mL, 4.62 mmol). The reaction mixture was allowed to warm to room temperature and the stirring was continued for 12 hours. After this time the mixture was transferred to a larger flask then celite and isopropyl alcohol were added. The mixture was stirred for 15 minutes and the precipitate was filtered, washed with acetone, and made alkaline by the addition of sat. NaHCO_{3 (aq)}. The solution was concentrated to remove organic solvents and washed with EtOAc (3 x 25 mL). The aqueous layer was acidified to a pH ~3 by the addition of solid citric acid and extracted with CH₂Cl₂ (5 x 25 mL). The combined extracts were washed with brine (3 x 15 mL) and dried over solid MgSO₄. The solvent was concentrated to 460 mg (59%) of a white solid, which could be recrystallized from Et₂O/Hexanes. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 10.41 (brs, 1H), 5.38 (d, J = 7.5 Hz, 1H), 4.32 (m, 1H), 4.10 (t, J = 7.3 Hz, 4H), 1.95 (m, 4H), 1.43 (s, 9H), 1.31 (dt, J = 7.0, 1.5 Hz, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23°C, δ): 190.58, 157.35, 62.47, 53.57, 28.49, 25.81, 22.44, 16.50 ppm.

4.1.7. 2-tert-butoxycarbonylamino-4-(diethoxyphosphoryl)-4-fluorobutyric acid ((2R)-4b)

Similar procedures for the synthesis of (2R)-4a were followed using 3b (125 mg, 0.326 mmol), 1 mL of acetone, and Jones’ reagent (0.25 mL, 0.625 mmol) to yield 40 mg (48%) of (2R)-4b as a white solid. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 5.90 (m, 1H), 5.50 (m, 2H), 5.00 (dt, J = 46.4, 8.8 Hz, 1H), 4.31 (m, 1H), 4.17 (m, 4H), 2.38 (m, 2H), 1.41 (s, 9H), 1.32 (dt, J = 7.0, 2.2 Hz, 6H) ppm.

4.1.8.1 2-benzyloxymethyloxirane ((2S)-5)

To a stirring mixture of BnBr (0.72 mL, 6 mmol), 60% NaH (193 mg, 4.8 mmol) suspended in mineral oil, and 5 mL of DMF was added R-glycidol (0.26 mL, 4 mmol) dissolved in 3 mL of DMF, dropwise and at 0 °C. The solution was added via syringe over 30 minutes at which time the reaction was allowed to stir for an additional 3 hours while warming to room temperature. The crude material was diluted with 50 mL of EtOAc and washed with NH₄Cl (3×50 mL), NaHCO₃ (3×50 mL), LiBr (3×25 mL) and brine (3×50 mL). The organic layer was dried over Na₂SO₄, filtered and concentrated to clear oil. The crude oil was purified by flash chromatography (~200 mL SiO₂, 1:4 EtOAc/ Hexanes) to yield 515 mg (78%) of clear oil. R_{f(1:4 EtOAc/Hex.)} = 0.31. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.31 (m, 5H), 4.56 (dd, J = 18.7, 12.1 Hz, 2H), 3.74 (dd, J = 11.4, 2.9 Hz, 1H), 3.39 (dd, J = 11.4, 5.9 Hz, 1H), 3.14 (dq, J = 2.9, 0.9 Hz, 1H), 2.73 (t, J = 4.6 Hz, 1H), 2.57 (dd, J = 5.1, 2.6 Hz, 1H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 137.83, 128.21, 128.19, 127.49, 72.96, 70.65, 50.58, 43.85 ppm.

4.1.8.2 2-benzyloxymethyloxirane ((2R)-5)

The above procedures for the formation of (2S)-5 were used to form 1.232 g (75%) of the title compound from S-glycidol (0.66 mL, 10.0 mmol). Rf_{(1:4 EtOAc/Hex.)} = 0.31. ¹H and ¹³C NMR data were consistent with (2S)-5.

4.1.9.1 General Procedure II: Nucleophilic Epoxide Opening ((3S)-6)

To a solution of diethyl methylphosphonate (2.04 mL, 14.1 mmol) dissolved in THF (14 mL), stirring at −78 °C, was added 2.5M n-BuLi (5.65 mL, 14.1 mmol) dropwise. The mixture was stirred for 15 minutes at −78 °C and (3S)-5 (773 mg, 4.71 mmol) dissolved in THF (2.4 mL) followed by BF₃·OEt₂ (2.32 mL, 18.8 mmol) were added dropwise at −78 °C. Stirring was continued at −78 °C for two additional hours before the reaction was quenched by the dropwise addition of NH₄Cl_(aq) (~2 mL). The mixture was allowed to warm to room temperature overnight, concentrated to a yellow oil, and purified by column chromatography (~300 mL of SiO₂, 1:1 acetone/chloroform) to give 1.424 g (96%) of a faintly yellowed liquid. Rf_{(1:1 acetone/chloroform)} = 0.39. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.34 (m, 5H), 4.55 (s, 2H), 4.10 (m, 4H), 3.85 (m, 1H), 3.49 (dd, J = 9.5, 3.3 Hz, 1H), 3.38 (dd, J = 9.7, 7.3 Hz, 1H), 1.85 (m, 4H), 1.31 (t, J = 7.0 Hz, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 137.74, 127.81, 127.12, 127.07, 127.00, 73.63, 72.69, 69.20 (d, J = 16.6 Hz), 60.99 (d, J = 6.6 Hz), 26.12 (d, J = 4.5 Hz), 22.03, 20.15, 15.93 (d, J = 6.0 Hz) ppm. [α]²³_D = −11.9° (c = 1.13, MeOH).

4.1.9.2 (4-benzyloxy-3-hydroxybutyl)phosphonic acid diethyl ester ((3R)-6)

Compound (2R)-5 (1.232 g, 7.50 mmol) was converted to (3R)-6 as in the above reaction to yield 1.311 g (55%) of the title compound as a faintly yellowed liquid. _{Rf(1:1 acetone/chloroform)} = 0.39. See (3S)-6 for corresponding ¹H and ¹³C NMR data. [α]²³_D = +10.4° (c = 1.06, MeOH).

