Synthesis of Structures Related to Antifreeze Glycoproteins

Final Thesis

Synthesis of Structures Related to

Antifreeze Glycoproteins

Timmy Fyrner

Department of Physics and Measurement Technology

Final Thesis

Synthesis of Structures Related to

Antifreeze Glycoproteins

Timmy Fyrner

LITH-IFM-EX--05/1416—SE

Examinator: Peter Konradsson Supervisor: Markus Hederos

Datum

Date 2005-06-03

Avdelning, institution

Division, Department

Chemistry

Department of Physics and Measurement Technology Linköping University

URL för elektronisk version

ISBN

ISRN: LITH-IFM-EX--05/1416--SE

_________________________________________________________________

Serietitel och serienummer ISSN

Title of series, numbering ______________________________

Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Titel Title

Synthesis of Structures Related to Antifreeze Glycoproteins

Författare Author Timmy Fyrner Nyckelord Keyword Sammanfattning Abstract

In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-galactopyranoside, and acceptor, ethyl 4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-1-thio-glucopyranoside, using silver triflate activation. Subsequent epimerization to a Gal-GalN disaccharide was achieved using Moffatt oxidation followed by L-selectride® reduction. The tripeptide was synthesized in a short and convenient manner using solid phase peptide synthesis with immobilized Fmoc-Ala on Wang® resins as starting point.

A

BSTRACT

In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and

a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzoyl-3,4,6-tri-O-benzyl-α-D

-galactopyranoside, and acceptor, ethyl 4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D

-1-thio-glucopyranoside, using silver triflate activation. Subsequent epimerization to a Gal-GalN disaccharide was achieved using Moffatt oxidation followed by L-selectride® reduction. The tripeptide was synthesized in a short and convenient manner using solid phase peptide synthesis with immobilized Fmoc-Ala on Wang® resins as starting point.

A

BBREVIATIONS

DCU N,N’-Dicyclohexyl urea

DIPEA N,N-Diisopropylethylamine

DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethylsulfoxide Dowex-H+ Dowex-H+ ion-exchange resin

DTBMP 2,6-di-tert.butyl-4-methylpyridine EtOAc Ethylacetate

Et2O Diethyl ether

FC Flash chromatography Gal Galactose

HOBt 1-Hydroxybenzotriazole

HOAc Acetic Acid

MS Molecular sieves

NMR Nuclear magnetic resonance

rt. Room temperature

TBTU O-Benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium tetrafluoroborate

tBu tert.butyl TEA Triethylamine TFA Trifluoroacetic acid TFAA Trifluoroacetic acid anhydride TfN3 Triflylazide

Tf2O Trifluoromethanesulfonic anhydride THF Tetrahydrofuran

Thr Threonine

TLC Thin layer chromatography MeCN Acetonitrile

AgOTf Silver trifluoromethanesulfonate (silver triflate) Glc Glucose

C

ONTENTS

1.2. CARBOHYDRATES - NUMBERING AND ANOMERIC EFFECT... 2

1.3. AIM OF PROJECT - TARGET MOLECULES AND SYNTHETIC STRATEGIES ... 4

1.3.1. Oligosaccharide Synthesis ... 4

(I) Formation of an efficient 2-O-acyl galactopyranoside ... 5

(II) Coupling of acceptor 4 with donor A ... 5

(III) Epimerization of C4 in dissaccharide B to a Gal-Gal compound C... 6

(IV) Conversion of the N-phthalimido group ... 8

1.3.2. Peptide Synthesis ... 8

2. RESULTS AND DISCUSSION... 9

2.1. PREPARATION OF A 2-O-ACYL GALACTOPYRANOSIDE DONOR ... 9

2.1.1. Synthesis of compound 8 ... 9

2.1.2. Glycosylation between 2-O-acetyl donor and acceptor 4... 10

2.1.3. Transformation of compound 8 to a 2-O-benzoyl donor 10... 10

2.2. GLYCOSYLATION BETWEEN 2-O-BENZOYL DONOR 11 AND ACCEPTOR 4 ... 11

2.3. SYNTHESIS OF A DERIVATIVE AVAILABLE FOR EPIMERIZATION... 12

2.4. EPIMERIZATION TO Gal β(1→3)GalNPhth ... 13

2.5. SYNTHESIS OF A FULLY BENZYLATED Galβ(1→3)GalNPhth DERIVATIVE... 14

2.5.1. Synthesis of Galβ(1→3)GalN3 derivative... 16

2.6. SYNTHESIS OF THE tBu-Ala-Thr-Ala-Fmoc TRIPEPTIDE ... 17

3. SUMMARY AND FUTURE PROSPECTS ... 21

4. EXPERIMENTAL ... 23

5. APPENDIX ... 33

5.1. APPENDIX A, SYNTHETIC ROUTE TO ACHIEVE Galβ(1→3)GlcNPhth 12... 33

5.2. APPENDIX B, SYNTHETIC ROUTE TO ACHIEVE Galβ(1→3)GalN3 2... 34

6. ACKNOWLEDGEMENT ... 35

1. I

NTRODUCTION

1.1. A

Antifreeze glycoproteins are essential for the surviving of many marine teleost fishes in polar and subpolar seawaters, where the temperature consistently are below the freezing point of physiological solutions. The AFGPs function is to inhibit the growth of ice crystals in the bloodstream of these fishes. Genetic studies have shown that AFGPs found in the two geographically distinct fish species Antarctic notothenioids and Arctic cod have evolved independently, a rare example of convergent molecular evolution.1 The difference between the melting- and freezing point of the ice crystals termed thermal hysteresis (TH), is used to detect and quantify the antifreeze activity.2 Although very little is known about the specific mechanism of the AFGPs during the depression of ice crystallization, several studies have been made to identify structure-function relationship of active AFGP derivatives. The glycoproteins consist of repeating tripeptide units (Ala-Thr-Ala)n (n ≥ 2), from which there are only minor natural variation. The hydroxyl group of the threonine residue is glycosylated to a Galβ(1→3)GalNAcα- moiety (Figure 1). Due to the difficulties in isolating sufficient quantities of pure native AFGPs, important chemical strategies in synthesis of AFGPs have been developed.3

Figure 1. Stucture of a native AFGP (n ≥ 2).

AFGP derivatives have been synthesized with various structure modifications both on the tripeptide- and the disaccharide moiety to probe which residues are important for antifreeze activity. Recently it was observed that the highest TH is found when the chain length is between two- and five tripeptides long. No significant increase in activity was recorded as the chain was prolonged. When the threonine amino acid in the tripeptide was exchanged to a serenine residue, the TH was lost which indicates the importance of the methyl group on Thr for activity.4,5 Further studies from modifications on the carbohydrate moiety have revealed other interesting structure-function relationships. For example, a β-O-linked glycoprotein was

H N _N H O O H N O O O AcHN OH O HO HO OH OH O HO n≥ 2

2

designed to probe the importance of the terminal α-glycoside linkage. Although weak interactions between the glycopeptide and nucleated ice were found, the complete lack of TH in the Galβ(1→3)GalNAcβ AFGPs attest the essential nature of the α-linkage.2 Even the β(1→3)-disaccharide linkage is essential for activity.4,5 Acetylation of the hydroxyl groups on the disaccharide eliminated the TH properties, showing that at least some of the hydroxyl groups with proton donating properties are important for function.4 AFGP analogues have been synthesized to examine the importance of the NHAc group at C2 of the GalNAc residue, finding the NHAc-group necessary for TH activity. Interestingly, a GalNAc monosaccharide AFGP analogue has also been shown to interact with preformed ice crystals.2 To summarize, the structure-function relation seems to be very delicate. As the mechanism still is missing on a molecular level, synthesis of AFGPs and analogues thereof will hopefully increase the understanding of these complex molecules. This could in the long-term lead to the development of new commercial applications. Using AFGPs in the cryosurgery field by increasing the destruction of solid tumors is one interesting area where AFGPs have special interest.6 It would also be possible to use AFGPs as food additives and thereby depress formation of large ice-crystals.7,8,9,10 Ideas for commercial applications of AFGPs are constantly under development.

1.2. C

Pyranoside (i.e. six-membered) rings are numbered from C1 to C6 in a clockwise manner where C1 is the anomeric carbon. The non-bonding electrons of the ring oxygen influence the C1 (anomeric center, Figure 2), making it chemically different from the other carbon atoms. At the anomeric center the substituent can have either α (axial)- or β (equatorial)-configuration. Due to a better orbital overlap between the oxygen and C1 in the α-configuration this is more stable than the β. The β-anomer also has a more parallel dipole moment, contributing to a less stable configuration. In this thesis D-galactose and D-glucose

carbohydrate backbones were used. The difference between glucose and galactose is that the hydroxyl at C4 is equatorial in glucose and axial in galactose (Figure 2).

Figure 2. Numbering of a pyranoside ring and the difference in α/β- anomer. O HO HO OH OH OH 1 2 3 4 5 6 O HO HO OH OH OH Anomeric center α-glycoside O HO HO NH₂ OH OH β-glycoside Galactose Glucosamine

4

1.3. A

The long-term aim of this project was to synthesize compound 1 as a suitable derivative in the synthesis of the repeating glycopeptide unit found in AFGPs (Figure 3). Retrosynthetic analysis of compound 1 gave key blocks 2 and 3.

Figure 3. Retrosynthetic analysis of the monomeric antifreeze derivative.

