Synthesis of E. faecium wall teichoic acid fragments

Article abstract of DOI:10.1016/j.bmc.2016.03.019

The first synthesis of different Enterococcus faecium wall teichoic acid (WTA) fragments is presented. The structure of these major cell wall components was elucidated recently and it was shown that these glycerolphosphate (GroP) based polymers are built up from -6-(GalNAc-α(1–3)-GalNAc-β(1–2)-GroP)- repeating units. We assembled WTA fragments up to three repeating units in length, in two series that differ in the stereochemistry of the glycerolphosphate moiety. The key GalNAc-GalNAc-GroP synthons, required for the synthesis, were generated from galactosazide building blocks that were employed in highly stereoselective glycosylation reactions to furnish both the α- and β-configured linkages. By comparing the NMR spectra of the synthesized fragments with the isolated material it appears that the hereto undefined stereochemistry of the glycerol phosphate moiety is sn-glycerol-3-phosphate. The generated fragments will be valuable tools to study their immunological activity at the molecular level.

Full text of DOI:10.1016/j.bmc.2016.03.019

Accepted Manuscript
Synthesis of E. faecium wall teichoic acid fragments
Daan van der Es, Nadia A. Groenia, Diana Laverde, Herman S. Overkleeft,
Johannes Huebner, Gijsbert A. van der Marel, Jeroen D.C. Codée
PII:
S0968-0896(16)30168-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.019
BMC 12868
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 February 2016
7 March 2016
8 March 2016
Please cite this article as: Es, D.v.d., Groenia, N.A., Laverde, D., Overkleeft, H.S., Huebner, J., van der Marel, G.A.,
Codée, J.D.C., Synthesis of E. faecium wall teichoic acid fragments, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.03.019
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Synthesis of E. faecium wall teichoic acid fragments
Daan van der Esa, Nadia A. Groeniaa, Diana Laverdeb, Herman S. Overkleefta, Johannes
Huebnerb, Gijsbert A. van der Marela*, Jeroen D.C. Codéea*
aLeiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The
Netherlands
bDivision of Pediatric Infectious Diseases, Dr. von Hauner Children's Hospital, Ludwig-
Maximilians-University, Munich, Germany.
*Corresponding authors. Tel.: +31 71 527 4275 (J.D.C.C.).
E-mail addresses: jcodee@chem.leidenuniv.nl (J.D.C. Codée), marel_g@chem.leidenuniv.nl
(Gijsbert A. Van der Marel).
Keywords
E. faecium; wall teichoic acid; carbohydrate; glycosylation; phosphoramidite.
Abstract
The first synthesis of different E. faecium wall teichoic acids (WTA) fragments is presented.
The structure of these major cell wall components was elucidated recently and it was shown
that these glycerolphosphate (GroP) based polymers are built up from -6-(GalNAc-(1-3)-
GalNAc-(1-2)-GroP)- repeating units. We assembled WTA fragments up to three repeating
units in length, in two series that differ in the stereochemistry of the glycerolphosphate
moiety. The key GalNAc-GalNAc-GroP synthons, required for the synthesis, were generated
from galactosazide building blocks that were employed in highly stereoselective glycosylation
reactions to furnish both the - and -configured linkages. By comparing the NMR spectra of
the synthesized fragments with the isolated material it appears that the hereto undefined
stereochemistry of the glycerol phosphate moiety is sn-glycerol-3-phosphate. The generated
fragments will be valuable tools to study their immunological activity at the molecular level.

Graphical Abstract
Introduction
The rise of multi-drug resistant bacteria is a great threat to public health and the development
of novel therapeutic or prophylactic approaches represents a grand challenge to the medical
and scientific community. Amongst the most resistant bacterial species are enterococci, of
which the vancomycin resistant enterococci (VRE) are especially difficult to treat with
currently available antibiotics. Enterococcus faecium and Enterococcus faecalis are the most
prevalent enterococci cultured from humans, accounting for 90% of all clinical isolates and
they are responsible for a large part of nosocomial infections (14% in the USA in 2009-20101)
E. faecium, a commensal bacterium inhabiting the gastrointestinal tract,2 is responsible for
most VRE infections. Novel ways to combat these bacteria are urgently needed and therefore
different vaccination strategies are currently being developed.3,4 Teichoic acids (TAs), poly-
alditol phosphate based glycopolymers, are major components of the cell wall of Gram-
positive bacteria, accounting for up to 70% of the dry weight of the bacterial cell wall. Two
types of TAs are commonly present in the Gram-positive cell wall. Lipoteichoic acids (LTAs)
are generally built up from a poly-glycerol phosphate backbone with D-alanyl and
carbohydrate residues randomly appended to the glycerol’s C-2-OH. They are functionalized
with a glycolipid anchor, which inserts into the phospholipid cell membrane underneath the
peptidoglycan (PG) layer. Wall teichoic acids (WTAs) are covalently attached to the PG
layerand the structure of WTAs varies considerably between species and even different strains
from the same bacterium.5 LTA is a target for opsonic (i.e. protective) antibodies6 and we
have recently shown that synthetic fragments of E. faecalis LTA can be used as antigens in
combination with a BSA-carrier protein to provide a semi-synthetic vaccine modality.7 Serum
raised against this conjugate showed effective cross reactivity towards different E. faecalis
and E. faecium strains indicating than an LTA based vaccine could provide broad protection
against different enterococci and also several other gram-positive pathogens (such as group-B
streptococci, S. aureus and S. epidermidis).6 Some strains of E. faecium however are not

opsonized by anti-LTA antibodies. One possible explanation for this lack of killing is that the
E. faecium LTA is not or less available for antibodies, probably due to capsular
polysaccharides that cover LTA epitopes and prevent binding of antibodies and activation of
complement. WTAs are generally longer than LTAs and may also obscure the targets for the
opsonic antibodies.8 Very recently, the first structure of an enterococcal WTA was elucidated.
This structure was isolated from an E. faecium strain, resistant to anti-LTA opsonic
antibodies9 and is shown in Figure 1. It is built up from pseudotrisaccharide repeats,
composed of an -N-acetyl galactosamine that is linked to a -N-acetyl galactosamine which
in turn is attached to a glycerol phosphate residue. The stereochemistry of the glycerol moiety
has not been established.
Figure 1. Structure of E. faecium WTA (A) and the target structures of this study (B).
Synthetic teichoic acid fragments can be powerful tools to unravel the immunological mode
of action of these molecules. Not only can they be used as components in (semi)-synthetic
vaccines and employed for diagnostic purposes, they can also be used to probe the interaction
with other players of the immune system, such as C-type lectin receptors (CLRs),
carbohydrate receptors playing an important role in shaping the immune system, and Toll-like
receptors (TLRs), receptors responsible for detecting pathogen associated molecular patterns
(PAMPs). Therefore the structure depicted in Figure 1 is an attractive synthetic target. In
addition synthetic fragments may be used to assign the stereochemistry of the as-yet unknown
glycerol C-2. Here we report the assembly of a small library of E. faecium WTA structures,
encompassing mono-, di- and trimeric repeats of both glycerol stereoisomers. Compounds 1-

3 represent members of the sn-glycerol-1-phosphate family, where compound 4-6 are sn-
glycerol-3-phosphate based targets).
Results and discussion
We, and others, have described a number of approaches to generate synthetic TA fragments10,
including traditional solution phase studies11, light fluorous approaches12 and fully automated
solid phase13 assemblies. All of our syntheses hinged on the use of well-established synthetic
“DNA-chemistry”, employing phosphoramidite building blocks bearing a dimethoxytrityl
ether as a temporary protecting group for the nascent oligomer chain. To assemble the set of
target compounds in Figure 1, we again resorted to this strategy here. All target structures
were equipped with an aminohexanol spacer for conjugation purposes in the future. Thus, for
the assembly of WTAs 1-6, spacer phosphoramidite 9 and diastereomeric building blocks 7
and 8 were required. To minimize protecting group manipulations on far-advanced building
blocks, we reasoned that the use of galactosazide building blocks as precursors for the - and
-GalNAc-moieties would be beneficial. We decided to explore two routes towards key
pseudo trisaccharides 10 and 11. The first route builds up the target structure from the
glycerol end, first introducing the -galactosamine linkage and subsequently installing the -
GalN3-GalN3 linkage. The alternative route proceeds in opposite direction and it installs the
Gal--(1-3)-Gal linkage first, after which the glycerol moieties are attached.
Figure 2. Retrosynthetic analysis

The synthesis of the building blocks required for the assembly of the central glycerol-
disaccharide intermediate is depicted in Scheme 1. Both galactosazide synthons were
accessed from azidoselenogalactoside 13, which was readily obtained through an
azidoselenylation14 of commercially available 3,4,6-triacetylgalactal 12. To facilitate the
installation of the α-galactosamine bond, we investigated donors 14 and 15, featuring a bulky
TBDPS-ether at C-6.15–18 To synthesize these galactosides, triol 13 was selectively protected
on C-6 with a TBDPS group and the remaining alcohols were benzylated yielding
selenoglycoside 14. Hydrolysis of the anomeric selenoacetal and subsequent introduction of
the trifluoroimidate19 furnished donor 15 in 87% yield over 2 steps.
To install the -galactosamine bond we aimed to exploit the nitrile assisted glycosylation
system.20 We thus equipped building block 18 with a “permanent” benzyl ether at C-4 and C-
6 and a 2-naphthylmethyl (Nap) ether, which can be chemoselectively removed, at C-3. To
introduce the Nap at C-3, the C-4 and C-6 alcohols in 13 were first masked with a silylidene-
ketal yielding compound 16 in good yield. Subsequent naphthylation, desilylation and double
benzylation yielded 17 in 54% yield from 16. Donor 18 was obtained from 17 by executing
the above described hydrolysis-imidate introduction sequence in 80% yield. Acceptor 19 was
synthesized by selectively removing the naphthylmethyl group from 17 in 78% yield using
DDQ in a biphasic solvent system.
Scheme 1. Building block synthesis.

Reagents and conditions: a) TBDPSCl, imidazole, DMF, 90%; b) BnBr, NaH, DMF, quant; c) NIS, THF/H2O; d)
CF3C(=NPh)Cl , K2CO3, acetone, 87% over 2 steps; e) (t-Bu)2Si(OTf)2, DMF, 97%; f) NapBr, NaH, DMF, 71%; g)
NEt3•3HF, THF, 94%; h) BnBr, NaH, DMF, 83%; i) NIS, THF/H2O; j) CF3C(=NPh)Cl , K2CO3, acetone, 80% over 2
steps; k) DDQ, DCM:H2O, 78%; l) 2-aminoethyl diphenylborinate, KI, K2CO3, allylbromide, MeCN, 66%; m) 2-
aminoethyl diphenylborinate, KI, K2CO3, allylbromide, MeCN, 62%.
Glycerol acceptors 21 and 23 were synthesized by the regioselective introduction of an allyl-
ether on the primary alcohol of commercially available diols 20 and 22. Using a palladium-tin
bimetallic catalysis system as reported by Onomura21 we obtained 21 from 20 with excellent
regioselectivity in 84% yield. Scaling up this reaction proved troublesome and therefore we
investigated alternative procedures. Using a tin-ketal mediated allylation, 21 was obtained in
66% as a single regioisomer. A similar yield was obtained by employing a 2-aminoethyl
diphenylborinate-catalyzed allylation as described by Taylor and co-workers.22–24 Although
this procedure also gave 20% of the undesired C-2-allyl ether, the regioisomers were readily
separated and the reaction could be easily scaled up (62% yield on 6 mmol scale).
With all building blocks in hand we attended to the assembly of the glycerol-disaccharide
building blocks 35 and 36 (Scheme 2). We first explored the combination of selenophenyl
galactosides 14 and 16 in a pre-activation based glycosylation reaction. However, we found
that activation of seleno donor 14 using the diphenyl sulfoxide (Ph2SO)-triflic anhydride
(Tf2O) couple and the subsequent addition of selenogalactoside 16 led to a complex reaction
mixture. We therefore switched to a chemoselective approach in which imidate donor 15 was
paired with 16. This led to the desired disaccharide in good yield but with poor
stereoselectivity. Even though we employed diethyl ether, a commonly employed solvent to
promote the formation of cis-glycosidc linkages, the -linked disaccharide was predominantly
formed (/ = 1 : 3). Using dibenzyl acceptor 19 instead of 16, and changing the solvent to
dichloromethane significantly improved the stereoselectivity of the reaction and disaccharide
24 was obtained as a single diastereomer in 58% yield. For the crucial condensation of the
disaccharide donor and the glycerol acceptor we changed the anomeric seleno group into an
imidate donor (24 to 26) to stay close to the condensation condition devised by Mong and co-
workers. Gratifyingly, we found that condensation of the disaccharide imidate donor 26 with
glycerol acceptor 21 under the agency of a catalytic amount of triflic acid (TfOH) in the
presence of acetonitrile and propionitrile provided the target glycerol-disaccharide 10 as a
single diastereoisomer in 90% yield.
In the alternative route towards the glycerol-disaccharide building block donor 18 was
coupled to glycerol acceptor 21 using the aforementioned conditions to provide 27 with good
stereoselectivity (/ = 1 : 19) and in good yield (Scheme 2). To unmask the C-3-napthyl

