A facile synthesis of d -ribo-C20-phytosphingosine and its C2 epimer from d -ribose

Carbohydrate Research 346 (2011) 1728–1738

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A facile synthesis of D-ribo-C20-phytosphingosine and its C2 epimer from D-ribose Miroslava Martinková a,⇑, Jozef Gonda a, Kvetoslava Pomikalová a, Jozef Kozˇíšek b, Juraj Kuchár c a

Institute of Chemical Sciences, Department of Organic Chemistry, P.J. Šafárik University, Moyzesova 11, Sk-040 01 Košice, Slovak Republic Institute of Physical Chemistry and Chemical Physics, Department of Physical Chemistry, Slovak University of Technology, Radlinského 9, Sk-812 37 Bratislava, Slovak Republic c Institute of Chemical Sciences, Department of Inorganic Chemistry, P.J. Šafárik University, Moyzesova 11, Sk-040 01 Košice, Slovak Republic b

a r t i c l e

i n f o

Article history: Received 15 April 2011 Received in revised form 23 May 2011 Accepted 25 May 2011 Available online 2 June 2011 Keywords: Sphingolipids Phytosphingosine Overman rearrangement Trichloroacetamides

a b s t r a c t A facile synthetic route to D-ribo-C20-phytosphingosine 31 and its C2 epimer 32 is described. The Overman rearrangement of allylic trichloroacetimidates derived from the known ribose derivative 7 has been used as the key step. The subsequent functional group interconversions in rearranged products 14 and 15 followed by Wittig olefination, Pd/C-mediated reduction and the removal of protecting groups successfully constructed the final molecules. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Sphingolipids are ubiquitous building blocks of the plasma membranes in all eukaryotic cells and have been also found in some prokaryotic organisms.1 From a structural standpoint, mammalian sphingolipids derive mainly from the most predominant Derythro-sphingosine (1),2 although a small amount is generated from its saturated analogue sphinganine (2).3 On the other hand, with phytosphingosines the majority possess a C18 lipid chain, although C16–C22 homologues have been also found, depending on their original sources.4 These are the principal sphingoid bases in plants, some fungi and marine organisms but are also present in mammalian tissues.1,5 D-ribo-C18-Phytosphingosine (3) [(2S,3S,4R)2-aminooctadecane-1,3,4-triol, Fig. 1], originally isolated from the mushroom Amanita muscaria,6 represents the most abundant member of phytosphingosine family with significant biological functions in living organisms. For example, amide-linked derivatives of 3 are essential for the function of human skin,7 which form about 40% of the total ceramide content of the stratum corneum (the uppermost layer of the epidermis) and thus contribute to the generation of the water permeability barrier. Recent studies in Saccharomyces cerevisiae showed the key role of D-ribo-phytosphingosine (3) and its C20 molecular species in heat stress response.8 Moreover, the synthetic D-ribo-C18-phytosphingosinecontaining a-galactosyl ceramide, known as KRN7000, is reported ⇑ Corresponding author. Tel.: +421 55 6228332; fax: +421 55 6222124. E-mail address: [email protected] (M. Martinková). 0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2011.05.028

to exhibit significant immuno-stimulatory activity and antitumor properties.9 Last but not least, phytosphingosine 3 has served as the starting point for several efficient syntheses of the interesting natural compounds, such as 1,10 jaspine B11a,b (4, Fig. 1) and its C2 epimer.11c These interesting biological findings have stimulated many synthetic chemists, and various syntheses of D-ribo-phytosphingosine 3 and its stereoisomers have been reported, employing different approaches and starting materials.12,13 On the other hand, only a few reported syntheses in the phytosphingosine family have mentioned other alkyl chain lengths13n,x,14 than C18. For example, the natural4a–c D-ribo-C20-phytosphingosine 31 was prepared only as its N-benzoyl derivative,14a N,O-isopropylidene compound14b or tetraacetate.14c Therefore, we turned our attention to the development of a facile synthetic route to D-ribo-C20-phytosphingosine (31) and its C2 epimer 32 starting from D-ribose synthon 715 using the Overman rearrangement16 as the key reaction. Interestingly, none of the synthetic protocols of the various reported approaches12,13 towards the phytosphingosine family has employed D-ribose as the chiral starting material. 2. Results and discussion The known15 5-O-(tert-butyldimethylsilyl)-2,3-O-isopropylidene-D-ribofuranose (7) served as the starting material and was easily obtained on a multigram scale, applying modifications of the combined literature protocols.15,17 Its subsequent Wittig olefination (Ph3P@CHCO2Et, CH2Cl2, benzoic acid, reflux), followed

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carried out in o-xylene in the presence of K2CO319 in a sealed tube and afforded the rearranged products 14 and 15 (Table 1, entries 1, 3, 5, and 7). On the other hand, the considerable acceleration of the thermal Overman rearrangement mediated by microwave irradiation20 with an additional improvement of isolated yields was observed (Table 1, entries 2, 4, 6, and 8); however, it had practically no influence on the diastereoselectivity. Furthermore, the results obtained as presented in Table 1 suggest that the facial selectivity of the rearrangement is not dependent on the geometry of the double bond in the starting imidates. The moderate diastereoselectivity observed for both trichloroacetimidates (Z)-12i and (E)-13i is somewhat surprising. In order to rationalize the observed stereoselectivity in the Overman rearrangement, high-level density functional theory (DFT) calculations, which include electron correlation effects, were carried out. Geometries of the transition states were optimized using B3LYP/631G(d,p) with JAGUAR 7.7 programme.21 The nature of vacuum B3LYP transition states was verified with frequency calculations, yielding only one large imaginary frequency. Harmonic zero-point energy corrections at B3LYP/6-31G(d,p) obtained from the frequency calculations of the vacuum transition states were applied to the transition-state energies. Single-point energies were computed by the B3LYP density functional method and the cc-pvTZ basis set. The solvent effect was taken into account via a single-point calculation in a dielectric continuum representing o-xylene as the solvent. A standard Poisson–Boltzmann continuum solvation model was applied as implemented in JAGUAR 7.7.21 The Overman rearrangement of (E)-13i occurs via transition states TS1E, TS2E, TS3E and TS4E, with relative free energies 2.09, 1.65, 3.95, and 0 kcal/mol (Fig. 2). The process is concerted but asynchronous, and the calculated geometries are in good agreement with the results of Houk and co-workers.22 From the calculations, for the pathway (E)-13i?TS4E?14, the activation energy was found to be 1.65 kcal/mol lower than that for the pathway (E)-13i?TS2E?15. Thus, the predicted diastereomeric ratio of 14:15 at 200 °C was 85:15. Analogously, for cis-imidate (Z)-12i were located as transition structures TS1Z, TS2Z, TS3Z and TS4Z, with relative free energies 0.92, 1.48, 0, and 2.53 kcal/mol (Fig. 3), and for the pathway (Z)-12i?TS3E?14, the activation energy was found to be 0.92 kcal/mol lower than that for the pathway (Z)-12i?TS1E?15. The calculated diastereoisomeric ratio of 14:15 at 200 °C was 72:28. These results are in relatively good agreement with the experimental data (14:15  2:1, for both (E)12i and (Z)-13i) with the correct prediction of trichloroacetamide 14 as the predominant diastereoisomer. These results provide an

Figure 1. Various natural sphingoid bases.

by chromatographic separation of the products, provided a mixture of a,b-unsaturated esters18 (Z)-8 and (E)-9 (8:9 = 3:1, as determined by 1H NMR spectroscopy) in 93% yield (Scheme 1). Their structures, including geometries of the double bonds, were assigned by NMR spectroscopic analysis. On the other hand, Webb et al.18 reported the preparation of (Z)-8 and (E)-9 in a 7:1 ratio and in 80% yield. However, by using the same reaction conditions (Ph3P@CHCO2Et, DME, 18 h, rt), we obtained the corresponding esters in a 2:1 ratio and with ca. 50% conversion (determined by 1 H NMR spectroscopy). The remaining hydroxyl group at the C-6 position in (Z)-8 and (E)-9 was protected as the tert-butyldimethylsilyl ether (Z)-10 (TBDMSCl, imidazole, DMF, 95%) and (E)-11 (the same conditions, 71%, Scheme 1). Reduction of these fully protected esters (Z)-10 and (E)-11 with diisobutylaluminum hydride in CH2Cl2 afforded allylic alcohols (Z)-12 and (E)-13 in 98% and 96% isolated yields, respectively (Scheme 1). The substrates for the Overman rearrangement, the corresponding chiral allylic trichloroacetimidates (Z)-12i, (E)-13i, were obtained by the treatment of (Z)-12 and (E)-13 with NaH and trichloroacetonitrile and were used immediately in the next step without purification. With both imidates (Z)-12i, (E)-13i in hand, we then explored the thermal Overman rearrangement (Scheme 2), which was

O

OH

1

R O

a

OH CO2Et

O

O

O

OR1

R 1O

1

R O

d

O

(Z)-12, R2= H, 98% 2 (Z)-12i, R = -(C=NH)CCl3 (E)-13, R2= H, 96% (E)-13i, R2= -(C=NH)CCl3

O

(Z)-10, 95% (E)-11, 71%

OR1

NHCOCCl3

+ O 14

c CO 2Et

O

O

1

R O

O

OR2 O

OR 1

(Z)-8, 70% (E)-9, 23%

715 R1 = TBDMS

d

b

R1O

R 1O

OR1

O

NHCOCCl3

O 15

Scheme 1. Reagents and conditions: (a) Ph3P@CHCO2Et, benzoic acid, CH2Cl2, reflux; (b) TBDMSCl, imidazole, DMF, 70 °C; (c) DIBAL-H, CH2Cl2, 15 °C; (d) (i) Cl3CCN, NaH, THF, 0 °C; (ii) MW, o-xylene, K2CO3 (see Table 1).

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RO

OR

NHCOCCl3

O

RO

OR

O

O

14

OR

RO

OR

O

OH

RO

OR

OR

NHBoc COOH O

22, 59%

O

O

OR BocN

O

19, 82% c

c

RO

NHBoc OH

18, 91%

O

RO

b NHBoc

O

d

O

O

17, 92%

b OR

NHBoc

O

16, 73%

RO

R = TBDMS

a NHBoc

O

O 15

a RO

NHCOCCl3

O

OR BocN

O

RO

O

O

20, 94%

O

21, 79%

Scheme 2. Reagents and conditions: (a) (i) NaOH, EtOH–H2O, rt; (ii) Boc2O, Et3N, CH2Cl2, rt; (b) (i) O3, MeOH–CH2Cl2, 78 °C; (ii) NaBH4, 78 °C?rt; (c) 2,2-DMP, CSA, benzene, reflux; (d) NaIO4, RuCl3H2O, 2:2:3 CCl4–CH3CN–H2O, rt.