1.3.1. Oligosaccharide Synthesis

Synthetic strategy to compound 2 is outlined in Figure 4 and consisted of four main critical synthetic subjects:

(I) Formation of an efficient 2-O-acyl galactopyranoside donor A;

(II) Coupling of acceptor 411 _{with donor A to a Galβ(1→3)GlcNPhth disaccharide B;}

(III) Epimerization of C4 in disaccharide B to a Gal-Gal compound C;

(IV) Conversion of the N-phthalimido group to a non-participating azido group, C → 2.

A more thoroughly discussion of each of these subjects will be presented below.

O N3 BnO OBn O BnO BnO OBn OBn O SEt O O _H N O OH N H O NHFmoc H2N N H O O _H N O OH O O AcHN OH O HO HO OH OH O HO FmocHN _N H O O H N O O O O N₃ OBn O BnO BnO OBn OBn O BnO 1 2 3 Antifreeze Glycopeptide

Figure 4. Synthetic strategies to compound 2. For more information see text.

(I). A convenient way to achieve a β(1→3) disaccharide link in a glycosylation is to use the

neighboring group participation (NGP) effect. An 2-O-acyl in a donor has the ability to donate non-bonding electrons, blocking the axial approach, leading the attack to occur in an equatorial manner. Examples of substituents with the ability to participate are esters (e.g.

OAc), amides and imides (e.g. N-phthalimido). The donor A consists of an electrophilic part

which is often activated with a promoter. Normally both the α- or β-glycosidic bond would be formed. However, using a 2-O-acyl donor, the NGP-effect will steer to β-anomer. Various substituents affect donor reactivity different. Using benzylethers at position 3, 4 and 6 as in A, the reactivity of the donor will be significantly increased compared to using acetates or benzoates.

(II). In opposite to the donor, the acceptor 4 consists a nucleophilic part, for instance a free

hydroxyl group which has the ability to make a nucleophilic attack (Figure 5). The acceptor 4 was a compound derived from glucosamine, with a free secondary alcohol at C3. C2 contained a N-phthalimido group whereas C4 and C6 was protected with a benzylidene acetal (Figure 5). Due to a potential risk of H+-formation as the glycosylation proceed, presence of a non-nucleophilic base will be required. Thioglycosides are relatively inert until specific activation

O N₃ BnO OBn O BnO BnO OBn OBn O SEt 2 Non-NGP O SEt O NPhth O BnO BnO OBn OBn OBn BnO _O N _O ≡NPhth R= OAc or OBz L= Leaving group O BnO BnO OBn R O SEt HO NPhth O O Ph O SEt O NPhth O O Ph O BnO BnO OBn R 4 L

Site for epimerization Acceptor Donor NGP A B C

6

is wanted (glycopeptide formation). By using glucosamine instead of galactosamine as starting material in the acceptor synthesis, followed by epimerization to achieve the Gal-Gal backbone, the costs could be reduced by a 250 fold.

Figure 5. The acceptor 4 used in the synthesis of the Gal-Glc disaccharide.

(III). The first step in the epimerization of the glucosamine compound D (Figure 6) to

corresponding galactosamine compound H (Figure 7) involves an oxidation. Among the available methods for oxidation, the focus here was on the Pfitzner-Moffatt oxidation, which is accomplished using DMSO, pyridine, DCC and TFA (Figure 6). The purpose of DCC initiated with pyridinium triflate is to interact with DMSO to generate a more reactive intermediate. DMSO does not alone oxidize alcohols to carbonyls.12 The positively charged sulfur atom in E increases the acidity of the methyl groups to the extent that deprotonation occurs with ease to form the ylid F.13 The final step, proton abstraction from the carbon undergoing oxidation, is probably taking place intramoleculary.12 As the oxidation takes place, the stable urea derivative DCU is formed, driving the reaction toward the ketohexose G in combination with the formation of volatile Me2S (Figure 6).

O SEt HO N O O O O 4

Figure 6. Proposed mechanism in the Pfitzner-Moffatt oxidation of compound D.

The second step in the epimerization was a stereoselective reduction of 4-ketohexose G to galactosamine compound H (Figure 7) using L-selectride®. Compared to the smaller borohydride (i.e. NaBH4), alkylborohydrides have greater steric demands and are therefore

more stereoselective in situations where steric factors are controlling.14 The stereoselectivity of the hydride-transfer reagent during reduction is an important aspect. If the reducing agent is a sterically hindered hydride donor, empirically results show that an axial alcohol is most likely to be formed.

Figure 7. Selective hydride reaction (with bulky means e.g. L-selectride®_).

O S R2O NPhth OBz HO O S R2O NPhth OBz HO O S R₂O NPhth O G OBz B R₃ R3 R3 H M + R3= Bulky R_{3= H} D H B H Li L-selectride O BnO BnO OBn R1O R₂= R₁= OAc or OBz O S R2O NPhth HO OBz C6H11N C NC6H11 C6H11N C NC6H11 S O H C6H11N C NHC6H11 O SMe2 H - H - (C6H11NH)2CO O S R₂O NPhth O OBz Me₂S H H O S R₂O NPhth O OBz H S H2C Me SMe₂ O S R₂O NPhth O G OBz E F O BnO BnO OBn R1 R2= N H TFA D R1= OAc or OBz

8

If a less hindered donor is used, for example NaBH4, the equatorial alcohol is to be the major product. The reasons behind these results are not fully explained. However, empirically studies follow this pattern.

(IV). In glycosylation with a N-phthalimido group the β-glycopeptide link would be obtained

as main product. The glycosidic bond in native AFGPs is in the α-configuration. Therefore, the masked amine in the N-phthalimido group was converted to the non-participating azido group to give the natural anomer in future glycosylations (Figure 4).

1.3.2. Peptide Synthesis

Synthesis of tripeptide 3 (Figure 3) was performed using solid phase peptide synthesis (SPPS) (Figure 8). In this thesis, Fmoc N-terminus protected amino acids were used and the idea is based upon the ability to selectively deprotect the Fmoc without cleaving the amino acid from the resin.

Figure 8. Solid phase peptide synthesis based on Fmoc protected amino acids. O O _H N O OH N H O NHFmoc 3 O O NHFmoc

2. R

ESULTS AND

D

ISCUSSION

2.1. P

Crystalline per-acetylated β-galactopyranoside 5, easily achieved via acetylation of D

-galactose, was used as starting point in the synthesis of the galactopyranoside donor to be used in the Gal-Gal disaccharide formation. In order to steer the formation to the desired β(1→3) glycoside bond in this latter reaction, a donor with a 2-O-acyl protecting group is required.

Scheme 1. i) HBr/HOAc (33%, v/v); ii) TEA, Et4NBr, MeOH, CH2Cl2, 45ºC; iii) K2CO3, MeOH; iv) BnBr,

NaH, DMF.

Compound 7 with a 1,2-O-methoxyethylidene group has the ability to be selectively manipulated at positions 3, 4 and 6 and transformed to a donor with either a O-acetate- or

2-O-benzoate group. Accomplishing a protecting group conversion from acetates to

benzylethers on positions 3, 4 and 6, will increase the reactivity of the future donor and hopefully contribute to a higher yield of the disaccharide. The generation of the orthoester described by Asai and co-workers,15 was accomplished by the NGP-effect that an 2-O-acyl group posses (Scheme 1). Compound 5 was brominated with 33% HBr/HOAc (v/v) to give bromosugar 6 which was treated with TEA, Et4NBr and MeOH in CH2Cl2 to give compound 7 in 95% yield (step 5→7). The decomposition of the acid-labile orthoester was minimized by adding 0.1% TEA to the mobile phase when purified by FC. Compound 7 was deacetylated

O AcO AcO OAc OAc OAc O AcO AcO OAc OAc Br O AcO AcO O OAc O O 5 6 7 i ii 95% iii, iv O BnO BnO O OBn O O 8 Overall yield 79% 83%

10

and benzylated to give 8 in 83% yield after crystallization from EtOAc/hexane. To summarize, a short synthetic route to a 3,4,6-tri-O-benzylated derivative with a potential 2-O-acyl functionality (i.e. 1,2-O-orthoester), has been developed in very good yield.

2.1.2. Glycosylation between 2-O-acetyl galactopyranoside donor and phthalimido glucopyranoside acceptor 4

In the first attempt to synthesize the β(1→3) disaccharide, compound 8 was converted to corresponding 2-O-acetyl bromosugar using acetylbromide, Et4NBr and 4A MS in CH2Cl2. Coupling with compound 4 using AgOTf activation gave only traces of product (Scheme 2). Therefore this route was abandoned and we next focused on using a 2-O-benzoyl donor.

Scheme 2. Glycosylation with 2-O-acetyl donor. i) Et4NBr, AcBr, 4A MS, CH2Cl2. ii) DTBMP, AgOTf, 4A MS,

CH2Cl2, -50ºC.

2.1.3. Transformation of compound 8 to a 2-O-benzoyl donor 10

Instead of acetate at C2, a donor described by Hindsgaul and co-workers,16 with a 2-O-benzoyl group was synthesized. The 1,2-orthoester 8 was regioselectively opened to achieve a 1-OAc compound as major product. The preference for the 1-OAc compound over corresponding 2-OAc compound can be explained by the anomeric effect which will be largest with the acetate at the anomeric position. Transformation of the anomeric acetate to the bromosugar was planned using the same conditions as described in the synthesis of bromosugar 6. In order to achieve the desired β(1→3) link, the free 2-O-hydroxyl was benzoylated to utilize its NGP effect (Scheme 3).