ether we subjected 27 to a catalytic amount of HCl in hexafluoro-iso-propanol (HFIP),25
which produced galactosazide 28 in 63%. When DDQ was used in a biphasic DCM/H2O
solvent system, the primary benzyl ether on the glycerol moiety was also partly cleaved. This
side reaction could be circumvented by executing the oxidative cleavage in a phosphate
buffer26 at pH=7.4 to yield glycerol-galactoside acceptor 28 in 75% yield. To complete the
assembly of the glycerol-disaccharide intermediate, donor 15 was coupled to alcohol 28 to
provide the key building block 10 as a single anomer in 88%.
Overall, both assembly routes feature highly stereoselective glycosylation reactions and both
deliver the target glycerol-disaccharide in similar yields (Scheme 2). Because the first route
allows the introduction of both glycerol epimers from a single far-advanced synthon we
proceeded with this route to provide the diastereomeric glycerol-disaccharide 11. To this end
donor 26 and glycerol 23 were united using the acetonitrile/propionitrile glycosylation
conditions to provide glycerol-disaccharide 11 in good yield and stereoselectivity.
Scheme 2. Glycerol-disaccharide building block synthesis.

Reagents and conditions: a) TfOH, DCM, 0oC, 58%; b) NIS, THF/H2O; c) CF3(=NPh)Cl,
K2CO3, acetone, quant over 2 steps; d) 21 or 23, TfOH, MeCN/EtCN/DCM, -40oC, 10:90%
11:80%; e) TfOH, MeCN/EtCN/DCM, -40oC, 90%; f) DDQ, phosphate buffer, DCM, 75%;
g) 15, TfOH, DCM, 0oC, 88%; h) PMe3, dioxane/H2O; i) Ac2O, NEt3 , DCM, 29:59% over 2
steps, 30:76% over 2 steps; j) TBAF, THF, 31:77%, 32:89%; k) DMTrCl, NEt3, DCM,
33:89%, 34:86%; l) 1.Ir(COD)(Ph2MeP)2, THF; 2.NaHCO3, I2, H2O/THF, 35:70%, 36:80%;
m) di-isopropylethylamine, N,N-di-isopropylamino-2-cyanoethyl-chlorophosphite, DCM, 7:
85% 8:62%.
To convert 10 and 11 into the necessary phosphoramidite building blocks 7 and 8 we
transformed the azide functionalities using a Staudinger reduction and subsequent N-
acetylation provided 29 and 30. Removal of the silyl ether in 29 and 30 was followed by
installation of the dimethoxytrityl group to set the stage for the final de-allylation (Scheme 2).
To this end we employed Ir(COD)(Ph2MeP)2, briefly activated with hydrogen gas, to
isomerize the allyl ether into the enol ether and I2 in combination with aqueous NaHCO3 to
unmask the alcohol. These mild conditions do not affect the acid labile dimethoxytrityl ether.
Following this sequence of reactions glycerol-disaccharide 35 and epimeric glycerol-
disaccharide 36 were obtained in 50% and 60% yield over 3 steps from 29 and 30,
respectively. Finally, the alcohols were transformed into phosphoramidites 7 and 8.
With the building blocks in hand, we turned to the assembly of the oligomers. To this end we
first coupled the phosphoramidite functionalized spacer 911 with alcohols 35 and 36 (Scheme
3) using dicyanoimidazole as an activator. Subsequently we oxidized the intermediate
phosphite to the phosphotriester and unmasked the labile dimethoxytrityl ether with
trichloroacetic acid (TCA). Because we found that the use of pyridine/H2O in combination
with I2 for the oxidation of the phosphite intermediate led to partial removal of the cyanoethyl
group, we employed the (10-camphorsulfonyl)oxaziridine (CSO) oxidation method27,28 to
yield spacer-coupled products 37 and 40 in 72% and 80%yield. Elongation of these molecules
with phosphoramidites 7 and 8, gave the protected spacer functionalized dimers 38 and 41.
Because we could not separate the target products from the used excess of phosphoramidite
by size exclusion chromatography or conventional flash column chromatography we used
reversed phase automated flash column chromatography (20% – 100% MeOH in H2O) to
purify dimers 38 and 41. These were obtained in 62% and 64%, respectively. The trimers 39
and 42 were obtained in a similar fashion and isolated in 69% and 67% yield.

Scheme 3. Assembly of spacer equipped oligomers.
Reagents and conditions: a) DCI, 9, MeCN; b) DCI, 7 or 8, MeCN; c) CSO, MeCN; d) 3%
TCA, DCM, 37:72%, 40:80%, 38:62%, 41:64%, 39:69%, 42:67%.
The fully protected compounds were deprotected by a treatment with concentrated ammonia
and dioxane to remove the cyanoethyl protecting groups from the phosphotriesters followed
by a hydrogenation of the so-formed phosphodiesters (Scheme 4). To this end the oligomers
were treated with H2 using palladium black as the catalyst in a mixture of water/dioxane under
slightly acidic conditions. Progress of the reactions was monitored by NMR spectroscopy. If
the crude reaction mixtures still contained aromatic functionalities the products were
resubjected to the deprotection conditions. Final purification of the fully deprotected
oligomers was affected by HW-40 gel filtration, after which the compounds were transformed
into the sodium salts.

Scheme 4. Deprotection of the oligomers
Reagents and conditions: a) NH3(conc), dioxane; b) Pd(0), H2, AcOH, H2O, 1:73%, 2:57%,
3:44%, 4:45%, 5:49%, 6:56%.
The spectra of the diastereomeric mono- di- and trimer repeats are shown in Figure
3, alongside the spectrum for the E. faecium WTA obtained from natural sources as reported
by
Bychowska
et
al.9
When
the
spectra
are
compared
it
becomes
clear that the spectrum of the sn-3-phosphate trimer 6 resembles the spectrum of the natural
compound better than the spectrum of the corresponding sn-1-phosphate 3. Especially the set
of signal belonging to the -anomeric galactosamine residues ( = 4.65 ppm) is indicative.
Also the overall shape of the multiplet between 3.6-4.1 ppm is matched better to the spectrum
of the natural WTA in the spectrum of 6 than of its diastereomer 3. Based on the similarities
between the spectrum of 6 and the spectrum of the isolated WTA we tentatively assign the
stereocenter in E. faecium WTA as R and the stereochemistry of the glycerol as sn-glycerol-3-
phosphate. Of note, the biosynthesis of WTA29–31 and LTA32 use different glycerolphosphate
donors. Where LTA is assembled using phosphatidyl glycerol as building block, WTA is
synthesized from cytidine diphosphate glycerol. In the latter process, the glycerol (or PG)
alcohol of the nascent WTA chain attacks the pyrophosphate moiety and expels cytid ine
monophosphate as the leaving group.33 The stereochemistry is thus defined by the CDP-
glycerol building block that is used. The natural configuration of this biological synthon is sn-
glycerol-3-phosphate, which corroborates the assignment of the stereocenter using the NMR
data presented above.

Figure 3: Part of the NMR spectra (solvent D2O) of WTA fragments 1-6 and WTA isolated from E. faecalis. (A) sn-
glycerol-1-phosphate tri-, di- and monomer, (B) natural WTA, (C) sn-glycerol-3-phosphate tri-, di- and monomer.
The triplet signal at  = 2.95 ppm is the resonance of the spacer’s CH2-NH2 moiety.
Conclusion
In conclusion, we have assembled three E. faecalis WTA fragments alongside their three
glycerol epimers. The fragments were built using a synthetic strategy that relied on the use of
DMT-phosphoramidite based building blocks. The repeating unit building block, that featured
both an - and a -linked GalNAc moiety was assembled using galactosazide donors with
excellent stereoselectivity. Based on the NMR spectra of the assembled structures and
biosynthesis arguments we assign the stereochemistry of the glycerol moiety in E. faecium
WTA as sn-glycerol-3-phosphate. The synthesized structures will be evaluated in
immunological assays in which they will be probed as synthetic antigens. Discovery of a
potent antigen will pave the way to use the structures in (semi)-synthetic vaccine modalities.
Experimental
General
All chemicals (Acros, Fluka, Merck, Sigma-Aldrich, etc.) were used as received and reactions
were carried out dry, under an argon atmosphere, at ambient temperature, unless stated
otherwise. Column chromatography was performed on Screening Devices silica gel 60
(0.040-0.063 mm). TLC analysis was conducted on HPTLC aluminium sheets (Merck, silica

gel 60, F245). Compounds were visualized by UV absorption (245 nm), by spraying with
20% H2SO4 in ethanol or with a solution of (NH4)6Mo7O24·4H2O 25 g/l and
o
(NH4)4Ce(SO4)4·2H2O 10 g/l, in 10% aqueous H2SO4 followed by charring at +/- 140 C.
Some unsaturated compounds were visualized by spraying with a solution of KMnO4 (2%)
and K2CO3 (1%) in water. Optical rotation measurements ([α]D20) were performed on a Propol
automated polarimeter (Sodium D-line, λ = 589 nm) with a concentration of 10 mg/ml (c = 1),
1
unless stated otherwise. Infrared spectra were recorded on a Shimadzu FT-IR 8300. H, 13C
and 31P NMR spectra were recorded with a Bruker AV 400 (400, 101 and 162 MHz
respectively), a Bruker AV 500 (500 and 202 MHz respectively) or a Bruker DMX 600 (600
and 151 MHz respectively). NMR spectra were recorded in CDCl3 with chemical shift (δ)
relative to tetramethylsilane, unless stated otherwise. High resolution mass spectra were
recorded by direct injection (2 µl of a 2 µM solution in water/acetonitrile; 50/50; v/v and 0.1
% formic acid) on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an
electrospray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary
o
temperature 250 C) with resolution R = 60000 at m/z 400 (mass range m/z = 150-2000) and
dioctylphthalate (m/z = 391.28428) as a lock mass. The high resolution mass spectrometer
was calibrated prior to measurements with a calibration mixture (Thermo Finnigan).
Experimental procedures
General procedure for phosphoramidite coupling
The alcohol was coevaporated with acetonitrile four times, DCI (0.25M in MeCN) (1.5-3 eq),
acetonitrile (0.06 M) and 3Å molsieves were added and the mixture was stirred for 15 minutes
under an argon atmosphere. The phosphoramidite (1.5-3 eq) was added and the reaction was
stirred for 1-4 h at RT. CSO (0.5M in MeCN) (1.5-3 eq) was added and the reaction was
stirred for 5 minutes. The mixture was diluted with EtOAc, washed with brine, dried over
Na2SO4 and concentrated in vacuo. The residue was taken up in DCM (0.03 M). TCA (3% in
DCM) (5 eq) was added and the mixture was stirred for 20 min. H2O was added and the
mixture was stirred for 15 minutes. The reaction mixture was diluted with DCM and was
washed with a mixture of sat. aq. NaHCO3 and brine (1/1 v/v). The aqueous layer was
extracted with DCM and the combined organic layers were dried over Na2SO4 and
concentrated in vacuo.
General procedure for global deprotection
The oligomer was dissolved in a mixture of ammonia (conc.) and dioxane (2 mM, 1/1 v/v).
The mixture was stirred for 1h and was concentrated in vacuo yielding the phosphodiester