Table 1 Overman rearrangement imidates derived from alcohol (Z)-12 and (E)-13

a b c

Entry

Imidate

Conditionsa

Time (h)

Ratiob 14:15

Yieldc (%)

1 2 3 4 5 6 7 8

(Z)-12i (Z)-12i (Z)-12i (Z)-12i (E)-13i (E)-13i (E)-13i (E)-13i

D, 150 °C MW, 150 °C D, 200 °C MW, 200 °C D, 150 °C MW, 150 °C D, 200 °C MW, 200 °C

34 20 25 3 19.5 8 5 0.5

66:34 62:38 55:45 67:33 68:32 64:36 66:34 63:37

30 57 48 65 56 51 56 54

In o-xylene, in the presence of K2CO3. Ratio in the crude reaction mixture. Isolated combined yields of 14 and 15.

initial step in understanding of the rearrangement, and the observed diastereoselectivity seems to depend on many factors that are still to be explored. Although, the observed diastereoselectivity in the [3,3]sigmatropic rearrangement of both trichloroacetimidates was moderate (14:15  2:1 ratio, see Table 1), the relatively short synthetic pathway to trichloroacetamides 14 and 15 led us to utilize this strategy for the construction of 31 and its C2 epimer 32. The stereochemistry of the newly incorporated stereogenic centre bearing the amino function was unambiguously determined by single-crystal X-ray analysis of the more advanced derivative, carboxylic acid 22 (Scheme 2). This compound was prepared during our search for the optimal reaction conditions for transformation of the vinyl function into the hydroxymethyl moiety in 16 and

Figure 2. Transition structures for the rearrangement (E)-13i?14 + 15. Relative energies of transition states (in kcal/mol) and bond distances (in Å) are shown.

M. Martinková et al. / Carbohydrate Research 346 (2011) 1728–1738

1731

Figure 3. Transition structures for the rearrangement (Z)-12i?14 + 15. Relative energies of transition states (in kcal/mol) and bond distances (in Å) are shown.

17. The first protocol included oxidation of the terminal double bond to the carboxylic acid, its esterification and reduction of the ester function to an alcohol. The second protocol required ozonolysis followed by treatment with NaBH4 which was shown to be more convenient. Thus, we found that NaIO4/RuCl3-mediated oxidation of the vinyl function in 17 resulted in the formation of the crystalline acid 22 (59%, Scheme 2) whose single-crystal X-ray analysis (Fig. 4) clearly showed that the C-2 stereocentre in 22 possesses the (S)-configuration and consequently revealed that the minor isomer of the rearrangement 15 must be the (R)-isomer. In order to obtain desired structures 23 and 24 suitable for the final coupling reaction, the trichloroacetyl moiety in both rearranged products 14 and 15 was removed with sodium hydroxide in ethanol23 to afford the free amines, which were isolated as their N-Boc derivatives 16 and 17 in 73% and 92% yields, respectively (Scheme 2). Compounds 16 and 17 were converted to the protected aminopolyols 20 and 21 by a manner as described in Scheme 2. Thus, the ozonolysis of 16 and 17, followed by NaBH4 reduction, successfully afforded primary alcohols 18 and 19 in 91% and 82% isolated yields (two reaction steps from 16 and 17, Scheme 2). The formation of the oxazolidine ring in 18 and 19 was achieved under mild conditions with 2,2-dimethoxypropane in refluxing benzene24 and catalytic amounts of camphorsulfonic acid (CSA), providing 20 and 21 in 94% and 79% yields (Scheme 2). Exposure of 20 and 21 to tetrabutylammonium fluoride (TBAF) in dry tetra-

hydrofuran produced the corresponding diols 23 (93%) and 24 (88%) (Scheme 3). Their oxidative cleavage with NaIO4 afforded aldehydes, which were used immediately in the next reaction step without purification to avoid problems connected with their possible instability. Wittig olefination of these crude aldehydes with pentadecytriphenylphosphonium bromide (C15H31PPh3Br; prepared from the commercial 1-bromopentadecane and triphenylphosphine in refluxing toluene for 21.5 h, 92% yield), employing LHMDS25 (freshly prepared from n-BuLi and NH(SiMe3)2) as a base, resulted in the formation of barely separable mixtures of olefins 25 (Z:E = 92:8) and 26 (Z:E = 91:9 ratio, determined by 1H NMR spectroscopy) in 82% and 69% yields, respectively (Scheme 3). Small amounts of the mixtures of 25 and 26 were separated by column chromatography to give only (Z)-olefins 25a and 26a in pure form. Geometries of the double bonds in these derivatives were identified through the vinyl proton coupling constant values (Jcis = 10.6 Hz for 25a and Jcis = 10.4 Hz for 26a). Finally, hydrogenation of the mixtures of compounds 25 and 26 was carried out under standard H2–Pd/C conditions to produce the saturated derivatives 27 and 28 in 92% and 86% isolated yields, respectively (Scheme 3). Deprotection of 27 and 28 (TFA/H2O) gave the corresponding TFA salts 29 (96%) and 30 (97%). Treatment of both salts with 1% solution of sodium hydroxide in CH3OH afforded the desired D-ribo-C20-phytosphingosine (31) (86%) and its 2-epi-congener 32 (87%) (Scheme 4). The structure of 31 was

Figure 4. ORTEP structure of 22 showing the crystallographic numbering.

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20 a

a

OH BocN

O

HO

O

4.1. General methods O

23, 93%

O

24, 88%

b

b

BocN

H3C(H2C)13

4. Experimental

OH BocN

O

HO O

ious lengths of the chain, starting from the appropriate sugar templates.

21

O

O

BocN

H3C(H2C)13

O

25, 82%

O

O

O

26 69%

c

c

BocN

BocN

O

H3C(H2C)14

O

H3C(H2C)14 O

O

O

O

28, 86%

27, 92%

Scheme 3. Reagents and conditions: (a) Bu4NF, THF, 0 °C?rt; (b) (i) NaIO4, CH3OH– H2O, rt; (ii) LHMDS, C15H31PPh3Br, THF, rt; (c) H2, Pd/C, EtOH, rt.

further confirmed by conversion to its tetraacetyl derivative 33 (Scheme 4). 3. Conclusions In summary, a facile approach towards D-ribo-C20-phytosphingosine (31) and its 2-epi-congener 32 was achieved employing Overman rearrangement of the allylic trichloroacetimidates (Z)12i and (E)-13i as the key reaction. The alkyl chains of the truncated amino alcohols were extended to the desired length via a Wittig reaction, and the resulting unsaturated derivatives 25 and 26 with three contiguous stereogenic centres after catalytic hydrogenation and deprotection with TFA gave the final molecules. This work demonstrates that our synthetic protocol is probably applicable to the synthesis for other possible stereoisomers of 3 with var-

a

a OH

4.2. Ethyl (4S,5R,6R,2Z)-7-[(tert-butyldimethylsilyl)oxy]-6hydroxy-4,5-(isopropylidenedioxy)hept-2-enoate (8) and ethyl (4S,5R,6R,2E)-7-[(tert-butyldimethylsilyl)oxy]-6hydroxy-4,5-(isopropylidenedioxy)hept-2-enoate (9)

28

27

NH2

CH3(CH2)14

OH OH

OH

NH2

CH3(CH2)14

OH OH 32, 87%

31, 86%

OAc NHAc CH3(CH2)14

All commercial reagents were used in the highest available purity from Sigma–Aldrich, Fluka, E. Merck or Acros Organics without further purification. Solvents were dried and purified before use according to standard procedures. For flash column chromatography on silica gel, Kieselgel 60 (0.040–0.063 mm, 230–400 mesh, E. Merck) was used. Solvents for flash chromatography (hexane, ethyl acetate, methanol, dichloromethane) were distilled before using. Thin-layer chromatography was run on E. Merck Silica Gel 60 F254 analytical plates; detection was carried out with either ultraviolet light (254 nm) or by spraying with a solution of phosphomolybdic acid, a basic potassium permanganate solution, or a solution of concentrated H2SO4, with subsequent heating. 1H and 13 C NMR spectra were recorded in CDCl3, CD3OD and C6D6 on a Varian Mercury Plus 400 FT NMR (400.13 MHz for 1H and 100.6 MHz for 13C) or on a Varian Premium Compact 600 (599.87 MHz for 1H and 150.84 MHz for 13C) spectrometers using TMS as the internal reference. For 1H d-values are given in parts per million (ppm) relative to TMS (d = 0.0) or C6D6 (d = 7.15) and for 13C relative to CDCl3 (d = 77.0), CD3OD (d = 49.05) and C6D6 (d = 128.02). The multiplicity of the 13C NMR signals concerning the 13C–1H coupling was determined by the DEPT method. Chemical shifts (in ppm) and coupling constants (in Hz) were obtained by first-order analysis; assignments were derived from COSY and H/C correlation spectra. Infrared (IR) spectra were measured with a Nicolet Avatar 330 FTIR spectrometer as KBr pellets and expressed in m values (cm1). Optical rotations were measured on a P3002 Krüss or P-2000 Jasco polarimeter and reported as follows: [a]D (c in grams per 100 mL, solvent). Melting points were recorded on a Kofler hot block, and are uncorrected. Microwave reactions were carried out on the focused microwave system (CEM Discover). The temperature content of the vessel was monitored using a calibrated infrared sensor mounted under the vessel. At the end of all reactions the contents of vessel were cooled rapidly using a stream of compressed air. Small quantities of reagents (lL) were measured with appropriate syringes (Hamilton). All reactions were performed under an atmosphere of nitrogen, unless otherwise noted.

OAc OAc 33

Scheme 4. Reagents and conditions: (a) (i) TFA–H2O, rt; 29, 96%, 30, 97%; (ii) 1% NaOH, CH3OH, rt; (b) Ac2O, pyridine, DMAP, 94%.

To a solution of 715 (12.1 g, 39.7 mmol) in dry CH2Cl2 (266 mL) were successively added benzoic acid (0.49 g, 4.0 mmol) and the stabilized ylid, Ph3P@CHCO2Et, (24.9 g, 71.5 mmol) at room temperature, and the resulting mixture was stirred for 24 h at reflux. After the starting compound was completely consumed (judged by TLC), the reaction was stopped and allowed to cool to room temperature, and the solvent was evaporated. The residue was diluted with hexane (50 mL), the salts were filtered off, the solvent was removed under reduced pressure and the residue was chromatographed on silica gel (15:1 hexane–EtOAc) to afford 10.5 g (70%) of (Z)-8 and 3.44 g (23%) of (E)-9 as colourless oils. 1 Compound (Z)-8: ½a25 D +77.5 (c 0.57, CHCl3). H NMR (CDCl3, 400 MHz): d 0.06 (s, 6H, 2  CH3), 0.88 (s, 9H, 3  CH3), 1.27 (t, J = 7.1 Hz, 3H, CH3), 1.35 (s, 3H, CH3), 1.46 (s, 3H, CH3), 2.70 (d, J6,OH = 4.3 Hz, 1H, OH), 3.56–3.61 (m, 1H, H-6), 3.67 (dd,

