synthesis of structures related to antifreeze glycoproteins18342/fulltext01.pdf · 2008-05-28 ·...
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Final Thesis
Synthesis of Structures Related to
Antifreeze Glycoproteins
Timmy Fyrner
LITH-IFM-EX--05/1416—SE
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 TechnologyLinköping 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.
i
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.
ii
iii
ABBREVIATIONS
Ala Alanine Ac Acetate Bz Benzoyl Bn Benzyl DCC N,N’-Dicyclohexylcarbodiimide 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 TsOH p-Toluenesulfonic acid
iv
v
CONTENTS
ABSTRACT ............................................................................................................................................................ i
ABBREVIATIONS...............................................................................................................................................iii
CONTENTS ........................................................................................................................................................... v
1. INTRODUCTION ............................................................................................................................................. 1
1.1. ANTIFREEZE GLYCOPROTEINS ........................................................................................................... 1
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
7. REFERENCES ................................................................................................................................................ 37
1
1. INTRODUCTION
1.1. ANTIFREEZE GLYCOPROTEINS
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
HN N
H
OO
HN
O
O
O
AcHN
OH
OHO
HOOH
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. CARBOHYDRATES - NUMBERING AND ANOMERIC EFFECT
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).
3
Figure 2. Numbering of a pyranoside ring and the difference in α/β- anomer.
OHO
HO
OHOH
OH
1
23
456
OHO
HO
OHOH
OHAnomeric center
α-glycoside
OHO
HONH2
OH
OHβ-glycoside
Galactose Glucosamine
4
1.3. AIM OF PROJECT - TARGET MOLECULES AND SYNTHETIC
STRATEGIES
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
OBnO
BnOOBn
OBn
O SEtO
O HN
O
OH
NH
ONHFmoc
H2N NH
OO
HN
OOH
O
O
AcHN
OH
OHO
HOOH
OH
O
HO
FmocHN NH
OO
HN
OO
O
O
N3
OBn
OBnO
BnOOBn
OBn
O
BnO
1
2 3
Antifreeze Glycopeptide
5
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
OBnO
BnOOBn
OBn
O SEt
2
Non-NGP
OSEtO
NPhth
OBnO
BnO
OBn
OBn
OBnBnO NOO
≡NPhth
R= OAc or OBzL= Leaving group
OBnO
BnO
OBn
R
OSEtHO
NPhth
OO
Ph
OSEtO
NPhth
OOPh 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).
OSEtHO
NOO
OO
4
7
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®).
OSR2O
NPhth
OBzHO
OSR2O
NPhth
OBz
HO
OSR2O
NPhth
O
G
OBz
BR3R3
R3H
M +
R3= Bulky
R3= H
D
H
BH
LiL-selectride
OBnO
BnO
OBn
R1O
R2=
R1= OAc or OBz
OSR2O
NPhth
HO
OBz
C6H11N C NC6H11 C6H11N C NC6H11
SO
HC6H11N C NHC6H11
OSMe2
H
- H- (C6H11NH)2CO
OSR2O
NPhth
O
OBzMe2SH
H
OSR2O
NPhth
O
OBzHS
H2C
Me
SMe2
OSR2O
NPhth
O
G
OBz
EF
OBnO
BnO
OBn
R1
R2=
NH 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.
OO H
N
O
OH
NH
ONHFmoc
3
OO
NHFmoc
Solid phase peptide synthesis
9
2. RESULTS AND DISCUSSION
2.1. PREPARATION OF A 2-O-ACYL GALACTOPYRANOSIDE DONOR
2.1.1. Synthesis of compound 8
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 2-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
OAcO
AcOOAc
OAc
OAc
OAcO
AcOOAc
OAc
Br
OAcO
AcOO
OAc
O
O5 67
i ii95%
iii, iv
OBnO
BnOO
OBn
O
O
8
Overall yield79% 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).
OBnO
BnOO
OBn
O
O8
OBnO
BnOOAc
OBn
Br
OSEtHO
NOO
OO
4
OSEtO
NPhth
OOPh O
BnO
BnO
OBn
AcO
+i
ii
11
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. GLYCOSYLATION BETWEEN 2-O-BENZOYL GALACTOPYRANOSIDE
DONOR 11 AND PHTHALIMIDO ACCEPTOR 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
OBnO
BnO
OBn
BzOBr
OSEtHO
NOO
OO
+
11
OBnO
BnOBzO
OBn
OAc10 4
OSEtO
NPhth
OOPh O
BnO
BnO
OBn
OBz
12
85%
i
ii
OBnO
BnOO
OBn
O
O
8
OBnO
BnOOH
OBn
OAc
9
OBnO
BnOBzO
OBn
OAc
10
i ii77%
12
of the benzylidene acetal during glycosylation, the sterically demanding and non-nucleophilic
base DTBMP, was used.17
2.3. SYNTHESIS OF A DERIVATIVE AVAILABLE FOR EPIMERIZATION
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).
OSEtO
NPhth
OOPh O
BnO
BnO
OBn
OBz
12
OSEtO
NPhth
HOOBnO
BnO
OBn
OBz
13
OH
OSEtO
NPhth
HOOBnO
BnO
OBn
OBz
14
OBz
63%
94%
i
ii
13
2.4. EPIMERIZATION OF Galβ(1→3)GlcNPhth TO Gal β(1→3)GalNPhth
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 1H-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).