4.1.10.1 (3-azido-4-benzyloxybutyl)phosphonic acid diethyl ester ((3R)-7)

To (3S)-6 (0.992 g, 3.136 mmol) dissolved in CH₂Cl₂ (4 mL) at 0 °C was added polymer-bound- Ph₃P (3.1 g, ~9.3 mmol), followed by DPPA (0.74 mL, 3.450 mmol) then DIAD (0.68 mL, 3.450 mmol) via syringe. The reaction mixture was allowed to warm to room temperature and was stirred overnight. By morning no starting material was present by TLC analysis. The reaction mixture was filtered through celite, which was rinsed with methanol, and the filtrate was concentrated to about 2 g of crude yellow oil. Further purification was acheived by column chromatography (~250 mL of SiO₂, 1:9 acetone/chloroform) to yield 952 mg (96%) of clear oil. Rf_{(1:9 acetone/chloroform)} = 0.31. ¹H NMR (300 MHz, CDCl₃, 23 °C,δ): 7.34 (m, 5H), 4.56 (s, 2H), 4.09 (m, 4H), 3.56 (m, 3H), 1.80 (m, 4H), 1.32 (dt, J = 7.0, 1.1 Hz, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 137.78, 128.62, 128.51, 127.98, 127.76, 73.54, 72.58, 61.87, 24.37, 23.33, 21.44, 16.61 (d, J = 6.1 Hz) ppm.

4.1.10.2 (3-azido-4-benzyloxybutyl)phosphonic acid diethyl ester ((3S)-7)

Compound (3R)-6 (741 mg, 2.342 mmol) was converted to (3S)-7 as the above reaction to yield 585 mg (73% isolated yield) of the title compound. Rf_{(1:9 acetone/chloroform)} = 0.31. See (3S)-7 for corresponding ¹H and ¹³C NMR data.

4.1.11.1 (4-benzyloxy-3-tert-butoxycarbonylaminobutyl)phosphonic acid diethyl ester ((3R)-8)

Compound (3R)-7 (1.412 g, 4.137 mmol) was stirred in 32 mL of methanol and Boc₂O (0.993 g, 4.551 mmol) followed by Lindlar’s catalyst (285 mg, 20% by weight) were added. The reaction mixture was stirred vigorously and a balloon of H_2(g) was affixed. The apparatus was purged numerous times by cycling between vacuum (5×5 min) and H_2(g). Finally H₂ atmosphere was applied and maintained for 24h at room temperature. At this time no starting material was visible by TLC analysis and the reaction mixture was filtered through Celite 545 with the aid of methanol. The solvent was condensed and the resultant clear oil was purified by column chromatography (~150mL of SiO₂, 1:9 acetone/chloroform) to yield 1.324 g (77%) of clear oil. Rf_{(5% MeOH in CHCl3)} = 0.35. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.30 (m, 5H), 4.94 (d, J = 8.5 Hz, 1H), 4.48 (d, J = 4.2 Hz, 2H), 4.06 (m, 4H), 3.74 (m, 1H), 3.45 (d, J = 3.5 Hz, 2H), 1.81 (m, 4H), 1.42 (s, 9H), 1.29 (t, J = 6.9 Hz, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 155.63, 137.95, 128.38, 128.22, 127.70, 127.59, 79.25, 73.15, 71.73, 61.54 (d, J = 6.8 Hz), 50.65 (d, J = 19.3 Hz), 28.35, 25.33, 23.34, 21.46, 16.45 (d, J = 6.8 Hz) ppm.

4.1.11.2 (4-benzyloxy-3-tert-butoxycarbonylaminobutyl)phosphonic acid diethyl ester ((3S)-8)

Compound (3S)-7 was converted to (3S)-8 by the above method to yield 605 mg of a clear oil, which was >95% pure by NMR and was carried on to reaction 4.1.12.2 without further purification. Rf_{(5% MeOH in CHCl3)} = 0.35. See (3S)-8 for corresponding ¹H and ¹³C NMR data.

4.1.12.1 (3-tert-butoxycarbonylamino-4-hydroxybutyl)phosphonic acid diethyl ester ((3R)-9)

General Procedure I was utilized for the hydrogenolysis of compound (3R)-8 (1.201 g, 2.891 mmol) to give 2.714 g (94%) of (3R)-9 as a clear oil. No further purification was necessary. Rf_{(5% MeOH in CHCl3)} = 0.09. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 5.33 (d, J = 6.9 Hz), 4.00 (m, 5H), 3.52 (m, 2H), 1.69 (brm, 5H), 1.34 (s, 9H), 1.23 (t, J = 6.9 Hz, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 156.20, 79.25, 64.31, 61.76, 57.30, 52.74 (d, J_P = 19.3 Hz), 33.31 (d, J_P = 15.5 Hz), 28.44, 24.32, 23.17, 16.46 (d, J_P = 5.8 Hz) ppm. [α]²³_D = −10.8° (c ≈ 1.00, MeOH).

4.1.12.2 (3-tert-butoxycarbonylamino-4-hydroxybutyl)phosphonic acid diethyl ester ((3S)-9)

The crude material from (3S)-8 (605 mg, 1.4 mmol) was converted to (3S)-9 by General Procedure I. The resultant oil was purified by flash chromatography (100 mL SiO₂, 5% methanol in chloroform) to yield 347 mg (72% over two steps) as clear oil. Rf_{(5% MeOH in CHCl3)} = 0.09. See (3R)-9 for corresponding ¹H and ¹³C NMR data. [α]²³_D = +10.4° (c = 1.00, MeOH).

4.1.12.3 (3-tert-butoxycarbonylamino-4-hydroxybutyl)-phosphonic acid diethyl ester ((3R)-9) from (3R)-2a

To a solution of compound 2a (5.211 g, 14.340 mmol) in EtOH (145 mL) was added 10% Pd/C (2.8 g, 20% ^w_/w) [caution: Pd/C may spark on contact with ground glass joint; funnel and joint were rinsed with EtOH prior to attaching ground glass apparatus.] The round-bottomed flask was affixed with a three-way adapter w/stopcock. The adapter was fitted with a balloon of H₂ and a vacuum hose. The system was purged of air and filled with H₂ repeatedly in five minute increments. After 3 cycles the vacuum was removed and mixture was opened to H₂ atmosphere. After 1.5 days the reaction had preceded ~60% by TLC and a second portion of Pd/C (1.4 g, 10% ^w_/w) was added as well as more EtOH (50 mL). Following another 1.5 days the reaction had progressed to one spot by TLC analysis. The mixture was filtered over celite and washed with MeOH (4×50 mL). The solvent was evaporated and NMR analysis showed the resultant clear oil (4.72 g, 14.34 mmol) to be >95% pure. ¹H and ¹³C NMR are consistent with data reported for 9a from 8a. [α]²³_D = −10.2° (c = 1.00, MeOH). HRMS (ES+) calculated m/z = 326.1733, experimental m/z = 326.1737.