O BnO BnO O OBn O O 8 O BnO BnO OAc OBn Br O SEt HO N O O O O 4 O SEt O NPhth O O Ph O BnO BnO OBn AcO + i ii

Scheme 3. i) HOAc (95%, v/v); ii) BzCl, pyridine.

Thus, compound 8 was treated with 95% (v/v) aq. HOAc to produce a free hydroxyl group at C2. The resulting secondary alcohol 9 was benzoylated with BzCl in pyridine and crystallized from Et2O/hexane to give compound 10 in 77 % yield (8→10).

2.2. G

DONOR

11

4

Although more stable than its 2-O-acetyl analogue, the 2-O-benzoyl bromosugar was found to be highly unstable and unsuitable for storing longer periods. Fortunately, both the conversion to bromosugar and sequent coupling to compound 4 could be optimized leading to a new disaccharide (Scheme 4).

Scheme 4. i) HBr/HOAc (33%, v/v), CH2Cl2; ii) 4, DTBMP, AgOTf, 4A MS, CH2Cl2, -33ºC.

Treating compound 10 with 33% (v/v) HBr/HOAc (2.5% HBr in the solution) gave α-bromosugar 11 according to 1H-NMR. This conversion was found very delicate and the suggested solution concentration of HBr was empirically determined. Compound 11 was glycosylated with 4 to give disaccharide 12 in 85% yield (11 + 4 → 12). To avoid hydrolysis

O BnO BnO OBn BzO Br O SEt HO N O O O O + 11 O BnO BnO BzO OBn OAc 10 4 O SEt O NPhth O O Ph O BnO BnO OBn OBz 12 85% i ii O BnO BnO O OBn O O 8 O BnO BnO OH OBn OAc 9 O BnO BnO BzO OBn OAc 10 i ii 77%

12

of the benzylidene acetal during glycosylation, the sterically demanding and non-nucleophilic base DTBMP, was used.17

2.3. S

To get a single free hydroxylic group at C4 of the glucose moiety, the benzylidene acetal in compound 12 was hydrolyzed to give a 4,6-diol which was selectively protected with a benzoyl group at C6 (Scheme 5).

Scheme 5. i) p-TsOH, CH2Cl2/MeOH (1:1); ii) BzCl, pyridine, 0ºC.

Two different approaches for hydrolysis of the acetal were tried where the first method involved ethylene glycol in CH2Cl2/TFA (5:1) with poor results. A better yield of diol 13 was obtained by treating compound 12 with p-TsOH in CH2Cl2/MeOH (63% yield, 12→13). This was followed by a regioselective protection of 13 with BzCl in pyridine to give compound 14 (94% yield, 13→14). O SEt O NPhth O O Ph O BnO BnO OBn OBz 12 O SEt O NPhth HO O BnO BnO OBn OBz 13 OH O SEt O NPhth HO O BnO BnO OBn OBz 14 OBz 63% 94% i ii

2.4. E

G

Oxidation of the 4-OH hydroxylic group in 14 to corresponding 4-ketosugar was performed using the method described by Moffatt and co-workers.18,19 The idea was to use a very sterically demanding alkylborohydride to obtain an equatorially hydride delivering to the Galβ(1→3)GalN-disaccharide (Scheme 6). To prove the right configuration after reduction, the hydroxyl group at C4 was benzoylated to give the characteristic downfield shift of H4 in 1_H-NMR.

Scheme 6. i) pyridine, TFA, DCC, CH2Cl2/DMSO (1:1); ii) L-selectride®, THF, -15ºC; iii) BzCl, pyridine, 0ºC.

Compound 14 was oxidized with pyridine, TFA and DCC in CH2Cl2/DMSO (1:1) to achieve ketohexose 15. After complete oxidation, the crude product was dissolved in Et2O and DCU were filtered off, to minimize possible interactions during the reduction. Notice, the 4-ketohexose was not stable enough to be purified by FC. The stereoselective reduction of 15 was performed by the action of L-selectride® in THF at -15ºC. Galacto derivative 16 was isolated in 67% yield (14→16). In the next step, compound 16 was benzoylated with BzCl in pyridine (84% yield, 16→17) to give a doublet (J = 3.0 Hz) at 5.83 ppm from the 4-OBz GalNPhth proton (Figure 9).

O SEt O NPhth HO O BnO BnO OBn OBz 14 OBz O SEt O NPhth O O BnO BnO OBn OBz 15 OBz O SEt O NPhth O BnO BnO OBn OBz 16 OBz HO O SEt O NPhth BzO O BnO BnO OBn OBz 17 OBz i ii iii 67% 84%

14

Figure 9. 1_{H-NMR of compound 17 showing the H}

4 doublet at 5.83 ppm, providing evidence of success in the

epimerization.

2.5. S

Cleavage of the benzoyl groups in 16 was found very slow (Scheme 7). 1H-NMR analysis showed that this problem was most concentrated to the Bz-group on C’2. A partial explanation for this finding could be sterical hindered methoxide ion by the N-phthalimido-group, leading to slower deprotection at this position. By increasing the amount of NaOMe and the reaction temperature, the 2’-OBz was cleaved. Unfortunately, under these conditions opening of the N-phthalimido-ring occurred to a high extent. This problem was solved by ring closing the NPhth-ring with TFAA in pyridine described by Chernyak and co-workers.17 With this reagent, the C4, C6, C’2 positions were trifluoroacetylated but could very easy, compared to the benzoyl groups, be removed with a catalytic amount of NaOMe.

O SEt O NPhth BzO O BnO BnO OBn OBz OBz ppm 6.00 5.50 5.00 4.50 4.00 H4 ppm 6.00 5.50 5.00 4.50 4.00 ppm 6.00 5.50 5.00 4.50 4.00 ppm 6.00 5.50 5.00 4.50 4.00 H4

Scheme 7. i) NaOMe, CH2Cl2/MeOH (1:4); ii) TFAA, pyridine, -10ºC; iii) NaOMe, CH2Cl2/MeOH (1:2).

Compound 16 was deprotected with NaOMe in CH2Cl2/MeOH (1:4) to give compound 18. The open NPhth was closed with TFAA in pyridine at -10ºC to achieve compound 19. The trifluoroacetylated 19 was deprotected with a catalytic amount of NaOMe in CH2Cl2/MeOH (1:2) to give 20 (75% yield, 16→20).

The difficulties during the deprotection at the C’2-position in 16 were reflected in the next step where the three hydroxylic groups in 20 were to be protected with benzylethers (Scheme 8). Three different approaches were tried to achieve fully benzylated disaccharide 21 from triol 20 (Table 1). The first method was to treat compound 20 with NaH, BnBr in DMF followed by heating at 40ºC (entry i). The reaction was found to be very difficult to go to completion, it stopped at the 4,6-dibenzyl derivative even though more BnBr and NaH were added. The second method described by Iversen and co-workers,20 involved benzyl-2,2,2-trichloroacetimidate and activation with triflic acid (TfOH) in CH2Cl2 (entry ii). The tribenzylated product was produced to a very low extent. The last method described by Ágoston and co-workes,21 was the most satisfying, compound 20 was benzylated using KI, BnBr, Ag2O, 4A MS in DMF to give 21 in 67% yield (entry iii and Scheme 8).

O SEt O N O O O BnO BnO OBn OBz 16 OBz HO O NH OH O BnO BnO OH OBn O SEt HO O OH O O SEt O N O O O BnO BnO OBn TFA TFA TFA O CF3 O ≡ TFA O SEt O N O O O BnO BnO OBn OH OH HO 18 19 20 75% ii i iii

16

Table 1. Results from attempts to benzylate compound 20.

Entry Reagents Mol. eq. benzylation agent Reaction times Yield of 21 (%)

i NaH, BnBr in DMF 16 24h ~25 ii Benzyl-2,2,2-trichloroacetimidate, TfOH in CH2Cl2 6 >96h Traces iii KI, BnBr, Ag2O, 4A MS in DMF 22 60h 67 Scheme 8. i) KI, BnBr, Ag2O, 4A MS, DMF.

2.5.1. Synthesis of a Galβ(1→3)GalN3 derivative suitable for glycopeptide

formation

Cleavage of the N-phthalimido-ring to achieve a secondary amine was accomplished using well-known reagents. The obtained 2-amino derivative was converted to an azide group following a protocol described by Vasella and co-workers22 using triflylazide to produce a Galβ(1→3)-2-azidoGal derivative (Scheme 9).

O SEt O NPhth HO O BnO BnO OBn OH 20 OH O NPhth OBn O BnO BnO OBn OBn O SEt BnO 21 i 67%

Scheme 9. i) n-butanol, ethylenediamine, 80ºC; ii) DMAP, TfN3, CH2Cl2.

Thus, compound 21 was treated with ethylenediamine in n-butanol at 80ºC to give 22 in 69% yield. A TfN3 solution was made with NaN3 and Tf2O in H2O and CH2Cl2 and used immediately to react with 22 in CH2Cl2 with the presence of DMAP to give 2 in 89% yield.

2.6. S

SPPS of the tripeptide was performed by using Wang® resins (p-benzyloxybenzyl alcohol linker on a polystyrene support). In this method base sensitive Fmoc-protected (fluorenyl-methyloxycarbonyl) amino acids are used. Commercial available Fmoc-Ala-Wang® resins were used as starting point to avoid problems with loading the resins with the first amino acid. The Fmoc group of the alanine residue was cleaved with piperidine in DMF to give an amino functionality I ready to be linked to a carboxylic group (see Scheme 10). Coupling to Fmoc-Thr-(tBu)-OH was accomplished using TBTU, HOBt and DIPEA in DMF to give dipeptide J. Again, the Fmoc group was cleaved from the N-terminal amino acid and the procedure was repeated using Fmoc-Ala (Scheme 10).