intermediate. The residue was flushed over a Dowex Na+ cation-exchange resin (type:
50WX4-200, stored on 0.5 M NaOH in H2O, flushed with H2O and MeOH before use)
column to end up in a mixture of water and dioxane (2 mM, 2/1 v/v). ~0.1ml AcOH was
added and the mixture was purged of oxygen. Pd-black (~20 mg) was added and
subsequently, the reaction mixture was treated with Hydrogen gas for 3 days. Celite was
added to the mixture and after short sonication (20-30 sec) the mixture was filtered over celite
and Chelex 100 resin and concentrated in vacuo The residue was purified by size-exclusion
chromatography (HW40, dimensions: 16/60 mm, eluent: 0.15 M NH4OAc). After repeated
lyophilization, the product was eluted through a small column containing Dowex Na+ cation-
exchange resin (type: 50WX4-200, stored on 0.5 M NaOH in H2O, flushed with H2O and
MeOH before use).
Phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-1-seleno-6-O-(tert-butyl-di-phenylsilyl)- α-D-
galactopyranoside (14)
To a cooled (0°C) solution of 13 (4.7 g, 13.5 mmol) in DMF (50 ml) imidazole (1.4 g, 20.3
mmol) and TBDPSCl (3.7 ml, 14 mmol) were added. After stirring for 10 minutes, TLC
analysis showed complete conversion of the starting material. H2O was added and the mixture
was diluted with Et2O. The organic layer was washed with H2O (5x), brine, dried over
MgSO4, filtered and concentrated in vacuo. Column chromatography yielded intermediate 2-
azido-2-deoxy-1-seleno-6-O-(tert-butyl-di-phenylsilyl)-α-D-galacto-pyranoside (7.03 g, 12.1
mmol) in 90% yield. TLC: Rf 0.8 (30% EtOAc/pentane); [α]D20 (CHCl3, c 1): +172; IR (neat,
cm-1): 3432, 2929, 2889, 2110, 1113, 1055, 738, 702; 1H NMR (400 MHz, CDCl3): δ = 7.42 –
7.15 (m, 15H), 5.96 (d, J = 5.2 Hz, 1H), 4.26 – 4.23 (m, 2H), 4.16 – 4.13 (m, 1H), 3.89 –
3.87 (m, 2H), 3.78 – 3.74 (m, 1H), 1.05 (s, 9H); 13C NMR (101 MHz, CDCl3): δ = 135.7,
135.6, 134.4, 132.7, 132.2, 130.2, 130.1, 129.2, 128.6, 128.0, 127.9, 127.8, 85.5,71.8, 71.6,
69.9, 64.4, 62.1, 26.9; HRMS: [C28H33N3O4SeSi + Na] requires 606.12997, found 606.12976.
To a cooled (0°C) solution of the intermediate (6.1 g, 10.5 mmol) and BnBr (3.1 ml, 26.3
mmol) in DMF (70 ml) NaH (60% disp.) (1.1 g, 26.3 mmol) was added in portions over 20
minutes. The mixture was allowed to warm up to RT while stirring overnight. The mixture
was cooled to 0°C and H2O was added. The mixture was diluted with Et2O, the organic layer
was washed with H2O (5x) and brine, dried over MgSO4 and concentrated in vacuo. Column
chromatography yielded compound 14 (8.0 g, 10.5 mmol) in >98% yield. TLC: Rf 0.9 (20%
20
EtOAc/pentane); [α]D (CHCl3, c 1): +131.6; IR (neat, cm-1): 3066, 2929, 2856, 2110, 1103,
1062, 738, 700; 1H NMR (400 MHz, CDCl3): δ = 7.41 – 7.05 (m, 25H), 5.82 (d, J = 5.2 Hz,

1H), 4.92 (d, J = 11.2 Hz, 1H), 4.78 (q, J = 11.6 Hz, 2H), 4.59 (d, J = 11.2 Hz, 1H), 4.34
(dd, J = 5.2 Hz, 10.2 Hz, 1H), 4.23 (t, J = 7.6 Hz, 1H), 4.10 (s, 1H), 3.85 – 3.80 (m, 1H), 3.72
(dd, J = 2.4 Hz, 10.2 Hz, 1H), 3.58 – 3.54 (m, 1H), 1.05 (s, 9H); 13C NMR (101 MHz,
CDCl3): δ = 138.4, 137.6, 135.6, 135.1, 133.2, 133.1, 130.0, 129.9, 129.1, 128.7, 128.4,
128.2, 128.1, 127.9, 127.8, 85.7, 80.4, 75.2, 73.3, 73.2, 72.7, 61.7, 61.2, 27.0 ; HRMS:
[C42H45N3O4SeSi + Na] requires 786.22411, found 786.22388.
2-azido-3,4-di-O-benzyl-2-deoxy-1-O-(N-phenyl-trifluoroacetimidoyl)-6-O-(tert-butyl-di-
phenylsilyl)-α/β-D-galactopyranoside (15)
To a cooled (0°C) solution of 14 (7.3 g, 9.5 mmol) in a mixture of H2O/THF (1:1 v/v, 85 ml)
NIS (2.4 g, 10.5 mmol) was added. The reaction was allowed to warm up towards RT and was
stirred for 1h. The reaction mixture was diluted with EtOAc, the organic layer was washed
with H2O, brine, dried over MgSO4 and concentrated in vacuo. Column chromatography
yielded intermediate 2-azido-3,4-di-O-benzyl-2-deoxy -6-O-(tert-butyl-di-phenylsilyl)-α/β- D-
galactopyranose (5.1 g, 8.3 mmol) in 87% yield. TLC: Rf 0.3 (10% EtOAc/pentane); IR (neat,
cm-1): 3389, 2956, 2929, 2856, 2110, 1112, 1060, 738, 700; 1H NMR (400 MHz, CDCl3): δ =
7.62 – 7.14 (m, 45H), 5.18 (d, J = 3.2 Hz, 1H), 4.93 – 4.90 (m, 2H), 4.74 – 4.54 (m, 6H), 4.34
(d, J = 8 Hz, 1H), 4.11 – 4.01 (m, 2H), 3.99 – 3.81 (m, 4H), 3.75 – 3.65 (m, 4H), 3.60 – 3.29
13
(m, 2H), 1.05 (s, 16H); C NMR (101 MHz, CDCl3): δ =138.5, 138.4, 137.7, 127.6, 135.6,
135.5, 133.2, 133.0, 129.9, 129.8, 129.1, 128.6, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.6, 96.5, 92.4, 80.7, 77.2, 74.9, 74.8, 73.6, 72.6, 72.4, 70.8, 64.9, 61.1, 61.8, 60.4, 26.9;
HRMS: [C36H41N3O5Si + Na] requires 646.27077, found 646.27081. To a stirred solution of
the intermediate (0.31 g, 0.5 mmol) in acetone (5 ml) K2CO3 (82 mg, 0.6 mmol) and N-phenyl
trifluoroacetimidoyl chloride (0.12 ml, 0.75 mmol) were added. After stirring for 2 days at RT
the reaction mixture was diluted with EtOAc and H2O. The organic layer was washed with
brine (2x), dried over MgSO4 and concentrated in vacuo. Column chromatography yielded
compound 15 (0.4 g, 0.5 mmol) in >98% yield. TLC: Rf 0.8 (10% EtOAc/pentane); IR (neat,
cm-1): 2953, 2858, 2113, 1716, 1209, 1163, 1112, 738, 696; 1H NMR (400 MHz, CDCl3): δ =
7.63 – 6.74 (m, 43H), 6.25 (s, 1H), 5.49 (s, 0.7H), 4.92 – 4.87 (m, 2H), 4.80 – 4.69 (m, 4H),
4.59 – 4.55 (m, 2H), 4.07 – 4.04 (m, 1H), 4.01 – 3.99 (m, 1H), 3.96 – 3.93 (m, 1H), 3.89 –
13
3.87 (m, 3H), 3.82 – 3.72 (m, 4H), 3.38 – 3.35 (m, 2H),1.04 (s, 15H); C NMR (101 MHz,
CDCl3): δ = 143.6, 143.5, 138.5, 137.6, 135.7, 133.5, 133.4, 130.0, 129.9, 129.4, 128.8,
128.7, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 124.5, 120.8, 119.7, 119.6, 96.4, 95.1,

81.1, 77.8, 76.4, 75.1, 75.0, 74.1, 73.5, 73.1, 72.9, 72.7, 62.7, 62.5, 62.4, 59.6, 27.1; HRMS:
[C44H45F3N4O5Si + Na+] requires 817.30035, found 817.30042.
Phenyl 2-azido-4,6-O-silylidene-2-deoxy-1-seleno-α-D-galactopyranoside (16)
To a cooled (-40°C) solution of 13 (0.17 g, 0.5 mmol) in dry DMF (2 ml) di-tert-butylsilyl
bis(trifluoromethanesulphonate) (0.17 ml, 0.5 mmol) was added. The mixture was stirred for
10 minutes, pyridine (0.12 ml, 1.5 mmol) was added and the mixture was stirred for 20
additional minutes at -40°C. Et2O and H2O were added and the organic layer was washed with
H2O (5x) and brine (2x). The aqueous layers were extracted with Et2O, and the combined
organic layers were dried over MgSO4 and concentrated in vacuo. Column chromatography
yielded compound 16 (0.24 g, 0.49 mmol) in 97% yield. TLC: Rf 0.7 (10% EtOAc/pentane);
[α]D20 (CHCl3): +196; 1H NMR (400 MHz, CDCl3): δ = 7.56 – 7.53 (m, 2H), 7.29 – 7.25 (m,
3H), 5.93 (m, J = 5.2 Hz, 1H), 4.48 – 4.47 (m, 1H), 4.30 – 4.26 (m, 1H), 4.18 (s, 1H), 4.04 –
4.00 (m, 2H), 3.81 – 3.77 (m, 1H), 2.81 (d, J = 10.4 Hz, 1H), 1.06 (s, 9H), 1.03 (s, 9H); 13C
NMR (101 MHz, CDCl3): δ = 134.4, 134.0, 129.2, 128.4, 127.9, 85.4, 72.3, 71.8, 69.8, 66.7,
62.1, 27.6, 27.3; HRMS: [C20H31N3O4SeSi + NH4] requires 501.15983, found 501.15244.
Phenyl
2-azido-4,6-di-O-benzyl-2-deoxy-3-O-naphthylmethyl-1-seleno-α-D-
galactopyranoside (17)
To a cooled (0°C) solution of 16 (0.24 g, 0.49 mmol) in DMF (2 ml) napthyl bromide (0.12 g,
0.54 mmol) and NaH (60% disp.) (0.03 g, 0.68 mmol) were added. After stirring for 2h, H2O
and Et2O were added, the organic layer was washed with H2O (5x) and brine, dried over
MgSO4 and concentrated in vacuo. Column chromatography yielded naphthylated
intermediate 2-azido-4,6-O-silylidene-2-deoxy-3-O-napthyl-1-seleno-α-D-galacto-pyranoside
(0.35 g, 0.22 mmol) in 71% yield. TLC: Rf 0.82 (10% EtOAc/pentane); [α]D20 (CHCl3): +154;
1
IR (neat, cm-1): 3055, 2931, 2856, 2112, 1473, 1080, 823, 731, 690; H NMR (400 MHz,
CDCl3): δ = 7.89 – 7.78 (m, 4H), 7.56 – 7.32 (m, 3H), 7.48 – 7.46 (m, 2H), 7.27 – 7.22 (m,
4H), 5.95 (d, J =5.2 Hz, 1H), 4.78 (q, J = 11.6 Hz, 2H), 4.59 – 4.57 (m, 1H), 4.35 (dd, J = 5.2
Hz, 10 Hz, 1H), 4.21 – 4.18 (m, 1H), 4.01, 3.96 (m, 2H), 3.68 (dd, J = 2.8 Hz, 10.2 Hz, 1H),
1.06 (s, 9H), 1.04 (s, 9H); 13C NMR (101 MHz, CDCl3): δ = 135.2, 134.5, 133.4, 133.2,
129.4, 129.2, 128.6, 128.5, 128.3, 128.0, 127.9, 127.8, 126.7, 126.4, 126.3, 126.1, 125.9,
125.6, 85.9, 78.8, 71.0, 70.5, 70.3, 67.0, 59.8, 28.0, 27.4 ; HRMS: [C31H39N3O4SeSi + Na]
requires 648.17697, found 648.17651. To a stirred solution of the intermediate (0.22 g, 0.35
mmol) in THF (7 ml) was added Et3N●3HF (0.17 ml, 1.05 mmol) . The mixture was stirred
for 2h at RT, after which the reaction was diluted with EtOAc and washed with H2O (1x). The