M. Martinková et al. / Carbohydrate Research 346 (2011) 1728–1738

J7,7 = 10.1 Hz, J7,6 = 5.7 Hz, 1H, H-7), 3.76 (dd, J7,7 = 10.1 Hz, J7,6 = 3.0 Hz, 1H, H-7), 4.17 (q, J = 7.1 Hz, 2H, CH2), 4.24 (dd, J5,6 = 8.6 Hz, J5,4 = 6.3 Hz, H-5), 5.75 (ddd, J4,3 = 8.6 Hz, J4,5 = 6.3 Hz, J4,2 = 1.3 Hz, 1H, H-4), 5.96 (dd, J2,3 = 11.7 Hz, J2,4 = 1.3 Hz, 1H, H2), 6.27 (dd, J3,2 = 11.7 Hz, J3,4 = 8.6 Hz, 1H, H-3). 13C NMR (CDCl3, 100 MHz): d 5.4 (2  CH3), 14.1 (CH3), 18.3 (C), 25.4 (CH3), 25.9 (3  CH3), 27.9 (CH3), 60.5 (CH2), 64.3 (CH2), 70.0 (CH), 73.8 (CH), 78.0 (CH), 109.1 (C), 122.2 (CH), 144.5 (CH), 166.1 (C@O). Anal. Calcd for C18H34O6Si: C, 57.72; H, 9.15. Found: C, 57.85; H, 9.03. 1 Compound (E)-9: ½a25 D +34.8 (c 0.46, CHCl3). H NMR (CDCl3, 400 MHz): d 0.08 (s, 3H, CH3), 0.09 (s, 3H, CH3), 0.91 (s, 9H, 3  CH3), 1.29 (t, J = 7.1 Hz, 3H, CH3), 1.37 (s, 3H, CH3), 1.49 (s, 3H, CH3), 2.55 (d, J6,OH = 5.6 Hz, 1H, OH), 3.55 (dtd, J6,5 = 9.4 Hz, J6,OH = 5.6 Hz, J6,7 = 5.5 Hz, J6,7 = 3.2 Hz, 1H, H-6), 3.66 (dd, J7,7 = 10.0 Hz, J7,6 = 5.5 Hz, 1H, H-7), 3.79 (dd, J7,7 = 10.0 Hz, J7,6 = 3.2 Hz, 1H, H-7), 4.13 (dd, J5,6 = 9.4 Hz, J5,4 = 6.7 Hz, 1H, H-5), 4.21 (q, J = 7.1 Hz, 2H, CH2), 4.85 (ddd, J4,5 = 6.7 Hz, J4,3 = 4.9 Hz, J4,2 = 1.7 Hz, 1H, H-4), 6.15 (dd, J2,3 = 15.6 Hz, J2,4 = 1.7 Hz, 1H, H2), 7.12 (dd, J3,2 = 15.6 Hz, J3,4 = 4.9 Hz, 1H, H-3). 13C NMR (CDCl3, 100 MHz): d 5.5 (CH3), 5.4 (CH3), 14.2 (CH3), 18.3 (C), 25.3 (CH3), 25.9 (3  CH3), 27.6 (CH3), 60.4 (CH2), 64.3 (CH2), 69.6 (CH), 76.9 (CH), 77.5 (CH), 109.5 (C), 122.3 (CH), 143.9 (CH), 166.2 (C@O). Anal. Calcd for C18H34O6Si: C, 57.72; H, 9.15. Found: C, 57.91; H, 9.01. 4.3. Ethyl (4S,5S,6R,2Z)-6,7-bis[(tert-butyldimethylsilyl)oxy]4,5-(isopropylidenedioxy)hept-2-enoate (10) To a solution of (Z)-8 (10.4 g, 27.9 mmol) in dry DMF (18.5 mL) were successively added imidazole (3.79 g, 55.7 mmol) and tertbutydimethylsilyl chloride (5.88 g, 39.0 mmol) at room temperature, and the resulting mixture was stirred at 70 °C. After 4 h no starting material was detected (judged by TLC), the stirring was stopped and the mixture was allowed to cool to room temperature. The mixture was then partitioned between ice water (190 mL) and Et2O (240 mL). The organic layer was dried over Na2SO4, the solvent was evaporated in vacuo, and the residue was purified by flash chromatography on silica gel (70:1 hexane–EtOAc). This procedure yielded 12.9 g (95%) of (Z)-10 as a colourless oil: ½a25 D +81.5 (c 0.26, CHCl3). 1H NMR (CDCl3, 400 MHz): d 0.04 (s, 6H, 2  CH3), 0.06 (s, 6H, 2  CH3), 0.87 (s, 9H, 3  CH3), 0.89 (s, 9H, 3  CH3), 1.29 (t, J = 7.1 Hz, 3H, CH3), 1.36 (s, 3H, CH3), 1.48 (s, 3H, CH3), 3.56 (dd, J7,7 = 10.7 Hz, J7,6 = 5.6 Hz, 1H, H-7), 3.60 (dd, J7,7 = 10.7 Hz, J7,6 = 4.5 Hz, 1H, H-7), 3.83 (m, 1H, H-6), 4.17 (q, J = 7.1 Hz, 2H, CH2), 4.45 (dd, J5,4 = 7.1 Hz, J5,6 = 3.7 Hz, 1H, H-5), 5.77 (ddd, J4,3 = 8.7 Hz, J4,5 = 7.1 Hz, J4,2 = 1.3 Hz, 1H, H-4), 5.87 (dd, J2,3 = 11.6 Hz, J2,4 = 1.3 Hz, 1H, H-2), 6.39 (dd, J3,2 = 11.6 Hz, J3,4 = 8.7 Hz, 1H, H-3). 13C NMR (CDCl3, 100 MHz): d 5.5 (CH3), 5.4 (CH3), 4.6 (CH3), 4.3 (CH3), 14.2 (CH3), 18.1 (C), 18.4 (C), 24.8 (CH3), 25.9 (3  CH3), 26.0 (3  CH3), 27.3 (CH3), 60.3 (CH2), 65.0 (CH2), 72.8 (CH), 73.2 (CH), 79.6 (CH), 108.3 (C), 120.7 (CH), 146.4 (CH), 165.7 (C@O). Anal. Calcd for C24H48O6Si2: C, 58.97; H, 9.90. Found: C, 58.90; H, 9.97. 4.4. Ethyl (4S,5S,6R,2E)-6,7-bis[(tert-butyldimethylsilyl)oxy]4,5-(isopropylidenedioxy)hept-2-enoate (11) Using the same procedure as described for the preparation of derivative (Z)-10, ester (E)-9 (2.21 g, 5.90 mmol), imidazole (0.80 g, 11.8 mmol) and TBDMSCl (1.25 g, 8.29 mmol) afforded after flash chromatography on silica gel (70:1 hexane–EtOAc) 2.05 g (71%) of compound (E)-11 as a colourless oil: ½a25 D 60.6 (c 0.20, CHCl3). 1H NMR (CDCl3, 400 MHz): d 0.06 (s, 6H, 2  CH3), 0.10 (s, 3H, CH3), 0.11 (s, 3H, CH3), 0.89 (s, 9H, 3  CH3), 0.90 (s, 9H, 3  CH3), 1.28 (t, J = 7.1 Hz, 3H, CH3), 1.37 (s, 3H, CH3), 1.50 (s, 3H, CH3), 3.68 (dd, J7,7 = 10.9 Hz, J7,6 = 3.8 Hz, 1H, H-7), 3.72

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(dd, J7,7 = 10.9 Hz, J7,6 = 3.3 Hz, H-7), 3.78 (dt, J6,5 = 7.1 Hz, J6,7 = 3.5 Hz, J6,7 = 3.5 Hz, 1H, H-6), 4.19 (q, J = 7.1 Hz, 2H, CH2), 4.33 (t, J5,6 = 6.9 Hz, J5,4 = 6.9 Hz, 1H, H-5), 4.74 (1H, ddd, J4,5 = 6.6 Hz, J4,3 = 5.1 Hz, J4,2 = 1.6 Hz, H-4), 6.08 (dd, J2,3 = 15.6 Hz, J2,4 = 1.6 Hz, 1H, H-2), 7.10 (dd, J3,2 = 15.6 Hz, J3,4 = 5.1 Hz, 1H, H3). 13C NMR (CDCl3, 100 MHz): d 5.6 (CH3), 5.4 (CH3), 4.8 (CH3), 3.9 (CH3), 14.3 (CH3), 18.2 (C), 18.4 (C), 25.4 (CH3), 25.9 (6  CH3), 27.7 (CH3), 60.3 (CH2), 64.6 (CH), 72.5 (CH), 76.5 (CH), 77.6 (CH), 108.8 (C), 122.3 (CH), 145.5 (CH), 166.2 (C@O). Anal. Calcd for C24H48O6Si2: C, 58.97; H, 9.90. Found: C, 59.05; H, 9.84. 4.5. (4S,5S,6R,2Z)-6,7-Bis[(tert-butyldimethylsilyl)oxy]-4,5(isopropylidenedioxy)hept-2-en-1-ol (12) Diisobutylaluminum hydride (79.5 mL of a 1.2 M toluene solution, 95.4 mmol) was added dropwise to a solution of ester (Z)10 (12.9 g, 26.4 mmol) in dry CH2Cl2 (120 mL) for 1 h at 15 °C. The resulting mixture was stirred at the same temperature for another 15 min and then quenched with MeOH (18.7 mL). The mixture was warmed to room temperature and poured into a 30% aq K/Na tartrate (397 mL). After stirring for 1 h, the mixture was then extracted with CH2Cl2 (3  267 mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated under reduced pressure, and the residue was subjected to flash chromatography on silica gel (7:1 hexane–EtOAc) to give 11.6 g (98%) of alcohol 1 (Z)-12 as a colourless oil: ½a25 D 24.8 (c 0.23, CHCl3). H NMR (CDCl3, 400 MHz): d 0.06 (s, 9H, 3  CH3), 0.08 (s, 3H, CH3), 0.87 (s, 9H, 3  CH3), 0.90 (s, 9H, 3  CH3), 1.34 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.86 (t, J1,OH = 5.5 Hz, 1H, OH), 3.66 (dd, J7,7 = 10.8 Hz, J7,6 = 4.7 Hz, 1H, H-7), 3.72 (dd, J7,7 = 10.8 Hz, J7,6 = 3.9 Hz, 1H, H7), 3.86 (ddd, J6,5 = 6.4 Hz, J6,7 = 4.7 Hz, J6,7 = 3.9 Hz, 1H, H-6), 4.16–4.25 (m, 2H, H-1, H-5), 4.30–4.36 (m, 1H, H-1), 4.95 (m, 1H, H-4), 5.79–5.87 (m, 2H, H-2, H-3). 13C NMR (CDCl3, 100 MHz): d 5.4 (2  CH3), 4.5 (CH3), 4.0 (CH3), 18.2 (C), 18.4 (C), 25.3 (CH3), 25.9 (6  CH3), 27.8 (CH3), 58.7 (CH2), 64.8 (CH2), 72.4 (CH), 72.7 (CH), 77.7 (CH), 108.1 (C), 128.8 (CH), 132.5 (CH). Anal. Calcd for C22H46O5Si2: C, 59.14; H, 10.38. Found: C, 59.03; H, 10.48. 4.6. (4S,5S,6R,2E)-6,7-Bis[(tert-butyldimethylsilyl)oxy]-4,5(isopropylidenedioxy)hept-2-en-1-ol (13) According to the same procedure described for the preparation of (Z)-12, ester (E)-11 (2.04 g, 4.17 mmol) was transformed to compound (E)-13 (1.79 g, 96%, 7:1 hexane–EtOAc); ½a25 D 62.3 (c 0.39, CHCl3). 1H NMR (CDCl3, 400 MHz): d 0.05 (s, 6H, 2  CH3), 0.06 (s, 3H, CH3), 0.09 (s, 3H, CH3), 0.88 (s, 9H, 3  CH3), 0.90 (s, 9H, 3  CH3), 1.35 (s, 3H, CH3), 1.47 (s, 3H, CH3), 3.67 (dd, J7,7 = 10.8 Hz, J7,6 = 4.3 Hz, 1H, H-7), 3.73 (dd, J7,7 = 10.8 Hz, J7,6 = 3.5 Hz, 1H, H-7), 3.81 (ddd, J6,5 = 6.9 Hz, J6,7 = 4.3 Hz, J6,7 = 3.5 Hz, H-6), 4.17 (m, 2H, 2  H-1), 4.24 (dd, J5,6 = 6.9 Hz, J5,4 = 6.4 Hz, 1H, H5), 4.60–4.63 (m, 1H, H-4), 5.89–5.91 (m, 2H, H-2, H-3). 13C NMR (CDCl3, 100 MHz): d 5.5 (CH3), 5.4 (CH3), 4.6 (CH3), 3.9 (CH3), 18.2 (C), 18.4 (C), 25.4 (CH3), 25.9 (6  CH3), 27.9 (CH3), 63.1 (CH2), 64.7 (CH2), 72.5 (CH), 77.5 (CH), 77.9 (CH), 108.1 (C), 128.4 (CH), 132.6 (CH). Anal. Calcd for C22H46O5Si2: C, 59.14; H, 10.38. Found: C, 59.29; H, 10.20. 4.7. N-{[(3S,4S,5S,6R)-6,7-Bis[(tert-butyldimethylsilyl)oxy]-4,5(isopropylidenedioxy)hept-1-en-3-yl]}-2,2,2-trichloroacetamide (14) and N-{[(3R,4S,5S,6R)-6,7-bis[(tert-butyldimethylsilyl)oxy]4,5-(isopropylidenedioxy)hept-1-en-3-yl]}-2,2,2-trichloroacetamide (15) 4.7.1. Microwave-assisted synthesis To a suspension of NaH (1.13 g, 47.1 mmol, 60% dispersion in mineral oil, freed of oil with anhydrous THF) in THF (34.5 mL),