O N OBn O BnO BnO OBn OBn O SEt BnO O O O NH2 OBn O BnO BnO OBn OBn O SEt BnO O N3 OBn O BnO BnO OBn OBn O SEt BnO 21 22 2 i ii 69% 89%

18

Scheme 10. i) 20% Piperidine in DMF; ii) Fmoc-Thr(tBu)-OH, TBTU, HOBt, DIPEA, DMF; iii) 20% Piperidine in DMF; iv) Fmoc-Ala-OH, TBTU, HOBt, DIPEA, DMF; v) TFA/Et3SiH/CH2Cl2 (95:5:5).

The tripeptide L was cleaved from the solid support using a mixture of TFA/Et3SiH/CH2Cl2 (95:5:5) for 2h. Besides giving a free carboxylic group at the tripeptide’s C-terminus, the tert.butyl of the threonine hydroxyl was deprotected to give tripeptide 23 in 90% yield (I→23). The C-terminus Ala was protected with a tBu-group using DCC, tBuOH and CuCl in THF/CH2Cl2 (1:1). Target compound 3 was achieved in 75% yield (23→3) (Scheme 11).

O O NHFmoc O O ≡ Fmoc O O NH₂ + HO O NHFmoc OtBu O O _H N O NHFmoc OtBu O O _H N O NH2 OtBu + HO O NHFmoc O O _H N O N H OtBu O NHFmoc HO O _H N O N H O NHFmoc J L 23 i ii iii iv v OH O OH Polymer Linker = I K 90%

Scheme 11. i)DCC, tBuOH, CuCl in THF/CH2Cl2 (1:1) HO O _H N O N H O NHFmoc 23 OH tBuO O _H N O N H O NHFmoc OH 3 75% i

3. S

UMMARY AND

F

UTURE

P

ROSPECTS

To summarize, a synthetic route to the disaccharide, Ethyl 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, has

been developed. Furthermore, a tBu-Ala-Thr-Ala-Fmoc tripeptide was conveniently obtained using SPPS. These derivatives will be useful in future synthesis of glycopeptide 1, a block opening for synthesis of AFGPs and analogues.

4. E

XPERIMENTAL

G

Organic phases were dried over MgSO4(s), filtered and concentrated in vacuo at 45ºC. THF was distilled over sodium wire while toluene and CH2Cl2 were distilled over calciumhydride. All dried solvents were further collected onto 4A predried MS (MERCK). DMF and MeCN were dried with 4A predried MS (MERCK). TLC: 0.25 mm precoated silica-gel plates (Merck 60 F254), detection by UV-abs. and/or with PAA-dip (EtOH (95% v/v, 744 mL), H2SO4 (conc., 27.6 mL), HOAc (100% v/v, 8.4 mL), p-anisaldehyde (20.4 mL)); or AMC (ammoniummolybdat (50 g), cerium(IV)sulphate (1 g), 10% aq. H2SO4 (1000 mL)) followed by heating at ~250ºC. FC: silica gel MERCK 60 (0.040-0.063 mm). 1H- and 13C-NMR spectra were recorded on a Varian Mercury 300 MHz instrument at 25ºC in CDCl3, MeOH-d4 or acetone-d6. Matrix assisted laser desorption ionization - Time of flight (MALDI-TOF) mass spectroscopy was recorded on a Voyager-DE STR Biochemistry Workstation, in a positive mode, using a matrix (α-cyano-4-hydroxy-trans-cinnamic acid in 0.1% TFA/acetonitrile (1:1). Melting points were recorded with a Gallenkamp melting point apparatus.

3,4,6-Tri-O-acetyl-1,2-O-(methoxyethylidene)-α-D-galactopyranoside (7)

Penta-O-acetyl-β-D-galactopyranoside 5 (25.0 g, 64.1 mmol) and HBr in HOAc (50 mL, 33%,

v/v) were stirred for 2.5 h. The reaction mixture was evaporated and co-concentrated with toluene to afford crude 6 (Rf = 0.56 toluene/EtOAc 2:1). Without purification, the obtained

solid was dissolved in CH2Cl2 (200 mL) and TEA (18.0 mL, 129 mmol), Et4NBr (10.3 g, 31.9 mmol) and MeOH (2.74 mL, 67.6 mmol) were added. The mixture was stirred for 19 h at 45°C when the organic phase was washed with brine, dried and concentrated to give crude compound 7. FC (toluene/EtOAc 2:1 + 0.1% TEA) gave 7 (22.0 g, 60.7 mmol, 95%) as a diastereomeric mixture (exo:endo 4:1). Rf = (0.43 toluene/EtOAc 2:1 + 0.1% TEA). 1H-NMR

(300 MHz, CDCl3) exo: δ 5.85 (d, 1 H, J = 4.7 Hz, H1), 5.48 (m, 1 H, H4), 5.11 (dd, 1 H, J = 3.3, 6.8 Hz, H3), 4.36 (m, 2 H), 4.20 (m, 2 H), 3.33 (s, 3 H, OCH3), 2.15 (s, 3 H, CH3CO), 2.11(s, 3 H, CH3CO), 2.10 (s, 3 H, CH3CO), 1.70 (s, 3 H, CH3); endo: δ 5.47 (d, 1 H, J = 5.2 Hz, H1), 5.48 (m, 2 H), 5.48 (m, 1 H), 4.20 (m, 3 H), 3.41 (s, 3 H, OCH3), 2.39 (s, 3 H, CH3), 2.15 (s, 3 H, CH3CO), 2.11 (s, 3 H, CH3CO), 2.10 (s, 3 H, CH3CO), 1.62 (s, 3 H, CH3); 13 C-NMR (75.4 MHz CDCl3) exo: δ 170.2 (COCH3), 169.7 (COCH3), 169.5 (COCH3), 121.4 (C), 97.3 (C1), 73.9, 71.2, 68.9, 65.8 (C2-5), 61.2 (C6), 49.8 (OCH3), 23.2 (CH3), 20.4 (COCH3),

24

20.4 (COCH3), 20.3 (COCH3). endo: δ 170.1 (COCH3), 169.6 (COCH3), 169.5 (COCH3), 121.7 (C), 97.8 (C1), 73.4, 71.4, 68.8, 66.0(C2-5), 61.3 (C6), 50.6 (OCH3), 22.1(CH3), 20.4 (COCH3), 20.4 (COCH3), 20.3 (COCH3).

3,4,6-Tri-O-benzyl-1,2-O-(methoxyethylidene)-α-D-galactopyranoside (8)

To a stirred solution of compound 7 (24.66 g, 68.12 mmol) in MeOH (50 mL), K2CO3 (0.47 g, 3.41 mmol) was added. After 5 h the reaction mixture was evaporated and co-concentrated with toluene to give a crude white solid (Rf = 0.44 (MeOH/EtOAc 1:9)). The obtained residue

was dissolved in dry DMF (50 mL) whereupon BnBr (32.41 mL, 46.60 g, 272.48 mmol) and NaH (11.89 g, 272.48 mmol, 55% dispersed in oil) were added at 0ºC. The ice bath was removed and after 16 h, the reaction was quenched with MeOH (5 mL) and diluted with toluene. The organic phase was washed with brine, dried and concentrated, followed by FC (toluene + 0.1% TEA → toluene/EtOAc 1:1 + 0.1% TEA) to give 8 (28.64 g, 56.54 mmol, 83%) as a diastereomeric mixture (exo:endo 4:1). Rf = 0.40 (toluene/EtOAc 9:1 + 0.1% TEA).

Mp 87ºC (from EtOAc/hexane).1H-NMR (300 MHz, CDCl3) exo: δ 7.38-7.21 (m, 15 H, aromatic), 5.71 (d, 1 H, J = 4.4 Hz, H1), 4.89 (d, 1 H, J = 11.4 Hz, CH2Ph), 4.77 (d, 1 H, J = 12.2 Hz, CH2Ph), 4.63 (d, 1 H, J = 12.2 Hz, CH2Ph), 4.58 (d, 1 H, J = 11.4 Hz, CH2Ph), 4.47 (d, 1 H, J = 11.8 Hz, CH2Ph), 4.45 (m, 1 H, H2), 4.43 (d, 1 H, J = 11.8 Hz, CH2Ph), 4.02 (m, 1 H, H3), 3.97 (m, 1 H, H4), 3.59 (m, 3 H, H5-6ab), 3.23 (s, 3 H, OCH3), 1.55 (s, 3 H, CH3) endo: δ 5.54 (d, 1 H, J = 4.7 Hz, H1), 4.89 (d, 1 H , CH2Ph), 4.77 (d, 1 H, CH2Ph), 4.32 (dd, 1 H, J = 6.2, 4.7 Hz, H2), 4.00 (m, 1 H, H4), 3.88 (dd, 1 H, J = 6.2, 2.5 Hz, H3), 3.59 (m, 3 H, H5-6ab), 3.25 (s, 3 H, OCH3), 1.53 (s, 3 H, CH3). 13C-NMR (75.4 MHz CDCl3) exo: δ 138.3, 138.0, 137.8 (aromatic C), 128.4, 128.4, 128.3, 127.9, 127.9, 127.7, 127.7, 127.6, 127.5 (aromatic C), 122.0 (Cq), 97.7 (C1), 80.2, 79.8, 74.5, 73.5, 73.1, 72.9, 71.3(C2-5, 3 CH2Ph), 67.9 (C6) 49.6 (OCH3), 24.5 (CH3) endo: δ 138.3, 138.1, 137.8 (aromatic C), 128.4-127.5, 127.5 (9 aromatic C), 122.4 (Cq), 97.0 (C1), 80.2, 79.0, 74.5, 73.5, 73.2, 73.0, 72.9, 71.5 (C2-5, 3 CH2Ph), 67.9 (C6) 50.0 (OCH3), 23.3 (CH3).