aqueous layer was extracted with EtOAc (2x), and the combined organic layers were dried
over MgSO4 and concentrated in vacuo. Column chromatography yielded intermediate diol 2-
azido-2-deoxy-3-O-napthyl-1-seleno-α-D-galacto-pyranoside (0.16 g, 0.33 mmol) in 94%
20
yield. TLC: Rf 0.4 (40% EtOAc/pentane); [α]D (CHCl3, c 1): +228; IR (neat, cm-1): 3419,
1
2885, 2108, 1080, 1066, 817, 740, 690; H NMR (400 MHz, CDCl3): δ = 7.78 – 6.99 (m,
13H), 5.90 (d, J = 5.2 Hz, 1H), 4.86 – 4.78 (m, 2H), 4.21 (dd, J = 5.2 Hz, 10 Hz, 1H), 4.16 –
4.13 (m, 1H), 4.12 – 4.02 (m, 1H), 3.82 – 3.76 (m, 1H), 3.71 – 3.65 (m, 1H), 2.98 (s, 1H),
2.18 (s, 1H); 13C NMR (101 MHz, CDCl3): δ = 134.4, 133.3, 133.2, 133.1, 129.3, 128.7,
128.2, 128.1, 127.9, 127.1, 126.5, 126.5, 125.7, 84.7, 78.8, 72.3, 72.2, 67.2, 62.8, 60.3;
HRMS: [C23H23N3O4Se + Na] requires 508.07474, found 508.07443. To a cooled (0°C)
solution of the intermediate (1.4 g, 3.4 mmol) and BnBr (1.0 ml, 8.5 mmol) in DMF (23 ml)
NaH (60% disp.) (0.34 g, 8.5 mmol) was added. After stirring overnight, H2O was added at
0°C and the mixture was diluted with Et2O. The organic layer was washed with H2O (2x) and
brine, dried over MgSO4 and concentrated in vacuo. Column chromatography yielded
20
compound 17 (1.67 g, 2.8 mmol) in 83% yield. TLC: Rf 0.8 (10% EtOAc/pentane); [α]D
(CHCl3, c 1): +172; IR (neat, cm-1): 3057, 3030, 2908, 2866, 2108, 1099, 1066, 817, 738,
696; 1H NMR (400 MHz, CDCl3): δ = 7.86 – 7.15 (m, 23H), 5.94 (d, J = 5.2 Hz, 1H), 4.97 –
4.55 (m, 4H), 4.43 – 4.36 (m, 4H), 4.08 – 4.07 (m, 1H), 4.77 (dd, J = 2.4 Hz, 10.6 Hz, 1H),
3.63 – 3.58 (m, 1H), 3.47 – 3.41 (m, 1H); 13C NMR (101 MHz, CDCl3): 138.3, 137.9, 135.0,
134.8, 133.4, 133.2, 129.1, 128.5, 128.4, 128.1, 127.9, 127.8, 127.7, 126.6, 126.3, 126.2,
125.7, 85.5, 80.3, 75.0, 73.5, 73.2, 72.5, 72.0, 68.4, 61.2; HRMS: [C37H35N3O4Se + Na]
requires 688.16883, found 688.16858.
2-azido-4,6-di-O-benzyl-2-deoxy-3-O-naphthylmethyl-1-O-(N-phenyl-
trifluoroacetimidoyl)-α/β-D-galactopyranoside (18)
To a cooled (0°C) solution of 17 (0.84 g, 1.4 mmol) in a mixture of THF/H2O (14 ml, 1/1,
v/v) NIS (0.35 g, 1.54 mmol) was added. The reaction was allowed to warm up to RT and
stirred overnight. The mixture was diluted with EtOAc, washed with Na2S2O3 (10% solution
in H2O), H2O, brine, dried over MgSO4 and concentrated in vacuo. Column chromatography
yielded intermediate 2-azido-4,6-di-O-benzyl-2-deoxy-3-O-napthyl-α/β-D-galactopyranose
(0.64 g, 1.4 mmol) in >98% yield. TLC: Rf 0.4 (30% EtOAc/pentane); IR (neat, cm-1): 3389,
1
3059, 2916, 2868, 2108, 1096, 1059, 818, 746, 696; H NMR (400 MHz, CDCl3): δ = 7.80 –
7.01 (m, 29H), 5.28 (d, J = 2.8 Hz, 1H), 4.87 – 4.30 (m, 10H), 4.15 – 4.12 (m, 1H), 3.99 –
3.82 (m, 4H), 3.77 – 3.76 (m, 1H), 3.56 – 3.52 (m, 2H), 3.48 – 3.43 (m, 1H), 3.37 – 3.34 (m,

1H), 3.28 – 3.25 (m, 1H); 13C NMR (101 MHz, CDCl3): δ = 138.0, 137.9, 137.4, 137.1,
135.0, 134.9, 133.2, 133.1, 132.9, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127., 127.3,
126.8, 126.4, 126.3, 126.1, 126.0, 125.9, 125.6, 96.2, 92.1, 80.6, 77.2, 74.5, 73.5, 73.4, 73.3,
73.2, 72.3, 72.2, 72.1, 69.3, 69.2, 68.5, 64.3, 60.2; HRMS: [C31H31N3O5 + Na] requires
548.21559, found 548.21535. To a stirred solution of the intermediate (0.31 g, 0.5 mmol) in
acetone (5 ml) K2CO3 (0.082 g, 0.6 mmol) and N-phenyl trifluoroacetimidoyl chloride (0.12
ml, 0.75 mmol) were added. After stirring for 4 days at RT, MgSO4 was added, the mixture
was filtered and concentrated in vacuo. Column chromatography yielded imidate 18 (0.32 g,
0.4 mmol) in 80% yield. TLC: Rf 0.75 (10% EtOAc/pentane); IR (neat, cm-1): 3061, 3030,
2918, 2886, 2113, 1716, 1209, 1161, 1112, 736, 696; 1H NMR (400 MHz, CDCl3): δ = 7.84 –
7.90 (m, 10H), 7.51 – 7.46 (m, 8H), 7.31 -7.21 (m, 28H), 7.08 – 7.03 (m, 3H), 6.81 – 6.79 (m,
4H), 6.32 (s, 1H), 5.42 (d, J = 7.2 Hz, 1H), 4.93 – 4.84 (m, 6H), 4.62 – 4.40 (m, 6H), 4.17 –
13
4.00 (m, 5H), 3.94 – 3.93 (m, 1H), 3.68 – 3.36 (m, 6H); C NMR (101 MHz, CDCl3): δ =
135.4, 133.6, 129.4, 129.1, 128.8, 128.4, 128.0, 127.8, 127.0, 126.5, 126.1, 124.7, 124.4
119.8, 119.5, 96.4, 96.1, 81.1, 77.9, 75.2, 75.1, 75.0, 73.9, 73.8, 73.6, 73.1, 72.9, 72.6, 62.7,
59.8 ; HRMS: [C39H35F3N4O5 + Na] requires 719.24518, found 719.24518.
Phenyl 2-azido-4,6-di-O-benzyl-2-deoxy-1-seleno-α-D-galactopyranoside (19)
To a stirred solution of 17 (0.71 g, 1.2 mmol) in a mixture of DCM/H2O (14 ml, 4/1, v/v),
DDQ (0.63 g, 2.8 mmol) was added. The mixture was stirred for 2h, after which TLC analysis
showed conversion of the starting material and formation of a lower running byproduct.
Na2S2O3 (10% solution in H2O) was added, the mixture was diluted with DCM and the layers
were separated. The organic layer was washed with sat. aq. NaHCO3 (2x), dried over MgSO4
and concentrated in vacuo. Column chromatography yielded compound 19 (0.49 g, 0.94
mmol) in 78% yield. TLC: Rf 0.61 (20% EtOAc/pentane); [α]D20 (CHCl3, c 1): +174; IR (neat,
cm- 1): 3442, 3062, 2922, 2106, 1089, 1055, 731, 692; 1H NMR (400 MHz, CDCl3): δ = 7.30
– 6.79 (m, 15H), 5.90 (d, J = 5.2 Hz, 1H), 4.71 – 4.59 (m, 2H), 4.52 – 4.40 (m, 3H), 4.04 (dd,
J = 5.2 Hz, 10.4 Hz, 1H), 3.89 – 3.88 (m, 1H), 3.76 – 3.70 (m, 1H), 3.67 – 3.62 (m, 1H), 3.51
13
– 3.45 (m, 1H), 2.52 (s, 1H); C NMR (101 MHz, CDCl3): δ = 137.9, 137.7, 134.7, 134.3,
129.3, 129.1, 128.6, 128.5, 128.5, 128.2, 128.1, 128.0, 127.9, 127.8, 125.3, 85.2, 76.1, 75.4,
73.4, 68.3, 68.1, 62.6 ; HRMS: [C26H27N3O4Se + Na] requires 548.10607, found 548.10583.
1-O-allyl-3-O-benzyl-sn-glycerol (21)
Bimetalic catalysis: To a stirred solution of 20 (0.09 g, 0.5 mmol), DPPB (0.05 g, 0.13
mmol), Pd(OAc)2 (0.011 g, 0.05 mmol), Me2SnCl2 (0.011 g, 0.05 mmol), and Cs2CO3 (0.24 g,

0.75 mmol) in DCM (3 ml), allylacetate (0.16 ml, 1.5 mmol) was added. After stirring
overnight at RT, H2O was added and the mixture was extracted with EtOAc. The organic
layer was washed with H2O (2x), brine, dried over MgSO4 and concentrated in vacuo. The
residue was purified by column chromatography, yielding compound 21 (0.09 g, 0.42 mmol)
in 84% yield.
Tin ketal: A mixture of 20 (0.09 g, 0.5 mmol) and dibutyltinoxide (0.12 g, 0.5 mmol) was
refluxed in toluene (10 ml) for 3h. The mixture was concentrated in vacuo, taken up in DMF
(3 ml) and CsF (0.11 g, 0.725 mmol) and allylbromide (0.056 ml, 0.65 mmol) were added.
The reaction was stirred for 1h, and refluxed for 1h, after which TLC analysis indicated
complete conversion of the starting material. The mixture was concentrated in vacuo, taken up
in EtOAc, dried over MgSO4 and concentrated in vacuo. The residue was purified by column
chromatography, yielding compound 21 (0.073 g, 0.33 mmol) in 66% yield.
Borinate catalysis: To a stirred solution of 20 (0.09 g, 0.5 mmol), 2-aminoethyl
diphenylborinate (0.011 g, 0.05 mmol), KI (0.08 g, 0.5 mmol), K2CO3 (0.08 g, 0.55 mmol) in
CH3CN (2.5 ml) under an argon atmosphere, allyl bromide (0.065 ml, 0.75 mmol) was added,
and the mixture was heated to 60°C. After stirring overnight, TLC analysis indicated
complete conversion of the starting material. The mixture was diluted with EtOAc, washed
with H2O (2x), brine, dried over MgSO4 and concentrated in vacuo. The residue was purified
by column chromatography, yielding compound 21 (0.073 g, 0.33 mmol) in 66% yield. TLC:
1
Rf 0.6 (50% EtOAc/pentane); H NMR (400 MHz, CDCl3): δ = 7.35 – 7.24 (m, 5H), 5.89
(ddd, J = 22.5 Hz, 10.8 Hz, 5.6 Hz, 1H), 5.27 – 5.15 (m, 2H), 4.53 (s, 2H), 3.94 (d, J = 5.6
Hz, 3H), 3.55 – 3.43 (m, 4H), 2.89 (s, 1H, OH); 13C NMR (101 MHz, CDCl3): δ = 138.0,
134.5, 128.4, 127.7, 117.2, 73.4, 72.3, 71.4, 71.3, 69.5.
3-O-allyl-1-O-benzyl-sn-glycerol (23)
To a stirred solution of 22 (1.8 g, 9.9 mmol), 2-aminoethyl diphenylborinate (0.22 g, 0.99
mmol), KI (1.64 g, 9.9 mmol), K2CO3 (1.5 g, 10.9 mmol) in CH3CN (50 ml) under an argon
atmosphere, allyl bromide (1.3 ml, 14.8 mmol) was added, and the mixture was heated to
60°C. After stirring overnight, TLC analysis indicated complete conversion of the starting
material. The mixture was diluted with EtOAc, washed with H2O (2x), brine, dried over
MgSO4 and concentrated in vacuo. The residue was purified by column chromatography,
yielding compound 23 (1.4 g, 6.1 mmol) in 62% yield. TLC: Rf 0.6 (50% EtOAc/pentane); 1H
NMR (400 MHz, CDCl3): δ = 7.35 – 7.24 (m, 5H), 5.89 (ddd, J = 22.5 Hz, 10.8 Hz, 5.6 Hz,

