Synthesis and characterization of some atypical sphingoid bases

Synthesis and Characterization of some Atypical Sphingoid Bases

1

, 2

1

3

1

Essa M. Saied , Thuy Linh-Stella Le , T. Hornemann and Christoph Arenz *

1

Institute for Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin,

Germany.

2

Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt

3

University of Zurich; University Hospital of Zurich, Switzerland

*

to whom correspondence should be submitted: arenzchr@hu-Berlin.de

Abstract

Sphingolipids are ubiquitous and abundant components of all eukaryotic and some

prokaryotic organisms. Sphingolipids show a large structural variety not only between the

different species, but also within an individual cell. This variety is not limited to alterations in

the polar headgroups of e.g. glycosphingolipids, but also affects the lipophilic anchors

comprised of different fatty acids on the one hand and different sphingoid bases on the other

hand. The structural variations within different sphingoid bases e.g. in pathogens can be used

to identify novel biomarkers and drug targets and the specific change in the profile of

common and uncommon sphingolipids are associated with pathological conditions like

diabetes or cancer. Therefore, the emerging field of sphingolipidomics is dedicated to collect

data on the sphingolipidome of a cell and hence to assign changes therein to certain states of a

cell or to pathological conditions. This powerful tool however is still limited by the

availability of structural information about the individual lipid species as well as by the

availability of appropriate internal standards for quantification. Herein we describe the

synthesis of a variety of 1-deoxy-sphingoid bases. 1-DeoxySphingolipids have recently

acquired significant attention due to its pathological role in the rare inherited neuropathy,

HSAN1 but also as predictive biomarkers in diabetes type II. Some of the compounds

synthesized and characterized herein, have been used and will be used to elucidate the correct

structure of these disease-related lipids and their metabolites.

1

Introduction

Sphingolipids are important components of mammalian cells. They are defined by a sphingoid

base, usually a C-18 2-amino-1,3-diol, that is mostly N-acylated to form ceramide, which in

1

turn serves as a lipid anchor for a variety of polar head groups. Such polar head groups

include phosphoryl cho line (forming sphingomyelins) or a variety of several hundred mono-

2

,3

and oligosaccharides. The latter species are grouped as glycosphingolipids (GSL) and

1

contribute to the cell’s glycocalyx and thus cell-cell and cell-ligand interactions. For

1

4

example, GSL determine the blood groups of the A,B, 0 system . The acidic GSL, the

5

ganglioside GM1, is used by cholera toxin to enter target cells and the ganglioside GM3

6

modulates the activity of the insulin receptor. While the variety of both, the sphingolipid

head groups and the acyl chains have been subject to intense research in the past, the

existence of structural variations in the sphingoid base has been much less appreciated so far.

Indeed, most organisms contain a predominant sphingoid base, like the “canonical”

7

sphingosine (2S,3 R,4E-2-aminooctadec-4-ene-1,3-diol) in mammals or phytosphingosine in

8

plants and fungi. Typically, the first and rate limiting step of sphingolipid biosynthesis is the

condensation of serine and palmitoyl-CoA to form 3-Ketosphinganine, which is catalyzed by

the enzyme serine-palmitoyl-CoA transferase (SPT), which is conserved in all eukaryotes

8

examined so far. 3-Ketosphinganine is then reduced to sphinganine (also termed

“

dihydrosphingosine”) followed by N-acylation and desaturation at the 4 position. In

mammalian cells, this desaturation occurs always after N-acylation, meaning that sphingosine

SO) is not an intermediate of de novo biosynthesis, but rather of the catabolism of complex

(

9

sphingolipids, . SO itself can be again re-acylated to ceramide or terminally degraded.

Variations to the sphingoid base are mostly introduced downstream of the SPT reaction.

Interestingly, studies in pathogenic fungi revealed the abundance of unusual sphingoid bases,

which derive from individual biosynthetic pathways, formed by specialized e nzymes that

1

0,11

might provide interesting pharmacological targets for antifungal therapies.

Additional

examples for atypical sphingoid bases are 1-deoxy-sphingoid bases spisolusine or the

xestoaminols, which are formed by the use of alanine over serine and which have been

originally isolated from a variety of marine animals. 1-Deoxy-sphingoid bases showed

pronounced cytotoxic properties to certain cancer cell lines and were tested as an

1

2

experimental cancer therapy. Later it was demonstrated that pathologically elevated 1-

deoxysphingolipids cause the rare inherited neuropathy HSAN1 which has been attributed to

several mutations in the SPT genes. These mutations induce a permanent shift in the substrate

affinity of SPT leading to an increased utilization of alanine over its canonical substrate

1

3

serine. In a recent study, a branched 1-deoxysphinganine was synthesized from 2-

carboxylaziridine and it was shown that this analogue of C. elegans sphingolipids did not

1

4

rescue a genetic sphingoid base depletion. In addition, 1-deoxysphingolipids are also

elevated in type 2 d iabetes mellitus and might thereby also involved in the frequent diabetic

1

5,16

polyneuropathy

Although the exact molecular mechanism for 1-deoxysphingolipid

formation in diabetes is still unknown. 1-Deoxysphingolipids lacks the primary hydroxyl

group of canonical sphingoid bases which prevents the attachment of a polar head group and

the formation of complex sphingolipids. As a consequence, 1-deoxysphinglipids cannot be

degraded over the canonical pathway as the catabolic intermediate sphingosine-1-phosphate

(

S1P) cannot be formed. Instead, 1-deoxysphingolipids seem to follow different metabolic

routes. Recent studies showed that the LC retention time of a synthetic 1-deoxysphingosine

standard bearing the canonical 4-5 trans double bond did not match with the naturally

occurring 1-deoxysphingosine, However, both lipids were isobaric, suggesting an altered

1

7

double bond position in 1-deoxysphingosine compared to its canonical counterparts. By

comparing the retention time of natural 1-deoxysphingosine with those of synthetic standard

compounds with incrementally shifted double bond position, we observed a step-wise

approximation of retention times and finally identified the structure of deoxysphingosine as

2

S,3R, 14Z-2-aminooctadec-14-ene-3-ol. This structure was further confirmed by MS

fragmentation studies on dimethyl disulfide derivatized 1-deoxysphingosine and ozonization-

1

7

MS.

In the present report, we describe the concise and efficient synthesis of a set of

deoxysphingosine derivatives, which can be used as potential biomarkers for human disease

and different pathogens. Most of these compounds either have been confirmed as naturally-

occurring metabolites or are currently under further investigation.

2

Results and Discussion

The canonical sphingosine is synthesized from an activated fatty acid and L-serine. In seminal

works, S. Garner and coworkers synthesized the protected serinal derivative known as

1

8

“

Garner’s aldehyde”, which can be nucleophilically attacked by metallated terminal alkynes

1

9

to form the respective substituted propargyl alcohols. Partial hydrogenation of the latter to

the corresponding E-alkenes yields the protected sphingosines in good yields. The

diasteroselectivity of the nucleophilic addition with concomitant hydroxyl group formation

2

0

was later improved by the use of HMPA and other auxiliaries. An alternative strategy to

2

1

synthesize the canonical sphingosine has been developed by Yamamoto et al. The group

reacted the Weinreb amide of protected L-serine with vinyl magnesium bromide to form the

respective , unsatured ketone. The latter was then stereospecifically reduced to the

corresponding substituted allyl alcohol, followed by olefin metathesis. The great advantage of

this approach is the free choice of distal residues from one common precursor molecule.

Therefore, this approach has been widely used since then to synthesize sphingolipid

2

2-24

derivatives modified in the sphingosine portion.

Following the same rationale, the

synthesis of a few deoxysphingosine derivates starting from L-alanine instead of L-serine

2

5-27

have been previously reported.

Indeed, in the present manuscript, all deoxysphingolipids

were derived from the common precursor 1, respectively. Analogously, the

deoxymethylsphingosine derivative 50 was derived from the respective glycine derivative 47.

In the course of our investigations, we envisioned to synthesize the most important

intermediates and products of the deoxysphingolipid metabolism in humans. Therefore, we

first synthesized 3-keto-1-deoxysphinganine 3, the direct product of the SPT reaction by

reaction of the precursor 1 with in situ generated pentadecane magnesium bromide, followed

by acid-mediated deprotection (scheme 1).

Scheme 1. Synthesis of 3-keto-1-deoxysphinganine analogue. Reagents and conditions: a)

Boc

2

O, 1M NaOH, dioxane, -10°C to r.t., 12h. b) N,O-dimethylhydroxylamine HCl,

O, reflux,

EDCI.HCl, DCM, -15°C, 4h. c) (i) Mg, 1,2-DBE (drops), 1-bromopentadecane, Et

2

h, and then (ii) 1, DCM:Et

2

O; 0°C-rt, 3h. d) AcCl, MeOH, 0°C, 2h.

3

During our biochemical investigations, we noticed that the HPLC retention time of the

orthopthalimide (OPA) derivative of deoxysphingosine taken from patient cells was slightly

1

7

different from the retention time of the commercial 4E-deoxysphingosine. Since the m/z

values were identical, we reasoned that the position of the double bond in “natural”

deoxysphingosine from patients might be shifted. Noteworthy, during sphingolipid

biosynthesis, the double bond is introduced only after N-acylation of the saturated

sphinganine (equal to dihydrosphingosine) to form ceramide, which then can be hydrolysed

again to form sphingosine. Therefore, an altered binding of N-Acyldeoxysphinganine to the

respective desaturase could result in a shifted position of the double bond. To follow this

hypothesis, we aimed at synthesizing deoxysphingosine isomers differing in the position of

the double bond. Towards this goal, 1 was reacted with different -alkenyl Grignard reagents,

which were freshly prepared from the respective alkenyl bromides (scheme 2). The resulting

-desaturated -amino ketones were subjected to reduction with TBLAH. The bulky N-Boc

substituent at the pre-defined stereo centre allows stereo-selective reduction of resulting α-

aminoketones to the desired D-erythro configured 2-amino-3-hydroxy motif. Next, the latter

intermediates were reacted with terminal olefins of different length in a metathesis reaction to

yield the desired C-18 deoxysphingosine isomers 16-19 after acid-mediated deprotection

(

scheme 2). Very recently, this set of deoxysphingosine isomers was used to systematically

explore the strengths and limitations of differential mobility spectrometry (DMS) and other

key techniques for mass spectrometry based lipidomics, for differentiation of subtle structural

2

8

changes between different lipid isomers.

Scheme 2. Synthesis of novel E-1-deoxysphingosine analogues. Reagents and conditions: a)

i) Mg, 1,2-DBE (drops), 1-bromo-alkene, Et O, reflux, 2h, and then (ii) 2, MeMgBr,

DCM:Et O, 0°C-rt, 3-4h. b) TBLAH, EtOH, -78°C, 2-3h, (anti:syn 4:1- 9:1). c) Cross partner

alkene, p-benzoquinone (10 mol%), Grubbs 2 generation cat., deuterated-chloroform, reflux,

-12h. d) 4M HCl-Dioxane, 0°C-rt, 1-2h.

(

2

nd

4

In addition to the mono-unsaturated 1-deoxysphingosine, two doubly unsaturated 1-deoxy-

sphingosines were hypothesized to be natural downstream metabolites of 1-deoxy-

2

9

sphingosine and they were synthesized accordingly. Noteworthy, doubly unsaturated

sphingosines also termed sphingadienes (sphing-4E, 14Z-diene) have been known for more

3

0-33

than 40 years, and have been isolated from a number of mammalian sources.

Therefore,

4

we aimed at synthesizing hypothetical 1-deoxysphingadienes having the same or a similar

signature of double bonds. Towards this goal, the precursor 1 was again reacted with alkenyl

Grignard reagents as to set the position of the first double bond, either in the “canonical”

position (Scheme 3, n=0) or in a position, shifted by one carbon atom (n=1). Subsequently,

the olefin metathesis reactions were carried out using terminal alkenyl bromides resulting in

the C-13 intermediates 24 and 25. Next, the latter were reacted with 1-pentyne in butyl

lithium solution, respectively, to yield the desired C-18 backbone. Finally, the precursors

were hydrogenated using Lindlar’s catalyst to convert the triple bonds into Z-alkenes thus

obtaining after acid-mediated deprotection the desired deoxysphingadienes 32 and 33.