Acetyl-2-O-benzoyl-3,4,6-tri-O-benzyl-α-D-galactopyranoside (10)

Compound 8 (27.14 g, 53.57 mmol) was treated with aq. HOAc (100 mL, 95%, v/v) for 1 h, diluted with CH2Cl2, washed with sat. aq. NaHCO3 several times, dried, filtered and concentrated to give crude compound 9 (Rf = 0.45 toluene/EtOAc 2:1). Without further

15.06 g, 107.14 mmol). After 2 h the reaction mixture was diluted with EtOAc, washed with 0.1 M aq. HCl, dried, filtered and concentrated. Crystallization from Et2O/hexane gave 10 (24.60 g, 41.22 mmol, 77%) as white crystals. Rf = 0.37 (toluene/EtOAc 9:1). Mp 101ºC

(from EtOAc/hexane). 1H-NMR (300 MHz, CDCl3) δ 7.99-7.96 (m, 2 H, aromatic), 7.61-7.55 (m, 1 H, aromatic), 7.47-7.41 (m, 2 H, aromatic), 7.39-7.24 (m, 15 H, aromatic) 6.48 (d, 1 H, J = 3.6 Hz, H1), 5.78 (dd, 1 H, J = 3.6, 10.2 Hz, H2), 4.99 (d, 1 H, J = 11.4 Hz, CH2Ph), 4.73 (d, 1 H, J = 12.2 Hz, CH2Ph), 4.67 (d, 1 H, J = 12.2 Hz, CH2Ph), 4.62 (d, 1 H, J = 11.4 Hz, CH2Ph), 4.50 (d, 1 H, J = 11.7 Hz, CH2Ph), 4.44 (d, 1 H, J = 11.7 Hz, CH2Ph), 4.15 (m, 1 H, J = 2.8 Hz, H4), 4.11 (m, 1 H, H5), 4.07 (dd, 1 H, J = 2.8, 10.2 Hz, H3), 3.68 (m, 2 H, H6ab), 2.07 (s, 3 H, COCH3). 13C-NMR (75.4 MHz, CDCl3) δ 169.0 (COCH3), 165.4 (COPh), 138.3, 137.9, 137.7, 133.1 (4 aromatic), 129.8, 129.6, 128.5, 128.4, 128.4, 128.3, 128.2 128.0, 127.9, 127.7, 127.6, 127.5 (12 aromatic), 90.5 (C1), 76.5, 74.9, 73.9, 73.6, 72.4, 72.0, 69.8, 68.3 (3 OCH2Ph, C2-6), 20.9 (COCH3).

Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-D -galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-1-thio-glucopyranoside (12)

To a stirred solution of compound 10 (1.00 g, 1.68 mmol) in dry CH2Cl2 (10 mL), HBr in HOAc (1.27 mL, 6.70 mmol, 33%, v/v) was added. After 25 min, the mixture was diluted with CH2Cl2 (60 mL), evaporated and co-concentrated with toluene to afford crude 11 (Rf =

0.58 (toluene/EtOAc 9:1)). Bromosugar 11 was dissolved in dry CH2Cl2 (25 mL) and 4 (0.46 g, 1.05 mmol), 2,6-di-tert.-butyl-4-methylpyridine (0.34 g, 1.68 g) and 4A MS were added. After 15 min at -33ºC, AgOTf (0.43 g, 1.68 mmol) dissolved in toluene (3 mL) was added. The solution was allowed to reach rt. during 1 h when TEA (4.67 mL, 33.53 mmol) was added. The mixture was diluted with CH2Cl2 and filtered through Celite® 521 AW, washed with H2O, dried, filtered and concentrated. FC (toluene/EtOAC 12:1 → EtOAc) gave 12 (0.87 g, 0.89 mmol, 85%) as a white solid. Rf = 0.51 (toluene/EtOAc 6:1). 1H-NMR (300 MHz,

CDCl3) δ 7.98-6.93 (29 H, m, aromatic), 5.56 (s, 1 H, CHPh), 5.43 (dd, 1 H, J = 8.1, 10.0 Hz, H’2), 5.23 (d, 1 H, J = 10.9 Hz, H1), 4.87 (d, 1 H, J = 11.7 Hz, CH2Ph), 4.76 (dd, 1 H, J = 9.6, 8.5 Hz, H3), 4.69 (d, 1 H, J = 8.1 Hz, H’1), 4.55 (d, 1 H, J = 11.7 Hz, CH2Ph), 4.45 (d, 1 H, J = 12.4 Hz, CH2Ph), 4.43 (t, 1 H, J = 10.9 Hz, H2), 4.40-4.34 (m, 1 H, H6), 4.24 (d, 1 H, J = 12.4 Hz, CH2Ph), 4.21 (s, 2 H, 2 CH2Ph), 3.92 (m, 1 H, H4), 3.88 (m, 1 H, H’4), 3.81 (t, 1 H, J = 10.2, H5), 3.69 (m, 1 H, H6), 3.63 (m, 1 H, H’5), 3.41 (dd, 1 H, J = 2.7, 10.0 Hz, H’3), 3.30 (m, 2 H, H’6ab), 2.70-2.57 (m, 2 H, SCH2CH3), 1.14 (t, 3 H, J = 7.6 Hz, SCH2CH3). 13C-NMR (75.4 MHz, CDCl3) δ 168.3 (NPhth), 166.9 (NPhth), 164.8 (COPh), 138.7, 138.0, 137.9,

26 137.7, 133.9, 132.6, 131.4-127.5, 126.4, 123.8, 122.8 (23 aromatic), 101.6 (CHPh), 101.0 (C’1), 81.8, 81.8, 80.3, 76.9, 74.6, 73.7, 73.6, 73.1, 72.1, 71.4, 70.8, 68.9, 68.4 (C’2-6, C1, 3-6, 3 CH2Ph), 54.1 (C2), 23.8 (SCH2CH3), 21.7 (COCH3), 15.0 (SCH2CH3). Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-N-phthalimido-β-D-1-thio-glucopyranoside (13)

To a stirred solution of compound 12 (10.80 g, 11.04 mmol) in CH2Cl2/MeOH (1:1, 60 mL),

p-TsOH (3.15 g, 16.56 mmol) was added. After 4 h the mixture was diluted with CH2Cl2,

washed with sat. aq. NaHCO3, dried, filtered and concentrated. FC (toluene/EtOAc 6:1 → EtOAc) gave 13 (6.38 g, 7.17 mmol, 63%) as a white solid. Rf = 0.29 (toluene/EtOAc 2:1).

1_{H-NMR (300 MHz, CDCl3) δ 7.55-7.52 (m, 2 H, aromatic), 7.46-7.10 (m, 18 H, aromatic),} 7.07- 7.02 (m, 2 H, aromatic), 6.97-6.94 (m, 2 H, aromatic), 5.55 (dd, 1 H, J = 10.2, 8.0 Hz, H’2), 5.13 (d, 1 H, J = 10.7 Hz, H1), 4.89 (d, 1 H, J = 11.8 Hz, CH2Ph), 4.87-4.84 (m, 1 H, CH2Ph), 4.59 (d, 1 H, J = 8.0 Hz, H’1), 4.55-4.42 (m, 4 H, H3, 3 CH2Ph), 4.32 (d, 1 H, J = 11.8 Hz, CH2Ph), 4.29 (t, 1 H, J = 11.4 Hz, H2) 3.93 (dd, 1 H, J = 3.2, 11.5 Hz, H’6), 3.83 (d, 1 H, J = 2.9 Hz, H’4), 3.77 (dd, 1 H, J = 5.8, 11.5 Hz, H’6), 3.72-3.66 (m, 3 H, H4, H6ab), 3.55 (dd, 1 H, J = 2.9, 10.2 Hz, H’3), 3.43 (dd, 1 H, J = 8.5, 13.2 Hz, H5), 2.58 (m, 2 H, SCH2CH3), 1.10 (t, 3 H, J = 7.6 Hz, SCH2CH3). 13C-NMR (75.4 MHz, CDCl3) δ 168.4 (NPhth), 166.8 (NPhth), 164.4 (COPh), 137.8, 137.2, 137.0, 133.8, 133.4, 132.4, 130.9, 130.8, 129.5, 129.4, 128.9, 128.4-127.4, 125.2, 123.3, 122.5 (19 aromatic), 101.3 (C’1), 83.1, 81.2, 80.0, 79.6, 74.2, 73.9, 73.7, 72.1, 72.0, 71.9, 71.2 (C’2-5, C1, C3-6, 3 CH2Ph), 68.9 (C’6), 63.3 (C’2-5, C1, C3-6, 3 CH2Ph), 53.6 (C2), 23.7 (SCH2CH3), 14.7 (SCH2CH3). Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-N-phthalimido-6-O-benzoyl-β-D-1-thio-glucopyranoside (14)

