Cyanopivaloyl Ester in the Automated Solid-Phase Synthesis of Oligorhamnans

Article abstract of DOI:10.1021/acs.joc.7b02511

The development of effective protecting group chemistry is an important driving force behind the progress in the synthesis of complex oligosaccharides. Automated solid-phase synthesis is an attractive technique for the rapid assembly of oligosaccharides, built up of repetitive elements. The fact that (harsh) reagents are used in excess in multiple reaction cycles makes this technique extra demanding on the protecting groups used. Here, the synthesis of a set of oligorhamnan fragments is reported using the cyanopivaloyl (PIVCN) ester to ensure effective neighboring group participation during the glycosylation events. The PIVCN group combines the favorable characteristics of the parent pivaloyl (PIV) ester, stability, minimal migratory aptitude, minimal orthoester formation, while it can be cleaved under mild conditions. We show that the remote CN group in the PIVCN renders the PIVCN carbonyl more electropositive and thus susceptible to nucleophilic cleavage. This feature is built upon in the automated solid-phase assembly of the oligorhamnan fragments. Where the use of a PIV-protected building block failed because of incomplete cleavage, PIVCN-protected synthons performed well and allowed the generation of oligorhamnans, up to 16 monosaccharides in length.

Full text of DOI:10.1021/acs.joc.7b02511

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Featured Article
The Cyanopivaloyl Ester in the Automated
Solid Phase Synthesis of Oligorhamnans
Anne Geert Volbeda, Jeanine van Mechelen, Nico Meeuwenoord,
Herman S. Overkleeft, Gijsbert A. van der Marel, and Jeroen D. C. Codée
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02511 • Publication Date (Web): 17 Nov 2017
Downloaded from http://pubs.acs.org on November 17, 2017
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The Cyanopivaloyl Ester in the Automated Solid Phase Synthesis of Oligorhamnans
Anne G. Volbeda, Jeanine van Mechelen, Nico Meeuwenoord, Herman S. Overkleeft,
Gijsbert A. van der Marel, Jeroen D. C. Codée*
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The
Netherlands
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jcodee@chem.leidenuniv.nl
Abstract: The development of effective protecting group chemistry is an important driving
force behind the progress in the synthesis of complex oligosaccharides. Automated solid
phase synthesis is an attractive technique for the rapid assembly of oligosaccharides, built
up of repetitive elements. The fact that (harsh) reagents are used in excess in multiple
reaction cycles make this technique extra demanding on the protecting groups used. Here,
the synthesis of a set of oligorhamnan fragments is reported using the cyanopivaloyl
(
PivCN) ester, to ensure effective neighboring group participation during the glycosylation
events. The PivCN group combines the favorable characteristics of the parent pivaloyl (Piv)
ester, stability, minimal migratory aptitude, minimal orthoester formation, while it can be
cleaved under mild conditions. We show that the remote CNꢀgroup in the PivCN renders
the PivCN carbonyl more electropositive and thus susceptible to nucleophilic cleavage.
This feature is built upon in the automated solid phase assembly of the oligorhamnan
fragments. Where the use of a Pivꢀprotected building block failed because of incomplete
cleavage, PivCNꢀprotected synthons performed well and allowed the generation of
oligorhamnans, up to 16 monosaccharides in length.
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Introduction
The advent of automated solid phase synthesis approaches for the assembly of nucleic acids
and peptides has transformed the way chemists generate (fragments of) these biopolymers,
and the rapid access to these molecules has revolutionized the life sciences. The automated
solid phase synthesis of oligosaccharides is significantly more complex than the assembly
of the other two biopolymers and as a result its development has been significantly slower.
Nonetheless, there has been significant progress in the field of automated solid phase
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oligosaccharide synthesis over the last decade. A commercial synthesizer is now
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available, and there are continuous efforts to build improved machines. Ever more
complex molecules are being assembled in an automated manner and recent highlights
include the assembly of libraries of plantꢀderived branched arabinoꢀxylan and xyloglucan
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structures, hyaluronic acid fragments up to 15 monosaccharides in length, a 50ꢀmer
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polymannoside, a set of dermatan and keratan sulfates, a set of αꢀglucans and a
collection of mannuronic acid alginates, built up to 12 βꢀmannuronic acid residues
1
1
linkages. These synthetic successes have shown that linear and branches structures can be
assembled in an automated means and that both 1,2ꢀtrans and 1,2ꢀcis linkages can be
reliably installed using solid phase chemistry. The method is especially attractive for the
generation of libraries of oligosaccharides and oligosaccharides featuring repetitive
elements.
The key to any successful oligosaccharide synthesis campaign is the protection group
strategy used. Permanent protecting groups should be able to withstand all conditions used
throughout the assembly route, while temporary protecting groups have to be removed
selectively without touching any other functionalities in the molecule. The requirements for
protecting groups in automated solid phase oligosaccharide synthesis are even more
strenuous as they have to withstand glycosylation and deprotection steps repeatedly, under
harsher conditions than used in traditional solution phase experiments, because often an
excess of reagents is used to drive reactions to completion. The introduction of new
protecting groups and protecting group chemistry will be crucial for the further
development of automated solid phase oligosaccharide synthesis.
We introduced the cyanopivaloyl (PivCN) group as an attractive participating group that
12
allows for the reliable construction of 1,2ꢀtransꢀglycosidic linkages. It features the
favourable characteristics of the pivaloyl ester ꢀstability, effective neighboring group
participation, minimal othoester formation and migratory aptitudeꢀ while it circumvents the
drawbacks of the parent pivaloyl group ꢀits problematic removal at the end of the synthesisꢀ
as it can be removed by reduction of the cyano group to the corresponding amine, which
can engage in an effective intramolecular ring closure to cleave the ester function. Thus,
removal of the cyanopivaloyl group can be effected in tandem with the removal of benzyl
ethers, commonly used as permanent protecting groups.
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These favourable characteristics should make the cyanopivaloyl group an attractive
protecting group to be used in automated synthesis. To probe its effectiveness in an
automated solid phase setting, we explored its use in the assembly of a set of
oligorhamnosides, up to 16 monosaccharides in length (See Figure 1). These target
structures represent fragments of the backbone of the cell wall polysaccharide of Group A
streptococcus (GAS), a Gramꢀpositive bacterium, which is the cause of various infections
(
pharyngitidis, necrotizing fasciitis) and which is found responsible for rheumatic fever,
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causing hundreds of thousands of deaths every year in developing countries.
The GAS
polyrhamnose backbone is decorated with Nꢀacetyl glucosamine appendages at the
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rhamnosyl Cꢀ3 hydroxyl. The potential use of this naturally occurring polysaccharide in
conjugate vaccines may be thwarted by the potentially autoimmunogenic GlcNAc epitopes
and it has been suggested that the nonꢀmammalian “bare” poly rhamnose backbone, devoid
of GlcNAc groups, may be an attractive structure for a GAS vaccine. Wellꢀdefined
fragments of the polyrhamnose backbone will be valuable in the generation of semiꢀ
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synthetic vaccines and therefore represent attractive synthetic targets.
The repetitive
nature of these molecules makes them very well suited for an automated synthesis
approach.
Figure 1. Synthetic approach described in this work
O
Bn
N
O
OH
O
O
PhN
CF
3
3
O
NH
2
O
O
O
BnO
automated
synthesis
HO
O
O
OR
OH
O
O
4
: n=4
BnO
HO
1
: R = Piv
5: n=5
6: n=6
7: n=7
8: n=8
BnO
HO
OLev
O
2: R = PivCN
O
n
H
PivCN =
N
Results and discussion
The synthetic strategy ꢀand testꢀcase for the cyanopivaloyl groupꢀ for the assembly of the
oligorhamnosides is depicted in Scheme 1. In this study, a commerical Glyconeer 2.1
synthesizer was used for the automated assembly. The oligosaccharides are built on a
23
polystyrene resin equipped with a linker system, that provides the target structures with an
aminopentanol spacer after global deprotection. The amine in the linker system is protected
with a benzyl and a modified Cbz protecting group. The Cbzꢀpart is connected to the solid
support via a base labile ester linkage. Disaccharide building blocks were used in this study
bearing an imidate as anomeric leaving group and a levulinoyl group as orthogonal
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temporary group, as these functionalities have proven very effective in various previous
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automated solid phase assembly procedures. Dimer donors were to be used because acyl
groups at the axial Cꢀ2ꢀhydroxyl of rhamnosides are prone to migrate to the equatorial Cꢀ3ꢀ
hydroxyl group, when this functionality is unmasked during the synthesis. Partial migration
of protecting groups will lead to complex and inseparable mixtures after several coupling
rounds. Two different dimer building blocks were explored: the first (dimer 1) carrying a
permanent pivaloyl ester at the Cꢀ2ꢀhydroxyl, the second (building block 2) with a
cyanopivaloyl at this position.
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The linkerꢀfunctionalized resin 3, is obtained in 7 steps, from 1,4ꢀbenzeneꢀdimethanol,
following an improved route of synthesis, originally developed by Czechura et al., as
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depicted in Scheme 1. After silylation of one hydroxyl group (30% yield), the remaining
hydroxyl is transformed into an active carbonate by reaction of compound 9 with paraꢀ
nitrophenylchloroformate and reacted with Nꢀbenzylꢀ5ꢀaminopentanol to yield compound
1
0. Installation of the dimethoxytrityl group proceeded uneventfully but because
purification of the fully protected linked system from excess reagent proved troublesome,
the TBS group was directly removed. Compound 11 was obtained pure in quantitative yield
over two steps yield on 16 mmol scale.
Scheme 1. Generation of the linker equipped resin.
OR
Bn
b, c
O
d
O
N
ODMTr
HO
TBSO
O
N
OH
O
OH
Bn
R = H
a
10
11
9
: R = TBS
OH
OMe
O
O
O
O
O
Bn
N
HO
MeO
O
e
f
O
OR
OH
OH
OH
O
O
OMe
O
HO
O
MeO
O
O
O
1
2
g
13: R = DMTr
3: R = H
Reagents and conditions: a) TBDMSꢀCl, imidazole, DMF, 0°C (30%); b) paraꢀnitrophenylchloroformate, pyridine,
°C, c) NꢀBenzylꢀ5ꢀaminopentanol, DIPEA, DMF, 0°C (90%); d) i. DMTrꢀCl, pyridine, 0°C, ii. TBAF, THF, 0°C
100%); e) TMSCHN , MeOH, THF; f) 11, DIC, DMAP, DCM, then MeOH; g) TCA, DCM.
0
(
2
Next the linker was conjugated to the carboxylic acid functionalized polystyrene resin.
Because the loading of the commercially available resin was too high (2.19 mmol/g), the
amount of carboxylic acid groups was first reduced by treatment of the resin with TMSꢀ
2
4,25
diazomethane.
Afterwards the resulting resin was coupled with the DMTꢀprotected
linker. Removal of the DMTr group was achieved by a TCA/DCM treatment, after which
the loading was determined to be 0.44ꢀ0.47 mmol/g.