Confirming our hypothesis, the 1-deoxy-sphingadiene 32 was recently shown to be a

2

9

downstream metabolite of 1-deoxysphingosine.

Scheme 3. Synthesis of novel 1-deoxysphingadiene analogues. Reagents and conditions: a)

freshly prepared Grignard reagent, THF, 0°C-rt, 4-8h. b) TBLAH, EtOH, -78°C, 1-2h,

(

anti:syn 9:1, for 22 and 4:1 for 23). c) Cross partner 1-bromo-alkene, p-benzoquinone (10

nd

mol%), Grubbs 2 generation cat., d-chloroform, reflux, 4-8h. d) 2,2-dimethoxypropane, p-

TsOH (cat.), toluene, reflux, 2-5h. e) (i) 1-Pentyne, tert-BuLi, THF, -78°C, 1h, and then (ii)

HHMPA, 26 or 27, THF, -78°C-rt, 4-6h f) H

2

, EDA, Lindlar catalyst, 5% DMF in EtOAc,

0

°C-rt, 16h. g) 4M HCl-Dioxane, 0°C-rt, 1-2h.

The canonical sphingoid base sphingosine is either N-acylated to form ceramide and its

derivatives or phosphorylated to sphingosine-1-phosphate, which then in turn can be

terminally degraded to hexadecenal and phosphorylethanolamine. In contrast, 1-

deoxysphingolipids cannot be phosphorylated and are therefore believed to be dead end

metabolites. However, by pulse-chase experiments in combination with high resolution and

high accuracy mass spectrometry it was demonstrated recently, that 1-deoxysphingosines are

instead, catabolized by a different pathway. This metabolism includes hydroxylation reactions

and it is likely mediated by enzymes of the cytochrome P450A/F family which are generally

2

9

described as -hydoxylases Therefore, a -hydroxylated deoxysphinganine 40 was

synthesized as a potential metabolite of this pathway (scheme 4). Towards this end, a

tetrahydropyran protected pentadecen-1-ol 37 was synthesized by bromide substitution of

partially protected pentadecan-1,15-diol 35 followed by elimination of HBr. The resulting

olefin 37 was subjected to cross metathesis with the literature known intermediate 22 (Scheme

5

26

3

). Selective reduction and deprotection of obtained 38 yielded the expected -hydroxy-1-

deoxysphinganine 40. Up to now however, only an isomer bis-hydroxylated at the 4- and 14-

2

9

position has been experimentally confirmed so far.

Scheme 4. Synthesis of ɷ-hydroxy-1-deoxysphinganine analogue. Reagents and

conditions: a) LiAlH , THF, -20°C-rt, 12h. b) DHP, PTSA (cat.), THF, 0°C-rt, 2h. c) CBr

PPh , DCM, 0°C-rt, 2h. d) t-BuOK, THF, reflux, 12h. e) 22, p-benzoquinone (10 mol%),

Grubbs 2 generation cat., d-chloroform, reflux, 8h. f) H

6h. g) 4M HCl-Dioxane, 0°C-rt, 3h.

4

,

3

nd

2

, Pd/C, AcOH (drops), MeOH, rt,

1

In addition to alanine, also glycine can used as an alternative substrate in the SPT reaction,

although to a minor extent. This reaction affords another 1-deoxysphingoid base (1-

12

deoxymethylsphinganine) which is devoid of the complete hydroxymethylene group. Up to

now, the structure of the unsaturated product 1-deoxymethyl-sphingosine is not known. In

analogy to 1-deoxySO, we synthesized a 1-deoxymethyl-sphingosine isomer with a Z-

configured double bond between carbons 13 and 14 (scheme 5). This was achieved by first

synthesizing, in analogy to its longer counterpart 36, the intermediate 42, which was reacted

with lithiated 1-pentyne to form the protected intermediate 43. Hydrogenation in the presence

of Lindlar’s catalyst afforded the Z-configured alkene 44, which after deprotection was

converted to the terminal bromide 46. In situ generation of the respective Grignard reagent

allowed reaction with the Weinreb-type amide of glycine 47. Stereoselective reduction and

acidic deprotection of the resulting intermediate 49 yielded the final hypothetical

deoxymethylsphingosine 50, which awaits experimental confirmation.

Scheme 5. Synthesis of 14Z-deoxymethyl-sphingosine analogue. Reagents and conditions:

a) DHP, PTSA (cat.), THF:DCM, 0°C-rt, 9h. b) CBr

4

, PPh , DCM, 0°C-rt, 2h. c) (i) 1-

3

Pentyne, tert-BuLi, THF, -78°C, 1h, and then (ii) HHMPA, 42, THF, -78°C-rt, 6h d) H ,

2

6

EDA, Lindlar catalyst, 5% DMF in EtOAc, 0°C, 6h. e) PPTS (cat.), EtOH, 62°C, 2h. f) (i)

Mg, 1,2-DBE (drops), Et O, reflux, 2h, and then (ii) 47, DCM:Et O; reflux, 2h. g) TBLAH,

EtOH, -78°C, 90min. h) 4M HCl-Dioxane, 0°C-rt, 2h.

2

3

Conclusion

Herein we report efficient and practical synthesis of a variety of different natural and

hypothesized deoxysphingoid bases, including saturated, hydroxylated and mono- or di-

unsubstituted entities. These compounds have been and will be used for investigation in the

metabolism of disease related deoxysphingolipids. Furthermore, these derivatives can be used

as standard compounds for MS-based sphingolipidomics or as clinical biomarkers for diabetes

type II or diabetic neuropathy. Noteworthy, similar compounds like the deoxysphingoid base

3

4

spisolusine from the mussel Spisula polynyma or, 3-epi-xestoaminol C from Xiphophora

3

5

36

chondrophylla and their derivatives are being investigated against cancer or mycobacterial

3

5

infections. The methods developed here are paving the way for the synthesis of many more

variants of this highly exciting compound class.

4

Experimental

.1 General

4

Unless otherwise specified, all reactions were carried out in oven-dried (>120°C) glassware

equipped with a magnetic stir bar and a rubber septum under a positive pressure of argon. Air-

or moisture-sensitive reagents were transferred to the reaction vessel under positive pressure

of argon via syringe or stainless steel cannula. Reactions were run at room temperature (20-

2

5°C) unless otherwise noted in the experimental procedure, and reported reaction

temperatures refer to the external temperatures measured for the bath in which the reaction

vessel was immersed. Heating was obtained through the use of a silicone oil bath. For

reactions run below room temperature, the term “-78°C” refers to a bath of acetone and dry

ice, “-20°C” refers to a slurry of sodium chloride and ice-water bath, and “0°C” refers to an

ice-water bath. Intermediate temperatures were obtained by the careful addition of dry ice to

an acetone bath at periodic intervals to maintain the desired temperature. “Concentration”

refers to the removal of solvent using a rotary evaporator equipped with a portable vacuum

pump. Removal of residual solvents was accomplished by evacuation of the container for a

period of 12-20 hours using a high vacuum line.

Unless otherwise specified, all the commercially available reagents were purchased from

Sigma-Aldrich, Fluka or Acros and used without further purification. Air and/or moisture

sensitive reactions were carried out in well dried glassware under an argon atmosphere with

dry, freshly distilled solvents using standard syringe-cannula/septa techniques. All the

solvents were used after distillation by standard methods. The petroleum ether used

throughout this study had a boiling range of 40–60°C. Reaction progress was monitored with

analytical normal-phase thin layer chromatography (NP TLC). All synthesized compounds

were purified to homogeneity as judged by TLC analysis, mass spectrometry (MS) analysis

and nuclear magnetic resonance (NMR) spectroscopic analysis. The thin layer

7

chromatography studies were performed on pre-coated silica gel 60-F254 on aluminum sheets

Merck KGaA) and spots were detected by UV illumination (254 nm), and/or spraying with

.3% ninhydrin solution, ceric ammonium molybdate (Seebach reagent) solution or KMnO4

(

1

solution followed by heating. Preparative flash column chromatography was performed

manually using glass columns of different size packed with Silica Gel 60M (0.04-0.063 mm)

as stationary phase with indicated eluent systems in parenthesis following the description of

purification. Solvent ratios for chromatography and R

f

values are reported in v/v% ratios.

1

13

The structure of all synthesized compounds was confirmed with H NMR, C NMR, DEPT

1

13

and MS analyses. ( H, C) NMR spectra of the synthesized compounds were recorded on

1

Bruker Avance -300 and Bruker Avance-500 nuclear magnetic resonance spectrometers ( H at

1

3

00 or 500 MHz and C at 75 or 126 MHz) as a solution in deuterated methanol (MeOD),

deuterated chloroform (CDCl ) or deuterated water (D O) at 27°C, unless otherwise indicated.

H NMR spectra are reported as chemical shifts (δ) in parts per million (ppm) as referenced to

the residual protons in the deuterated solvent (d-chloroform: δ 7.27 ppm; d-methanol: δ

.31ppm, unless otherwise noted). Peak multiplicities are designed by the following

3

2

1

3

abbreviations: s = singlet; d= doublet; t = triplet; q = quartet; dd = doublet of doublets, ddd =

doublet of doublet of doublets, dt = doublet of triplets, qd = quartet of doublets, m = multiplet;

br = broad. The coupling constants (J) are quoted in hertz (Hz). The reported chemical shifts

are tabulated in the following format: δ (ppm) (multiplicity, coupling constant (s), number of

1

3

protons). C NMR chemical shifts are expressed in parts per million (ppm) and referenced to

the residual solvent peak (d-chloroform: δ 77.16 ppm; d-methanol δ 49.05 ppm, unless

otherwise indicated). High-resolution mass spectroscopy (HRMS) experiments were recorded

on a Hewlett-Packard GCMS 5995-A mass spectrometer equipped with standard electrospray

ionization (ESI) in the positive and negative ion detection modes. The major observable

molecular ion and selected fragments and clusters have been reported.

4

.2 Synthesis of 3-keto-1-deoxysphinganine analogue

.2.1 Synthesis of (S)-tert-butyl-(3-oxooctadecan-2-yl)carbamate 2

The Grignard solution of 1-bromopentadecane was synthesized as follows; To a mixture of

magnesium turnings (0.23 g, 20 mmol) in anhydrous Et O (6 mL) under an argon atmosphere

2

was added drops of 1,2-dibromoethane. After the resulting mixture was allowed to stir at the

same conditions for 20 min, a solution of 1-bromopentadecane (1.2 g, 4 mmol, in 2 mL of

anhydrous Et O) was added dropwise over 20min. The reaction mixture was then allowed to

2

stir for additional 3h at 35°C to afford a transparent 1-pentadecylmagnesium bromide solution

which was immediately used in the next step.

To a stirred solution of Weinreb amide derivative 1 (0.23 g, 1 mmol) in anhydrous Et O (10

2

mL) at -20°C under an argon atmosphere was added a freshly prepared 1-

pentadecylmagnesium bromide solution (8 mL,4 mmol, 0.5M solution in Et

2

O) dropwise over

3

0 min. After being stirred at ambient temperature for additional 2h (as monitored by TLC

analysis; cylcohexane / EtOAc 7:3; Rf (adduct)= 0.2; Rf (product)=0.73; visualized with 1.3%

ninhydrine solution), the reaction mixture was subsequently cooled again to 0°C and

successfully quenched with an ice-cold 1M HCl solution (40 mL). The resulting mixture was

diluted with diethylether (50 mL), and the layer were separated. The aqueous layer was

8

extracted with diethylether (3x50 mL), and the combined organic phases were washed with

saturated NaHCO solution (80 mL) and brine (80 mL), dried over anhydrous Na SO , filtered

and concentrated in vacuo. Purification of the obtained residue by flash column

3

2

4

chromatography over silica gel using cylcohexane and ethylacetate as eluents (from 5-15%

ethylacetate in cyclohexane) afforded compound 2 as colorless solid. Yield: 245 mg (64 %).

