Synthesis of C-Glycosyl Amino Acid Building Blocks Suitable for the Solid-Phase Synthesis of Multivalent Glycopeptide Mimics

Article abstract of DOI:10.1002/ejoc.202000587

Five C-glycosyl functionalized lysine building blocks, featuring C-glycosidic derivatives of α-rhamnose, α-mannose, α-galactose, β-galactose, and β-N-acetyl glucosamine have been designed and synthesized. These derivatives, equipped with acid-labile protecting groups, are eminently suitable for solid-phase synthesis of multivalent glycopeptides. The lysine building blocks were prepared from C-allyl glycosides that underwent a Grubbs cross-metathesis with an acrylate, followed by a reduction of the C=C double bond in the resulting α,β-unsaturated esters, and liberation of the carboxylate to allow condensation with a lysine side chain. The thus obtained C-glycosides, five in total, were applied in the solid-phase peptide synthesis (SPPS) of three glycopeptides, showing the potential of the described building blocks in the assembly of well-defined mimics of homo- and heteromultivalent glycopeptides and glycoclusters.

Full text of DOI:10.1002/ejoc.202000587

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Synthesis of C-glycosyl amino acid building blocks suitable
for solid-phase synthesis of multivalent glycopeptide mimics
Authors: Niels Reintjens, Tony Koemans, Nick Zilverschoon, Riccardo
Castelli, Robert Cordfunke, Jan-Wouter Drijfhout, Nico
Meeuwenoord, Herman Overkleeft, Dmitri Filippov, Gijsbert
Van der Marel, and Jeroen D. C. Codée
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000587
Link to VoR: https://doi.org/10.1002/ejoc.202000587
01/2020

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
Synthesis of C-glycosyl amino acid building blocks suitable
for solid-phase synthesis of multivalent glycopeptide mimics
Niels R. M. Reintjensa, Tony S. Koemansa, Nick Zilverschoona, Riccardo Castellia, Robert A. Cordfunkeb,
Jan Wouter Drijfhoutb, Nico J. Meeuwenoorda, Herman S. Overkleefta, Dmitri V. Filippova, Gijsbert A. van
der Marela and Jeroen D. C. Codéea*
[a]
[b]
Dr. N. R. M. Reintjens, T. S. Koemans, N. Zilverschoon, Dr. R. Castelli, N. J. Meeuwenoord, Prof. Dr. H. S. Overkleeft, Dr. D. V. Filippov, Prof. Dr. G. A.
van der Marel, Dr. J. D. C. Codée
Leiden Institute of Chemistry, Leiden University
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: jcodee@chem.leidenuniv.nl
R. A. Cordfunke, Dr. J. W. Drijfhout
Dept. of Immunohematology and Blood Transfusion,
Leiden University Medical Center, Leiden University
Albinusdreef 2, 2333 ZA Leiden (The Netherlands)
Supporting information for this article is given via a link at the end of the document
Abstract: Five C-glycosyl functionalized lysine building blocks,
featuring C-glycosidic derivatives of α-rhamnose, α-mannose, α-
galactose, β-galactose and β-N-acetyl glucosamine have been
designed and synthesized. These derivatives, equipped with acid-
labile protecting groups, are eminently suitable for solid-phase
synthesis of multivalent glycopeptides. The lysine building blocks
were prepared from C-allyl glycosides that underwent a Grubbs cross-
metathesis with an acrylate, followed by a reduction of the C=C double
bond in the resulting α,β-unsaturated esters and liberation of the
carboxylate to allow condensation with a lysine side chain. The thus
obtained C-glycosides, five in total, were applied in the solid-phase
peptide synthesis (SPPS) of three glycopeptides, showing the
potential of the described building blocks in the assembly of well-
defined mimics of homo- and heteromultivalent glycopeptides and
glycoclusters.
glycan structures[7–9], but also their physical properties can be
changed and tuned.[4,10,11] Another example is the exploitation of
the abundance of the circulating anti-L-rhamnose antibodies in
human blood,[12,13] that can be recruited using multivalent
rhamnose conjugates.[14–18]
We reasoned that the development of automated solid phase
techniques to generate clusters of (different) carbohydrates would
be attractive to rapidly assemble multivalent glycoconjugates.[6]
Ponader et al. developed a solid-phase synthesis method to
obtain homo- and hetero-multivalent glycooligomers using
alkyne-functionalized building blocks functionalized by a Cu-
catalyzed azide-alkyne [2+3] cycloaddition with mannose,
galactose or glucose synthons equipped with an azide.[19] This
approach, however, cannot be used to install different glycan
entities and requires “post-assembly” synthetic steps making the
approach overall more elaborate. In addition, O-glycosides[20] are
generated that can be degraded enzymatically. To prevent
hydrolysis of the glycosidic linkage, C-glycosides[21,22] have been
developed and incorporated into C-glycosyl amino acid building
blocks[6,23–26] allowing the online solid-phase peptide synthesis
(SPPS) of glycopeptides.
Introduction
Carbohydrates are involved in various inter- and intracellular
recognition events and can be recognized by lectins leading to a
row of biological processes. Lectins function as pattern
recognition receptors. In innate immunity they promote the
secretion of cytokines and in adaptive immunity they contribute to
endocytosis.[1,2] Examples of these receptors are DC-SIGN and
the mannose receptor, both C-type lectins that are present on
dendritic cells. Since the binding interactions between lectins and
their carbohydrate binding partners are often relatively weak,
strong interactions depend on the multivalent binding. Therefore,
many multivalent carbohydrate structures such as polymers,
glycoconjugates and dendrimers have been designed,
synthesized and evaluated for the development of new
therapeutics and more efficient vaccine therapies.[2] For instance,
mannosylated polymers and peptides have been used as
therapeutics against HIV, SARS and influenza virus as well as in
cancer immunotherapeutic strategies.[3–6] Such multivalent
conjugates can not only be tailored to effectively mimic complex
Here, we report on the development of a methodology for the
generation of a library of C-glycoside functionalized lysine SPPS
building blocks, including α-rhamnose 1, α-mannose 2, α-
galactose 3, β-galactose 4, and β-N-acetylglucosamine
5
functionalized lysine synthons, as depicted in Figure 1A. These
building blocks have been designed to be suitable for
contemporary Fmoc-based SPPS chemistry and can be used for
the synthesis of homo- and heteromultivalent glycomimetics. The
synthesis of building blocks 1-5 hinges on the introduction of an
anomeric C-allyl group[27], cross-metathesis to install a carboxylic
acid functionality and condensation with a suitably protected
lysine (See Figure 1B). The monosaccharides are protected with
acid-labile trityl, p-methoxybenzyl, isopropylidene, and/or
benzylidene groups to allow a one-step protocol in the final stage
of the SPPS that simultaneously removes all protecting groups
and releases the glycopeptides or glycoclusters from the resin.
1
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10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
Figure 1. A) Structures of the C-glycoside SPPS building blocks 1-5; B) Synthesis of the C-glycosidic SPPS building blocks; PG: protecting group.
Installation of the required carboxylic acid functionality was
achieved by a cross-metathesis with benzyl acrylate under the
influence of Grubbs 2nd generation catalyst to give the α,β-
unsaturated ester in excellent yield (Scheme 1B). The double
Results and Discussion
bond was chemoselectively reduced with NaBH4 and ruthenium
trichloride.[30,31] Saponification of the ester then set the stage for
the condensation with Fmoc-L-lysine-OMe. Under the influence of
HCTU and DIPEA, C-rhamnose 9 and the lysine building block
were condensed in 80% yield. Subsequent hydrolysis of the
methyl ester with LiOH at 0°C gave Fmoc-protected C-rhamnose-
functionalized lysine building block 1 in 25% yield over 9 steps.
The first step in the optimized synthesis route[6] towards
mannose building block 2 comprised the C-allylation at the
anomeric position of mannose. Girard et al. achieved a
stereoselective allylation using a Sakurai-type reaction on per-
The synthesis of the target C-glycosyl lysine building blocks is
depicted in Scheme 1. The preparation of C-rhamnose 6 started
with the treatment of peracetylated rhamnopyranose with
allyltrimethylsilane under the agency of BF2OTf·OEt2 as Lewis
acid[28] generated in situ from BF3·OEt2 and TMSOTf, which
afforded the allyl rhamnoside 6 as an inseparable 5/1 α/β-mixture.
This mixture was resolved in the following manner. Following
sodium methoxide-mediated deacetylation, treatment of the
mixture of rhamnosyl-C-glycosides with N-bromosuccinimide led
to the corresponding mixture of bromonium ions, as shown in
Figure 2. Nucleophilic attack of the C-2-OH in the β-rhamnoside
on the formed bromonium ion occurred rapidly. The reaction of
the C-2-OH in the α-rhamnose, in contrast, is hampered, because
of the requirement to adopt an energetically unfavorable 4C1
conformation and the formation of the trans-fused 5,6-bicyclic ring
system (see Figure 2). The cyclized β-product and unreacted α-
rhamnose could be readily separated via column chromatography,
giving pure α-compound 7 in 86% over three steps.[29] Next, triol
7 was alkylated with p-methoxybenzyl chloride in the presence of
sodium hydride to provide the fully protected C-rhamnoside 8.
benzylated
methyl
α-D-mannopyranoside.[32]
However,
debenzylation and purification proved to be problematic when
performed on a larger scale. Therefore, an alternative procedure
was followed in which per-acetyl-D-mannose was treated with a
mixture of allyltrimethylsilane, BF3·OEt2, and TMSOTf in MeCN to
give the desired allyl mannoside as a 4.2/1 α/β mixture (Scheme
1A). Known methods[33,34] to separate the α/β mixture of C-allyl
mannose could not be reproduced on a large scale, and therefore
a procedure, analogous to the one described above for the
rhamnose synthon, was used. The primary alcohol in the crude
α/β-C-allyl mannose was protected with a trityl, to produce a
mixture of 10 and 11 (55% yield over four steps). Next, the two
anomers were treated with N-bromosuccinimide in THF. This led
to the selective formation of the cyclic β-anomer that could be
separated from α-C-allyl mannose 11, which was isolated in 91%
yield. Based on our previous synthesis of a mannose-6-C-
phosphonate building block[35] the C-2-OH and C-3-OH were
masked with an isopropylidene ketal, and, next, by the installation
of a p-methoxybenzyl at the C-4-OH to give fully protected 12.
This compound was transformed into the required C-mannosyl
lysine building block following the sequence of reactions
performed for the rhamnose building block. Thus, a cross-
Figure 2. Cyclization of C-rhamnose to obtain pure 7.
2
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10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
Scheme 1. A) Synthesis of C-allyl building blocks. Reagents and conditions: a) allyltrimethylsilane, BF3·OEt2, TMSOTf, MeCN, 6: 86%, 22: 58%; b) i. NaOMe,
MeOH; ii. N-bromosuccinimide, THF, 3 h, then Na2S2O3, 79% over two steps; c) p-methoxybenzyl chloride, NaH, TBAI, DMF, 80%; d) i. Ac2O, pyridine, ii.
allyltrimethylsilane, BF3·OEt2, TMSOTf, MeCN; iii. NaOMe, MeOH; iv. TrtCl, Et3N, DMF, 60°C, 55% over four steps; e) N-bromosuccinimide, THF, 3 h, 91%; f) i. p-
toluenesulfonic acid, 2,2-dimethoxypropane, 93%; ii. p-methoxybenzyl chloride, NaH, DMF, 97%; g) allyltrimethylsilane, BF3·OEt2, CH3NO2, 89%; h) NaOMe, MeOH,
91%; i) TrtCl, Et3N, DMF, 60°C, 79%; j) p-methoxybenzyl chloride, NaH, DMF, 17: 52%, 18: 28%; k) i. tetrachlorophthalic anhydride, NaOMe, Et3N, MeOH, 50°C; ii.
Ac2O, pyridine, 51% over two steps; l) AcCl, MeOH, 94%; m) benzaldehyde dimethylacetal, p-toluenesulfonic acid, DMF/MeCN, 60°C, 87%; n) i. ethylene diamine,
EtOH, 90°C; ii. Ac2O, NaHCO3, THF/H2O, 83% over two steps; o) p-methoxybenzyl-2,2,2-trichloroacetimidate, TfOH, THF, 78%; B) Synthesis of C-glycoside-
functionalized lysines 1-5. Reagents and conditions: p) i. benzyl acrylate, Grubbs 2nd gen. catalyst, DCM, 50°C; ii. NaBH4, RuCl3, MeOH, DCE, 40°C; iii. LiOH,
THF/MeOH/H2O, 40°C, 9: 89 over three steps; q) i. methyl acrylate, CuI, Grubbs 2nd gen. catalyst, DCE, 50°C; ii. NaBH4, RuCl3, MeOH, DCE, 45°C; iii. LiOH,
THF/H2O/MeOH or THF/H2O, 40°C, 13: 70%, 19: 67%, 20: 65%, 27: 68% over three steps; r) i. Fmoc-L-Lys-OMe, HCTU, DIPEA, DMF; ii. LiOH, THF/H2O, 0°C,
71%, 1: 57%, 2: 54%, 3: 41%, 4: 45% over two steps; s) i. Fmoc-L-Lys-OMe, HCTU, DIPEA, DMF; ii. LiOH, THF/H2O, then 1 M HCl, then NaHCO3, Fmoc N-
hydroxysuccinimide ester, 5: 91% over two steps.
metathesis of 12 with methyl acrylate was followed by a reduction
of the obtained α,β-unsaturated ester with NaBH4 and ruthenium
trichloride to give the desired intermediate in 73% yield over the
two steps. Saponification of the methyl ester using LiOH yielded
acid 13, which was condensed with Fmoc-L-lysine-OMe under the
influence of HCTU and DIPEA. The obtained methyl ester was
then treated with LiOH at 0°C to obtain to SPPS building block 2
in 17% yield over 12 steps.
The synthesis of galactose SPPS building blocks 3 and 4 starts
with the C-glycosylation of acetyl 2,3,4,6-tetra-O-acetyl-β-D-
galactopyranose with allyltrimethylsilane. Performing this reaction
in MeCN gave 14 as a 6/1 α/β mixture (98% yield), while in
nitromethane a 2/1 α/β mixture (89% yield) was obtained. Of note,
these reactions show, in line with literature precedents,[36,37] that
neighbouring group participation of the C-2-O-acetate is not a
decisive factor in determining the stereochemical outcome of
3
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10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
these reactions. The preference for the formation of the α-product
can be accounted for by the reactivity of the galactopyranosyl
oxocarbenium ion.[38] Deacetylation of 15 with sodium methoxide
and subsequent tritylation of the primary alcohol with TrtCl and
Et3N, produced compound 16 as an inseparable α/β mixture. In
this case, treatment with NBS did not result in a selective
cyclization as both the α- and the β-compound underwent the
cyclization at the same rate. Fortunately, after alkylation with p-
methoxybenzyl chloride, the anomers could be separated by silica
gel column chromatography yielding α-anomer 17 and β-anomer
18 in diastereomerically pure forms. Both anomers were
subjected to the previously described cross-metathesis-
reduction-saponification reaction sequence to furnish acids 19
and 20 in good overall yield. The acids were condensed with
Fmoc-L-lysine-OMe in the presence of HCTU and DIPEA,
followed again by carefully hydrolysis with LiOH at 0°C, providing
galactose SPPS building blocks 3 and 4 in respectively 9% and
6% yield over 9 steps.
assembly of glycopeptides is the fact that one does not have to
rely on post-assembly azide-alkyne click-chemistry, allowing the
use of these functionalities for other conjugation purposes. Thus,
using standard Fmoc-SPPS protocols, immobilized peptide 28
was generated carrying an α-Gal, β-Gal and α-Man appendage.
Cleavage of the glycopeptide from the resin was achieved by
treatment with a cocktail of TFA/TIS/H2O (95/2.5/2.5 v/v/v) for
three hours. Triisopropylsilane (TIS) and H2O effectively
scavenged the cations released upon acidic deprotection of the
glycosyl lysine moieties. After precipitation of the peptide from
Et2O, it was purified by RP-HPLC to give 29 (3.4 mg) in 5% yield.