The synthesis of the required dirhamnosyl building blocks is depicted in Scheme 2 and
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26
12
started by coupling imidate donor 14 and acceptor 15 /16 , using a catalytic amount of
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TfOH. This led to disaccharides 17 and 18, which could both be purified by crystallization
from hot ethanol. The thioglycosides 17/18 were transformed into the corresponding
imidate donors by treatment with Nꢀbromosuccinimide in acetone/water and subsequent
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installation of the Nꢀphenyl trifluoroacetimidoyl functionality.
Scheme 2. Synthesis of donor 1 and 2.
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Reagents and conditions: a) 14, TfOH, DCM, 0°C (17: 69%, 18: 88%); b) NBS, acetone/H
then ClC(=NPh)CF , Cs CO , acetone, 0°C (1: 88%, 2: 79%); c) NIS, TFA, DCM, 0°C (20a: 73%) then
ClC(=NPh)CF , Cs CO , acetone, 0°C (2: 79%) .
2
O (19: 65%, 20: 89%),
3
2
3
3
2
3
With the required building blocks in hand the assembly of the oligosaccharides was started.
As a first research objective, the assembly of a decasaccharide was targeted employing the
pivaloyl protected building block 1. Previously developed glycosylation and deprotection
6,11
conditions
were applied to couple donor 1 to resin 3 (3 x 3 equivalents donor, 0.2
equivalents TfOH with respect to the donor, 30 min at 0°C, Scheme 3), followed by
removal of the Lev group (3 x 5 equivalents H NNH •AcOH, 10 min at 40°C). After five
2
2
coupling/deprotection cycles, the resin was subjected to cleavage conditions (a catalytic
amount of NaOMe in a mixture of THF/MeOH).
Scheme 3. Automated synthesis of decamer 21 and 22 and hexadecamer 23.
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O
Bn
N
PhN
CF
3
O
OH
O
O
O
O
BnO
3
O
OR
O
BnO
automated
synthesis
[a-b]5 or 8
BnO
OLev
1: R = Piv
: R = PivCN
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O
O
O
N
O
O
N
O
Bn
OH
Bn
OH
O
O
BnO
BnO
O
O
OR
OR
24: R = Piv or H, n=5, 4, 3, 2
5: R = H, n=4
26: R = H, n=5
7: R = H, n=6
O
21: R = Piv or H, n=5
2: R = PivCN or H, n=5
3: R = PivCN or H, n=8
d or e
O
BnO
BnO
2
2
2
BnO
BnO
O
O
2
n
n
28: R = H, n=7
29: R = H, n=8
H
H
O
O
NH
2
HO
f
O
OH
4
5
6
: n=4
: n=5
: n=6
O
HO
HO
O
H
7: n=7
: n=8
8
n
Reagents and conditions: a) 3 equiv. 1 or 2, 0.3 equiv. TfOH, DCM, 0°C, 3 cycles; b) 8 equiv. H
2 2
NNH ·AcOH,
pyr/AcOH, 40°C, 3 cycles; c) NaOMe, MeOH/THF; d) NaOMe, MeOH/THF (25: 9%, 26: 26%, 27: 6%, 28: 9%,
29: 9% starting from resin 3); e) NaOH (aq), MeOH/Dioxane, 40°C; f) H
(4: 69%, 5: 57%, 6: 27%, 7: 92%, 8: 50%)
2 2 2
, Pd(OH )/C, AcOH, H O/THF/tBuOH
The crude decasaccharide 21 was analyzed by LCꢀMS and the obtained LCꢀspectrum is
shown in Figure 2. A complex mixture was obtained, which was the result of incomplete
glycosylation reactions and removal of some of the pivaloyl esters. Unfortunately, it proved
to be impossible to remove all pivaloyl esters, even under harsh basic conditions (NaOH in
MeOH/dioxane at elevated temperature (conditions e, Scheme 3), and the desired
decasaccharide could not be obtained from the complex reaction mixture (figure 2B). The
use of pivaloyl funtionalized donor 1 was therefore not further explored and attention was
switched to the use of its PivCN counterpart 2.
2
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Figure 2. LCꢀchromatograms of the crude products cleaved from the resin before and after prolonged base
treatment. A: 21; B: 24; C: 22; D: 26; E: 23; F: 29.
A
B
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C
26
D
E
25
28
29
F
27
Reagents and conditions: A) diphenyl column, 50ꢀ90% B; B) diphenyl column, 50ꢀ90% B; C) C4 column,
0ꢀ90% B; D) C4 column, 50ꢀ90% B; E) diphenyl column, 50ꢀ98% B; F) diphenyl column, 50ꢀ98% B.
5
When donor 2 was used for the assembly of decasaccharide 22 again a complex product
29
mixture arose after cleavage of the products form the resin (Figure 2C). It was noted
however that a significantly larger portion of the PivCNꢀgroups had been removed from the
target structures in comparison to the pivaloyl decasaccharide mixture. This indicates that
the cyano group in the PivCN ester render the ester carbonyl more electrophilic, as a result
of its electron withdrawing character, even though it is separated from the carbonyl by two
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carbon atoms. This also suggested that the PivCN groups could potentially be removed by
an additional and/or elongated base treatment. To explore this possibility, the crude mixture
was resubjected to basic conditions and progress of the reaction was monitored by LCꢀMS.
The LCꢀchromatogram of the mixture that was finally obtained is shown in Figure 2D, and
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it shows the presence of only two products. The major product in the mixture proved to be
the desired decasaccharide 26, while the minor other peak corresponds to the
octasaccharide (25). Purification of the target compound was readily achieved from this
mixture and the target decasaccharide was obtained in 26% overall yield after 12 steps
(
89% per step).
Driven by this success, a hexadecasaccharide was synthesized by running eight
coupling/deprotection cycles using donor 2. After cleavage of the products from the resin,
again a complex mixture was obtained (Figure 2E). Subjection of this mixture to an
additional base treatment led to complete cleavage of all PivCN groups and Figure 2F
depicts the LCꢀchromatogram of the resulting mixture. From this mixture, the target
hexadecarhamnoside 29 was obtained in 9% yield (18 steps, 87% per step) alongside the
dodecaꢀ and tetradecasaccharide deletion sequences, 27 and 28, respectively.
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To complete the syntheses of the oligorhamnosides, all obtained partially protected
oligorhamnosides (25ꢀ29) were subjected to hydrogenolysis over Pd(OH) /C in
2
H O/THF/tBuOH, to remove all benzyl groups and liberate the alcohols and the amine
2
functionality on the spacer. Gel filtration (HW40, eluted with NH OAc) yielded the fully
4
deprotected octaꢀ, decaꢀ, dodeca, tetradeca and hexadecasaccharides (4ꢀ8) in
multimilligram quantities.
Conclusion
We have introduced the cyanopivaloyl (PivCN) ester as an effective protecting group for
solid phase oligosaccharide synthesis. This novel protecting group was probed in the
assembly of a series of oligorhamnosides, alongside its pivaloyl counterpart. It was found
that cleavage of the protected oligosaccharides from the resin was accompanied by partial
cleavage of the pivaloyl groups. Complete removal of all pivaloyl groups however proved
to be difficult, underscoring the problems often encountered with this bulky ester. The
cyanopivaloyl ester on the other hand could be effectively cleaved under basic conditions,
as a result of the remote electronꢀwithdrawing cyano group, which renders the ester
carbonyl group more electrophilic. The favorable cleavage characteristics of the PivCNꢀ
group in combination with the favorable properties of the pivaloylꢀtype esters (minimal
orthoester formation during glycosylations, minimal migration, stability) make the PivCNꢀ
group an attractive asset in the toolbox of the synthetic chemist. Here, it has proven its
merits in the automated solid phase assembly of GAS related oligorhamnosides of
considerable length.
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Experimental
General experimental procedures. All solvents used under anhydrous conditions were stored over 4Å molecular
1
13
sieves except for methanol which was stored over 3Å molecular sieves. H and C NMR spectra were recorded on
a 400/100, 500/125, 600/150, or a 850/214 MHz spectrometer. Chemical shifts (δ) are given in ppm relative to
tetramethylsilane as internal standard. Coupling constants are given in Hz. All individual signals were assigned
ꢀ1
using 2DꢀNMR spectroscopy, HHꢀCOSY, HSQC, and HMBC. IR spectra are reported in cm , and recorded on a
Shimadzu FTIRꢀ8300 or a PerkinElmer universal attenuated total reflectance (UATR; Single Reflection Diamond)
Spectrum Two instrument. Solvents used for workup and column chromatography were of technical grade from
Sigma Aldrich, Boom, Biosolve or Honeywell and used directly. Unless stated otherwise, solvents were removed
by rotary evaporation under reduced pressure at 40 °C. All chemicals were used as received unless stated
otherwise. Reactions were monitored by TLC‐analysis using Merck 25 DC plastikfolien 60 F254 with detection by
10
11
12
13
14
15
16
17
18
19
20
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23
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37
38
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
spraying with 20% H
sulfuric acid or by spraying with a solution of ninhydrin (3 g/L) in EtOH / AcOH (20/1 v/v), or by dipping in
anisaldehyde (10 mL in 180 mL EtOH / 10 mL H SO ) followed by charring at approx. 150 °C. Column
2 4 4 6 7 2 4 4 4 4 2
SO in EtOH, (NH ) Mo O24·4H O (25 g/L) and (NH ) Ce(SO ) ·2H O (10 g/L) in 10%
2
4
chromatography was performed on Fluka silicagel (0.04 – 0.063 mm). For LCꢀMS analysis a Agilent
Technologies 1260 Infinity LC system (detection simultaneously at 214 and 254 nm) coupled to a Agilent
Technologies 6120 Quadrupole LC/MS, using an analytical Vydac C4 column (Alltech, 50 x 4.60 mm, 5 µm) or a
Vydac Diphenyl (Alltech, 150 x 4.60 mm, 5 µm) in combination eluents A: H
2
O; B: MeCN and C: 1% aq. TFA.
For HPLC, a Gilson HPLC system in combination with eluents A: H O (0.1% TFA); B: MeCN as the solvent
2
system using a Vydac C4 HPLC column (Grace, 250 x 10 mm, 5 µm). High resolution mass spectra were recorded
by direct injection (2 ꢁL of a 2 ꢁM solution in water/acetonitrile; 50/50; v/v and 0.1% formic acid) on a mass
spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electrospray ion source in positive mode (source
voltage 3.5 kV, sheath gas flow 10, capillary temperature 250 °C) with resolution R = 60000 at m/z 400 (mass
range m/z = 150‐2000) and dioctylphthalate (m/z = 391.2842) as a “lock mass”. The high resolution mass
spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). Maldi spectra
were recorded on an Ultraflextreme MALDIꢀTOF (Bruker Daltonics), equipped with SmartbeamꢀII laser, to
measure the samples in reflectron positive ion mode. The MALDIꢀTOF was calibrated using a peptide calibration
standard prior to measurement. 1 ꢁl of 2,5ꢀdihydroxybenzoic acid (2,5ꢀDHB; Bruker Daltonics) matrix (20 mg/mL
in ACN/water; 50:50 (v/v)) was applied on a 384ꢀMTP target plate (Bruker Daltonics, Bremen, Germany) and airꢀ
dried. Subsequently, 1 ꢁl of compound water solution was spotted on the plate and the spots were left to dry prior
MALDIꢀTOF analysis.
