One pot synthesis of thio -glycosides via aziridine opening reactions

Article abstract of DOI:10.1039/d0ob01956a

A one-pot aziridine opening reaction by glycosyl thiols generated in situ from the corresponding anomeric thio-acetates affords thio-glycosides with a pseudo-disaccharide structure and an N-linked tether. The scope of the one-pot aziridine opening reaction was explored on a series of mono- and disaccharides, creating a class of pseudo-glycosidic compounds with potential for further functionalization. Unexpected anomerization of glycosyl thiols was observed under the reaction conditions and the influence of temperature, base and solvent on the isomerization was investigated. Single isomers were obtained in good to acceptable yields for mannose, rhamnose and sialic acid derivatives. The class of thio-glycomimetics synthesized can potentially be recognized by various lectins, while presenting hydrolytic and enzymatic stability. The nitrogen functionality incorporated in the glycomimetics can be exploited for further functionalization, including tethering to linkers, scaffolds or peptide residues.

Full text of DOI:10.1039/d0ob01956a

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One pot synthesis of thio-glycosides via aziridine
opening reactions†
Cite this: DOI: 10.1039/d0ob01956a
Nives Hribernik,
Alice Tamburrini, Ermelinda Falletta and Anna Bernardi
*
A one-pot aziridine opening reaction by glycosyl thiols generated in situ from the corresponding anome-
ric thio-acetates affords thio-glycosides with a pseudo-disaccharide structure and an N-linked tether.
The scope of the one-pot aziridine opening reaction was explored on a series of mono- and disacchar-
ides, creating a class of pseudo-glycosidic compounds with potential for further functionalization.
Unexpected anomerization of glycosyl thiols was observed under the reaction conditions and the
influence of temperature, base and solvent on the isomerization was investigated. Single isomers were
obtained in good to acceptable yields for mannose, rhamnose and sialic acid derivatives. The class of
thio-glycomimetics synthesized can potentially be recognized by various lectins, while presenting hydro-
lytic and enzymatic stability. The nitrogen functionality incorporated in the glycomimetics can be
exploited for further functionalization, including tethering to linkers, scaffolds or peptide residues.
Received 23rd September 2020,
Accepted 12th November 2020
DOI: 10.1039/d0ob01956a
rsc.li/obc
but is significantly more stable to enzymatic hydrolysis18 and
arguably simpler to synthesize.
Introduction
It is well known that interactions between carbohydrates and
Inspired by the potential of this system, we aimed at develop-
proteins play an important role in physiological and pathologi- ing a one-pot procedure for opening the corresponding aziri-
cal processes of major relevance and yet the therapeutic use of dine 4. This approach would afford N-linked-pseudo-thio-disac-
carbohydrates is limited by their difficult synthesis, their low charides such as 5. Natural N-linked glycans, which represent
intrinsic activity and their chemical nature, which does not fit one of the most common covalent modification of proteins,19
expectations for drug-like molecules.1 To overcome at least are generally connected to an Asn side-chain through a
some of these drawbacks, chemically modified analogues of β-GlcNAc residue. Thus, here we are not trying to reproduce or
natural carbohydrates, so called glycomimetics, have been pro- mimic the structure of these glycans, but are using a totally arti-
posed.2 The development of functional carbohydrate mimics ficial structure (an α-Man mimic) to allow the rapid generation
has been tackled either by structure-based design,1,3–13 or by of hydrolytically stable pseudo-glycoconjugates that can be
discovery campaigns, often planned taking advantage of tested in a variety of fashions against relevant carbohydrate
microarray technology.14–16 The latter approach has been fru- binding proteins in drug discovery programs. Initially, we devel-
strated by the cumbersome synthesis of oligosaccharides, that oped the approach using the mannosyl thio-acetate 1 as the
severely limits the diversity and the number of compounds model compound. We then further explored the scope of the
that can be rapidly generated.
one-pot aziridine opening reaction on various mono- and disac-
We have recently reported that the one-pot opening reaction charides, to create a class of glycomimetics (N-linked-pseudo-
of epoxide 2 by the glycosyl thiol generated in situ from 2,3,4,6- thio-disaccharides) with improved stability and the potential of
tetra-O-acetyl-1-S-acetyl-α-D-mannopyranose 1 provides a facile being recognized by various lectins. While looking into the reac-
access to the 1,2-dimannoside mimic 3, which is produced as tion scope, we observed interesting phenomena connected to
a single isomer resulting from exclusive trans-diaxial opening the configurational stability of anomeric thiols, some of which
of the epoxide (Scheme 1).17 The pseudo-thio-1,2-dimannoside unexpectedly underwent anomeric isomerization under the azir-
3 binds to the dendritic cell receptor DC-SIGN with an affinity idine opening conditions. Our results are reported below.
comparable to that of the natural disaccharide Manα(1,2)Man,
Results
Università degli Studi di Milano, Dipartimento di Chimica, via Golgi 19,
Synthesis of peracetylated glycosyl thio-acetates
20133 Milano, Italy. E-mail: anna.bernardi@unimi.it
Various methods for the synthesis of peracetylated glycosyl
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0ob01956a
thio-acetates have been reported. To simplify the synthesis
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Scheme 1 One-pot synthesis of the pseudo-thio-1,2-dimannoside 3 by epoxide opening (from ref. 17) and the planned approach to the aziridine-
opening process (this article).
and at the same time achieve satisfying yields, we selected series of different acylating agents and conditions were
one-pot procedures with conditions tailored for each sugar explored (Scheme 3). Reaction of 13 with acyl chlorides and
individually. The conditions used are summarized in pyridine did not afford the expected amide. Rather, products
Scheme 2 and described in detail in the ESI.† Briefly, the 1-S- resulting from nucleophilic opening of the aziridine by chlor-
acetyl derivatives of mannose (Man), rhamnose (Rha), ide ions were identified by MS analysis of the crude reaction
glucose (Glc), galactose (Gal) and lactose (Lac) (Scheme 2, 1 mixtures.26–28 Clean amide formation could be obtained either
and 6–9) were obtained by acid catalysed acetylation of the by carbodiimide-promoted coupling, or using activated esters.
free sugar, followed by reaction with thioacetic acid (AcSH) The reaction occurred smoothly, including with functionalized
under TMSOTf or BF3 catalysis (two step, one pot).20,21 and hindered acyl donors 17–20 (Scheme 3), that could be
The 1-S-acetyl derivatives of sialic acid (Neu5Ac) and used to install appropriate linkers for further (pseudo)-glyco-
N-acetylglucosamine (GlcNAc)22 (Scheme 2, 10–11) were syn- conjugation of the fully-formed mimics.
thesized by reaction of AcSK with the corresponding peracety-
One-pot aziridine opening
lated anomeric chloride, generated in situ by reaction of the
free sugar with AcCl. In most cases, the peracetylated glycosyl
thio-acetates were obtained as single isomers and, when
not, the two isomers were chromatographically separated
(see ESI†).
The aglycone moiety 4 used in this study was carefully selected
for its symmetry properties and high conformational stability,
both imparted by the 1,2-trans-dicarboxy substituents.29
Additionally, aziridines 4 lack potential chelating groups that
have been shown to impinge on the regio- and stereocontrol of
nucleophilic substitutions of both cyclitol epoxides and
Synthesis of aziridine 4
The required aziridine 4 was prepared as a single enantiomer aziridines.30,31 Thus we expected a single reaction product to
by N–H aziridination of (1S,2S)-1,2-dicarbomethoxy-4-cyclohex- be formed by trans-diaxial opening of the aziridine, in keeping
ene 1223 using the method recently introduced by Falck and with the results of the analogous reaction of epoxide 2 with 1-
co-workers.24 The resulting N–H aziridine 13 can then be S-acetyl-α-D-mannopyranose 1.17
transformed in a variety of amides and carbamates of general
Indeed, reaction of 1 with 4a (Scheme 4) proceeded
formula 4 (Scheme 3). Falck’s N–H aziridination occurs via smoothly under the conditions established for epoxide 2
homogenous Rhodium catalysis (Rh2(esp)2, Du Bois’ catalyst (1.9 mol equiv. of Et2NH in DMF at room temperature).
14) and uses O-(2,4-dinitrophenyl)hydroxylamine (DPH 15) as Product 5a was obtained in 82% yield as a single isomer from
the aminating agent, with no external oxidants. The reaction is a completely selective trans-diaxial opening process, preser-
operationally simple, fast and scalable, but careful optimiz- ving, as expected, the α-configuration of mannose. The
ation of the reaction conditions was required to avoid dimeri- product configuration was fully confirmed by coupling con-
zation of 13, a process which occurs rapidly at room tempera- stant analysis and NOESY, as previously described for 3.17 To
ture to afford 16 (Scheme 3). Under optimal conditions (6 h at further explore the role of the nitrogen protecting group in this
0 °C, addition of 10% catalyst and DPH after 3 h) on a reaction, substrates 4b–d were also examined (Scheme 4).
