Development of sphingosine kinase (SPHK) inhibitors and the role of sphingolipids in adult stem cell proliferation and differentiation 2

These compounds are aimed to be more specific inhibitors for SPHK, and even isotype-specific SPHK1 vs.. The inhibitors of human SPHK used in this study were designed as the analogues of

CHAPTER 2 DEVELOPMENT AND EVALUATION OF HUMAN

SPHK INHIBITORS

As discussed in Chapter 1, SPHK and its product S1P, play important role in many cellular processes, such as in the regulation of intracellular calcium signals, in angiogenesis and control of cell adhesion molecule expression, and chemotaxis More particularly, in immune cells, it was shown that the SPHK/S1P pathway may promote

inflammation by triggering the release of proinflammatory mediators (Taha et al., 2006,

Melendez, 2008) SPHK and S1P are very tightly involved in several pathological processes, indicating that the SPHK/S1P pathway represents an interesting target for the development of novel therapeutics In particular, compounds having the ability to modulate the levels of S1P would have a high potential for the treatment of diseases wherein S1P is believed to be involved, such as cardiovascular diseases-including atherosclerosis, thrombosis and dyslipidemia, diabetes including type I and type II, stroke, autoimmune and inflammatory diseases such as multiple sclerosis, psoriasis and inflammatory arthritis, allergic diseases such as asthma and dermatitis, T helper-1 related diseases, chronic obstructive pulmonary disease, cancer and neurodegenerative disorders

(Kuokkanen et al., 1997; Nair et al., 1977; Enlund et al., 1999; Hong et al., 1999; Xia et

al 2000)

Another important potential of SPHK inhibitors is that, they may be capable to promote the stem cells differentiation, therefore, shortening the incubation duration for stem cells differentiating into pure subpopulation(s), as discussed in Chapter 1

Unfortunately, there are no specific inhibitors for SPHK commercially available yet

not specific to SPHK It was found to inhibit not only two isozymes of human SPHKs (SPHK1 & SPHK2), but other kinases such as PKC (Igarashi and Hakomori 1989;

Igarashi et al., 1989) Some attempts to produce inhibitors of SPHK have been carried out yielding promising compounds (French et al., 2003, Kim et al., 2005), but they are

lack of proper validation

In this study, novel inhibitors were designed and synthesized based on the natural substrate of SPHK (D-erythro-Sphingosine) These compounds are aimed to be more specific inhibitors for SPHK, and even isotype-specific (SPHK1 vs SPHK2) With the newly developed inhibitors, a better understanding of the role of SPHK in the process of stem cells differentiation will be studied as well In this chapter, only the compounds synthesis and evaluation are discussed Compounds function on stem cells differentiation will be addressed in Chapter 3

The inhibitors of human SPHK used in this study were designed as the analogues of the

natural substrate, D-erythro-sphingosine, by modifying several functional chemical

groups of it The synthetic compounds were then evaluated for their efficiency and specificity to inhibit human exogenous and endogenous SPHK; the compounds were also tested for their potential cytotoxicity effects, and counter screened against other kinases including DAGK and PKCα The materials and methods are addressed below, followed

by the results and discussion

2.1 MATERIALS AND METHODS

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich, Singapore

2.1.1 Synthesis of Human SPHK Inhibitors

2.1.1.1 Compounds Designed As Analogues of Sphingosine

Earlier studies have shown that D-erythro-sphingosine (Figure 2.1a) is a natural substrate for SPHKs The design of the compounds was based on investigations of: 1) changing the fatty acid carbon chain length; 2) converting the existing double bond to either a single or triple bond, or 3) changing the hydroxyl group to other functionalities (Figure 2.1b) Scheme 1 summarises the experimental design, including compounds structures and key procedures to synthesize them During the first round testing, only six compounds were finally synthesized and evaluated

It should be noticed that Scheme 1 is the synthesis flow for the whole project of developing SPHK inhibitors, which has been filed as a patent, while this thesis only covered six analogues described here However, in order to keep the story as a whole one, Scheme 1 is still used here to describe the flow

(a)

The functional groups which would be modified

(b)

(1) (2)

(3)