To a stirred solution of compound 13 (2.97 g, 3.34 mmol) in pyridine (10 mL) at 0ºC, BzCl (0.58 mL, 0.70 g, 5.01 mmol) was added. After 1 h the mixture was diluted with toluene, washed with aq. 1 M HCl and sat. aq. NaHCO3, dried and concentrated. FC (toluene/EtOAC 12:1 → 2:1) gave 14 (3.10 g, 3.12 mmol, 94%) as a colorless oil. Rf = 0.48 (toluene/EtOAc

4:1). 1H-NMR (300 MHz, CDCl3) δ 8.14-8.07 (m, 2 H, aromatic), 7.60-7.05 (m, 23 H, aromatic), 7.05-7.02 (m, 2 H, aromatic), 6.94-6.93 (m, 2 H, aromatic) 5.57 (dd, 1 H, J = 8.0, 10.0 Hz, H’2), 5.14 (d, 1 H, J = 10.4 Hz, H1), 4.89 (m, 2 H, 2 CH2Ph), 4.75-4.29 (m, 9 H, H’1,

H2, H3, H6ab, 4 CH2Ph), 3.90-3.76 (m, 3 H, H’4, H’6ab), 3.72-3.65 (m, 2 H, H4, H’5), 3.54 (dd, 1 H, J = 2.7, 10.0 Hz, H’3), 3.38 (dd, 1 H, J = 8.2, 12.6 Hz, H5), 2.63-2.47 (m, 2 H, SCH2CH3), 1.08 (t, 3 H, J = 7.4 Hz, SCH2CH3). 13C-NMR (75.4 MHz, CDCl3) δ 168.5 (NPhth), 166.8 (NPhth), 166.3 (COPh), 164.5 (COPh), 137.8, 137.1, 136.9, 133.8, 133.6, 133.4, 132.9, 132.4, 130.9, 130.8, 130.1, 130.0, 129.7, 129.5, 129.3, 129.0, 128.4-127.6, 125.2, 123.4, 122.5 (23 aromatic) 101.4 (C’1), 83.3, 81.0, 79.9, 77.7, 74.2, 73.9, 73.7, 72.0, 71.9, 71.8, 69.8, 68.8, 63.9 (C1, 3-6, C’2-6, 3 CH2Ph), 53.6 (C2), 23.7 (SCH2CH3), 14.8 (SCH2CH3). Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-N-phthalimido-6-O-benzoyl-β-D-1-thio-galactopyranoside (16)

To a stirred solution of compound 14 (2.81 g, 2.83 mmol) in CH2Cl2/DMSO (1:1, 40 mL), pyridine (0.45 mL, 5.65 mmol), TFA (0.21 mL, 0.32 g, 2.83 mmol) and DCC (1.46 g, 7.07 mmol) were added. After 20 h, the mixture was diluted with CH2Cl2, filtered through Celite® 521 AW, washed with H2O, dried and concentrated. Crude 15 was dissolved in Et2O, and DCU was filtered off whereupon the filtrate was concentrated. L-selectride® was added to the crude ketosugar dissolved in THF (100 mL) at -15ºC. The mixture was stirred for 15 min, diluted with CH2Cl2 and the organic phase washed sequently with H2O, aq. 0.1 M HCl, sat. aq. NaHCO3, dried and concentrated. FC (toluene/EtOAC 9:1 → 6:1) gave 16 (1.87 g, 1.88 mmol, 67%) as a colorless oil. Rf = 0.40 (toluene/EtOAc 4:1). 1H-NMR (300 MHz, CDCl3) δ

8.06-8.03 (m, 2 H, aromatic), 7.58-6.98 (m, 27 H, aromatic), 5.53 (dd, 1 H, J = 8.5, 9.5 Hz, H’2), 5.15 (d, 1 H, J = 9.6 Hz, H1), 4.91 (d, 1 H, J = 11.8 Hz, CH2Ph), 4.72-4.30 (m, 11 H, H’1, H2, H3, H5, H6ab, 5 CH2Ph), 4.02-3.98 (m, 1 H, H4), 3.92-3.91 (m, 1 H, H’4), 3.67-3.55 (m, 4 H, H’3, H’5, H’6ab), 2.74-2.51 (m, 2 H, SCH2CH3), 1.22 (t, 3 H, J = 7.4 Hz, SCH2CH3). 13_{C-NMR (75.4 MHz, CDCl3) δ 168.5 (NPhth), 166.9 (NPhth), 166.3 (COPh), 164.3 (COPh),} 101.5 (C’1), 81.1, 79.4, 78.5, 75.8, 74.5, 73.9, 73.6, 72.2, 71.6, 71.3, 68.7, 67.6, 64.3 (C’2-6, C1, C3-6, 3 CH2Ph), 50.1 (C2), 23.7 (SCH2CH3), 14.8 (SCH2CH3). Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-N-phthalimido-4,6-di-O-benzoyl-β-D-1-thio-galactopyranoside (17)

To a stirred solution of compound 16 (13 mg, 14 µmol) in pyridine (1.0 mL) at 0ºC, BzCl (3.12 µl, 27.0 µmol) was added. After 17 h the mixture was diluted with toluene, washed with aq. 1 M HCl and sat. aq. NaHCO3, dried and concentrated. FC (toluene → toluene/EtOAc

28

6:1) gave 17 (12 mg, 11 µmol, 84%) as a colorless oil. Rf = 0.56 (toluene/EtOAc 4:1). 1

H-NMR (300 MHz, CDCl3) δ 8.14-8.03 (m, 6 H, aromatic), 7.65-7.07 (m, 24 H, aromatic), 7.02-6.91 (m, 4 H, aromatic), 5.83 (d, 1 H, J = 3.0 Hz, H4), 5.35 (dd, 1 H, J = 7.8, 9.9 Hz, H’2), 5.29 (d, 1 H, J = 10.2 Hz, H1), 4.91-4.76 (m, 3 H, H2, H5, H6ab, 6 CH2Ph), 4.63 (d, 1 H, J = 7.9 Hz, H’1), 4.61-4.56 (m, 1 H, H2, H5, H6ab, 6 CH2Ph), 4.45-4.11 (m, 7 H, H2, H5, H6ab, 6 CH2Ph), 3.87 (d, 1 H, J = 2.2 Hz, H’4), 3.55-3.34 (m, 4 H, H’3, H’5, H’6ab), 2.73-2.53 (m, 2 H, SCH2CH3), 1.12 (t, 3 H, J = 7.4 Hz, SCH2CH3). 13C-NMR (75.4 MHz, CDCl3) δ 168.5 (NPhth), 166.9 (NPhth), 166.2 (2 COPh), 164.4 (COPh), 138.6, 137.9, 137.3, 133.7-121.4 (27 aromatic), 101.3 (C’1), 81.6, 79.4, 75.9, 75.6, 74.1, 73.5, 73.3, 72.0, 71.6, 71.1, 70.1, 67.7, 64.0 (C’2-6, C1, C3-6, 3 CH2Ph), 51.2 (C2), 24.2 (SCH2CH3), 14.9 (SCH2CH3). Ethyl 3,4,6-tri-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-N-phthalimido-β-D -1-thio-galactopyranoside (20)

To a stirred solution of compound 16 (1.64 g, 1.65 mmol) in CH2Cl2/MeOH (25 mL, 1:4), NaOMe (0.515 g, 9.53 mmol) dissolved in MeOH (2 mL) was added. After 31 h, the mixture was neutralized with Dowex-H+, diluted with CH2Cl2, filtered and concentrated. The crude compound 18 was dissolved in pyridine (30 mL) and TFAA (15.5 mL) was added at -10ºC and left over night to attain rt. The mixture was diluted with CH2Cl2, washed sequently with H2O, 10% aq. CuSO4, aq. 1 M HCl, sat. aq. NaHCO3, H2O, dried and concentrated. To a stirred solution of the crude obtained trifluoroacetylated compound 19 in CH2Cl2/MeOH (60 mL, 1:2), NaOMe (0.054 g, 1.00 mmol) dissolved in MeOH (1 mL) was added. After 1.5 h, the mixture was neutralized with Dowex-H+, filtered and concentrated followed by FC (toluene/EtOAc 2:1 → EtOAc) to give 20 (0.982 g, 1.25 mmol, 75%) as a colorless oil. Rf =

0.30 (toluene/EtOAc 1:1). 1H-NMR (300 MHz, CDCl3) δ 7.82 (m, 2 H, aromatic), 7.70-7.67 (m, 2 H, aromatic), 7.29 (15 H, m, aromatic), 5.26 (d, 1 H, J = 10.4 Hz, H1), 4.82 (d, 1 H, J = 11.5 Hz, CH2Ph), 4.70 (t, 1 H, J = 10.4 Hz, H2), 4.62-4.36 (m, 6 H, H3, 5 CH2Ph), 4.23-4.19 (m, 2 H, H’1, H4), 3.90-3.66 (m, 5 H, H’2, H’4, H5, H6ab), 3.47-3.37 (m, 3 H, H’5, H’6ab), 3.21 (dd, 1 H, J = 2.7, 9.9 Hz, H’3), 2.79-2.62 (m, 2 H, SCH2CH3), 1.19 (t, 3 H, J = 7.4 Hz, SCH2CH3) 13C-NMR (75.4 MHz, CDCl3) δ 168.7 (NPhth), 167.9 (NPhth), 138.1, 137.8, 137.7, 133.9, 133.7, 131.7, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7, 127.4, 123.6, 123.0 (15 aromatic), 103.8 (C’1), 81.5, 81.0, 78.1, 78.0, 74.6, 73.5, 73.3, 72.7, 72.1, 70.5, 68.6, 68.2 (C1, C3-5, C’2-6, 3 CH2Ph), 62.3 (C6), 50.6 (C2), 23.4 (SCH2CH3), 14.8 (SCH2CH3).