1.0 mmol scale no dimer was observed and the reaction yield Reaction of 1 with 4b failed to afford the desired aziridine
was 90%, after Boc-protection of 13 to afford 4a (Scheme 3). opening product (5b), rather the thiolate anion generated
Boc protection was performed using di-t-butyl-dicarbonate and in situ from 1 displaced the terminal chloride of the linker, to
adapting the conditions reported by Mordini and co-workers give 21.32 Steric hindrance of the primary chloride, as in 4c,
for a similar substrate.25
abolished this side reaction, and restored the aziridine
Acylation of the aziridine nitrogen protects the molecule opening pathway giving 5c in almost quantitative yields.
against polymerization, while preserving its reactivity as an Reaction of the corresponding azide-bearing compound 4d
electrophile in a nucleophilic substitution reaction. The nitro- was more sluggish, but microwave irradiation of the reaction
gen protecting group can serve multiple purposes in the mixture at 60 °C for 2 h afforded 5d in 68% yield. The same
design of the glycomimetic structures of our interest, thus a product was obtained in 71% yield from 5a by Boc removal
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Scheme 2 Synthesis of peracetylated glycosyl thio-acetates 1 and 6–11.
(TFA, quant) followed by acylation of crude 22 with the p-nitro- afforded 23 with 92% isolated yield (0.3 mmol scale, Table 2,
phenyl ester 20, thus establishing 5a as an appropriate inter- entry 2) as a single α isomer, as established by NOESY experi-
mediate for later stage elaboration of the scaffold linker ments (see ESI†).
(Scheme 4).
In the same conditions, however, reaction of the β-Glc
We then moved on to investigate the scope of the aziridine thioacetate 7 afforded both the β- and α-isomers β-24 and α-24
opening approach using the mono- and disaccharide anomeric (Scheme 6) in 2 : 1 ratio, as estimated by integration of the
thio-acetates 6–11 and the N-Boc-aziridine 4a as the model sub- anomeric proton signals at 4.72 and 5.73 ppm, respectively.
strate (Scheme 5). The reactions were performed under the The two isomers were separated chromatographically
conditions established with mannose and their course was fol- (iPr2O : EtOAc eluent), and analysis of coupling constants sup-
lowed by LC-MS and/or 1H NMR. LC-MS revealed that the ported by NOESY-NMR and MS data undoubtedly confirmed
expected products were formed in most cases within 1 h at their structure and anomeric configuration. Similarly, the dis-
room temperature. The main by-product observed was the gly- ulphide by-product 29 was obtained as an anomeric mixture
cosyl disulphide, as exemplified by 29 for the gluco series (see ESI†). This was rather unexpected, because most literature
(Scheme 6). Reaction of 4a with the α-Rha derivative 6 claims configurational stability of glycosyl thiols, particularly
(Scheme 5) under the conditions established for mannose under basic conditions.33–35
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Scheme 3 Synthesis and acylation of the N–H aziridine 13, to afford the N-acyl aziridines 4a–d.
Further experimentation with 7 was based on an analysis of concentration, base concentration and solvent on the relative
the putative reaction mechanism depicted in Scheme 6: nucleo- rate of these reactions. The amount of anomeric isomerization
philic attack of Et2NH on the thioester generates Et2NAc and strongly depended on temperature: at 0 °C (Table 1, entry 3) the
the free thiol 30, in equilibrium with the thiolate β-31 under the ratio changed to 10 : 1 in favour of the β product β-24, but the
basic reaction conditions. The thiolate can either attack the azir- overall yield of 24 decreased. Reducing the amount of base and/
idine, in an SN2-like process, or equilibrate, presumably via or the substrate concentration did not have an effect on the β : α
ring-opening (mutarotation conditions) to α-31. Additionally, ratio, but also slowed down the SN2 reaction process, thus
both β and α thiol/thiolate can dimerize to the disulphide, increasing the amount of disulphide by-product (e.g. compare
which was found as an anomeric mixture in the reaction crude. entries 1 and 2, 3 and 4, 3 and 5). Changing the solvent from
Thus, we examined the role of reaction temperature, substrate DMF to CH2Cl2 (entry 6) gave low yields (16%) of almost pure
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Scheme 4 Opening reaction of aziridines 4a–d with 1-S-acetyl-α-D-mannopyranose 1. The azide-bearing product 5d can also be obtained upon
acylation of 22 deriving from the N-Boc opening product 5a.
β-product, but dimerization of the glycosyl thiol was the major
Upon suggestion of a referee, we also examined whether the
result. Using both acetonitrile or DMF/CH2Cl2 mixtures of presence of aziridine 4 has an influence on the anomerization
various composition (from 7/3 to 9/1) the reaction was slow and process. To this end, β-Glc thioacetate 7 was treated with
low yields of β : α = 3 : 1 were obtained (not shown in the table). Et2NH (1.5 mol equiv.) in DMF overnight and, upon quench-
Thus, it appears that low temperatures (0 °C) favour aziridine ing, a mixture of 2 steroisomeric disulphides was identified in
opening over anomerization. A high concentration of substrate the crude reaction product. This result is indeed expected,
is also beneficial, by increasing the rate of the nucleophilic sub- based on the anomeric isomerizarition mechanism proposed
stitution. A solvent of lower polarity as CH2Cl2 appears to in Scheme 6. However, when the thiol 30 was dissolved in
reduce anomeric isomerization, but it also slows down the SN2 degassed DMF and treated with 23% DIPEA for 5 h, it was
reaction, thus resulting in low yields and extensive dimerization recovered unchanged. Upon addition of the aziridine 4 to the
of the thiol. Reducing the amount of base has no influence on reaction mixture, formation of both β-24 and α-24 was
the selectivity, but again favors dimerization over the formation observed in the reaction crude. Thus, the anomerization
of the aziridine opening product 24.
mechanism of the thiol/thiolate appears to be somewhat more
As an alternative, the one-pot aziridine procedure could be complex than suggested in Scheme 6 and strongly dependent
split in two consecutive reactions. Thus, 7 was selectively dea- on the reaction conditions. This aspect will surely deserve
cetylated at the anomeric position (DTT35) and then the free further investigations, that we will report in due course.
thiol 30 was used in the reaction with 4a (Scheme 7). When
In conclusion, the highest yields of aziridine opening
using the free thiol, a nucleophilic base is no longer needed product 24 were obtained using the reaction conditions devel-
and a sub-stoichiometric amount (0.3 mol equiv.) of the bulky oped for mannose and operating at room temperature. Under
non-nucleophilic base iPr2NEt (DIPEA) was used. Both the β- these conditions, a high isomerization rate was observed.
and α-isomers were still formed in 3 : 1 ratio (34% yield). In Performing the reaction at 0 °C, improved the β-24 : α-24 ratio
the absence of base, no product was formed and the to synthetically useful levels (10 : 1), but reduced the yield to
β-configuration of the starting thiol was preserved, which indi- 34%, due to extensive formation of the disulphide dimer. For
cates that formation of the thiolate is required for both the all the other thio-glycosides, thus, we stuck to the conditions
aziridine opening reaction and the isomerization.
adopted for mannose, adjusting the reaction temperature as
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Scheme 5 Scope of the aziridine opening reaction.
required by the reactivity of the individual substrate (see parable mixture of products which contains the expected
Table 2). α-thioacetate 10 (Scheme 5), together with the glycal and acetyl
Further exploring the scope of the reaction, the GlcNAc disulphide products.36 Using this mixture in the aziridine
derivative 11 (Scheme 5) gave ca. 19% of a 2 : 1 β : α mixture at opening reaction afforded a crude which, upon chromato-
room temperature (Table 2, entry 9). At 0 °C, the β : α ratio graphic purification, yielded 27 (56%), together with ca. 17%
improved marginally (3 : 1) and the yields of the reaction glycal (Table 2, entry 8). The mixture was purified by HPLC for
became so low that it was not worth optimizing further. analytical purposes and NMR analysis confirmed that 27 was
Surprisingly, the anomeric isomerization of β-galactose 8 and obtained as a single α-anomer, as shown by the signal of the
β-lactose 9 showed lower dependence on temperature in the H3eq proton, which appears at 2.70 ppm as a doublet of
interval examined (0 °C to room temperature) and 20%–30% doublet (Jgem = 12.8 Hz, J3eq-4 = 4.5 Hz)37 and by the signal of
of the α-isomer was formed in all cases (Table 2, entries 5 and the C1 carbon in a proton not decoupled 13C NMR spectra,
6). The reaction of the N-acetylneuraminic acid derivative 10 which appears at 168.4 ppm as a doublet of quartets with a
was complicated by the heterogeneity of the starting material. coupling constant JC1-H3ax = 3.9 Hz (see ESI†).38 Thus, as for
Thioacetylation of Neu5Ac is known to yield a basically inse- mannose and rhamnose, the Neu5Ac thiol is configurationally
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Scheme 6 Mechanism of the aziridine opening reaction.