Figure 2.1 (a) sphingosine (b) Positions of modifications on sphingosine

OH H

R HO

Scheme 1: Synthesis of Analogues of Sphingosine

2.1.1.2 Chemical Experimental Procedures for the Synthesis of Compounds

2.1.1.2.1 Starting Materials and Apparatus

All chemical reagents and solvents were obtained from Sigma Aldrich, Merck, Lancaster,

or Fluka, and were used without further purification Analytical thin layer chromatography (TLC) was carried out on pre-coated silica plates (Merck silica gel 60, F254) and visualized with UV light or stained with phosphomolybdic acid (PMA) stain

Flash column chromatography was performed with silica (Merck, 70-230 mesh) 1H NMR and 13C NMR spectra were measured on a Bruker ACF 300 or AMX 500 Fourier Transform spectrometer Chemical shifts were reported in parts per million (δ), relative to the internal standard of tetramethylsilane (TMS) The signals observed were described as follows: s (singlet), d (doublet), t (triplet), m (multiplet) The number of protons (n) for a given resonance was indicated as nH Mass spectra were performed on a Finnigan/MAT LCQ mass spectrometer under electron spray ionization (ESI) Optical rotations were determined with a JASCO DCP-1000 digital polarimeter and were the average of at least

10 measurements The purity of all synthesized compounds was >95% as estimated by 1H NMR analysis

As described in the Scheme 1 above, starting from dimethyloxazolidine-3-carboxylate, also known as Garner’s aldehyde 1, and using the

S-(-)-1,1-dimethylethyl-4-formyl-2,2-synthetic route shown in Scheme 1, six analogues of sphingosine were synthesized

In the first step, acetylides of various chain lengths, obtained from the treatment of

alkynes with BuLi, were coupled diastereoselectively with 1 (Scheme 1) giving both the

erythro-isomer 2a and the threo-isomer 2b (Garner et al., 1996) Subsequent ring opening

of 2a using Amberlyst 15 yielded 3a, in high yields (Herold et al., 1988)

2.1.1.2.2 Synthesis Procedure for the Six Compounds as described in the Scheme 1

Synthesis of compounds 2a

((S)-tert-Butyl-4-((R)-1-hydroxy-3-alkyl-2-ynyl)-2,2-dimethyloxazolidine- 3-carboxylate), and 2b

((S)-tert-Butyl-4-((S)-1-hydroxy-3-alkyl-2-ynyl)-2,2-dimethyloxazolidine-3-carboxylate)

Garner’s aldehyde 1 (0.432 ml, 2 mmol) in tetrahydrofuran (12 ml) was added, via a

cannula, to a solution of the respective alkyne (2.72 mmol) and BuLi (1.45 ml, 2.32

mmol) in tetrahydrofuran (25 ml), at -23 ºC The reaction mixture was stirred under nitrogen for 4 hours at -23 ºC When TLC indicated the complete consumption of the starting materials, the reaction mixture was quenched with distilled water (10ml) and extracted with ethyl acetate The combined organic layer was washed with ammonium chloride, dried over magnesium sulfate, filtered, concentrated and purified by column

chromatography (ethyl acetate: hexane = 1:8) to yield 2a and 2b

Synthesis of Compound 3a (tert-Butyl-(2S,3R)-1,3-dihydroxyalk-4-yn-2-ylcarbamate)

Compound 2a (0.2534 mmol) was dissolved in anhydrous methanol (15 ml) Amberlyst

15 resin (1.2 wt eq) was added and the reaction mixture was allowed to shake for 48 hours After which, the reaction mixture was filtered through Celite and evaporated in vacuo, and the residue was purified by filtration through silica gel with Hexane/ethyl

acetate 1:1 to give 3a

2.1.2 Evaluation of the Synthetic Human SPHK Inhibitors

2.1.2.1 Compound Function on Exogenous SPHK Activity

After the six analogues of sphingosine were synthesized, the inhibitory function of them

on SPHK activity was first investigated by an in vitro SPHK assay (addressed in Section 2.1.2.1.5 SPHK Assay) using cell lysate which predominantly contained SPHK1 or

SPHK2 protein CHO cells were transfected to over-express SPHK1 or SPHK2, to study the function of the synthetic compounds on the specific inhibitory potential against SPHK1 or SPHK2 activity

2.1.2.1.1 CHO cells culture

CHO cells were cultured in Dulbeco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated FBS (GIBCO), 1% 2mM L-glutamine, 10mg/mL streptomycin and 10U/mL penicillin The cells were cultured in an incubator at 37°C, 5% CO2 in a humidified environment