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pre-cooled to 0 °C, was added alcohol (Z)-12 (9.49 g, 21.2 mmol) in THF (34.5 mL). After stirring at 0 °C for 30 min, trichloroacetonitrile (2.59 mL, 25.8 mmol) was added dropwise, and the resulting mixture was stirred for further 20 min at the same temperature. The insoluble materials were removed by filtration through a small pad of Celite, the solvent was removed under reduced pressure, and the crude trichloroacetimidate (Z)-12i was used immediately in the next step without further purification to avoid problems of its possible instability. Crude imidate (Z)-12i (0.25 g, 0.423 mmol) was weighed into a 10-mL glass pressure microwave tube equipped with a magnetic stirbar. o-Xylene (5.7 mL) and K2CO3 (66.8 mg, 0.483 mmol) were added, the tube was closed with a silicone septum, and the reaction mixture was subjected to microwave irradiation (Table 1). Evaporation of the solvent and chromatography on silica gel (70:1 hexane–EtOAc) gave 0.108 g (43%) of 14 and 54 mg (21.6%) of 15 (Table 1, entry 4) This procedure was repeated several times using the same amount of the starting material. Using the same procedure, alcohol (E)-13 (1.78 g 3.98 mmol) was converted to a crude imidate (E)-13i that after microwave irradiation (Table 1) and chromatography on silica gel (70:1 hexane– EtOAc) afforded the corresponding rearranged products 14 (0.65 g, 34%) and 15 (0.38 g, 20%; Table 1, entry 8). 4.7.2. Conventional method To a solution of the crude imidate (Z)-12i or E)-13i (0.25 g, 0.423 mmol) in o-xylene (5.7 mL) was added K2CO3 (66.8 mg, 0.483 mmol), and the resulting mixture was heated in a sealed tube (Table 1, entries 1, 3, 5, and 7). Evaporation of the solvent and chromatography on silica gel (70:1 hexane–EtOAc) afforded trichloroacetamides 14 and 15 (for the combined yields, see, Table 1, entries 1, 3, 5, and 7). Diastereoisomer 14: mp 27–28 °C; ½a25 D 72.6 (c 0.20, CHCl3). 1 H NMR (CDCl3, 400 MHz): d 0.06 (s, 6H, 2  CH3), 0.15 (s, 3H, CH3), 0.20 (s, 3H, CH3), 0.90 (s, 18H, 6  CH3), 1.35 (s, 3H, CH3), 1.47 (s, 3H, CH3), 3.70 (dd, J7,7 = 11.1 Hz, J7,6 = 3.5 Hz, 1H, H-7), 3.80 (dd, J7,7 = 11.1 Hz, J7,6 = 2.8 Hz, H-7), 4.10 (dt, J6,5 = 8.6 Hz, J6,7 = 3.2 Hz, J6,7 = 3.2 Hz, 1H, H-6), 4.30 (dd, J4,5 = 6.7 Hz, J4,3 = 2.2 Hz, 1H, H-4), 4.38 (dd, J5,6 = 8.6 Hz, J5,4 = 6.7 Hz, 1H, H-5), 4.72–4.76 (m, 1H, H-3), 5.28 (m, 1H, H-1cis), 5.35 (td, J1trans,2 = 17.3 Hz, J1trans,1cis = 1.2 Hz, J1trans,3 = 1.2 Hz, 1H, H-1trans), 6.03 (ddd, J2,1trans = 17.3 Hz, J2,1cis = 10.5 Hz, J2,3 = 5.8 Hz, 1H, H-2), 7.03 (br d, J3,NH = 8.7 Hz, 1H, NH). 13C NMR (CDCl3, 100 MHz): d 5.6 (CH3), 5.4 (CH3), 4.5 (CH3), 3.6 (CH3), 18.2 (C), 18.4 (C), 25.1 (CH3), 25.9 (6  CH3), 26.3 (CH3), 54.1 (CH), 64.5 (CH2), 71.5 (CH), 75.6 (CH), 78.3 (CH), 92.7 (C), 108.9 (C), 118.3 (CH2), 134.0 (CH), 160.7 (C@O). Anal. Calcd for C24H46Cl3NO5Si2: C, 48.76; H, 7.84; N, 2.37. Found: C, 48.85; H, 7.64; N, 2.21. Diastereoisomer 15: a colourless oil; ½a25 D +26.2 (c 0.25, CHCl3). 1 H NMR (CDCl3, 400 MHz): d 0.04 (s, 6H, 2  CH3), 0.15 (s, 3H, CH3), 0.19 (s, 3H, CH3), 0.89 (s, 9H, 3  CH3), 0.90 (s, 9H, 3  CH3), 1.38 (s, 3H, CH3), 1.55 (s, 3H, CH3), 3.67 (dd, J7,7 = 11.3 Hz, J7,6 = 3.8 Hz, 1H, H-7), 3.76–3.82 (m, 2H, H-6, H-7), 4.32–4.37 (m, 2H, H-4, H-5), 4.76 (m, 1H, H-3), 5.23–5.30 (m, 2H, 2  H-1), 5.83 (ddd, J2,1trans = 17.1 Hz, J2,1cis = 10.4 Hz, J2,3 = 5.8 Hz, 1H, H-2), 7.13 (br d, J3,NH = 8.0 Hz, 1H, NH). 13C NMR (100 MHz, CDCl3): d 5.6 (CH3), 5.4 (CH3), 4.7 (CH3), 3.4 (CH3), 18.2 (C), 18.4 (C), 23.9 (CH3), 25.9 (3  CH3), 26.0 (3  CH3), 26.6 (CH3), 52.5 (CH), 64.6 (CH2), 72.2 (CH), 75.4 (CH), 77.9 (CH), 92.8 (C), 108.4 (C), 116.7 (CH2), 134.9 (CH), 160.6 (C@O). Anal. Calcd for C24H46Cl3NO5Si2: C, 48.76; H, 7.84; N, 2.37. Found: C, 48.60; H, 7.91; N, 2.25. 4.8. tert-Butyl [(3S,4S,5S,6R)-6,7-bis[(tert-butyldimethylsilyl)oxy]-4,5-(isopropylidenedioxy)hept-1-en-3-yl]carbamate (16) To a solution of 14 (4.33 g, 7.32 mmol) in EtOH (39 mL) was added a 6 M aq NaOH (36.6 mL) at room temperature. After stirring