1H), 5.27 – 5.15 (m, 2H), 4.53 (s, 2H), 3.94 (d, J = 5.6 Hz, 3H), 3.55 – 3.43 (m, 4H), 2.89 (s,
13
1H, OH); C NMR (101 MHz, CDCl3): δ = 138.0, 134.5, 128.4, 127.7, 117.2, 73.4, 72.3,
71.4, 71.3, 69.5.
Phenyl
3-O-(2-azido-3,4-O-di-benzyl-2-deoxy-6-O-(tert-butyl-di-phenylsilyl)-α-D-
galactopyranosyl)-2-azido-4,6-O-di-benzyl-2-deoxy-1-seleno-α-D-galactopyranoside (24)
Donor 15 (6.86 g, 8.6 mmol) and acceptor 19 (5.43 g, 10.4 mmol) were coevaporated with
toluene (2x) and dissolved in DCM (85 ml). Molecular sieves (3Å) were added and the
mixture was stirred at 0°C for 10 minutes. TfOH (0.15 ml, 1.7 mmol) was added and the
mixture was stirred for 30 minutes. After addition of NEt3, DCM was added and the organic
layer was washed with H2O, brine, was dried over MgSO4 and concentrated in vacuo. Column
chromatography yielded compound 24 (5.7 g, 5.0 mmol) in 58% yield as a single anomer.
20
TLC: Rf 0.4 (10% EtOAc/pentane); [α]D (CHCl3, c 1): +102; IR (neat, cm-1): 2927, 2856,
1
2110, 1726, 1112, 1051, 738, 700; H NMR (400 MHz, CDCl3): δ = 7.68 – 7.15 (m, 35H),
5.98 (d, J = 5.2 Hz, 1H), 5.20 (d, J = 3.6 Hz, 1H), 5.05 – 4.73 (m, 4H), 4.58 – 4.52 (m, 2H),
4.44 – 4.41 (m, 1H), 4.37 (s, 2H), 4.31 – 4.72 (m, 1H), 4.22 – 4.15 (m, 3H), 4.08 – 4.00 (m,
2H), 3.95 – 3.90 (m, 1H), 3.87 – 3.81 (m, 2H), 3.60 – 3.56 (m, 1H), 3.38 – 3.32 (m, 1H), 1.10
13
(s, 9H); C NMR (101 MHz, CDCl3): δ = 138.5, 138.3, 137.8, 137.6, 135.6, 135.0, 134.9,
133.4, 133.0, 131.0, 129.9, 129.1, 128.9, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 127.9,
127.8, 127.6, 127.5, 94.4 (C-1’), 84.5 (C- 1), 77.5, 75.2, 75.0 (C-3’), 74.9, 73.7, 73.4, 72.7,
71.7, 71.6, 71.5, 68.0, 61.9, 61.0, 59.6, 27.2; HRMS: [C62H66N6O8SeSi + 2H]2+ requires
566.20158, found 566.19305.
3-O-(2-azido-3,4-O-di-benzyl-2-deoxy-6-O-(tert-butyl-di-phenylsilyl)-α-D-
galactopyranosyl)-2-azido-4,6-O-di-benzyl-2-deoxy-α/β-D-galactopyranose (25)
To a cooled (0°C) solution of 24 (0.06 g, 0.05 mmol) in THF/H2O (0.5 ml, 1/1, v/v,) was
added NIS (0.012 g, 0.05 mmol) . The mixture was allowed to warm up to RT and stirred 2h,
after which Na2S2O3 (10% solution in H2O) was added to the reaction and it was diluted with
EtOAc. The organic layer was washed with H2O (2x), brine, dried over MgSO4 and
concentrated in vacuo. Column chromatography yielded compound 25 (0.05 g, 0.05 mmol) in
>98% yield. TLC: Rf 0.2 (20% EtOAc/pentane); IR (neat, cm-1): 3390, 3030, 2927, 2856,
1
2108, 1496, 1103, 1047, 1026, 734, 696; H NMR (400 MHz, CDCl3): δ = 7.65 – 7.15 (m,
80H), 5.34 (d, J = 3.2 Hz, 1H), 5.13 (d, J = 3.2 Hz, 2H), 5.03 – 5.01 (m, 2H), 4.91 – 4.87 (m,
2H), 4.76 – 4.73 (m, 4H), 4.56 – 4.52 (m, 5H), 4.47 – 4.33 (m, 3H), 4.18 – 4.02 (m, 8H), 3.91
13
– 3.72 (m, 6H), 3.59 – 3.40 (m, 4H) 1.05 (s, 20H); C NMR (101 MHz, CDCl3): δ = 138.5,

138.4, 138.3, 138.1, 137.7, 137.5, 135.6, 133.4, 133.3, 133.1, 129.9, 129.8, 128.9, 128.7,
129.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 96.6, 95.7,
94.3, 92.3, 77.7, 77.4, 75.5, 75.1 – 74.6, 74.0, 73.7, 73.6, 73.5, 73.4, 72.7, 72.6, 71.9, 70.5,
69.6, 69.0, 62.7, 62.0, 60.3, 60.0, 59.7, 27.0; HRMS: [C56H2N6O9Si + Na] requires
952.95447, found 952.42582.
3-O-(2-azido-3,4-O-di-benzyl-2-deoxy-6-O-(tert-butyl-di-phenylsilyl)-α-D-
galactopyranosyl)-2-azido-4,6-O-di-benzyl-2-deoxy-1-O-(N-phenyl-trifluoroace-
timidoyl)-α/β-D-galactopyranoside (26)
To a stirred solution of 25 (0.05 g, 0.05 mmol) in acetone (0.5 ml) was added K2CO3 (8 mg,
0.06 mmol) and N-phenyl trifluoroacetimidoyl chloride (0.01 ml, 0.07 mmol) were added.
The reaction mixture was stirred for 3 days under a dry atmosphere at RT. The reaction
mixture was dried over MgSO4 and concentrated in vacuo. Column chromatography yielded
compound 26 (0.056 g, 0.05 mmol) in >98% yield. TLC: Rf 0.76 (20% EtOAc/pentane); IR
(neat, cm-1): 3311, 2954, 2924, 2854, 2112, 1714, 1207, 1151, 1112, 731, 696; 1H NMR (400
MHz, CDCl3): δ = 7.65 – 6.78 (m, 35H), 5.17 – 5.16 (m, 1H), 5.06 – 5.03 (m, 1H), 4.94 –
4.89 (m, 1H), 4.80 – 4.75 (m, 3H), 4.59 – 4.55 (m, 3H), 4.46 – 4.37 (m, 1H), 4.15 – 3.91 (m,
6H), 3.78 – 3.58 (m, 5H), 1.07 (s, 9H); 13C NMR (101 MHz, CDCl3): δ = 138.3, 138.2, 137.6,
137.5, 135.6, 133.3, 133.1, 129.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.9, 127.8, 127.6, 124.5, 119.4, 94.5, 77.53, 75.6, 75.1, 74.9 74.5, 73.6, 73.6, 72.7,
72.0, 70.4, 67.8, 62.7, 61.7, 59.7, 27.0; HRMS: [C64H66F3N7O9Si + Na] requires 1184.45356,
found 1184.45410.
2-O-(2-azido-4,6-di-O-benzyl-2-deoxy-3-O-naphthylmethyl-β-D-galactopyranosyl)-1-O-
allyl-3-O-benzyl-sn-glycerol (27)
Donor 18 (1.67 g, 2.4 mmol) and acceptor 21 (0.80 g, 3.6 mmol) were coevaporated with
toluene (2x) and dissolved in a mixture of acetonitrile, propionitrile and DCM (24 ml, 2:1:1
v/v/v). Molecular sieves (3Å) were added and the mixture was stirred at -40°C for 10 minutes.
TfOH (45 µl, 0.5 mmol) was added and the mixture was stirred for 20 minutes. After addition
of NEt3, DCM was added and the organic layer was washed with H2O, brine, was dried over
MgSO4 and concentrated in vacuo. Size exclusion chromatography yielded compound 27 (1.6
g, 2.2 mmol) in 90% yield (α/β = 1 : 19). TLC: Rf 0.85 (20% EtOAc/pentane); [α]D20 (CHCl3,
1
c 1): -8; IR (neat, cm-1): 3061, 2922, 2860, 2110, 1454, 1064, 817, 732, 696; H NMR (400
MHz, CDCl3): δ = 7.85 – 7.80 (m, 4H), 7.51 – 7.46 (m, 3H), 7.32 – 7.22 (m, 16H), 5.90 (ddd,
J = 22.7 Hz, 10.4 Hz, 5.6 Hz, 1H), 5.29 – 5.24 (m, 1H), 5.16 – 5.13 (m, 1H), 4.93 – 4.90 (m,

1H), 4.82 (s, 2H), 4.67 – 4.37 (m, 6H),4.06 – 4.00 (m, 3H), 3.89 – 3.84 (m, 2H), 3.71 – 3.45
(m, 7H), 3.32 (dd, J = 2.8 Hz, 10.4 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ = 139.1, 138.9,
138.1, 127.8, 133.3, 131.9, 131.7, 128.6, 128.4, 128.4, 128.3, 128.1, 127.9, 127.8, 127.7,
127.6, 126.6, 126.3, 126.1, 125.8, 116.9, 102.7, 80.6, 78.1, 74.4, 73.6, 73.5, 73.4, 72.7, 72.5,
72.4, 71.0, 70.3, 68.6, 63.5; HRMS: [C44H47N3O7 + Na] requires 752.33062, found
752.33026.
2-O-(2-azido-4,6-di-O-benzyl-2-deoxy-β-D-galactopyranosyl)-1-O-allyl-3-O-benzyl-sn-
glycerol (28)
HCl/HFIP method: To a stirred solution of 27 (0.46 g, 0.6 mmol) in a mixture of HFIP/DCM
(7 ml, 1/1 v/v) was added a solution of HCl (3.5 ml, 0.2M, 0.7 mmol) in HFIP. The mixture
was stirred for 30 min, after which TLC analysis indicated complete conversion of the starting
material. The mixture was poured into sat. aq. NaHCO3, DCM was added and the layers were
separated. The organic layer was washed with sat. aq. NaHCO3, brine, dried over MgSO4 and
concentrated in vacuo. Column chromatography yielded compound 28 (0.21 g, 0.35 mmol) in
63% yield.
Buffered DDQ method: To a cooled (0°C) solution of 27 (0.42 g, 0.57 mmol) in DCM (20
ml) a phosphate buffer (2 ml, pH=7.5, 10 mM) was added. DDQ (0.41 g, 1.8 mmol) was
added over 1h in small portions, after which the mixture was allowed to warm up to RT and
was stirred for 30 min. The mixture was diluted with NaHCO3 and the aqueous layer was
extracted with DCM (2x). The combined organic layers were dried over MgSO4 and
concentrated in vacuo. Column chromatography yielded compound 28 (0.25 g, 0.42 mmol) in
20
75% yield. TLC: Rf 0.8 (20% EtOAc/pentane); [α]D (CHCl3, c 1): +42; IR (neat, cm-1):
1
3447, 2916, 2863, 2110, 1454, 1064, 910, 732, 696; H NMR (400 MHz, CDCl3): δ = 7.34 –
7.21 (m, 15H), 5.90 (ddd, J =22.6 Hz, 10.6 Hz, 5.6 Hz, 1H), 4.71 – 4.65 (m, 2H), 4.55 – 4.41
(m, 5H), 4.13 – 3.96 (m, 3H), 3.78 (d, J = 3.2 Hz, 1H), 3.72 – 3.35 (m, 7H), 3.43 -3.35 (m,
13
1H), 2.41 (d, J = 7.2 Hz, 1H); C NMR (101 MHz, CDCl3): δ = 138.3, 138.2, 137.7, 134.4,
128.6, 128.5, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 116.9, 102.6, 78.0, 75.2,
75.2, 73.5, 73.4, 73.3, 72.6, 71.0, 70.2, 68.3, 64.8; HRMS: [C33H39N3O7 + Na] requires
612.28602, found 612.28746.
2-O-(3-O-(2-azido-3,4-O-di-benzyl-2-deoxy-6-O-(tert-butyl-di-phenylsilyl)-α-D-
galactopyranosyl)-2-azido-4,6-O-di-benzyl-2-deoxy-β-D-galactopyranosyl)-1-O-allyl-3-O-
benzyl-sn-glycerol (10)