1

R

f

: 0.61 (cylcohexane /EtOAc 8:2, visualized with 1.3% ninhydrine solution). H NMR

(

CDCl

3

, 500 MHz, ppm) δ 5.28 (d, J = 6.0 Hz, 1H), 4.30 (p, J = 7.0 Hz, 1H), 2.55 – 2.40 (m,

2

H), 1.62 – 1.54 (m, 2H), 1.43 (s, 9H), 1.31 (d, J = 7.2 Hz, 3H), 1.29 – 1.21 (m, 24H), 0.87 (t,

13

J = 7.0 Hz, 3H).. C NMR (CDCl

3

, 126 MHz, ppm) δ 209.79, 155.18, 79.65, 55.02, 39.21,

3

1.93, 29.70, 29.67, 29.60, 29.45, 29.37, 29.22, 28.35, 23.59, 22.69, 17.97, 14.13. ESI-MS

+

=

m/z calcd for C23

H

46NO [M+H] 384.34; observed: 384.3.

3

4

.2.2 Synthesis of 2-(S)-aminooctadecan-3-one hydrochloride 3

To a stirred solution of compound 2 (191 mg, 0.5 mmol) in methanol (5mL) at 0°C was added

dropwise AcCl (355 µL, 5 mmol). After the resulting reaction mixture was allowed to stir at

ambient temperature for 2h, the resultant white solid was filtered off and subsequently washed

with diethylether and dried to provide 3-ketodeoxysphinganine as a colorless solid. The

obtained crude product was further purified by flash column chromatography over silica gel

using dichloromethane and isopropanol as eluents (from 0-5% isopropanol in

dichloromethane) to afford compound 47 as white solid. Yield: 110 mg (78 %). R

f

: 0.42

solution). H NMR (MeOD, 500 MHz,

ppm) δ 4.17 (q, J = 7.3 Hz, 1H), 2.68 (dt, J = 17.7, 7.3 Hz, 1H), 2.59 (dt, J = 17.7, 7.2 Hz,

1

(

EtOAc/ iso-propanol 9:1, visualized with KMnO

4

1

3

H), 1.68 – 1.60 (m, 2H), 1.54 (d, J = 7.4 Hz, 3H), 1.38 – 1.26 (m, 24H), 0.92 (t, J = 7.0 Hz,

1

3

H). C NMR (MeOD, 126 MHz, ppm) δ 207.44, 55.85, 39.21, 33.10, 30.80, 30.74, 30.63,

0.56, 30.50, 30.16, 24.31, 23.76, 15.75, 14.47, 2.31. ESI-HRMS: m/z calcd for C18

H37NONa

+

[

M+Na] 306.2773; observed 306.2768.

4

.3 Synthesis of E-1-deoxysphingosine derivatives

.3.1 Synthesis of (S)- tert-butyl-(3-oxoalken-2-yl)carbamates

The Grignard solutions were synthesized from the corresponding 1-bromo-ω-alkenes as

follows: A mixture of magnesium turnings (5 equiv.) in anhydrous Et O (1M) under an argon

atmosphere was treated with drops of 1,2-dibromoethane, followed by 1-bromoalkene (1

equiv., 1M solution in anhydrous Et O). The reaction mixture was allowed to stir at 35°C for

-6h to yield a transparent Grignard solution which was immediately used in the next step.

To a stirred solution of compound 1 (1 equiv.) in anhydrous Et O (1M) at -15°C under an

2

3

2

argon atmosphere was added a solution of methylmagnesium bromide (0.9 equiv., 3M in

diethylether). After being stirred for 10min at the same conditions, the resulting mixture was

treated dropwise with a freshly prepared Grignard solution over 30 min (3-4 equiv., 1M

solution in Et O, which was synthesized form the corresponding 1-bromoalkene as described

2

above). The resulting reaction mixture was allowed to stir for 2-3h at ambient temperature (as

monitored by TLC analysis; visualized with 1.3% ninhydrin solution), and subsequently

quenched with an ice-cold 1M HCl solution. The resulting mixture was diluted with

diethylether and the layers were separated. The aqueous layer was extracted with diethylether

(

3times), and the combined organic extracts were subsequently washed with saturated

9

NaHCO

3

solution and brine, dried over anhydrous Na

2

SO , filtered and concentrated under

4

reduced pressure. The resultant residue was purified by flash column chromatography over

silica gel using cylcohexane and ethylacetate as eluents (from 5-15% ethylacetate in

cyclohexane) to afford the desired product.

4

.3.1.1 (S)- tert-butyl-(3-oxonon-8-en-2-yl)carbamate 4

Starting from compound 1 (0.7 g, 3 mmol) and 6-bromohexene (1.46 g, 9 mmol), the title

compound 4 was obtained as white solid after flash column chromatography. Yield: 620 mg

1

(

81%). R

f

: 0.58 (cyclohexane/ethylacetate 7:3, visualized with KMnO

4

solution). H NMR

CDCl3, 500 MHz, ppm): δ 5.78 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.27 (d, J = 5.7 Hz, 1H),

4

1

.99 (ddd, J = 17.1, 3.5, 1.7 Hz, 1H), 4.94 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H), 4.34 – 4.26 (m,

H), 2.56 – 2.42 (m, 2H), 2.09 – 2.02 (m, 2H), 1.61 (dt, J = 20.6, 7.5 Hz, 2H), 1.47 – 1.36 (m,

1H), 1.31 (d, J = 7.2 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 209.73, 155.32, 138.50,

13

14.88, 79.82, 55.15, 39.10, 33.59, 28.52, 28.47, 23.13, 18.05. ESI-HRMS: m/z calcd for

+

C

14

H26NO

3

[M+H] 256.1913; observed 256.1922.

4

.3.1.2 (S)- tert-butyl-(3-oxotridec-12-en-2-yl)carbamate 5

Starting from compound 1 (0.57 g, 2.44 mmol) and 10-bromodecene (1.9 g, 8.54 mmol), the

title compound 5 was obtained as white waxy-solid after flash column chromatography.

Yield: 540 mg (71%). R

solution). H NMR (CDCl3, 500 MHz, ppm): δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.27 (d,

f

: 0.49 (cyclohexane/ethylacetate 9:1, visualized with KMnO

4

1

J = 5.6 Hz, 1H), 4.98 (ddd, J = 17.1, 3.6, 1.6 Hz, 1H), 4.92 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H),

4

.30 (p, J = 7.0 Hz, 1H), 2.55 – 2.40 (m, 2H), 2.06 – 1.99 (m, 2H), 1.64 – 1.55 (m, 2H), 1.43

13

(

s, 9H), 1.35 (dd, J = 13.9, 7.0 Hz, 2H), 1.31 (d, J = 7.2 Hz, 3H), 1.29-1.24 (m, 8H). C NMR

CDCl3, 126 MHz, ppm): δ 209.90, 155.31, 139.31, 114.29, 79.78, 55.15, 39.32, 33.91, 29.43,

2

9.40, 29.31, 29.18, 29.01, 28.48, 23.69, 18.10. ESI-HRMS: m/z calcd for C18

H

33NO Na

3

+

[

M+Na] 334.2358; observed 334.2351.

4

.3.1.3 (S)- tert-butyl-(3-oxotetradec-13-en-2-yl)carbamate 6

Starting from compound 1 (0.49 g, 2.11 mmol) and 11-bromoundecene (1.7 g, 7.4 mmol), the

title compound 6 was obtained as colorless oil after flash column chromatography. Yield: 590

1

f

mg (86%). R : 0.49 (cyclohexane/ethylacetate 9:1, visualized with KMnO

4

solution). H

NMR (CDCl3, 500 MHz, ppm): δ 5.80 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 5.28 (d, J = 5.7 Hz,

1

H), 4.98 (ddd, J = 17.1, 3.6, 1.6 Hz, 1H), 4.95 – 4.90 (m, 1H), 4.30 (p, J = 7.0 Hz, 1H), 2.55

2.40 (m, 2H), 2.07 – 1.98 (m, 2H), 1.62 – 1.55 (m, 2H), 1.43 (s, 9H), 1.40 – 1.22 (m, 15H).

C NMR (CDCl3, 126 MHz, ppm): δ 209.90, 155.31, 139.36, 114.26, 79.78, 55.15, 39.33,

–

1

3

3.94, 29.54, 29.51, 29.48, 29.33, 29.23, 29.05, 28.49, 23.70, 18.11. ESI-HRMS: m/z calcd

+

for C19

H35NO

3

Na [M+H] 348.2515; observed 348.2499.

4

.3.1.4 (S)- tert-butyl-(3-oxopentadec-14-en-2-yl)carbamate 7

Starting from compound 1 (0.59 g, 2.53 mmol) and 12-bromododecene (2.5 g, 10.12 mmol),

the title compound 7 was obtained as colorless oil after flash column chromatography. Yield:

1

6

f

80 mg (79%). R : 0.50 (cyclohexane/ethylacetate 9:1, visualized with KMnO

4

solution). H

NMR (CDCl3, 500 MHz, ppm): δ 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.28 (d, J = 5.9 Hz,

1

7

H), 4.98 (ddd, J = 17.1, 3.7, 1.6 Hz, 1H), 4.92 (ddt, J = 10.1, 2.3, 1.2 Hz, 1H), 4.30 (p, J =

.0 Hz, 1H), 2.55 – 2.40 (m, 2H), 2.06 – 2.00 (m, 2H), 1.59 (dd, J = 14.3, 7.1 Hz, 2H), 1.43 (s,

10

1

3

9

1

2

4

H), 1.40 – 1.21 (m, 17H). C NMR (CDCl3, 126 MHz, ppm): δ 209.91, 155.31, 139.39,

14.24, 79.78, 55.15, 39.34, 33.95, 29.65, 29.59, 29.55, 29.49, 29.34, 29.26, 29.06, 28.48,

3.71, 18.11. ESI-HRMS: m/z calcd for C20

.3.2 Synthesis of (2S,3R)-tert-butyl-(3-hydroxyalken-2-yl)carbamates

+

H38NO

3

[M+H] 340.2852; observed 340.2860.

A stirred solution of ketone derivative (1 equiv.) in absolute ethanol (0.4M) at -78°C under an

argon atmosphere was treated with lithium tri-(tert-butoxy)-aluminum hydride (2.25 equiv.)

portionwise; the addition rate was adjusted so as to keep the internal temperature below -65°C

and it took 30 min to complete. After being stirred at the same conditions for additional 2h (as

monitored by TLC analysis for almost complete reduction of the adduct), the reaction was

carefully quenched with an ice-cold 1M HCl solution; again so as to keep the internal

temperature below -65°C. The resulting mixture was allowed to gradually warm to ambient

temperature and diluted with ethylacetate. The layers were separated and the aqueous layer

was subsequently extracted several times with ethylacetate (4 times). The combined organic

layers were sequentially washed with saturated NaHCO

Na SO , filtered and concentrated in vacuo. Purification of the resultant residue by flash

column chromatography over silica gel using cyclohexane and ethylacetate as eluents (from

3

solution, brine, dried over anhydrous

2

4

1

5-28% ethylacetate in cyclohexane) provided the desired product.

4

.3.2.1 (2S,3R)-tert-butyl-(3-hydroxynon-8-en-2-yl)carbamate 8

Starting from compound 4 (0.45 g, 1.8 mmol), the title compound 8 was obtained as white

waxy-solid after flash column chromatography. Yield: 340 mg (74%, anti:syn 4:1). R : 0.45

solution). H NMR (CDCl3, 500 MHz,

f

1

(

cyclohexane/ethylacetate 7:3, visualized with KMnO

4

ppm): δ 5.73 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.93 (ddd, J = 17.1, 3.6, 1.6 Hz, 1H), 4.87 (ddt,

J = 10.2, 2.1, 1.2 Hz, 1H), 4.70 (br.s, 1H), 3.63 (m, 1H), 3.60 – 3.54 (m, 1H), 2.02 – 1.96 (m,

1

3

2

1

H), 1.50 – 1.23 (m, 15H), 1.01 (d, J = 6.8 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ

56.06, 138.93, 114.61, 79.62, 74.56, 50.74, 33.80, 33.39, 29.04, 28.54, 25.66, 14.53. ESI-

+

HRMS: m/z calcd for C14

H28NO

3

[M+H] 258.2069; observed 258.2074.