4.1.12.3 (3-tert-butoxycarbonylamino-4-hydroxybutyl-phosphonic acid diethyl ester ((3S)-9) from (3S)-2a

The above procedure was repeated with (3S)-2a (1 g, 2.75 mmol), 10% Pd/C (1.5 g then 0.75 g) in EtOH (75 mL then 25 mL) to yield 0.905 g ( > 95%) of clear oil. The physical data was consistent with that of compound (3S)-9 derived from (3S)-8. HRMS (ES+) calculated m/z = 326.1733, experimental m/z = 326.1729.

4.1.13.1 TEMPO Oxidation: ((2R)-4a)

To a mixture of (3R)-9 (181 mg, 0.72 mmol) and iodosobenzenediacetate (430 mg, 1.335 mmol) and NaHCO₃ (1.44 mmol) stirring in 22 mL of 1:1 MeCN/H₂O was added 2,2,6,6-tetramethyl-1-piperidinyloxy (20 mg, 0.128 mmol). The mixture was stirred vigorously at room temperature for three hours then diluted with 10 mL of chloroform and extracted with Na₂CO₃. Ethyl acetate was added to the aqueous layer and the solution was acidified with 1N HCl and extracted (5×15 mL) with EtOAc. The organic layers were combined, dried over Na₂SO_4(s), filtered and evaporated to an amorphous solid. The desired material was used without further purification in the next series of reactions. Rf_{(AcOH/MeOH/CH2Cl2) = 0.40.} ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 9.91 (brs, 1H), 5.41 (m, 1H), 4.31 (m, 1H), 4.08 (m, 4H), 2.02 (m, 4H), 1.41 (s, 9H), 1.29 (m, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 173.79, 135.57, 80.15, 62.53 (d, J_P = 6.0Hz), 57.58, 53.52, 28.41, 25.66, 22.40, 20.51, 16.46 (d, J_P = 6.0 Hz) ppm. HRMS (ES+) calculated m/z = 340.1525, experimental m/z = 340.1514.

4.1.13.2 RuCl₃/NaIO₄ Oxidation: ((2S)-4a)

To (3S)-9 (231 mg, 0.710 mmol) in 1.4 mL of CCl₄, 1.4 mL of MeCN, and 2.2 mL of H₂O was added NaIO₄ (456 mg, 2.130 mmol) followed by RuCl₃ hydrate (3 mg, 0.016 mmol). By TLC analysis all the alcohol was consumed in 5 minutes (presumably to the aldehyde; Rf_{(1:9 MeOH/CHCl3)} ≈ 1). After 1.5 hours, only a baseline spot was visible by TLC and the reaction was diluted with H₂O and extracted with CHCl₃ (5×15 mL). The organic layers were combined and dried over MgSO₄, then concentrated and purified by flash chromatography (~75 mL of SiO₂, 10 to 25% MeOH in CH₂Cl₂) to yield 184 mg (76%) of an amorphous white solid. Rf_{(AcOH/MeOH/CH2Cl2)} = 0.40. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 11.32 (s, 1H), 5.39 (d, J = 7.2 Hz), 4.23 (m, 1H), 4.03 (dq, J = 7.2, 2.4 Hz, 4H), 1.93 (m, 4H), 1.35 (s, 9H), 1.23 (t, J = 7.0 Hz, 6H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 173.81, 155.59, 79.94, 62.30, 53.32 (d, J = 19.8 Hz), 28.29, 25.55, 22.30, 20.40, 16.33 (d, J = 6.1 Hz) ppm. HRMS (ES+) calculated m/z = 340.1525, experimental m/z = 340.1525.

4.1.14.1 General Procedure III: PyBOP Condensation (11a)

To a solution of the protected amino acid (2R)-4a (50 mg,0.147 mmol) in dry CH₂Cl₂ (4 mL) was added PyBOP (77 mg, 0.147 mmol) and DIEA (0.03 mL, 0.147 mmol), followed by p-decylaniline (34 mg, 0.147 mmol). The reaction was stirred at room temperature for 12 hours then concentrated and purified by column chromatography (0 to 20% acetone in chloroform) to 35 mg of clear oil. Rf_{(1:1 EtOAc/Hex.)}=0.15. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 9.16 (s, 1H), 7.46 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 5.69 (d, J = 6.5 Hz, 1H), 4.44 (m, J = 7.3, 1.2 Hz, 1H), 4.12 (m, 4H), 2.54 (t, J = 7.7 Hz, 2H), 2.07 (m, J = 7.7 Hz, 2H), 1.88 (m, 2H), 1.56 (p, J = 6.9 Hz, 2H), 1.44 (s, 9H), 1.32 (dt, J = 11.9, 7.3 Hz, 6H), 0.87 (t, J = 6.9 Hz, 3H) ppm.

4.1.14.2 [3-tert-butoxycarbonylamino-3-(4-octylphenylcarbamoyl)propyl]-phosphonic acid diethyl ester (11b)

General Procedure III was used to convert (2R)-4a (50 mg, 0.147 mmol) and p-octylaniline (0.05 mL, 0.147 mmol) to the title compound. The crude material was purified through flash chromatography (~ 50 mL of SiO₂, 1:9 Acetone/ CHCl₃) to give 54 mg (70%) of clear oil. Rf_{(1:9 Acetone/ CHCl3)} = 0.2. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 9.22 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 5.77 (d, J = 8.1 Hz, 1H), 4.44 (m, 1H), 4.10 (m, 4H), 2.53 (t, J = 7.9 Hz, 2H), 2.09 (m, 2H), 1.87 (m, 2H), 1.55 (p, J = 6.8 Hz, 2H), 1.43 (s, 9H), 1.31 (dt, J = 10.6, 7.0 Hz, 6H), 0.86 (t, J = 7.0, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 169.75, 155.96, 139.21, 135.76, 128.99, 120.12, 80.30, 62.29 (d, J = 22.2 Hz), 35.63, 32.13, 31.78, 29.72, 29.51, 28.59, 26.52 (d, J = 5.0 Hz), 23.08, 22.91, 21.20, 16.67 (d, J = 7.6 Hz), 14.35 ppm.