(4ꢀtertꢀButylꢀdimethylꢀsiloxylmethylphenyl)methanol (9) 1,4ꢀbenzeneꢀdimethanol (8.29 g, 60 mmol, 1.0 equiv.)
was dissolved in 25 mL DMF and cooled to 0°C followed by the addition of imidazole (10.2 g, 150 mmol, 2.5
equiv.). A solution of tertꢀbutyldimethylsilyl chloride (9.13 g, 60.6 mmol, 1.01 equiv.) in 40 mL DMF was added
dropwise and the reaction was allowed to stir overnight. After TLC analysis showed complete consumption of the
starting material, the mixture was dilute with Et
The organic phase was dried over MgSO
2
O and washed subsequently with H
2
O (2x) and sat. aq. NaCl (1x).
4
and concentrated in vacuo. Purification using flash column
chromatography (PE/EtOAc, 9:1 ꢀ 6:1) yielded the title compound as a colorless oil (4.73 g, 18.7 mmol, 30%).
1
TLC: R
f
0.39 (PE/EtOAc, 6/1, v/v); H NMR (400 MHz, CDCl
3
): δ 7.33 (s, 4H), 4.74 (s, 2H), 4.68 (d, 2H, J=3.1
31
Hz), 1.67ꢀ1.50 (m, 2H), 0.94 (s, 9H), 0.10 (s, 6H). Analytical data are identical to literature values.
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NꢀBenzylꢀ5ꢀaminopentanol Benzaldehyde (10.67 mL, 104.6 mmol, 1.01 equiv.) was added to a solution of 5ꢀ
aminopentalnol (11.3 mL, 104.0 mmol, 1.0 equiv.) in 150 mL EtOH. The solution was heated to 50°C under
reduced pressure until all solvent was removed. The crude mixture was coꢀevaporated twice with anhydrous
toluene, dissolved in MeOH (200 mL) and cooled to 0°C. NaBH
portions and the solution was allowed to stir at 0°C for 70 minutes. After stirring for another 2 hours, the solution
was cooled to 0°C followed by addition of 4,5 mL AcOH. A 1.2M K CO (aq) solution (135 mL) was added and
the mixture was diluted with Et O. The organic layer was dried over MgSO
4
(4.82 g, 124.7 mmol, 1.2 equiv.) was added in
2
3
2
4
and concentrated in vacuo.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Purification using flash column chromatography yielded the linker in 62% yield (12.4 g, 64 mmol); H NMR (400
MHz, CDCl
3
): δ 7.41ꢀ7.15 (m, 5H), 3.78 (s, 2H), 3.62 (t, 2H, J=6.4, 6.4 Hz), 2.64 (t, 2H, J=7.0, 7.0 Hz), 1.92 (s,
13
2
4
H), 1.72ꢀ1.47 (m, 4H), 1.47ꢀ1.25 (m, 2H); C NMR (101 MHz, CDCl
3
): δ 140.2, 128.5, 128.3, 127.1, 62.7, 54.1,
32
9.3, 42.0, 32.6, 29.7, 29.1, 23.5. Analytical data are identical to literature values.
5ꢀ(benzyl(4ꢀtertꢀbutyldimethylsilyl)oxymethylbenzyloxycarbonyl)amino)pentanol (10) Silylether 9 (4.73 g,
18.7 mmol, 1.0 equiv.) was dissolved in dry DCM (125 mL) and cooled to 0°C. Pyridine (3.0 mL, 37.5 mmol, 2.0
equiv.) was added followed by addition of paraꢀnitrophenylchloroformate (4.53 g, 22.5 mmol, 1.2 equiv.) after
which the solution was allowed to warm up to RT and stirred overnight. The reaction was concentrated in vacuo
and coevaporated with toluene. The crude compound was dissolved in DMF (75 mL) and cooled to 0°C. To this
mixture was added Nꢀbenzylꢀ5ꢀaminopentanol (4.78 g, 23.0 mmol, 1.23 equiv.) in DMF (20 mL) followed by
addition of DIPEA (4.23 mL, 24.4 mmol, 1.3 equiv.) The reaction mixture was stirred overnight, diluted with
Et2O and washed with H
were washed multiple times with sat. aq. NaHCO
in vacuo. Column purification (PE/EtOAc, 8:1 ꢀ 3:1) yielded the title compound (8.02 g, 17.0 mmol, 90%). IR
2
O. The aqeous layer wash back extracted with Et
2
O, and the combined organic layers
3
. The solution was dried over MgSO
4
, filtered and concentrated
–1
1
(neat): 1083, 1249, 1417, 1454, 1681, 1695, 2856, 2929, 2949, 3062, 3387, 3437 cm ; H NMR (500 MHz,
CDCl ) δ 7.21ꢀ7.10 (m, 9H, CHarom), 5.06 (s, 2H, CH Cbz), 4.63 (s, 2H, CH Bn), 4.39 (s, 2H, CH Cbz), 3.46 (s,
H, CH ), 1.41ꢀ1.20 (s, 6H, 3x CH ), 0.85 (s, 9H, 3x CH TBDMS), 0.00 (s, 6H, 2x CH
): δ 141.5, 138.3, 135.7 (Cq), 128.7, 128.1, 127.5, 126.4 (CHarom), 67.3
), 50.7 (CH ), 32.6 (CH ), 26.1 (3x CH TDBDMS), 23.2 (CH ), ꢀ5.1 (2x CH
Si 472.2878, found 472.2877.
3
2
2
2
2
2
), 3.15 (s, 2H, CH
2
2
3
3
13
TBDMS); C NMR (126 MHz, CDCl
3
(
CH
2
), 65.0 (CH
2
), 62.9 (CH
2
2
2
3
2
3
+
TBDMS); HRMS: [M+H] calcd. for C27
H42NO
4
5
ꢀ(benzyl(4ꢀhydroxymethylbenzyloxycarbonyl)amino)pentyl dimethoxytrityl ether (11) Silylether 10 (7.50 g,
15.9 mmol, 1.0 equiv.) was coevaporated twice with pyridine under an argon atmosphere, before being dissolved
in pyridine (160 mL) and cooled to 0°C. To the mixture was added DMTrꢀCl (5.92 g, 17.5 mmol, 1.1 equiv.) and it
was allowed to stir overnight. After overnight stirring, TLC analysis (Hexans/EtOAc, 4:1) showed conversion of
the starting material to a high running spot. The mixture was concentrated, dissolved in EtOAc and washed twice
2 4
with H O, dried over MgSO and concentrated in vacuo. The intermediate was coevaporated with toluene,
dissolved in THF (160 mL) and cooled to 0°C. TBAF (1.0 M in THF, 25 mL, 1.6 equiv.) was added and the green
coloured reaction was stirred for 5h after which it was concentrated. The compound was dissolved in EtOAc,
washed subsequently with H
concentrated in vacuo. Purification by column chromatography (Tol/EtOAc + Et
protected linker (10.2 g, 15.9 mmol, 100%). IR (neat): 1031, 1246, 1300, 1417, 1506, 1606, 1693, 2835, 2864,
2
O, sat. aq. NaHCO
3
and sat. aq. NaCl. The organic layer was dried over MgSO
4
and
3
N, 9:1 ꢀ 4:1) yielded DMTr
–1 1
3
2931, 3030, 3059, 3415, 3441 cm ; H NMR (400 MHz, CDCl , T=328K) δ 7.41 (d, J = 7.6 Hz, 2H, CHarom),
7
2
.35ꢀ7.07 (m, 16H, CHarom), 6.80 (d, J = 8.5 Hz, 4H, CHarom), 5.13 (s, 2H, CH
2
Cbz), 4.63 (s, 2H, CH
2
Bn), 4.46 (s,
13
H, CH Cbz), 3.76 (s, 6H, 2x CH OMe), 3.21 (s, 2H, CH ), 3.02 (s, 2H, CH
2
3
2
2
), 1.69ꢀ1.24 (m, 6H, 3x CH
2
);
C
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NMR (101 MHz, CDCl
13.2 (CHarom), 67.1 (CH
for C42 45NO Na 682.3139, found 682.3139.
3
) δ 158.6, 145.6, 140.9, 136.9 (Cq), 130.2, 128.6, 128.4, 128.2, 127.8, 127.4, 127.1, 126.7,
+
1
2
), 65.1 (CH
2
), 63.4 (CH
2
), 55.3 (OMe), 29.9 (CH
2
), 23.8 (CH
2
); HRMS: [M+Na] calcd.
H
6
Synthesis of aminopentanolꢀfunctionalized Polystyrene (12)
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12
13
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17
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60
Carboxy Polystyrene (Rapp polymer, 5 g, 2,19 mmol/g, 11 mmol) was added to a fritted seringe and swollen in 32
mL DCM. The resin was purged with argon after which it was washed with DCM (3x), alternating DCM and
hexane (3x), and DCM (2x). The resin was dried in vacuo at 45°C overnight. The dried resin was suspended in 60
3 2
mL THF and MeOH (1.03 mL, 25.4 mmol, 3 equiv. with respect to Me SiCHN ) was added. The suspension was
shaked for 10 min followed by addition of Me SiCHN (4.24 mL of 2.0M solution in hexanes, 8.47 mmol, 0.77
3
2
equiv. with respect to the resin), whereupon the solution turned yellow. The reaction was allowed to shake
overnight, after which it became colorless. The solution was filtered, and resin 12 was washed with DCM (4x),
hexanes (4x) and THF (4x), and dried in vacuo at 45°C.
Carboxy Polystyrene 12 (Rapp polymer, 5g, ~0.51 mmol/g, 2.54 mmol) was swollen in DCM (60 mL) and the
suspension was shaken for 1h. The solution was filtered and DCM (40 mL) was added to the resin. Compound 11
(5.04 g, 7.64 mmol, 3 equiv.) was coevaporated twice with toluene under argon, dissolved in DCM (8.5 mL),
alongwith addition of DIC (1.20 mL, 7.64 mmol, 3 equiv.) and DMAP (0.03 g, 0.25 mmol, 0.1 equiv.). An
additional rinse with 5 mL DCM was performed before the resin was allowed to shake overnight. Then, MeOH
(0.6 mL) was added and the suspension was shaken again. The mixture was filtered, and resin 13 was washed with
alternating DCM and hexanes (4x), followed by DCM (3x). The resin was dried in vacuo at 45°C.
Solid support (3) DMTꢀfunctionalized resin 13 (5 g) was loaded into a fritted funnel and washed with 3% TCA
(w/v in DCM, 60 mL) and shaken for 5 min. The orange solution was filtered and the procedure was repeated 4x.