Elongation of immobilized peptide 28 with building block 5 proved
challenging due to the poor solubility of the building block. The
use of a condensation cocktail of 5, HCTU and DIPEA in DMSO
proved effective and after a double treatment of 28 and ensuing
cleavage and RP-HPLC purification target peptide 30 was
obtained in 2% yield. To illustrate the use of the C-rhamnosyl
lysine building block in a relevant larger oligopeptide, we
The last C-glycosyl lysine building block to be synthesized
comprised glucosamine synthon 5. En route to this building block,
a TCP protecting group was installed on glucosamine, which was
followed by acetylation to give donor 21. Fuchss et al. reported a
synthesis of 22 in which they first transformed acetyl donor 21 into
the corresponding α-fluoride, which was then used to
stereoselectively install the C-allyl group.[39] To shorten the
functionalized
the
ovalbumin
derived
peptide
LEQLESIINFEKLAAAAAK, harboring the MHC-I epitope
SIINFEKL, which can be used as a model antigen with six C-
rhamnoses. After an automated synthesis of immobilized peptide
31, it was elongated at the N-terminus with six rhamnose-
functionalized lysines to obtain the protected resin-bound
conjugate. After TFA/TIS/H2O-mediated cleavage and RP-HPLC
synthesis of 22, donor 21 was used directly for the C-glycosylation. purification, conjugate 32 (2.6 mg) was obtained in a 6% yield,
After substantial optimization of the reaction conditions, it was
found that sonication of 21 with allyltrimethylsilane (5.0 eq.), and
BF3·OEt2 (5.0 eq.) and TMSOTf (1.0 eq.) delivered the C-
glycoside 22 in 58% yield on a 40 mmol scale. Deacetylation with
in situ generated HCl (0.8 eq.) gave triol 23 in 94%. The use of
more HCl or the use of sodium methoxide resulted in lower yields
as a result of the ring-opening of the TCP protecting group.
Subsequent installation of the benzylidene protecting group gave
alcohol 24 in 87%. Removal of the TCP protecting group with
ethylene diamine followed by selective N-acetylation gave 25 in
83% yield. Alkylation of the C-3-OH was accomplished by
treatment of 25 with p-methoxybenzyl-2,2,2-trichloroacetimidate
and a catalytic amount of TfOH giving 26 in 78% yield.
Subsequently, cross-metathesis with methyl acrylate, reduction of
the resulting double bond and hydrolysis of the obtained methyl
ester led to acid 27 in a 68% yield over three steps. After coupling
with Fmoc-L-lysine-OMe, the obtained methyl ester was isolated
by crystallization. Selective hydrolysis of the methyl ester in the
fully protected amino acid C-glycoside proved challenging due to
poor solubility. To solve this problem, the reaction was performed
at room temperature, while closely monitoring the conversion with
LC-MS. As partial Fmoc cleavage could not be prevented, the
mixture was quenched with 1 M HCl and then treated with
NaHCO3 and Fmoc-N-hydroxysuccinimide ester to reinstall the
Fmoc-protecting group. Subsequent precipitation with Et2O and
recrystallization from MeOH/DCM/Et2O gave SPPS building block
5 in 91% yield, completing the set of target compounds in 8% yield
over 13 steps.
showing the applicability of the C-rhamnosyl building block in the
synthesis of the more complex peptides and the compatibility of
the protecting group strategy with common Fmoc-SPPS for the
generation of C-glycosylated peptides
Conclusion
With the increasing interest in glycosylated polymers, dendrimers
and peptides, more effective tools are required for their assembly.
In line with this, we have here developed glycosylated lysine
building blocks that can be used in (automated) solid-phase
peptide syntheses. Five C-glycosyl lysine SPPS building blocks,
featuring α-rhamnose, α-mannose, α-galactose, β-galactose and
β-N-acetyl glucosamine moiety have been synthesized and their
application in SPPS is described. The building blocks were
equipped with solely acid-labile protecting groups to allow
deprotection of the moieties, concomitantly with the release of the
peptides from the resin, increasing assembly efficiency. Key steps
in the synthesis of the glycosyl amino acids are the installation of
a C-allyl functionality on the carbohydrates, the ensuing Grubbs
cross-metathesis with an acrylate synthon, and the
chemoselective reduction of the C=C double bond in the α,β-
unsaturated ester. The C-allylation reactions proceeded to
provide mixtures of α- and β-anomers. The undesired β-anomers
of the C-allyl rhamnoside and C-allyl mannoside could be
separated from the target α-anomers using a selective cyclization
reaction of the β-anomer using N-bromosuccinimide. The
application of the C-glycosyl lysines was investigated by the
synthesis of a model peptide in which the building blocks were
combined with an azido lysine. Although the solubility of the
GlcNAc-amino acid initially posed a challenge, conditions have
been found to effectively use the building blocks in an SPPS
assembly. The C-rhamnose building block was used in the
assembly of a larger antigenic peptide, containing the MHC-I
With the target C-glycosyl building blocks in hand, we set out
to probe their efficacy in SPPS. To this end, the four C-glycoside
SPPS building blocks (α-Man 2, α-Gal 3, β-Gal 4, and β-GlcNAc
5) were used in the synthesis of glycopeptides 29 and 30
(Scheme 2), which also featured a 6-azido lysine, to illustrate the
compatibility with these two functionalities. One of the major
advantages of the use of the building blocks designed here for the
4
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10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
Scheme 2. SPPS synthesis of glycopeptides 29, 30, and 32. Reagents and conditions: a) i. 20% piperidine, DMF; ii. Fmoc SPPS cycle for Lys(α-C-Man)-Lys(β-C-
Gal)-Lys(α-C-Gal)-Lys(N3)-Lys(Boc); iii. 20% piperidine, DMF; b) Ac2O, DIPEA, DMF; c) TFA/TIS/H2O (95/2.5/2.5 v/v/v), 3h; d) RP-HPLC; e) i. 5, HCTU, DIPEA,
DMSO, 50°C, 2h; ii. 5, HCTU, DIPEA, DMSO, overnight; iii. 20% piperidine, DMF f) i. Fmoc SPPS cycle for LEQLESIINFEKLAAAAA; ii. 20% piperidine, NMP; g) i.
1, PyBOP, NMM, NMP; ii. 20% piperidine, NMP; iii. repeat of conditions i. and ii. five more times; h) TFA/TIS/H2O (93/2/5 v/v/v). Yield glycopeptides: 29) 3.4 mg,
5%; 30) 1.8 mg, 2%; 32) 2.6 mg, 6%.
Optical rotations were measured on an Anton Paar Modular Circular
Polarimeter MCP 100/150. High resolution mass spectra were recorded
on a Synapt G2-Si or a Q Exactive HF Orbitrap equipped with an electron
spray ion source positive mode. Infrared spectra were recorded on a
Perkin Elmer Spectrum 2 FT-IR.
epitope, SIINFEKL, to deliver a conjugate that can be used as a
model antigen, functionalized with an antibody targeting
rhamnose cluster. The developed protecting group strategy
allows one to combine the building blocks with many other
functionalities in the target peptides, such as azide and alkyne
click handles. The described building blocks will enable the rapid
assembly of libraries of well-defined mimics for homo- and hetero-
oligovalent glycopeptides and glycoclusters and the chemistry
described can be applied to generate other C-glycosyl amino
acids, featuring different monosaccharides.
General procedure glycopeptides synthesis. The automated solid-
phase peptide synthesis was performed on a Protein Technologies
Tribute-UV IR Peptide Synthesizer applying Fmoc based protocol starting
from Tentagel S RAM resin (loading 0.22 mmol/g). The synthesis was
continued with Fmoc-amino acids specific for each peptide. The
consecutive steps performed in each cycle for HCTU chemistry: 1)
Deprotection of the Fmoc-group with 20% piperidine in DMF for 10 min; 2)
DMF wash; 3) Coupling of the appropriate amino acid using a four-fold
excess. Generally, the Fmoc amino acid (1.0 mmol) was dissolved in 0.2
M HCTU in DMF (5 mL), the resulting solution was transferred to the
reaction vessel followed by 0.5 mL of 0.5 M DIPEA in DMF to initiate the
coupling. The reaction vessel was then shaken for 30 min at 50°C; 4) DMF
wash; 5) capping with 10% Ac2O in 0.1 M DIPEA in DMF; 6) DMF wash;
7) DCM wash. Aliquots of resin of the obtained sequences were checked
on an analytical Agilent Technologies 1260 Infinity system with a Gemini 3
µm, C18, 110 Å, 50 x 4.6 mm column. 30 μmol resin was washed with
DMF, DCM and dried after the last synthesis step followed by a treatment
for 180 minutes with 0.6 mL cleavage cocktail of 95% TFA, 2.5% TIS and
2.5% H2O. The suspension was filtered, the resin washed with 0.6 mL of
the cleavage cocktail, and the combined TFA solutions were added
dropwise to cold Et2O and stored at -20°C overnight. The obtained
suspension of the product in Et2O was centrifuged, Et2O was removed and
the precipitant was dissolved in CH3CN/H2O/tBuOH (1/1/1 v/v/v).
Purification was performed on a Gilson GX-281 preparative RP-HPLC with
a Gemini-NX 5u, C18, 110 Å, 250 x 10.0 mm column.
Experimental Section
General experimental. All reagents were of commercial grade and used
as received unless stated otherwise. Reaction solvents were of analytical
grade and when used under anhydrous conditions stored over flame-dried
3Å molecular sieves. All moisture and oxygen sensitive reactions were
performed under an argon atmosphere. Column chromatography was
performed on silica gel (Screening Devices BV, 40-63 µm, 60 Å). For TLC
analysis, pre-coated silica gel aluminum sheets (Merck, silica gel 60, F254)
were used with detection by UV-absorption (254/366 nm) where applicable.
Compounds were visualized on TLC by staining with one of the following
TLC stain solutions: (NH4)6Mo7O24·H2O (25 g/L), (NH4)4Ce(SO4)4·2H2O
(10 g/L) and 10% H2SO4 in H2O; bromocresol (0.4 g/L) in EtOH; KMnO4
(7.5 g/L), K2CO3 (50 g/L) in H2O. Staining was followed by charring at
~150°C. 1H and 13C NMR spectra were recorded on a Bruker AV-400
(400/100/162 MHz) spectrometer or
a Bruker AV-500 Ultrashield
(500/126/202 MHz) spectrometer and all individual signals were assigned
using 2D-NMR spectroscopy. Chemical shifts are given in ppm (δ) relative
to TMS (0 ppm) in CDCl3 or via the solvent residual peak. Coupling
constants (J) are given in Hz. LC-MS analysis were done on an Agilent
Technologies 1260 Infinity system with a C18 Gemini 3 µm, C18, 110 Å,
50 x 4.6 mm column. Absorbance was measured at 214 nm and 256 nm
and an Agilent Technologies 6120 Quadrupole mass spectrometer was
used as detector. Peptides and conjugates were purified with a Gilson GX-
281 preparative HPLC with a Gemini-NX 5u, C18, 110 Å, 250 x 10.0 mm
column. Peptide fragments were synthesized with automated solid phase
peptide synthesis on an Applied Biosystems 433A Peptide Synthesizer.
General procedure rhamnose-conjugate synthesis. The synthesis of
the peptide components of the constructs was performed as has been
described before.[40] In short, the peptides were synthesized using solid-
phase peptide synthesis on a Tentagel S Ac resin (Rapp, Tübingen) using
a Syro II peptide synthesizer (MultiSyntech, Witten, Germany). Normal
couplings (1.5 h - 2 h) were performed using Fmoc amino acids carrying
acid labile side chain protection groups (where required). Activation of
Fmoc amino acids was performed with PyBop and NMM, azidopropionic
5
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
acid was coupled using the succinimidyl ester. Fmoc deprotection was
performed with 20% vol piperidine in NMP. Washings were performed with
NMP. Cleavage from the resin and side chain deprotection was performed
with TFA containing 5% vol water and 2% vol triethylsilane. Purification
was performed with rpHPLC. Analysis of the purified peptide was
performed with UPLC-MS (Acquity, Waters) and showed the expected
molecular masses.
another portion of sodium hydride (60% dispersion in mineral oil, 0.40 g,
10 mmol, 1.0 eq.) was added and the reaction was stirred overnight. The
reaction was quenched with MeOH at 0°C, diluted with H2O and extracted
with DCM. The organic layer was dried over MgSO4, filtered and
concentrated in vacuo. Purification by column chromatography (1020%
Et2O in pentane) gave compound 8 (4.4 g, 8.0 mmol, 79%) as a white solid.
Rf: 0.84 (8/2 pentane/EtOAc); [훼]2D0 +20.0° (c = 2.0, DCM); 1H NMR (CDCl3,
400 MHz, HH-COSY, HSQC): δ 7.32 (d, 2H, Ar), 7.27 (d, 4H, Ar), 6.93 –
6.84 (m, 6H, Ar), 5.79 – 5.66 (m, 1H, CH2-CH=CH2), 5.08 – 4.98 (m, 2H,
CH2-CH=CH2), 4.78 (d, 1H, J = 10.8 Hz, CHH PMB), 4.65 – 4.49 (m, 5H,
2x CH2 PMB, CHH PMB), 4.05 – 3.97 (m, 1H, H-1), 3.79 (s, 9H, 3x CH3
PMB), 3.73 (dd, 1H, J = 7.9, 3.1 Hz, H-3), 3.70 – 3.65 (m, 1H, H-4), 3.62
(t, 1H, J = 3.3 Hz, H-5), 3.60 – 3.54 (m, 1H, H-2), 2.42 – 2.32 (m, 1H, CHH-
CH=CH2), 2.30 – 2.20 (m, 1H, CHH-CH=CH2), 1.34 (d, 3H, J = 6.3 Hz, H-
6); 13C-APT NMR (CDCl3, 101 MHz, HSQC): δ 159.1 (Cq Ar), 134.2 (CH2-
CH=CH2), 130.5, 130.3, 130.2 (Cq Ar), 129.5, 129.5, 129.3, 128.3 (Ar),
116.9 (CH2-CH=CH2), 113.6, 113.6, 113.5 (Ar), 79.5 (C-2), 77.3 (C-3), 74.5
(C-5), 74.0 (CH2 PMB), 72.7 (C-1), 71.4, 71.1 (CH2 PMB), 69.5 (C-4), 64.4
(CH2 PMB), 55.0 (CH3 PMB), 34.2 (CH2-CH=CH2), 17.9 (C-6); FT-IR (neat,
cm-1): 2934, 2836, 2360, 1641, 1612, 1586, 1512, 1464, 1421, 1358, 1302,
1245, 1173, 1079, 1033, 917, 820, 783, 755, 710, 668, 637, 587, 517;
HRMS: [M+Na]+ calcd. For C33H40O7Na: 571.2672, found 571.2670.
3-(2,3,4-tri-O-acetyl-α/β-L-rhamnosyl)-1-propene
(6)
After
co-
evaporating with toluene (4x), acetyl 2,3,4-tri-O-acetyl-α/β-L-
rhamnopyranoside (44.7 g, 134 mmol, 1.0 eq.) was dissolved in dry MeCN
(0.25 L) under an argon atmosphere, followed by the addition of
allyltrimethylsilane (44 mL, 0.28 mol, 2.0 eq.). After cooling the mixture to
0°C, BF3·OEt2 (35 mL, 0.28 mmol, 2.0 eq.) and TMSOTf (2.3 mL, 13 mmol,
0.10 eq.) were added and the reaction was allowed to warm-up to room
temperature overnight. Upon completion determined by TLC analysis, the
reaction was cooled to 0°C and slowly quenched with Et3N. The mixture
was diluted with sat. aq. NaHCO3 and extracted with EtOAc (2x). The
combined organic layers were dried over MgSO4, filtered and concentrated
in vacuo. Purification by column chromatography (10%16% EtOAc in
pentane) gave compound 6 (36.2 g, 115 mmol, 86%, α/β ratio: 5/1) as a
1
yellow oil. Rf: 0.40 (7/2 pentane/EtOAc); H NMR (CDCl3, 400 MHz, HH-
COSY, HSQC): δ 5.75 – 5.58 (m, 1H, CH2-CH=CH2), 5.13 – 5.07 (m, 2H,
H-2, H-3), 5.06 – 5.00 (m, 1H, CH2-CH=CHH), 4.97 – 4.85 (m, 2H, H-4,
CH2-CH=CHH), 3.88 – 3.81 (m, 1H, H-1), 3.70 – 3.61 (m, 1H, H-5), 2.48 –
2.38 (m, 1H, CHH-CH=CH2), 2.35 – 2.23 (m, 1H, CHH-CH=CH2), 2.01 (s,
3H, Ac), 1.95 (s, 3H, Ac), 1.90 (s, 3H, Ac), 1.12 (d, 3H, J = 6.3 Hz, CH3-
6).13C-APT NMR (CDCl3, 101 MHz, HSQC): δ 170.5, 170.3, 170.0 (C=O),
133.0 (CH2-CH=CH2), 118.3 (CH2-CH=CH2), 74.5 (C-1), 71.6 (C-4), 70.5
(C-3), 69.2 (C-2), 68.3 (C-5), 33.8 (CH2-CH=CH2), 21.1, 21.0, 20.8 (CH3
Ac), 17.7 (CH3-6); FT-IR (neat, cm-1): 2983, 2361, 1745, 1371, 1222, 1051,
668; HRMS: [M+Na]+ calcd. for C15H22O7Na: 337.1263, found 337.1264.