4

.3.2.2 (2S,3R)-tert-butyl-(3-hydroxytridec-12-en-2-yl)carbamate 9

Starting from compound 5 (0.46 g, 1.47 mmol), the title compound 9 was obtained as

colourless oil after flash column chromatography. Yield: 410 mg (89%, anti:syn 9:1). R

f

: 0.46

1

(

cyclohexane/ethylacetate 8:2, visualized with KMnO

4

solution). H NMR (CDCl3, 500 MHz,

ppm): δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.98 (ddd, J = 17.1, 3.6, 1.6 Hz, 1H), 4.92 (ddt,

J = 10.1, 2.2, 1.1 Hz, 1H), 4.77 (br.s, 1H), 3.69 (m, 1H), 3.63 (m, 1H), 2.06 – 2.00 (m, 2H),

1

3

1

.44 (s, 9H), 1.41 – 1.24 (m, 14H), 1.07 (d, J = 6.8 Hz, 3H). C NMR (CDCl3, 126 MHz,

ppm): δ 156.03, 139.36, 114.26, 79.58, 74.61, 50.72, 33.94, 33.59, 29.78, 29.63, 29.54, 29.24,

+

2

9.05, 28.55, 26.17, 14.49. ESI-HRMS: m/z calcd for C18

H

35NO Na [M+Na] 336.2515;

3

observed 336.2521.

4

.3.2.3 (2S,3R)-tert-butyl-(3-hydroxytetradec-13-en-2-yl)carbamate 10

Starting from compound 6 (0.43 g, 1.32 mmol), the title compound 10 was obtained as

colourless oil after flash column chromatography. Yield: 360 mg (83%, anti:syn 9:1). R

f

: 0.46

1

(

cyclohexane/ethylacetate 8:2, visualized with KMnO

4

solution). H NMR (CDCl3, 500 MHz,

ppm): δ 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 4.98 (ddd, J = 17.2, 3.7, 1.6 Hz, 1H), 4.92 (ddt,

J = 10.2, 2.3, 1.2 Hz, 1H), 4.77 (br.s, 1H), 3.69 (m, 1H), 3.65 – 3.61 (m, 1H), 2.03 (tt, J = 8.1,

11

1

3

1

2

.3 Hz, 2H), 1.44 (s, 9H), 1.40 – 1.24 (m, 16H), 1.07 (d, J = 6.8 Hz, 3H). C NMR (CDCl3,

26 MHz, ppm): δ 156.01, 139.39, 114.24, 79.58, 74.60, 50.71, 33.94, 33.60, 29.79, 29.67,

9.65, 29.60, 29.26, 29.06, 28.54, 26.18, 14.48. ESI-HRMS: m/z calcd for C19

H

37NO Na

3

+

[

M+Na] 350.2671; observed 350.2665.

4

.3.2.4 (2S,3R)-tert-butyl-(3-hydroxypentadec-14-en-2-yl)carbamate 11

Starting from compound 7 (0.42 g, 1.25 mmol), the title compound 11 was obtained as

colourless oil after flash column chromatography. Yield: 315 mg (74%, anti:syn 4:1). R

f

: 0.46

1

(

cyclohexane/ethylacetate 8:2, visualized with KMnO

4

solution). H NMR (CDCl3, 500 MHz,

ppm): δ 5.81 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 4.99 (ddd, J = 17.1, 3.7, 1.6 Hz, 1H), 4.92 (ddt,

J = 10.2, 2.2, 1.2 Hz, 1H), 4.77 (br.s, 1H), 3.67 (d, J = 12.7 Hz, 1H), 3.65 – 3.61 (m, 1H), 2.03

1

3

(

tt, J = 8.1, 1.3 Hz, 2H), 1.44 (s, 9H), 1.41 – 1.23 (m, 18H), 1.07 (d, J = 6.8 Hz, 3H). C

NMR (CDCl3, 126 MHz, ppm): δ 156.01, 139.41, 114.23, 79.58, 74.61, 50.70, 33.96, 33.60,

9.80, 29.69, 29.62, 29.28, 29.07, 28.54, 26.18, 14.48. ESI-HRMS: m/z calcd for C20

2

[

H

40NO

3

+

M+H] 342.3008; observed 342.3014.

4

.3.3 Synthesis of (2S,3R, E)-tert-butyl-(3-hydroxyalken-2-yl)carbamates

To a stirred solution of aminoalcohol derivative (1 equiv.) in dry deuterated-chloroform

0.2M) under an argon atmosphere was added the cross partner (3-4 equiv.). After the

(

resulting mixture was heated under reflux at 61°C, a catalytic amount of p-benzoquinone (10

nd

mol%) was added followed by Grubbs Catalyst 2 Generation (3-7 mol%) in one portion.

The resulting reaction mixture was allowed to stir under reflux for additional 4-12h and was

monitored by TLC analysis until no further change in the composition of the reaction mixture

(

visualized with KMNO ). The solvent was subsequently removed under reduced pressure

4

and the resultant residue was directly purified by flash column chromatography over silica gel

using cyclohexane and ethylacetate as eluents (from 10-20% ethylacetate in cyclohexane) to

afford the desired product.

4

.3.3.1 (2S,3R, 8E)-tert-butyl-3-hydroxynonen-2-yl)carbamate 12

Starting from compound 8 (150 mg, 0.58 mmol) and 1-undecene (360 mg, 2.33 mmol), the

title compound 12 was obtained as colorless oil after flash column chromatography. Yield:

1

f

32 mg (59%). R : 0.6 (cyclohexane/ethylacetate 7:3, visualized with KMnO

4

solution). H

NMR (CDCl3, 500 MHz, ppm): δ 5.42 – 5.33 (m, 2H), 4.76 (br.s, 1H), 3.70 (dd, J = 13.4, 6.4

Hz, 1H), 3.66 – 3.60 (m, 1H), 2.03 – 1.93 (m, 4H), 1.44 (s, 9H), 1.41 – 1.23 (m, 20H), 1.07

1

3

(

d, J = 6.8 Hz, 3H), 0.88 (t, J = 7.0 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 155.88,

1

2

30.40, 130.33, 79.46, 74.49, 50.58, 33.46, 32.60, 32.58, 31.83, 31.41, 29.64, 29.53, 29.44,

9.34, 29.14, 28.41, 26.05, 22.55, 14.35, 14.10. ESI-HRMS: m/z calcd for C23

H46NO

3

+

[

M+H] 384.3478; observed 384.3485.

4

.3.3.2 (2S,3R, 12E)-tert-butyl-3-hydroxytridecen-2-yl)carbamate 13

Starting from compound 9 (160 mg, 0.51 mmol) and 1-heptene (206 mg, 2.1 mmol), the title

compound 9 was obtained as pale yellow oil after flash column chromatography. Yield: 144

1

mg (73%). R

f

: 0.6 (cyclohexane/ethylacetate 7:3, visualized with KMnO

4

solution). H NMR

(

CDCl3, 500 MHz, ppm): δ 5.44 – 5.30 (m, 2H), 4.76 (br.s, 1H), 3.68 (m, 1H), 3.66 – 3.60 (m,

1

H), 2.05 – 1.93 (m, 4H), 1.44 (s, 9H), 1.42 – 1.22 (m, 20H), 1.07 (d, J = 6.8 Hz, 3H), 0.87 (t,

12

1

3

J = 7.0 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 156.02, 130.88, 130.03, 79.61, 74.57,

5

1

4

0.76, 33.44, 32.74, 32.61, 32.05, 29.78, 29.75, 29.68, 29.49, 29.34, 28.55, 25.68, 22.83,

+

4.51, 14.26. ESI-HRMS: m/z calcd for C23

H

45NO Na [M+Na] 406.3297; observed

3

06.3305.

4

.3.3.3 (2S,3R, 13E)-tert-butyl-3-hydroxytetradecen-2-yl)carbamate 14

Starting from compound 10 (150 mg, 0.45 mmol) and 1-hexene (153 mg, 1.82 mmol), the title

compound 14 was obtained as colorless oil flash column chromatography. Yield: 116 mg

1

(

1

67%). R

f

: 0.6 (cyclohexane/ethylacetate 7:3, visualized with KMnO

4

solution). H NMR

CDCl3, 500 MHz, ppm): δ 5.42 – 5.31 (m, 2H), 4.76 (br.s, 1H), 3.69 (m, 1H), 3.66 – 3.60 (m,

H), 2.04 – 1.93 (m, 4H), 1.44 (s, 9H), 1.41 – 1.23 (m, 20H), 1.07 (d, J = 6.8 Hz, 3H), 0.88 (t,

J = 7.1 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 156.03, 130.49, 130.46, 79.59, 74.63,

1

3

5

1

0.72, 33.61, 32.75, 32.42, 31.98, 29.82, 29.80, 29.69, 29.64, 29.30, 28.55, 26.19, 22.33,

+

4.49, 14.11. ESI-HRMS: m/z calcd for C23

H

46NO [M+H] 384.3478; observed 384.3469.

3

4

.3.3.4 (2S,3R, 14E)-tert-butyl-3-hydroxypentadecen-2-yl)carbamate 15

Starting from compound 11 (180 mg, 0.53 mmol) and 1-pentene (150 mg, 2.12 mmol), the

title compound 15 was obtained as colorless oil after flash column chromatography. Yield:

1

f

03 mg (51%). R : 0.6 (cyclohexane/ethylacetate 7:3, visualized with KMnO

4

solution). H

NMR (CDCl3, 500 MHz, ppm): δ 5.43 – 5.33 (m, 2H), 4.74 (br.s, 1H), 3.69 (m, 1H), 3.64 (m,

H), 2.03 – 1.92 (m, 4H), 1.44 (s, 9H), 1.41 – 1.24 (m, 20H), 1.08 (d, J = 6.8 Hz, 3H), 0.88 (t,

1

3

J = 7.4 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 156.04, 130.74, 130.25, 79.60, 74.64,

5

1

0.71, 34.86, 33.61, 32.77, 29.81, 29.75, 29.71, 29.66, 29.31, 28.55, 26.20, 22.90, 14.51,

+

3.81. ESI-HRMS: m/z calcd for C23

H

46NO [M+H] 384.3478; observed 384.3486.

3

4

.3.4 Synthesis of (2S,3R)-2-aminooctadecen-3-ol

A stirred solution of protected 1-deoxysphingosine (1 mmol) in 1, 4-dioxane (5 mL, 0.2M) at

°C was treated with a solution of 4N HCl-dioxane (5 mL). The resulting reaction mixture

0

was allowed to stir for 30 min at 0°C, gradually warmed to ambient temperature and stirred

for additional 60-90 min (as judged by TLC analysis for almost a complete deprotection;

visualized with 1.3% ninhydrine). The reaction mixture was diluted with CH

subsequently quenched with a saturated NaHCO solution (to pH ≈8.0). The resulting mixture

was then extracted with CH Cl (4 times) and the combined organic layers were washed with

water and brine, dried over anhydrous Na SO , filtered and concentrated under reduced

2

Cl

2

and

3

2

4

pressure. Purification of the crude product by flash column chromatography over silica gel

using ethylacetate and isopropanol as eluents (from 0-7% isopropanol in ethylacetate)

provided the desired product.

4

.3.4.1 (2S,3R,8E)-2-aminooctadecen-3-ol 16

Starting from compound 12 (102 mg, 0.27 mmol), the title compound 16 was obtained as

yellow oil after flash column chromatography. Yield: 47 mg (62%). R : 0.45 (EtOAc/iso-

solution). H NMR (CDCl3, 500 MHz, ppm): δ 5.44 –

.32 (m, 2H), 3.82 (m, 1H), 3.29 (m, 1H), 2.04 – 1.90 (m, 4H), 1.31 (m, J = 46.6, 34.6 Hz,

f

1

propanol 4:1, visualized with KMnO

5

2

4

13

3H), 0.87 (t, J = 6.9 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 130.52, 130.43, 70.85,

13

5

1.68, 32.73, 32.43, 31.98, 31.56, 29.82, 29.62, 29.49, 29.32, 25.87, 22.69, 14.21, 11.19. ESI-

+

HRMS: m/z calcd for C18H38NO [M+H] 284.2953; observed 284.2961.