After the TCA washes, the orange resin was washed 3x with DCM (60 mL), 3x with toluene (60 mL), 3x with
DCM/MeOH (60 mL), 1x with MeOH (60 mL) and 4x DCM (60 mL). The resin was dried in vacuo to a constant
weight of 4.22 g.
DMTrꢀassay (performed in duplicate) DMTꢀfunctionalized resin 13 (4.1 mg) was added to a 10 mL volumetric
flask and treated with 10 mL 3% TCA/DCM (w/v). A 1 mL aliquot was taken and diluted 100x with the 3%
TCA/DCM solution. Absorbance read at λ = 503 nm.
Loading calculation:
[
ꢀꢁꢂꢃꢄꢅꢀꢆꢇꢇꢈꢉꢊꢅ]
=
ꢍꢍꢎꢏꢈꢐꢑꢈꢒꢐꢑꢓꢏꢈꢔꢎꢏꢕꢖꢐꢎꢑ
ꢋꢌ
ꢉꢉꢘꢙꢈꢚꢛꢈꢜꢚꢛꢝꢙꢈꢞꢘꢙꢟꢠꢚꢘꢛ
ꢗ
=
=
ꢈꢤ × 10ꢈꢍꢥ = ꢍꢍꢎꢏꢈꢐꢑꢈꢐꢑꢐꢖꢐꢓꢏꢈꢔꢎꢏꢕꢖꢐꢎꢑ
ꢡꢘꢙꢟꢉꢢꢈꢝꢙꢚꢣꢟꢘꢠ
ꢀ
ꢇ.ꢆꢦꢋꢅꢀꢆꢇꢇꢈꢉꢊꢅ
= 0.180ꢈꢨꢍꢎꢏ
ꢋꢌꢈꢉꢊ⁄ꢧꢉꢘꢙ
ꢇ.ꢇꢇꢇꢆꢩꢈꢉꢉꢘꢙ
(
ꢅꢈ× 10ꢈꢍꢥ = 0.00180ꢈꢍꢍꢎꢏ
ꢆꢈꢉꢊ
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ꢇ.ꢇꢇꢆꢩꢇꢈꢉꢉꢘꢙ
ꢇ.ꢇꢇꢪꢆꢈꢫ
ꢍꢍꢎꢏ
ꢭ
ꢬ
Loading
=
= 0.44ꢈ
A loading of 0.44ꢀ0.47 mmol/g was determined.
Phenyl
4ꢀOꢀBenzylꢀ2ꢀO-Pivaloylꢀ3ꢀOꢀ(3,4ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀlevulinoylꢀαꢀLꢀrhamnopyranosyl)ꢀ1ꢀthioꢀαꢀLꢀ
26
rhamnopyranoside (17) Compound 15 (4.96 g, 11.52 mmol, 1.0 equiv.) and imidate donor 14 (7.56 g, 13.32
mmol, 1.2 equiv.) were coevaporated twice with anhydrous toluene under an argon atmosphere, after which they
were dissolved in dry DCM (56 mL). The mixture was stirred on activated molecular sieves for 20 min at RT, and
then cooled to 0°C. TfOH (0.1 mL, 1.12 mmol, 0.1 equiv.) was added, and, after 135 min, TLC analysis showed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
complete consumption of the acceptor, the reaction was quenched by addition of 0.3 mL Et
diluted with EtOAc, washed subsequently with sat. aq. NaHCO and sat. aq. NaCl, dried over MgSO
concentrated in vacuo. Crystallization from hot EtOH (5.65 g, 6.61 mmol), followed by a second crystallization of
the mother liquid yielded the disaccharide as white crystals (6.65 g, 7.78 mmol, 69%). M.p. 145°C. R 0.69
PE/EtOAc, 2/1, v/v); IR (neat): 918, 987, 1026, 1039, 1060, 1082, 1138, 1454, 1479, 1732, 2873, 2910, 2933,
3
N. The mixture was
3
4
, and
f
(
–
1 1
3
2974 cm ; H NMR (500 MHz, CDCl ): δ 7.47ꢀ7.41 (m, 2H, CHarom), 7.36 (d, J = 4.4 Hz, 4H, CHarom), 7.37ꢀ7.16
(m, 13H, CHarom), 5.39 (dd, J = 3.3, 1.8 Hz, 1H, Hꢀ2’), 5.35 (d, J = 1.8 Hz, 1H, Hꢀ1), 5.29 (dd, J = 3.2, 1.8 Hz,
1H, Hꢀ2), 5.06 (d, J = 1.9 Hz, 1H, Hꢀ1’), 4.91 (d, J = 11.5 Hz, 1H, CHH Bn), 4.78 (d, J = 10.8 Hz, 1H, CHH Bn),
4
3
.67ꢀ4.54 (m, 3H, CHH, CH
.1 Hz, 1H, Hꢀ3), 3.81 (dd, J = 9.3, 3.4 Hz, 1H, Hꢀ3’), 3.73ꢀ3.64 (m, 1H, Hꢀ5’), 3.49 (t, J = 9.5 Hz, 1H, Hꢀ4), 3.41
Lev), 2.13 (s, 3H, CH Lev), 1.33ꢀ1.22 (m, 6H, 2x CH ꢀ6),
): δ 206.0 (C=O Lev), 177.3, 171.8 (C=O Lev, Piv), 138.7,
37.8, 137.7, 133.6 (Cq), 132.1, 129.0, 128.5, 128.4, 128.3, 128.2, 127.9, 127.7, 127.6, 127.5, 127.4 (CHarom), 99.7
Bn), 74.8 (CH Bn), 73.6 (Cꢀ2), 71.5
Lev), 29.8 (CH Lev), 28.1 (CH Lev),
62NO11S 872.4038, found 872.4045.
2
Bn), 4.46 (d, J = 11.5 Hz, 1H, CHH Bn), 4.26ꢀ4.16 (m, 1H, Hꢀ5), 4.11 (dd, J = 9.4,
(
t, J = 9.4 Hz, 1H, Hꢀ4’), 2.69ꢀ2.65 (m, 4H, 2x CH
2
3
3
13
1
1
3 3
.20 (s, 9H, 3x CH Piv); C NMR (126 MHz, CDCl
(
Cꢀ1’), 85.6 (Cꢀ1), 80.4 (Cꢀ4), 79.6 (Cꢀ4’), 77.7 (Cꢀ3), 77.2 (Cꢀ3’), 75.5 (CH
2
2
(CH
2
Bn), 69.3 (Cꢀ5), 69.1 (Cꢀ2’), 68.5 (Cꢀ5’), 39.0 (Cq Piv), 38.0 (CH
2
3
2
+
27.1 (CH
3
Piv), 17.9 (Cꢀ6), 17.8 (Cꢀ6’). HRMS: [M+H] calcd. for C49
H
4
ꢀOꢀBenzylꢀ2ꢀOꢀPivaloylꢀ3ꢀOꢀ(3,4ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀlevulinoylꢀαꢀ
Lꢀrhamnopyranosyl)ꢀ)ꢀα/βꢀLꢀ
rhamnopyranoside (19) Compound 17 (2.76 g, 3.23 mmol, 1.0 equiv.) was dissolved in acetone/H
2
O (3:1, 16
mL) and cooled to 0°C. NBS (1.73 g, 9.69 mmol, 3.0 equiv.) was added and the reaction was stirred overnight.
TLC analysis showed conversion of the starting material to a lower running spot and the mixture was quenched
with sat. aq. Na
NaHCO , dried over MgSO
1:1) yielded the title hemiacetal (1.60 g, 2.10 mmol, 65%). Spectroscopic data are reported for the major (α)
2
S
2
O
3
. The mixture was diluted with EtOAc and the organic layer was washed with sat. aq.
3
4
and concentrated in vacuo. Purification by column chromatography (PE/EtOAc, 4:1
→
isomer. R
f
0.26 (PE/EtOAc, 2/1, v/v); IR (neat): 1064, 1082, 1134, 1163, 1363, 1708, 1776, 2875, 2933, 2974,
ꢀ1
1
3
4
381 cm ; H NMR (500 MHz, CDCl
3
): δ 7.39ꢀ7.15 (m, 15H, CHarom), 5.36 (dd, J = 3.1, 1.8 Hz, 1H, Hꢀ2’), 5.05ꢀ
.99 (m, 3H, Hꢀ1, Hꢀ1’, Hꢀ2), 4.90 (d, J = 11.4 Hz, 1H, CHH Bn), 4.74 (d, J = 10.8 Hz, 1H, CHH Bn), 4.66ꢀ4.51
Bn), 4.43 (d, J = 11.5 Hz, 1H, CHH Bn), 4.16 (dd, J = 9.5, 3.0 Hz, 1H, Hꢀ3’), 3.97ꢀ3.87
(m, 1H, Hꢀ5), 3.79 (dd, J = 9.3, 3.3 Hz, 1H, Hꢀ3), 3.71ꢀ3.62 (m, 1H, Hꢀ5’), 3.48 (s, 1H, OH), 3.38 (dt, J = 9.4, 6.7
Hz, 2H, Hꢀ4, Hꢀ4’), 2.73ꢀ2.60 (m, 4H, 2x CH Lev), 2.15 (s, 3H, CH Lev), 1.29ꢀ1.17 (m, 15H, 2x CH Cꢀ6, Cꢀ6’,
) δ 206.5 (C=O Lev), 177.7, 171.9 (C=O Lev, Piv), 138.8, 138.8, 137.9,
37.9 (Cq), 128.6, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.1, 128.0, 128.0, 127.8, 127.7, 127.6, 127.6, 127.5
CHarom), 99.7 (Cꢀ1’), 91.7 (Cꢀ1), 80.4, 79.7 (Cꢀ4, Cꢀ4’), 77.3 (Cꢀ3’), 77.1 (Cꢀ3), 75.5 (CH Bn), 74.9 (CH Bn),
Lev), 29.9 (CH Lev), 28.2
(
m, 3H, CHH Bn, CH
2
2
3
3
13
3 3
3x CH Piv); C NMR (126 MHz, CDCl
1
(
2
2
7
2.5 (Cꢀ2), 71.6 (CH
2
Bn), 69.4 (Cꢀ2’), 68.4, 67.7 (Cꢀ5, Cꢀ5’), 39.0 (Cq), 38.1 (CH
2
3
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7
8
9
+
(
CH
2
Lev), 27.2 (3x CH
3
Piv), 18.2, 17.9 (2x CH
3
Cꢀ6, Cꢀ6’); HRMS: [M+Na] calcd. for C43
H
54
O
12Na 785.3508,
found 785.3511.