*Only data given for the α-anomer.
3-(2,3,4-tri-O-p-methoxybenzyl-α-L-rhamnosyl)-butanoic acid (9)
Compound 8 (25.1 g, 45.8 mmol, 1.0 eq.) and benzyl acrylate (19.3 mL,
128 mmol, 2.8 eq.) were co-evaporated with toluene (1x) under argon
atmosphere. The mixture was dissolved in DCM (0.23 L) and the flask was
shielded from light with aluminum foil. Grubbs 2nd gen. catalyst (0.78 g,
0.92 mmol, 0.02 eq.) was added and the reaction was continued to reflux
overnight at 50°C. Upon completion determined by TLC analysis, the
reaction mixture was filtered over Celite® and concentrated in vacuo.
Purification by column chromatography (1025% EtOAc in pentane) gave
the alkene intermediate (28.9 g, 42.2 mmol, 92%) as a white solid. The
obtained alkene (15.4 g, 22.6 mmol, 1.0 eq.) was co-evaporated with
toluene (1x) under argon atmosphere and dissolved in DCE (90 mL). RuCl3
(0.89 g, 4.2 mmol, 0.19 eq.) was added and the argon balloon was
replaced with an empty balloon. The reaction was cooled to 0°C, NaBH4
(2.7 g, 72.3 mmol, 3.2eq.) was added, after which MeOH (9.15 mL) was
carefully added. The mixture was heated to 40°C for 4.5 hours,
subsequently quenched with MeOH at 0°C. The reaction mixture was
diluted with DCM and washed with brine (1x). The organic layer was dried
over MgSO4, filtered and concentrated in vacuo. Purification by column
chromatography (14% acetone in DCM) yielded the intermediate (13.3
g, 22.6 mmol, 86%) as a transparent oil. 22.7 g of the intermediate (33.2
mmol, 1.0 eq.) was dissolved in a mixture of THF/MeOH/H2O (7/2/1 v/v/v,
0.11 L). The reaction was cooled to 0°C and LiOH.H2O (3.5 g, 83 mmol,
2.5 eq.) was added. The reaction was heated to 40°C for 4 hours, after
which TLC analysis showed full conversion of the starting material. The
reaction mixture was acidified with 1 M HCl to pH = 4-5 and extracted with
DCM (2x). The combined organic layers were dried over MgSO4, filtered
and concentrated in vacuo. Purification by column chromatography
(120% acetone in DCM + 0.1% AcOH) addorded the title compound
(18.3 g, 31 mmol, 96%) as a yellow oil. Rf: 0.32 (4/1 DCM/acetone); [훼]D20
3-(α-L-rhamnosyl)-1-propene (7) Compound 6 (36.3 g, 115 mmol, 1.0 eq.,
α/β ratio: 5/1) was co-evaporated with toluene (3x) under argon
atmosphere and dissolved in MeOH (0.58 L). Sodium methoxide (5.4 M in
MeOH, 2.2 mL, 12 mmol, 0.1 eq.) was added and the solution was stirred
for 2 hours, after which TLC analysis showed complete conversion of the
starting material. The reaction mixture was acidified by the addition of
amberlite H+ resin, filtered and concentrated in vacuo. The obtained
residue was co-evaporated with toluene (1x) under argon atmosphere and
dissolved in THF (1.2 L). N-bromosuccinimide (10 g, 55 mmol, 0.48 eq.)
was added and the reaction was stirred for 3 hours, after which the reaction
was quenched with an aqueous solution of Na2S2O3 (4.4 M, 40 mL). The
mixture was further diluted with toluene and concentrated in vacuo. The
crude product was embedded on silica and purified by column
chromatography (28% MeOH in DCM) yielding compound 7 (14.2 g,
75.4 mmol, 79%) as a white solid. Rf: 0.24 (9/1 DCM/MeOH); [훼]2D0 +18.0°
(c = 1.0, MeOH); 1H NMR (MeOD, 400 MHz, HH-COSY, HSQC): δ 5.89 –
5.75 (m, 1H, CH2-CH=CH2), 5.17 – 5.03 (m, 2H, CH2-CH=CH2), 3.91 –
3.82 (m, 1H, H-1), 3.81 – 3.75 (m, 1H, H-2), 3.65 (dd, 1H, J = 8.9, 3.4 Hz,
H-3), 3.54 – 3.45 (m, 1H, H-5), 3.40 (t, 1H, J = 8.9 Hz, H-4), 2.54 – 2.42
(m, 1H, CHH-CH=CH2), 2.38 – 2.26 (m, 1H, CHH-CH=CH2), 1.24 (d, 3H,
J = 6.1 Hz, CH3-6); 13C-APT NMR (MeOD, 101 MHz, HSQC): δ 135.9
(CH2-CH=CH2), 117.4 (CH2-CH=CH2), 78.5 (C-1), 74.3 (C-4), 72.4 (C-3),
72.3 (C-2), 71.0 (C-5), 34.7 (CH2-CH=CH2), 18.3 (CH3-6); FT-IR (neat, cm-
1): 3371, 2977, 2934, 2361, 1644, 1418, 1253, 1140, 1057, 981, 916, 825,
779, 668, 550; HRMS: [M+Na]+ calcd. for C9H16O4Na: 211.0946, found
211.0944.
1
+37.0° (c = 1.0, DCM); H NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ
7.30 – 7.18 (m, 6H, Ar), 6.90 – 6.81 (m, 6H, Ar), 4.72 (d, 1H, J = 10.9 Hz,
CHH PMB), 4.58 – 4.48 (m, 5H, 2x CH2 PMB, CHH PMB), 3.92 – 3.85 (m,
1H, H-1), 3.80 (s, 9H, 3x CH3 PMB ), 3.67 (dd, 1H, J = 7.7, 3.1 Hz, H-3),
3.63 – 3.56 (m, 1H, H-5), 3.54 – 3.48 (m, 2H, H-2, H-4), 2.32 (t, 2H, J =
7.1 Hz, CH2-9), 1.77 – 1.63 (m, 1H, CHH-8), 1.63 – 1.51 (m, 2H, CHH-8,
CHH-7), 1.47 – 1.36 (m, 1H, CHH-7), 1.30 (d, 3H, J = 6.3 Hz, CH3-6); 13C-
APT NMR (CDCl3, 101 MHz, HSQC): δ 178.9 (C=O), 159.3, 130.7, 130.6,
130.4 (Cq PMB), 129.8, 129.7, 129.5, 113.9, 113.9 (Ar), 79.7 (C-2), 77.9
(C-3), 75.6 (C-4), 74.2 (CH2 PMB), 73.0 (C-1), 71.8, 71.4 (CH2 PMB), 69.6
(C-5), 55.4 (CH3 PMB), 33.5 (CH2-9), 28.7 (CH2-7), 21.2 (CH2-8), 18.1
(CH3-6); FT-IR (neat, cm-1): 2934, 2836, 1721, 1707, 1611, 1586, 1512,
1463, 1359, 1302, 1245, 1173, 1108, 1077, 1032, 819, 756, 710, 637, 584,
516; HRMS: [M+Na]+ calcd. for C34H42O9Na: 617.2727, found 617.2736.
3-(2,3,4-tri-O-p-methoxybenzyl-α-L-rhamnosyl)-1-propene
(8)
Compound 7 (1.92 g, 10.2 mmol, 1.0 eq.) was co-evaporated with toluene
(1x) under argon atmosphere and dissolved in DMF (0.10 L). Sodium
hydride (60% dispersion in mineral oil, 1.47 g, 36.5 mmol, 3.6 eq.) was
added at 0°C. After 20 minutes, p-methoxybenzyl chloride (5.0 mL, 37
mmol, 3.6 eq.) and TBAI (0.38 g, 1.0 mmol, 0.1 eq.) were added. The
reaction was allowed to warm-up to room temperature. After 6 hours,
6
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
Nα-Fmoc-Nε-[butan-4-(2,3,4-tri-O-p-methoxybenzyl-α-L-rhamnosyl)-
amide]-L-lysine (1) Compound 9 (3.0 g, 5.0 mmol, 1.0 eq.) and Fmoc-L-
lysine-OMe (2.4 g, 6.3 mmol, 1.3 eq.) were co-evaporated with toluene
(2x) under argon atmosphere. The mixture was dissolved in DMF (25 mL).
HCTU (2.49 g, 6.0 mmol, 1.2 eq.) and DIPEA (2.6 mL, 15 mmol, 3.0 eq.)
were subsequently added at 0°C. The reaction was allowed to warm-up to
room temperature and stirred for 5 hours. The reaction was quenched with
H2O and diluted with EtOAc. The organic layer was subsequently washed
with 1 M HCl (2x), sat. aq. NaHCO3 (1x) and brine (1x). The organic layer
was dried over MgSO4, filtered and concentrated in vacuo. Purification by
column chromatography (1070% acetone in DCM) gave the
intermediate (3.8 g, 4.0 mmol, 80%) as a white solid, of which 2.8 g (2.9
mmol, 1.0 eq.) was dissolved in THF (40 mL) and cooled to 0°C. An
aqueous solution of LiOH (0.30 M, 19 mL, 5.7 mmol, 2.0 eq.) was slowly
added. After 40 minutes, the solution was diluted with EtOAc and acidified
with 1 M HCl. The aqueous layer was extracted with EtOAc (2x) and the
combined organic layers were dried over MgSO4, filtered and concentrated
in vacuo. Purification by column chromatography (1525% acetone in
DCM + 0.1% AcOH) affored the title compound (1.94 g, 2.06 mmol, 71%)
as a white solid. Rf: 0.26 (4/1 DCM/acetone + 0.1% AcOH); [훼]2D0 +39.0°
(c = 1.0, DCM); 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ 7.76 (d,
2H, J = 7.6 Hz, Ar), 7.62 (t, 2H, J = 7.3 Hz, Ar), 7.43 – 7.36 (m, 2H, Ar),
7.35 – 7.18 (m, 9H, Ar, Ar), 6.91 – 6.82 (m, 6H, Ar), 5.89 (t, 1H, J = 5.8 Hz,
NH), 5.78 (d, 1H, J = 7.9 Hz, NHFmoc), 4.71 (d, 1H, J = 10.9 Hz, CHH
PMB), 4.61 – 4.46 (m, 5H, 2x CH2 PMB, CHH PMB), 4.44 – 4.34 (m, 3H,
CH2 Fmoc, CH L-Lys), 4.22 (t, 1H, J = 7.0 Hz CH Fmoc), 3.96 – 3.88 (m,
1H, H-1), 3.81 (s, 9H, 3x CH3 PMB), 3.73 – 3.62 (m, 2H, H-3, H-5), 3.59 –
3.50 (m, 2H, H-2, H-4), 3.31 – 3.17 (m, 2H, CH2 ε-L-Lys), 2.16 (t, 2H, CH2-
9), 1.95 – 1.84 (m, 1H, CHH-8), 1.83 – 1.75 (m, 1H, CHH-8), 1.74 – 1.64
(m, 1H, CHH-7), 1.64 – 1.35 (m, 7H, CHH-7, 3x CH2 β/γ/δ-L-Lys), 1.32 (d,
3H, J = 6.4 Hz, CH3-6); 13C-APT NMR (CDCl3, 101 MHz, HSQC): δ 174.2,
173.7 (C=O), 159.4, 159.3, 156.2, 144.0, 143.9, 141.4, 130.5, 130.4, 129.8
(Cq Ar), 129.6, 127.8, 127.2, 125.3, 120.1, 113.9 (Ar), 79.3 (C-2), 77.3 (C-
3), 75.6 (C-4), 74.0 (CH2 PMB), 72.7 (C-1), 71.8, 71.5 (CH2 PMB), 70.0 (C-
5), 67.1 (CH2 Fmoc), 55.4 (CH3 PMB), 53.6 (CH L-Lys), 47.2 (CH Fmoc),
39.1 (CH2 ε-L-Lys), 36.1 (CH2-9), 31.8 (CH2-7), 29.0, 28.7, 22.3, 22.0 (CH2-
8, CH2 β/γ/δ-L-Lys), 17.9 (CH3-6); FT-IR (neat, cm-1): 2930, 1719, 1612,
1512, 1451, 1302, 1247, 1174, 1080, 1034, 821, 741; LC-MS: Rt = 9.03
min (Gemini C18, 10-90% MeCN, 12.5 min run); ESI-MS (m/z): [M+Na]+
calcd. for C61H66N2O11Na: 967.4, found 967.4; HRMS: [M+Na]+ calcd. for
C55H64N2O12Na: 967.4357, found 967.4385.
The reaction mixture was filtered and concentrated in vacuo, which gave
a mixture of the fully deacetylated intermediates (47.2 g, max. 231 mmol)
as an oil. After co-evaporating with dioxane (1x) under an argon
atmosphere, the residue was dissolved in DMF (0.77 L). Trityl chloride
(100 g, 348 mmol, 1.5 eq.) and Et3N (80 mL, 0.57 mol, 2.5 eq.) were added
and the suspension was heated to 60°C. After stirring for 2.5 h, TLC
analysis showed complete conversion of the starting material. The reaction
mixture was cooled to room temperature, diluted with H2O and extracted
with EtOAc (2x). The combined organic layers were dried over MgSO4,
filtered and concentrated in vacuo. After purification by column
chromatography (2040% EtOAc in pentane) compounds 10 and 11 (74.5
g, 167 mmol, 55% over four steps) were obtained as a foam with an α/β
ratio of 4.2/1. Rf: 0.46 (1/4 pentane/EtOAc); See compound 11 for analysis.
3-(6-O-trityl-α-D-mannopyranosyl)-1-propene (11)
A
solution of
compound 10 and 11 (31.4 g, 70.3 mmol, 1.0 eq., α/β: 4.2/1) and N-
bromosuccinimide (6.3 g, 35 mmol, 0.5 eq.) in THF (0.70 L) was stirred for
2 h. The reaction was quenched by the addition of sat. aq. Na2S2O3 (0.50
L). After stirring for an additional 10 minutes, the mixture was further
diluted with sat. aq. Na2S2O3 (0.25 L) and extracted with DCM (1x). The
organic layer was washed with sat. aq. NaHCO3 (1x), dried over Na2SO4,
filtered and concentrated in vacuo. Purification by column chromatography
(1060% acetone in DCM + 0.1% Et3N) yielded the title compound (23.1
g, 51.7 mmol, 91%) as a white foam. Rf: 0.42 (7/3 DCM/acetone); [훼]D25
-
18.2° (c = 0.72, CHCl3); 1H NMR (CD3CN, 400 MHz, HH-COSY, HSQC):
δ 7.54 – 7.44 (m, 6H, Ar), 7.36 – 7.29 (m, 6H, Ar), 7.29 – 7.23 (m, 3H, Ar),
6.04 – 5.90 (m, 1H, CH2-CH=CH2), 5.26 – 5.11 (m, 2H, CH2-CH=CH2),
3.88 (ddd, 1H, J = 9.5, 5.4, 2.5 Hz, H-1), 3.71 – 3.64 (m, 2H, H-2, H-5),
3.60 (ddd, 1H, J = 9.3, 6.0, 3.5 Hz, H-4), 3.45 – 3.37 (m, 1H, H-3), 3.25 –
3.12 (m, 3H, CH2-6, OH), 3.05 (t, 2H, J = 4.6 Hz, 2x OH), 2.60 – 2.51 (m,
1H, CHH-CH=CH2), 2.35 – 2.27 (m, 1H, CHH-CH=CH2); 13C-APT NMR
(CD3CN, 101 MHz, HSQC): δ 145.3 (Cq Trt), 136.3 (CH2-CH=CH2), 129.6,
128.8, 128.0 (Ar), 117.3 (CH2-CH=CH2), 87.2 (Cq Trt), 77.4 (C-1), 74.4 (C-
5), 72.5 (C-4), 71.6 (C-2), 69.7 (C-3), 65.1 (CH2-6), 34.4 (CH2-CH=CH2);
FT-IR (neat, cm-1): 3402, 3060, 2928, 1708, 1643, 1597, 1490, 1449, 1221,
1073, 1033, 989, 901, 827, 765, 748, 701, 633, 529; HRMS: [M+Na]+ calcd.
for C28H30O5Na: 469.1991, found 496.1991.