Finally, the thio-rhamno conjugate 23 was successfully used
in the preparation of an N-linked pseudo-glycosylaminoacid
scaffold, a viable building block for solid phase pseudo-glyco-
peptide synthesis (Scheme 8). After removal of the Boc protect-
ing group (TFA, quant), the resulting amine was coupled in
solution with the side chain carboxy group of an appropriately
protected glutamic acid 32, using HATU as the coupling agent.
The pseudo-glycosylaminoacid 33 was isolated in 92% yield
after chromatography.
Table 1 Opening reaction of aziridine 4a with 1-S-acetyl-β-D-glucopyr-
anose 7 a
Et2NH
(mol equiv.)
Yieldc
(%)
Entry
T (°C)
β-24 : α-24b
29b (%)
1
2
3
20
20
0
0
0
1.9
1.4
1.9
1.4
1.9
1.9
2 : 1
2 : 1
10 : 1
10 : 1
10 : 1
20 : 1
61
43
34
28
24
16
<5
37
41
61
63
71
4
5d
6e
20
a Unless otherwise noted, all reactions were performed on a 0.06 mmol
scale, with a 0.65 M concentration of 4a in DMF and 1.3 mol equiv. of
7 for 4 h with the amount of base and at the temperature indicated.
Discussion and conclusions
1
b As judged by H NMR of the crude. c Isolated, combined yields of the
two anomeric products 24. d 0.3 M concentration of 4a. e Reaction per-
formed in CH2Cl2.
A new synthetic approach was devised for the synthesis of thio-
glycosides with the structure of N-linked pseudo-disaccharides
through aziridine opening. In the developed procedure, for-
mation of the thiol and opening of the aziridine are combined
stable under the reaction conditions. The results obtained in in a one-pot reaction, providing an efficient and operationally
the screening (yields, anomeric ratios and optimal reaction simple alternative to classical glycosylation methods. Under
conditions) for the glycosyl thio-acetates 1 and 6–11 are sum- the conditions of the aziridine opening reaction, glycosyl
marized in Table 2.
thiols of glucose, N-acetylglucosamine, galactose and lactose
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Scheme 7 Model studies with glucose derivative 7. Two step conditions: synthesis of the free thiol 30 and reaction with 4a.
Table 2 Scope of the aziridine opening reactiona
remains relatively unclear. The configurational stability of gly-
cosyl thiols could be partially explained by poor orbital overlap
between the anomeric carbon and the sulphur atom, which
does not favor opening and subsequent mutarotation of the
pyranose ring.39 However, it was shown that in aqueous media
mutarotation of free glycosyl thiols occurs at lower and neutral
pH, while it is almost completely blocked under basic con-
ditions. An exception is 1-thio-D-mannopyranose that was
observed to mutarotate under both acidic and basic con-
ditions.40 The pH dependence of mutarotation is a result of
steric, electronic (anomeric) and solvation effects. Accordingly,
there are a few reports of mutarotation during 1-S-glycosylation
and similar reactions.39,41,42 Anomerization of glycosyl thiols
was also noticed in reactions with Lewis acids such as TiCl4
and SnCl4. In uronic acids such anomerization is particularly
fast, presumably favoured by coordination of the
Entry
Sugar
T (°C)
Product
β/α ratiob (yield %c)
1
2
3
4
5
6
7
8
9
Man 1
Rha 6
Glc 7
Glc 7
Gal 8
Gal 8
Lac 9
Neu5Ac 10
GlcNAc 11
20
20
0
20
0
20
0
20
20
5a
23
24
24
25
25
26
27
28
α only (82)
α only (92)
10 : 1 (34)
2 : 1 (61)
4.5 : 1 (36)
3 : 1 (44)
5 : 1 (42)
α only (56)d
2 : 1 (19)
a All reactions were performed with a 0.65 M concentration of aziridine
4a and 1.9 mol equiv. of Et2NH in DMF, at the indicated temperature.
b Evaluated by 1H NMR. c Isolated, both isomers. d Contains 17%
glycal.
showed anomeric isomerization. Particularly for glucose, the C-1 heteroatom and C-6 carbonyl group to the Lewis acid.
anomerization proved to be temperature, base and solvent However, conditions have been found to exploit protecting
dependant. As one of the main advantages of glycosyl thiols is groups to achieve similar results with many other
that – once formed – they are believed to retain their anomeric monosaccharides.43,44 To the best of our knowledge, anomeric
configuration, the finding is of high importance for future epimerization under (slightly) basic conditions such as the
studies on thio-derivatives of carbohydrates.
ones employed in our system has never been observed. Yet our
In principle, there are two major pathways through which data clearly show that it occurs for all the β-glycosyl thiols that
anomeric isomerization of glycosyl thiols can occur, mutarota- we have examined, at least in DMF. The configurational stabi-
tion or Lewis acids promoted epimerization. While the mutar- lity observed for α-mannosyl and α-rhamnosyl thiols may be
otation process of reducing sugars (1-hydroxyaldoses) has been related to the axial configuration of the C2 hydroxy group, an
extensively explored, the mutarotation of glycosyl thiols still element which is known to affect anomerization equilibria.45
Scheme 8 Synthesis of 33, a building block for solid phase pseudo-glycopeptide synthesis.
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The mechanism and the origin of stereoselectivity in this reac- Initiator+ system. NMR experiments were recorded on a Bruker
tion will deserve further attention. AVANCE-400 MHz instrument at 298 K. Chemical shifts (δ) are
Despite this unforeseen hurdle, the one-pot aziridine reported in ppm. The 1H and 13C NMR resonances of com-
opening reaction by glycosyl thio-acetates could be used in a pounds were assigned with the assistance of COSY, HSQC and
number of cases, including mannose, rhamnose and Neu5Ac, in some cases NOESY experiments. Multiplicities are assigned
to synthesize in good-to-acceptable yields mimics of thio-disac- as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
charides outfitted with a N-linked tether. These molecules are mult. (for multiplets encompassing more than one proton).
equipped for easy conjugation and, at the same time, charac-
Mass spectra were recorded on Apex II ICR FTMS (ESI
terized by hydrolytic stability both of the thio-glycosidic ionization-HRMS), Waters Micromass Q-TOF (ESI ionization-
linkage and of the conjugation handle. As an example, we HRMS) or Thermo Fischer LCQ apparatus (ESI ionization). The
described here the thio-mannosyl derivatives 5b–d, which are mass spectrometer was operated with electrospray ionization
fully hydrolytically stable analogues of Manα(1,2)Man ready for in the positive ion mode. Full-scan mass spectra were recorded
conjugation. The of Manα(1,2)Man disaccharide is a well- in the mass/charge (m/z) range of 50–2000. Liquid chromato-
known binding epitope of immune system C-type lectins, graphy-mass spectrometry (LC-MS) analyses were carried out
including DC-SIGN.17,23 We also described the thio-rhamnosy- on a Thermo Fisher LCQ Fleet ion trap mass spectrometer
lated glutamic acid derivative 33, which can be used in the syn- equipped with a UPLC UltiMate™ 3000 system containing UV
thesis of pseudo-rhamnopeptides. Rhamnosyl glycoconjugates detector. A Zorbax RX-C18 (2.1 × 150 mm–5 µm) was used as
have been recently described as important tools for the devel- column. The column oven was maintained at 30 °C. 5 µL of
opment of novel immunotherapeutics.46–51
each sample solution were eluted using a binary gradient
Carbohydrate-binding proteins (lectins) have been difficult elution consisting of 0.1% formic acid in water (solvent A) and
to target selectively, since they often share broad selectivity for 0.1% formic acid in acetonitrile (solvent B) as follows: from
individual monosaccharides. One of the effective strategies 2% to 95% B in 25 min, 95% B kept for 10 min, then the
described so far uses a monosaccharide, acting as the lectin eluent composition was brought at 2% B in 5 min. The flow
anchor and connected to an aglycone. The aglycone is rate was 0.25 mL min−1. The mass spectrometer was operated
designed to host additional functionalities and orient them to with electrospray ionization in the positive ion mode. Full-scan
secondary interaction sites in the lectin binding region, which mass spectra were recorded in the mass/charge (m/z) range of
are often more easily differentiated between different protein 50–2000. Specific optical rotation values were measured using
targets.52 We believe that the synthetic approach described in a Perkin-Elmer 241, at 589 nm in a 1 dm cell. The following
this paper, which exploits the expeditious glycosylation of azir- abbreviations are used: DCC (N,N′-dicyclohexylcarbodiimide),
idine-containing scaffolds, will be instrumental for the fast DMF
(N,N′-dimethylformamide),
DMAP
(4-dimethyl-
synthesis of (pseudo)-glycosylated libraries of compounds that aminopyridine), TFA (trifluoroacetic acid), DPH (2,4-dinitro-
could be used for the identification of selective lectin ligands.
phenylhydroxylamine 15), Rh2(esp)2 (Du Bois’ catalyst 14), Hex
(hexane). The olefin 12 was synthesized as previously
described.23 The peracetylated glycosyl thio-acetates 1–9 were
obtained as described in ref.20,21 The Neu5Ac and GlcNAc
derivatives 10 and 11 were synthesized according to ref. 22.