4

.3.4.2 (2S,3R,12E)-2-aminooctadecen-3-ol 17

Starting from compound 13 (112 mg, 0.29 mmol), the title compound 17 was obtained as

yellow oil after flash column chromatography. Yield: 61 mg (71%). R : 0.45 (EtOAc/iso-

500 MHz, ppm): δ 5.46 –

.28 (m, 2H), 3.66 (m, 1H), 3.22 (m, 1H), 2.07 – 1.88 (m, 4H), 1.53 – 1.20 (m, 20H), 1.17 (d,

f

1

propanol 4:1, visualized with KMnO

5

J = 6.7 Hz, 3H), 0.85 (t, J = 6.4 Hz, 3H). C NMR (MeOD

4

solution). H NMR (MeOD

,

13

,

126 MHz, ppm): δ 131.71,

1

2

31.13, 71.56, 52.49, 33.81, 33.61, 33.47, 33.01, 30.72, 30.68, 30.56, 30.42, 30.21, 26.39,

3.69, 14.46, 12.01. ESI-HRMS: m/z calcd for C18H38NO [M+H] 284.2953; observed

84.2947.

+

4

.3.4.3 (2S,3R,13E)-2-aminooctadecen-3-ol 18

Starting from compound 14 (86 mg, 0.22 mmol), the title compound 18 was obtained as pale

yellow oil after flash column chromatography. Yield: 53 mg (83%). R : 0.45 (EtOAc/iso-

solution). H NMR (CDCl3, 500 MHz, ppm): δ 5.45 –

.32 (m, 2H), 3.86 (m, 1H), 3.36 (m, 1H), 2.05-1.92 (m, 4H), 1.52 – 1.19 (m, 23H), 0.88 (t, J

f

1

propanol 4:1, visualized with KMnO

4

5

=

1

3

7.0 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 130.46, 130.00, 70.93, 51.84, 32.90,

3

2.76, 32.43, 31.97, 29.82, 29.70, 29.64, 29.56, 29.32, 25.91, 22.33, 14.09, 11.40. ESI-

+

HRMS: m/z calcd for C18H37NONa [M+Na] 306.2773; observed 306.2781.

4

.3.4.4 (2S,3R,14E)-2-aminooctadecen-3-ol 19

Starting from compound 15 (77 mg, 0.2 mmol), the title compound 19 was obtained as pale

yellow oil after flash column chromatography. Yield: 39 mg (69%). R : 0.45 (EtOAc/iso-

solution). H NMR (CDCl3, 500 MHz, ppm): δ 5.46 –

.31 (m, 2H), 4.28 (br.s, 1H), 3.85 (m, 1H), 3.32 (m, 1H), 2.05-1.91 (m, 4H), 1.53 – 1.20 (m,

f

1

propanol 4:1, visualized with KMnO

4

5

2

5

13

3H), 0.88 (t, J = 7.3 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 130.71, 130.24, 70.70,

1.62, 34.86, 32.86, 32.78, 29.83, 29.78, 29.68, 29.62, 29.35, 22.89, 13.79, 11.44. ESI-

+

HRMS: m/z calcd for C18

H

38NO [M+H] 284.2953; observed 284.2959.

4

.4 Synthesis of 1-deoxysphingadiene derivatives

.4.1 Synthesis of (S)-tert-butyl-(3-oxoalken-2-yl) carbamates

To a stirred solution of compound 1 (1 equiv.) in dry THF (0.25M) at -20°C under an argon

atmosphere was added dropwise a freshly prepared solution of Grignard reagent (3-4 equiv.,

1

2

M solution in THF); the addition rate was adjusted so as to keep the internal temperature at -

0 °C and it took 45 min to complete. After being stirred at the same conditions for 30 min

and for additional 4-8h at ambient temperature (as judged by TLC analysis for almost a

complete reaction), the reaction mixture was carefully poured to an ice-cold 1M HCl solution

to quench; again so as to keep the temperature below -10°C and it took 40 min to complete.

The resultant mixture was subsequently extracted several times with ethylacetate, and the

combined organic extracts were washed with saturated NaHCO

anhydrous Na SO , filtered and concentrated under reduced pressure. Purification of the

resultant residue by flash column chromatography over silica gel using cyclohexane and

3

solution, brine, dried over

2

4

14

ethylacetate as eluents (from 9-15% ethylacetate in cyclohexane) afforded the desired

product.

2

6

4

.4.1.1 (S)-tert-butyl-3-oxopent-4-en-2-ylcarbamate 20

Starting from compound 1 (1.2 g, 5.1 mmol), the title compound 20 was obtained as white

solid after flash column chromatography. Yield: 0.64 g (64 %). R : 0.69

cyclohexane/ethylacetate 3:2, visualized with 1.3% ninhydrine solution). H NMR (CDCl3,

f

1

(

5

00 MHz, ppm): δ 6.48 (dd, J = 17.4, 9.9 Hz, 1H). 6.37 (dd, J = 17.4, 1.9 Hz, 1H), 5.88 (dd, J

=

10.2, 1.9 Hz, 1H), 5.37 (br s, 1H), 4.54-4.67 (m, 1H), 1.44 (s, 9H), 1.33 (d, J = 6.9 Hz, 3H).

1

3

C NMR (CDCl3, 126 MHz, ppm): δ 198.68, 155.23, 132.91, 130.34, 79.51, 53.20, 28.63,

+

1

4

8.42. ESI-HRMS m/z calcd for C10

H17NO

3

Na [M+Na] 222.1106; observed 222.1102.

.4.1.2 (S)-tert-butyl-(3-oxohex-5-en-2-yl)carbamate 21

Starting from compound 1 (1.2 g, 5.1 mmol), the title compound 21 was obtained as pale

yellow solid after flash column chromatography. Yield: 0.78 g (72 %). R : 0.69

f

1

(

cyclohexane/ethylacetate 3:2, visualized with 1.3% ninhydrine solution). H NMR (CDCl3,

5

1

2

00 MHz, ppm): δ 5.85 (ddt, J = 17.1, 10.2, 6.9 Hz, 1H), 5.15 (ddd, J = 10.2, 2.7, 1.3 Hz, 1H),

.10 (dq, J = 17.2, 1.5 Hz, 1H), 4.30 (p, J = 7.0 Hz, 1H), 3.28 – 3.16 (m, 2H), 1.38 (s, 9H),

1

3

.27 (d, J = 7.2 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 207.58, 155.29, 129.93,

+

19.46, 79.96, 54.91, 44.14, 28.46, 17.82. ESI-HRMS m/z calcd for C12

H

22NO [M+H]

3

28.1600; observed 228.1593.

4

.4.2 Synthesis of (2S,3R)-tert-butyl-(3-hydroxypent-4-en-2-yl)carbamates

A stirred solution of ketone derivative (1 equiv.) in anhydrous ethanol (0.5M) at -78°C under

an argon atmosphere was treated with lithium tri-(tert-butoxy)-aluminum hydride (2.25

equiv.) in portionwise; the addition rate was adjusted so as to keep the internal temperature

below -65°C and it took 30 min to complete. After the resulting reaction mixture was stirred

at the same conditions for 90-180 min (as monitored by TLC analysis for almost complete

reduction of the adduct), an ice-cold 1M HCl solution was added dropwise to quench; again

so as to keep the internal temperature below -65°C and it took 30 min to complete. The

resulting mixture was allowed to gradually warm to ambient temperature and was diluted with

ethylacetate. The layers were separated and the aqueous layer was subsequently extracted

several times with ethylacetate. The combined organic layers were sequentially washed with

saturated NaHCO

3

solution, brine, dried over anhydrous Na

2

SO , filtered and concentrated in

4

vacuo. Flash column chromatography of the resultant crude mixture over silica gel using

cyclohexane and ethylacetate as eluents (from 10-25% ethylacetate in cyclohexane) provided

the desired product.

2

6

4

.4.2.1 (2S,3R)-tert-butyl-(3-hydroxypent-4-en-2-yl)carbamate 22

Starting from compound 20 (0.55 g, 2.8 mmol), the title compound 22 was obtained as white

solid after flash column chromatography. Yield: 0.49 g (88%, anti:syn 9:1) . R : 0.54

solution). H NMR (CDCl3, 500 MHz,

ppm): δ 5.85 (ddd, J= 17.2, 10.6, 5.5 Hz, 1H), 5.33 (dd, J = 17.2, 1.6 Hz, 1H), 5.24 (dd, J =

0.5, 1.5 Hz, 1H), 4.76-4.62 (m, 1H), 4.23-4.16 (m, 1H), 2.85 (br. s, 1H), 1.45 (s, 9H), 1.09

f

1

(

cyclohexane/ethylacetate 3:2, visualized with KMnO

4

1

(

13

d, J = 6.9 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 156.46, 136.98, 116.68, 79.89,

15

+

7

5.92, 50.96, 28.50, 15.46. ESI-HRMS: m/z calcd for C10

H

19NO Na [M+Na] 224.1263;

3

observed 224.1259.

4

.4.2.2 (2S,3R)-tert-butyl-(3-hydroxyhex-5-en-2-yl)carbamate 23

Starting from compound 21 (0.7 g, 3.3 mmol), the title compound 23 was obtained as white

waxy-solid after flash column chromatography. Yield: 0.54 g (76%, anti:syn 4:1). R : 0.54

solution). H NMR (CDCl3, 500 MHz,

ppm): δ 5.83 (ddt, J = 17.1, 10.2, 7.2 Hz, 1H), 5.17 – 5.10 (m, 2H), 4.78 (br.s, 1H), 3.70 (m,

f

1

(

cyclohexane/ethylacetate 3:2, visualized with KMnO

4

1

3

2

H), 2.27 – 2.12 (m, 2H), 1.44 (s, 9H), 1.11 (d, J = 6.8 Hz, 3H). C NMR (CDCl3, 126 MHz,

ppm): δ 155.93, 134.83, 118.24, 79.65, 73.45, 50.40, 38.44, 28.53, 14.74. ESI-HRMS: m/z

+

calcd for C12

H23NO

3

Na [M+Na] 252.1576; observed 252.1571.

4

.4.3 Synthesis of (2S,3R,E)-tert-butyl (13-bromo-3-hydroxytridecen-2-yl)carbamate

A stirred mixture of aminoalcohol derivative (1 equiv.) and the cross partner (4-5 equiv.) in

anhydrous deuterated-chloroform (0.3M) under an argon atmosphere was treated with a

nd

catalytic amount of p-benzoquinone (10 mol%) followed by Grubbs Catalyst 2 Generation

(

8

7 mol%, in one portion). After the resulting reaction mixture was stirred under reflux for 4-

h (until no further change in the composition of the reaction mixture as monitored by TLC

analysis, visualized with 1.3% ninhydrine solution), the solvent was subsequently removed

under reduced pressure. Purification of the obtained residue by flash column chromatography

over silica gel using cyclohexane and ethylacetate as eluents (from 10-25% ethylacetate in

cyclohexane) afforded the desired product.

4

.4.3.1 (2S,3R,4E)-tert-butyl-(13-bromo-3-hydroxytridec-4-en-2-yl)carbamate 24

Starting from compound 22 (350 mg, 1.73 mmol) and 10-bromo-1-decene (1.9 g, 8.7 mmol),

the title compound 24 was obtained as pale yellow oil after flash column chromatography.