3-(2,3-O-isopropylidene-4-O-p-methoxybenzyl-6-O-trityl-α-D-
mannopyranosyl)-1-propene (12) Compound 11 (43.9 g, 98.3 mmol, 1.0
eq.) was dissolved in 2,2-dimethoxypropane (0.50 L) and cooled to 0°C.
p-Toluenesulfonic acid (2.88 g, 15.1 mmol, 0.15 eq.) was added and the
reaction mixture was stirred for 10 minutes, after which TLC analysis
showed complete conversion of the starting material. The reaction was
quenched by the addition of Et3N (7 mL), diluted with DCM and washed
with a mixture of sat. aq. NAHCO3/brine (1/1, v/v, 1x). The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. Purification by
column chromatography (1050% Et2O in pentane + 0.1% Et3N) gave the
intermediate (44.6 g, 89.1 mmol, 91%) as a clear oil. After co-evaporating
with toluene (2x) under an argon atmosphere, the intermediate (49.9 g,
102.5 mmol, 1.0 eq.) was dissolved in DMF (0.50 L) and cooled to 0°C.
Sodium hydride (60% dispersion in mineral oil, 4.95 g, 123 mmol, 1.2 eq.)
and p-methoxybenzyl chloride (17.0 mL, 125 mmol, 1.2 eq.) were added
and the suspension was allowed to warm-up up to room temperature after
20 minutes. After stirring at room temperature for an additional hour, TLC
analysis showed complete conversion of the starting material. The reaction
was quenched by the addition of MeOH at 0°C, diluted with Et2O and
washed with H2O (2x). The organic layer was dried over Na2SO4, filtered
and concentrated in vacuo. Purification by column chromatography
(520% Et2O in pentane + 0.1% Et3N) yielded the title compound (60.3 g,
99.4 mmol, 97%) as a clear oil. Rf: 0.63 (pentane/Et2O); [훼]2D5 +12.7° (c =
0.67, CHCl3); 1H NMR (CD3CN, 400 MHz, HH-COSY, HSQC): δ 7.49 –
7.44 (m, 6H, Ar), 7.35 – 7.24 (m, 9H, Ar), 6.96 – 6.92 (m, 2H, Ar), 6.80 –
6.75 (m, 2H, Ar), 6.06 – 5.93 (m, 1H, CH2-CH=CH2), 5.24 – 5.10 (m, 2H,
CH2-CH=CH2), 4.62 (d, 1H, J = 11.0 Hz, CHH PMB), 4.31 – 4.21 (m, 2H,
H-3, CHH PMB), 4.08 (dd, 1H, J = 6.4, 5.4 Hz, H-2), 3.99 – 3.92 (m, 1H,
H-1), 3.75 (s, 3H, CH3 PMB), 3.70 – 3.60 (m, 2H, H-4, H-5), 3.31 (dd, 1H,
J = 9.9, 2.1 Hz, CHH-6), 3.08 (dd, 1H, J = 9.8, 5.0 Hz, CHH-6), 2.42 (t, 2H,
J = 6.9 Hz, CH2-CH=CH2), 1.46 (s, 3H, CH3 isopropylidene), 1.34 (s, 3H,
3-(6-O-trityl-α/β-D-mannopyranosyl)-1-propene (10 + 11) D-Mannose
(52.3 g, 302 mmol, 1.0 eq.) was dissolved in pyridine (0.43 L) and the
reaction mixture was cooled to 0°C. Acetic anhydride (0.20 L, 2.1 mol, 7.0
eq.) and DMAP (3.69 g, 30.2 mmol, 0.1 eq.) were added. After stirring for
25 minutes, the solution was allowed to warm-up to room temperature and
stirring was continued overnight. The mixture was subsequently cooled to
0°C and quenched with MeOH. The solution was diluted with EtOAc and
washed with 1 M HCl (5x). The organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was co-evaporated with toluene (2x),
which gave acetyl 2,3,4,6-tetra-O-acetyl-α/β-D-mannopyranoside as a
clear oil which solidified on bench in quantitative yield (124 g). The
intermediate was co-evaporated with toluene (2x) and dissolved in MeCN
(1.20 L) under an argon atmosphere. After cooling the mixture to 0°C,
allyltrimethylsilane (95 mL, 0.62 mol, 2.0 eq.), BF3∙OEt2 (0.19 L, 1.5 mol,
4.9 eq.) and TMSOTf (11 mL, 62 mmol, 0.2 eq.) were added, respectively.
After stirring for 30 minutes, the reaction mixture was allowed to warm-up
to room temperature and stirring continued for 3 days. The reaction mixture
was cooled to 0°C, diluted with EtOAc and quenched with Et3N to pH 8.
The organic layer was washed with sat. aq. NaHCO3 (1x), dried over
MgSO4, filtered and concentrated in vacuo. Purification by column
chromatography (1060% Et2O in pentane) gave a mixture (86.3 g) of 3-
(α/β-D-mannopyranosyl)-1-propene and unreacted acetyl 2,3,4,6-tetra-O-
acetyl-α/β-D-mannopyranoside. After dissolving the mixture in MeOH
(0.60 L), sodium methoxide (5.4 M in MeOH, 22 mL, 0.12 mol, 0.4 eq.) was
added and the solution was stirred for 1.5 hours. TLC analysis showed
complete conversion into a lower running spot (Rf = 0.19 (MeOH/DCM: 1/9
v/v) and the reaction was quenched using amberlite H+ resin to pH 2-3.
7
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
CH3 isopropylidene); 13C-APT NMR (CD3CN, 101 MHz, HSQC): δ 160.1
(Cq PMB), 145.1 (Cq Trt), 135.9 (CH2-CH=CH2), 131.3 (Cq PMB), 130.5,
129.6, 128.8, 128.1 (Ar), 117.6 (CH2-CH=CH2), 114.4 (Ar), 109.9 (Cq
isopropylidene), 87.2 (Cq Trt), 79.2 (C-3), 77.2 (C-2), 76.5 (C-4), 73.7 (C-
1), 73.2 (CH2 PMB), 73.0 (C-5), 64.4 (CH2-6), 55.8 (CH3 PMB), 37.4 (CH2-
CH=CH2), 28.1, 26.0 (CH3 isopropylidene); FT-IR (neat, cm-1): 2987, 2934,
1613, 1514, 1491, 1449, 1381, 1302, 1247, 1212, 1172, 1069, 1034, 1002,
915, 868, 822, 765, 747, 704, 633, 518; HRMS: [M+Na]+ calcd. for
C39H42O6Na: 629.2879, found 629.2881.
for 30 minutes, after which the mixture was diluted with EtOAc and
acidified by the addition of 1 M HCl to pH = 3-4. The mixture was washed
with brine (1x) and the aqueous layer was extracted with EtOAc (1x). The
combined organic layers were dried over Na2SO4, filtered and
concentrated in vacuo. After purification by column chromatography
(18% MeOH in DCM) the title compound (1.73 g, 1.72 mmol, 57%) was
obtained as a white foam. Rf: 0.61 (9/1 DCM/MeOH); [훼]2D5 +20.8° (c = 0.62,
CHCl3); 1H NMR (MeOD, 400 MHz, HH-COSY, HSQC): δ 7.77 (d, 2H, J =
7.5 Hz, Ar), 7.69 – 7.63 (m, 2H, Ar), 7.45 – 7.34 (m, 8H, Ar), 7.33 – 7.18
(m, 11H, Ar), 6.95 – 6.88 (m, 2H, Ar), 6.75 – 6.68 (m, 2H, Ar), 4.61 (d, 1H,
J = 11.0 Hz, CHH PMB), 4.34 (d, 2H, J = 7.0 Hz, CH2 Fmoc), 4.29 (d, 1H,
J = 11.1 Hz, CHH PMB), 4.25 – 4.16 (m, 2H, H-3, CH Fmoc), 4.09 (dd, 1H,
J = 9.4, 4.6 Hz, CH L-Lys), 4.01 (t, 1H, J = 6.4 Hz, H-2), 3.81 (dd, 1H, J =
7.7, 5.3 Hz, H-1), 3.75 (s, 3H, CH3 PMB), 3.69 (dd, 1H, J = 9.5, 7.3 Hz, H-
4), 3.62 – 3.55 (m, 1H, H-5), 3.35 – 3.30 (m, 1H, CHH-6), 3.11 (t, 2H, J =
6.7 Hz, CH2 ε-L-Lys), 3.05 (dd, 1H, J = 9.9, 5.2 Hz, CHH-6), 2.26 – 2.16
(m, 2H, CH2-9), 1.98 – 1.86 (m, 1H, CHH-8), 1.86 – 1.68 (m, 2H, CHH-8,
CHH-7), 1.68 – 1.56 (m, 3H, CHH-7, 1x CH2 β/γ/δ-L-Lys), 1.50 – 1.30 (m,
10H, 2x CH2 β/γ/δ-L-Lys, 2x CH3 isopropylidene); 13C-APT NMR (MeOD,
101 MHz, HSQC): δ 175.8, 160.7 (C=O), 145.3, 142.6, 131.3 (Cq Ar), 130.8,
130.0, 128.8, 128.2, 128.1, 126.3, 120.9, 114.5 (Cq Ar), 110.5 (Cq
isopropylidene), 87.8 (Cq Trt), 80.0 (C-3), 78.4 (C-2), 76.6 (C-4), 74.0 (C-
5), 73.8 (C-1), 73.6 (CH2 PMB), 67.8 (CH2 Fmoc), 64.6 (CH2-6), 55.7 (CH3
PMB), 55.4 (CH L-Lys) 48.4 (CH Fmoc), 40.1 (CH2 ε-L-Lys), 36.8 (CH2-9),
32.9 (CH2-7), 30.3, 29.9 (CH2 β/γ/δ-L-Lys), 28.0, 25.7 (CH3 isopropylidene),
24.3 (CH2 β/γ/δ-L-Lys), 23.3 (CH2-8); FT-IR (neat, cm-1): 3330, 2934, 1716,
1612, 1513, 1449, 1381, 1302, 1246, 1213, 1179, 1160, 1067, 1033, 1002,
900, 865, 822, 760, 735, 701, 646, 632, 621, 516; LC-MS: Rt = 13.48 min
(Vydac 219TP 5 µm Diphenyl, 10 - 90% MeCN, 21 min run); ESI-MS (m/z):
[M+Na]+ calcd. for C61H66N2O11Na: 1025.5, found 1025.4; HRMS: [M+H]+
calcd. for C61H67O2N11: 1003.47394, found 1003.47380.
4-(2,3-O-isopropylidene-4-O-p-methoxybenzyl-6-O-trityl-α-D-
mannopyranosyl)-butanoic acid (13) Compound 12 (5.7 g, 9.4 mmol,
1.0 eq.) was co-evaporated with toluene (2x) under an argon atmosphere,
before being dissolved in dry DCE (0.10 L). Methyl acrylate (2.4 mL, 26
mmol, 2.8 eq.), CuI (0.28 g, 1.5 mmol, 0.16 eq.) and Grubbs 2nd generation
catalyst (0.32 g, 0.38 mmol, 0.04 eq.) were added and the flask was
covered in aluminum foil. The suspension was heated to 50°C and stirred
for 48 hours, after which it was concentrated in vacuo and co-evaporated
with toluene (3x). Purification by column chromatography (1070% Et2O
in pentane) afforded the intermediate (4.9 g, 7.4 mmol, 1.0 eq.), which was
co-evaporated with toluene (2x) under an argon atmosphere and dissolved
in dry DCE (37 mL). Two empty balloons were placed on the flask, followed
by the addition of RuCl3 (0.29 g, 1.4 mmol, 0.19 eq.) and NaBH4 (0.89 g,
24 mmol, 3.2 eq.) at 0°C. Methanol (6.0 mL, 0.15 mol, 20 eq.) was carefully
added to the suspension over 20 minutes, after which the mixture was
allowed to warm-up up to room temperature over 20 minutes. The mixture
was subsequently heated to 45°C for 4 hours. The reaction mixture was
cooled to room temperature, diluted with brine, filtered over celite and
extracted with DCM (2x). The combined organic layers were dried over
Na2SO4, filtered and concentrated in vacuo. Purification by column
chromatography (2060% Et2O in pentane) gave the intermediate (4.6 g,
6.9 mmol, 73% over two steps), which was dissolved in a mixture of
THF/H2O/MeOH (7/1/2, v/v/v, 35 mL). LiOH (0.87 g, 21 mmol, 3.0 eq.) was
added and the mixture was heated to 40°C for 8 hours, after which TLC
analysis showed complete conversion of the starting material. The reaction
mixture was cooled to 0°C, acidified with 1 M HCl to pH = 6, diluted with
H2O and extracted with DCM (2x). The combined organic layers were dried
over Na2SO4, filtered and concentrated in vacuo. The title compound was
obtained (4.3 g, 6.6 mmol, 96%). Rf: 0.85 (9/1 DCM/MeOH); [훼]2D5 +16.0°
(c = 0.43, CHCl3); 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ 7.51
– 7.43 (m, 6H, Ar), 7.34 – 7.20 (m, 9H), 7.01 – 6.92 (m, 2H, Ar), 6.79 –
6.71 (m, 2H, Ar), 4.69 (d, 1H, J = 10.9 Hz, CHH PMB), 4.34 (d, 1H, J =
10.9 Hz, CHH PMB), 4.26 (t, 1H, J = 6.9 Hz, H-3), 4.03 (t, 1H, J = 6.5 Hz,
H-2), 3.91 – 3.81 (m, 1H, H-1), 3.79 (s, 3H, CH3 PMB), 3.78 – 3.65 (m, 2H,
H-4, H-5), 3.38 (dd, 1H, J = 9.9, 2.1 Hz, CHH-6), 3.18 (dd, 1H, J = 9.9, 4.9
Hz, CHH-6), 2.43 (t, 2H, J = 7.2 Hz, CH2-9), 2.00 (m, 1H, CHH-8), 1.85 –
1.63 (m, 3H, CHH-8, CH2-7), 1.50 (s, 3H, CH3 isopropylidene), 1.38 (s, 3H,
CH3 isopropylidene); 13C-APT NMR (CDCl3, 101 MHz, HSQC): δ 179.2
(C=O), 159.2, 144.1, 130.2 (Cq Ar), 129.8, 128.9, 127.9, 127.0, 113.7 (Ar),
109.4 (Cq isopropylidene), 86.5 (Cq Trt), 78.8 (C-3), 77.1 (C-2), 75.6 (C-4),
72.9 (C-5), 72.8 (CH2 PMB), 72.6 (C-1), 63.4 (CH2-6), 55.4 (CH3 PMB),
33.8 (CH2-9), 32.0 (CH2-7), 27.8, 25.7 (CH3 isopropylidene), 20.8 (CH2-8);
FT-IR (neat, cm-1): 3058, 2987, 2934, 1707, 1612, 1586, 1513, 1490, 1449,
1381, 1302, 1245, 1216, 1160, 1068, 1034, 1002, 900, 866, 822, 777, 765,
737, 703, 644, 632; HRMS: [M+Na]+ calcd. for C40H44O8Na: 675.29284,
found 675.29260.
3-(2,3,4,6-tetra-O-acetyl-α/β-D-galactopyranosyl)-1-propene
(14)
Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (23.7 g, 60.8 mmol, 1.0
eq.) was co-evaporated with toluene (2x) under an argon atmosphere and
dissolved in CH3NO2 (0.24 L). Allyltrimethylsilane (20 mL, 0.13 mol, 2.1
eq.) was added, followed by the addition of BF3∙OEt2 (23 mL, 0.18 mol, 3.0
eq.) at 0°C. The yellow solution was allowed to stir at room temperature
for 3 days. The reaction was quenched by the addition of sat. aq. NaHCO3
at 0°C, diluted with EtOAc and washed with brine (1x). The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. Purification by
column chromatography (1050% Et2O in pentane) afforded the title
compound (20.1 g, 54.0 mmol, 89%) as a yellow oil with an α/β ratio of 2/1.