Ethyl 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-N-phthalimido-4,6-di-O-benzyl-β-D-1-thio-galactopyranoside (21)

To a stirred solution of 20 (50 mg, 0.064 mmol) in dried DMF (1 mL), KI (0.064 g, 0.386 mmol), BnBr (0.5 mL, 0.719 g, 4.20 mmol), Ag2O (0.18 g, 0.76 mmol) and 4A MS were added. After 64 h, the reaction mixture was diluted with CH2Cl2, washed with H2O, dried and concentrated. FC (toluene/EtOAc 12:1 → 4:1) gave 21 (0.042 g, 0.043 mmol, 67%) as a colorless oil. Rf = 0.57 (toluene/EtOAc 4:1). 1H-NMR (300 MHz, CDCl3) δ 7.76-7.74 (m, 1

H, aromatic), 7.62-7.57 (m, 1 H, aromatic), 7.49-6.96 (m, 32 H, aromatic), 5.17 (d, 1 H, J = 10.4 Hz, H1), 5.03 (d, 1 H, J = 12.4 Hz, CH2Ph), 4.92 (d, 1 H, J = 11.5 Hz, CH2Ph), 4.89 (t, 1 H, J = 10.4 Hz, H2), 4.75 (dd, 1 H, J = 2.2, 10.4 Hz, H3), 4.73 (d, 1 H, J = 12.4 Hz, CH2Ph), 4.59-4.34 (m, 10 H, H’1, 9 CH2Ph), 4.17 (d, 1 H, J = 2.2 Hz, H4), 3.87 (d, 1 H, J = 2.7 Hz, H’4), 3.79 (t, 1 H, J = 5.9 Hz, H5), 3.71-3.62 (m, 3 H, H’2, H6ab), 3.58-3.45 (m, 3 H, H’5, H’6ab), 3.36 (dd, 1 H, J = 2.6, 9.6 Hz, H’3), 2.76-2.59 (m, 2 H, SCH2CH3), 1.17 (t, 3H, 7.23 Hz, SCH2CH3). 13C-NMR (75.4 MHz, CDCl3) δ 169.1 (NPhth), 167.8 (NPhth), 104.9 (C’1), 81.9, 81.5, 79.2, 78.1, 77.9, 75.7, 74.5, 74.4, 74.0, 73.7, 73.6, 73.2, 73.1, 72.8 (C1, C3-5, C’2-5, 6 CH2Ph), 69.5 (C6), 68.6 (C’6), 51.4 (C2), 23.7 (SCH2CH3), 14.8 (SCH2CH3). MALDI-TOF Calcd for C64H65NO11S: [M+Na]+ 1078.4. Found: [M+Na]+ 1078.6.

Ethyl 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-amino-4,6-di-O-benzyl-β-D-1-thio-galactopyranoside (22)

Compound 21 (0.218 g, 0.206 mmol) was dissolved in n-butanol/ethylenediamine (7:2, 14 mL) and stirred for 24 h at 80ºC. The mixture was evaporated and co-concentrated with toluene to afford crude 22. FC (EtOAc/MeOH 19:1 + 0.1% TEA → EtOAc/MeOH 9:1 + 0.1% TEA) gave 22 (0.132 g, 0.143 mmol, 69%). Rf = 0.80 (EtOAc/MeOH 9:1 + 0.1% TEA).

1_{H-NMR (300 MHz, CDCl3) δ 7.35-7.19 (m, 30 H, aromatic), 5.02-4.57 (m, 9 H, H’1, 12} CH2Ph), 4.47-4.34 (m, 4 H, H’1, 12 CH2Ph), 4.24 (d, 1 H, J = 10.4 Hz, H1), 3.95 (d, 1 H, J = 3.0 Hz, H4), 3.89 (d, 1 H, J = 2.7 Hz, H’4), 3.85 (dd, 1 H, J = 7.7, 9.6 Hz, H’2), 3.71-3.43 (m, 8 H, H3, H5, H6ab, H’3, H’5, H’6ab), 3.28 (t, 1 H, J = 9.6 Hz, H2), 2.94-2.65 (m, 2 H, SCH2CH3), 1.27 (t, 3 H, J = 7.4 Hz, SCH2CH3). 13C-NMR (75.4 MHz, CDCl3) δ 139.1, 138.8, 138.4, 138.2, 138.1, 137.9 (6 aromatic), 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 127.9, 127.8, 127.7, 127.7, 127.7, 127.7, 127.5, 127.5, 127.5, 127.4, 127.3, 127.1 (18 aromatic), 105.2 (C’1), 86.9, 85.9, 82.3, 79.8, 77.7, 75.4, 74.9, 74.5, 73.9, 73.7, 73.5, 73.3, 73.3, 72.7 (C’2-5, C1, C3-5, 6 CH2Ph), 69.5 (C6), 68.6 (C’6), 51.4 (C2), 23.7 (SCH2CH3), 14.8 (SCH2CH3).

30

Ethyl 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside (2)

To a stirred solution of NaN3 (2.1 g, 31.8 mmol) in H2O (5.2 mL), CH2Cl2 (6.5 mL) was added at 0ºC under nitrogen. Tf2O (1.0 mL, 5.9 mmol) was added over 10 min. After 3 h, the two phases were separated and the water phase was extracted with CH2Cl2 (2x 2.6 mL). The combined organic layers were washed with sat. aq. NaHCO3 (5.2 mL), H2O (5.2 mL), dried and filtered to give a 0.5 M TfN3 in CH2Cl2. To a stirred solution of compound 22 (0.132 g, 0.143 mmol) and DMAP (0.053 g, 0.435 mmol) in dry CH2Cl2, the TfN3 solution (2.4 mL) was added dropwise. After 20 h, the mixture was diluted with EtOAc, evaporated and co-concentrated with toluene to afford crude 2. FC (toluene/EtOAC 12:1 → toluene/EtOAC 4:1) gave 2 (0.121 g, 0.127 mmol, 89%). Rf = 0.64 (toluene/EtOAC 4:1). 1H-NMR (300 MHz,

CDCl3) δ 7.38-7.18 (m, 30 H, aromatic), 5.02-4.92 (m, 3 H, H’1, 12 CH2Ph), 4.80-4.55 (6 H, m, H’1, 12 CH2Ph), 4.44-4.33 (m, 4 H, H’1, 12 CH2Ph), 4.29 (d, 1 H, J = 10.2 Hz, H1) 3.96 (d, 1 H, J = 2.5 Hz, H’4), 3.91 (d, 1 H, J = 2.7 Hz, H4), 3.86 (dd, 1 H, J = 7.7, 9.9 Hz, H’2), 3.78 (t, 1 H, J = 9.9 Hz, H2), 3.65-3.40 (m, 8 H, H3, H5, H6ab, H’3, H’5, H’6ab), 2.82-2.68 (m, 2 H, SCH2CH3), 1.31 (t, 3 H, J = 7.4 Hz, SCH2CH3). 13C-NMR (75.4 MHz, CDCl3) δ 138.8, 138.7, 138.5, 138.4, 138.0, 137.8 (6 aromatic), 128.4-127.3 (18 aromatic), 104.8 (C’1), 85.1, 82.1, 81.2, 79.5, 77.6 (C’2-5, C1, C3-5), 75.2 (CH2Ph), 75.0 (C’2-5, C1, C3-5), 74.5 (CH2Ph), 74.2 (CH2Ph), 73.9 (C’2-5, C1, C3-5), 73.5 (CH2Ph), 73.4 (C’2-5, C1, C3-5), 73.3 (CH2Ph), 73.1 (CH2Ph ), 69.2 (C6), 68.9 (C’6), 63.2 (C2), 24.6 (SCH2CH3), 15.0 (SCH2CH3). Ala-Thr-Ala-Fmoc (23) I Kaiser test - Prepared solutions:

(1) Ninhydrin (5.00 g) dissolved in tBuOH (100 mL). (2) Liquefied phenol (80.00 g)

dissolved in tBuOH (20 mL). (3) Potassium cyanide (2 mL, aq. 0.001 M) in pyridine (98 mL).

-Performance:

A few resin beads were washed several times with ethanol and tranfered to a small glass tube and 2 drops of each solution above were added, mixed well and heated to 120ºC for 4-6 min. A positive test (contains free amines) was indicated by blue resin beads.

II SPPS

Used solutions:

(1) TBTU (3.21 g, 10.00 mmol, 0.5 M) and HOBt (1.35 g, 10.00 mmol, 0.5 M) were

dissolved in DMF (20 mL). (2) DIPEA (3.49 mL, 20.00 mmol, 1 M) was dissolved in DMF (20 mL). (3) TFA (4.52 mL, 3.12 mmol) and Et3SiH (0.24 mL, 1.50 mmol) dissolved in CH2Cl2 (0.24 mL) (95:5:5). (4) 20% piperidine in DMF (50 mL).