2.1.2.1.2 Over-expression of SPHK1 and SPHK2 in CHO Cells

A plasmid containing the SPHK1-cDNA fused to an enhanced green fluorescence protein

cDNA (EGFP-SPHK1) has previously been developed in the laboratory (Melendez et al.,

2000) The plasmid details are shown in Figure 2.2 The construct was made using the restrictive sites for the enzymes NheI and EcoRI, and inserting the SPHK1 cDNA

Figure 2.2 Map of EGFP-SPHK1 plasmid

The SPHK2 clone was purchased from iDNA Technology (Open Biosystems), with SPHK2 cDNA was inserted into a pCMV-SPORT6 vector

Both plasmids were amplified in DH5α (E.coli) system and purified using QIAfilter Midi

Cartridges (QIAGEN)

Over-expression of SPHK1 and SPHK2 was achieved by transfecting the SPHK1 or SPHK2 plasmids into CHO cells using Lipofectamine TM 2000 (Invitrogen), following the instructions provided by the manufacturer Briefly, CHO cells were transfected with 24μg plasmid/DNA using LipofectamineTM 2000 (Invitrogen) as a carrier, following the instructions provided The cells had been plated in 100mm tissue culture dishes Transfection efficiency for SPHK1 was detected by fluorescence microscope (by detecting green fluorescence) and SPHK activity assay For SPHK2, since there is no reporter system (like GFP) in the plasmid, the transfection efficiency was measured by SPHK activity assay comparing transfected to un-transfected cell lysates The SPHK activity assay utilizes P32-γ-ATP to generate a radio-labeled product (S1P), the gold-

standard method to measure and quantify SPHK activity in vitro More details about this

assay will be addressed below

2.1.2.1.3 Cell lysis

CHO cells were collected and re-suspended in suitable amount of SPHK buffer (described below) Cells were lysed by several cycles of freeze-thawing and total cell lysates were obtained from the supernatant after centrifugation Protein concentration in the cell lysates was measured using the Bradford assay

2.1.2.1.4 Protein Quantification by Bradford Assay

Bradford assay was used to quantify proteins in cell lysates It is a rapid and accurate method commonly used to determine the total protein concentration of a sample The assay is based on the notion that the absorbance maximum for an acidic solution, of Coomassie Brilliant Blue G-250, shifts from 465 nm to 595 nm when it binds to proteins Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a

visible color change Within the linear range of the assay (~5-25μg/mL), the more protein present, the more the Coomassie Blue binds changing the absorbance of the sample After getting cell lysates from the CHO cells, over-expressing SPHK1 or SPHK, compounds were tested for their inhibitory function on these two proteins using the SPHK assay (described below)

2.1.2.1.5 SPHK Assay

2.1.2.1.5.1 Buffers, Solutions and Substrate Preparation

D-erythro-sphingosine was prepared in ethanol at 50mM in a screw-capped glass tube and store at -70°C

Bovine serum albumin (BSA) (tissue culture grade) was prepared in PBS to generate a final concentration of 4mg/ml

20mM ATP was freshly prepared in 200mM MgCl2 solution

γ[32P]ATP (10mCi/ml) RedivueTM was purchased from GE Healthcare Bio-Sciences and the final working concentration was 2μCi/sample

SPHK buffer: 20mM Tris-HCl (pH7.4), containing 20% glycerol, 1mM mercaptoethanol, 1mM EDTA, 1mM sodium orthovanadate (SOV), 1mM PMSF, 1mM Aprotinin, 1mM Leupeptin and 1mM Pepstatin A The buffer was stored at 4℃ and the protease inhibitors (Aprotinin, Leupeptin, Pepstatin A, PMSF and SOV) were freshly added each time before using

Substrate preparation: 1mM D-erythro-sphingosine was prepared by mixing 20μl sphingosine (50mM) with 1ml BSA (4mg/ml) This solution was routinely vortexed (1-2 min) before using it, to generate sphingosine-BSA complexes These complexes ensure

enough contact area for sphingosine to react with SPHK as sphingosine alone is not readily phosphorylated by SPHKs

Radio-labeled ATP/Mg2+ mixture was fleshly prepared, before each experiment, by mixing 10mCi/ml [32P]-γ-ATP with unlabeled ATP-MgCl2 (5mM ATP “cold”) followed by vortex, to generate a mixture of “hot” and “cold” ATP of 2μCi/sample