Experimental details and characterization of these known
Experimental section
General
Chemicals were purchased from commercial sources and used compounds are reported as ESI.†
without further purification, unless otherwise indicated. When
anhydrous conditions were required, the reactions were per-
Synthesis of aziridine 13
formed under nitrogen or argon atmosphere. Anhydrous sol- The reaction was performed in previously well-dried glassware
vents were purchased from Sigma-Aldrich® with a content of flushed with Ar. Olefin 12 (200 mg, 1.01 mmol) was dissolved
water ≤0.005%. Triethylamine (TEA), methanol and dichloro- in CF3CH2OH (6.7 ml, 0.15 M) and transferred into the flask
methane were dried over calcium hydride, THF was dried over under Ar. The solution was cooled to 0 °C and then Rh2(esp)2
sodium/benzophenone
and
freshly
distilled.
N,N- (7.7 mg, 0.010 mmol) and DPH (241 mg, 1.21 mmol) were
Dimethylformamide (DMF) was dried over 4 Å molecular added. The reaction mixture was stirred at 0 °C (ice bath)
sieves. Reactions were monitored by analytical thin-layer under Ar atmosphere for 6 h. After 3 h additional portions of
chromatography (TLC) performed on Silica Gel 60 F254 plates Rh2(esp)2 (7.7 mg, 0.010 mmol) and DPH (40 mg, 0.20 mmol)
(Merck), and TLC Silica gel 60 RP-18 F254s (Merck) with UV were added. After 6 h the reaction mixture was diluted with
detection (254 nm and 365 nm) and/or staining with CH2Cl2 (10 ml) and washed with sat. NaHCO3 (10 ml), then
ammonium molybdate acid solution, potassium permanga- H2O was added (10 ml) to dissolve any forming salts. The
nate alkaline solution or ninhydrin. Flash column chromato- aqueous phase was washed twice with CH2Cl2. Combined
graphy was performed using silica gel 60 (40–63 μm, Merck). organic phases were washed with brine and dried over Na2SO4,
Automated flash chromatography was performed with Biotage filtered and concentrated under vacuum. The crude product
Isolera Prime system and SNAP ULTRA cartridges were was used directly and without further purification in the
employed. Microwave irradiation was performed by a Biotage N-protection reaction to avoid decomposition and dimeriza-
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tion that occur during chromatographic purification. For Me), 27.5 (C6), 26.3 (C3); MS (ESI) calcd for C15H23NO6 [M +
analytical purposes, chromatographic purification (95 : 5 Na]+ m/z: 336.14; found m/z: 336.18.
CH2Cl2 : MeOH) was performed on a batch obtaining aziridine
Synthesis of N-acyl-aziridine 4b
13 in 68% (110 mg, 0.52 mmol) yield starting from 150 mg of
olefin 12. Rf = 0.2 (CH2Cl2/MeOH 95 : 5); 1H NMR (400 MHz,
To a solution of the free aziridine 13 (32 mg, 0.15 mmol) in dry
THF (750 μl, 0.2 M), 3-chloropropionic acid 17 (19.5 mg,
0.18 mmol) and DCC (37 mg, 0.18 mmol) were added at room
temperature. After 2.5 h, the white precipitate was filtered over
celite, washing with CH2Cl2 and finally the solvents were
removed under reduced pressure. Amide 4b was isolated
through a chromatographic column (1 : 1 Hex : EtOAc) in 73%
yield (34 mg, 0.11 mmol). Rf = 0.3 (Hex/EtOAc 1 : 1); 1H NMR
(400 MHz, CDCl3): δ 3.77 (t, J8-7 = 6.4 Hz, 2H, CH2Cl), 3.68 (s,
3H, OMe), 3.67 (s, 3H, OMe), 2.91–2.78 (mult., 5H, H4, H5, H1,
CH2-linker), 2.64 (ddd, J2-3ax = J2-1 = 11.1 Hz, J2-3eq = 6.6 Hz, 1H,
H2), 2.44 (ddd, J6eq-6ax = 14.3 Hz, J6eq-1 = 4.2 Hz, J6eq-5 = 1.3 Hz,
1H, H6eq), 2.36 (dt, J3eq-3ax = 14.8 Hz, J3eq-2 = J3eq-4 = 6.4 Hz, 1H,
CDCl3): δ 3.66 (s, 3H, OMe), 3.64 (s, 3H, OMe), 2.78 (td, J1-6ax
J1-2 = 11.4 Hz, J1-6eq = 4.6 Hz, 1H, H1), 2.54 (td, J2-1 = J2-3ax
=
=
,
11.4 Hz, J2-3eq = 6.5 Hz, 1H, H2), 2.39–2.33 (mult., 4H, H3eq
H
6eq, H4, H5), 1.86–1.72 (mult., 2H, H3ax, H6ax); 13C NMR
(100 MHz, CDCl3): δ 175.9 (CO), 175.0 (CO), 51.9 (OMe), 51.9
(OMe), 41.3 (C2), 38.2 (C1), 29.7 (C4), 28.0 (C6), 27.5 (C3), 27.2
(C5); MS (ESI) calcd for C10H15NO4 [M + H]+ m/z: 214.11; found
m/z: 214.11.
The dimer 16 was also isolated by chromatography.
Characterization of the dimer 16: Rf = 0.39 (CH2Cl2/MeOH
9 : 1); 1H NMR (400 MHz, CDCl3): δ 3.71–3.64 (mult., 12H,
OMe), 3.21 (td, J1-6ax = J1-2 = 9.9 Hz, J1-6eq = 4.4 Hz, 1H, H1),
3.10–3.01 (mult., 2H, H2, H4), 2.80 (td, J10-9ax = J10-11 = 10.3
Hz, J10-9eq = 5.0 Hz, 1H, H10), 2.52 (td, J11-12ax = J11-10 = 10.3
H3eq), 2.03 (ddd, J3ax-3eq = 15.1 Hz, J3ax-2 = 10.8 Hz, J3ax-4 = 0.7
Hz, 1H, H3ax), 1.88 (ddd, J6ax-6eq = 14 Hz, J6ax-1 = 10.2 Hz, J6ax-5
=
0.9 Hz, 1H, H6ax); MS (ESI) calcd for C13H18ClNO5 [M + Na]+ m/
z: 326.09 (100%), 328.07 (32%); found m/z: 326.38, 328.37.
Hz, J11-12eq = 6.3 Hz, 1H, H11), 2.34–2.14 (mult., 3H, H12eq
,
,
H
H
9eq, H3eq), 1.96–1.66 (mult., 5H, H12ax, H6eq, H6ax, H9ax
3ax), 1.66–1.60 (m, 1H, H7), 1.37–1.31 (m, 1H, H5); 13C
Synthesis of N-acyl-aziridine 4c
NMR (100 MHz, CDCl3): δ 175.9 (CO), 175.70 (CO), 175.3
(CO), 174.9 (CO), 69.4 (C5), 52.2 (2 × OMe), 52.1 (2 × OMe),
50.0 (C4), 41.1 (C1), 40.1 (C10), 39.5 (C2), 39.0 (C11), 38.6
(C7), 34.5 (C8), 31.7 (C6), 28.4 (C3), 27.9 (C9), 27.2 (C12).; MS
(ESI) calcd for C20H30N2O8 [M + H]+ m/z: 427.21; found m/z:
427.59.
The free aziridine 13 (28 mg, 0.13 mmol) was dissolved in
freshly distilled THF (650 μl). 3-Chloro-2,2-dimethylpropionic
acid 18 (21 mg, 0.16 mmol) and DCC (32 mg, 0.16 mmol) were
added in this order at room temperature and under nitrogen.