Rf: 0.41 (1/1 pentane/Et2O); 1H NMR (CDCl3, 400 MHz, HH-COSY,
HSQC): δ 5.80 – 5.69 (m, 1H, CH2-CH=CH2), 5.43 – 5.38 (m, 1H, H-4),
5.26 (dd, 1H, J = 9.3, 5.0 Hz, H-2), 5.20 (dd, 1H, J = 9.4, 3.2 Hz, H-3), 5.12
– 5.06 (m, 2H, CH2-CH=CH2), 4.33 – 4.25 (m, 1H, H-1), 4.24 – 4.14 (m,
1H, CHH-6), 4.14 – 4.01 (m, 2H, H-5, CHH-6), 2.54 – 2.38 (m, 1H, CHH-
CH=CH2), 2.35 – 2.21 (m, 1H, CHH-CH=CH2), 2.11 (s, 3H, CH3 Ac), 2.06
(s, 3H, CH3 Ac), 2.05 – 2.00 (m, 6H, 2x CH3 Ac); 13C-APT NMR (CDCl3,
101 MHz, HSQC): δ 170.7, 170.2, 170.1, 170.0 (C=O), 133.4 (CH2-
CH=CH2), 117.8 (CH2-CH=CH2), 71.6 (C-1), 68.4 (C-5), 68.0 (C-2), 67.8
(C-3), 67.7 (C-4), 61.6 (CH2-6), 31.0 (CH2-CH=CH2), 20.9, 20.9, 20.8, 20.8,
20.8 (CH3 Ac); FT-IR (neat, cm-1): 2978, 1740, 1644, 1434, 1369, 1212,
1044, 909, 601; HRMS: [M+Na]+ calcd for C17H24O9Na: 395.1318, found
395.1316. *NMR analysis only given for the α-anomer.
Nα-Fmoc-Nε-[butan-4-(2,3-O-isopropylidene-4-O-p-methoxybenzyl-6-
O-trityl-α-D-mannopyranosyl)-amide]-L-lysine (2) Compound 13 (3.8 g,
5.8 mmol, 1.0 eq.) and Fmoc-L-lysine-OMe (2.9 g, 7.0 mmol, 1.2 eq.) were
dissolved in DMF (30 mL). HCTU (2.9 g, 7.0 mmol, 1.2 eq.) and DIPEA
(3.0 mL, 17 mmol, 3.0 eq.) were added and the solution was stirred for 4
hours. The reaction mixture was diluted with EtOAc and washed with 1 M
HCl (1x), sat. aq. NaHCO3 (1x), brine (1x). The organic layer was dried
over Na2SO4, filtered and concentrated in vacuo. Purification by column
chromatography (230% acetone in DCM) yielded the intermediate (5.6
g, 5.5 mmol, 95%) of which 3.05 g (3.00 mmol, 1.0 eq.) was dissolved in
THF (30 mL) and cooled to 0°C. An aqueous solution of LiOH (0.30 M, 20
mL, 6.0 mmol, 2.0 eq.) was added and the mixture was stirred vigorously
3-(α/β-D-galactopyranosyl)-1-propene (15) Compound 14 (20.0 g, 53.8
mmol, 1.0 eq.) was dissolved in MeOH (0.11 L). Sodium methoxide (5.4 M
in MeOH, 4.0 mL, 22 mmol, 0.40 eq.) was added and the solution was
stirred for 3 hours, after which it was acidified by the addition of amberlite
H+ resin. The mixture was filtered and concentrated in vacuo. The title
compound (10.0 g, 49.2 mmol, 91%) was obtained as a yellow foam and
used without further purification. Rf: 0.13 (9/1 DCM/MeOH); 1H NMR
(MeOD, 400 MHz, HH-COSY, HSQC): δ 5.93 – 5.82 (m, 1H, CH2-
CH=CH2), 5.17 – 5.06 (m, 2H, CH2-CH=CH2), 4.03 – 3.93 (m, 2H, H-1, H-
2), 3.93 – 3.85 (m, 1H, H-3), 3.80 – 3.62 (m, 4H, H-4, H-5, CH2-6), 2.52 –
2.32 (m, 2H, CH2-CH=CH2); 13C-APT NMR (MeOD, 101 MHz, HSQC): δ
136.7 (CH2-CH=CH2), 116.9 (CH2-CH=CH2), 75.6 (C-1), 74.0, 71.9 (C-4,
8
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
C-5), 70.1 (C-3), 70.0 (C-2), 61.9 (CH2-6), 31.0 (CH2-CH=CH2); FT-IR
(neat, cm-1): 3352, 2919, 1642, 1416, 1073, 914, 515; HRMS: [M+Na]+
calcd for C9H16O5Na: 227.0895, found 227.0894. *NMR analysis only
given for the α-anomer.
Hz, CHH PMB), 4.27 (d, 1H, J = 10.7 Hz, CHH PMB), 3.93 (dd, 1H, J =
2.9, 1.1 Hz, H-4), 3.82 – 3.71 (m, 9H, 3x CH3 PMB), 3.63 – 3.54 (m, 2H,
H-3, H-5), 3.43 (t, 1H, J = 9.3 Hz, H-2), 3.34 – 3.19 (m, 2H, H-1, CHH-6),
2.89 (dd, 1H, J = 9.3, 5.6 Hz, CHH-6), 2.60 – 2.49 (m, 1H, CHH-CH=CH2),
2.23 – 2.11 (m, 2H, CHH-CH=CH2); 13C-APT NMR (CD3CN, 101 MHz,
HSQC): δ 160.3, 160.2, 160.1, 145.1 (Cq Ar), 136.5 (CH2-CH=CH2), 132.0,
131.9, 131.9 (Cq Ar), 130.7, 130.6, 130.5, 129.6, 128.8, 128.1 (Ar), 116.9
(CH2-CH=CH2), 114.7, 114.5, 114.4 (Ar), 87.5 (Cq Trt), 85.2 (C-3), 79.7 (C-
1), 78.9 (C-2), 78.0 (C-5), 75.4 (C-4), 75.3, 74.9, 72.3 (CH2 PMB), 64.7
(CH2-6), 55.9, 55.9 (CH3 PMB), 37.1 (CH2-CH=CH2); FT-IR (neat, cm-1):
2907, 1613, 1583, 1513, 1491, 1449, 1362, 1302, 1248, 1173, 1076, 1034,
915, 821, 747, 707, 633, 518; HRMS: [M+Na]+ calcd for C52H54O8Na:
829.3716, found 829.3740.
3-(6-O-trityl-α/β-D-galactopyranosyl)-1-propene (16) Trityl chloride (21
g, 75 mmol, 1.3 eq.) and Et3N (17 mL, 0.12 mol, 2.5 eq.) were added to a
solution of compound 15 (10.0 g, 48.9 mmol, 1.0 eq.) in DMF (0.16 L). The
mixture was heated to 60°C overnight. The mixture was diluted with EtOAc
and washed with brine (3x). The organic layer was dried over Na2SO4,
filtered and concentrated in vacuo. Purification by column chromatography
(30100% EtOAc in pentane) gave the title compound (17.3 g, 38.7 mmol,
79%). Rf: 0.36 (3/7 pentane/EtOAc); 1H NMR (CD3CN, 400 MHz, HH-
COSY, HSQC): δ 7.50 – 7.44 (m, 6H, Trt), 7.36 – 7.29 (m, 6H, Trt), 7.29 –
7.23 (m, 3H, Trt), 6.04 – 5.87 (m, 1H, CH2-CH=CH2), 5.24 – 5.03 (m, 2H,
CH2-CH=CH2), 3.96 – 3.89 (m, 1H, H-1), 3.89 – 3.83 (m, 1H, H-5), 3.81 –
3.72 (m, 2H, H-2, H-4), 3.64 – 3.53 (m, 1H, H-3), 3.33 – 3.12 (m, 3H, CHH-
6, 2x OH), 3.11 – 3.04 (m, 1H, CHH-6), 2.91 – 2.82 (m, 1H, OH), 2.52 –
2.42 (m, 1H, CHH-CH=CH2), 2.39 – 2.29 (m, 1H, CHH-CH=CH2); 13C-APT
NMR (CD3CN, 101 MHz, HSQC): δ 145.3 (Cq Trt), 137.0 (CH2-CH=CH2),
129.6, 128.8, 128.0 (Ar), 116.9 (CH2-CH=CH2), 87.3 (Cq Trt), 75.4 (C-1),
71.4 (C-5), 70.8 (C-3), 70.2, 69.7 (C-2, C-4), 64.3 (CH2-6),30.2 (CH2-
CH=CH2); FT-IR (neat, cm-1): 3391, 3059, 2929, 1642, 1597, 1490, 1448,
1265, 1222, 1153, 1069, 988, 901, 823, 762, 737, 704, 650, 632, 580, 536;
HRMS: [M+Na]+ calcd for C28H30O5Na: 469.1991, found 469.1988. *NMR
analysis only given for the α-anomer.
4-(2,3,4-tri-O-p-methoxybenzyl-6-O-trityl-α-D-galactopyranosyl)-
butanoic acid (19) Compound 17 (15.2 g, 18.8 mmol, 1.0 eq.) was co-
evaporated with toluene (2x) under an argon atmosphere before being
dissolved in dry DCE (0.19 L). Methyl acrylate (4.8 mL, 53 mmol, 2.8 eq.),
CuI (0.54 g, 2.8 mmol, 0.15 eq.) and Grubbs 2nd generation catalyst (0.63
g, 0.74 mmol, 0.04 eq.) were added and the flask was covered in aluminum
foil. The suspension was heated to 50°C for 48 hours, after which it was
concentrated in vacuo and co-evaporated with toluene (2x) under an argon
atmosphere. The obtained residue was dissolved in dry DCE (0.10 L) and
cooled to 0°C. Two empty balloons were placed on the flask, followed by
the addition of RuCl3 (0.74 g, 3.6 mmol, 0.19 eq.) and NaBH4 (2.3 g, 61
mmol, 3.2 eq.). Methanol (15 mL, 0.37 mol, 20 eq.) was carefully added to
the suspension over 20 minutes, after which the mixture was allowed to
warm-up up to room temperature over 30 minutes. The mixture was
subsequently heated to 45°C for 6 hours. The reaction mixture was cooled
to room temperature, diluted with brine and extracted with DCM (3x). The
combined organic layers were dried over Na2SO4, filtered and
concentrated in vacuo. Purification by column chromatography (2070%
Et2O in pentane) gave the intermediate (11.8 g, 13.6 mmol, 73% over two
steps) of which 11.7 g (13.5 mmol, 1.0 eq.) was dissolved in a mixture of
THF/H2O (4/1, v/v, 0.14 L), followed by the addition of LiOH (1.7 g, 41
mmol, 3.0 eq.). The mixture was heated to 40°C for 30 hours. The reaction
mixture was cooled to 0°C, acidified with 3 M HCl to pH = 4/5, diluted with
H2O and extracted with DCM (2x). The combined organic layers were dried
over Na2SO4, filtered and concentrated in vacuo. The title compound was
obtained (10.5 g, 12.4 mmol, 92%). Rf: 0.84 (9/1 DCM/MeOH); [훼]D25
+25.6° (c = 0.90, CHCl3); 1H NMR (CD3CN, 500 MHz, HH-COSY, HSQC):
δ 7.51 – 7.47 (m, 6H, Ar), 7.33 (t, 6H, J = 7.4 Hz, Ar), 7.30 – 7.24 (m, 5H,
Ar), 7.18 (d, 2H, J = 8.5 Hz, Ar), 7.10 (d, 2H, J = 8.6 Hz, Ar), 6.92 (d, 2H,
J = 8.6 Hz, Ar), 6.90 – 6.84 (m, 4H, Ar), 4.59 – 4.46 (m, 5H, 2x CH2 PMB,
1x CHH PMB), 4.39 (d, 1H, J = 11.1 Hz, CHH PMB), 4.04 – 3.97 (m, 1H,
H-5), 3.94 – 3.90 (m, 1H, H-4), 3.84 – 3.77 (m, 10H, H-1, 3x CH3 PMB),
3.76 (dd, 1H, J = 6.9, 2.8 Hz, H-3), 3.69 – 3.64 (m, 1H, H-2), 3.64 – 3.58
(m, 1H, CHH-6), 3.24 (dd, 1H, J = 10.5, 3.4 Hz, CHH-6), 2.38 (t, 2H, J =
7.0 Hz, CH2-9), 1.83 – 1.68 (m, 2H, CHH-7, CHH-8), 1.64 – 1.55 (m, 2H,
CHH-7, CHH-8); 13C-bbdec NMR (CD3CN, 126 MHz, HSQC): δ 175.7
(C=O), 160.5, 160.4, 160.3, 145.5 (Cq Ar), 132.2, 132.0, 132.0, 130.6,
130.4, 130.1, 129.7, 128.8, 128.0, 114.9, 114.8, 114.8 (Ar), 87.6 (Cq Trt),
77.4 (C-2, C-3), 75.6 (C-4), 73.7 (C-5), 73.3 (CH2 PMB), 71.7 (C-1), 63.0
(CH2-6), 56.1, 56.1 (CH3 PMB), 34.4 (CH2-9), 27.8, 22.4 (CH2-7, CH2-8);
FT-IR (neat, cm-1): 2937, 1707, 1612, 1513, 1449, 1302, 1248, 1173, 1087,
1034, 821, 707, 633; HRMS: [M+Na]+ calcd. for C53H56O10Na: 875.37657,
found 875.37656.
3-(2,3,4-tri-O-p-methoxybenzyl-6-O-trityl-α-D-galactopyranosyl)-1-
propene (17) Triol 16 (16.4 g, 36.8 mmol, 1.0 eq.) was co-evaporated with
toluene (2x) under an argon atmosphere and dissolved in DMF (0.37 L).
Sodium hydride (60% dispersion in mineral oil, 5.3 g, 0.13 mol, 3.5 eq.)
was added at 0°C over 30 minutes. After 1 hour, p-methoxybenzyl chloride
(18 mL, 0.13 mol, 3.5 eq.) and tetrabutylammonium iodide (1.4 g, 3.8 mmol,
0.10 eq.) were added. Another portion of sodium hydride (60% dispersion
in mineral oil, 2.3 g, 58 mmol, 1.6 eq.) was added after 1 hour and the
mixture was stirred at room temperature overnight. The reaction was
quenched with MeOH at 0°C, diluted with Et2O and washed H2O (3x). The
organic layer was dried over Na2SO4, filtered and concentrated in vacuo.
Purification by column chromatography (1030% Et2O in pentane) gave
compound 17 (15.5 g, 19.2 mmol, 52%) and compound 18 (8.23 g, 10.3
mmol, 28%). Rf: 0.26 (7/3 pentane/Et2O); [훼]2D5 +35.9° (c = 0.44, CHCl3);
1H NMR (CD3CN, 500 MHz, HH-COSY, HSQC): δ 7.47 – 7.41 (m, 6H, Ar),
7.34 – 7.21 (m, 11H, Ar), 7.09 (dd, 4H, J = 19.9, 8.5 Hz, Ar), 6.90 (d, 2H,
J = 8.6 Hz, Ar), 6.86 – 6.81 (m, 4H, Ar), 5.87 – 5.76 (m, 1H, CH2-CH=CH2),
5.17 – 5.03 (m, 2H, CH2-CH=CH2), 4.52 – 4.42 (m, 5H, 2x CH2 PMB, 1x
CHH PMB), 4.34 (d, 1H, J = 11.2 Hz, CHH PMB), 4.09 – 4.03 (m, 1H, H-
5), 3.91 (dd, 1H, J = 4.3, 3.0 Hz, H-4), 3.82 – 3.74 (m, 11H, H-1, H-3, 3x
CH3 PMB), 3.62 – 3.55 (m, 2H, H-2, CHH-6), 3.17 (dd, 1H, J = 10.6, 3.2
Hz, CHH-6), 2.45 – 2.36 (m, 1H, CHH-CH=CH2), 2.36 – 2.27 (m, 1H, CHH-
CH=CH2); 13C-bbdec NMR (CD3CN, 126 MHz, HSQC): δ 160.7, 160.6,
160.5, 145.7 (Cq Ar), 136.8 (CH2-CH=CH2), 132.3, 132.1, 130.8, 130.5,
130.2, 129.9, 128.9, 128.1 (Ar), 117.2 (CH2-CH=CH2), 115.0, 114.9 (Ar),
87.6 (Cq Trt), 77.2 (C-2), 76.8 (C-3), 75.3 (C-4), 74.4 (C-5), 73.4, 73.0 (CH2
PMB), 71.2 (C-1), 62.5 (CH2-6), 56.2 (CH3 PMB), 33.8 (CH2-CH=CH2); FT-
IR (neat, cm-1): 2934, 2906, 2836, 1612, 1586, 1513, 1491, 1464, 1449,
1355, 1302, 1247, 1173, 1091, 1034, 995, 916, 821, 765, 748, 707, 649,
633, 568, 516; HRMS: [M+Na]+ calcd for C52H54O8Na: 829.3716, found
829.3735.