2.1.2.1.5.2 Reaction Procedures

80μg of cell lysate, over-expressing SPHK1 or SPHK2, was placed into conical glass tubes and the tubes were placed on ice, 10μl of sphingosine-BSA complex was added, with or without the synthesized compounds Compounds, as well as DMS (used as a control), were tested at five different concentrations: 10μM, 25μM, 50μM, 75μM, and 100μM The reaction mixture was supplemented with SPHK buffer to a final volume of 190μl, finally 10μl of ATP mixture was added and the tubes were then vortexed Reactions were started by placing the samples at 37°C, and reactions were terminated

after 30 minutes To terminate the reactions, 20μl of HCl (1N) was added To extract the

lipids, 0.8ml of chloroform: methanol: HCl (100:200:1, v/v) mixture was added to the samples, mixed and left to stand at room temperature for 5-10 minutes The organic and aqueous phases were separated by adding 240μl of chloroform and 240 μl of 2N KCl The mixture was then vortexed, and rested at room temperature for 5-10 min, followed by

a 5-10 min centrifugation at 400g 50μl of samples from the organic phase, which contain radio-labeled S1P, were spotted onto a TLC plate (silica G50, Watman), using a positive displacement pipette After the sample spots were completely dry, the TLC plate was placed into a TLC chamber containing the resolution solvent: 1-butanol:methanol:acetic acid:water (80:20:10:20, v/v) The organic solvent mixture helps to run the lipid

components in the samples, which are then separated in the TLC plate The plate was run

in the chamber until the solvent front reached 2cm from the top of the plate, then removed from the chamber and air dried in a fume hood Radioactive signal on the TLC plate was captured by exposing the plate to a TyphoonTM scanner and the intensity of the radioactive signals were visualized and quantified using a TyphoonTM phosphor-imager

2.1.2.2 Compounds Function on Endogenous Human SPHK1 Activity

After detecting compound function on over-expressed SPHK1 and SPHK2 containing lysates, their inhibitory function on endogenous SPHK activity was investigated, this assay also helps to evaluate the membrane penetration ability of the compounds

In this study, U937 cells, differentiated into macrophages, were used, as the lab has previously shown that the anaphylatoxin C5a stimulates SPHK1 in these cells, without stimulating SPHK2

2.1.2.2.1 Human Histiocytic Lymphoma U-937 Cells culture

The U-937 cell line was purchased from ATCC (Rockville, MD) It is a human leukemic pro-monocyte lymphoma cell line The cells were cultured in RPMI 1640 supplemented with 10% FBS (GIBCO, Invitrogen Singapore), 2mM glutamine, 10U/ml penicillin and 10mg/ml streptomycin at 37C, 5% carbon dioxide in a humidified atmosphere

The cells were differentiated into a more macrophages adding 1mM of dbcAMP to the culture media and continuing the culture for further 48 hours

2.1.2.2.2 Compounds Function on endogenous SPHK1 activity

Differentiated U937 cells were pretreated with 10μM of each of the synthesized compounds, or with 10μM of DMS, for 30 minutes prior to stimulation Cells were then stimulated with 5nM of C5a and warmed to 37°C for different the times indicated in the

figures Stimulation was stopped by adding v/v of ice-cold 1x PBS The cell samples

were collected (by centrifugation) and lysed (Section 2.1.2.1.3 Cell lysis), and enzyme activity was detected by SPHK assay stated above in Section 2.1.2.1.5 (SPHK assay)

2.1.2.3 Compounds Specificity Testing

As was stated in the beginning of this chapter, one of the motivations for synthesizing

new inhibitors for SPHK was the present lack of specific inhibitors Therefore, the newly

synthetic compounds were tested not only on SPHK, but on some other enzymes like DAGK and PKC for their specificity

The most widely used SPHK inhibitor DMS, also inhibits DAGK and some PKC isoforms, suggesting that any novel SPHK inhibitors may possibly also inhibit DAGK and PKC Therefore bacterial DAGK and human PKCα were chosen as counter-screens for compounds specificity testing

2.1.2.3.1 DAGK Assay

DAGK assay was established and described by Bollag and Griner et al (1998) In this

assay, DAGK is incubated with its substrate (diacylglycerol), and a mixture of “cold” and radio-labeled ATP, the radio-labeled lipid product generated reflects the amount of DAGK activity It is similar to the SPHK assay which was described above