From 28: Donor 15 (0.75 g, 1.3 mmol) and acceptor 28 (1.35 g, 1.7 mmol) were
coevaporated with toluene (2x) and dissolved in DCM (15 ml). Molecular sieves (3Å) were
added and the mixture was stirred at 0°C for 10 minutes. TfOH (23 µl, 0.26 mmol) was added
and the mixture was stirred for 30 minutes. After addition of NEt3, DCM was added and the
organic layer was washed with H2O, brine, was dried over MgSO4 and concentrated in vacuo.
Column chromatography yielded compound 10 (1.36 g, 1.14 mmol) in 88% yield as α-anomer
only.
From 26: Donor 26 (0.06 g, 0.05 mmol) and acceptor 21 (0.02 g, 0.1 mmol) were
coevaporated with toluene (2x) and dissolved in a mixture of acetonitrile, propionitrile and
DCM (0.5 ml, 2:1:1 v/v/v). Molecular sieves (3Å) were added and the mixture was stirred at -
40°C for 10 minutes. TfOH (1 µl, 10 µmol) was added and the mixture was stirred for 20
minutes. After addition of NEt3, DCM was added and the organic layer was washed with
H2O, brine, was dried over MgSO4 and concentrated in vacuo. Size exclusion chromatography
yielded compound 10 (0.37 g, 0.03 mmol) in 70% yield (α/β = 1 : 19). TLC: Rf 0.6 (10%
20
EtOAc/toluene); [α]D (CHCl3, c 1): +54; IR (neat, cm-1): 2929, 2910, 2856, 2110, 1454,
1
1103, 1078, 1028, 736, 700; H NMR (400 MHz, CDCl3): δ = 7.65 – 7.62 (m, 4H), 7.43 –
7.20 (m, 31H), 5.87 (ddd, J = 22.4 Hz, 10.4 Hz, 5.6 Hz, 1H), 5.25 – 5.21 (m, 1H), 5.14 -5.01
(m, 2H), 5.02 (d, J = 12 Hz, 1H), 4.88 (d, J = 11.2 Hz, 1H), 4.76 (s, 2H), 4.56 – 4.51 (m, 4H),
4.40 – 4.38 (m, 3H), 4.22 – 4.20 (m, 1H), 4.13 – 4.10 (m, 1H), 4.04 – 3.98 (m, 5H), 3.90 –
13
3.82 (m,2H), 3.76 – 3.47 (m, 9H), 3.44 – 3.41 (m, 1H), 1.06 (s, 9H); C NMR (101 MHz,
CDCl3): δ = 138.5, 138.4, 137.9, 137.7, 135.7, 135.6, 134.9, 133.5, 133.2, 129.9, 129.8,
128.8, 128.7, 128.6, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.4, 116.9,
102.6, 94.5, 78.4, 77.7, 75.6, 75.0, 74.7, 73.7, 73.6, 73.4, 73.3, 72.6, 72.5, 71.8, 70.8, 70.4,
70.3, 68.4, 62.8, 62.7, 59.9, 27.0; HRMS: [C69H78N6O11Si + Na] requires 1217.53900, found
1217.53846.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-tert-butyl-di-phenylsilyl-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)-1-O-allyl-3-O-
benzyl-sn-glycerol (29)
Di-azido-pseudotrisaccharide 10 (0.93 g, 0.78 mmol) was dissolved in a mixture of dioxane
(4.5 ml) and water (0.9 ml). PMe3 (1M in THF, 3 ml, 3 mmol) was added to the mixture and it
was stirred for 0.5h. The mixture was concentrated, coevaporated with toluene and dissolved
in DCM (10 ml). Triethylamine (0.33 ml, 2.3 mmol) and Ac2O (0.14 ml, 1.5 mmol) were
added and the mixture was stirred for 1.25h. The organic layer was washed with sat.aq.

NaHCO3 and brine, dried over MgSO4 and concentrated in vacuo. Column chromatography
yielded di-acetylated pseudotrisaccharide 29 (0.56 g, 0.46 mmol) in 59% yield. TLC: Rf=0.3
20
(50% EtOAc/pentane); [α]D (CHCl3, c 0.2): +113; IR (neat, cm-1): 2859, 1661, 1454, 1112,
1
1056, 1027, 696, 667; H NMR (400 MHz, CDCl3) δ 7.69 – 7.54 (m, 6H), 7.48 – 7.37 (m,
8H), 7.37 – 7.21 (m, 44H), 7.18 (s, 5H), 5.82 (ddd, J = 22.7, 10.0, 4.7 Hz, 3H), 5.55 (d, J =
8.1 Hz, 1H), 5.26 – 5.17 (m, 2H), 5.15 – 5.09 (m, 3H), 4.94 (d, J = 11.7 Hz, 1H), 4.86 – 4.68
(m, 4H), 4.64 (d, J = 12.1 Hz, 1H), 4.55 (d, J = 12.2 Hz, 1H), 4.52 – 4.44 (m, 9H), 4.44 – 4.33
(m, 3H), 4.31 – 4.19 (m, 1H), 3.97 – 3.86 (m, 8H), 3.81 (s, 4H), 3.74 – 3.57 (m, 9H), 3.57 –
3.41 (m, 10H), 1.86 (s, 3H), 1.51 (s, 3H), 1.08 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 170.1,
170.0, 138.3, 138.0, 137.5, 135.6, 135.4, 135.3, 134.3, 132.9, 132.9, 129.8, 129.8, 128.4,
128.3, 128.3, 128.2, 128.1, 128.1, 128.0, 128.0, 127.9, 127.8, 127.7, 127.6, 127.6, 127.5,
127.5, 127.4, 127.4, 127.2, 126.7, 126.6, 117.1, 102.1, 93.9, 78.5, 77.9, 77.3, 77.0, 76.7, 75.7,
74.1, 73.9, 73.2, 73.1, 73.1, 72.9, 72.3, 72.1, 71.8, 71.1, 71.1, 69.9, 67.9, 63.5, 51.9, 48.7,
26.9, 23.5, 22.7, 19.1; HRMS: [C73H86N2O13Si +H] requires 1227.59719, found 1227.59871.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-α-D-galactopyranosyl]-2-N-acetamido-4,6-di-
O-benzyl-β-D-galactopyranosyl)-1-O-allyl-3-O-benzyl-sn-glycerol (31)
Silyl-protected pseudotrisaccharide 29 (0.6 g, 0.5 mmol) was coevaporated with toluene and
was dissolved in THF (10 ml). TBAF (1M in THF, 0.54 ml, 0.54 mmol) was added and the
reaction was stirred for 30min. The mixture was concentrated and subsequent column
chromatography yielded alcohol 31 (0.37 g, 0.37 mmol) in 77% yield. TLC: Rf=0.1 (50%
20
EtOAc/pentane); [α]D (CHCl3, c 0.2): +61; IR (neat, cm-1): 3331, 3063, 2912, 2870, 1699,
1645, 1538, 1118, 1014, 734, 695; 1H NMR (400 MHz, Acetone-d6) δ 7.47 – 7.21 (m, 32H),
7.16 (dd, J = 21.5, 9.0 Hz, 2H), 5.96 – 5.84 (m, 1H), 5.26 (dd, J = 17.8, 2.4 Hz, 2H), 5.12 (d,
J = 10.5 Hz, 1H), 4.95 (dd, J = 21.3, 11.5 Hz, 2H), 4.79 (d, J = 11.5 Hz, 1H), 4.74 – 4.66 (m,
2H), 4.66 – 4.58 (m, 2H), 4.56 – 4.42 (m, 4H), 4.39 – 4.28 (m, 1H), 4.15 (s, 1H), 4.10 (t, J =
6.3 Hz, 1H), 4.03 – 3.92 (m, 6H), 3.87 (dd, J = 11.2, 2.2 Hz, 1H), 3.81 (dd, J = 10.6, 7.0 Hz,
1H), 3.75 – 3.57 (m, 7H), 3.57 – 3.49 (m, 4H), 1.97 (s, 3H), 1.70 (s, 3H); 13C NMR (101
MHz, Acetone-d6) δ 170.7, 170.3, 140.1, 140.1, 139.7, 139.7, 139.4, 136.2, 129.0, 129.0,
128.9, 128.9, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 116.4, 103.0, 95.7, 78.6, 78.3,
77.0, 75.1, 75.0, 74.6, 74.0, 73.6, 73.5, 73.4, 72.6, 72.6, 72.5, 71.1, 70.8, 69.7, 62.4, 52.2,
50.3, 30.4, 30.2, 30.0, 29.8, 29.6, 29.5, 23.7, 23.0; HRMS: [C57H68N2O13 +H] requires
989.47942, found 989.48077.

2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-{4,4’-di-methoxy-tri-phenylmethyl}-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)-1-O-allyl-3-O-
benzyl-sn-glycerol (33)
Pseudotrisaccharide 31 (0.26 g, 0.26 mmol) was coevaporated with toluene three times and
was dissolved in DCM (2.6 ml). Triethylamine (0.06 ml, 0.4 mmol) and 4,4’-dimethoxytrityl-
chloride (0.098 g, 0.3 mmol) were added and the mixture was stirred for 23h. Water was
added and the organic phase was washed with brine, dried over Na2SO4 and concentrated in
vacuo. Column chromatography with neutralized silica yielded protected pseudotrisaccharide
20
33 (0.30 g, 0.24 mmol) in 89% yield. TLC: Rf=0.2 (50% EtOAc/pentane); [α]D (CHCl3, c
0.2): +43; IR (neat, cm-1): 3032,2861, 1669, 1508, 1247, 1061, 1027, 736, 696; 1H NMR (400
MHz, Acetone-d6) δ 7.53 (d, J = 7.5 Hz, 2H), 7.46 (d, J = 7.3 Hz, 5H), 7.42 – 7.18 (m, 39H),
7.12 (dd, J = 6.5, 2.9 Hz, 3H), 6.91 (dd, J = 8.9, 5.4 Hz, 4H), 6.59 (d, J = 8.8 Hz, 1H), 5.92 –
5.79 (m, 1H), 5.35 (d, J = 3.5 Hz, 1H), 5.24 (dd, J = 17.3, 1.8 Hz, 1H), 5.09 (dd, J = 10.5, 1.7
Hz, 1H), 4.99 (d, J = 11.5 Hz, 1H), 4.93 – 4.86 (m, 1H), 4.83 (d, J = 10.3 Hz, 1H), 4.79 (s,
1H), 4.74 (ddd, J = 12.1, 9.0, 3.5 Hz, 2H), 4.68 (d, J = 11.7 Hz, 1H), 4.61 (d, J = 11.5 Hz,
1H), 4.53 (s, 2H), 4.52 – 4.41 (m, 3H), 4.36 (q, J = 8.7 Hz, 1H), 4.31 – 4.24 (m, 1H), 4.19 (s,
1H), 4.15 (dd, J = 10.9, 2.3 Hz, 1H), 4.10 – 4.03 (m, 3H), 3.98 – 3.90 (m, 3H), 3.77 (s, 9H),
3.73 – 3.65 (m, 4H), 3.65 – 3.53 (m, 7H), 3.26 (s, 2H), 3.14 (dd, J = 9.1, 4.7 Hz, 1H), 1.92 (s,
3H), 1.72 (s, 3H); 13C NMR (101 MHz, Acetone-d6) δ 170.4, 170.1, 159.4, 146.0, 139.9,
139.8, 139.6, 139.6, 139.2, 136.8, 136.4, 136.0, 131.0, 130.9, 129.0, 129.0, 128.9, 128.9,
128.7, 128.6, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 127.9, 127.5, 116.7, 113.9, 113.8,
102.9, 94.8, 86.9, 79.0, 78.4, 76.2, 75.1, 74.8, 74.7, 74.3, 73.6, 73.5, 72.8, 72.6, 72.6, 71.4,
71.1, 70.8, 69.5, 64.4, 55.5, 52.6, 50.0, 30.2, 30.0, 29.8, 29.6, 29.5, 23.8, 23.0; HRMS:
[C78H86N2O15 +H] requires 1291.61010, found 1291.61161.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-{4,4’-di-methoxy-tri-phenylmethyl}-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)-3-O-benzyl-sn-
glycerol (35)
Allyl-protected pseudotrisaccharide 33 (0.44, 0.34 mmol) was coevaporated with toluene
three times and was dissolved in distilled THF (3.5 ml). The mixture was purged of oxygen
by
bubbling
through
argon
for
10
minutes.
(1,5-
Cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexafluorophosphate (0.035 g, 0.04
mmol) was added and H gas was bubbled through for 5 seconds. Argon was applied and the
2
reaction mixture was stirred for 2.5h, yielding the isomerised intermediate. The reaction