4-(2,3,4-tri-O-p-methoxybenzyl-6-O-trityl-β-D-galactopyranosyl)-
butanoic acid (20) Allyl 18 (8.0 g, 9.9 mmol, 1.0 eq.) was co-evaporated
with toluene (2x) under an argon atmosphere before being dissolved in dry
DCE (0.10 L). Methyl acrylate (2.6 mL, 29 mmol, 2.9 eq.), CuI (0.29 g, 1.5
mmol, 0.15 eq.) and Grubbs 2nd generation catalyst (0.34 g, 0.40 mmol,
0.04 eq.) were added and the flask was covered in aluminum foil. The
suspension was heated to 50°C for 48 hours, after which it was
concentrated in vacuo and co-evaporated with toluene (2x) under an argon
atmosphere. The obtained residue was dissolved in dry DCE (50 mL) and
cooled to 0°C. Two empty balloons were placed on the flask, followed by
the addition of RuCl3 (0.39 g, 1.9 mmol, 0.19 eq.) and NaBH4 (1.2 g, 32
3-(2,3,4-tri-O-p-methoxybenzyl-6-O-trityl-β-D-galactopyranosyl)-1-
propene (18) See experimental of compound 17. Purification gave
compound 18 (8.23 g, 10.3 mmol, 28%). Rf: 0.33 (7/3 pentane/Et2O); [훼]D25
+9.3° (c = 0.52, CHCl3); 1H NMR (CD3CN, 400 MHz, HH-COSY, HSQC):
δ 7.47 – 7.41 (m, 6H, Ar), 7.38 – 7.20 (m, 13H, Ar), 6.99 – 6.95 (m, 2H,
Ar), 6.92 – 6.85 (m, 4H, Ar), 6.79 – 6.74 (m, 2H, Ar), 5.94 – 5.82 (m, 1H,
CH2-CH=CH2), 5.13 – 4.97 (m, 2H, CH2-CH=CH2), 4.78 (d, 1H, J = 10.4
Hz, CHH PMB), 4.72 (d, 1H, J = 11.3 Hz, CHH PMB), 4.67 (d, 1H, J = 10.7
Hz, CHH PMB), 4.60 (d, 1H, J = 11.3 Hz, CHH PMB), 4.51 (d, 1H, J = 10.5
9
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
mmol, 3.2 eq.). Methanol (8.0 mL, 0.18 mol, 20 eq.) was carefully added
to the suspension over 30 minutes, after which the mixture was allowed to
warm-up up to room temperature over 15 minutes. The mixture was
subsequently heated to 45°C for 5 hours. The reaction mixture was cooled
to room temperature, diluted with brine and extracted with DCM (3x). The
combined organic layers were dried over Na2SO4, filtered and
concentrated in vacuo. NMR analysis showed still 20% alkene present,
therefore the 2nd step was repeated using the same reaction conditions
and heated for 7 hours. Purification by column chromatography (2070%
Et2O in pentane) afforded the intermediate (5.8 g, 6.7 mmol, 68% over two
steps), which was was dissolved in a mixture of THF/H2O (4/1, v/v, 65 mL),
followed by the addition of LiOH (0.85 g, 20 mmol, 3.0 eq.). The mixture
was heated to 40°C for 30 hours. The reaction mixture was cooled to 0°C,
acidified with 3 M HCl to pH = 4/5, diluted with H2O and extracted with
DCM (2x). The combined organic layers were dried over Na2SO4, filtered
and concentrated in vacuo. The title compound was obtained (5.5 g, 6.4
mmol, 96%). Rf: 0.89 (9/1 DCM/MeOH); [훼]2D5 +3.6° (c = 0.53, CHCl3); 1H
NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ 7.44 – 7.39 (m, 6H, Ar), 7.34
– 7.20 (m, 13H, Ar), 7.09 – 7.04 (m, 2H, Ar), 6.93 – 6.82 (m, 4H, Ar), 6.77
– 6.71 (m, 2H, Ar), 4.84 (d, 1H, J = 10.4 Hz, CHH PMB), 4.73 (d, 1H, J =
11.3 Hz, CHH PMB), 4.68 – 4.60 (m, 2H, CH2 PMB), 4.54 (d, 1H, J = 10.5
Hz, CHH PMB), 4.45 (d, 1H, J = 11.3 Hz, CHH PMB), 3.89 (dd, 1H, J =
2.7, 1.0 Hz, H-4), 3.82 (s, 3H, CH3 PMB), 3.80 – 3.76 (m, 6H, 2x CH3 PMB),
3.60 – 3.41 (m, 3H, H-2, H-2, H-3, CHH-6), 3.31 (t, 1H, J = 6.2 Hz, H-5),
3.17 – 3.06 (m, 2H, H-1, CHH-6), 2.34 (t, 2H, J = 7.2 Hz, CH2-9), 1.93 –
1.81 (m, 2H, CHH-8, CHH-7), 1.74 – 1.62 (m, 1H, CHH-8), 1.52 – 1.42 (m,
1H, CHH-7); 13C-APT NMR (CDCl3, 101 MHz, HSQC): δ 179.2 (C=O),
159.4, 159.3, 159.1, 144.1, 131.0, 130.8, 130.7 (Cq Ar), 130.0, 129.8,
129.3, 128.8, 128.0, 127.1, 113.9, 113.6 (Ar), 86.9 (Cq Trt), 84.7 (C-3),
79.4 (C-1), 78.7 (C-2), 77.6 (C-5), 75.2, 73.9 (CH2 PMB), 73.9 (C-4), 72.1
(CH2 PMB), 63.3 (CH2-6), 55.4, 55.4, 55.4 (CH3 PMB), 33.9 (CH2-9), 31.0
(CH2-7), 21.1 (CH2-8); FT-IR (neat, cm-1): 2935, 1707, 1612, 1586, 1513,
1449, 1302, 1247, 1173, 1075, 1033, 821, 748, 706, 633; HRMS: [M+Na]+
calcd. for C53H56O10Na: 875.37657, found 875.37650.
30.1, 28.2, 23.8, 23.4 (CH2-7/8, 3x CH2 β/γ/δ-L-Lys); FT-IR (neat, cm-1):
2935, 1720, 1612, 1513, 1449, 1302, 1248, 1174, 1088, 1034, 822, 761,
743, 707, 633; LC-MS: Rt = 7.68 min (Vydac 219TP 5 µm Diphenyl, 50 -
90% MeCN, 21 min run); ESI-MS (m/z): [M+Na]+ calcd. for C74H78N2O13Na:
1225.5, found 1225.5; HRMS: [M+H]+ calcd. for C74H79O13N2: 1203.55767,
found 1203.55754.
Nα-Fmoc-Nε-[butan-4-(2,3,4-tri-O-p-methoxybenzyl-6-O-trityl-β-D-
galactopyranosyl)-amide]-L-lysine (4) Compound 20 (3.4 g, 4.0 mmol,
1.0 eq.) and Fmoc-L-lysine-OMe (2.0 g, 4.8 mmol, 1.2 eq.) were dissolved
in DMF (20 mL). HCTU (2.0 g, 4.8 mmol, 1.2 eq.) and DIPEA (2.1 mL, 12
mmol, 3.0 eq.) were added and the solution was stirred for 2 hours. The
reaction mixture was diluted with EtOAc and washed with 1 M HCl (1x),
sat. aq. NaHCO3 (1x), brine (1x). The organic layer was dried over Na2SO4,
filtered and concentrated in vacuo. Purification by column chromatography
(3080% EtOAc in pentane) gave the intermediate (4.5 g, 3.7 mmol, 93%)
as an oil, of which 4.0 g (3.3 mmol, 1.0 eq.) was dissolved in THF (37 mL)
and cooled to 0°C. An aqueous solution of LiOH (0.30 M, 22 mL, 6.6 mmol,
2.0 eq.) was added and the suspension was stirred vigorously for 75
minutes, after which the obtained solution was acidified by the addition of
1 M HCl to pH = 5-6 and diluted with brine. The mixture was extracted with
EtOAc (2x) and the organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. After purification by column chromatography
(28% MeOH in DCM), the title compound (1.9 g, 1.6 mmol, 48%) was
obtained as a white foam. Rf: 0.64 (9/1 DCM/MeOH); [훼]2D5 +35.6° (c = 0.41,
CHCl3); 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ 7.74 (d, 2H, J =
7.6 Hz, Ar), 7.60 (t, 2H, J = 7.2 Hz, Ar), 7.44 – 7.33 (m, 8H, Ar), 7.33 – 7.18
(m, 15H, Ar), 7.05 – 6.99 (m, 2H, Ar), 6.93 – 6.87 (m, 2H, Ar), 6.87 – 6.81
(m, 2H, Ar), 6.76 – 6.69 (m, 2H, Ar), 5.78 – 5.67 (m, 2H, NH, NHFmoc),
4.84 (d, 1H, J = 10.5 Hz, CHH PMB), 4.74 (d, 1H, J = 11.2 Hz, CHH PMB),
4.65 (s, 2H, CH2 PMB), 4.55 (d, 1H, J = 10.5 Hz, CHH PMB), 4.44 (d, 1H,
J = 11.3 Hz, CHH PMB), 4.36 (d, 2H, J = 7.2 Hz, CH2 Fmoc), 4.33 – 4.25
(m, 1H, CH L-Lys), 4.20 (t, 1H, J = 7.1 Hz, CH Fmoc), 3.84 (d, 1H, J = 2.8
Hz, H-4), 3.83 – 3.73 (m, 9H, 3x CH3 PMB), 3.63 (t, 1H, J = 9.3 Hz, H-2),
3.50 (dd, 1H, J = 9.3, 2.8 Hz, H-3), 3.43 (dd, 1H, J = 9.6, 6.4 Hz, CHH-6),
3.32 (t, 1H, J = 6.2 Hz, H-5), 3.19 – 3.10 (m, 2H, H-1, CHH ε-L-Lys), 3.10
– 2.95 (m, 2H, CHH-6, CHH ε-L-Lys), 2.30 – 2.11 (m, 2H, CH2-9), 1.92 –
1.37 (m, 6H, CH2-7, CH2-8, 1x CH2 β/γ/δ-L-Lys), 1.36 – 1.09 (m, 4H, 2x
CH2 β/γ/δ-L-Lys); 13C-APT NMR (CDCl3, 101 MHz, HSQC): δ 174.0, 159.3
(C=O), 159.3, 159.2, 156.1, 144.1, 143.9, 141.4, 130.6, 130.4 (Cq Ar),
130.0, 129.3, 128.7, 128.0, 127.8, 127.2, 127.2, 125.3, 120.0, 113.9, 113.9,
113.6 (Ar), 87.0 (Cq Trt), 84.5 (C-3), 80.2 (C-1), 78.5 (C-2), 77.7 (C-5), 75.2
(CH2 PMB), 74.0 (C-4), 73.9, 72.2 (CH2 PMB), 67.0 (CH2 Fmoc), 63.6
(CH2-6), 55.4, 55.3 (CH3 PMB), 53.6 (CH L-Lys), 47.2 (CH Fmoc), 38.9
(CH2 ε-L-Lys), 36.4 (CH2-9), 31.7 (CH2 β/γ/δ-L-Lys), 30.3 (CH2-7), 28.8,
22.9 (CH2 β/γ/δ-L-Lys), 21.8 (CH2-8); FT-IR (neat, cm-1): 2935, 1717, 1612,
1586, 1512, 1449, 1302, 1246, 1173, 1153, 1074, 1032, 900, 821, 760,
735, 704, 651, 633, 621, 541, 516; LC-MS: Rt = 7.96 min (Vydac 219TP 5
µm Diphenyl, 50 - 90% MeCN, 21 min run); ESI-MS (m/z): [M+Na]+ calcd.
for C74H78N2O13Na: 1225.5, found 1225.6; HRMS: [M+H]+ calcd. for
C74H79O13N2: 1203.55767, found 1203.55765.
Nα-Fmoc-Nε-[butan-4-(2,3,4-tri-O-p-methoxybenzyl-6-O-trityl-α-D-
galactopyranosyl)-amide]-L-lysine (3) Compound 19 (4.3 g, 5.0 mmol,
1.0 eq.) and Fmoc-L-lysine-OMe (2.5 g, 6.0 mmol, 1.2 eq.) were dissolved
in DMF (25 mL). HCTU (2.5 g, 6.0 mmol, 1.2 eq.) and DIPEA (2.6 mL, 15
mmol, 3.0 eq.) were added and the solution was stirred for 2 hours. The
reaction mixture was diluted with EtOAc and washed with 1 M HCl (1x),
sat. aq. NaHCO3 (1x), brine (1x). The organic layer was dried over Na2SO4,
filtered and concentrated in vacuo. Purification by column chromatography
(4080% EtOAc in pentane) gave the intermediate (5.5 g, 4.5 mmol, 90%)
as an oil, of which 4.8 g (4.0 mmol, 1.0 eq.) was dissolved in THF (55 mL)
and cooled to 0°C. An aqueous solution of LiOH (0.30 M, 27 mL, 8.1 mmol,
2.0 eq.) was added and the suspension was stirred vigorously for 1 hour,
after which the obtained solution was acidified by the addition of 1 M HCl
to pH = 5-6 and diluted with brine. The mixture was extracted with EtOAc
(1x) and the organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. After purification by column chromatography
(26% MeOH in DCM), the title compound (2.2 g, 1.8 mmol, 46%) was
obtained as a white foam. Rf: 0.70 (9/1 DCM/MeOH); [훼]2D5 +15.8° (c = 1.1,
CHCl3); 1H NMR (CD3CN, 500 MHz, HH-COSY, HSQC): δ 7.80 (d, 2H, J
= 7.4 Hz, Ar), 7.64 (d, 2H, J = 7.1 Hz, Ar), 7.46 – 7.36 (m, 8H, Ar), 7.34 –
7.17 (m, 13H, Ar), 7.10 (d, 2H, J = 8.3 Hz, Ar), 7.04 (d, 2H, J = 8.4 Hz, Ar),
6.84 (dd, 6H, J = 23.1, 8.0 Hz, Ar), 6.23 (br, 1H, NH), 5.92 (br, 1H,
NHFmoc), 4.51 – 4.38 (m, 5H, 2x CH2 PMB, 1x CHH PMB), 4.38 – 4.29
(m, 3H, CHH PMB, CH2 Fmoc), 4.21 (t, 1H, J = 6.7 Hz, CH Fmoc), 4.11
(br, 1H, CH L-Lys), 3.97 – 3.90 (m, 1H, H-5), 3.88 – 3.82 (m, 1H, H-4), 3.82
– 3.73 (m, 9H, 3x CH3 PMB), 3.73 – 3.65 (m, 2H, H-1, H-3), 3.60 – 3.48
(m, 2H, H-2, CHH-6), 3.23 – 3.07 (m, 3H, CHH-6, CH2 ε-L-Lys), 2.17 – 2.10
(m, 2H, CH2-9), 1.84 – 1.27 (m, 10H, 2x CH2-7/8, 3x CH2 β/γ/δ-L-Lys); 13C-
bbdec NMR (CD3CN, 126 MHz, HSQC): δ 174.1, 160.7 (C=O), 160.5,
160.5, 145.6, 145.4, 145.4, 142.4, 132.4, 132.1 (Cq Ar), 130.8, 130.5,
130.3, 129.9, 129.0, 128.9, 128.3, 128.2, 126.4, 121.1, 115.0, 114.9 (Ar),
87.7 (Cq Trt), 77.5, 77.3 (C-2, C-3), 75.6 (C-4), 74.0 (C-5), 73.4, 73.3, 73.2
(CH2 PMB), 71.7 (C-1), 67.6 (CH2 Fmoc), 63.0 (CH2-6), 56.2 (CH3 PMB),
55.2 (CH L-Lys), 48.4 (CH Fmoc), 39.7 (CH2 ε-L-Lys), 37.1 (CH2-9), 32.3,
1,3,4,6-tetra-O-acetyl-2-deoxy-2-tetrachlorophthalimido-α-D-
glucopyranoside (21) Glucosamine hydrochloride (21.6 g, 100 mmol, 1.0
eq.) was added to a solution of sodium methoxide (1.0 M in MeOH, 0.10
L, 1.0 eq.) at room temperature and the obtained solution was stirred for
10 minutes, followed by the addition of tetrachlorophthalic anhydride (14.3
g, 50.0 mmol, 0.5 eq.). After 20 minutes, additional tetrachlorophthalic
anhydride (14.3 g, 50.0 mmol, 0.5 eq.) and Et3N (10 mL, 0.10 mol, 1.0 eq.)