for 8 h, the resulting mixture was poured into Et2O (80 mL). The aqueous phase was extracted with further portions of Et2O (2  80 mL), the combined organic layers were dried over Na2SO4, the solvent was evaporated in vacuo, and the crude amine was immediately used in the subsequent reaction. To a solution of the crude amine in CH2Cl2 (9.1 mL) were successively added Et3N (1.04 mL, 7.40 mmol) and Boc2O (3.20 g, 14.66 mmol) at room temperature. After 17 h, no starting material was detected (TLC) in the reaction mixture, which was then diluted with CH2Cl2 (98 mL) and washed with a 1 M aq solution of KHSO4 (66 mL) and a 1 M aq solution of NaHCO3 (66 mL). The organic layer was dried over Na2SO4, the solvent was removed under reduced pressure, and the residue was subjected to flash chromatography on silica gel (70:1 hexane–EtOAc) to give 2.90 g (73%) of crystalline 1 carbamate 16: mp 51–52 °C; ½a25 D 24.6 (c 0.30, CHCl3). H NMR (CDCl3, 400 MHz): d 0.05 (s, 3H, CH3), 0.06 (s, 3H, CH3), 0.15 (s, 3H, CH3), 0.18 (s, 3H, CH3), 0.90 (s, 18H, 6  CH3), 1.32 (s, 3H, CH3), 1.43 (s, 12H, 4  CH3), 3.68 (dd, J7,7 = 11.0 Hz, J7,6 = 4.2 Hz, 1H, H-7), 3.79 (dd, J7,7 = 11.0 Hz, J7,6 = 3.2 Hz, 1H, H-7), 4.10 (ddd, J6,5 = 7.9 Hz, J6,7 = 4.2 Hz, J6,7 = 3.2 Hz, 1H, H-6), 4.19 (dd, J4,5 = 6.4 Hz, J4,3 = 3.6 Hz, 1H, H-4), 4.28 (dd, J5,6 = 7.9 Hz, J5,4 = 6.4 Hz, 1H, H-5), 4.36 (m, 1H, H-3), 5.07 (m, 1H, NH), 5.18 (dt, J1cis,2 = 10.5 Hz, J1trans,1cis = 1.5 Hz, J1cis,3 = 1.5 Hz, 1H, H-1cis), 5.25 (m, 1H, H-1trans), 5.95 (ddd, J2,1trans = 17.3 Hz, J2,1cis = 10.5 Hz, J2,3 = 5.6 Hz, 1H, H-2). 13C NMR (CDCl3, 100 MHz): d 5.5 (CH3), 5.4 (CH3), 4.4 (CH3), 3.7 (CH3), 18.2 (C), 18.4 (C), 25.3 (CH3), 25.9 (3  CH3), 26.0 (3  CH3), 26.5 (CH3), 28.4 (3  CH3), 53.4 (CH), 64.8 (CH2), 71.7 (CH), 76.4 (CH), 79.1 (CH), 79.3 (C), 108.3 (C), 116.4 (CH2), 136.5 (CH), 154.9 (C@O). Anal. Calcd for C27H55NO6Si2: C, 59.40; H, 10.16; N, 2.57. Found: C, 59.59; H, 10.04; N, 2.45. 4.9. tert-Butyl [(3R,4S,5S,6R)-6,7-bis[(tert-butyldimethylsilyl)oxy]-4,5-(isopropylidenedioxy)hept-1-en-3-yl]carbamate (17) According to the same procedure described for the preparation of 16, compound 15 (2.16 g, 3.65 mmol) was converted into carbamate 17 (1.83 g, 92%, colourless oil, 70:1 hexane–EtOAc): ½a25 D 50.1 (c 0.29, CHCl3). 1H NMR (CDCl3, 400 MHz): d 0.06 (s, 6H, 2  CH3), 0.16 (s, 3H, CH3), 0.18 (s, 3H, CH3), 0.89 (s, 9H, 3  CH3), 0.90 (s, 9H, 3  CH3), 1.34 (s, 3H, CH3), 1.43 (s, 9H, 3  CH3), 1.50 (s, 3H, CH3), 3.70 (m, 1H, H-7), 3.81 (dd, J7,7 = 11.1 Hz, J7,6 = 1.9 Hz, 1H, H-7), 4.00 (m, 1H, H-6), 4.24 (m, 1H, H-4), 4.29 (dd, J5,6 = 8.9 Hz, J5,4 = 7.0 Hz, H-5), 4.45 (m, 1H, H3), 4.89 (br d, J3,NH = 8.1 Hz, 1H, NH), 5.14–5.21 (m, 2H, 2  H-1), 5.81 (ddd, J2,1trans = 16.8 Hz, J2,1cis = 10.3 Hz, J2,3 = 5.5 Hz, 1H, H-2). 13 C NMR (CDCl3, 100 MHz): d 5.6 (CH3), 5.3 (CH3), 4.7 (CH3), 3.6 (CH3), 18.2 (C), 18.4 (C), 24.2 (CH3), 25.9 (3  CH3), 26.0 (3  CH3), 26.5 (CH3), 28.4 (3  CH3), 51.6 (CH), 64.9 (CH2), 72.0 (CH), 75.8 (CH), 78.5 (CH), 79.0 (C), 108.0 (C), 115.0 (CH2), 137.5 (CH), 154.9 (C@O). Anal. Calcd for C27H55NO6Si2: C, 59.40; H, 10.16; N, 2.57. Found: C, 59.53; H, 10.29; N, 2.68. 4.10. (2S,3S,4S,5R)-2-[(tert-Butoxycarbonyl)amino]-5,6-bis[(tert-butyldimethylsilyl)oxy]-3,4-(isopropylidenedioxy)hexanoic acid (22) To a suspension of 17 (0.49 g, 0.90 mmol) in 2:2:3 CCl4–CH3CN– H2O (8.7 mL) were successively added NaIO4 (0.960 g, 4.49 mmol) and ruthenium trichloride hydrate (8.90 mg, 0.043 mmol) at room temperature. After 18 h no starting compound was detected (TLC) in the reaction mixture, which was then extracted with CH2Cl2 (2  20 mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated in vacuo, and the residue was purified through a short column of silica gel (3:1 hexane–EtOAc) to afford 0.30 g (59%) of crystalline acid 22: mp 154–155 °C; ½a25 D +122.7

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(c 0.18, CHCl3). IR (KBr) 3400, 2966, 2930, 2858, 1752, 1715, 1498, 1369, 1256, 1212, 1162, 1096, 1062. 1H NMR (CDCl3, 400 MHz, 60 °C): d 0.06 (s, 6H, 2  CH3), 0.15 (s, 3H, CH3), 0.17 (s, 3H, CH3), 0.91 (s, 18H, 6  CH3), 1.35 (s, 3H, CH3), 1.45 (s, 9H, 3  CH3), 1.49 (s, 3H, CH3), 3.71 (dd, J6,6 = 11.1 Hz, J6,5 = 3.5 Hz, 1H, H-6), 3.82 (dd, J6,6 = 11.1 Hz, J6,5 = 2.4 Hz, 1H, H-6), 4.00 (m, 1H, H-5), 4.36 (dd, J4,5 = 8.8 Hz, J4,3 = 6.9 Hz, 1H, H-4), 4.60 (m, 1H, H-2), 4.70 (m, 1H, H-3), 5.08 (1H, m, NH). 13C NMR (CD3OD, 100 MHz): d 5.2 (CH3), 5.0 (CH3), 4.3 (CH3), 3.0 (CH3), 19.2 (C), 19.4 (C), 24.5 (CH3), 26.6 (3  CH3), 26.7 (3  CH3), 26.8 (CH3), 28.8 (3  CH3), 54.7 (CH), 66.3 (CH2), 73.9 (CH), 77.2 (CH), 78.0 (CH), 81.0 (C), 109.9 (C), 157.3 (C@O), 174.3 (COOH). Anal. Calcd for C26H53NO8Si2: C, 55.38; H, 9.47; N, 2.48. Found: C, 55.49; H, 9.57; N, 2.33. 4.11. tert-Butyl [(2S,3S,4S,5R)-5,6-bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-3,4-(isopropylidenedioxy)hexan-2-yl]carbamate (18) Ozone was introduced into a solution of 16 (2.75 g, 5.04 mmol) in 5:1 MeOH–CH2Cl2 (189 mL) at 78 °C for 1 h. This resulted in the formation of a slightly blue solution. Dry N2 was passed through the cold solution in order to remove the excess ozone. Then NaBH4 (0.86 g, 22.7 mmol) was added, and the resulting mixture was stirred at 78 °C for 30 min. The mixture was allowed to warm to room temperature, and the stirring was continual for another 30 min. The solvent was evaporated, and the residue was partitioned between EtOAc (125 mL) and a satd aq solution of NH4Cl (75 mL). The aqueous phase was extracted with further portions of EtOAc (2  125 mL), the combined organic layers were dried over Na2SO4, the solvent was evaporated, and the residue was chromatographed on silica gel (7:1 hexane–EtOAc) to yield 2.52 g (91%) of 18 as a colourless oil: ½a25 D +78.7 (c 0.30, CHCl3). 1 H NMR (CDCl3, 400 MHz): d 0.07 (s, 6H, 2  CH3), 0.15 (s, 3H, CH3), 0.16 (s, 3H, CH3), 0.90 (s, 18H, 6  CH3), 1.33 (s, 3H, CH3), 1.43 (s, 9H, 3  CH3), 1.44 (s, 3H, CH3), 3.40 (br s, 1H, OH), 3.71 (dd, J6,6 = 10.8 Hz, J6,5 = 4.5 Hz, 1H, H-6), 3.75–3.84 (m, 4H, 2  H1, H-2, H-6), 4.09 (td, J5,4 = 5.9 Hz, J5,6 = 4.5 Hz, J5,6 = 4.5 Hz, 1H, H-5), 4.23–4.29 (m, 2H, H-3, H-4), 5.48 (m, 1H, NH). 13C NMR (CDCl3, 100 MHz): d 5.5 (CH3), 5.4 (CH3), 4.5 (CH3), 4.0 (CH3), 18.2 (C), 18.3 (C), 25.1 (CH3), 25.9 (6  CH3), 27.0 (CH3), 28.3 (3  CH3), 52.8 (CH), 63.9 (CH2), 64.6 (CH2), 72.4 (CH), 76.8 (CH), 77.4 (CH), 79.7 (C), 108.0 (C), 156.2 (C@O). Anal. Calcd for C26H55NO7Si2: C, 56.79; H, 10.08; N, 2.55. Found: C, 56.64; H, 10.16; N, 2.49. 4.12. tert-Butyl [(2R,3S,4S,5R)-5,6-bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-3,4-(isopropylidenedioxy)hexan-2-yl]carbamate (19) Using the same procedure as described for the preparation of 18, compound 17 (1.33 g, 2.44 mmol) was converted to alcohol 19 (1.10 g, 82%, a colourless oil, 7:1 hexane–EtOAc): ½a25 D +56.6 (c 0.17, CHCl3). 1H NMR (CDCl3, 400 MHz): d 0.06 (s, 6H, 2  CH3), 0.15 (s, 3H, CH3), 0.18 (s, 3H, CH3), 0.89 (s, 9H, 3  CH3), 0.90 (s, 9H, 3  CH3), 1.34 (s, 3H, CH3), 1.44 (s, 9H, 3  CH3), 1.49 (s, 3H, CH3), 2.59 (m, 1H, OH), 3.65–3.75 (m, 3H, 2  H-1, H-6), 3.80 (dd, J6,6 = 11.1 Hz, J6,5 = 2.1 Hz, 1H, H-6), 3.94 (m, 1H, H-5), 4.01 (m, 1H, H-2), 4.26–4.33 (m, 2H, H-3, H-4), 4.99 (br d, 1H, J2,NH = 7.4 Hz, NH). 13C NMR (CDCl3, 100 MHz): d 5.6 (CH3), 5.3 (CH3), 4.8 (CH3), 3.6 (CH3), 18.2 (C), 18.4 (C), 24.3 (CH3), 25.9 (3  CH3), 26.0 (3  CH3), 26.6 (CH3), 28.4 (3  CH3), 51.2 (CH), 64.9 (CH2), 66.0 (CH2), 72.1 (CH), 75.9 (CH), 76.5 (CH), 79.6 (C), 108.2 (C), 156.2 (C@O). Anal. Calcd for C26H55NO7Si2: C, 56.79; H, 10.08; N, 2.55. Found: C, 56.88; H, 10.00; N, 2.62.