4.1.14.3 [3-tert-butoxycarbonylamino-3-(3-heptylphenylcarbamoyl)propyl]-phosphonic acid diethyl ester (11c)

General Procedure III was used to convert (2R)-4a (50 mg, 0.147 mmol) and m-heptylaniline³⁹ (0.28 mg, 0.147 mmol) to the title compound. The crude material was purified through flash chromatography (~50 mL of SiO₂, 1:3 Acetone/ CHCl₃) to give 35 mg (46%) of clear oil. Rf_{(1:3 Acetone/ CHCl3)}=0.55. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 9.22 (s, 1H), 7.40 (s, 1H), 7.39 (d, J = 9.2 Hz, 1H), 7.19 (t, J = 7.7 Hz, 1H), 6.91 (d, J = 7.3 Hz, 1H), 5.68 (d, J = 7.3 Hz, 1H), 4.46 (m, 1H), 4.11 (m, 4H), 2.55 (t, J = 7.7 Hz, 2H), 2.07 (m, 2H), 1.89 (m, 2H), 1.58 (p, J = 7.3 Hz, 2H), 1.44 (s, 9H), 1.32 (dt, J = 11.9, 6.9 Hz, 6H), 0.86 (t, J = 6.9 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 169.72, 144.14, 138.00, 128.90, 124.61, 123.12, 119.93, 117.28, 78.50, 36.21, 62.15, 36.21, 31.99, 31.67, 29.53, 29.38, 28.52, 26.44, 22.58, 21.18, 16.64, 14.29 ppm.

4.1.14.4 [3-tert-butoxycarbonylamino-3-(4-octylphenylcarbamoyl)propyl]-phosphonic acid diethyl ester (11d)

General Procedure III was used to convert (2R)-4a (1.122 g, 3.307 mmol) and m-octylaniline (0.815 g, 3.968 mmol) to the title compound. The crude material was purified through flash chromatography (~ 300 mL of SiO₂, 1:9 acetone/CHCl₃) to give 1.134 g (65%) of translucent oil. R_{f (3:7 acetone/CHCl3)} = 0.58. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 9.28 (s, 1H), 7.41 (s, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.18 (t, J = 8.1 Hz, 1H), 6.90 (d, J = 7.7 Hz, 1H), 5.74 (d, J = 8.1 Hz, 1H), 4.46 (m, 1H), 4.12 (ddt, J = 24.6, 7.3, 3.5 Hz, 4H), 2.54 (t, J = 7.3 Hz, 2H), 2.07 (sep., J = 6.5 Hz, 2H), 1.90 (dq, J = 18.4, 13.5, 6.5 Hz, 2H), 1.57 (p, J = 7.7 Hz, 2H), 1.43 (m, 9H), 1.31 (dt, J = 10.0, 7.3 Hz, 6H), 1.28 (m, 12H), 0.86 (t, J = 6.5 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 169.77, 144.07, 138.04, 128.85, 124.53, 120.33, 119.94, 118.11, 117.29, 80.20, 62.36, 62.04, 54.79, 36.20, 32.06, 31.65, 30.07, 29.66, 29.56, 29.43, 29.13, 28.51, 26.46, 23.03, 22.83, 22.78, 21.16, 16.64, 16.55, 14.28 ppm.

4.1.14.5 [3-tert-butoxycarbonylamino-3-(4-octylphenylcarbamoyl)propyl]-phosphonic acid diethyl ester (11e)

General Procedure III was used to convert (2S)-4a (50 mg, 0.147 mmol) and p-octylaniline (0.05 mL, 0.147 mmol) to the title compound. The crude material was purified through flash chromatography (~ 50 mL of SiO₂, 1:9 Acetone/ CHCl₃) to give 54 mg (70%) of clear oil. Rf_{(1:9 Acetone/ CHCl3)} = 0.2. See 11b for corresponding ¹H and ¹³C NMR data.

4.1.14.6 [3-tert-butoxycarbonylamino-3-(3-octylphenylcarbamoyl)propyl]-phosphonic acid diethyl ester (11f)

General Procedure III was used to convert (2S)-4a (34 mg, 0.100 mmol) and m-octylaniline³⁹ (0.02 mL, 0.100 mmol) to the title compound. The crude material was purified through flash chromatography (~50 mL of SiO₂, 1:1 EtOAc/ Hex.) to give 23 mg (45%) of an off-white solid. Rf_{(1:1 EtOAc/Hex.)}=0.10. See 11d for corresponding ¹H and ¹³C NMR data.

4.1.15.1 General Procedure IV: Phosphonate Diester Deprotection (12a)

The phosphonate diester 11a (35 mg, 0.063 mmol) was dissolved in 0.65 mL of CH₂Cl₂ to which bromotrimethylsilane (0.08 mL, 0.630 mmol) was added via syringe. The solution was allowed to stir for four to six hours. The off-white solution was then concentrated and reconstituted in > 0.65 mL of 95% methanol in water. The new solution was allowed to stir for four hours to ensure hydrolysis and was concentrated and co-evaporated with methanol and diethyl ether until a pasty solid was attained. The compound was triturated to a fine white solid on addition of water. The solid was filtered through a fine fritted funnel and washed with water (3×) followed by cold pentane (3×) to yield 31 mg of the desired compound as a white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.51 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 4.12 (t, J = 6.4 Hz, 1H), 2.58 (t, J = 7.7 Hz, 2H), 2.24 (m, 2H), 1.86 (m, 2H), 1.60 (p, J = 6.7 Hz, 2H), 1.28 (m, 12H), 0.90 (t, J = 7.0 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 141.04, 130.04, 121.40, 114.14, 56.36, 36.47, 33.21, 32.88, 30.89, 30.86, 30.74, 30.60, 30.41, 23.88, 14.59 ppm. MS (ESI+) m/z 399 [M+H]⁺.