After 4 h under stirring, the mixture was filtered over celite and
washed with CH2Cl2. Flash chromatography (1 : 1 Hex : EtOAc)
afforded the amide 4c in 67% yield (29 mg, 0.087 mmol). Rf =
Synthesis of the N-Boc-aziridine 4a
1
0.4 (Hex/EtOAc 1 : 1); H NMR (400 MHz, CDCl3): δ 3.69 (s, 3H,
To a solution of the free aziridine 13 (crude from the previous
step, 1.01 mmol) dissolved in freshly distilled CH2Cl2 under
N2 atmosphere, DMAP (a few crystals) were added. The mixture
was cooled to 0 °C and Et3N (freshly distilled, 0.84 ml,
6.06 mmol) and Boc2O (1.2 ml, 5.05 mmol) were added. After
1 h under stirring at 0 °C, the reaction was allowed to reach
room temperature, then quenched with H2O. The resulting
mixture was diluted with a saturated solution of NH4Cl; the
aqueous phase extracted twice with EtOAc. The organic layers
were washed with saturated solutions of KHSO4, Na2CO3 and
NaCl in this order and finally dried over Na2SO4, filtered and
concentrated in vacuum. Purification by flash chromatography
(7 : 3 Hex : EtOAc) afforded the t-butylcarbamate 4a as a yellow
waxy solid in 90% yield (0.29 g, 0.91 mmol). Rf = 0.33 (Hex/
OMe), 3.67 (s, 3H, OMe), 3.61 (AB system, Japp = 14.7 Hz, 2H,
CH2Cl), 2.93–2.82 (mult., 3H, H4, H5, H1), 2.67 (td, J2-3ax = J2-1
=
10.1 Hz, J2-3eq = 6.6 Hz, 1H, H2), 2.44 (ddd, J6eq-6ax = 14.5 Hz,
J6eq-1 = 4.8 Hz, J6eq-5 = 1.3 Hz, 1H, H6eq), 2.48–2.31 (m, 1H, H3eq),
2.02 (ddd, J3ax-3eq = 14.5 Hz, J3ax-2 = 10.4 Hz, J3ax-4 = 1 Hz, 1H,
H
H
3ax), 1.9 (ddd, J6ax-6eq = 14 Hz, J6ax-1 = 11 Hz, J6ax-5 = 3.3 Hz, 1H,
6ax), 1.34 (s, 6H, 2 × CH3 linker); 13C NMR (100 MHz, CDCl3): δ
188.7 (CO amide), 174.9 (CO), 174.3 (CO), 52.0 (CH2Cl), 52.0 (2
× OMe), 40.4 (C2), 38.1 (C1), 36.7 (C5), 34.0 (C4), 29.6 (CMe2),
26.4 (C6), 25.5 (C3), 24.1, 23.9 (2 × CH3 linker); MS (ESI) calcd
for C15H22ClNO5 [M + Na]+ m/z: 354.12 (100%), 356.11 (33%);
found m/z: 354.43, 356.41.
Synthesis of the activated ester 20
EtOAc 7 : 3); [α]2D6 (CHCl3, c 1.05): +34; 1H NMR (400 MHz, 2,2-Dimethyl-3-chloro-propanoic acid 18 (400 mg, 2.97 mmol)
CDCl3): δ 3.66 (s, 3H, OMe), 3.65 (s, 3H, OMe), 2.82 (td, J1-6ax was dissolved in dry DMF (14.6 ml) and NaN3 (990 mg,
=
J1-2 = 11.5 Hz, J1-6eq = 4.5 Hz, 1H, H1), 2.72–2.67 (m, 1H, H5), 15.20 mmol) was added. The mixture was stirred at 50 °C
2.62 (td, J4-3eq = J4-5 = 6.4 Hz, J4-3ax = 1.0 Hz 1H, H4), 2.54 (td, under nitrogen atmosphere. After 3 days, the reaction was
J2-1 = J2-3ax = 11.5 Hz, J2-3eq = 6.4 Hz, 1H, H2), 2.45 (ddd, J6eq-6ax cooled to room temperature, diluted with CH2Cl2 and filtered
= 14.2 Hz, J6eq-1 = 4.6 Hz, J6eq-5 = 1.6 Hz, 1H, H6eq), 2.35 (dt, over a celite pad to remove unreacted NaN3 and the generated
J3eq-3ax = 14.9 Hz, J3eq-4 = J3eq-2 = 6.4 Hz, 1H, H3eq), 1.95 (ddd, salts (NaCl). The solvent was evaporated at reduced pressure
J3ax-3eq = 14.9 Hz, J3ax-2 = 11.8 Hz, J3ax-4 = 1.0 Hz, 1H, H3ax), 1.74 giving 3-azido-2,2-dimethylpropanoic acid 19 in quantitative
(ddd, J6ax-6eq = 14.5 Hz, J6ax-1 = 11.4 Hz, J6ax-5 = 3.0 Hz, 1H, yield (425 mg). 1H NMR (400 MHz, CDCl3): δ 3.42 (s, 2H, CH2),
H
6ax), 1.43 (s, 9H, tBu); 13C NMR (100 MHz, CDCl3): δ 175.4 1.25 (s, 6H, 2 × CH3).
(CO), 174.5 (CO), 162.3 (CO, carbamate), 81.3 (CIV Boc), 52.0 (2 To a solution of crude acid 19 (311 mg, 2.17 mmol) in dry
× OMe), 40.8 (C2), 38.3 (C1), 37.0 (C5), 34.5 (C4), 28.0 (tBu 3 × DMF (7.2 ml, 0.3 M) p-nitrophenyl trifluoacetate (766 mg,
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3.25 mmol) and dry pyridine (350 μl) were added. The mixture TLC (eluents based on different ratios of H2O/MeOH) and,
was warmed to 60 °C and stirred overnight. The following day, after completion, the solvent and the excess MeNH2 were
the reaction was cooled to room temperature and solvent and removed under reduced pressure. The products were separated
pyridine were removed under vacuum. Compound 20 was iso- from the N-methylacetamide by-product using automated
lated in 73% (419 mg, 1.58 mmol) yield through a chromato- flash chromatography (Ultra HP-Sphere C18 cartridges) in
graphic column (8 : 2 Hex : EtOAc). Rf = 0.4 (Hex/EtOAc 8 : 2); reverse phase (isocratic elution, 1 : 1 H2O : MeOH).
1H NMR (400 MHz, CDCl3): δ 8.28 (d, J = 8.9 Hz, 2H, Har), 7.27
Synthesis of the pseudo-thio-dimannoside 5a
CH3); 13C NMR (100 MHz, CDCl3): δ 173.5 (CO), 155.5 (Car), Prepared from peracetylated thio-mannose
(d, J = 9.2 Hz, 2H, Har), 3.57 (s, 2H, CH2N3), 1.4 (s, 6H, 2 ×
1
(37 mg,
145.6 (Car), 125.3 (2 × CHar), 122.5 (2 × CHar), 59.59 (CH2), 44.4 0.091 mmol) and aziridine 4a (22 mg, 0.070 mmol) according
(C(CH3)2), 23.03 (2 × CH3).
to the general procedure and purified by flash chromatography
(1 : 1 Hex : EtOAc) to give 5a in 82% yield (39 mg, 0.058 mmol).
Synthesis of N-acyl-aziridine 4d
1
Rf = 0.33 (Hex/EtOAc 1 : 1); [α]1D9 (CHCl3, c 1.95): +59; H NMR
Using DCC: To a solution of 13 (38 mg, 0.18 mmol) in dry THF (400 MHz, CDCl3): δ 5.40–5.34 (mult., 2H, H1, H2), 5.30 (dd,
(890 μl, 0.2 M), carboxylic acid 19 (31 mg, 0.21 mmol) was J4-5 = 10.2 Hz, J4-3 = 9.8 Hz, 1H, H4), 5.21 (dd, J3-4 = 9.8 Hz, J3-2
added and then DCC (44 mg, 0.21 mmol). After 3 h under stir- = 2.9 Hz, 1H, H3), 4.80–4.70 (m, 1H, NH), 4.44–4.29 (mult., 2H,
ring at room temperature, the mixture was filtered over celite H5, H6a), 4.17–4.07 (m, 1H, H6b), 3.95–3.83 (m, 1H, H4′), 3.71
and washed with CH2Cl2. The chromatographic column (s, 3H, OMe), 3.70, (s, 3H, OMe), 3.31–3.21 (m, 1H, H5′),
afforded compound 4d in 30% yield (18 mg, 0.053 mmol). 3.09–3.00 (m, 1H, H1′), 2.88–2.77 (m, 1H, H2′), 2.21–1.95
Using the activated ester 20: To a solution of aziridine 13 (mult., 3H, H6′eq, H3′eq, H6′ax), 2.16 (s, 3H, OAc), 2.08 (s, 3H,
(39 mg, 0.18 mmol) in dry DMF (720 μl, 0.25 M) and pyridine OAc), 2.04 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.91–1.80 (m, 1H, H3′
(18 μl) the activated ester 20 (63 mg, 0.24 mmol) was added. ax), 1.43 (s, 9H, tBu); 13C NMR (100 MHz, CDCl3): δ 173.7 (CO),
The reaction proceeded at 60 °C under stirring for 3 h. The 173.7 (CO), 170.6 (CO), 169.9 (CO), 169.9 (CO), 169.7 (CO),
mixture was cooled to room temperature and then solvent and 169.6 (CO, carbammate), 82.1 (C1), 80.0 (CIV Boc), 70.9 (C2),
pyridine were removed under reduced pressure. Flash chrom- 69.4 (C3), 69.3 (C5), 66.1 (C4), 62.5 (C6), 52.3 (OMe), 52.2 (OMe),
atography (1 : 1 Hex : EtOAc) allowed to isolate compound 4d 49.0 (C4′), 44.5 (C5′), 39.9 (C1′, C2′), 29.7 (C6′), 29.1 (C3′), 28.3
in 60% yield (36 mg, 0.11 mmol). Rf = 0.38 (Hex/EtOAc 1 : 1); (tBu, 3 × Me), 20.9 (OAc), 20.7 (OAc), 20.7 (OAc), 20.6 (OAc); MS
1H NMR (400 MHz, CDCl3): δ 3.69 (s, 3H, OMe), 3.67 (s, 3H, (ESI) calcd for C29H43NO15S [M + Na]+ m/z: 700.24; found m/z:
OMe), 3.43 (AB system, Japp = 14.7 Hz, 2H, CH2N3), 2.90–2.79 701.01.