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4.13. tert-Butyl (4S)-4[(10 S,20 S,30 R)-30 ,40 -bis[(tert-butyldimethylsilyl)oxy]-10 ,20 -(isopropylidenedioxy)butyl]-2,2-dimethyloxazolidine-3-carboxylate (20) To a solution of 18 (2.50 g, 4.55 mmol) in dry benzene (15.8 mL) were added 2,2-dimethoxypropane (7.34 mL, 59.2 mmol) and camphor sulfonic acid (CSA, 15.8 mg, 0.068 mmol) at room temperature, and the resulting mixture was stirred for 2 h at reflux. After the starting material was completely consumed (judged by TLC), the reaction mixture was allowed to cool to room temperature, then poured into H2O (53 mL). The aqueous phase was extracted with Et2O (2  80 mL). The combined organic layers were dried over Na2SO4, the solvent was removed under reduced pressure, and the residue was purified by flash chromatography on silica gel (40:1 hexane–EtOAc) to yield 2.52 g (94%) of compound 20 as a colourless oil: ½a25 40.4 (c 0.45, CHCl3). 1H NMR (CDCl3, D 400 MHz, 60 °C): d 0.06 (s, 6H, 2  CH3), 0.12 (s, 3H, CH3), 0.13 (s, 3H, CH3), 0.90 (s, 9H, 3  CH3), 0.92 (s, 9H, 3  CH3), 1.33 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.48 (br s, 12H, 4  CH3), 1.58 (s, 3H, CH3), 3.69 (dd, J40 ,40 = 10.7 Hz, J40 ,30 = 4.5 Hz, 1H, H-40 ), 3.78 (m, 1H, H-40 ), 3.85–3.90 (m, 2H, H-30 , H-5), 4.16–4.26 (m, 2H, H-5, H-4), 4.30 (t, J20 ,30 = 6.5 Hz, J20 ,10 = 6.5 Hz, 1H, H-20 ), 4.61 (m, 1H, H-10 ). 13 C NMR (CDCl3, 100 MHz, 60 °C): d 5.4 (2  CH3), 4.2 (2  CH3), 18.3 (C), 18.5 (C), 25.0 (CH3), 25.9 (3  CH3), 26.2 (4  CH3), 26.4 (CH3), 28.6 (4  CH3), 58.0 (CH), 65.0 (2  CH2), 72.9 (CH), 76.4 (2  CH), 79.7 (C), 93.8 (C), 107.6 (C), 152.1 (C@O). Anal. Calcd for C29H59NO7Si2: C, 59.04; H, 10.08; N, 2.37. Found: C, 59.15; H, 10.01; N, 2.46. 4.14. tert-Butyl (4R)-4[(10 S,20 S,30 R)-30 ,40 -bis[(tert-butyldimethylsilyl)oxy]-10 ,20 -(isopropylidenedioxy)butyl]-2,2-dimethyloxazolidine-3-carboxylate (21) According to the same procedure described for the preparation of 20, compound 19 (0.970 g, 1.76 mmol) and 2,2-DMP (2.84 mL, 22.9 mmol) afforded after flash chromatography on silica gel (50:1 hexane–EtOAc) 0.82 g (79%) of compound 21 as a colourless oil: 1 ½a25 D +13.8 (c 0.38, CHCl3). H NMR (CDCl3, 400 MHz): d 0.05 (s, 3H, CH3), 0.06 (s, 3H, CH3), 0.11 (s, 3H, CH3), 0.12 (s, 3H, CH3), 0.90 (s, 18H, 6  CH3), 1.29 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.47 (s, 9H, 3  CH3), 1.53 (s, 3H, CH3), 1.63 (s, 3H, CH3), 3.67 (m, 1H, H-5), 3.78–3.83 (m, 3H, H-30 , 2  H-40 ), 3.88 (m, 1H, H-5), 3.97 (m, 1H, H-20 ), 4.24 (m, 1H, H-10 ), 4.40 (dd, J4,10 = 10.5 Hz, J4,5 = 5.0 Hz, 1H, H-4). 13C NMR (CDCl3, 100 MHz): d 5.5 (CH3), 5.4 (CH3), 3.6 (CH3), 3.5 (CH3), 18.5 (2  C), 23.4 (CH3), 25.1 (CH3), 25.9 (3  CH3), 26.2 (3  CH3), 27.8 (CH3), 28.1 (CH3), 28.3 (3  CH3), 55.3 (CH), 64.4 (CH2), 66.9 (CH2), 72.7 (CH), 76.7 (CH), 78.3 (CH), 79.3 (C), 93.9 (C), 107.6 (C), 152.5 (C@O). Anal. Calcd for C29H59NO7Si2: C, 59.04; H, 10.08; N, 2.37. Found: C, 59.14; H, 9.95; N, 2.49. 4.15. tert-Butyl (4S)-4[(10 S,20 R,30 R)-30 ,40 -dihydroxy-10 ,20 (isopropylidenedioxy)butyl]-2,2-dimethyloxazolidine-3carboxylate (23) To a solution of 20 (2.28 g, 3.87 mmol) in dry THF (38.4 mL) was added dropwise a 1 M solution of Bu4NF (7.69 mL, 7.73 mmol) in THF at 0 °C. The resulting reaction mixture was stirred for a further 10 min at 0 °C and then at room temperature for 1 h 50 min. The solvent was evaporated in vacuo, and the residue was partitioned between EtOAc (39 mL) and water (44.5 mL). The aqueous phase was extracted with further portions of EtOAc (2  20 mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated, and the crude product was subjected to flash chromatography on silica gel (1:1 hexane–EtOAc) to afford 1.30 g (93%) of 1 diol 23 as a colourless syrup: ½a25 D 22.6 (c 0.50, CHCl3). H NMR (CDCl3, 400 MHz, 60 °C): d 1.33 (s, 3H, CH3), 1.44 (s, 3H, CH3),

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1.49 (s, 9H, 3  CH3), 1.50 (s, 3H, CH3), 1.58 (s, 3H, CH3), 3.62–3.65 (m, 1H, H-30 ), 3.69 (dd, J40 ,40 = 11.0 Hz, J40 ,30 = 5.5 Hz, 1H, H-40 ), 3.82 (dd, J40 ,40 = 11.0 Hz, J40 ,30 = 3.5 Hz, 1H, H-40 ), 3.97 (dd, J5,5 = 8.9 Hz, J5,4 = 6.8 Hz, 1H, H-5), 4.16 (m, 1H, H-20 ), 4.21 (dd, J5,5 = 8.9 Hz, J5,4 = 2.4 Hz, 1H, H-5), 4.43–4.47 (m, 2H, H-4, H-10 ). 13C NMR (CDCl3, 100 MHz, 60 °C): d 24.8 (CH3), 25.1 (CH3), 26.3 (CH3), 27.0 (CH3), 28.4 (3  CH3), 56.2 (CH), 64.4 (CH2), 64.9 (CH2), 69.8 (CH), 77.2 (CH), 77.9 (CH), 80.9 (C), 93.9 (C), 108.3 (C), 153.0 (C@O). Anal. Calcd for C17H31NO7: C, 56.49; H, 8.65; N, 3.88. Found: C, 56.57; H, 8.510; N, 3.96. 4.16. tert-Butyl (4R)-4[(10 S,20 R,30 R)-30 ,40 -dihydroxy-10 ,20 (isopropylidenedioxy)butyl]-2,2-dimethyloxazolidine-3carboxylate (24) Using the same procedure as described for the preparation of 23, compound 21 (0.820 g, 1.39 mmol) and a 1 M solution of Bu4NF (2.76 mL, 2.76 mmol) in THF yielded after flash chromatography on silica gel (1:1 hexane–EtOAc) 0.44 g (88%) of crystalline diol 24: mp 176–177 °C; ½a25 +56.8 (c 0.20, CHCl3). 1H NMR (CDCl3, D 400 MHz): d 1.32 (s, 3H, CH3), 1.42 (s, 3H, CH3), 1.47 (s, 9H, 3  CH3), 1.52 (s, 3H, CH3), 1.63 (s, 3H, CH3), 3.73–3.86 (m, 4H, H-5, H-30 , 2  H-40 ), 3.91–3.97 (m, 2H, H-5, H-20 ), 4.30 (dd, J10 ,4 = 10.4 Hz, J10 ,20 = 5.3 Hz, 1H, H-10 ), 4.49 (m, 1H, H-4). 13C NMR (CDCl3, 100 MHz): d 25.0 (CH3), 28.0 (CH3), 28.3 (5  CH3), 55.6 (CH), 64.2 (CH2), 66.7 (CH2), 69.0 (CH), 76.8 (CH), 78.7 (CH), 79.6 (C), 94.0 (C), 108.4 (C), 152.7 (C@O). Anal. Calcd for C17H31NO7: C, 56.49; H, 8.65; N, 3.88. Found: C, 56.38; H, 8.79; N, 3.80. 4.17. tert-Butyl (4S)-4[(10 S,20 R,30 Z)-10 ,20 -(isopropylidenedioxy)octadec-3-enyl]-2,2-dimethyloxazolidine-3-carboxylate (25a) and tert-butyl (4S)-4[(10 S,20 R,30 E)-10 ,20 -(isopropylidenedioxy)octadec-3-enyl]-2,2-dimethyloxazolidine-3-carboxylate (25b) A solution of diol 23 (1.20 g, 3.32 mmol) in MeOH (5.4 mL) was treated with a solution of NaIO4 (0.850 g, 3.97 mmol) in H2O (5.4 mL). The resulting mixture was stirred at room temperature for 45 min and then diluted with a small volume of CH2Cl2. The insoluble materials were removed by filtration, the solvent was evaporated, and the crude aldehyde was used immediately in the subsequent reaction without further purification. To a solution of 1,1,1,3,3,3-hexametyldisilazane (1.97 mL, 9.56 mmol) in dry THF (9.8 mL) was added n-BuLi (5.77 mL, 9.23 mmol of a 1.6 M solution in n-hexane) at room temperature. The solution of lithium hexamethyldisilazide (LHMDS) thus generated was treated with pentadecyltriphenylphosphonium bromide (3.79 g, 6.85 mmol), and the resulting dark mixture was stirred at room temperature for 15 min. Then a solution of the crude aldehyde (1.09 g, 3.31 mmol) in dry THF (9.8 mL) was added dropwise, and the reaction mixture was stirred at room temperature. After 40 min, the starting material was completely consumed (judged by TLC), the mixture was poured into satd aq NH4Cl (69 mL) and extracted with EtOAc (2  98 mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated, and the residue was purified by flash chromatography on silica gel (30:1 hexane–EtOAc) to afford 1.43 g (82%) of a mixture of isomers 25 as a colourless oil. A small amount of the mixture of olefins was separated by column chromatography on silica gel (30:1 hexane–EtOAc) to give only (Z)-isomer 25a in pure form. 1 (Z)-Isomer 25a: mp 30.5–31 °C; ½a25 D 29.7 (c 0.19, CHCl3). H NMR (C6D6, 400 MHz, 75 °C): d 0.89 (t, J = 6.8 Hz, 3H, CH3), 1.27– 1.32 (m, 30H, 2  CH3, 12  CH2), 1.42 (s, 9H, 3  CH3), 1.54 (s, 3H, CH3), 1.79 (s, 3H, CH3), 2.08 (m, 2H, 2  H-50 ), 3.81 (m, 1H, H-5), 4.03 (m, 1H, H-4), 4.23 (dd, J5,5 = 8.6 Hz, J5,4 = 2.6 Hz, 1H, H5), 4.94 (m, 1H, H-10 ), 5.11 (m, 1H, H-20 ), 5.35 (m, 1H, H-30 ), 5.48 (m, 1H, H-40 ). 13C NMR (C6D6, 100 MHz, 75 °C): d 14.2 (CH3), 23.0