Initially, 15μl of 60mU/ml DAGK and different amounts of DAG was mixed with ATP, including radio-labeled ATP (2μCi/sample), for optimizing the amount of DAG that should be used The radio-labeled ATP (2μCi/sample) used was a mixture of 10mCi/ml radio-labeled ATP and 5mM non-radio-labeled ATP The optimized DAG concentration was determined by the lowest amount tested that generated detectable clear signals

After the amount of DAG was optimized, the DAGK assay was carried out as a counter screening for all of the synthetic compounds Compounds, as well as DMS as a control, were tested at five concentrations: 10μM, 25μM, 50μM, 75μM, and 100μM The lipids were separated using the TLC method as for the SPHK assay, and the results were visualized and quantified using TyphoonTM phosphor-imager

2.1.2.3.2 PKC Assay

Another counter screening in this study was to evaluate the compounds on PKC activity PepTag® assay for non-radioactive detection of PKC (Promega) was used This PKC assay is a non-radioactive assay, which utilizes fluorescent peptide substrates that are highly specific for PKCs The method is very simple: the active PKC phosphorylates its specific substrate, and thus the peptide net charge was altered from +1 to -1 Electrophoresis is then used to separate the phosphorylated and non-phosphorylated peptides, as the change in the net charge will change the migratory properties of the peptide during electrophoresis

In this study, recombinant human PKC alpha was used Our compounds, as well as DMS

as a control, were tested at five different concentrations: 10μM, 25μM, 50μM, 75μM, and 100μM in this assay The assay was carried out following the protocol manufacturer provided Briefly, 5μl of PKC reaction 5xBuffer, 5μl of PepTag C1 peptide (0.4μg/μl), and sonicated PKC activator 5X Solution were mixed with deionized water to make up a final volume of 25μl, to prepare a reaction solution mixture Initially, the reaction mixture was incubated for 2 minutes at 30°C 5ng human PKC alpha was immediately added into the pre-warmed reaction mixture and incubated at 30°C for 30 minutes The incubation was terminated by placing the reaction tubes in a 95°C heat block for 10

minutes 1μl of 80% glycerol was then added and mixed to ensure the samples remaining

in the wells when running electrophoresis, and then the samples were loaded on a 0.8% agarose gel After which phosphorylated and non-phosphorylated peptides were separated

by standard electrophoresis, corresponding bands can be excised for quantification by spectrophotometry, densitometry or spectrofluorometry

2.1.2.4 Compounds Cytotoxicity Testing

25μM of compounds and DMS were tested for their cytotoxicity in U937 cells

U937 cell were incubated with 25μM of compounds or DMS for 24 hours and 48 hours

in 96-well plates (5,000 or 10,000 cells/well) The ratio of dead cells versus total cells was measured by cell counting using trypan blue

2.1.3 Statistical Analysis

Results are expressed as mean ± SD Significance between mean values was determined

by Student’s t-test Samples were analyzed by two-sample equal variance, and two-tailed distribution, with a value of P< 0.05 considered significant

2.2 RESULTS

2.2.1 Synthesis of D-erythro-sphingosine Analogues

Six analogues of sphingosine were finally synthesized for the first round testing, and their analytical data are listed below The six compounds structures and yields are summarized

in table 2.1

(S)-tert-Butyl-4-((R)-1-hydroxyhept-2-ynyl)-2,2-dimethyloxazolidine-3-carboxylate

(2a1) [α]25 = -44.28° (c = 99.5 x 10-3 g/ml, CH Cl ); 1H NMR (500 MHz, C D ) δ4.71

(m, 1H), 4.18 (m, 1H), 3.84 (m, 2H), 2.10 (t, 2H), 1.75 (s, 3H), 1.53 (s, 3H), 1.46 (s, 9H), 1.41 (m, 4H), 0.87 (t, 3H); 13C NMR (500 MHz, C6D6) δ 154.8, 95.6, 86.6, 81.3, 80.1, 64.8, 63.8, 63.1, 30.4, 28.9, 27.8, 27.7, 25.6, 25.3, 21.6, 18.2, 13.2; HRMS calculated for

C17H29O4N+ Na: 334.1994, found 334.1989 Yield: 76.4%

(S)-tert-Butyl-4-((R)-1-hydroxyundec-2-ynyl)-2,2-dimethyloxazolidine-3-carboxylate