4ꢀOꢀBenzylꢀ2ꢀOꢀPivaloylꢀ3ꢀOꢀ(3,4ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀlevulinoylꢀαꢀ
trifluoroacetimidoyl)ꢀ α/βꢀ ꢀrhamnopyranoside (1) To a solution of hemiacetal 19 (1.66 g, 2.18 mmol, 1.0
equiv.) in acetone (11 mL) at 0°C were added Nꢀphenyl trifluoroacetimidoyl chloride (0.41 mL, 2.62 mmol, 1.2
equiv.) followed by Cs CO (1.07 g, 3.27 mmol, 1.5 equiv.). The solution was allowed to stir for 3h after which it
was filtered over Celite and concentrated in vacuo. Purification by column chromatography (PE/EtOAc, 6:1 →
:1) yielded the title compound as a clear yellow oil (1.81 g, 4.15 mmol, 88%). Spectroscopic data are reported for
the major (α) isomer. R 0.67 (PE/EtOAc, 2/1, v/v); IR (neat): 989, 1028, 1116, 1138, 1207, 1454, 1597, 1716,
Lꢀrhamnopyranosyl)ꢀ1ꢀ(Nꢀphenylꢀ
L
2
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
f
ꢀ1
1
1
3
737, 2908, 2976 cm ; H NMR (400 MHz, CDCl ): δ 7.49ꢀ7.22 (m, 17H, CHarom), 7.08 (t, J = 7.5 Hz, 1H,
CHarom), 6.81 (d, J = 7.7 Hz, 2H, CHarom), 6.08 (s, 1H, Hꢀ1), 5.39 (dd, J = 3.0, 1.8 Hz, 1H, Hꢀ2’), 5.22 (s, 1H, Hꢀ
2), 5.07 (s, 1H, Hꢀ1’), 4.92 (d, J = 11.4 Hz, 1H, CHH Bn), 4.77 (d, J = 10.7 Hz, 1H, CHH Bn), 4.69ꢀ4.54 (m, 3H,
CHH Bn, CH
2
Bn), 4.47 (d, J = 11.5 Hz, 1H, CHH Bn), 4.22ꢀ4.15 (m, 1H, Hꢀ3), 3.90ꢀ3.75 (m, 2H, Hꢀ5, Hꢀ3’ ),
3
.75ꢀ3.63 (m, 1H, Hꢀ5’), 3.49 (t, J = 9.5 Hz, 1H, Hꢀ4), 3.41 (t, J = 9.3 Hz, 1H, Hꢀ4’), 2.75ꢀ2.60 (m, 4H, 2x CH
2
13
Lev), 2.15 (s, 3H, CH
3
Lev), 1.39ꢀ1.11 (m, 15H, 2x CH
3
Cꢀ6, Cꢀ6’, 3x CH
3
Piv); C NMR (101 MHz, CDCl
3
): δ
2
1
06.2 (C=O Lev), 177.3, 171.9 (C=O Lev, Piv), 143.4, 138.8, 137.9, 137.5 (Cq), 128.8, 128.6, 128.5, 128.4, 128.3,
28.2, 128.1, 127.8, 127.6, 127.5, 124.5, 119.4 (CHarom), 99.8 (Cꢀ1’), 79.7 (Cꢀ4’), 77.3 (Cꢀ3), 76.6 (Cꢀ3’), 75.8
(
CH
Lev), 29.9 (CH
for C51 NO12Na 956.3803, found 956.3809.
2
Bn), 74.9 (CH
2
Bn), 71.7 (CH
2
Bn), 70.5 (Cꢀ2), 70.5 (Cꢀ5), 69.4 (Cꢀ2’), 68.7 (Cꢀ5’), 39.1 (Cq), 38.1 (CH
2
+
3
Lev), 28.2 (CH
2
Lev), 27.2 (3x CH
3
Piv), 18.2, 17.9 (2x CH
3
Cꢀ6, Cꢀ6’); HRMS: [M+Na] calcd.
58 3
H F
Phenyl
3ꢀOꢀ(3,4ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀlevulinoylꢀαꢀ
Lꢀrhamnopyranosyl)ꢀ4ꢀOꢀbenzylꢀ2ꢀOꢀ(3ꢀcyanoꢀ2,2ꢀ
dimethylpropanoyl)ꢀ1ꢀthioꢀαꢀ
L
ꢀrhamnopyranoside (18) Imidate donor 14 (4.00 g, 6.51 mmol, 1.1 equiv.) and
acceptor 16 (2.70 g, 5.92 mmol, 1.0 equiv.) were coevaporated two times with anhydrous toluene under an argon
atmosphere before being dissolved in distilled DCM (59 mL) and the mixture was stirred at room temperature for
3
0 min over activated molecular sieves (3Å). The reaction was cooled to 0 °C and TfOH (0.05 mL, 0.59 mmol, 0.1
equiv.) was added. After 50 min the reaction was quenched by addition of 1.0 mL Et N. The reaction mixture was
diluted with Et O and washed with sat. aq. NaHCO , H O and sat. aq. NaCl. The organic layer was dried over
MgSO and concentrated in vacuo. A quick column purification (PE/EtOAc, 6:1 ꢀ 1:1) followed by
3
2
3
2
4
12
crystallization from hot EtOH yielded the target disaccharide as a white powder (4.57 g, 5.19 mmol, 88%). M.p.
106°C.
4
ꢀOꢀBenzylꢀ2ꢀOꢀ(3ꢀcyanoꢀ2,2ꢀdimethylpropanoyl)ꢀ3ꢀOꢀ(3,4ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀlevulinoylꢀαꢀ
rhamnopyranosyl)ꢀ)ꢀα/βꢀ ꢀrhamnopyranoside (20) Compound 18 (0.260 g, 0.295 mmol, 1.0 equiv.) was
dissolved in acetone/H O (1.2 mL/0.4 mL) and cooled to 0°C. NBS (0.16 g, 0.899 mmol, 3.0 equiv.) was added
and the reaction was stirred for 3h, after which TLC analysis showed conversion of the starting material in a lower
running spot. The reaction was quenched by addition of sat. aq. Na , and diluted with EtOAc. The organic
layer was washed with sat. aq. NaHCO , sat. aq, NaCl, dried over MgSO and concentrated in vacuo. Column
Lꢀ
L
2
2 2 3
S O
3
4
purification (PE/EtOAc, 4:1 ꢀ 1:1) yielded the hemiacetal (0.209 g, 0.264 mmol, 89%). Spectroscopic data are
1
reported for the major (α) isomer. H NMR (500 MHz, CDCl
3
): δ 7.39ꢀ7.20 (m, 15H, CHarom), 5.37 (dd, J = 3.0,
1
.9 Hz, 1H, Hꢀ2’), 5.06 (dd, J = 3.1, 1.9 Hz, 1H, Hꢀ2), 5.02 (m, 2H, Hꢀ1, Hꢀ1’), 4.91 (d, J = 11.2 Hz, 1H, CHH
Bn), 4.76 (d, J = 10.9 Hz, 1H, CHH Bn), 4.68ꢀ4.54 (m, 3H, CH Bn, CHH Bn), 4.46 (d, J = 11.9 Hz, 1H, CHH
2
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Bn), 4.18 (dd, J = 9.5, 3.2 Hz, 1H, Hꢀ2), 4.01ꢀ3.89 (m, 1H, Hꢀ5 or Hꢀ5’), 3.77 (dd, J = 9.2, 3.3 Hz, 1H, Hꢀ3’),
3
2 2
.62ꢀ3.51 (m, 1H, Hꢀ5 or Hꢀ5’), 3.39 (m, 2H, Hꢀ4, Hꢀ4’), 2.78ꢀ2.58 (m, 4H, CH Lev), 2.58ꢀ2.40 (m, 2H, CH
13
3 3 3 3
PivCN), 2.15 (s, 3H, CH Lev), 1.37ꢀ1.17 (m, 12H, 2x CH PivCN, 2x CH ꢀ6); C NMR (126 MHz, CDCl ): δ
2
1
7
6
06.5 (C=O Lev), 174.3, 171.9 (C=O Lev, PivCN), 138.6, 137.9, 137.8 (Cq), 128.6, 128.4, 128.4, 128.0, 128.0,
27.9, 127.8, 127.8, 127.7 (CHarom), 117.4 (CN), 99.8 (Cꢀ1’), 91.4 (Cꢀ1), 80.3 (Cꢀ4 or Cꢀ4’), 79.7 (Cꢀ4 or Cꢀ4’),
7.0 (Cꢀ3’), 76.6 (Cꢀ3), 75.6 (CH Bn), 75.0 (CH Bn), 73.6 (Cꢀ2), 71.3 (CH2 Bn), 69.1 (Cꢀ2’), 68.5 (Cꢀ5 or Cꢀ5’),
7.8 (Cꢀ5 or Cꢀ5’), 41.0 (Cq), 38.1 (CH Lev), 29.9 (CH Lev), 29.6 (CH Lev), 28.2 (CH PivCN), 27.8 (CH
PivCN), 18.1, 17.9 (2x CH Cꢀ6, Cꢀ6’);
2
2
2
3
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
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2
3
4
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7
8
9
0
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2
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8
9
0
1
2
3
4
5
6
7
8
9
0
PivCN), 24.9, 24.8 (2x CH
3
3
Anomeric PivCN (20a) Compound 18 (3.65 g, 4.15 mmol, 1 equiv.) was dissolved in DCM (40 mL) and cooled
to 0°C. NIS (1.03 g, 4.57 mmol, 1.1 equiv.) was added followed by the dropwise addition of TFA (0.35 mL, 4.57
mmol, 1.1 equiv.), after which the reaction turned purple. After 340 min, the reaction was quenched by addition of
5
0 mL sat. aq. Na
aqeaous layers were extracted 2x with DCM and the combined organic layers were washed with sat. aq. NaCl,
dried over MgSO and concentrated in vacuo. Column purification (PE/EtOAc, 4:1 ꢀ 1:1) resulted a mixture of
2 2 3 3
S O . The mixture was diluted with 60 mL DCM and washed with 60 mL sat. aq. NaHCO . The
4
2
0 and 20a 73% yield (2.37 g, 3.01 mmol). R
f
0.35 (PE/EtOAc, 2/1, v/v); IR (neat): 733, 839, 988, 1040, 1063,
1
1
135, 1363, 1454, 1497, 1717, 1737, 2933, 2976; H NMR (500 MHz, Chloroformꢀd) δ 7.41ꢀ7.20 (m, 40H), 6.07
(d, J = 2.2 Hz, 1H), 5.38 (dd, J = 3.3, 1.6 Hz, 3H), 5.13 (t, J = 2.8 Hz, 1H), 5.10ꢀ5.05 (m, 3H), 5.05ꢀ5.01 (m, 3H),
4.92 (dd, J = 11.2, 2.7 Hz, 3H), 4.81ꢀ4.73 (m, 3H), 4.67ꢀ4.54 (m, 8H), 4.53ꢀ4.43 (m, 3H), 4.18 (dd, J = 9.5, 3.2
Hz, 1H), 4.13 (dd, J = 9.3, 3.3 Hz, 1H), 3.86ꢀ3.72 (m, 4H), 3.63ꢀ3.49 (m, 4H), 3.45ꢀ3.34 (m, 4H), 2.75ꢀ2.60 (m,
13
1
2
1
1
1
7
1H), 2.56ꢀ2.40 (m, 5H), 2.16 (d, J = 2.8 Hz, 8H), 1.37ꢀ1.22 (m, 33H). C NMR (126 MHz, CDCl
3
) δ 206.4,
06.3, 173.9, 171.9, 171.9, 155.6, 155.2, 138.0, 138.0, 137.9, 137.9, 137.8, 137.3, 128.7, 128.7, 128.7, 128.6,
28.6, 128.6, 128.5, 128.5, 128.5, 128.5, 128.5, 128.5, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3,
28.3, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9, 127.9, 127.9, 127.9, 127.9, 127.8, 127.8, 127.8, 127.8, 127.8,
27.7, 127.7, 127.7, 127.7, 127.7, 127.6, 127.5, 117.8, 117.4, 117.2, 115.4, 113.1, 75.9, 75.8, 75.6, 75.1, 75.1,
4.9, 74.8, 72.1, 71.5, 71.4, 71.4, 71.3, 70.6, 69.7, 69.1, 68.9, 41.1, 41.1, 38.2, 38.1, 38.1, 29.8, 29.7, 28.2, 28.2,
+
28.2, 28.0, 27.9, 27.8, 27.8, 18.1, 18.1, 18.0. HRMS: [M+ NH
4
]
calcd. for C44
H
57
N
2
O12 805.3906, found
805.3908.