(CH3), 25.1 (2  CH3), 26.9 (CH3), 26.9 (CH2), 28.7 (3  CH3), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2), 29.9 (CH2), 30.0 (CH2), 30.1 (6  CH2), 32.3 (CH2), 58.7 (CH), 64.4 (CH2), 74.4 (CH), 77.7 (CH), 79.5 (C), 94.2 (C), 108.5 (C), 127.0 (CH), 134.3 (CH), 152.2 (C@O). Anal. Calcd for C31H57NO5: C, 71.08; H, 10.97; N, 2.67. Found: C, 71.19; H, 10.88; N, 2.74. 4.18. tert-Butyl (4R)-4[(10 S,20 R,30 Z)-10 ,20 -(isopropylidenedioxy)octadec-3-enyl]-2,2-dimethyloxazolidine-3-carboxylate (26a) and tert-butyl (4R)-4[(10 S,20 R,30 E)-10 ,20 -(isopropylidenedioxy)octadec-3-enyl]-2,2-dimethyloxazolidine-3-carboxylate (26b) According to the same procedure described for the preparation of 25, compound 24 (0.440 g, 1.22 mmol) was converted to a mixture of olefins 26 (0.44 g, 69%, colourless oil, 30:1 hexane–EtOAc). A small amount of the mixture of 26 was separated by column chromatography on silica gel (30:1 hexane–EtOAc) to give only the (Z)-isomer 26a in pure form. 1 (Z)-Isomer 26a: colourless oil; ½a25 D 9.3 (c 0.45, CHCl3). H NMR (CDCl3, 600 MHz): d 0.88 (t, J = 6.9 Hz, 3H, CH3), 1.26–1.31 (m, 24H, 12  CH2), 1.35 (s, 3H, CH3), 1.48 (s, 9H, 3  CH3), 1.50 (s, 3H, CH3), 1.52 (s, 3H, CH3), 1.63 (s, 3H, CH3), 2.00 (m, 1H, H-50 ), 2.14 (m, 1H, H-50 ), 3.52 (m, 1H, H-5), 3.82 (dd, J5,5 = 9.0 Hz, J5,4 = 4.7 Hz, 1H, H5), 4.19 (m, 2H, H-4, H-10 ), 4.91 (m, 1H, H-20 ), 5.42 (m, 1H, H-30 ), 5.63 (dt, J40 ,30 = 10.4 Hz, J40 ,50 = 7.5 Hz, J40 ,50 = 7.5 Hz, 1H, H-40 ). 13C NMR (CDCl3, 100 MHz): d 14.1 (CH3), 22.7 (CH2), 25.1 (CH3), 27.6 (CH2), 27.8 (CH3), 28.3 (5  CH3), 29.3 (CH2), 29.4 (CH2), 29.5 (2  CH2), 29.6 (2  CH2), 29.7 (4  CH2), 31.9 (CH2), 56.4 (CH), 65.6 (CH2), 72.5 (CH), 78.6 (CH), 79.6 (C), 94.1 (C), 108.7 (C), 125.6 (CH), 135.0 (CH), 152.6 (C@O). Anal. Calcd for C31H57NO5: C, 71.08; H, 10.97; N, 2.67. Found: C, 70.94; H, 11.09; N, 2.55. 4.19. tert-Butyl (4S)-4[(10 S,20 R)-10 ,20 -(isopropylidenedioxy)octadecyl]-2,2-dimethyloxazolidine-3-carboxylate (27) To a solution of the mixture of olefins 25 (1.00 g, 1.91 mmol) in dry EtOH (15.2 mL) was added 10% palladium-on-carbon (134 mg), and the resulting mixture was stirred under a hydrogen atmosphere at room temperature. After 1.5 h, no starting material was detected (TLC) in the reaction mixture. The catalyst was then filtered off, the solvent was evaporated, and the residue was purified by flash chromatography on silica gel (30:1 hexane–EtOAc) to give 0.92 g (92%) of 27 as a colourless oil: ½a25 D 36.5 (c 0.86, CHCl3). IR (KBr) mmax (cm1) 2926, 2854, 1707, 1691, 1390, 1365, 1257, 1211, 1176, 1097, 1086. 1H NMR (CDCl3, 400 MHz, 60 °C): d 0.88 (t, J = 6.8 Hz, 3H, CH3), 1.27 (s, 30H, 15  CH2), 1.34 (s, 3H, CH3), 1.48 (s, 15H, 5  CH3), 1.59 (s, 3H, CH3), 3.88–3.91 (m, 2H, H-4, H-5), 4.15–4.21 (m, 2H, H-20 , H-5), 4.48 (m, 1H, H-10 ). 13C NMR (CDCl3, 100 MHz, 60 °C): d 14.0 (CH3), 22.7 (CH2), 25.2 (2  CH3), 26.6 (CH3), 26.8 (CH3), 28.6 (3  CH3), 29.3 (CH2), 29.5 (CH2), 29.6 (2  CH2), 29.7 (8  CH2), 30.1 (CH2), 31.9 (CH2), 57.9 (CH), 64.0 (CH2), 77.1 (CH), 77.2 (CH), 80.1 (C), 93.8 (C), 107.8 (C), 152.3 (C@O). Anal. Calcd for C31H59NO5: C, 70.81; H, 11.31; N, 2.66. Found: C, 70.72; H, 11.42; N, 2.54. 4.20. tert-Butyl (4R)-4[(10 S,20 R)-10 ,20 -(isopropylidenedioxy)octadecyl]-2,2-dimethyloxazolidine-3-carboxylate (28) According to the same procedure described for the preparation of 27, a mixture of the isomers 26 (0.44 g, 0.84 mmol) was converted to compound 28 (0.38 g, 86%, a colourless oil, 30:1 hexane–EtOAc): ½a25 +27.8 (c 0.54, CHCl3). 1H NMR (CDCl3, D 400 MHz): d 0.88 (t, J = 6.7 Hz, 3H, CH3), 1.26 (m, 30H, 15  CH2), 1.31 (s, 3H, CH3), 1.46 (s, 3H, CH3), 1.48 (s, 9H, 3  CH3), 1.52 (s, 3H, CH3), 1.63 (s, 3H, CH3), 3.61 (m, 1H, H-5), 3.93 (dd, J5,5 = 8.4 Hz, J5,4 = 4.6 Hz, 1H, H-5), 4.05 (m, 1H, H-20 ), 4.15–4.22

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(m, 2H, H-10 , H-4). 13C NMR (CDCl3, 100 MHz): d 14.1 (CH3), 22.7 (CH2), 23.8 (CH3), 25.5 (CH3), 27.8 (CH3), 28.3 (4  CH3), 29.4 (CH2), 29.5 (CH2), 29.6 (2  CH2), 29.7 (9  CH2), 31.9 (CH2), 56.1 (CH), 65.6 (CH2), 77.2 (CH), 78.2 (CH), 79.6 (C), 94.0 (C), 108.1 (C), 152.5 (C@O). Anal. Calcd for C31H59NO5: C, 70.81; H, 11.31; N, 2.66. Found: C, 70.92; H, 11.23; N, 2.75. 4.21. TFA salt of (2S,3S,4R)-2-aminoicosane-1,3,4-triol (29) A solution of 27 (50 mg, 0.095 mmol) in 0.27 mL of 10:3 TFA– H2O was stirred for 15 min at room temperature. The solvent was removed and the residue was dried under vacuum to provide the desired product 29 (42 mg, 96%) as a white amorphous solid: 1 mp 94–96.5 °C; ½a25 ) D +21.3 (c 0.39, CH3OH); IR (KBr) mmax (cm 1 3423, 2956, 2918, 2850, 1651, 1221, 1176, 1165. H NMR (400 MHz, CD3OD): d 0.86 (t, J = 6.9 Hz, 3H, CH3), 1.25 (s, 28H, 14  CH2), 1.51–1.55 (m, 1H, CH2), 1.73–1.79 (m, 1H, CH2), 3.39– 3.52 (m, 3H, H-2, H-3, H-4), 3.72 (dd, J1,1 = 11.5 Hz, J1,2 = 8.3 Hz, H-1), 3.89 (dd, J1,1 = 11.5 Hz, J1,2 = 3.7 Hz, H-1). 13C NMR (CD3OD, 100 MHz): d 14.5 (CH3), 23.8 (CH2), 26.3 (CH2), 30.5 (CH2), 30.8 (2  CH2), 30.9 (8  CH2), 33.1 (CH2), 35.5 (CH2), 56.5 (CH), 58.9 (CH2), 73.4 (CH), 73.5 (CH). Anal. Calcd for C22H44F3NO5: C, 57.49; H, 9.65; N, 3.05. Found: C, 57.38; H, 9.76; N, 3.13. 4.22. TFA salt of (2R,3S,4R)-2-aminoicosane-1,3,4-triol (30) Using the same procedure as described for the preparation of 29, compound 28 (54 mg, 0.103 mmol) was converted to compound 30 (46 mg, 97%, white amorphous solids); mp 91.5–93.5 °C; ½a25 D +6.4 (c 0.47, CH3OH); IR (KBr) mmax (cm1) 3319, 2955, 2918, 2850, 1672, 1209, 1192, 1130. 1H NMR (CD3OD, 400 MHz): d 0.87 (t, J = 6.8 Hz, 3H, CH3), 1.26–1.42 (m, 28H, 13  CH2, H-5, H-6), 1.50– 1.54 (m, 1H, H-6), 1.58–1.64 (m, 1H, H-5), 3.43–3.49 (m, 2H, H-2, H-3), 3.56–3.61 (m, 1H, H-4), 3.68 (dd, J1,1 = 11.3 Hz, J1,2 = 8.0 Hz, H-1), 3.74 (dd, J1,1 = 11.3 Hz, J1,2 = 5.3 Hz, H-1). 13C NMR (CD3OD, 100 MHz): d 14.5 (CH3), 23.8 (CH2), 26.7 (CH2), 30.5 (CH2), 30.8 (10  CH2), 33.1 (CH2), 34.8 (CH2), 55.2 (CH), 61.7 (CH2), 71.1 (CH), 74.2 (CH). Anal. Calcd for C22H44F3NO5: C, 57.49; H, 9.65; N, 3.05. Found: C, 57.58; H, 9.53; N, 3.10. 4.23. (2S,3S,4R)-2-Aminoicosane-1,3,4-triol (D-ribo-C20phytosphingosine) (31) A solution of 29 (45.0 mg, 0.098 mmol) in CH3OH (3 mL) was titrated to pH 8 with NaOH solution (1% in CH3OH, 0.2 mL) at room temperature. The solvent was evaporated under reduced pressure, and the resulting white powder was washed with water. The aqueous layer was separated, and the residue was dried under vacuum to give 29 mg (86%) of compound 31 as white solids: mp 101– 1 103 °C; ½a25 ) 3360, D +11.4 (c 0.14, CH3OH); IR (KBr) mmax (cm 1 2956, 2918, 2848, 1599, 1468, 1080, 1038, 721. H NMR (CD3OD, 600 MHz): d 0.89 (t, J = 7.0 Hz, 3H, CH3), 1.28–1.40 (m, 28H, 13  CH2, H-5, H-6), 1.52–1.57 (m, 1H, H-6), 1.71–1.76 (m, 1H, H-5), 2.95 (ddd, J2,1 = 6.7 Hz, J2,3 = 5.5 Hz, J2,1 = 4.1 Hz, 1H, H-2), 3.33 (dd, J3,4 = 7.8 Hz, J3,2 = 5.5 Hz, 1H, H-3), 3.51 (td, J4,5 = 8.5 Hz, J4,3 = 8.4 Hz, J4,5 = 2.6 Hz, 1H, H-4), 3.56 (dd, J1,1 = 10.9 Hz, J1,2 = 6.7 Hz, 1H, H-1), 3.75 (dd, J1,1 = 10.9 Hz, J1.2 = 4.1 Hz, 1H, H-1). 13C NMR (CD3OD, 150 MHz): d 14.5 (CH3), 23.8 (CH2), 26.7 (CH2), 30.5 (CH2), 30.8 (CH2), 30.9 (8  CH2), 31.0 (CH2), 33.1 (CH2), 34.8 (CH2), 55.9 (CH), 64.1 (CH2), 74.5 (CH), 76.5 (CH). Anal. Calcd for C20H43NO3: C, 69.51; H, 12.54; N, 4.05. Found: C, 69.42; H, 12.63; N, 4.12. 4.24. (2R,3S,4R)-2-Aminoicosane-1,3,4-triol (32) According to the same procedure described for the preparation of 31, compound 30 (29.0 mg, 0.063 mmol) was converted to