(mult., 3H, H1, H5, H4), 2.66 (td, J2-3ax = J2-1 = 10.6 Hz, J2-3eq
=
Product 5a (19 mg, 0.028 mmol) was deacetylated following
6.7 Hz, 1H, H2), 2.44 (ddd, J6eq-6ax = 14.3 Hz, J6eq-1 = 4.8 Hz, the general procedure for deacetylation (reaction time 4 h). The
J6eq-5 = 1.3 Hz, 1H, H6eq), 2.36 (ddd, J3eq-3ax = 15 Hz, J3eq-2 = 6.9 deacetylated product was obtained after purification in 70%
Hz, J3eq-4 = 6.4 Hz, 1H, H3eq), 2.01 (ddd, J3ax-3eq = 14.9 Hz, J3ax-2 yield (10 mg, 0.020 mmol). Rf = 0.24 (H2O/MeOH 1 : 1) [α]D18
1
= 10.7 Hz, J3ax-4 = 0.6 Hz, 1H, H3ax), 1.26 (s, 6H, 2 CH3 linker); (MeOH, c 0.5): +113; H NMR (400 MHz, CD3OD): δ 5.34 (d, J1-2
13C NMR (100 MHz, CDCl3): δ 189.5 (CO amide), 175.1 (CO), = 1 Hz, 1H, H1), 3.96–3.81 (mult., 4H, H2, H5, H4′, H6a),
174.4 (CO), 60.3 (CH2N3), 52.2 (OMe), 52.1 (OMe), 40.5 (C2), 3.78–3.71 (dd, 1H, J6a-6b = 12.5 Hz, J6b-5 = 5.8 Hz, H6b), 3.68 (s,
38.3 (C1), 36.9 (C5), 34.2 (C4), 29.8 (C(CH3)2 linker), 26.6 (C6), 3H, OMe), 3.68 (s, 3H, OMe), 3.66–3.59 (mult., 2H, H4, H3),
25.7 (C3), 23.9 (CH3 linker), 23.7 (CH3 linker); MS (ESI) calcd 3.24–3.17 (m, 1H, H5′), 3.06–2.94 (mult., 2H, H1′, H2′), 2.25–2.11
for C15H22N4O5 [M + Na]+ m/z: 361.15; found m/z: 361.61.
(m, 1H, H6′eq), 2.11–1.97 (mult., 2H, H6′ax, H3′eq), 1.9–1.8 (m,
1H, H3′ax), 1.45 (s, 9H, tBu); 13C NMR (100 MHz, CD3OD): δ
176.1 (CO), 176.0 (CO), 157.2 (CO, carbammate), 86.4 (C1), 80.4
General procedure for the opening reaction of aziridine 4a
Peracetylated thio-glycoside (1.3 eq.) and aziridine (1 eq.) were (CIV Boc),75.4 (C5), 73.8 (C2), 73.2 (C3), 68.8 (C4), 62.9 (C6), 52.5
dissolved in dry DMF (0.65 M) under N2 atmosphere at the (2 × OMe), 50.7 (C4′), 46.0 (C5′), 41.5 (C1′), 41.0 (C2′), 30.3 (C6′),
temperature indicated in Table 2 and Et2NH (1.9 eq.) was 30.3 (C3′), 28.8 (tBu, 3 × Me). MS (ESI) calcd for C21H35NO11S [M
added. The reaction mixture was stirred for 4 h at the tempera- + Na] + m/z: 532.18; found: 532.51; HR-MS (ESI) calcd for
ture indicated in Table 2, then the reaction mixture was C21H35NO11S [M + Na] + m/z: 532.1829; found 532.1835.
diluted with EtOAc and washed with 1 M HCl. The organic
Synthesis of the pseudo thio-disaccharide 23
phase was washed three times with H2O and the aqueous
phases were additionally extracted with EtOAc. The combined Prepared from peracetylated thio-rhamnose
6 (126 mg,
organic phases were dried over Na2SO4, filtered and concen- 0.36 mmol) and aziridine 4a (87 mg, 0.28 mmol) according to
trated under vacuum.
the general procedure and purified by automated flash
chromatography (Biotage Isolera, SNAP Cartridge, gradient
30–50% EtOAc/Hex) to give 23 as a single α isomer in 92% yield
General method for deacetylation of the sugar moiety
The substrates were dissolved in a MeNH2/EtOH solution (4 M) (158 mg, 0.26 mmol) as colourless oil. Rf = 0.34 (Hex/EtOAc
at room temperature in order to have a final solution 0.05 M of 1 : 1); [α]2D1 (CHCl3, c 1.00): −71; 1H NMR (400 MHz, CDCl3) δ
the substrate. The reactions were monitored by reverse-phase 5.37–5.31 (m, 1H, H2), 5.31 (s, 1H, H1), 5.19–5.11 (m, 1H, H3),
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5.13–5.03 (m, 1H, H4), 4.79 (s, 1H, NH), 4.23 (dt, J5-4 = 12.3 Hz, Synthesis of the pseudo thio-disaccharide 25
J5-Me = 6.3 Hz, 1H, H5), 3.90–3.83 (m, 1H, H4′), 3.70 (s, 3H,
OMe), 3.68 (s, 3H, OMe), 3.35–3.28 (m, 1H, H5′), 3.05–2.99 (m,
Prepared from peracetylated thio-galactose
8
(51 mg,
0.13 mmol) and aziridine 4a (30 mg, 0.096 mmol) according to
the general procedure and purified by flash chromatography
(1 : 1 Hex : EtOAc) to give 25 as a mixture of α and β isomers in
44% (29 mg, 0.042 mmol) yield as colourless oil. The β and α
isomers were separated by a second flash chromatography
(8 : 1 iPr2O : EtOAc) to give pure β-25 in 34% yield (22 mg,
0.033 mmol). β-25: Rf = 0.33 (iPr2O/EtOAc 8 : 2); 1H NMR
(400 MHz, CDCl3) δ 5.45 (dd, J4-3 = J4-5 = 3.4 Hz, 1H, H4), 5.24
1H, H1′), 2.84–2.73 (m, 1H, H2′), 2.14 (s, 3H, OAc), 2.06 (s, 3H,
OAc), 2.17–1.93 (mult., 3H, H3′eq, H6′ax, H6′eq), 1.97 (s, 3H, OAc),
1.96–1.85 (m, 1H, H3′ax), 1.43 (s, 9H, tBu), 1.24 (d, JMe-5 = 6.3 Hz,
3H, Me); 13C NMR (101 MHz, CDCl3) δ 175.0 (CO), 174.4 (CO),
170.4 (CO), 170.3 (CO), 170.3 (CO), 83.8 (C1), 71.7 (C2), 71.6 (C4),
69.9 (C3), 67.9 (C5), 52.7 (OMe), 52.6 (OMe), 49.8 (C4′), 45.7 (C5′),
40.3 (C2′), 40.1 (C1′), 30.1 (C6′), 29.3 (C3′), 28.7 (tBu-3 × Me), 21.3
(OAc), 21.2 (OAc), 21.1 (OAc), 17.6 (Me); JH1–C1 = 170.3 (HSQC
without 13C decoupling); LC-MS (Rt = 20.41 min) calcd for
C27H41NO13S [M + Na]+ m/z: 642.23; found m/z: 641.78. MS
(HRMS): calcd for C27H41NO13S [M + Na]+ m/z: 642.2196; found
m/z: 642.2195.
(dd, J2-1 = J2-3 = 10.0 Hz, 1H, H2), 5.05 (dd, J3-2 = 10.0 Hz, J3-4
=
3.4 Hz, 1H, H3), 4.86 (m, 1H, NH), 4.69 (d, J1-2 = 10.0 Hz, 1H,
H1), 4.13 (mult., 2H, H6a, H6b), 3.98 (m, 1H, H5), 3.87–3.77 (m,
1H, H4′), 3.70 (s, 3H, OMe), 3.69 (s, 3H, OMe), 3.39–3.28 (m,
1H, H5′), 3.02–2.93 (m, 1H, H1′), 2.81–2.69 (m, 1H, H2′), 2.17 (s,
3H, OAc), 2.14–2.09 (mult., 3H, H3′eq, H6′ax, H6′eq), 2.04 (s, 3H,
OAc), 2.03 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.89–1.82 (m, 1H, H3′
ax), 1.45 (s, 9H, tBu); 13C NMR (101 MHz, CDCl3) δ 174.0 (CO),
170.4 (CO), 170.4 (CO), 170.2 (CO), 170.1 (CO), 169.7 (CO), 84.4
(C1), 74.5 (C5), 72.0 (C3), 67.5 (C4), 67.3 (C2), 61.3 (C6), 52.3
(OMe), 52.2 (OMe), 49.7 (C4′), 43.4 (C5′), 40.0 (C2′, C1′), 29.8 (C3′,
C6′), 28.5 (tBu-3 × Me), 20.9 (OAc), 20.8 (OAc), 20.7 (OAc), 20.7
(OAc); LC-MS (Rt = 19.51 min) calcd for C29H43NO15S [M + Na]+
m/z: 700.24; found m/z: 699.74; MS (HRMS): calcd for
C29H43NO15S [M + Na]+ m/z: 700.2251; found m/z: 700.2259.