4.1.15.2 [3-amino-3-(4-octylphenylcarbamoyl)propyl]-phosphonic acid (12b – VPC44152)

General Procedure IV was used to globally deprotect compound 11b in formation of the title compound as 47 mg (100%) of an off-white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.51 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 4.13 (t, J = 6.5 Hz, 1H), 2.59 (t, J = 7.9 Hz, 1H), 2.24 (m, 2H), 1.88 (m, 2H), 1.60 (p, J = 6.2 Hz, 2H), 1.31 (m, 10H), 0.89 (t, J = 7.0 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 140.90, 136.48, 129.69, 121.25, 66.69, 57.13, 36.32, 33.02, 32.72, 30.56, 30.41, 30.27, 23.71, 22.89, 22.46, 14.43 ppm. MS (APCI) m/z = 371.9 [M+1]⁺, 370.80 [M, 100%]⁺. Elemental CHN: calculated C = 47.90%, H = 7.15%, N = 6.21%; found C = 48.21%, H = 7.25%, N = 6.02%.

4.1.15.3 [3-amino-3-(3-heptylphenylcarbamoyl)propyl]-phosphonic acid (12c)

General Procedure IV was used to globally deprotect compound 11c in formation of the title compound as 25 mg (84%) of a white solid. ¹H NMR (300 MHz, (CD₃)₂SO, 23 °C, δ): 8.26 (m, 1H), 7.42 (m, 1H), 7.26 (t, J = 6.8 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 3.98 (m, 1H), 2.55 (m, 2H), 2..03 (m, 2H), 1.60 (m, 4H), 1.26 (m, 8H), 0.85 (m, 3H) ppm. ¹³C NMR (300 MHz, (CD₃)SO, 23 °C, δ): 166.79, 143.28, 137.83, 128.94, 124.37, 119.27, 116.95, 53.25, 35.17, 31.27, 30.87, 28.60, 28.54, 25.36, 24.32, 22.51, 22.11, 13.99 ppm. MS (ESI+) m/z 357 [M+H]⁺.

4.1.15.4 [3-amino-3-(3-octylphenylcarbamoyl)propyl]-phosphonic acid (12d – VPC44116)

General Procedure IV was used to globally deprotect compound 12d in formation of the title compound as 28 mg (90%) of a white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.45 (m, 2H), 7.23 (t, J = 7.8 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 4.15 (m, 1H), 2.59 (t, J = 7.3 Hz, 2H), 2.25 (m, 2H), 1.89 (m, 2H), 1.61 (m, 2H), 1.31 (m, 10H), 0.89 (t, J = 6.8 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 167.99, 139.32, 130.01, 126.19, 121.28, 119.44, 118.72, 55.39, 55.20, 37.04, 33.17, 32.77, 30.74, 30.47, 29.72, 27.11, 25.42, 23.87, 23.61, 14.58 ppm. MS (APCI) m/z = 371.9 [M+1]⁺, 370.80 [M, 100%]⁺. HRMS (ES+) calculated m/z = 371.2100, experimental m/z = 371.2108. Elemental CHN: calculated C = 47.90%, H = 7.15%, N = 6.21%; found C = 48.11%, H = 7.14%, N = 6.14%.

4.1.15.5 [3-amino-3-(4-octylphenylcarbamoyl)propyl]-phosphonic acid (12e)

General Procedure IV was used to globally deprotect compound 11e in formation of the title compound as 25 mg (96%) of a white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.47 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 4.25 (m, 1H), 3.95 (t, J = 6.8 Hz, 1H), 2.57 (t, J = 7.5 Hz, 2H), 2.22 (m, 2H), 1.97 (m, 1H), 1.84 (m, 2H), 1.58 (m , 2H), 1.26 (m, 10H), 0.87 (t, J = 6.6 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 140.90, 136.48, 129.69, 121.25, 66.69, 57.13, 36.32, 33.02, 32.72, 30.56, 30.41, 30.27, 23.71, 22.89, 22.46, 14.43 ppm. MS (ESI+) m/z 371 [M+H]⁺.

4.1.15.6 [3-amino-3-(3-octylphenylcarbamoyl)propyl]-phosphonic acid (12f)

General Procedure IV was used to globally deprotect compound 11f in formation of the title compound as 20 mg (100%) of a white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.44 (s, 1H), 7.42 (d, J = 10.0 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 7.8 Hz, 1H), 4.08 (t, J = 6.6 Hz, 1H), 2.60 (t, J = 7.3 Hz, 2H), 2.23 (m, 2H), 1.89 (m, 2H), 1.61 (quin., J = 6.8Hz, 2H), 1.32 (m, 10H), 0.89 (t, J = 6.6 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 167.99, 139.32, 130.01, 126.19, 121.28, 119.44, 118.72, 55.39, 55.20, 37.04, 33.17, 32.77, 30.74, 30.47, 29.72, 27.11, 25.42, 23.87, 23.61, 14.58 ppm. MS (ESI+) m/z 371 [M+H]⁺.

4.1.15.7 [3-amino-3-(4-decylphenylcarbamoyl)-2-fluoropropyl]-phosphonic acid (13)

Compounds (3R)-4b and p-decylaniline were converted to the title compound by application of first; General Procedure III to yield 22 mg (24%) of a clear liquid, Rf_{(1:1 EtOAc/Hexanes)}=0.21. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 8.78 (d, J = 38.9, 1H), 7.43 (d, J = 7.7 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 5.56 (d, J = 22.6 Hz, 1H), 4.99 (m, 1H), 4.56 (m, 1H), 4.22 (m, 4H), 2.55 (t, J = 7.9 Hz, 2H), 2.40 (m, 2H), 1.56 (p, J = 7.0 Hz, 2H), 1.45 (s, 9H), 1.36 (dt, J = 7.2, 7.0 Hz, 6H), 1.25 (m, 12H), 0.87 (t, J = 6.6 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 129.01, 120.11, 100.16, 63.98, 63.47, 51.81, 51.50, 25.59, 32.10, 31.74, 29.83, 29.81, 29.71, 29.53, 29.45, 28.50, 28.48, 22.89, 16.63, 14.33 ppm. General Procedure IV was then implemented to give 19 mg (100%) of the title compound as a white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.50 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 5.02 (m, 1H), 4.26 (m, 1H), 4.11 (m, 1H), 2.59 (t, J = 7.6 Hz, 2H), 2.39 (m, 1H), 1.60 (p, J = 7.0 Hz, 2H), 1.29 (m, 12H), 0.90 (t, J = 6.6 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 166.71, 140.61, 135.83, 129.57, 120.97, 36.04, 32.64, 32.29, 30.32, 30.21, 30.05, 29.89, 23.36, 16.81, 14.37 ppm. MS (ESI+) m/z 417 [M+H]⁺.