4
ꢀOꢀBenzylꢀ2ꢀOꢀ(3ꢀcyanoꢀ2,2ꢀdimethylpropanoyl)ꢀ3ꢀOꢀ(3,4ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀlevulinoylꢀαꢀLꢀ
rhamnopyranosyl)ꢀ1ꢀ(Nꢀphenylꢀtrifluoroacetimidoyl)ꢀ α/βꢀLꢀrhamnopyranoside (2) To a solution of mixture
hemiacetal 20 and 20a (4.15 g, 5.27 mmol, 1 equiv.) in acetone (26 mL) at 0°C were added Nꢀphenyl
trifluoroacetimidoyl chloride (0.98 mL, 6.32 mmol, 1.2 equiv.) followed by Cs CO (2.57 g, 7.9 mmol, 1.5 equiv.).
The solution was allowed to stir over night after which it was diluted with EtOAc and washed subsequently with
O and sat. aq. NaCl. The organic layer was dried over MgSO and concentrated in vacuo. Purification by
column chromatography (PE/EtOAc, 6:1 ꢀ 1:1) yielded the title compound as a clear yellow oil (3.98 g, 4.15
2
3
H
2
4
mmol, 79%). R
f
0.69 (PE/EtOAc, 2/1, v/v); IR (neat): 751, 1044, 1137, 1119, 1137, 1364, 1453, 1597, 1720, 1741,
ꢀ
1
1
2
1
1
4
4
1
935 cm ; Spectroscopic data are reported for the major (α) isomer. H NMR (500 MHz, CDCl
3
): δ 7.39ꢀ7.18 (m,
7H, CHarom), 7.12ꢀ7.04 (m, 1H, CHarom), 6.83ꢀ6.77 (m, 2H, CHarom), 6.00 (s, 1H, Hꢀ1), 5.38 (dd, J = 3.3, 1.9 Hz,
H, Hꢀ2’), 5.24 (dd, J = 3.3, 2.0 Hz, 1H, Hꢀ2), 5.06 (d, J = 2.0 Hz, 1H, Hꢀ1), 4.90 (d, J = 11.3 Hz, 1H, CHH Bn),
.79 (d, J = 10.9 Hz, 1H, CHH Bn), 4.66ꢀ4.54 (m, 3H, CHH, CH2 Bn), 4.48 (dd, J = 11.8, 3.2 Hz, 1H, CHH Bn),
.18 (dd, J = 9.5, 3.2 Hz, 1H, Hꢀ3), 3.92ꢀ3.81 (m, 1H, Hꢀ5), 3.79 (dd, J = 9.1, 3.4 Hz, 1H, Hꢀ3’), 3.67ꢀ3.57 (m,
2
H, Hꢀ5’), 3.53 (t, J = 9.5 Hz, 1H, Hꢀ4), 3.41 (t, J = 9.3 Hz, 1H, Hꢀ4’), 2.72ꢀ2.55 (m, 4H, CH Lev), 2.54ꢀ2.40 (m,
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H, CH
CDCl ) δ 205.8 (C=O Lev), 173.9, 171.8 (C=O Lev, PivCN), 143.4, 138.8, 138.2, 137.7 (Cq), 128.9, 128.7, 128.6,
28.5, 128.5, 128.4, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 124.7, 124.6, 119.5, 119.4,
17.1 (CHarom), 100.0 (Cꢀ1’), 93.7 (Cꢀ1), 79.9 (Cꢀ4), 79.8 (Cꢀ4’), 77.3 (Cꢀ3’), 76.9 (Cꢀ3), 75.8 (CH Bn), 75.1
Bn), 71.8 (Cꢀ2), 71.6 (CH Bn), 70.9 (Cꢀ5), 69.4 (Cꢀ2’), 68.9 (Cꢀ5’), 41.1 (Cq), 38.2 (CH Lev), 29.8 (CH
Lev), 28.4 (CH Lev), 28.0 (CH PivCN), 25.0, 24.9 (2x CH PivCN), 18.2 (Cꢀ6), 18.0 (Cꢀ6’); HRMS: [M+NH
calcd. C52 12 976.4202, found 976.4205.
2 3 3 3
PivCN), 2.14 (s, 3H, CH Lev), 1.39ꢀ1.21 (m, 12H, 2x CH PivCN, 2x CH ꢀ6); C NMR (126 MHz,
3
1
1
2
(CH
2
2
2
3
+
2
2
3
4
]
61 3 3
H F N O
10
11
12
13
14
15
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methods for automated synthesis
The washing solvents are preꢀdried 24h before use on 4Å molecular sieves and are of HPLC grade. Activator and
deblock solutions are freshly prepared using the preꢀdried solvents.
Activator: 0.09 M trifluoromethanesulfonic acid in DCE
Deblock: 0.12 M hydrazine acetate in pyridine/AcOH (4/1, v/v)
Method A. Agitation of the resin during washing
After addition of the appropriate solvent, an argonꢀflow is applied from the bottom of the RV, suspending the resin
in solution. The argonꢀflow is applied for 15 seconds after which the RV is emptied to the waste.
Method B. Agitation of the resin during coupling/deblock
After addition of the solvent, an argonꢀflow is applied from the bottom of the RV for 10 s, suspending the resin in
the solution. After 10 s, the argon flow is interrupted, and the resin is allowed to settle for 20 s.
Method C. Swelling of the resin
Dry resin is applied to the RV and washed with DCM (3x), alternating THF/Hexane (3x), THF (1x) and DCM
(3x).
Method D. Coupling cycle
The resin is suspended in DCM. The RV is emptied, followed by addition of the building block solution (1 mL)
while being agitated. The delivery line is flushed with an additional 0.5 mL DCM to the RV. The temperature is
set to 0°C while employing method B. A 10 min pause is started, after which the activator solution (300 µL) is
added, keeping the temperature below 0 °C. The delivery line is flushed with an additional 0.5 mL DCM to the
RV. Method B is applied for 1h, after which the RV is emptied and the mixture is collected in the fraction
collector. The resin is washed with DCM (3x 2 mL), and the washes are drained to the fraction collector.
Method E. Deblock cycle
The resin is washed with DMF (4x 3 mL), running method A. The deblock solution is added (3 mL) and the
temperature is set to 40 °C, followed by a 5 min incubation applying method B. The temperature is kept at 40 °C,
after which the solid support is incubated 10 minutes applying protocol B. Then the RV is emptied to the waste.
The resin is washed with DMF (3x 3 mL), running method A.
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Method F. Washing of the resin after coupling
The temperature is set to 20 °C. The resin is washed with MeOH (3x 2 mL), alternating THF/Hexane (6x 2 mL),
THF (2x 2 mL), DCM (5x 3 mL), all applying method A.
Method G. Washing of the resin after deblock
The temperature is set to 20 °C. The resin is washed with DMF (4x 3 mL), DCM (4x 3 mL), alternating
THF/Hexane (6x 3 mL), 0.01M AcOH in THF (6x 3 mL), THF (4x 3 mL) and DCM (8x 5 mL).
0
1
2
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8
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0
1
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0
1
2
3
4
5
6
7
8
9
0
Method H. Suspending the resin for isolation
To the dry resin is added a mixture of DCM/MeOH (3:2; 5 mL), after which the resin is agitated for 15 s. The
suspended resin is collected from the RV. The procedure is repeated four times.
Method
C
# Cycles
Description
Swelling of the resin
Time
60 min
15 min
Temperature
1
RT
Coupling: 3 equiv. donor, 0.3 equiv.
TfOH
D
1
0 °C
E
F
3
3
1
Washing of the resin after coupling
20 °C
40 °C
20 °C
Deblock: 8 equiv. H
2 2
NNH ·AcOH
G
Washing of the resin after deblock
Automated synthesis of rhamnose fragments. The reaction vessel is charged with CarboxyPolystyrene 3 (100
mg, 45 µmol) and method C was applied to prepare the resin for synthesis. Then method D and E for coupling and
deprotection are repeated 5 times for decasaccharide 22 and 8 times to obtain hexadecasaccharide 23. Method H
was used to isolate the resin from the reaction vessel. The resin was dried overnight. After cleavage from solid
support the rhamnose fragments were analyzed by LC/MS.
Decarhamnoside (22) The dry resin was charged in a syringe with screw cap and suspended in THF/MeOH (2
mL, 1:1) followed by addition of NaOMe (0.08 mL, 0.54M NaOMe/MeOH, 1 equiv.). The resin was shaked
overnight. The solution was filtered and the remaining resin was washed with MeOH (5x 4 mL). The combined
filtrate and washes were neutralized with 2ꢀ3 drops of AcOH and concentrated in vacuo. The cleavage procedure
was repeated once to obtain the mixture containing 22 as an amorphous solid (0.161 g).
Hexadecarhamnoside (23) The dry resin was charged in a syringe with screw cap and suspended in THF/MeOH
(2 mL, 1:1) followed by addition of NaOMe (0.08 mL, 0.54M NaOMe/MeOH, 1 equiv.). The resin was shaked
overnight. The solution was filtered and the remaining resin was washed with MeOH (5x 4 mL). The combined
filtrate and washes were neutralized with 2ꢀ3 drops of AcOH and concentrated in vacuo. The cleavage procedure
was repeated once to obtain the mixture containing 23 as an amorphous solid (0.198 g).
General procedure for complete removal of PivCN groups. The crude rhamnoside mixture was dissolved in
THF/MeOH (0.6ꢀ2 mL, 1:1), and treated with a 0.54M NaOMe/MeOH (0.7ꢀ2 equiv.) solution. The reaction was
monitored by LC/MS and allowed to stir overnight. Additional 0.54M NaOMe/MeOH was added when LC/MS
analysis indicated incomplete removal of the PivCN groups. If the deprotection proceeded slowly, the mixture was
neutralized, concentrated in vacuo and treated with the conditions mentioned vide supra. Purification by size
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exclusion chromatography (LH20, eluted with DCM/MeOH, 1/1,v/v) or HPLC yielded the target rhamnoside
fragments.