Synthesis of the pseudo thio-disaccharide 24
Prepared from peracetylated thio-glucose
7
(34 mg,
0.083 mmol) and aziridine 4a (20 mg, 0.064 mmol) according
to the general procedure and purified by flash chromatography
(1 : 1 Hex : EtOAc) to give 24 in 61% yield (26 mg, 0.039 mmol)
as colourless oil (entry 4 in Table 2). The β and α isomers
could be separated by flash chromatography (8 : 1
iPr2O : EtOAc). β-24: Rf = 0.22 (iPr2O/EtOAc 8 : 1); 1H NMR
(400 MHz, CDCl3) δ 5.23 (dd, J3-4 = J3-2 = 10 Hz, 1H, H3), 5.13
(dd, J4-3 = J4-5 = 10 Hz, 1H, H4), 5.04 (dd, J2-1 = J2-3 = 10 Hz, 1H,
H2), 4.87 (m, 1H, NH), 4.72 (d, J1-2 = 10 Hz, 1H, H1), 4.27 (dd,
J6a-6b = 12.4 Hz, J6a-5 = 4.5 Hz, 1H, H6a), 4.15 (dd, J6b-6a = 12.4
Synthesis of the pseudo thio-disaccharide 26
Hz, J6b-5 = 2.4 Hz, 1H, H6b), 3.93–3.80 (m, 1H, H4′), 3.76 (ddd, Prepared from peracetylated thio-lactose
9
(56 mg,
J5-4 = 10.0 Hz, J5-6a = 4.5 Hz, J5-6b = 2.4 Hz, 1H, H5), 3.69 (s, 6H, 0.080 mmol) and aziridine 4a (19 mg, 0.062 mmol) according
2 × OMe), 3.42–3.26 (m, 1H, H5′), 2.99–2.86 (m, 1H, H1′), to the general procedure and purified by flash chromatography
2.81–2.70 (m, 1H, H2′), 2.24–2.08 (mult., 3H, H3′eq, H6′eq, H6′ax), (3 : 2 Hex : EtOAc) to give 26 as a 5 : 1 β : α anomeric mixture in
2.07 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.00 (s, 42% yield (25 mg, 0.026 mmol) as colourless oil. Rf = 0.14
3H, OAc), 1.91–1.81 (m, 1H, H3′ax), 1.45 (s, 9H, tBu); 13C NMR (Hex/EtOAc 1 : 1); 1H NMR (400 MHz, CDCl3) β-26 5.35–5.33
(101 MHz, CDCl3) δ 174.0 (CO), 171.8 (CO), 171.7 (CO), 170.8 (m, 1H, H10), 5.21 (dd, J3-4 = J3-2 = 9.2 Hz, 1H, H3), 5.10 (dd, J8-9
(CO), 170.3 (CO), 169.5 (CO), 83.7 (C1), 76.0 (C5), 74.1 (C3), 70.2 = 10.4 Hz, J8-7 = 7.9 Hz, 1H, H8), 5.02–4.97 (m, 1H, H2), 4.95
(C2), 68.3 (C4), 62.0 (C6), 52.4 (OMe), 52.3 (OMe), 48.2* (C4′), (dd, J9-8 = 10.4 Hz, J9-10 = 3.2 Hz, 1H, H9), 4.86 (d, JNH-4′ = 6.9
42.1* (C5′), 40.1 (C1′, C2′), 29.8 (C3′, C6′), 28.5 (tBu-3 × Me), 20.9 Hz, 1H, NH), 4.67 (d, J1β-2β = 10.1 Hz, 1H, H1β), 4.49 (mult., 2H,
(OAc), 20.9 (OAc), 20.8 (OAc), 20.8 (OAc). * These signals are H7, H12a), 4.15–4.01 (mult., 3H, H12b, H6a, H6b), 3.91–3.81
better visible in the HSQC spectrum; LC-MS (Rt = 19.64 min) (mult., 3H, H11, H4, H5), 3.70 (s, 1H, H4′), 3.68 (2× s, 6H, 2 ×
calcd for C29H43NO15S [M + Na]+ m/z: 700.24; found m/z: OMe), 3.37–3.30 (m, 1H, H5′), 2.96–2.87 (m, 1H, H1′), 2.82–2.70
699.81; MS (HRMS): calcd for C29H43NO15S [M + Na]+ m/z: (m, 1H, H2′), 2.15 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.12–2.05
700.2251; found m/z: 700.2249. α-24: 1H NMR (400 MHz, (mult., 3H, H3′eq, H6′ax, H6′eq), 2.06 (s, 6H, 2 × OAc), 2.04 (s, 9H,
CDCl3) δ 5.73 (d, J2-1 = 5.7 Hz, 1H, H1), 5.35 (dd, J3-2 = J3-4 = 9.8 3 × OAc), 1.96 (s, 3H, OAc), 1.89–1.81 (m, 1H, H3′ax), 1.44 (s,
Hz, 1H, H3), 5.14–4.99 (mult., 2H, H4, H2), 4.75 (s, 1H, NH), 9H, tBu), α-26 δ 5.62 (d, J1α-2α = 5.7 Hz, 1H, H1α); β-26 13C NMR
4.48–4.39 (m, 1H, H5), 4.34 (dd, J6a-6b = 12.5 Hz, J6a-5 = 4.6 Hz, (101 MHz, CDCl3) δ 174.7 (CO), 174.0 (CO), 170.5 (CO), 170.4
1H, H6a), 4.19–4.09 (m, 1H, H6b), 3.92–3.83 (m, 1H, H4′), 3.71 (CO), 170.3 (CO), 170.2 (CO), 169.8 (CO), 169.8 (CO), 169.2
(s, 3H, OMe), 3.70 (s, 3H, OMe), 3.24–3.15 (m, 1H, H5′), (CO), 83.5 (C1), 76.8 (C5), 76.1 (C11), 74.0 (C3), 71.2 (C4), 70.8
3.11–2.98 (m, 1H, H1′), 2.88–2.73 (m, 1H, H2′), 2.09 (s, 3H, (C9), 70.5 (C2), 69.2 (C8), 66.7 (C10), 62.1 (C12), 60.9 (C6),
OAc), 2.08 (s, 3H, OAc), 2.10–2.00 (mult., 3H, H3′eq, H6′eq, H6′ 52.3 (OMe), 52.2 (OMe), 49.7 (C4′), 43.1 (C5′), 40.1 (C1′, C2′),
ax), 2.03 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.92–1.84 (m, 1H, H3′ 32.1 (C6′), 29.8 (C3′), 28.5 (tBu-3 × Me), 21.0 (OAc), 20.9 (OAc),
ax), 1.44 (s, 9H, tBu); 13C NMR (HSQC) (101 MHz, CDCl3) δ 81.2 20.9 (OAc), 20.9 (OAc), 20.8 (OAc), 20.8 (OAc), 20.6 (OAc);
(C1), 70.2 (C3), 67.8 (C5, C2, C4), 61.6 (C6), 51.9 (2 × OMe), 49.2 LC-MS (Rt
= 20.56 min (β), 20.66 min (α)) calcd for
(C4′), 42.5 (C5′), 39.7 (C1′, C2′), 29.0 (C3′, C6′), 29.0(tBu-3 × Me), C41H59NO23S [M + Na]+ m/z: 988.32; found m/z: 987.62; MS
20.6 (4 × OAc); LC-MS (Rt = 19.87 min) calcd for C29H43NO15S (HRMS): calcd for C41H59NO23S [M + Na]+ m/z: 988.3096; found
[M + Na]+ m/z: 700.24; found m/z: 699.81.
m/z: 988.3090.