4.1.16.1 General Procedure V: Standard Mitsunobu Conditions (14a)

To a solution of p-octylphenol (1.000 g, 4.847 mmol) in 5 mL of anhydrous THF was added (R)-(+)- glycidol (0.35 mL, 5.331 mmol) and Ph₃P (1.398 g, 5.331 mmol). The reaction mixture was cooled to 0 °C and DIAD (1.05 mL, 5.331 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 24 hours. A white precipitate formed upon addition of Et₂O and was filtered off through a fine fritted funnel. The filtrate was concentrated and purified by column chromatography (~200 mL SiO₂, CHCl₃) to give 1.023 g (80%) of 14a as colorless oil. Rf_(CHCl3)=0.66. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.09 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 4.19 (dd, J = 11.2, 3.3 Hz, 1H), 3.95 (dd, J = 11.0, 5.5 Hz, 1H), 3.35 (p, J = 4.8 Hz, 1H), 2.90 (t, J = 4.2 Hz, 1H), 2.75 (dd, J = 4.8, 2.6 Hz, 1H), 2.54 (t, J = 7.5 Hz, 2H), 1.56 (m, 2H), 1.28 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 156.70, 135.92, 129.51, 114.63, 69.02, 50.44, 45.01, 35.26, 32.10, 31.93, 29.70, 29.49, 22.89, 14.33 ppm.

4.1.16.2 2-(4-octylphenoxymethyl)-oxirane (14b)

The title compound was formed, through General Procedure V, starting with 1.000 g of (S)-(-)-glycidol, as 1.073 g (84%) of a clear oil following column chromatography (~200 mL SiO₂, CHCl₃) Rf_(CHCl3)=0.66. ¹H and ¹³C NMR were consistent with those of epimer 14a.

4.1.17.1 [3-hydroxy-4-(4-octylphenoxy)-butyl]-phosphonic acid diethyl ester (15a)

General Procedure II was used to transform 14a (1.023 g, 3.899 mmol) to 1.400 g (87%) of 15a as a clear oil after column chromatography (~300 mL SiO₂, 1:4 acetone/chloroform). Rf_{(1:4 acetone/chloroform)} =0.33. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.09 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 4.11 (m, 4H), 4.02 (m, 1H), 3.90 (m, 2H), 2.53 (t, J = 7.7 Hz, 2H), 1.92 (m, 4H), 1.56 (p, J = 8.1 Hz, 2H), 1.30 (m, 16H), 0.87 (t, J = 6.9 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 156.70, 135.89, 129.54, 114.54, 71.77, 70.08, 62.02, 35.26, 32.11, 31.95, 29.70, 29.49, 26.59, 23.12, 22.89, 21.23, 16.69, 14.33 ppm.

4.1.17.2 [3-hydroxy-4-(4-octylphenoxy)-butyl]-phosphonic acid diethyl ester (15b)

General Procedure II was used to transform 14b (1.073 g, 4.089 mmol) to 1.215 g (72%) of 15b as a clear oil after column chromatography (~300 mL SiO₂, 1:4 acetone/chloroform). Rf_{(1:4 acetone/chloroform)} =0.33.

4.1.18.1 General Procedure VI: Azide Formation with Free Phosphine (16a)

To a stirring solution of 15a (500 mg, 1.206 mmol) in 1.6 mL of CH₂Cl₂ at 0 °C was added Ph₃P (348 mg, 1.327 mmol). The solution was stirred for 15 minutes and then DIAD (0.26 mL, 1.327 mmol) and DPPA (0.29 mL, 1.327 mmol) were added consecutively, dropwise. The reaction was allowed to warm to room temperature and stirred overnight. Mixture was concentrated and purified by chromatography (~150 mL SiO₂, 1:9 Acetone/CHCl₃) followed by (~100 mL SiO₂, 50% to 80% Et₂O in petroleum ether) to yield 518 mg (98%) of non-viscous clear liquid. Rf_{(1:9 Acetone/CHCl3)}=0.44. 1H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.04 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H), 4.05 (m, 4H), 3.95 (m, 2H), 3.75 (m, 1H), 2.48 (t, J = 7.9 Hz, 2H), 1.80 (m, 4H), 1.51 (p, J = 7.3 Hz, 2H), 1.27 (t, J = 7.0 Hz, 6H), 1.23 (m, 10H), 0.82 (t, J = 6.6 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 156.11, 135.80, 129.29, 114.30, 70.45, 61.67, 60.95, 35.01, 31.84, 31.68, 29.44, 29.22, 24.16, 23.10, 22.63, 21.21, 16.41, 14.06 ppm.

4.1.18.2 [3-azido-4-(4-octylphenoxy)-butyl]-phosphonic acid diethyl ester (16b)

General Procedure VI was used to form the title compound from alcohol 15b. Purification consisted of two chromatography steps. First a column (~250 mL of SiO₂, 1:9 Acetone/CHCl₃) was run to yield the desired product with OPPh₃. The crude material was then run through a short plug (~50 mL of SiO₂) with CHCl₃ as the eluent. The phosphine oxide did not more over this system and the plug yielded 354 mg (67%) of a clear liquid. Data were consistent with that of 16a.

4.1.19.1 [3-amino-4-(4-octylphenoxy)-butyl]-phosphonic acid diethyl ester (17a)

General Procedure I was used to reduce the azide 16a to 488 mg of amine 17a as a yellow liquid. Further purification was not necessary and the product was carried on to the final deprotection. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 6.97 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 8.1 Hz, 2H), 5.43 (m, 2H), 3.99 (m, 4H), 3.90 (m, 2H), 2.43 (t, J = 7.9 Hz, 2H), 1.89 (m, 4H), 1.47 (p, J = 7.0 Hz, 2 H), 1.21 (m, 16H), 0.79 (t, J = 6.7 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 168.83, 156.23, 135.57, 129.18, 114.35, 69.57, 61.82, 51.05, 49.84, 34.96, 31.81, 31.66, 29.41, 29.20, 24.83, 22.73, 22.58, 20.85, 16.34, 14.03 ppm.