Yield: 460 mg (68%). R

f

: 0.6 (cyclohexane/ethylacetate 7:3, visualized with 1.3% ninhydrine

solution). H NMR (CDCl3, 500 MHz, ppm): δ 5.70 (dtd, J = 7.7, 6.7, 1.0 Hz, 1H), 5.42 (ddt, J

15.4, 6.5, 1.2 Hz, 1H), 4.68 (d, J = 5.7 Hz, 1H), 4.10 (d, J = 2.5 Hz, 1H), 3.84 – 3.71 (m,

H), 3.40 (t, J = 6.8 Hz, 2H), 2.07 – 1.99 (m, 2H), 1.90 – 1.79 (m, 2H), 1.43 (s, 9H), 1.40 –

1

=

1

3

.25 (m, 8H), 1.06 (d, J = 6.9 Hz, 3H). C NMR (CDCl3, 126 MHz, ppm): δ 156.40, 133.99,

28.58, 79.74, 75.86, 51.19, 34.15, 32.92, 32.45, 29.37, 29.23, 29.14, 28.82, 28.52, 28.26,

5.61. ESI-HRMS: m/z calcd for C18

-

H33NO

3

Br [M-H] 390.1644; observed 390.1656.

4

.4.3.2 (2S,3R,5E)-tert-butyl-(13-bromo-3-hydroxytridec-5-en-2-yl)carbamate 25

Starting from compound 23 (460 mg, 2.14 mmol) and 9-bromo-1-nonene (2.2 g, 10.7 mmol),

the title compound 25 was obtained as pale yellow oil after flash column chromatography.

Yield: 510 mg (61%). R

solution). H NMR (CDCl3, 500 MHz, ppm): δ 5.53 (dt, J = 13.5, 6.6 Hz, 1H), 5.40 (dt, J =

f

: 0.6 (cyclohexane/ethylacetate 7:3, visualized with 1.3% ninhydrine

1

2

5.1, 7.4 Hz, 1H), 4.78 (br.s, 1H), 3.68 (m, 1H), 3.64 (m, 1H), 3.40 (t, J = 6.9 Hz, 2H), 2.22 –

.08 (m, 2H), 2.01 (dd, J = 14.2, 7.1 Hz, 3H), 1.88 – 1.81 (m, 2H), 1.44 (s, 9H), 1.42 – 1.26

1

3

(

m, 8H), 1.10 (d, J = 6.8 Hz, 2H). C NMR (CDCl3, 126 MHz, ppm): δ 155.87, 134.65,

1

25.86, 79.52, 73.64, 50.26, 37.34, 34.16, 32.92, 32.69, 29.39, 29.08, 28.72, 28.55, 28.24,

+

4.68. ESI-HRMS: m/z calcd for C18

H

34NO BrNa [M+Na] 414.1620; observed 414.1614.

3

16

4

.4.4 Synthesis of (4S,5R)-tert-butyl-5-((E)-10-bromodecen-1-yl)-2,2,4-trimethyl-

oxazolidine-3-carboxylate

A stirred solution of aminoalcohol derivative (1 equiv.) in anhydrous toluene (0.15M) under

an argon atmosphere was treated with 2,2-dimethoxypropane (2.2 equiv.), followed by a

catalytic amount of p-toluensulfonic acid monohydrate (10 mol%). After being stirred under

reflux in a Dean-Stark apparatus for 2-5h (as judged by the TLC analysis, visualized with

KMnO

ethylacetate, and successfully quenched with saturated NaHCO

separated, and the aqueous layer was extracted several times with ethylacetate. The combined

organic layers were washed with brine, dried over anhydrous Na SO , filtered and

4

solution), the reaction mixture was cooled to ambient temperature, diluted with

3

solution. The layers were

2

4

concentrated under reduced pressure. The resultant crude mixture was immediately purified

by flash column chromatography over silica gel using cyclohexane and ethylacetate as eluents

(

from 0% to 10% ethylacetate in cyclohexane) to afford the desired product.

4

.4.4.1 (4S,5R)-tert-butyl-5-((E)-10-bromodec-1-en-1-yl)-2,2,4-trimethyl-oxazolidine-3-

carboxylate 26

Starting from compound 24 (435 mg, 1.12 mmol), the title compound 26 was obtained as

colorless oil after flash column chromatography. Yield: 440 mg (91%). R

f

: 0.5

1

(

cyclohexane/EtOAc 9:1, visualized with 1.3% ninhydrine solution). H NMR (CDCl3, 500

MHz, ppm): δ 5.83 (ddd, J = 13.4, 6.8, 6.2 Hz, 1H), 5.44 (dd, J = 15.4, 7.8 Hz, 1H), 4.46 (dd,

J = 7.5, 5.6 Hz, 1H), 4.02 – 3.80 (m, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.07 (dd, J = 13.6, 6.8 Hz,

1

3

2

1

3

H), 1.89 – 1.79 (m, 2H), 1.63 – 1.28 (m, 26H), 1.10 (t, J = 7.1 Hz, 3H). C NMR (CDCl3,

26 MHz, ppm): δ 151.42, 136.89, 124.44, 92.83, 80.02, 79.54, 77.55, 56.26, 34.02, 32.79,

2.38, 29.36, 29.19, 28.81, 28.65, 25.15, 14.84. ESI-HRMS: m/z calcd for C21

H

39NO Br

3

+

[

M+H] 432.2113; observed 432.2118.

4

.4.4.2 (4S,5R)-tert-butyl-5-((E)-10-bromodec-2-en-1-yl)-2,2,4-trimethyl-oxazolidine-3-

carboxylate 27

Starting from compound 25 (485 mg, 1.24 mmol), the title compound 27 was obtained as pale

yellow oil after flash column chromatography.

Yield: 370 mg (69%). R

solution). H NMR (CDCl3, 500 MHz, ppm): δ 5.51 – 5.43 (m, 1H), 5.28 (dt, J = 13.1, 6.0 Hz,

f

: 0.5 (cyclohexane/EtOAc 9:1, visualized with 1.3% ninhydrine

1

3

1

3

H), 3.97 – 3.72 (m, 2H), 3.34 (t, J = 6.8 Hz, 2H), 2.36 – 2.09 (m, 2H), 2.07 – 1.86 (m, 2H),

.82 – 1.75 (m, 2H), 1.54 – 1.39 (m, 15H), 1.38 – 1.18 (m, 8H), 1.04 (dd, J = 14.0, 6.3 Hz,

H). C NMR (CDCl3, 126 MHz, ppm): δ 151.96, 151.63*, 133.53, 133.46*, 124.88,

1

3

24.79*, 92.67, 92.24*, 79.90, 79.25*, 76.25, 76.01*, 55.17, 55.13*, 33.99, 32.78, 32.56,

2.37*, 32.15, 29.19, 28.88*, 28.55, 28.48*, 28.11, 25.00, 23.86*, 14.33, 13.63* (*Asterisk

+

denotes minor conformer peaks). ESI-HRMS: m/z calcd for C21

H39NO

3

Br [M+H] 432.2113;

observed 432.2125.

4

.4.5 Synthesis of (4S,5R)-tert-butyl-2,2,4-trimethyl-5-((E)-pentadecen-11-yn-1-

yl)oxazolidine-3-carboxylate

A stirred solution of 1-pentyne (2.1 equiv.) in anhydrous THF (0.2M) at -78°C under an argon

atmosphere was treated with a solution of t-BuLi (1.9 equiv. 1.7M solution in pentane) in

dropwise over a period of 40 min; the rate of addition was adjusted so as to keep the internal

17

temperature below -65°C. After stirred for 90 min at the same conditions, the formed lithated

-pentyne was treated with hexamethylphosphoramide (4.5 equiv.), followed by dropwise

1

addition of a solution of bromo derivative (1 equiv., 0.25M solution in anhydrous THF) over a

period of 20 min. The resulting reaction mixture was allowed to stir for 1h at -45°C, and for

additional 4-6h at ambient temperature (as monitored by TLC analysis, visualized with

Seebach reagent), before it was carefully quenched with an ice-cold NH Cl solution (150

4

mL). The resulting mixture was diluted with diethylether and the layers were separated. The

aqueous phase was extracted several times with diethylether, and the combined organic

phases were consecutively washed with saturated NaHCO

3

solution and brine, dried over

anhydrous Na SO , filtered, and concentrated under in vacuo. Purification of the resultant

2

4

residue by flash column chromatography over silica gel using petroleum ether and

ethylacetate as eluents (from 0-15% ethylacetate in petroleum ether) afforded the desired

product.

4

.4.5.1 (4S,5R)-tert-butyl-2,2,4-trimethyl-5-((E)-pentadec-1-en-11-yn-1-yl)-oxazolidine-3-

carboxylate 28

Starting from compound 26 (420 mg, 0.96 mmol), the title compound 28 was obtained as

colorless oil after flash column chromatography. Yield: 255 mg (63 %). R

f

: 0.52 (Pet. ether/

, 500 MHz, ppm) δ 5.83 (dt, J

14.8, 6.7 Hz, 1H), 5.44 (dd, J = 15.4, 7.8 Hz, 1H), 4.46 (dd, J = 7.5, 5.7 Hz, 1H), 4.02 – 3.81

m, 1H), 2.12 (dtt, J = 7.2, 4.6, 2.4 Hz, 4H), 2.09 – 2.03 (m, 2H), 1.67 – 1.43 (m, 21H), 1.41 –

1

EtOAc 95:5, visualized with Seebach reagent). H NMR (CDCl

3

=

(

1

3

1

7

2

.23 (m, 10H), 1.11 (dd, J = 14.1, 6.4 Hz, 3H), 0.96 (t, J = 7.3 Hz, 3H). C NMR (CDCl

26 MHz, ppm) δ 152.03, 151.77*, 137.10, 124.51, 124.32*, 92.88, 80.50, 80.20*, 80.01,

3

,

9.38, 77.98, 77.71*, 56.49, 56.42*, 32.56, 29.47, 29.29, 29.21, 29.02*, 28.97, 28.67, 28.43,

7.53*, 25.29, 24.15*, 22.70, 20.91, 18.88, 15.63, 14.97*, 13.61 (*Asterisk denotes minor

+

conformer peaks). ESI-HRMS: m/z calcd for C26

H46NO

3

[M+H] 420.3478; observed

4

20.3467.

4

.4.5.2 (4S,5R)-tert-butyl-2,2,4-trimethyl-5-((E)-pentadec-2-en-11-yn-1-yl)-oxazolidine-3-

carboxylate 29

Starting from compound 27 (350 mg, 0.81 mmol), the title compound 29 was obtained as pale

yellow oil after flash column chromatography. Yield: 184 mg (54 %). R

f

: 0.52 (Pet. ether/

, 500 MHz, ppm) δ 5.60 –

.50 (m, 1H), 5.39 – 5.29 (m, 1H), 4.03 – 3.78 (m, 2H), 2.41 – 2.17 (m, 2H), 2.16 – 2.09 (m,

H), 2.08 – 1.95 (m, 2H), 1.60 – 1.44 (m, 19H), 1.42 – 1.24 (m, 8H), 1.10 (dd, J = 14.8, 6.3

1

EtOAc 95:5, visualized with Seebach reagent). H NMR (CDCl

3

5

2

1

3

Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H). C NMR (CDCl

3

, 126 MHz, ppm) δ 152.07, 151.76*,

1

5

2

33.82, 133.74*, 124.88, 124.80*, 92.79, 92.34*, 80.48, 80.21*, 79.96, 79.34*, 76.41, 76.16*,

5.31, 55.26*, 32.76, 32.52*, 32.30, 29.44, 29.41, 29.29, 29.16*, 29.12, 28.94, 28.69, 28.61*,

8.37, 27.45, 25.13, 23.98*, 22.69, 20.90, 18.87, 14.46, 13.76*, 13.62 (*Asterisk denotes

+

minor conformer peaks). ESI-HRMS: m/z calcd for C26

H45NO

3

Na [M+Na] 442.3297;

observed 442.3302.

18

4

.4.6 Synthesis of (4S,5R)-tert-butyl-2,2,4-trimethyl-5-((E,11Z)-pentadeca-1,11-dien-1-

yl)oxazolidine-3-carboxylate

To a stirred solution of alkyne derivative (1 equiv.) in anhydrous mixture of

ethylacetate:DMF (95:5, 0.05M) under an argon atmosphere was added ethylenediamine (0.9

equiv.), followed by a catalytic amount of Lindlar catalyst. After the reaction vessel was

evacuated and then backfilled with hydrogen gas, the resulting reaction mixture was allowed

to stir for 16h at ambient temperature (as monitored by TLC analysis; visualized with Seebach

reagent). The mixture was subsequently filtered through a pad of Celite, and the combined

filtrates were concentrated under in vacuo. Flash column chromatography of the resultant oily

residue over silica gel using petroleum ether and ethylacetate as eluents (from 0-10%

ethylacetate in petroleum ether) provided the desired compound.