The Fmoc-Ala-Wang® resin (1.000 g, 0.670 mmol) was added to a peptide synthesis syringe, washed with DMF (3x25 mL) and flushed under nitrogen. 20% piperidine in DMF (2x11 mL) was added to the mixture, vigorously stirred for 5 min each time to achieve crude deprotected

I. The polymer was washed with DMF (3x50 mL) and CH2Cl2 (25 mL). Kaiser test = positive. Fmoc-Thr-(tBu)-OH (1.07 g, 2.68 mmol), solution 1 (3 mL) and solution 2 (3 mL) were added and the syringe was vigorously stirred for 2.5 h. The reaction mixture was washed with DMF (4x25 mL) and CH2Cl2 (10 mL) to afford crude dipeptide J. Kaiser test = negative. 20% piperidine in DMF (2x11 mL) was added to the mixture and was stirred for 5 min each time to achieve crude K. The polymer was washed with DMF (4x25 mL) and CH2Cl2 (10 mL). Kaiser test = positive. Fmoc-Ala-OH (0.834 g, 2.68 mmol), solution 1 (3 mL) and solution 2 (3 mL) were added and stirred overnight. The reaction mixture was washed sequently with DMF (100 mL), CH2Cl2 (50 mL), MeOH (50 mL), CH2Cl2 (50 mL), MeOH (10 mL) and dried to afford crude tripeptide L. Kaiser test = negative. The polymer was treated with

solution 3 (5 mL) and vigorously stirred for 2 h. The tripeptide was washed out with CH2Cl2 (50 mL), MeOH (50 mL) and the solution was evaporated and co-concentrated with toluene to afford crude 23. FC (CHCl3/MeOH 5:1 + 1% HOAc) gave 23 (0.291 g, 0.603 mmol, 90%). Rf

= 0.43 (CH3Cl/MeOH 5:1 + 0.1% HOAc). 1H-NMR (300 MHz, acetone-d6) δ 7.74 (d, 2 H aromatic), 7.60 (m, 2 H, aromatic), 7.32 (m, 4 H aromatic), 4.36 (m, 4 H, 5 CH, CH2), 4.20 (m, 3 H, 5 CH, CH2), 1.38 (d, 6 H, 2 CH3), 1.16 (d, 3 H, CH3). 13C-NMR (75.4 MHz, acetone-d6) δ 174.8 (CO), 174.5 (CO), 171.2 (CO), 157.6 (CO), 145.1, 142.2, 128.2, 128.1, 126.2, 120.8 (aromatic), 67.9, 67.6, 58.9, 51.9, 51.9, 48.0 (5 CH, CH2), 19.6 (CH3), 18.3 (CH3), 17.8 (CH3). MALDI-TOF Calcd for C25H29N3O7: [M+Na]+ 506.2. Found: [M+Na]+ 506.2.

tBuOH-Ala-Thr-AlaNHFmoc (3)

Copper(I) chloride (1 mg) was added to DCC (96 mg, 0.466 mmol) and tBuOH (45 mg, 0.606 mmol). The mixture was stirred for 3 days, diluted with CH2Cl2 (2 mL) and added dropwise to a solution of tripeptide 23 (50 mg, 0.10 mmol) dissolved in THF (2 mL). The mixture was

32

stirred overnight, diluted with CH2Cl2 and washed with brine. The organic phase was dried, filtered and concentrated to give a white solid which was dissolved in acetone and placed in the fridge for 1 h. The white precipitate was filtered off and the filtrate was concentrated. FC (CHCl3/MeOH 15:1) gave protected tripeptide 3 (40 mg, 0.075 mmol, 75%) as a colorless oil. 13_{C-NMR (75.4 MHz, MeOH-d4/ CDCl3 1:1) δ 173.6 (CO), 171.4 (CO), 169.8 (CO), 156.5} (CO), 143.2, 140.6, 127.0, 126.4, 124.3, 119.1 (aromatic), 81.2 (Cq), 66.4, 66.3, 57.4, 50.6, 47.8, 46.4 (5 CH, CH2), 32.9 (3 CH3), 18.2 (CH3), 16.6 (CH3), 16.2 (CH3).

5. A

PPENDIX

5.1

A

12

Appendix A. i) HBr/HOAc (33%, v/v); ii) TEA, Et4NBr, MeOH, CH2Cl2, 45ºC; iii) K2CO3, MeOH; iv) BnBr,

NaH, DMF; v) HOAc (95%, v/v); vi) BzCl, pyridine; vii) HBr/HOAc (33%, v/v), CH2Cl2; viii) 4, DTBMP,

AgOTf, 4A MS, CH2Cl2, -33ºC. O AcO AcO OAc OAc OAc 5 O AcO AcO OAc OAc Br O AcO AcO O OAc O O 6 7 O BnO BnO O OBn O O 8 95% 83% O BnO BnO OH OBn OAc 9 O BnO BnO BzO OBn OAc 10 77% O BnO BnO OBn BzO Br 11 O SEt HO NPhth O O Ph + 4 O SEt O NPhth O O Ph O BnO BnO OBn OBz 12 85% ii iii,iv v vi vii viii i

34

5.2.

A

2

Appendix B. i) p-TsOH, CH2Cl2/MeOH (1:1); ii) BzCl, pyridine, 0ºC; iii) pyridine, TFA, DCC, CH2Cl2/DMSO

(1:1); iv) L-selectride®_{, THF, -15ºC; v) BzCl, pyridine, 0ºC; vi) NaOMe, CH}

2Cl2/MeOH (1:4); vii) TFAA,

pyridine, -10ºC; viii) NaOMe, CH2Cl2/MeOH (1:2); ix) KI, BnBr, Ag2O, 4A MS, DMF; x) n-butanol,

ethylenediamine, 80ºC; xi) DMAP, TfN3, CH2Cl2. O SEt O NPhth O O Ph O BnO BnO OBn OBz 12 O SEt O NPhth HO O BnO BnO OBn OBz 13 OH O SEt O NPhth HO O BnO BnO OBn OBz 14 OBz 63% 94% O SEt O NPhth O O BnO BnO OBn OBz 15 OBz O SEt O NPhth O BnO BnO OBn OBz 16 OBz HO O SEt O NPhth BzO O BnO BnO OBn OBz 17 OBz 84% 67% O SEt O NPhth O BnO BnO OBn OH OH HO 20 O NPhth OBn O BnO BnO OBn OBn O SEt BnO 21 67% O NH2 OBn O BnO BnO OBn OBn O SEt BnO O N3 OBn O BnO BnO OBn OBn O SEt BnO 22 2 69% 89% 75% i ii iii iv v

vi, vii, viii

ix

x

6. A

CKNOWLEDGEMENT

Prof. Peter Konradsson for giving me the opportunity to participating in this project.

PhD. student Markus Hederos for excellent supervision through this project and for being a good friend.

PhD. student Andreas Åslund for the help during this project.

7. R

EFERENCES

1. Harding M. M.; Anderberg P. I.; Haymet A. D. J. Eur. J. Biochem. 2003, 270, 1381-1392.

2. Tachibana Y.; Fletcher G. L.; Fujitani N.; Tsuda S.; Monde K.; Nishimura S. I. Angew. Chem. Int. Ed. 2004, 43, 856-862.

3. Wu Y.; Banoub J.; Goddard S. V.; Kao M. H.; Fletcher G. L. Comp. Biochem. Physiol. Part B 2001, 128, 265.

4. Komatsu, S.K., DeVries, A.L.; Feeney, R.E. J. Biol. Chem. 1970, 245, 2909-2913.

5. Ahmed, A. I.; Osuga, D. T.; Feeney, R. E. J. Biol. Chem. 1973, 248, 8524-8527. 6. Rubinsky, B. Ann. Rev. Biomed. Eng. 2000, 2, 157-187.

7. Fletcher, G.L., Goddard, S.V. and Wu, Y.L. Chemtech 1999, 29, 17-28.

8. Feeney, R.E. & Yeh, Y. Trends Food Sci. Technol. 1998, 9, 102-106.

9. Clemmings, J.F., Zoerb, H.F., Rosenwald, D.R.; Huang, V.T. Trends Food Sci. Technol. 1992, 8, 425.

10. Griffith, M. & Ewart, K.V. Biotechn. Adv. 1995, 13, 375-402.

11. (a) Lönn, H. Carbohydr. Res. 1985, 139, 105-113. (b) Kerekgyarto, J.; van der Jos, G. M.; Kamerling, J. P.; Liptak, A.; Vilegenhart, J. F. G. Carbohydr. Res. 1993, 238, 135-146.

12. Peter Collins, Robin Ferrier. Monosaccharides 1995, 290-293. 13. Roger W. Binkley. Modern Carbohydrate Chemistry 1988, 225-228.

14. (a) Brown H. C.; Krishnamurthy S. J. Am. Chem. Soc. 1972, 94, 7159; (b) Krishnamurthy S.; Brown H. C. J. Am. Chem. Soc. 1976, 98, 3383.

15. Asai, N.; Fusetani, N.; Matsunaga, S. J. Nat. Prod. 2001, 64, 1210-1215.

16. Hindsgaul, O.; Norberg, T.; Pendu, J. L.; Lemieux, R. U. Carbohydr. Res. 1982, 109, 109-142. 17. Chernyak, A.; Oscarson, S. Carbohydr. Res. 2000, 329, 309-316.

18. Pfitzner, k. E.; Moffatt, J. G. J. Am. Chem. Soc. 1965, 87, 5661-5670.

19. Pfitzner, k. E.; Moffatt J. G. J. Am. Chem. Soc. 1965, 87, 5670-5678.

20. Iversen, T.; Bundle, D. R. J.C.S. Chem. Comm. 1981, 1077, 1240-1241.

21. Ágoston, K.; Kerékgyárto. J.; Hajkó. J.; Batta. G.; Lefeber. D. J.; Kamerling. J. P.; Vliegenthart. F. G. Chem. Eur. J. 2002, 8, No. 1, 151-161.