compound 32 (19 mg, 87%, white solids); mp 66–67 °C; ½a25 D +14.2 (c 0.09, pyridine); IR (KBr) mmax (cm1) 3419, 2918, 2850, 1 1633, 1468, 1128, 1049, 721. H NMR (CD3OD, 600 MHz): d 0.89 (t, J = 7.0 Hz, 3H, CH3), 1.28–1.41 (m, 28H, 13  CH2, H-5, H-6), 1.50–1.57 (m, 1H, H-6), 1.64–1.69 (m, 1H, H-5), 3.18 (ddd, J2,1 = 7.4 Hz, J2,1 = 5.8 Hz, J2,3 = 2.5 Hz, 1H, H-2), 3.41 (dd, J3,4 = 6.8 Hz, J3,2 = 2.5 Hz, 1H, H-3), 3.55–3.58 (m, 2H, H-1, H-4), 3.63 (dd, J1,1 = 10.9 Hz, J1,2 = 5.8 Hz, 1H, H-1). 1H NMR (CD3OD, 150 MHz): d 14.5 (CH3), 23.8 (CH2), 26.8 (CH2), 30.5 (CH2), 30.8 (9  CH2), 30.9 (CH2), 33.1 (CH2), 34.9 (CH2), 54.4 (CH), 64.4 (CH2), 73.5 (CH), 73.7 (CH). Anal. Calcd for C20H43NO3: C, 69.51; H, 12.54; N, 4.05. Found: C, 69.62; H, 12.42; N, 4.09. 4.25. D-ribo-C20-Phytosphingosine tetraacetate (33) To a solution of 31 (15 mg, 43.4 lmol) in dry pyridine (1.4 mL) were added Ac2O (0.08 mL, 0.85 mmol) and DMAP (2.65 mg, 21.7 lmol) at room temperature. After 6 h, no starting compound was detected (TLC) in the reaction mixture. The solvent was evaporated under reduced pressure, and the residue was subjected to flash chromatography on silica gel (1:1 hexane–EtOAc) to afford 21 mg (94%) of crystalline tetraacetate 33: mp 48–49 °C (lit.14c 14c mp 54–55 °C); ½a25 ½a25 D +29.5 (c 0.22, CHCl3) [lit. D +25.0 (c 1, 1 CHCl3)]. H NMR (CDCl3, 600 MHz): d 0.88 (t, J = 7.0 Hz, 3H, CH3), 1.25–1.43 (m, 28H, 14  CH2), 1.59–1.68 (m, 2H, H-5), 2.03 (s, 3H, CH3), 2.05 (s, 6H, 2  CH3), 2.08 (s, 3H, CH3), 4.00 (dd, J1,1 = 11.7 Hz, J1,2 = 3.0 Hz, 1H, H-1), 4.29 (dd, J1,1 = 11.7 Hz, J1,2 = 4.8 Hz, 1H, H-1), 4.47 (m, 1H, H-2), 4.94 (dt, J4,5 = 10.0 Hz, J4,5 = 3.1 Hz, J4,3 = 3.1 Hz, 1H, H-4), 5.10 (dd, J3,2 = 8.3 Hz, J3,4 = 3.1 Hz, 1H, H-3), 5.97 (d, J2,NH = 9.3 Hz, 1H, NH). 13C NMR (CDCl3, 150 MHz): d 14.1 (CH3), 20.7 (CH3), 20.8 (CH3), 21.0 (CH3), 22.7 (CH2), 23.3 (CH3), 25.5 (CH2), 28.2 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (3  CH2), 29.7 (5  CH2), 31.9 (CH2), 47.6 (CH), 62.8 (CH2), 72.0 (CH), 73.0 (CH), 169.7 (C@O), 170.1 (C@O), 170.8 (C@O), 171.1 (C@O). Anal. Calcd for C28H51NO7: C, 65.47; H, 10.01; N, 2.73. Found: C, 65.39; H, 10.12; N, 2.65.

Table 2 Crystal data and structure refinement parameters for compound 22 22 Empirical formula Formula weight Temperature, T (K) Wavelength, k (Å) Crystal system Space group

C26H53NO8Si2 563.87 100(2) 0.71073 Orthorhombic P212121

a (Å) b (Å) c (Å) V (Å3) Formula per unit cell, Z Dcalcd (g/cm3) Absorption coefficient, l (mm1) F (0 0 0) Crystal size (mm) h range for data collection (°) Index ranges

7.14150(10) 17.0506(4) 28.2216(6) 3436.46(12) 4 1.090 0.143 1232 0.797  0.094  0.075 3.41–26.50 8 6 h 6 8 21 6 k 6 21 35 6 l 6 35 7097 (0.1142) Analytical 0.991 and 0.938 Full-matrix least-squares on F2 7097/0/353 0.913 R1 = 0.0437, wR2 = 0.0792 R1 = 0.0821, wR2 = 0.0862 0.592 and 0.286

Independent reflections (Rint) Absorption correction Max. and min. transmission Refinement method Data/restraints/parameters Goodness-of-fit on F2 Final R indices [I > 2r(I)] R indices (all data) Largest diff. peak and hole (e/Å3)

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4.26. X-ray techniques Single crystals of 22 suitable for X-ray diffraction were obtained from Et2O by slow evaporation at room temperature. The intensities were collected at 100 K on a Oxford Diffraction0 Gemini R CCD diffractometer using Mo Ka radiation (k = 0.71073 Å A). Selected crystallographic and other relevant data for the compound 22 are listed in Table 2. The structure was solved by direct methods.26 All non-hydrogen atoms were refined anisotropically by full-matrix least squares calculations based on F2.24 All hydrogen atoms were included in calculated positions as riding atoms, with SHEL26 XL97 defaults. The PLATON27 programme was used for structure analysis and molecular and crystal structure drawings.

12.

13.

4.27. Supplementary data Complete crystallographic data for the structural analysis have been deposited with the Cambridge Crystallographic Data Centre, CCDC No. 805003. These data can be obtained free of charge from the Director, Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1223 336033; e-mail: [email protected] or via: www.ccdc.cam.ac.uk). Acknowledgements The present work was supported by the Grant Agency (Nos. 1/ 0100/09 and 1/0433/11) of the Ministry of Education, Slovak Republic. NMR measurements provided by the Slovak State Programme Project No. 2003SP200280203 are gratefully acknowledged. References 1. (a) Merrill, A. H., Jr.; Sweeley, C. C. In Vance, D. E., Vance, J., Eds.; Biochemistry of Lipids: Lipoproteins and Membranes; Elsevier: Amsterdam, 1996; pp 309– 339; (b) Merrill, A. H., Jr.; Sandhoff, K. In Vance, D. E., Vance, J., Eds.; Biochemistry of Lipids: Lipoproteins and Membranes; Elsevier: Amsterdam, 2002; pp 373–407; (c) Hanada, K. Biochim. Biophys. Acta 2003, 1632, 16–30; (d) Rao, R. P.; Acharya, J. A. Prostaglandins Other Lipid Mediat. 2008, 85, 1–16. and references cited therein. 2. For a review of syntheses of sphingosine, see: Koskinen, P. M.; Koskinen, A. M. P. Synthesis 1998, 1075–1091. 3. For recent review of syntheses of sphinganines, see: Howell, A. R.; So, R. C.; Richardson, S. K. Tetrahedron 2004, 60, 11327–11347. 4. (a) Proštenik, M.; Stanac´ev, N. Zˇ. Chem. Ber. 1958, 91, 961–965; (b) Oda, T.; Kamiya, H. Chem. Pharm. Bull. 1958, 6, 682–687; (c) Sweelley, C. C.; Moscatelli, E. A. J. Lipid Res. 1959, 1, 40–47; (d) Karlsson, K.-A.; Holm, G. A. L. Acta Chim. Scand. 1965, 19, 2423–2425; (e) Carter, H. E.; Gaver, R. C.; Yu, R. K. Biochem. Biophys. Res. Commun. 1966, 22, 316–320; (f) Karlsson, K.-A.; Samuelsson, B. E.; Steen, G. O. Acta Chim. Scand. 1968, 22, 1361–1363. 5. Holst, O. In Glycoscience: Chemistry and Chemical Biology; Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds., 1st ed.; Springer: New York, 2001; pp 2083–2106. 6. Zellner, J. Monatsh. Chem. 1911, 133–142. 7. (a) Wertz, P. W.; Miethke, M. C.; Long, S. A.; Strauss, J. S.; Downing, D. T. J. Invest. Dermatol. 1985, 84, 410–412; (b) Wertz, P. W.; Swartzendruber, D. C.; Madison, K. C.; Downing, D. T. J. Invest. Dermatol. 1987, 89, 419–425; (c) Ponec, M.; Weerheim, A.; Lankhorst, P.; Wertz, P. J. Invest. Dermatol. 2003, 120, 581–588. 8. (a) Wells, G. B.; Dickson, R. C.; Lester, R. L. J. Biol. Chem. 1998, 273, 7235–7243; (b) Chung, N.; Jenkins, G.; Hannun, Y. A.; Heitman, J.; Obeid, L. M. J. Biol. Chem. 2000, 275, 17229–17232. 9. (a) Henón, E.; Dauchez, M.; Haudrechy, A.; Banchet, A. Tetrahedron 2008, 64, 9480–9489. and references cited therein; (b) Liu, Z.; Byun, H.-S.; Bittman, R. Org. Lett. 2010, 12, 2974–2977. and references cited therein. 10. (a) van den Berg, R. J. B. H. N.; Korevaar, C. G. N.; van der Marel, G. A.; Overkleef, H. S.; van Boom, J. H. Tetrahedron Lett. 2002, 43, 8409–8412; (b) van den Berg, R. J. B. H. N.; Koeravaar, C. G. N.; Overkleeft, H. S.; van der Marel, G. A.; van Boom, J. H. J. Org. Chem. 2004, 69, 5699–5704. 11. (a) van den Berg, R. J. B. H. N.; Boltje, T. J.; Verhagen, C. P.; Litjens, R. E. J. N.; van der Marel, G. A.; Overkleeft, H. S. J. Org. Chem. 2006, 71, 836–839; (b) Lee, T.;

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