4.1.19.2 [3-amino-4-(4-octylphenoxy)-butyl]-phosphonic acid diethyl ester (17b)

General Procedure I was used to reduce the azide 16b to 333 mg of amine 17b as a light yellow liquid. Further purification was not necessary and the product was carried on to the final deprotection. Data were similar to that obtained for 17a.

4.1.20.1 [3-amino-4-(4-octylphenoxy)-butyl]-phosphonic acid (18a)

General Procedure IV was used to deprotect compound 17a in formation of the title compound as 500 mg (97%) of an off-white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.12 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.22 (dd, J = 10.4, 3.5 Hz, 1H), 4.07 (dd, J = 10.7, 6.1 Hz, 1H), 3.68 (m, 1H), 2.55 (t, J = 7.3 Hz, 2H), 2.09 (m, 2H), 1.88 (m, 2H), 1.57 (p, J = 7.7 Hz, 2H), 1.30 (m, 10H), 0.89 (t, J = 6.8 Hz, 3H) ppm. ¹³C NMR (300 MHz, CD₃OD, 23 °C, δ): 13764, 130.64, 115.70, 101.52, 68.14, 36.14, 33.18, 33.07, 30.73, 30.58, 30.39, 25.28, 24.70, 23.87, 14.58 ppm. MS (ESI+) m/z 358 [M+H]⁺.

4.1.20.2 [3-amino-4-(4-octylphenoxy)-butyl]-phosphonic acid (18b)

General Procedure IV was used to deprotect compound 17b in formation of the title compound as 278 mg (79%) of an off-white solid. ¹H NMR, ¹³C NMR, and MS (ESI+) were consistent with those obtained for compound 18a. Elemental CHN: calculated C = 60.49%, H = 9.02%, N = 3.92%; found C = 60.21%, H = 9.00%, N = 3.68%.

4.1.21. [3-hydroxy-4-(4-octylphenoxy)-butyl]-phosphonic acid (19)

General Procedure IV was used to deprotect compound 15a (25 mg, 0.060 mmol) in formation of the title compound. Further purification was performed by recrystallization from EtOAc and Hexanes to yield 22 mg (79%) of a white solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.07 (m, 2H), 6.85 (m, 2H), 3.90 (m, 3H), 2.53 (m, 2H), 1.95 (m, 2H), 1.78 (m, 2H), 1.29 (m, 10H), 0.89 (m, 3H) ppm. MS (ESI-) m/z 357 [M-H]⁻.

4.1.22. 4-methyl-N-(3-octylphenyl)-benzenesulfonamide (20)

To a stirring solution of m-octylaniline⁴⁰ (1.000 g, 4.87 mmol) in 5 mL of pyridine at 0 °C was added tosyl chloride (928 mg, 4.87 mmol). The reaction mixture was warmed to room temperature. After one hour, the mixture was diluted with EtOAc and washed with 1N HCl (3×), NaHCO₃ (2×), and brine (2×). The organic layer was dried over Na₂SO₄, filtered, and concentrated. The crude material was purified by column chromatography (~150 mL SiO₂, 1:19 Acetone/Chloroform) to yield 1.700 g (97%) of a white solid. Rf _{(1:19 Acetone/Chloroform)} = 0.70. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.72 (d, 2H), 7.59 (bs, 1H), 7.19 (d, J = 7.8 Hz, 2H), 7.03 (q, 4H), 2.51 (t, J = 7.2Hz, 2H), 2.34 (s, 3H), 1.54 (p, J = 7.6 Hz, 2H), 1.27 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 144.2, 140.5, 136.7, 134.7, 130.1, 129.7, 127.9, 122.4, 35.8, 32.4, 31.9, 30.0, 29.8, 23.2, 22.0, 14.6. MS (ESI) m/z 360 [M+H]⁺.

4.1.23. 4-methyl-N-(3-octylphenyl)-N-oxiranylmethylbenzenesulfonamide (21)

General Procedure V was used to couple 20 (0.809 g, 2.25 mmol) with (R)-(+)-glycidol (0.23 mL, 3.375 mmol) to form 644 mg (69%) of the title compound 21 after flash chromatography (~150 mL SiO₂, 1:4 EtOAc/Hexanes), as a clear oil. Rf_{(1:4 EtOAc/Hex.)} = 0.36. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.45 (d, J = 8.1 Hz, 2H), 7.20 (m, 2H), 7.07 (m, 1H), 7.02 (m, 1H), 6.89 (m, 1H), 6.82 (m, 1H), 3.65 (ddd, J = 35.2, 14.3, 5.3 Hz, 2H), 3.08 (m, 1H), 2.63 (m, 1H), 2.49 (t, J = 7.5 Hz, 2H), 2.37 (s, 3H), 2.16 (m, 1H), 1.48 (p, J = 6.8 Hz, 2H), 1.25 (m, 10H), 0.85 (t, J = 7.0 Hz, 3H). ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 144.01, 143.51, 139.60, 138.83, 129.39, 127.68, 126.10, 53.65, 50.24, 45.78, 35.56, 31.84, 31.24, 29.41, 29.15, 22.64, 21.47, 14.10 ppm.

4.1.24. {3-hydroxy-4-[(3-octylphenyl)-(toluene-4-sulfonyl)-amino]-butyl}-phosphonic acid diethyl ester (22)

General Procedure II completed the synthesis of 853 mg (97%) of compound 22 from 21 as a clear oil following column chromatography (SiO₂, 5 to 20% Acetone in CHCl₃). Rf_{(1:4 EtOAc/Hex.)} = 0.05. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.41 (dd, J = 8.1, 6.5 Hz, 2H), 7.18 (d, J = 7.7 Hz, 2H), 7.13 (d, J = 8.1, 1H), 7.04 (d, J = 7.7 Hz, 1H), 6.85 (m, 1H), 6.73 (m, 1H), 4.00 (m, 4H), 3.61 (m, 2H), 3.47 (m, 2H), 2.45 (t, J = 8.1Hz, 2H), 1.76 (m, 4H), 1.45 (m, 2H), 1.24 (m, 16H), 0.83 (t, J = 6.9 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 143.63, 139.60, 138.92, 134.77, 129.50, 129.44, 128.93, 128.31, 127.88, 126.04, 68.80 (d, J = 13.5 Hz), 61.74 (d, J = 5.8 Hz), 56.23 (d, J = 7.7 Hz), 35.66, 31.93, 31.35, 29.49, 29.30, 28.98, 27.10 (d, J = 4.8 Hz), 22.70, 21.59, 20.48, 16.47 (d, J = 5.8 Hz), 14.18 ppm.