Semiꢀprotected decarhamnoside (26) The crude rhamnoside mixture (0.162 g) was dissolved in THF/MeOH (4
mL, 1:1) and treated with 0.16 mL NaOMe (0.54M NaOMe/MeOH). After overnight stirring, LC/MS analysis
indicated incomplete removal of the PivCN groups, after which the mixture was neutralized with AcOH and
concentrated in vacuo. The mixture was redissolved in THF/MeOH (2 mL, 1:1), treated with 0.1 mL NaOMe
(0.54M NaOMe/MeOH) and stirred overnight. After overnight stirring, 0.08 mL NaOMe (0.54M NaOMe/MeOH)
was added, followed by 0.16 mL NaOMe (0.54M NaOMe/MeOH) after 6.5h, whereafter LC/MS analysis
indicated complete removal of the PivCN groups. The mixture was neutralized with AcOH and concentrated in
vacuo and coevaporated once with toluene. The target decarhamnoside was isolated using RPꢀHPLC purification
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(C4 column, gradient 70 ꢀ 90 , 20 min per run) as a white solid (37.4 mg, 11.8 µmol, 26% based on 45 µmol
–1 1
resin). IR (neat): 736, 1028, 1041, 1070, 1126, 1207, 1361, 1454, 1496, 1681, 2927, 3030, 3377 cm ; H NMR
500 MHz, MeCNꢀd , T=328K) δ 7.42ꢀ7.18 (m, 84H, CHarom), 5.11ꢀ5.06 (m, 4H, CH linkerꢀCBz, 2x Hꢀ1), 5.04 (s,
H, Hꢀ1), 4.98 (s, 1H, Hꢀ1), 4.87ꢀ4.49 (m, 33H), 4.45 (s, 2H, CH linker), 4.05ꢀ3.95 (m, 8H), 3.95ꢀ3.77 (m, 14H),
.72 (m, 4H), 3.66ꢀ3.51 (m, 2H), 3.52ꢀ3.34 (m, 9H), 3.32 (s, 1H), 3.21 (t, J = 7.3 Hz, 2H, CH linker), 3.06 (s,
linker), 1.35ꢀ1.24 (m, 7H, CH2 linker, CH ꢀ6), 1.24ꢀ1.11 (m, 17H, CH ꢀ6), 1.10ꢀ1.00
ꢀ6); C NMR (126 MHz, MeCNꢀd , T=328K) δ 140.1 (Cq), 129.6, 129.5, 129.5, 129.4, 129.4,
29.3, 129.2, 129.2, 129.1, 129.0, 129.0, 128.8, 128.8, 128.7, 128.7, 128.3, 128.0 (CHarom), 103.2, 103.0, 103.0,
03.0, 102.4, 101.2, 101.0 (10x Cꢀ1), 81.5, 81.4, 81.3, 81.2, 81.0, 80.9, 80.8, 80.8, 80.0 (10x Cꢀ3, 10x Cꢀ4), 77.4,
(
3
2
1
3
2
2
5H), 1.53ꢀ1.46 (m, 4H, CH
2
3
3
13
(m, 10H, CH
3
3
1
1
7
7.3, 77.2 (Cꢀ2), 76.1, 76.0, 75.9 (CH
), 51.5 (CH ), 30.1 (CH
44 3188.5173, found 3188.5121.
2
), 72.9, 72.3, 72.2 (CH
2
), 72.1 (10 xCꢀ2), 69.6, 69.6, 69.3, 69.0, 68.3, 67.8
+
(10x Cꢀ5), 64.8 (CH
2
2
2
), 24.4 (CH ), 18.8, 18.7, 18.7, 18.6 (10x CH ꢀ6). HRMS: [M+ NH ]
2
3
4
221 2
calcd. for C186H N O
Semiꢀprotected hexadecarhamnoside (29) The crude rhamnoside mixture (0.199 g) was dissolved in
THF/MeOH (2 mL, 1:1) and treated with 0.10 mL NaOMe (0.54M NaOMe/MeOH). After 2h, an additional 0.24
mL NaOMe (0.54M NaOMe/MeOH) was added, followed by another 0.10 mL after 4h. After overnight stirring,
LC/MS analysis indicated complete removal of all PivCN groups after which the mixture was neutralized by
addition of 2ꢀ3 drops AcOH. The mixture was concentrated in vacuo and coevapotrated with toluene once. The
target hexadecarhamnoside was isolated using RPꢀHPLC purification (C4 column, gradient 70 ꢀ 90 , 20 min per
run) as a white solid (20.3 mg, 4.2 µmol, 9.3% based on 45 µmol resin). IR (neat): 750, 1051, 1129, 1454, 1671,
ꢀ1 1
2
917 cm ; H NMR (600 MHz, MeCNꢀd
m, 8H), 5.03 (d, J = 1.9 Hz, 1H), 4.97 (d, J = 1.7 Hz, 1H), 4.87ꢀ4.47 (m, 60H), 4.44 (s, 2H), 4.08ꢀ3.93 (m, 16H),
3.94ꢀ3.80 (m, 24H), 3.80ꢀ3.63 (m, 8H), 3.63ꢀ3.51 (m, 4H), 3.51ꢀ3.34 (m, 16H), 3.33ꢀ3.24 (m, 2H), 3.20 (t, J = 7.3
3
, T=328K): δ 7.39 (d, J = 7.4 Hz, 2H), 7.36ꢀ7.17 (m, 129H), 5.12ꢀ5.04
(
13
Hz, 2H), 3.17ꢀ2.79 (m, 9H), 1.55ꢀ1.43 (m, 4H), 1.36ꢀ1.22 (m, 8H), 1.21ꢀ1.10 (m, 29H), 1.09ꢀ0.95 (m, 21H);
NMR (151 MHz, MeCNꢀd , T=328K): δ 140.1, 140.0, 140.0, 140.0, 129.6, 129.5, 129.4, 129.4, 129.4, 129.3,
29.3, 129.2, 129.1, 129.1, 129.0, 128.9, 128.9, 128.8, 128.7, 128.7, 128.7, 128.6, 128.6, 128.2, 127.9, 118.3,
C
3
1
1
7
1
03.1, 103.0, 102.9, 102.9, 102.9, 102.3, 102.3, 100.9, 81.4, 81.3, 81.3, 81.2, 81.2, 81.1, 81.0, 80.8, 80.7, 79.9,
7.3, 77.3, 77.2, 77.0, 76.0, 75.9, 75.9, 72.8, 72.8, 72.2, 72.1, 72.0, 69.5, 69.5, 69.2, 68.9, 64.7, 51.4, 30.0, 24.3,
+
8.8, 18.7, 18.6, 18.6, 18.6, 18.5, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9. MALDIꢀTOF m/z [M+Na] calc. for
285
C H331NO68Na 4878.2, found 4884.9.
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General procedure for the hydrogenation. The oligosaccharide was dissolved in H
2
O/THF/tBuOH (3:1.3:1.3)
for 5 min, after which
for 5 min. The solution was purged for 5
atmosphere overnight. The mixture was filtered over a Whatmann filter, and
O/THF/tBuOH mixture and H O.
followed by addition of several drops of AcOH. The solution was purged with N
Pd(OH) /C (10ꢀ20 mg) was added followed by another purge with N
min with H and kept under a H
rinsed with the H
2
2
2
2
2
2
2
0
1
2
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4
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0
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0
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7
8
9
0
Decarhamnoside (5) Compound 26 (19.1 mg, 6 µmol) was dissolved in H
and 4ꢀ5 drops of AcOH were added. The solution was purged with N for 5 min after which Pd(OH)
was added, followed by another purge with N for 5 min. The solution was purged for 5 min with H2 and kept
under a H atmosphere overnight. After overnight stirring, the mixture was filtered through a Whatmann filter and
concentrated in vacuo. Purification by size exclusion chromatography (LH20, eluted with MeOH/H O, 9/1,v/v)
and analysis by HꢀNMR indicated the presence of aromatic signals. The hydrogenation procedure was repeated
2
O/THF/tBuOH (1.6 mL, 3:1.3:1.3)
2
2
/C (20 mg)
2
2
2
once. Purification using gel filtration (HWꢀ40, eluted with NH
4
OAc) and subsequent lyophilization yielded the
1
target decarhamnoside as a white powder (5.3 mg, 3.4 µmol, 57%). H NMR (500 MHz, D
2
O): δ 5.11ꢀ5.06 (m,
4
H), 4.93 (d, J = 1.8 Hz, 1H), 4.87ꢀ4.81 (m, 4H), 4.05 (m, 4H), 3.99ꢀ3.93 (m, 5H), 3.91ꢀ3.87 (m, 1H), 3.86ꢀ3.80
(m, 4H), 3.78ꢀ3.70 (m, 9H), 3.70ꢀ3.56 (m, 8H), 3.49ꢀ3.31 (m, 11H), 2.89 (t, J = 7.6 Hz, 2H), 1.64ꢀ1.52 (m, 4H),
13
1.42ꢀ1.29 (m, 2H), 1.24ꢀ1.09 (m, 30H); C NMR (126 MHz, D
2
O): δ 102.5, 102.2, 102.2, 101.0, 101.0, 100.9,
9
6
9.7, 78.3, 78.2, 78.1, 77.7, 77.6, 77.6, 72.3, 72.1, 71.9, 71.8, 71.5, 70.3, 70.3, 70.1, 70.1, 70.1, 70.0, 69.5, 69.5,
+
9.4, 69.3, 68.8, 67.6, 39.5, 28.2, 26.7, 22.8, 22.6, 16.9, 16.8, 16.8, 16.7, 16.6; HRMS: [M+H] calcd. for
114NO41 1564.6861, found 1564.6873.