were added and the reaction was stirred at 50°C for 20 minutes. The
mixture was concentrated in vacuo. The residue was dissolved in pyridine
(98 mL), followed by slow addition of Ac2O (0.15 L, 1.6 mol, 16.0 eq.). The
resulting mixture was stirred for 16 hours at room temperature, after which
it was poured into ice water (0.15 L) and extracted with DCM (3x). The
combined organic layers were subsequently washed with a 1 M HCl (2x),
sat. aq. NaHCO3 (2x) and brine (1x). The organic layer was dried over
MgSO4, filtered, concentrated in vacuo and co-evaporated with toluene
(1x). Recrystallization in MeOH yielded the title compound (31.4 g, 51.0
mmol, 51%) as a white solid. Rf: 0.6 (3/2 pentane/EtOAc); [훼]2퐷0 = +96.6°
10
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
(c = 1.0, DCM); 1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ 6.48 (dd,
1H, J = 11.5, 9.1 Hz, H-3), 6.24 (d, 1H, J = 3.4 Hz, H-1), 5.15 (t, 1H, J =
10.1, 9.0 Hz, H-4), 4.70 (dd, 1H, J = 11.5, 3.4 Hz, H-2), 4.38 – 4.27 (m, 2H,
H-5, CHH-6), 4.13 (dd, 1H, J = 12.2, 1.8 Hz, CHH-6), 2.11 (s, 3H, CH3 Ac),
2.08 (s, 3H, CH3 Ac), 2.05 (s, 3H, CH3 Ac), 1.90 (s, 3H, CH3 Ac); 13C-APT
NMR (CDCl3, 101 MHz, HSQC): δ 170.8, 169.9, 169.8, 169.6 (C=O), 140.9
(Cq Ar), 130.3, 126.8 (C-Cl), 90.6 (C-1), 70.4 (C-5), 69.3 (C-4), 67.0 (C-3),
61.5 (CH2-6), 53.5 (C-2), 21.1, 20.9, 20.8, 20.8 (CH3 Ac); FT-IR (neat, cm-
1): 2965, 1750, 1731, 1385, 1370, 1219, 1154, 1081, 1040, 1015, 922, 794,
752, 740, 603, 540, 485; HRMS: [M+Na]+ calcd. for C22H19Cl4NO11Na
635.9610, found 635.9617.
mixture was heated to 60°C. After stirring overnight, the mixture was
cooled to 0°C and quenched with Et3N to pH = 7. The solution was diluted
with EtOAc and the organic layer washed with H2O (3x), dried over MgSO4,
filtered and concentrated in vacuo. Purification by column chromatography
(1040% Et2O in pentane) gave compound 24 (11.0 g, 19.7 mmol, 87%)
as a white solid. Rf: 0.8 (1/1 pentane/Et2O); [훼]2퐷0 = +17.5° (c = 1.0, DCM);
1H NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ 7.39 – 7.27 (m, 5H, Ar),
5.78 – 5.64 (m, 1H, CH2-CH=CH2), 5.49 (s, 1H, CH benzylidene), 4.94 (t,
2H, J = 9.4 Hz, CH2-CH=CH2), 4.60 (t, 1H, J = 10.2, 9.0 Hz, H-3), 4.33 (dd,
1H, J = 10.2, 4.7 Hz, CHH-6), 4.30 – 4.22 (m, 1H, H-1), 4.05 (t, 1H, J =
10.2 Hz, H-2), 3.70 (t, 1H, J = 10.1 Hz, CHH-6), 3.64 – 3.55 (m, 1H, H-5),
3.48 (t, 1H, J = 9.1 Hz, H-4), 3.19 (s, 1H, OH), 2.30 – 2.17 (m, 2H, CH2-
CH=CH2); 13C-APT NMR (CDCl3, 101 MHz, HSQC): δ 163.7, 163.2 (C=O),
140.4, 140.3 (C-Cl), 136.9 (Cq Ar), 133.0 (CH2-CH=CH2), 130.1, 129.8 (C-
Cl), 129.3, 128.3 (Ar), 127.1, 127.1 (C-Cl), 126.0 (Ar), 117.7 (CH2-
CH=CH2), 101.7 (CH benzylidene), 82.5 (C-4), 75.0 (C-1), 70.1 (C-5), 68.8
(CH2-6), 68.6 (C-3), 57.1 (C-2), 37.0 (CH2-CH=CH2); FT-IR (neat, cm-1):
3485, 2864, 1779, 1720, 1371, 1351, 1300, 1203, 1124, 1096, 988, 918,
790, 753, 740, 699, 643; HRMS: [M+H]+ calcd. for C24H20Cl4NO6 558.0039,
found 558.0047.
3-C-(3,4,6-tri-O-acetyl-2-deoxy-2-tetrachlorophthalimido-β-D-
glucopyranosyl)-1-propene (22) Compound 21 (24.6 g, 40.0 mmol, 1.0
eq.) was co-evaporated with toluene (3x) under an argon atmosphere. The
residue was dissolved in acetonitrile (0.24 L) and cooled to 0°C.
Allyltrimethylsilane (32 mL, 0.20 mol, 5.0 eq.) was added, followed by slow
addition of TMSOTf (7.2 mL, 40 mmol, 1.0 eq.) and BF3·OEt2 (25 mL, 0.20
mol, 5.0 eq.). The yellow suspension was sonicated for 90 minutes and
stirred for an additional hour at room temperature. The resulting brown
solution was cooled to 0°C and quenched with Et3N to pH = 7. The reaction
was diluted with EtOAc, washed with sat. aq. NaHCO3 (1x) and brine (1x).
The organic layer was dried over Na2SO4, filtered and concentrated in
vacuo. Purification by column chromatography (1050% Et2O in pentane)
yielded the title compound (13.9 g, 23.2 mmol, 58%) as a white foam. Rf:
0.5 (1/1 pentane/Et2O); [훼]2퐷0 = +74.4° (c = 1.0, DCM); 1H NMR (CDCl3,
400 MHz, HH-COSY, HSQC): δ 5.78 – 5.65 (m, 2H, H-3, CH2-CH=CH2),
5.12 (t, 1H, J = 10.2 Hz, H-4), 5.00 – 4.87 (m, 2H, CH2-CH=CH2), 4.47 –
4.34 (m, 1H, H-1), 4.27 (dd, 1H, J = 12.3, 4.9 Hz, CHH-6), 4.21 (t, 1H, J =
10.2 Hz, H-2), 4.10 (dd, 1H, J = 12.3, 2.3 Hz, CHH-6), 3.79 – 3.73 (m, 1H,
H-5), 2.26 (t, 2H, J = 6.8 Hz, CH2-CH=CH2), 2.09 (s, 3H, CH3 Ac), 2.01 (s,
3H, CH3 Ac), 1.86 (s, 3H, CH3 Ac); 13C-APT NMR (CDCl3, 101 MHz,
HSQC): δ 170.9, 170.8, 169.6, 163.5, 162.8 (C=O), 140.9, 140.6 (Cq Ar),
132.5 (CH2-CH=CH2), 130.2, 130.0, 127.1, 126.8 (C-Cl), 118.1 (CH2-
CH=CH2), 75.8 (C-5), 74.0 (C-1), 71.9 (C-3), 69.0 (C-4), 62.4 (CH2-6), 55.3
(C-2), 36.8 (CH2-CH=CH2), 20.9, 20.7, 20.6 (CH3 Ac); FT-IR (neat, cm-1):
2957, 1782 1746, 1724, 1384, 1370, 1352, 1226, 1150, 1047, 908, 791,
753, 740, 603; HRMS: [M+H]+ calcd. for C23H22Cl4NO9 596.0043, found
596.0045.
3-C-(2-deoxy-2-N-acetyl-4,6-O-di-benzylidene-β-D-glucopyranosyl)-1-
propene (25) To a solution of compound 24 (3.8 g, 6.8 mmol, 1.0 eq.) in
EtOH (70 mL) was added ethylenediamine (23 mL, 0.34 mol, 50 eq.) and
the reaction was heated to 90°C. After 16 hours, the reaction mixture was
diluted with toluene and concentrated in vacuo. The residue was co-
evaporated with toluene (3x) and embedded on silica gel. Purification by
column chromatography (25% MeOH in DCM) gave 3-C-(4,6-di-O-
benzylidene-2-deoxy-2-amine-β-D-glucopyranosyl)-1-propene (1.92 g,
6.59 mmol) as a yellow solid. Rf: 0.42 (1/9 MeOH/DCM). The obtained
amine (1.92 g, 6.59 mmol, 1.0 eq.) was dissolved in a mixture of THF/H2O
(1/1 v/v, 50 mL). Sodium bicarbonate (5.6 g, 66 mmol, 10 eq.) and Ac2O
(3.1 mL, 33 mmol, 5.0 eq.) were added. The mixture was stirred at room
temperature for 4 days, after which the reaction mixture was diluted with
EtOAc. The obtained suspension was filtered and the obtained pure title
compound was collected as a white solid. The filtrate was washed with sat.
aq. NaHCO3 (1x) and brine (1x). The organic layer was dried over MgSO4,
filtered and concentrated in vacuo. The remaining crude product was
crystallized using DCM/MeOH/pentane giving compound 25. The
remaining residue was embedded on silica and purified by column
chromatography (26% MeOH in DCM). The combined title compound
(1.87 g, 5.63 mmol, 83% over two steps) was collected as a white solid.
Rf: 0.5 (1/9 MeOH/DCM); [훼]퐷20 = -38.5° (c = 0.3, MeOH); 1H NMR (MeOD,
400 MHz, HH-COSY, HSQC): δ 7.53 – 7.46 (m, 2H, Ar), 7.38 – 7.30 (m,
3H, Ar), 5.93 – 5.80 (m, 1H, CH2-CH=CH2), 5.59 (s, 1H, CH benzylidene),
5.03 (t, 2H, J = 17.5, 8.7 Hz, CH2-CH=CH2), 4.25 (dd, 1H, J = 10.3, 5.0 Hz,
CHH-6), 3.75 (q, 2H, J = 11.5, 10.0 Hz, H-2, CHH-6), 3.66 (t, 1H, J = 9.7,
8.9 Hz, H-3), 3.50 (t, 1H, J = 9.1 Hz, H-4), 3.47 – 3.34 (m, 2H, H-1, H-5),
2.41 – 2.29 (m, 1H, CHH-CH=CH2), 2.26 – 2.13 (m, 1H, CHH-CH=CH2),
1.99 (s, 3H, CH3 Ac); 13C-APT NMR (MeOD, 101 MHz, HSQC): δ 173.7
(C=O), 139.2 (Cq Ar), 135.7 (CH2-CH=CH2), 129.9, 129.0, 127.5 (Ar),
117.2 (CH2-CH=CH2), 102.9 (CH benzylidene), 83.2 (C-4), 80.4 (C-1), 73.9
(C-3), 71.8 (C-5), 69.8 (CH2-6), 57.2 (C-2), 37.7 (CH2-CH=CH2), 22.9 (CH3
Ac); FT-IR (neat, cm-1): 3380, 2361, 1630, 1377, 1125, 1033, 999, 698;
HRMS: [M+H]+ calcd. for C18H24NO5 334.1655, found 334.1654.
3-C-(2-deoxy-2-tetrachlorophthalimido-β-D-glucopyranosyl)-1-
propene (23) Acetyl chloride (1.6 mL, 23 mmol, 0.8 eq.) was added to a
solution of compound 22 (17.1 g, 28.7 mmol, 1.0 eq.) in a mixture of
DCM/MeOH (1:4 v/v, 0.29 L) at 0°C. After stirring for 1 hour, the reaction
mixture was allowed to warm-up to room temperature and stirred for 72
hours. The mixture was diluted with toluene (30 mL) and concentrated in
vacuo. The residue was co-evaporated with toluene (2x) and purified by
column chromatography (110% MeOH in DCM) to obtain the title
compound (12.7 g, 26.9 mmol, 94%) as a white solid. Rf: 0.5 (1/9
MeOH/DCM); [훼]2퐷0 = +34.7° (c = 1.0, DCM); H NMR (CDCl3, 400 MHz,
1
HH-COSY, HSQC): δ 5.76 – 5.63 (m, 1H, CH2-CH=CH2), 4.88 (t, 2H, CH2-
CH=CH2), 4.24 (t, 1H, J = 10.5, 8.8 Hz, H-3), 4.21 – 4.13 (m, 1H, H-1),
3.91 (t, 1H, J = 10.3, 10.3 Hz, H-2), 3.86 – 3.77 (m, 2H, CH2-6), 3.56 (t,
1H, J = 9.2, 9.2 Hz, H-4), 3.52 – 3.43 (m, 3H, OH), 3.40 (dt, 1H, J = 9.6,
3.2, 3.2 Hz, H-5), 2.27 – 2.10 (m, 2H, CH2-CH=CH2); 13C-APT NMR (CDCl3,
101 MHz, HSQC): δ 163.9, 163.8 (C=O), 140.4, 140.4 (Cq Ar), 133.4 (CH2-
CH=CH2), 130.1, 129.7, 127.3, 127.3 (C-Cl), 117.5 (CH2-CH=CH2), 79.2
(C-5), 74.2 (C-1), 71.8 (C-3), 71.4 (C-4), 62.0 (CH2-6), 57.5 (C-2), 37.2
(CH2-CH=CH2); FT-IR (neat, cm-1): 3378, 2929, 1779, 1718, 1387, 1370,
1351, 1299, 1202, 1142, 1085, 1000, 919, 791, 753, 740, 643; HRMS:
[M+Na]+ calcd. for C17H15Cl4NO6Na 491.9551, found 491.9551.
3-(2-deoxy-2-N-acetyl-4,6-O-di-benzylidene-3-O-p-methoxybenzyl-β-
D-glucopyranosyl)-1-propene (26) Alcohol 25 (1.6 g, 4.9 mmol, 1.0 eq.)
was co-evaporated with toluene (1x) under an argon atmosphere, followed
by the addition of dry THF (0.12 L) and p-methoxybenzyl-2,2,2-
trichloroacetimidate (3.1 mL, 15 mmol, 3.0 eq.). The suspension was
cooled to 0°C and a solution of TfOH in THF (0.01 M, 50 mL, 0.50 mmol,
0.10 eq.) was added. After 15 minutes, the mixture was allowed to warm
up to room temperature and stirred for 3 hours. The obtained solution was
neutralized with Et3N and diluted with EtOAc and washed with sat. aq.
NaHCO3 (1x) and brine (1x). The organic layer was dried over Na2SO4,
filtered and concentrated in vacuo. The residue was embedded on silica
and purified by column chromatography (220% acetone in DCM). The
title compound was obtained (1.7 g, 3.8 mmol, 78%) as a white solid. Rf:
3-C-(4,6-di-O-benzylidene-2-deoxy-2-tetrachlorophthalimido-β-D-
glucopyranosyl)-1-propene (24) Compound 23 (10.6 g, 22.5 mmol, 1.0
eq.) was co-evaporated with toluene (3x) under an argon atmosphere. The
residue was dissolved in a mixture of DMF/acetonitrile (9:1 v/v, 113 mL).