4.1.25. {3-azido-4-[(3-octylphenyl)-(toluene-4-sulfonyl)-amino]-butyl}-phosphonic acid diethyl ester (23)

General Procedure VI transformed alcohol 22 (302 mg, 0.532 mmol) into azide 23. The crude material was purified twice by chromatography (~150 mL of SiO₂, 10%Acetone/CHCl₃) followed by (~100 mL of SiO₂, 7:3 EtOAc/Hexanes) to yield 267 mg (85%) as a clear oil. [Another 5% of the desired product was isolated with residual phosphine oxide.] Rf_{(1:9 Acetone/CHCl3)} = 0.43. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.38 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 7. Hz, 2H), 7.07 (d, J = 7.7 Hz, 1H), 6.86 (d, J = 13.2 Hz, 1H), 6.78 (m, 1H), 4.02 (m, 4H), 3.53 (m, 3H), 2.48 (t, J = 7.5 Hz, 2H), 2.37 (s, 3H), 1.75 (m, 4H), 1.46 (p, J = 7.0 Hz, 2H), 1.25 (m, 16H), 0.84 (t, J = 7.0 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 144.36, 143.80, 139.38, 134.55, 132.04, 129.48, 129.05, 128.45, 127.87, 125.73, 61.75 (d, J = 6.6 Hz), 61.32 (d, J = 16.1 Hz), 54.74, 35.68, 31.92, 31.33, 29.49, 29.28, 28.97, 28.56, 25.30 (d, J = 4.5 Hz), 22.70, 21.59, 16.49 (d, J = 6.0 Hz), 14.16 ppm.

4.1.26. {3-amino-4-[(3-octylphenyl)-(toluene-4-sulfonyl)-amino]-butyl}-phosphonic acid diethyl ester (24)

Compound 23 (267 mg, 0.450 mmol) was dissolved in 50 mL of a 20:1 MeOH/HCl solution. To this solution was added ~0.5 g of Pd(OH)₂ and the apparatus was assembled and experiment run analogous to that of General Procedure I. When no starting material remained (~four hours by TLC) the mixture was filtered over celite and washed with 2% Et₃N in MeOH. The filtrate was concentrated to yield 268 mg (100%) of the title compound as yellow oil. No further purification was required. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.26 (d, J = 7.9 Hz, 2H), 7.11 (d, J = 7.9 Hz, 2H), 7.06 (d, J = 7.7 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 6.72 (d, J = 7.7 Hz, 1H), 6.60 (m, 1H), 4.02 (bs, 2H), 3.87 (m, 4H), 3.30 (m, 2H), 2.63 (m, 1H), 2.35 (t, J = 7.7 Hz, 2H), 2.27 (s, 3H), 1.59 (m, 4H), 1.33 (m, 2H), 1.12 (m, 16H), 0.72 (t, J = 6.6 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 144.36, 143.95, 138.95, 137.75, 129.45, 128.99, 128.49, 128.07, 127.68, 125.79, 61.96 (d, J = 7.1 Hz), 56.05, 48.67 (t, J = 21.7), 35.48, 31.76, 31.22, 29.33, 29.13, 26.26 (d, J = 7.6 Hz), 22.52, 22.22, 21.29, 20.35, 16.08 (d, J = 6.6 Hz), 13.86 ppm.

4.1.27. [3-amino-4-(3-octylphenylamino)-butyl]-phosphonic acid diethyl ester (25)

A solution of Na_(s) (70 mg, 3.06 mmol) in 25 mL of NH_3(l) was contained in a 2-neck flask fitted with a cold finger cooled to −78 °C and stir bar. Compound 24 (182 mg, 0.306 mmol) diluted in a minimal amount of THF was quickly added to the solution and the reaction was allowed to proceed for no longer than 5 minutes, at which time ethanol was added under vigorous stirring and the reaction was allowed to warm to room temperature. The reaction mixture was concentrated to dryness, dissolved in EtOAc, washed with NaHCO₃ (3×) and brine (1×). The organic layer was dried then columned (~75 mL of SiO₂, 5% MeOH in CHCl₃). The desired product was found to be a clear oil, in 25 mg (25%) quantities. Starting material (28%) was also recovered. Rf_{(5% MeOH in CHCl3)} = 0.17. ¹H NMR (300 MHz, CDCl₃, 23 °C, δ): 7.07 (t, J = 7.5 Hz, 1H), 6.53 (t, J = 7.0 Hz, 1H), 6.44 (m, 2H), 4.08 (m, 4H), 3.70 (m, 1H), 3.11 (m, 3H), 2.50 (t, J = 7.9 Hz, 2H), 1.83 (m, 4H), 1.57 (p, J = 7.3 Hz, 2H), 1.31 (m, 16H), 0.87 (t, J = 7.0 Hz, 3H) ppm. ¹³C NMR (300 MHz, CDCl₃, 23 °C, δ): 157.35, 148.46, 129.30, 118.14, 113.44, 110.41, 62.05, 61.72, 58.20, 47.13, 36.41, 34.55, 32.10, 31.72, 29.92, 29.73, 29.64, 29.49, 28.18 (d, J = 8.6 Hz), 22.88, 16.69 (d, J = 7.1 Hz), 14.32 ppm.

4.1.28. [3-amino-4-(3-octylphenylamino)-butyl]-phosphonic acid (26)

The deprotection of 25 (23 mg, 0.056 mmol) was carried out by General Procedure IV to give 20 mg (83%) of the title compound as a colorless solid. ¹H NMR (300 MHz, CD₃OD, 23 °C, δ): 7.36 (dd, J = 7.9, 3.7 Hz, 1H), 7.26 (t, J = 1.7 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 3.70 (m, 3H), 2.65 (t, J = 7.5 Hz, 2H), 2.14 (m, 2H), 1.95 (m, 2H), 1.64 (p, J = 7.5 Hz, 2H), 1.31 (m, 10H), 0.89 (t, J = 7.0 Hz, 3H) ppm. MS (ESI+) m/z 357 [M+H]⁺.