mixture of was diluted with THF (5.5 ml) and sat. aq. NaHCO3 (6 ml) was added. Iodine
(0.13 g, 0.5 mmol) was added and the reaction was stirred for 1h. The mixture was diluted
with EtOAc, washed with sat. aq. Na2S2O3 and brine, dried over Na2SO4 and concentrated in
vacuo. Column chromatography yielded deprotected pseudotrisaccharide 35 (0.30 g, 0.24
20
mmol) in 70 % yield. TLC: Rf=0.1 (75% EtOAc/pentane); [α]D (CHCl3, c 0.2): +25; IR
1
(neat, cm-1): 3100, 2929, 2910, 1699, 1509, 1247, 829, 732, 697; H NMR (400 MHz,
Acetone-d6) δ 7.52 (d, J = 7.4 Hz, 2H), 7.49 – 7.42 (m, 6H), 7.42 – 7.19 (m, 42H), 7.11 (dd, J
= 6.6, 2.9 Hz, 3H), 6.99 (d, J = 8.3 Hz, 1H), 6.91 (dd, J = 8.9, 6.8 Hz, 4H), 5.36 (d, J = 3.6
Hz, 1H), 5.00 (d, J = 3.4 Hz, 1H), 4.98 (s, 1H), 4.83 (dd, J = 17.9, 11.6 Hz, 2H), 4.78 – 4.69
(m, 2H), 4.67 (s, 0H), 4.60 (d, J = 11.5 Hz, 1H), 4.51 (s, 2H), 4.46 (d, J = 6.7 Hz, 2H), 4.44 –
4.40 (m, 1H), 4.39 – 4.30 (m, 1H), 4.27 (t, J = 6.0 Hz, 1H), 4.22 – 4.17 (m, 1H), 4.16 – 4.09
(m, 1H), 4.09 – 4.04 (m, 2H), 4.02 – 3.94 (m, 5H), 3.76 (s, 10H), 3.74 – 3.65 (m, 4H), 3.65 –
3.52 (m, 5H), 3.34 (s, 2H), 3.15 (dd, J = 9.0, 5.0 Hz, 1H), 1.96 (s, 3H), 1.71 (s, 3H); 13C NMR
(101 MHz, Acetone-d6) δ 171.2, 170.2, 159.4, 159.4, 145.9, 139.9, 139.7, 139.6, 139.5, 139.2,
136.8, 136.4, 132.6, 132.5, 130.9, 130.8, 129.4, 129.3, 129.0, 129.0, 128.9, 128.9, 128.7,
128.6, 128.4, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.0, 127.9, 127.5, 113.9, 113.8,
103.0, 94.7, 86.9, 80.8, 78.3, 76.2, 75.0, 74.8, 74.7, 74.3, 73.6, 73.5, 72.8, 72.5, 71.4, 70.8,
69.5, 64.3, 63.5, 55.4, 52.9, 49.9, 30.2, 30.0, 29.8, 29.6, 29.5, 23.7, 23.0; HRMS:
[C75H82N2O15 +H] requires 1251.57880, found 1251.57979.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-{4,4’-di-methoxy-tri-phenylmethyl}-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)- 3-O-benzyl -1-
O-([N, N’-di-isopropylamino]-2-cyanoethyl-phosphite)-sn-glycerol (7)
Pseudotrisaccharide 35 (0.37 g, 0,29 mmol) was dissolved in DCM (3 ml).
Diisopropylethylamine (0.077 ml, 0.44 mmol) and 3Å molsieves were added and the resulting
solution was stirred for 45 minutes. N, N’-di-isopropylamino-2-cyanoethyl-chlorophosphite
(0.078 ml, 0.35 mmol) was added and the reaction mixture was stirred for 2h. Water was
added, the organic phase was washed with brine, dried over Na2SO4 and concentrated in
vacuo. Column chromatography with neutralized silica yielded phosphoramidite 7 (0.36 g,
0.25 mmol) in 85% yield. TLC: Rf=0.8 (80% EtOAc/pentane); 1H NMR (400 MHz, CD3CN)
δ 7.50 (d, J = 7.7 Hz, 2H), 7.45 – 7.22 (m, 42H), 7.13 – 7.03 (m, 3H), 6.93 – 6.76 (m, 4H),
6.39 – 6.29 (m, 1H), 5.44 (d, J = 2.4 Hz, 1H), 5.21 (s, 1H), 4.85 (dd, J = 11.1, 2.1 Hz, 1H),
4.81 – 4.71 (m, 3H), 4.59 (d, J = 11.6 Hz, 1H), 4.55 – 4.40 (m, 7H), 4.35 (d, J = 11.3 Hz, 1H),
4.19 – 4.14 (m, 1H), 4.14 – 4.03 (m, 3H), 4.03 – 3.92 (m, 3H), 3.83 – 3.53 (m, 17H), 3.47 (q,

J = 8.0, 7.2 Hz, 1H), 3.04 (td, J = 8.9, 4.4 Hz, 1H), 2.64 – 2.47 (m, 5H), 1.93 (d, J = 5.7 Hz,
3H), 1.70 (s, 3H), 1.26 – 1.09 (m, 12H); 13C NMR (101 MHz, CD3CN) δ 171.2, 170.8, 159.6,
146.1, 139.7, 139.6, 139.3, 136.9, 136.6, 131.2, 131.1, 129.4, 129.3, 129.3, 129.2, 129.1,
128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.4, 127.8, 119.6, 118.3, 114.1, 114.0, 102.4, 94.8,
87.1, 79.1, 78.3, 76.1, 76.0, 75.1, 75.0, 74.9, 74.3, 73.8, 73.7, 73.0, 72.7, 71.5, 70.6, 70.4,
69.6, 64.6, 61.0, 59.6, 59.4, 59.3, 59.1, 55.9, 52.4, 49.8, 43.9, 43.7, 25.1, 25.0, 23.9, 23.2,
21.0, 14.6; 31P NMR (162 MHz, CD3CN) δ 147.8, 147.7.
2-O-(3-O-[2-azido-3,4-di-O-benzyl-2-deoxy-6-O-tert-butyl-di-phenylsilyl-α-D-
galactopyranosyl]-2-azido-4,6-di-O-benzyl-2-deoxy-β-D-galactopyranosyl)-3-O-allyl-1-O-
benzyl-sn-glycerol (11)
Disaccharide donor 26 (2.33 g, 2.0 mmol) and glycerol acceptor 23 (0.67 g, 3.0 mmol) were
coevaporated with toluene (2x) and dissolved in a mixture of acetonitrile, propionitrile and
DCM (10 ml, 2:1:1 v/v/v). Molecular sieves (3Å) were added and the mixture was stirred at -
40°C for 10 minutes. TfOH (35 µl, 0.4 mmol) was added and the mixture was stirred for 30
minutes. After addition of NEt3, DCM was added and the organic layer was washed with
H2O, brine, was dried over MgSO4 and concentrated in vacuo. Size exclusion chromatography
yielded compound pseudotrisaccharide 11 (1.92 g, 1.61 mmol) in 80% yield as a single
anomer. TLC: Rf 0.6 (15% EtOAc/pentane); [α]D20 (CHCl3, c 0.2): +48; IR (neat, cm-1): 2915,
2856, 2109, 1454, 1207, 1097, 1028, 736; 1H NMR (400 MHz, CDCl3) δ 7.69 – 7.56 (m, 4H),
7.47 – 7.13 (m, 28H), 5.21 (dd, J = 17.2, 1.6 Hz, 1H), 5.17 – 5.07 (m, 2H), 5.03 (d, J = 11.2
Hz, 1H), 4.89 (d, J = 11.2 Hz, 1H), 4.76 (s, 1H), 4.61 – 4.49 (m, 4H), 4.40 (s, 2H), 4.24 (t, J =
6.6 Hz, 1H), 4.16 (dd, J = 10.6, 2.4 Hz, 1H), 4.09 – 3.99 (m, 3H), 3.96 (d, J = 5.3 Hz, 3H),
3.93 – 3.84 (m, 2H), 3.84 – 3.76 (m, 1H), 3.76 – 3.67 (m, 2H), 3.67 – 3.62 (m, 2H), 3.62 –
3.49 (m, 3H), 3.46 – 3.41 (m, 1H), 1.06 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 138.2, 138.1,
137.5, 137.3, 135.4, 135.3, 134.6, 133.1, 132.8, 129.7, 129.6, 128.6, 128.4, 128.3, 128.3,
128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.7, 127.6,
127.6, 127.6, 127.5, 127.5, 127.5, 127.4, 127.3, 116.6, 102.2, 94.2, 78.0, 77.4, 77.3, 77.0,
76.7, 75.4, 74.7, 74.4, 73.4, 73.3, 73.2, 73.0, 72.3, 72.1, 71.5, 70.5, 70.2, 69.9, 68.0, 62.5,
59.6, 26.7, 26.7, 19.1; HRMS: [C69H78N6O11Si + NH4] requires 1213.58658, found
1213.58828.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-tert-butyl-di-phenylsilyl-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)-3-O-allyl-1-O-
benzyl-sn-glycerol (30)