(2a2) [α]25 = -44.62° (c = 43.5 x 10-3 g/ml, CH2Cl2); 1H NMR (300 MHz, C6D6) δ4.75 (m, 1H), 4.10 (m, 1H), 3.78 (m, 2H), 2.04 (t, 2H), 1.68 (s, 3H), 1.44 (s, 3H), 1.37 (s, 9H), 1.20 (m, 12H), 0.90 (t, 3H); 13C NMR (300 MHz, C6D6) δ154.8, 95.6, 86.6, 81.3, 80.1, 65.9, 65.5, 64.8, 64.2, 63.9, 62.9, 32.8, 30.2, 30.1, 29.8, 29.6, 29.0, 26.7, 23.6, 19.7, 14.9; HRMS calculated for C21H37O4N + Na: 390.2615, found 390.2621 Yield: 12.46%

(S)-tert-Butyl-4-((S)-1-hydroxyhept-2-ynyl)-2,2-dimethyloxazolidine-3-carboxylate

(2b1) [α]25 = -49.16° (c = 84.0 x 10-3 g/ml, CH2Cl2); 1H NMR (300 MHz, C6D6) δ4.85 (m, 1H), 4.08 (m, 1H), 3.79 (m, 2H), 2.01 (t, 2H), 1.56 (s, 3H), 1.40 (s, 3H), 1.36 (s, 9H), 1.32 (m, 4H), 0.76 (t, 3H); 13C NMR (300 MHz, C6D6) δ 154.8, 95.6, 86.6, 81.3, 80.1, 65.9, 64.7, 64.2, 31.6, 29.1, 28.9, 28.8, 26.7, 26.4, 22.8, 19.3, 14.3; HRMS calculated for

C17H29O4 N + Na: 334.1994, found 334.1987 Yield: 15%

1.100 x 10-3 g/ml, DMSO); 1H NMR (300 MHz, CDCl3) δ4.59 (m, 1H), 4.06 (m, 1H), 3.74 (m, 2H), 2.21 (t, 2H), 1.44 (s, 9H), 1.24 (m, 4H), 0.89 (t, 3H); 13C NMR (300 MHz, CDCl3) δ 156.2, 87.9, 80.0, 64.4, 62.6, 55.7, 30.4, 28.3, 21.8, 18.3, 13.5; HRMS calculated for C14H25O4N + Na: 294.1676, found 294.1681 Yield: 64.44%

1.060 x 10-3 g/ml, DMSO); 1H NMR (300 MHz, CDCl3) δ4.57 (m, 1H), 4.08 (m, 1H),

3.72 (m, 2H), 2.18 (t, 2H), 1.43 (s, 9H), 1.24 (m, 12H), 0.85 (t, 3H); 13C NMR (300 MHz, CDCl3) δ 156.2, 87.9, 80.0, 64.3, 62.6, 55.8, 31.7, 29.1, 29.0, 28.8, 28.4, 28.2, 22.5, 18.6, 14.0; HRMS calculated for C18H33O4N + Na: 350.2302, found 350.2314 Yield: 72.98%

= 0.420 x 10-3 g/ml, DMSO); 1H NMR (300 MHz, CDCl3) δ4.58 (m, 1H), 4.05 (m, 1H), 3.70 (m, 2H), 2.19 (t, 2H), 1.44 (s, 9H), 1.24 (m, 22H), 0.86 (t, 3H); 13C NMR (300 MHz, CDCl3) δ 156.2, 88.0, 80.0, 64.4, 63.6, 62.6, 55.8, 31.8, 29.6, 29.3, 29.0, 28.8, 28.5, 28.3, 22.6, 18.6, 14.0; HRMS calculated for C23H43O4N + Na: 420.3084, found 420.3088 Yield: 63.88%

In fact, compound 3a3 was synthesized from compound 2a3 (followed the procedure

stated in the Section 2.1.1.2.2 Synthesis procedure for the six compounds) The reason

why compound 2a3 was not in the final list and not being tested further, is mainly because the yield of compound 2a3 is really low (~7%) Therefore, obtaining compound

3a3 requied a large amount of the starting material-Garner’s aldyhide, which is very

expensive Comparing the the structures of compounds 2a3 and 3a3, it is deduced that compound 3a3, which does not contain a (O-C-Nboc) ring, is more like to sphingosine, in terms of the structure similarity Therefore, all compound 2a3 obtained was converted into 3a3 in the first round testing, and no extra 2a3 was left that could be used in the