65
C H
Hexadecarhamnoside (8) Compound 29 (7.2 mg, 1.5 µmol) H
of AcOH were added. The solution was purged with N for 5 min after which Pd(OH)
followed by another purge with N for 5 min. The solution was purged for 5 min with H
atmosphere overnight. After overnight stirring, the mixture was filtered through a Whatmann filter and
2
O/THF/tBuOH (1.0 mL, 3:1.3:1.3) and 4ꢀ5 drops
/C (8 mg) was added,
and kept under a H
2
2
2
2
2
concentrated in vacuo. Purification using gel filtration (HWꢀ40, eluted with NH
4
OAc) and subsequent
1
lyophilization yielded the target hexadecarhamnoside as a white powder (1.8 mg, 0.75 µmol, 50%). H NMR (500
MHz, D
2
O): δ 5.26ꢀ5.12 (m, 7H), 5.04 (d, 2H, J = 1.5 Hz), 4.95 (s, 9H), 4.16 (s, 7H), 4.07 (s, 8H), 3.99 (s, 1H),
3
2
7
2
.95 (m, 7H), 3.88ꢀ3.68 (m, 25H), 3.59ꢀ3.43 (m, 17H), 2.99 (t, 2H, J = 7.5 Hz), 1.74ꢀ1.59 (m, 4H), 1.52ꢀ1.39 (m,
13
2
H), 1.35ꢀ1.20 (m, 48H); C NMR (126 MHz, D O): δ 102.4, 102.1, 102.1, 100.9, 100.9, 99.6, 78.2, 78.1, 78.0,
7.6, 77.4, 72.2, 72.0, 71.7, 71.4, 70.2, 70.1, 70.0, 70.0, 69.9, 69.4, 69.4, 69.3, 69.2, 68.7, 67.5, 39.4, 28.1, 26.6,
+
3.3, 22.5, 16.8, 16.7, 16.7, 16.6, 16.5. HRMS: [M+H] calcd. for C101
H174NO65 2442.0369, found 2442.0361.
Isolation of deletion fragments
1
Octarhamnoside (25) Obtained as byproduct from 22. (10.4 mg, 4.0 µmol). H NMR (500 MHz, MeCNꢀd3,
T=328K): δ 7.42 – 7.37 (m, 2H), 7.36 – 7.13 (m, 67H), 5.13 – 5.07 (m, 4H), 5.04 (d, J = 2.1 Hz, 1H), 4.98 (d, J =
1
3
.8 Hz, 1H), 4.88 – 4.73 (m, 10H), 4.72 – 4.52 (m, 20H), 4.45 (s, 2H), 4.06 – 3.96 (m, 7H), 3.95 – 3.78 (m, 13H),
.78 – 3.67 (m, 3H), 3.63 – 3.51 (m, 2H), 3.51 – 3.36 (m, 8H), 3.37 – 3.28 (m, 1H), 3.22 (t, J = 7.3 Hz, 2H), 3.05
13
(
s, 5H), 1.56 – 1.44 (m, 4H), 1.30 (s, 6H), 1.24 – 1.12 (m, 15H), 1.11 – 1.02 (m, 9H); C NMR (126 MHz,
MeCNꢀd3, T=328K): δ 140.1, 129.6, 129.6, 129.5, 129.4, 129.4, 129.3, 129.3, 129.2, 129.2, 129.1, 129.1, 129.0,
29.0, 128.9, 128.8, 128.8, 128.7, 128.7, 128.7, 128.6, 128.3, 128.3, 127.9, 111.2, 103.1, 102.9, 102.3, 101.0, 81.4,
1
81.3, 81.3, 81.3, 81.1, 81.1, 81.0, 80.9, 80.8, 79.9, 77.2, 77.1, 76.1, 75.9, 75.9, 72.8, 72.8, 72.3, 72.1, 72.1, 69.6,
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+
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2
9.5, 69.2, 69.0, 68.2, 67.7, 64.8, 18.8, 18.7, 18.6. HRMS: [M+H] calcd. For C153
H180NO36 2608.2246, found
608.2273.
Deprotected octarhamnoside (4) White solid after general hydrogenation procedure (1.35 mg, 1.06 µmol, 69%).
1
H NMR (500 MHz, D
2
O) δ 5.24 – 5.13 (m, 3H), 5.03 (s, 1H), 4.94 (s, 3H), 4.15 (s, 3H), 4.06 (s, 4H), 3.98 (s, 1H),
3
–
.97 – 3.90 (m, 3H), 3.87 – 3.66 (m, 14H), 3.60 – 3.39 (m, 9H), 2.99 (t, J = 7.4 Hz, 2H), 1.76 – 1.59 (m, 4H), 1.54
13
2
1.37 (m, 2H), 1.36 – 1.18 (m, 24H); C NMR (126 MHz, D O) δ 109.8, 102.5, 102.2, 100.9, 99.7, 78.1, 77.6,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
72.4, 72.1, 71.9, 71.8, 71.5, 70.3, 70.0, 69.5, 69.4, 69.3, 68.8, 67.6, 59.3, 39.5, 28.2, 26.7, 22.6, 16.8, 16.8, 9.3.
+
HRMS: [M+H] calcd. For C53
H94NO33 1272.5703, found 1272.5714.
1
Dodecarhamnoside (27) Obtained as byproduct from 23. (10.3 mg, 2.8 µmol). H NMR (600 MHz, MeCNꢀd
3
,
T=328K) δ 7.42 – 7.17 (m, 99H), 5.12 – 5.05 (m, 6H), 5.03 (d, J = 2.1 Hz, 1H), 4.97 (d, J = 2.0 Hz, 1H), 4.87 –
4
3
1
1
.48 (m, 44H), 4.45 (s, 2H), 4.05 – 3.94 (m, 12H), 3.93 – 3.76 (m, 19H), 3.76 – 3.66 (m, 6H), 3.64 – 3.51 (m, 3H),
.50 – 3.35 (m, 13H), 3.33 – 3.26 (m, 1H), 3.26 (s, 1H), 3.21 (t, J = 7.3 Hz, 3H), 1.54 – 1.44 (m, 4H), 1.29 (s, 9H),
13
.23 – 0.98 (m, 36H); C NMR (151 MHz, CD
3
CN) δ 143.0, 140.3, 140.1, 140.1, 140.0, 140.0, 140.0, 140.0,
39.8, 137.3, 129.6, 129.5, 129.4, 129.4, 129.4, 129.4, 129.4, 129.4, 129.3, 129.3, 129.3, 129.3, 129.2, 129.2,
129.1, 129.1, 129.1, 129.0, 129.0, 129.0, 128.9, 128.8, 128.8, 128.7, 128.7, 128.7, 128.7, 128.6, 128.6, 103.1,
103.0, 102.9, 102.9, 102.9, 102.9, 102.9, 102.3, 102.3, 101.0, 81.4, 81.3, 81.2, 81.2, 81.2, 81.1, 81.0, 80.9, 80.8,
7
1
6.1, 76.0, 76.0, 75.9, 75.9, 75.9, 72.8, 72.8, 72.7, 72.2, 69.5, 69.5, 68.2, 67.7, 64.7, 30.0, 27.7, 24.3, 18.7, 18.6,
+
8.6, 18.6, 18.5. MALDIꢀTOF m/z [M+Na] calc. for C219
H255NO52Na 3753.7, found 3756.4.
Deprotected dodecarhamnoside (6) White solid after general hydrogenation procedure (0.51 mg, 0.27 µmol,
1
2
4
7%). H NMR (500 MHz, D
2
O) δ 5.24 – 5.15 (m, 5H), 5.05 – 5.00 (m, 1H), 4.94 (s, 6H), 4.14 (d, J = 2.4 Hz, 6H),
.06 (s, 7H), 3.98 (s, 1H), 3.97 – 3.88 (m, 7H), 3.87 – 3.65 (m, 24H), 3.61 – 3.35 (m, 16H), 3.04 – 2.93 (m, 2H),
+
1.76 – 1.60 (m, 6H), 1.53 – 1.39 (m, 2H), 1.35 – 1.18 (m, 36H). HRMS: [M+H] calcd. for C77
1856.8019, found 1856.8064.
H134NO49
1
Tetradecarhamnoside (28) Obtained as byproduct from 23. (20 mg, 4.65 µmol). H NMR (600 MHz, MeCNꢀd
3
,
T=328K): δ 7.48 – 7.14 (m, 114H), 5.10 – 5.05 (m, 6H), 5.03 (d, J = 2.1 Hz, 1H), 4.97 (d, J = 2.0 Hz, 1H), 4.87 –
4
3
–
1
.48 (m, 46H), 4.45 (s, 2H), 4.06 – 3.94 (m, 12H), 3.94 – 3.77 (m, 20H), 3.71 (m, 6H), 3.64 – 3.50 (m, 3H), 3.50 –
.35 (m, 13H), 3.30 (s, 1H), 3.21 (t, J = 7.3 Hz, 2H), 3.16 – 3.01 (m, 7H), 1.50 (m, 4H), 1.36 – 1.24 (m, 5H), 1.24
1
3
0.97 (m, 42H); C NMR (151 MHz, CD
3
CN) δ 140.3, 140.1, 140.1, 140.0, 140.0, 140.0, 140.0, 140.0, 139.8,
37.3, 129.5, 129.5, 129.4, 129.4, 129.4, 129.4, 129.4, 129.3, 129.3, 129.3, 129.2, 129.2, 129.1, 129.1, 129.1,
129.1, 129.0, 129.0, 129.0, 128.8, 128.8, 128.7, 128.7, 128.7, 128.7, 128.7, 128.6, 128.6, 128.6, 103.1, 103.0,
102.9, 102.9, 102.9, 102.9, 102.3, 102.3, 101.0, 81.3, 81.2, 81.2, 81.1, 80.7, 76.1, 76.0, 75.9, 75.9, 75.9, 75.9, 72.8,
+
7
2.8, 72.7, 72.2, 72.1, 69.5, 69.5, 68.2, 67.7, 64.7, 30.0, 24.3, 18.6. MALDIꢀTOF m/z [M+K] calc. For
252
C H293KNO60 4332.0, found 4337.0.
Deprotected Tetradecarhamnoside (7) White solid after general hydrogenation procedure (4.7 mg, 2.2 µmol,
1
9
2%) H NMR (500 MHz, D
2
O): δ 5.23 – 5.14 (m, 6H), 5.04 (d, J = 1.7 Hz, 1H), 4.98 – 4.90 (m, 7H), 4.17 – 4.13
(m, 6H), 4.07 (s, 7H), 4.02 – 3.98 (m, 1H), 3.98 – 3.90 (m, 7H), 3.89 – 3.68 (m, 26H), 3.61 – 3.38 (m, 16H), 3.04
13
–
2
2.95 (m, 2H), 1.77 – 1.57 (m, 5H), 1.54 – 1.37 (m, 3H), 1.36 – 1.18 (m, 42H). C NMR (126 MHz, D O): δ
102.5, 102.1, 100.9, 99.6, 78.1, 78.0, 77.6, 77.4, 72.2, 72.0, 71.8, 71.7, 71.4, 70.2, 70.1, 69.9, 69.4, 69.3, 69.2,
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
6
2
8.7, 67.5, 39.4, 28.1, 26.6, 22.5, 16.8, 16.7, 16.5. HRMS: [M+H] calcd. For C89
H154NO57 2149.9211, found
149.9220.
0
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Acknowledgments
This work was supported by The Netherlands Organization of Scientific Research (NWO
Vidi grant to J.D.C.). We thank F. Lefeber, and K. Babu for their assistance with the NMR
experiments and F. Chiodo for his assistance with MALDIꢀTOF measurements.
Supporting Information
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Hꢀ and CꢀNMR spectra of all new compounds.
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For this synthesis, the reaction time for the glycosylation reaction was doubled (60 min instead of 30
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equivalents.
2 2
NNH ·AcOH used for deprotection of the Lev group was increased to 8
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