Di-azido-pseudotrisaccharide 11 (1.92 g, 1.6 mmol) was dissolved in a mixture of dioxane (9
ml) and water (1.8 ml). PMe3 (1M in THF, 6.4 ml, 6.4 mmol) was added to the mixture and it
was stirred for 1.7h. The mixture was concentrated, coevaporated with toluene and dissolved
in DCM (20 ml). Triethylamine (0.7 ml, 4.8 mmol) and Ac2O (0.45 ml, 4.8 mmol) were added
and the mixture was stirred for 47h. The organic layer was washed with sat.aq. NaHCO3,
water and brine, dried over MgSO4 and concentrated in vacuo. Column chromatography
yielded di-acetylated pseudotrisaccharide 30 (1.49 g, 1.2 mmol) in 76% yield. TLC: Rf=0.3
20
(50% EtOAc/pentane); [α]D (CHCl3, c 0.2): +44; IR (neat, cm-1): 3067, 2930, 2857, 1663,
1
1305, 1112, 1027, 732, 696; H NMR (400 MHz, Acetone-d6) δ 7.69 – 7.52 (m, 7H), 7.50 –
6.94 (m, 67H), 5.97 – 5.65 (m, 3H), 5.44 (d, J = 8.3 Hz, 1H), 5.29 – 5.04 (m, 5H), 4.93 (d, J =
11.7 Hz, 1H), 4.77 (td, J = 10.6, 3.4 Hz, 3H), 4.70 (d, J = 11.7 Hz, 1H), 4.63 (d, J = 12.1 Hz,
1H), 4.56 – 4.44 (m, 11H), 4.41 (d, J = 6.7 Hz, 3H), 4.30 – 4.19 (m, 2H), 4.08 (d, J = 7.1 Hz,
0H), 3.98 – 3.86 (m, 8H), 3.86 – 3.78 (m, 4H), 3.71 (td, J = 9.9, 8.8, 3.6 Hz, 5H), 3.68 – 3.39
13
(m, 19H), 1.74 (s, 3H), 1.51 (s, 3H), 1.07 (s, 9H); C NMR (101 MHz, Acetone-d6) δ 169.9,
138.2, 138.2, 137.9, 137.8, 137.5, 135.4, 135.2, 134.4, 132.8, 129.8, 129.7, 128.2, 128.2,
128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.4, 127.3, 127.2, 126.6,
126.5, 116.6, 101.7, 94.0, 78.2, 77.7, 77.3, 77.0, 76.7, 75.6, 74.0, 73.9, 73.1, 73.1, 73.0, 72.8,
72.2, 72.0, 71.7, 71.3, 70.7, 69.8, 67.9, 63.4, 51.8, 48.7, 26.8, 23.3, 22.6, 19.0; HRMS:
[C73H87N2O13Si + H] requires 1227.59719, found 1227.59868.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-α-D-galactopyranosyl]-2-N-acetamido-4,6-di-
O-benzyl-β-D-galactopyranosyl)-3-O-allyl-1-O-benzyl-sn-glycerol (32)
Silyl-protected pseudotrisaccharide 30 (1.53 g, 1.25 mmol) was coevaporated with toluene
and was dissolved in THF (15 ml). TBAF (1M in THF, 1.4 ml, 1.4 mmol) was added and the
reaction was stirred for 45 min. The mixture was concentrated and subsequent column
chromatography yielded alcohol 32 (1.1 g, 1.1 mmol) in 89% yield. TLC: Rf=0.1 (50%
20
EtOAc/pentane); [α]D (CHCl3, c 0.2): +39; IR (neat, cm-1): 3282, 3029, 2869, 2865, 1652,
1
1539, 1553, 1370, 1061, 735, 696; H NMR (400 MHz, Acetone-d6) δ 7.41 (d, J = 7.2 Hz,
6H), 7.38 – 7.21 (m, 43H), 7.14 (dd, J = 17.1, 9.0 Hz, 2H), 5.84 (ddt, J = 17.2, 10.6, 5.3 Hz,
1H), 5.22 (d, J = 3.1 Hz, 2H), 5.19 (q, J = 1.7 Hz, 1H), 5.06 (dq, J = 10.5, 1.4 Hz, 2H), 4.95
(dd, J = 19.9, 11.5 Hz, 3H), 4.83 – 4.71 (m, 2H), 4.67 (ddd, J = 11.9, 8.6, 3.7 Hz, 3H), 4.61
(dd, J = 11.4, 7.8 Hz, 3H), 4.54 (s, 3H), 4.51 (d, J = 3.3 Hz, 2H), 4.48 (d, J = 7.3 Hz, 2H),
4.20 – 4.12 (m, 3H), 4.07 (t, J = 6.2 Hz, 2H), 4.02 (d, J = 2.3 Hz, 2H), 3.99 – 3.91 (m, 7H),
3.86 (dd, J = 11.2, 2.6 Hz, 2H), 3.78 (dd, J = 10.6, 7.0 Hz, 2H), 3.72 – 3.65 (m, 4H), 3.65 –

3.61 (m, 3H), 3.58 (m, 7H), 3.49 (dd, J = 10.0, 6.5 Hz, 1H), 3.32 (s, 2H), 3.07 (s, 3H), 1.90 (s,
3H), 1.69 (s, 3H); 13C NMR (101 MHz, Acetone-d6) δ 206.2, 170.7, 170.2, 140.2, 140.2,
139.8, 139.7, 139.4, 136.2, 129.1, 129.0, 128.9, 128.7, 128.6, 128.5, 128.5, 128.3, 128.3,
128.2, 128.1, 116.3, 103.0, 95.8, 78.6, 78.3, 77.1, 75.1, 75.1, 74.7, 74.1, 73.7, 73.7, 73.5, 72.7,
72.5, 72.5, 71.2, 70.7, 69.7, 62.5, 52.3, 50.3, 30.4, 30.2, 30.0, 29.8, 29.6, 29.5, 29.3, 23.7,
23.0; HRMS: [C57H68N2O13 +H] requires 989.47942, found 989.48061.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-{4,4’-di-methoxy-tri-phenylmethyl}-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)-3-O-allyl-1-O-
benzyl-sn-glycerol (34)
Pseudotrisaccharide 32 (0.9 g, 0.9 mmol) was coevaporated with toluene three times and was
dissolved in DCM (9 ml). Triethylamine (0.2 ml, 1.4 mmol) and 4,4’-dimethoxytrityl-chloride
(0.34 g, 1.0 mmol) were added and the mixture was stirred for 2h. Water was added and the
organic phase was washed with brine, dried over Na2SO4 and concentrated in vacuo. Column
chromatography with neutralized silica yielded protected pseudotrisaccharide 34 (1.01 g, 0.78
20
mmol) in 86% yield. TLC: Rf=0.2 (50% EtOAc/pentane); [α]D (CHCl3, c 0.2): +40; IR
1
(neat, cm-1): 2930, 1699, 1508, 1247, 1060, 1027, 828, 736, 696; H NMR (400 MHz,
Acetone-d6) δ 7.56 (d, J = 7.7 Hz, 2H), 7.50 – 7.20 (m, 40H), 7.17 – 7.10 (m, 3H), 7.05 (d, J =
8.6 Hz, 1H), 6.92 (dd, J = 8.6, 5.9 Hz, 4H), 6.54 (d, J = 8.7 Hz, 1H), 5.95 – 5.82 (m, 1H), 5.38
(d, J = 3.4 Hz, 1H), 5.33 – 5.21 (m, 1H), 5.11 (d, J = 10.5 Hz, 1H), 4.98 (t, J = 9.5 Hz, 2H),
4.91 – 4.82 (m, 1H), 4.81 – 4.72 (m, 2H), 4.66 (dd, J = 16.4, 11.7 Hz, 2H), 4.55 – 4.44 (m,
6H), 4.43 – 4.27 (m, 2H), 4.20 (d, J = 8.3 Hz, 2H), 4.07 (d, J = 7.3 Hz, 2H), 4.01 – 3.91 (m,
4H), 3.81 (t, J = 6.3 Hz, 2H), 3.76 (s, 6H), 3.73 – 3.50 (m, 10H), 3.16 (dd, J = 9.0, 4.7 Hz,
1H), 1.85 (s, 3H), 1.75 (s, 3H); 13C NMR (101 MHz, Acetone-d6) δ 170.1, 170.0, 159.3,
145.9, 139.8, 139.7, 139.5, 139.4, 139.2, 136.8, 136.3, 136.0, 130.9, 130.8, 129.0, 128.9,
128.9, 128.7, 128.5, 128.5, 128.3, 128.3, 128.2, 128.1, 128.1, 128.1, 128.0, 127.9, 127.4,
116.4, 113.8, 113.8, 102.7, 95.0, 86.9, 78.8, 78.3, 76.3, 74.8, 74.7, 74.6, 74.2, 73.5, 72.8, 72.6,
72.4, 71.3, 70.9, 70.6, 69.5, 64.3, 55.4, 52.6, 50.0, 30.2, 30.0, 29.8, 29.6, 29.5, 23.7, 23.0;
HRMS: [C78H86N2O15 +H] requires 1291.61010, found 1291.61152.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-{4,4’-di-methoxy-tri-phenylmethyl}-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)-1-O-benzyl-sn-
glycerol (36)
Allyl-protected pseudotrisaccharide 34 (1.01 g, 0.78 mmol) was coevaporated with toluene
three times and was dissolved in distilled THF (5 ml). The mixture was purged of oxygen by

bubbling
through
argon
for
10
minutes.
(1,5-
Cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexafluorophosphate (0.02 g, 0.02
mmol) was added and H gas was bubbled through for 5 seconds. Argon was applied and the
2
reaction mixture was stirred for 2h, yielding the isomerised intermediate. The reaction
mixture of was diluted with THF (12.5 ml) and sat. aq. NaHCO3 (13.5 ml) was added. Iodine
(0.30 g, 1.2 mmol) was added and the reaction was stirred for 1.5h. The mixture was diluted
with EtOAc, washed with sat. aq. Na2S2O3 and brine, dried over Na2SO4 and concentrated in
vacuo. Column chromatography yielded deprotected pseudotrisaccharide 36 (0.78 g, 0.63
20
mmol) in 80 % yield. TLC: Rf=0.1 (75% EtOAc/pentane); [α]D (CHCl3, c 0.2): +28; IR
1
(neat, cm-1): 3061, 2862, 1674, 1508, 1248, 1053, 1026, 826, 735, 697; H NMR (400 MHz,
Acetone-d6) δ 7.51 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 7.4 Hz, 4H), 7.40 – 7.19 (m, 33H), 7.14 –
7.09 (m, 2H), 6.93 – 6.87 (m, 4H), 6.74 – 6.68 (m, 1H), 5.34 (d, J = 3.4 Hz, 1H), 4.97 (d, J =
11.4 Hz, 1H), 4.90 – 4.76 (m, 3H), 4.77 – 4.69 (m, 1H), 4.67 (d, J = 11.7 Hz, 1H), 4.58 (d, J =
11.4 Hz, 1H), 4.53 – 4.42 (m, 5H), 4.42 – 4.33 (m, 2H), 4.26 – 4.20 (m, 1H), 4.20 – 4.10 (m,
2H), 4.06 (s, 1H), 3.97 – 3.87 (m, 3H), 3.87 – 3.78 (m, 3H), 3.76 (s, 5H), 3.69 – 3.59 (m, 6H),
3.57 (d, J = 5.5 Hz, 2H), 3.53 – 3.42 (m, 2H), 3.35 (d, J = 4.9 Hz, 4H), 3.25 – 3.16 (m, 1H),
3.12 (dd, J = 9.1, 4.6 Hz, 1H), 1.85 (s, 3H), 1.72 (s, 3H); 13C NMR (101 MHz, Acetone-d6) δ
189.9, 189.4, 178.6, 165.1, 158.9, 158.8, 158.6, 158.3, 156.0, 155.6, 150.1, 150.0, 148.2,
148.2, 148.1, 147.9, 147.7, 147.7, 147.6, 147.6, 147.4, 147.4, 147.3, 147.3, 147.1, 146.7,
133.0, 133.0, 122.3, 114.0, 106.1, 101.8, 97.6, 95.4, 94.2, 94.0, 93.9, 93.6, 92.8, 92.7, 92.0,
91.7, 90.6, 90.1, 88.8, 83.7, 82.7, 74.6, 71.7, 69.1, 68.9, 49.5, 49.4, 49.2, 49.0, 48.8, 48.6,
48.4, 42.8, 42.2; HRMS: [C75H82N2O15 +H] requires 1251.57880, found 1251.58015.
2-O-(3-O-[2-N-acetamido-3,4-di-O-benzyl-6-O-{4,4’-di-methoxy-tri-phenylmethyl}-α-D-
galactopyranosyl]-2-N-acetamido-4,6-di-O-benzyl-β-D-galactopyranosyl)- 1-O-benzyl -3-
O-([N, N’-di-isopropylamino]-2-cyanoethyl-phosphite)-sn-glycerol (8)
Pseudotrisaccharide 36 (0.19 g, 0,15 mmol) was dissolved in DCM (1.5 ml).
Diisopropylethylamine (0.045 ml, 0.26 mmol) and 3Å molsieves were added and the resulting
solution was stirred for 45 minutes. N,N-di-isopropylamino-2-cyanoethyl-chlorophosphite
(0.043 ml, 0.20 mmol) was added and the reaction mixture was stirred for 1.6h. Water was
added, the organic phase was washed with brine, dried over Na2SO4 and concentrated in
vacuo. Column chromatography with neutralized silica yielded phosphoramidite 8 (0.13 g,
0.09 mmol) in 62% yield. TLC: Rf=0.8 (80% EtOAc/pentane); 1H NMR (400 MHz, CD3CN)
δ 7.48 (d, J = 7.5 Hz, 2H), 7.44 – 7.20 (m, 45H), 7.07 (dd, J = 6.3, 2.7 Hz, 3H), 6.93 – 6.82