4

3

.4.6.1 (4S,5R)-tert-butyl-2,2,4-trimethyl-5-((1E,11Z)-pentadeca-1,11-dien-1-yl)oxazolidine-

-carboxylate 30

Starting from compound 28 (237 mg, 0.56 mmol), the title compound 30 was obtained as pale

yellow oil after flash column chromatography. Yield: 192 mg (81 %). R : 0.56 (Pet. ether/

, 500 MHz, ppm) δ 5.83 (dt, J

14.9, 6.6 Hz, 1H), 5.44 (dd, J = 15.4, 7.9 Hz, 1H), 5.35 (ddd, J = 5.6, 3.6, 1.9 Hz, 2H), 4.46

dd, J = 7.5, 5.7 Hz, 1H)., 4.02 – 3.81 (m, 1H), 2.10 – 2.03 (m, 2H), 2.03 – 1.97 (m, 4H), 1.63

f

1

EtOAc 95:5, visualized with KmnO

4

solution). H NMR (CDCl

3

=

(

–

3

1

2

1

1.45 (m, 15H), 1.42 – 1.25 (m, 14H), 1.11 (dd, J = 14.2, 6.4 Hz, 3H), 0.90 (t, J = 7.4 Hz,

1

3

H). C NMR (CDCl , 126 MHz, ppm) δ 152.04, 151.77*, 137.13, 130.21, 129.80, 124.50,

3

24.31*, 92.89, 92.39*, 80.01, 79.38*, 78.00, 77.73*, 56.50, 56.43*, 32.57, 29.89, 29.60,

9.56*, 29.44, 29.33, 29.04, 28.67*, 28.43, 27.54*, 27.35, 25.31, 24.15*, 23.04, 15.64,

4.98*, 13.95 (*Asterisk denotes minor conformer peaks). ESI-HRMS: m/z calcd for

+

C

26

H47NO

3

Na [M+Na] 444.3454; observed 444.3459.

4

3

.4.6.2 (4S,5R)-tert-butyl-2,2,4-trimethyl-5-((2E,11Z)-pentadeca-2,11-dien-1-yl)oxazolidine-

-carboxylate 31

Starting from compound 29 (162 mg, 0.39 mmol), the title compound 31 was obtained as pale

yellow oil after flash column chromatography. Yield: 122 mg (75 %). R : 0.56 (Pet. ether/

, 500 MHz, ppm) δ 5.58 –

.50 (m, 1H), 5.40 – 5.29 (m, 3H), 4.03 – 3.79 (m, 2H), 2.40 – 2.14 (m, 2H), 2.07 – 1.95 (m,

H), 1.61 – 1.44 (m, 15H), 1.40 – 1.25 (m, 12H), 1.10 (dd, J = 15.1, 6.3 Hz, 3H), 0.90 (t, J =

.4 Hz, 3H). 13C NMR (CDCl , 126 MHz, ppm) δ 152.06, 151.76*, 133.85, 133.78*, 130.18,

f

1

EtOAc 95:5, visualized with KmnO

4

solution). H NMR (CDCl

3

5

6

7

1

3

2

3

29.81, 124.86, 124.77*, 92.79, 92.33*, 79.94, 79.32*, 76.41, 76.16*, 55.31, 55.26*, 32.78,

2.53*, 32.30, 29.89, 29.51, 29.43, 29.39, 29.23, 28.69, 28.62*, 28.37, 27.69, 27.46, 27.34,

5.14, 23.99*, 23.03, 14.47, 13.95*, 13.77 (*Asterisk denotes minor conformer peaks). ESI-

+

HRMS: m/z calcd for C26

H

48NO

3

[M+H] 422.3634; observed 422.3642.

4

.4.7 Synthesis of (2S,3R)-2-aminooctadecadien-3-ol

To a stirred solution of protected deoxysphingadiene (1 mmol) in 1,4- dioxane (5 mL) at 0°C

was added a solution of 4M HCl-dioxane (5 mL). After being stirred for 30 min at the same

conditions, and for additional 90 min at ambient temperature (as monitored by TLC,

visualized with KMnO

4

solution), the reaction mixture was concentrated under reduced

pressure. The resultant residue was taken up in diethylether (100 mL) and sequentially

19

washed with saturated NaHCO

3

solution (80 mL) and brine solution (80 mL), dried over

anhydrous Na SO , filtered and concentrated under reduced pressure. Purification of the

2

4

obtained residue by flash column chromatography silica gel using ethylacetate and

isopropanol as eluents (from 0-10% isopropanol in ethylacetate) yielded the desired 1-

deoxysphingadiene.

4

.4.7.1 (2S,3R,4E,14Z)-2-aminooctadeca-4,14-dien-3-ol 32

Starting from compound 30 (172 mg, 0.4 mmol), the title compound 32 was obtained as

yellow oil after flash column chromatography. Yield: 67 mg (59%). R : 0.32 (EtOAc/iso-

solution). H NMR (MeOD, 500 MHz, ppm) δ 5.84

dtd, J = 14.9, 6.8, 1.0 Hz, 1H), 5.46 (ddt, J = 15.5, 6.7, 1.4 Hz, 1H), 5.35 (ddd, J = 5.6, 3.7,

f

1

propanol 4:1, visualized with KMnO

(

4

1

.8 Hz, 2H), 4.19 (dd, J = 5.6, 3.8 Hz, 1H), 3.29 – 3.23 (m, 1H), 2.16 – 1.96 (m, 6H), 1.45 –

.28 (m, 14H), 1.21 (d, J = 6.8 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). C NMR (MeOD, 126 MHz,

1

3

ppm) δ 136.40, 131.00, 130.62, 128.35, 72.98, 52.80, 33.39, 30.85, 30.60, 30.32, 28.12,

-

2

3.95, 14.12, 13.31. ESI-HRMS: m/z calcd for C18H34NO [M-H] 280.2640; observed

80.2653.

4

.4.7.2 (2S,3R,5E,14Z)-2-aminooctadeca-5,14-dien-3-ol 33

Starting from compound 31 (100 mg, 0.24 mmol), the title compound 33 was obtained as

yellow oil after flash column chromatography. Yield: 44 mg (66%). R : 0.32 (EtOAc/iso-

solution). H NMR (MeOD, 500 MHz, ppm) δ 5.58 (dt,

f

1

propanol 4:1, visualized with KMnO

4

J = 15.0, 6.8 Hz, 1H), 5.47 – 5.39 (m, 1H), 5.39 – 5.32 (m, 2H), 3.76 – 3.72 (m, 1H), 3.28 (tt,

J = 6.7, 3.2 Hz, 1H), 2.28 – 2.13 (m, 2H), 2.10 – 1.99 (m, 6H), 1.41 – 1.29 (m, 14H), 1.22 (d,

13

J = 6.8 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). C NMR (MeOD, 126 MHz, ppm) δ 135.30,

1

2

3

31.00, 130.62, 126.14, 71.67, 51.87, 37.71, 33.69, 30.84, 30.53, 30.49, 30.29, 30.26, 28.12,

3.95, 14.11, 11.64. ESI-HRMS: m/z calcd for C18H35NONa [M+Na] 304.2616; observed

04.2623.

+

4

.5 Synthesis of ɷ-hydroxy-1-deoxysphinganine analogue

4

.5.1 Synthesis of 2-(pentadec-14-en-1-yloxy)tetrahydro-2H-pyran 37

A stirred solution of compound 36 (2.6 g, 6.6 mmol) in anhydrous THF (66 mL) at ambient

temperature under an argon atmosphere was treated with tert-BuOK (0.89 g, 7.9 mmol) in

portionwise over 30 min. After the resulting reaction mixture was allowed to stir under reflux

for 12h (as monitored by TLC analysis, cyclohexane/EtOAc 95:5; Rf(adduct) = 0.45; Rf(product)

.9; visualized with Seebach reagent), water (200 mL) was added to quench. The resulting

mixture was extracted with diethylether (3x100 mL), and the combined organic extracts were

washed with brine (200 mL), dried over anhydrous Na SO , filtered and concentrated under

=

0

2

4

reduced pressure. Purification of the resultant oil by flash column chromatography over silica

gel using petroleum ether as eluent to provided compound 37 as colorless oil. Yield: 1.7 g (83

1

%

). R

f

: 0.6 (100% petroleum ether, visualized with Seebach reagent). H NMR (CDCl

3

, 500

MHz, ppm) δ 5.81 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 4.99 (ddd, J = 17.1, 3.7, 1.6 Hz, 1H), 4.92

ddt, J = 10.2, 2.3, 1.2 Hz, 1H), 3.87 (ddd, J = 11.2, 7.5, 3.3 Hz, 1H), 3.72 (dt, J = 9.6, 6.9 Hz,

(

1

H), 3.52 – 3.46 (m, 1H), 3.38 (dt, J = 9.6, 6.7 Hz, 1H), 2.07 – 2.00 (m, 2H), 1.88 – 1.79 (m,

13

H), 1.75 – 1.68 (m, 1H), 1.61 – 1.48 (m, 6H), 1.41 – 1.23 (m, 20H). C NMR (CDCl , 126

3

MHz, ppm) δ 139.42, 114.21, 98.99, 67.85, 62.48, 33.97, 30.93, 29.90, 29.79, 29.75, 29.65,

20

+

2

9.30, 29.09, 26.39, 25.65, 19.84. ESI-MS m/z calcd for C20

H

38NaO [M+Na] 333.28;

2

observed 333.2.

4

.5.2 Synthesis of ((2S,3R,4E)-tert-butyl-3-hydroxy-18-((tetrahydro-2H-pyran-2-

yl)oxy)octadec-4-en-2-yl)-carbamate 38

A stirred solution of compound 22 (195 mg, 0.97 mmol) and the cross partner 37 (1.2 g, 3.9

mmol) in anhydrous d-chloroform (5 mL) under an argon atmosphere was treated with a

nd

catalytic amount of p-benzoquinone (10 mol%), followed by Grubbs Catalyst 2 Generation

(

7 mol %, in one portion). After the resulting reaction mixture was allowed to stir under reflux

for 8h (until the TLC analysis showed no further change in the composition of the reaction

mixture; visualized with 1.3% ninhydrine solution), the solvent was removed under reduced

pressure. The resultant residue was subsequently purified by flash column chromatography

using cyclohexane and ethylacetate as eluents (from 10-15% ethylacetate in cyclohexane) to

afford the desired product 38 as white-waxy solid. Yield: 340 mg (65%). R

(

f

: 0.55

cyclohexane / EtOAc 8:2, visualized with 1.3% ninhydrine solution). H NMR (CDCl

MHz, ppm): δ 5.70 (dtd, J = 14.9, 6.8 Hz, 1H), 5.42 (ddt, J = 15.4, 6.6 Hz, 1H), 4.66 (m, 1H),

1

3

, 500

4

3

7

1

.57 (dd, J = 4.4 Hz, 1H), 4.10 (m, 1H), 3.86 (ddd, J = 11.2, 7.5, 3.2 Hz, 1H), 3.78 (m, 1H),

.72 (dt, J = 9.6, 7.0 Hz, 1H), 3.52 – 3.47 (m, 1H), 3.37 (dt, J = 9.6, 6.7 Hz, 1H), 2.03 (q, J =

.1 Hz, 2H), 1.82 (dtd, J = 12.0, 8.4, 3.3 Hz, 1H), 1.75 – 1.67 (m, 1H), 1.61 – 1.48 (m, 6H),

13

.44 (s, 9H), 1.38 – 1.23 (m, 20H), 1.07 (d, J = 6.9 Hz, 3H); C NMR (CDCl , 126 MHz,

3

ppm): δ 156.42, 134.18, 128.45, 98.97, 79.74, 75.90, 67.84, 62.48, 51.18, 32.50, 30.92, 29.89,

9.78, 29.73, 29.62, 29.31, 28.52, 26.37, 25.64, 19.83, 15.65. ESI-MS m/z calcd for

[M+H]: 484.40; observed 484.3.