Benzylaldehyde dimethyl acetal (6.9 mL, 45 mmol, 2.0 eq.) and p-
toluenesulfonic acid (0.43 g, 2.3 mmol, 0.1 eq.) were added and the
11
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
0.45 (9/1 DCM/acetone); [훼]2D5 -58.4° (c = 0.32, CHCl3/MeOH 1/1); 1H
NMR (CDCl3, 400 MHz, HH-COSY, HSQC): δ 7.53 – 7.48 (m, 2H, Ar), 7.42
– 7.35 (m, 3H, Ar), 7.25 – 7.21 (m, 2H, Ar), 6.89 – 6.84 (m, 2H, Ar), 5.88 –
5.75 (m, 1H, CH2-CH=CH2), 5.58 (s, 1H, CH benzylidene), 5.15 – 5.00 (m,
3H, NH, CH2-CH=CH2), 4.82 (d, 1H, J = 11.8 Hz, CHH PMB), 4.61 (d, 1H,
J = 11.7 Hz, CHH PMB), 4.32 (dd, 1H, J = 10.4, 5.0 Hz, CHH-6), 3.80 (s,
3H, CH3 PMB), 3.77 – 3.64 (m, 4H, H-2, H-3, H-4, CHH-6), 3.51 – 3.44 (m,
1H, H-1), 3.44 – 3.37 (m, 1H, H-5), 2.39 – 2.31 (m, 1H, CHH-CH=CH2),
2.28 – 2.18 (m, 1H, CHH-CH=CH2), 1.90 (s, 3H, CH3 Ac); 13C-APT NMR
(CDCl3, 101 MHz, HSQC): δ 170.3 (C=O), 159.5, 137.6 (Cq Ar), 134.4
(CH2-CH=CH2), 130.6 (Cq Ar), 130.2, 129.1, 128.4, 126.1 (Ar), 117.2 (CH2-
CH=CH2), 113.9 (Ar), 101.2 (CH benzylidene), 83.0 (C-4), 79.2 (C-1), 77.8
(C-3), 73.5 (CH2 PMB), 70.5 (C-5), 69.0 (CH2-6), 55.4 (CH3 PMB), 55.1 (C-
2), 36.6 (CH2-CH=CH2), 23.7 (CH3 Ac); FT-IR (neat, cm-1): 3277, 2871,
1651, 1615, 1555, 1514, 1454, 1371, 1302, 1249, 1172, 1132, 1102, 1034,
1015, 962, 919, 821, 749, 697, 592, 516; HRMS: [M+H]+ calcd. for
C26H32O6N: 454.22241, found 454.22225.
MeOH/DCM/Et2O to obtain the intermediate (1.3 g, 1.5 mmol, 78%), of
which 0.92 g (1.1 mmol, 1.0 eq.) was suspended in THF (21 mL). An
aqueous solution of LiOH (0.30 M, 7.1 mL, 2.1 mmol, 2.0 eq.) was added
at room temperature and stirring was continued for 50 minutes, before the
obtained clear solution was neutralized with 1 M HCl (2.2 mL, 2.2 mmol,
2.0 eq.). NaHCO3 (0.36 g, 4.3 mmol, 4.0 eq.) and Fmoc N-
hydroxysuccinimide ester (0.72 g, 2.1 mmol, 2.0 eq.) were added and the
mixture was stirred vigorously for 2 hours. Upon completion of the reaction
determined by LC-MS, Et2O was added at 0°C to precipitate the crude
product. After filtration, the precipitate was purified by recrystallization
(MeOH/DCM/Et2O) to yield the title compound (0.88 g, 1.0 mmol, 91 %) as
a white solid. Rf: 0.40 (85/15 DCM/MeOH); [훼]2D5 +15.5° (c = 1.0,
CHCl3/MeOH 1/1); 1H NMR (MeOD/CDCl2: 1/1 v/v, 400 MHz, HH-COSY,
HSQC): δ 7.80 – 7.74 (m, 2H, Ar), 7.68 – 7.60 (m, 2H, Ar), 7.51 – 7.44 (m,
2H, Ar), 7.43 – 7.32 (m, 5H, Ar), 7.35 – 7.26 (m, 2H, Ar), 7.23 – 7.15 (m,
2H, Ar), 6.86 – 6.78 (m, 2H, Ar), 5.58 (s, 1H, CH benzylidene), 4.75 (d, 1H,
J = 11.3 Hz, CHH PMB), 4.59 – 4.54 (m, 1H, CHH PMB), 4.37 – 4.28 (m,
2H, CH2 Fmoc), 4.31 – 4.18 (m, 2H, CHH-6, CH Fmoc), 4.02 (dd, 1H, J =
7.0, 5.0 Hz, CH L-Lys), 3.81 – 3.72 (m, 4H, H-2, CH3 PMB), 3.74 – 3.59
(m, 3H, H-3, H-4, CHH-6), 3.44 – 3.32 (m, 2H, H-1, H-5), 3.20 – 3.08 (m,
2H, CH2 ε-L-Lys), 2.13 (t, 2H, J = 7.6 Hz, CH2-9), 1.91 (s, 3H, CH3 Ac),
1.86 – 1.29 (m, 10H, CH2-7, CH2-8, 3x CH2 β/γ/δ-L-Lys); 13C-APT NMR
(MeOD, 101 MHz, HSQC): δ 178.6, 175.2, 172.6 (C=O), 160.0, 157.3,
144.9, 144.7, 142.0, 138.5, 131.5 (Cq Ar), 130.2, 129.5, 128.8, 128.3,
127.7, 126.8, 125.8, 120.5, 114.2 (Ar), 101.9 (CH benzylidene), 83.3 (C-
4), 80.4 (C-3), 79.8 (C-1), 74.6 (CH2 PMB), 71.1 (C-5), 69.5 (CH2-6), 67.3
(CH2 Fmoc), 57.0 (CH L-Lys), 55.6 (CH3 PMB), 55.4 (C-2), 48.0 (CH Fmoc),
39.8 (CH2 ε-L-Lys), 36.6 (CH2-9), 33.4, 32.0, 29.5, 25.7, 23.4 (CH2-7/8, 3x
CH2 β/γ/δ-L-Lys), 23.0 (CH3 Ac), 22.6 (CH2-7/8, 3x CH2 β/γ/δ-L-Lys); FT-
IR (neat, cm-1): 3286, 1638, 1547, 1513, 1451, 1370, 1249, 1176, 1135,
1088, 1030, 820, 737, 696, 621, 541; LC-MS: Rt = 7.75 min (C18 Gemini,
10 - 90% MeCN, 11 min run); ESI-MS (m/z): [M+Na]+ calcd. for
C48H55N3O11Na: 872.4, found 872.4 [M+Na]+; HRMS: [M+H]+ calcd. for
C48H56O11N3: 850.39094, found 850.39103.
4-(2-deoxy-2-N-acetyl-4,6-O-di-benzylidene-3-O-p-methoxybenzyl-β-
D-glucopyranosyl)-butanoic acid (27) Compound 26 (1.4 g, 3.1 mmol,
1.0 eq.) was co-evaporated with toluene (2x) under an argon atmosphere
and dissolved in dry DCE (31 mL). Methyl acrylate (0.78 mL, 8.6 mmol, 2.8
eq.), CuI (90 mg, 0.47 mmol, 0.15 eq.) and Grubbs 2nd generation catalyst
(0.26 g, 0.31 mmol, 0.10 eq.) were added and the flask was covered in
aluminum foil. The suspension was heated to 50°C overnight, after which
it was concentrated in vacuo and co-evaporated with toluene (3x) under
an argon atmosphere. The residue was dissolved in dry DCE (31 mL) and
cooled to 0°C. RuCl3 (0.12 g, 0.58 mmol, 0.19 eq.) and NaBH4 (0.37 g, 9.8
mmol, 3.2 eq.) were added, and an empty balloon was placed on the flask.
Methanol (2.5 mL, 61 mmol, 20 eq.) was carefully added to the suspension
over 20 minutes, after which the mixture was allowed to warm-up up to
room temperature over 25 minutes. The mixture was subsequently heated
to 45°C for 7 hours. The reaction mixture was cooled to room temperature,
diluted with brine and extracted with DCM (3x). The combined organic
layers were dried over Na2SO4, filtered and concentrated in vacuo.
Purification by column chromatography (815% acetone in DCM) gave
the intermediate (1.1 g, 2.1 mmol, 68% over two steps), of which 1.0 g (1.0
g, 2.0 mmol, 1.0 eq.) was suspended in THF/H2O/MeOH (7/1/2, v/v/v, 20
mL). LiOH (0.26 g, 6.2 mmol, 3.1 eq.) was added and the mixture was
heated to 40°C for 3 hours, after which TLC analysis showed complete
conversion of the starting material. The reaction was diluted with EtOAc
and acidified with 1 M HCl to pH = 6-7, followed by the extraction of the
aqueous layer with EtOAc (1x) and DCM (1x). The combined organic
layers were dried over Na2SO4, filtered and concentrated in vacuo. The
title compound was obtained (1.0 g, 2.0 mmol, quant.). Rf: 0.45 (9/1
DCM/MeOH); [훼]2D5 -54.3° (c = 0.28, CHCl3/MeOH 1/1); 1H NMR (DMSO,
400 MHz, HH-COSY, HSQC): δ 11.99 (s, 1H, OH), 7.89 (d, 1H, J = 9.0 Hz,
NH), 7.47 – 7.34 (m, 5H, Ar), 7.18 (d, 2H, J = 8.7 Hz, Ar), 6.85 (d, 2H, J =
8.7 Hz, Ar), 5.68 (s, 1H, CH benzylidene), 4.64 (d, 1H, J = 11.3 Hz, CHH
PMB), 4.51 (d, 1H, J = 11.4 Hz, CHH PMB), 4.21 (dd, 1H, J = 10.1, 4.9 Hz,
CHH-6), 3.77 – 3.53 (m, 7H, H-2, H-3, H-4, CHH-6, CH3 PMB), 3.40 – 3.26
(m, 2H, H-1, H-5), 2.18 (t, 2H, J = 7.2 Hz, CH2-9), 1.83 (s, 3H, CH3 Ac),
1.73 – 1.62 (m, 1H, CHH-8), 1.58 – 1.41 (m, 2H, CHH-7, CHH-8), 1.34 –
1.22 (m, 1H, CHH-7); 13C-APT NMR (DMSO, 101 MHz, HSQC): δ 174.4
(C=O), 169.2, 158.6, 137.8, 130.9 (Cq Ar), 129.0, 128.7, 128.1, 126.0,
113.4 (Ar), 100.0 (CH benzylidene), 81.4 (C-4), 79.7 (C-1), 79.0 (C-3), 72.9
(CH2 PMB), 70.0 (C-5), 68.0 (CH2-6), 55.0 (CH3 PMB), 53.9 (C-2), 33.5
(CH2-9), 30.8 (CH2-7), 22.9 (CH3 Ac), 20.8 (CH2-8); FT-IR (neat, cm-1):
2870, 2428, 1725, 1597, 1516, 1489, 1366, 1302, 1274, 1251, 1223, 1172,
1136, 1109, 1088, 1041, 1020, 966, 923, 822, 747, 694, 606, 539; HRMS:
[M+H]+ calcd. for C27H34O8N1: 500.22789, found 500.22784.
Acetyl-Lys(Nε-[butan-4-(α-D-mannosyl)-amide])-Lys(Nε-[butan-4-(β-D-
galactosyl)-amide])-Lys(Nε-[butan-4-(α-D-galactosyl)-amide])-
Lys(N3)-Lys-NH2 (29) Tentagel S Ram resin on 100 µmol scale was
treated with 20% piperidine in DMF for 10 min and subsequently elongated
with Fmoc-Lys(Boc)-OH, Fmoc-Lys(N3)-OH, 3,
4 and 2 using 2.0
equivalents of each amino acid and 2 hours coupling time at 50°C. After
deprotection of the Fmoc, 50 µmol of the resin was capped by treatment
with a mixture of 20% Ac2O in 0.1 M DIPEA in DMF. The resin was washed
with DCM and treated with the standard cleavage cocktail (TFA/TIS/H2O,
95/2.5/2.5 v/v/v, 2.0 mL) for 3 hours. The suspension was filtered and the
product was precipitated with Et2O. After purification by RP-HPLC and
lyophilisation, conjugate 29 (3.4 mg, 2.4 µmol, 5%) was obtained as a
white solid. LC-MS: Rt = 6.88 min (C18 Gemini, 5 - 20% MeCN, 15 min
run); ESI-MS (m/z): [M+H]+ calcd. for C62H112N13O24: 1422.8, found
1422.8; HRMS: [M+2H]2+ calcd. for C62H113N13O24: 711.90052, found
711.89978.
Acetyl-Lys(Nε-[butan-4-(2-deoxy-2-N-acetyl-β-D-glucosyl)-amide])-
Lys(Nε-[butan-4-(α-D-mannosyl)-amide])-Lys(Nε-[butan-4-(β-D-
galactosyl)-amide])-Lys(Nε-[butan-4-(α-D-galactosyl)-amide])-
Lys(N3)-Lys-NH2 (30) The previously obtained 50 µmol pentamer on resin
(see 29) was treated with a mixture of 5 (2.0 eq.), HCTU (2.0 eq.), DIPEA
(4.0 eq.) in DMSO (2.0 mL) at 50 °C for 2 hours. LC-MS analysis showed
the presence of pentamer. Therefore the resin was treated with the same
mixture at room temperature overnight. After deprotection of the Fmoc, the
resin was capped by treatment with a mixture of 20% Ac2O in 0.1 M DIPEA
in DMF. The resin was washed with DCM and treated with the standard
cleavage cocktail (TFA/TIS/H2O, 95/2.5/2.5 v/v/v, 2.0 mL) for 3urs. The
suspension was filtered and the product was precipitated with Et2O. After
purification by RP-HPLC and lyophilisation, conjugate 30 (1.8 mg, 1.0 µmol,
2%) was obtained as a white solid. LC-MS: Rt = 7.33 min (C18 Gemini, 5
Nα-Fmoc-Nε-[butan-4-(2-deoxy-2-N-acetyl-4,6-O-di-benzylidene-3-O-
p-methoxybenzyl-β-D-glucopyranosyl)-amide]-L-lysine (5) Acid 27
(0.97 g, 1.9 mmol, 1.0 eq.) and Fmoc-L-lysine-OMe (0.98 g, 2.3 mmol, 1.2
eq.) were dissolved in DMF (20 mL), followed by the addition of HCTU
(0.96 g, 2.3 mmol, 1.2 eq.) and DIPEA (1.0 mL, 5.7 mmol, 3.0 eq.). The
reaction was stirred at room temperature for 4 hours, after which Et2O was
added. The precipitate was filtered and recrystallized with
- 20% MeCN, 15 min run); ESI-MS (m/z): [M+H]+ calcd. for C80H143N16O31
1824.0, found 1824.0; HRMS: [M+2H]2+ calcd. for C80H144N16O31
912.50862, found 912.50813.
:
:
12
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
Lys(Nε-[butan-4-(α-L-rhamnosyl)-amide])-Lys(Nε-[butan-4-(α-L-
rhamnosyl)-amide])-Lys(Nε-[butan-4-(α-L-rhamnosyl)-amide])-Lys(Nε-
[butan-4-(α-L-rhamnosyl)-amide])-Lys(Nε-[butan-4-(α-L-rhamnosyl)-
amide])-Lys(Nε-[butan-4-(α-L-rhamnosyl)-amide])-Leu-Glu-Gln-Leu-
Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-Ala-Ala-Ala-Ala-Ala-Lys-OH (32)
Tentagel S Ac resin loaded with LEQLESIINFEKLAAAAAK on 10 μmol
scale was elongated six times with 1 (3.0 eq., 2 hours coupling time). After
cleaving from the resin, the peptide was purified by RP-HPLC. After
lyophilisation, conjugate 32 (2.6 mg, 0.63 µmol, 6%) was obtained as a
white solid. UPLC-MS: Rt = 3.34 min (ACQUITY UPLC BEH C18, 5 - 75%
MeCN, 10 min run); MALDI-TOF MS (m/z): [M+Na]+ calcd. for
[24]
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4976–4978.
C189H324N35O65
C189H325O65N35: 1375.44212, found 1375.44806.
:
4124.9, found 4128.7; HRMS: [M+3H]3+ calcd. for
E. L. Myers, C. P. Butts, V. K. Aggarwal, Chem. Commun. 2006,
4434–4436.
Close monitoring of the reaction progress was required as too short
reaction times led to incomplete conversion of the β-rhamnose while
longer reaction times decreased the yield due to cyclization of the α-
rhamnose.
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This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000587
European Journal of Organic Chemistry
FULL PAPER
Table of Contents
Keyword: Glycopeptide
Reported is the synthesis of five C-glycosyl amino acids, which are stable against the acidic conditions used in solid phase peptide
synthesis. Key steps include the introduction of a C-allyl on the carbohydrate, a Grubbs cross-metathesis, reduction of the obtained
alkene and condensation with a protected lysine.
14
This article is protected by copyright. All rights reserved.