OSEtO
NPhth
HOOBnO
BnO
OBn
OBz
14
OBzO
SEtONPhth
OO
BnO
BnO
OBn
OBz
15
OBz
OSEtO
NPhth
OBnO
BnO
OBn
OBz
16
OBzHOO
SEtONPhth
BzOO
BnO
BnO
OBn
OBz
17
OBz
i
ii
iii
67%
84%
14
Figure 9. 1H-NMR of compound 17 showing the H4 doublet at 5.83 ppm, providing evidence of success in the
epimerization.
2.5. SYNTHESIS OF A FULLY BENZYLATED Galβ(1→3)GalNPhth
DERIVATIVE
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.
OSEtO
NPhth
BzOO
BnO
BnO
OBn
OBz
OBz
ppm4.004.505.005.506.00
H4
ppm4.004.505.005.506.00
ppm4.004.505.005.506.00
ppm4.004.505.005.506.00
H4
15
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).
OSEtO
NOO
OBnO
BnO
OBn
OBz
16
OBzHOO
NH
OH
OBnO
BnOOH
OBn
O SEt
HO
OOH
O
OSEtO
NOO
OBnO
BnO
OBn
TFA
TFATFA
O CF3
O
≡ TFA
OSEtO
NOO
OBnO
BnO
OBn
OH
OHHO
18
1920
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).
OSEtO
NPhth
HOO
BnO
BnO
OBn
OH
20
OHO
NPhth
OBn
OBnO
BnOOBn
OBn
O SEt
BnO
21
i67%
17
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. SYNTHESIS OF THE tBu-Ala-Thr-Ala-Fmoc TRIPEPTIDE
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
OBnO
BnOOBn
OBn
O SEt
BnO
OO
O
NH2
OBn
OBnO
BnOOBn
OBn
O SEt
BnO
O
N3
OBn
OBnO
BnOOBn
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).
OO
NHFmoc
O
O
≡ Fmoc
OO
NH2 + HO
ONHFmoc
OtBu
OO H
N
ONHFmoc
OtBu
OO H
N
ONH2
OtBu
+
HO
ONHFmoc
OO H
N
ONH
OtBuO
NHFmocHO
O HN
ONH
ONHFmoc
J
L 23
i
ii
iii
iv
v
OH
O OH
Polymer Linker
=
I
K
90%
19
Scheme 11. i)DCC, tBuOH, CuCl in THF/CH2Cl2 (1:1)
HOO H
N
ONH
ONHFmoc
23
OH
tBuOO H
N
ONH
ONHFmoc
OH
3
75%i
20
21
3. SUMMARY AND FUTURE PROSPECTS
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.
22
23
4. EXPERIMENTAL
GENERAL METHODS
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); 13C-
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
purification, compound 9 was dissolved in pyridine (60 mL) and treated with BzCl (12.4 mL,
25
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). 1H-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,
27
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). 13C-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). 1H-
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).
29
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). 1H-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.
31
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. 13C-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).
33
5. APPENDIX
5.1. APPENDIX A, SYNTHETIC ROUTE TO ACHIEVE Galβ(1→3)GlcNPhth 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.
OAcO
AcOOAc
OAc
OAc
5
OAcO
AcOOAc
OAc
Br
OAcO
AcOO
OAc
O
O6
7
OBnO
BnOO
OBn
O
O8
95% 83%
OBnO
BnOOH
OBn
OAc9
OBnO
BnOBzO
OBn
OAc10
77%
OBnO
BnO
OBn
BzOBr
11
OSEtHO
NPhth
OO
Ph+
4
OSEtO
NPhth
OOPh O
BnO
BnO
OBn
OBz
12
85%
ii iii,iv
v
vivii
viii
i
34
5.2. APPENDIX B, SYNTHETIC ROUTE TO ACHIEVE Galβ(1→3)GalN3 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, CH2Cl2/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.
OSEtO
NPhth
OOPh O
BnO
BnO
OBn
OBz12
OSEtO
NPhth
HOOBnO
BnO
OBn
OBz13
OH
OSEtO
NPhth
HOOBnO
BnO
OBn
OBz
14
OBz
63%
94%
OSEtO
NPhth
OO
BnO
BnO
OBn
OBz15
OBz
OSEtO
NPhth
OBnO
BnO
OBn
OBz16
OBzHOO
SEtONPhth
BzOO
BnO
BnO
OBn
OBz17
OBz
84%
67%
OSEtO
NPhth
OBnO
BnO
OBn
OH
OHHO
20
O
NPhth
OBn
OBnO
BnOOBn
OBn
O SEt
BnO
21
67%
O
NH2
OBn
OBnO
BnOOBn
OBn
O SEt
BnO
O
N3
OBn
OBnO
BnOOBn
OBn
O SEt
BnO
22
2
69%
89%
75%
i
ii
iii
iv
v
vi, vii, viii
ix
x
xi
35
6. ACKNOWLEDGEMENT
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.
Stefan Svensson for contributing knowledge.
36
37
7. REFERENCES
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43, 856-862. 3. Wu Y.; Banoub J.; Goddard S. V.; Kao M. H.; Fletcher G. L. Comp. Biochem. Physiol. Part B 2001, 128,
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