2

C

28

H53NO

5

4

.5.3 Synthesis of (2S,3R)- tert-butyl -(3,18-dihydroxyoctadecan-2-yl)carbamate 39

A stirred solution of compound 38 (305 mg, 0.63 mmol) in anhydrous THF:MeOH mixture (3

mL, 1:1) under an argon atmosphere at ambient temperature was treated with a catalytic

amount of 10ꢀ% Pd/C (6 mg) followed by 2-drops of acetic acid. After being evacuated, the

reaction vessel was backfilled with hydrogen gas and the resulting reaction mixture was

allowed to stir for 16h at the same conditions (as monitored by TLC analysis; cyclohexane /

EtOAc 8:2; Rf (adduct)= 0.55; Rf (product)=0.15; visualized with 1.3% ninhydrine solution). The

suspension was subsequently filtered through a short pad of Celite, and the combined filtrates

were concentrated under reduced pressure. The residue was subsequently purified by flash

column chromatography over silica gel using cyclohexane and ethylacetate as eluents (from

1

(

5-25% ethylacetate in cyclohexane) to afford compound 39 as white solid. Yield: 183 mg

72%, over the two steps). R : 0.42 (cyclohexane / EtOAc 3:2, visualized with 1.3%

ninhydrine solution). H NMR (CDCl , 500 MHz, ppm): δ 4.77 (m, 1H), 3.69 (m, 1H), 3.63

t, J = 6.7 Hz, 4H), 1.55 (dd, J = 14.7, 6.7 Hz, 3H), 1.44 (s, 11H), 1.39 – 1.23 (m, 31H), 1.07

f

1

3

(

3

1

3

d, J = 6.9 Hz, 3H); C NMR (CDCl

3.59, 32.94, 29.80, 29.74, 29.68, 29.56, 28.54, 26.18, 25.87, 14.48. ESI-MS m/z calcd for

[M+Na] 424.3.

3

, 126 MHz, ppm): δ 156.04, 79.60, 74.64, 63.23, 50.71,

C

23

H

47NNaO

4

.5.4 Synthesis of (2S,3R)- 2,18-diaminooctadecan-3-ol 40

A solution of 4N HCl-dioxane (3 mL) was added dropwise over 20 min to a stirred solution of

compound 39 (145 mg, 0.36 mmol) in in 1, 4-dioxane (3 mL) at 0°C. After the resulting

reaction mixture was allowed to stir for 1h at the same conditions and for additional 2h at

21

ambient temperature (as monitored by TLC analysis), a saturated NaHCO solution (20 mL)

3

was added to quench. The resultant mixture was extracted with diethylether (3x50mL), and

the combined organic phases were washed with brine (100 mL), dried over anhydrous

Na

2

SO

4

, filtered and concentrated under reduced pressure. Flash column chromatography of

the resultant crude product over silica gel using ethylacetate and isopropanol as eluents (from

0

6

-7% isopropanol in ethylacetate) afforded the desired product 40 as white-waxy solid. Yield:

1

6 mg (61%). R

f

: 0.48 (EtOAc/iso-propanol 4:1, visualized with KMnO solution). H NMR

4

(

MeOD, 500 MHz, ppm): δ 3.69 (ddd, J = 10.4, 5.3, 3.0 Hz, 1H), 3.54 (t, J = 6.7 Hz, 1H),

3

1

5

.26 (qd, J = 6.7, 3.0 Hz, 1H), 1.52 (dt, J = 13.7, 6.8 Hz, 1H), 1.47 – 1.41 (m, 1H), 1.31 (d, J =

5.3 Hz, 9H), 1.21 (d, J = 6.8 Hz, 1H). C NMR (MeOD, 126 MHz, ppm): δ 71.65, 63.01,

1

3

2.58, 34.00, 33.67, 30.76, 30.67, 30.61, 26.96, 12.02. ESI-HRMS: m/z calcd for

+

C

18

H39NO

2

Na [M+Na] 324.2878; observed 324.2884.

4

.6 Synthesis of 14Z-1-deoxymethylsphingosine analogue

4

.6.1 Synthesis of tert-butyl (Z)-(2-oxoheptadec-13-en-1-yl)-carbamate 48

The Grignard solution of (Z)-15-bromopentadec-4-ene 46 was synthesized as follows; A

mixture of magnesium turnings (0.36 g, 15 mmol) in anhydrous Et O (15 mL) under an argon

atmosphere was treated with drops of 1,2-dibromoethane, followed by a solution of 46 (0.87

g, 3 mmol, in 6 mL of anhydrous Et O). The reaction mixture was allowed to stir at 35°C for

0 min to afford (11Z)-pentadecenylmagnesium bromide solution, which was directly used in

the next step.

A freshly prepared (11Z)-pentadecenylmagnesium bromide solution was added dropwise to a

2

9

3

7

stirred solution of compound 47 (0.17 g, 0.75 mmol) in anhydrous Et O (7.5 mL) at -20°C

2

under an argon atmosphere. After being stirred for 2h at 35°C (as monitored by TLC analysis;

cyclohexane/ EtOAc 4:1; Rf (adduct)= 0.21; Rf (product)=0.64; visualized with 1.3% ninhydrine

solution), the reaction mixture was cooled again to 0°C and successfully quenched with an

ice-cold 1M HCl solution (40 mL). The resulting mixture was extracted with diethylether

(

3x50 mL), and the organic layers were combined, washed with saturated NaHCO

3

solution

80 mL) and brine (80 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo.

2

4

Purification of the resultant residue by flash column chromatography over silica gel using

cylcohexane and ethylacetate as eluents (from 5-15% ethylacetate in cyclohexane) yielded the

desired product 48 as white solid. Yield: 0.15 g (57 %). R

f

: 0.53 (cyclohexane/EtOAc 5:1,

visualized with 1.3% ninhydrine solution). H NMR (CDCl , 500 MHz, ppm) δ 5.36 (ddd, J =

.8, 3.7, 1.5 Hz, 2H), 5.29 (d, J = 6.1 Hz, 1H), 4.35 – 4.27 (m, 1H), 2.49 (qd, J = 17.0, 8.6 Hz,

H), 2.05 – 1.96 (m, 4H), 1.63 – 1.56 (m, 2H), 1.44 (s, 9H), 1.40 – 1.24 (m, 18H), 0.90 (t, J =

1

3

5

2

7

5

1

3

.4 Hz, 3H). C NMR (CDCl

3

, 126 MHz, ppm) δ 209.92, 155.31, 130.23, 129.78, 79.79,

5.15, 39.34, 29.89, 29.69, 29.65, 29.57, 29.50, 29.43, 29.34, 28.48, 27.35, 23.71, 23.03,

8.10, 13.96. ESI-MS m/z clcd for C22

+

H42NO

3

[M+H] : 368.32; observed 368.3.

4

.6.2 Synthesis of tert-butyl (R,Z)-(2-hydroxyheptadec-13-en-1-yl)carbamate 49

Lithium tri-(tert-butoxy)-aluminum hydride (0.12 g, 0.5 mmol) was added in portion-wise to a

stirred solution of compound 48 (0.11 g, 0.29 mmol) in absolute ethanol (3 mL) under an

argon atmosphere at -78°C; the addition rate was adjusted so as to keep the internal

temperature below -65°C. The resulting reaction mixture was allowed to stir for 90 min at -

22

7

0

8°C (as monitored by TLC analysis; cyclohexane/EtOAc 8:2; Rf(adduct) = 0.64; Rf(product) =

.47; visualized with 1.3% ninhydrine solution), before it was quenched with an ice-cold 1M

HCl solution (60 mL); the quenching rate was adjusted so as to keep the internal temperature

below -55°C. After being stirred at ambient temperature for 1h, the resulting mixture was

diluted with ethylacetate (80 mL) and the layers were separated. The aqueous layer was

extracted with ethylacetate (3×50 mL), and the combined organic layers were washed with

saturated NaHCO

3

solution (80 mL) and brine (80 mL), dried over anhydrous Na

2

SO , filtered

4

and concentrated under reduced pressure. Flash column chromatography of the resultant crude

mixture over silica gel using cyclohexane and ethylacetate as eluents (from 10-20 %

ethylacetate in cyclohexane) provided the desired compound 49 as colorless oil. Yield: 75 mg

1

(

69 %). R

NMR (CDCl

.74 – 3.59 (m, 2H), 2.06 – 1.98 (m, 4H), 1.45 (s, 9H), 1.40 – 1.24 (m, 18H), 1.08 (d, J = 6.8

f

: 0.47 (cyclohexane/EtOAc 5:1, visualized with 1.3% ninhydrine solution). H

3

, 500 MHz, ppm) δ 5.36 (ddd, J = 5.8, 4.1, 2.2 Hz, 2H), 4.78 (d, J = 4.0 Hz, 1H),

3

1

3

Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). C NMR (CDCl

3

, 126 MHz, ppm) δ 156.01, 130.25,

1

2

3

29.76, 79.56, 74.61, 50.72, 33.60, 29.91, 29.81, 29.75, 29.71, 29.70, 29.68, 29.45, 29.43,

+

8.55, 27.36, 26.19, 23.03, 14.48, 13.95. ESI-MS m/z clcd for C22

H

43NNaO [M+Na]

3

92.31; observed 392.31.

4

.6.3 Synthesis of (2R,13Z)- 1-aminoheptadec-13-en-2-ol 50

A stirred solution of compound 49 (64 mg, 0.17 mmol) in 1,4- dioxane (2 mL) at 0°C was

treated with a solution of 4M HCl-dioxane (2 mL) over a period of 10 min. The resulting

reaction mixture was allowed to stir for 1h at the same conditions, gradually warmed to

ambient temperature, and followed by TLC analysis. After being stirred for additional 2h (as

monitored by TLC, Pet. ether/EtOAc 4:1; Rf (adduct)= 0.47; Rf (product)=0.0; visualized with

KMnO

resultant residue was dissolved in with diethylether (50 mL) and sequentially washed with

saturated NaHCO solution (40 mL), water (30 mL), and brine solution (40 mL). The organic

layer was subsequently dried over anhydrous Na SO , filtered and concentrated under reduced

4

solution), the reaction mixture was concentrated under reduced pressure. The

3

2

4

pressure. Flash column chromatography of the obtained crude product over silica gel using

ethylacetate and isopropanol as eluents (from 0-7% isopropanol in ethylacetate) provided the

desired compound 50 as white-waxy solid. Yield: 27 mg (58%). R

propanol 5:1, visualized with 1.3% ninhydrine). H NMR (MeOD, 500 MHz, ppm) δ 5.34

f

: 0.36 (EtOAc/iso-

1

(

ddd, J = 5.9, 3.3 Hz, 2H), 3.57 (dd, J = 11.5, 8.9 Hz, 1H), 2.82 – 2.54 (m, 2H), 2.07 – 1.93

m, 4H), 1.52 – 1.28 (m, 20H), 0.91 (t, J = 7.4 Hz, 3H). C NMR (MeOD, 126 MHz, ppm) δ

1

3

1

2

31.03, 130.60, 72.01, 47.65, 36.01, 33.65, 30.86, 30.78, 30.74, 30.65, 30.35, 30.30, 28.14,

6.67, 23.96, 14.14. ESI-MS m/z clcd for C17H36NO [M+H] : 270.28; observed 270.2.

+

Acknowledgements

T.H. wishes to thank the 7th Framework Program of the European Commission (ªRESOLVEº, Project

number 305707) and the Swiss National Foundation SNF (Project 31003A_153390/1 for financial

support.

23

A Supplementary data

Supplementary data associated with this article can be found, in the online version, at

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25

Graphical abstract

26

Article Doi

Synthesis and characterization of some atypical sphingoid bases

DOI: 10.1016/j.bmc.2018.06.031

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Article abstract of DOI:10.1016/j.bmc.2018.06.031

Full text of DOI:10.1016/j.bmc.2018.06.031

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