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Synthesis of the pseudo thio-disaccharide 27
the general procedure described for the synthesis of 5a and
purified by flash chromatography (1 : 1 Hex : EtOAc) to give 5c
in 96% yield (60 mg, 0.086 mmol). Rf = 0.2 (Hex/EtOAc 1 : 1);
[α]1D9 (CHCl3, c 1.22): +55; 1H NMR (400 MHz, CDCl3): δ
5.96–5.89 (m, 1H, NH), 5.41 (d, J1-2 = 1.5 Hz, 1H, H1), 5.34 (dd,
J2-3 = 3.5 Hz, J2-1 = 1.5 Hz, 1H, H2), 5.29 (t, J4-3 = J4-5 = 9.7 Hz,
1H, H4), 5.20 (dd, J3-4 = 9.7 Hz, J3-2 = 3.5, 1H, H3), 4.42–4.30
(mult., 2H, H5, H6a), 4.21–4.07 (mult., 2H, H4′, H6b), 3.72 (s,
3H, OMe), 3.70, (s, 3H, OMe), 3.58 (AB system, Japp = 10.7 Hz,
2H, CH2Cl), 3.33–3.27 (m, 1H, H5′), 3.08 (ddd, J1′-2′ = 13.5 Hz,
J1′-6′ax = 8.8 Hz, J1′-6′eq = 5.34 Hz, 1H, H1′), 2.87–2.78 (m, 1H,
H2′), 2.23 (ddd, J3′eq-3′ax = 14.1 Hz, J3′eq-2′ = 10 Hz, J3′eq-4′ = 4.4
Hz, 1H, H3′eq), 2.18–1.94 (mult., 2H, H6′eq, H6′ax), 2.15 (s, 3H,
OAc), 2.07 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.97 (s, 3H, OAc),
1.92–1.82 (m, 1H, H3′ax), 1.28 (s, 3H, CH3 linker), 1.27 (s, 3H,
CH3 linker); 13C NMR (100 MHz, CDCl3): δ 174.34 (CO), 174.22
(CO), 173.56 (CO), 170.76 (CO), 169.94 (CO), 169.83 (CO),
169.72 (CO), 81.80 (C1), 70.95 (C2), 69.52 (C3), 69.44 (C5), 66.26
(C4), 62.59 (C6), 60.4 (C(CH3)2 linker), 52.82 (CH2 linker), 52.42
(OMe), 52.29 (OMe), 48.16 (C4′), 43.67 (C5′), 40.21 (C1′), 40.03
(C2′), 29.35 (C6′), 28.76 (C3′), 23.63 (CH3 linker), 23.37 (CH3
linker), 20.96 (OAc), 20.77 (OAc), 20.73 (OAc), 20.66 (OAc); MS
(ESI) calcd for C29H42ClNO14S [M + Na]+ m/z: 718.19; found m/
z: 718.6.
Prepared from peracetylated thio-sialic acid 10 36 (68 mg,
0.12 mmol) and aziridine 4a (30 mg, 0.096 mmol) according to
the general procedure and purified by flash chromatography
(3 : 1 CH2Cl2 : acetone) to give 27 as a single isomer in 56%
yield (containing 17% glycal as estimated by NMR) as yellow
waxy solid. Rf = 0.33 (CH2Cl2 : acetone = 3 : 1); 1H NMR
(400 MHz, CDCl3) δ 5.51–5.44 (m, 1H, H7), 5.43–5.36 (mult.,
2H, H8, NH), 5.06 (d, J = 11.0 Hz, 1H, NH), 4.84 (td, J4-5 = J4-3ax
= 11.0 Hz, J4-3eq = 4.5 Hz, 1H, H4), 4.28–4.18 (m, 1H, H9),
4.28–4.13 (mult., 2H, H9a, H9b), 4.02 (ddd, J5-NH = J5-4 = J5-6
=
11.0 Hz, 1H, H5), 3.91–3.86 (m, 1H, H6), 3.85 (s, 3H, COOMe),
3.73 (s, 3H, COOMe), 3.71 (s, 3H, COOMe), 3.60–3.51 (m, 1H,
H4′), 3.13–3.07 (m, 1H, H5′), 3.07–3.01 (m, 1H, H1′), 2.91–2.84
(m, 1H, H2′), 2.70 (dd, J3eq-3ax = 12.8 Hz, J3eq-4 = 4.5 Hz, 1H,
H
3eq), 2.20 (s, 3H, OAc), 2.14 (s, 3H, OAc), 2.06 (s, 3H, OAc),
2.18–2.04 (mult., 4H, H3′eq, H3′ax, H6′eq, H6′ax), 2.03 (s, 3H,
OAc), 2.00–1.89 (m, 1H, H3ax), 1.87 (s, 3H, NHAc), 1.47 (s, 9H,
tBu); 13C NMR (HSQC) (101 MHz, CDCl3) δ 72.3 (C6), 68.2 (C4),
66.2 (C7), 66.0 (C8), 60.2 (C9), 51.3 (OMe), 50.6 (OMe), 50.5
(OMe), 47.4 (C5), 41.3 (C2′), 38.6 (C1′, C5′), 36.6 (C3), 29.6 (C3′,
C6′), 27.1 (tBu-3 × Me), 21.8 (NHAc), 19.8 (Ac), 19.4 (Ac), 19.3 (2
× Ac); LC-MS (Rt = 17.99 min) calcd for C35H52N2O18S [M +
Na]+ m/z: 843.29; found m/z: 842.71; MS (HRMS) calcd for
C35H52N2O18S [M + Na]+ m/z: 843.2834; found m/z: 843.2820.
Product 5c (22 mg, 0.032 mmol) was deacetylated according
to the general procedure for acetylation (reaction time 3 h).
The deacetylated product was obtained in 62% (10.5 mg,
0.020 mmol) yield after purification. Rf = 0.31 (H2O/MeOH
1 : 1) [α]2D0 (MeOH, c 0.47): +110; 1H NMR (400 MHz, CD3OD): δ
Synthesis of the pseudo thio-disaccharide 28
Prepared from peracetylated thio-N-acetylglucosamine 11
(34 mg, 0.083 mmol) and aziridine 4a (20 mg, 0.064 mmol)
according to the general procedure and purified by flash
chromatography (9 : 1 CH2Cl2 : MeOH) to give 28 as an inse-
parable anomeric mixture in 19% yield (8 mg, 0.012 mmol)
as yellow waxy solid. Rf = 0.08 (Hex/EtOAc 1 : 1); 1H NMR
(400 MHz, CDCl3) β-28 δ 5.63 (d, JNH-2 = 9.3 Hz, 1H, NH),
5.18–5.09 (mult., 2H, H3, H4), 4.92–4.84 (m, 1H, NH), 4.75 (d,
J1β-2β = 10.4 Hz, 1H, H1β), 4.40–4.34 (m, 1H, H2), 4.26 (dd,
J6a-6b = 12.1 Hz, J6a-5 = 4.3 Hz, 1H, H6a), 4.18–4.13 (m, 1H,
5.34 (d, J1-2 = 1.2 Hz, 1H, H1), 4.1 (ddd, J4′-3′ax = 8.2 Hz, J4′-3′eq
=
7.3 Hz, J4′-5′ = 4.2 Hz, 1H, H4′), 3.93–3.82 (mult., 3H, H5, H2,
H6a), 3.77–3.56 (mult., 5H, H6b, H4, H3, CH2Cl), 3.72 (s, 3H,
OMe), 3.71 (s, 3H, OMe), 3.22–3.15 (mult., 2H, H5′, H2′),
3.15–3.09 (m, 1H, H1′), 2.34 (ddd, J6′eq-6′ax = 14.4 Hz, J6′eq-1′
=
7.7 Hz, J6′eq-5′ = 3.6 Hz, 1H, H6′eq), 2.19–2.10 (m, 1H, H3′eq),
2.02 (ddd, J6′ax-6′eq = 14.2 Hz, J6′ax-5′ = 8.1 Hz, J6′ax-1′ = 4.3 Hz,
1H, H6′ax), 1.88 (ddd, J3′ax-3′eq = 14.2 Hz, J3′ax-4′ = 7.7 Hz, J3′ax-2′
=
4.7 Hz, 1H, H3′ax), 1.28 (s, 6H, 2 × CH3 linker); 13C NMR
(100 MHz, CD3OD): δ 177.2 (CO), 175.7 (CO), 175.6 (CO), 85.3
(C1), 75.5 (C5), 73.9 (C2), 73.3 (C3), 68.9 (C4), 63.0 (C6), 53.5
(CH2Cl), 52.8 (OMe), 52.7 (OMe), 49.9 (C4′), 45.1 (C5′), 42.0
(C1′), 41.6 (C2′), 31.4 (C6′), 30.5 (C3′), 24.0 (CH3 linker), 23.8
(CH3 linker); HR-MS (ESI) calcd for C21H34ClNO10S [M + Na] +
m/z: 550.1490; found: 550.1495.
H
6b), 3.92–3.79 (m, 1H, H4′), 3.76–3.74 (m, 1H, H5), 3.71 (s,
3H, COOMe), 3.68 (s, 3H, COOMe), 3.35–3.30 (m, 1H, H5′),
2.97–2.90 (m, 1H, H1′), 2.75–2.68 (m, 1H, H2′), 2.13–2.01
(mult., 3H, H3′eq, H6′ax, H6′eq), 2.09 (s, 3H, OAc), 2.04 (s, 3H,
OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.98 (s, 3H, OAc),
1.88–1.83 (m, 1H, H3′ax) 1.45 (s, 9H, Boc); α-28 δ 5.52 (d, J1α-2α
= 5.2 Hz, 1H, H1α); 13C NMR (HSQC) (101 MHz, CDCl3) δ 67.9
(C3), 73.7 (C4), 85.2 (C1), 68.0 (C2), 61.3 (C6), 52.1 (OMe), 52.0 Synthesis of the pseudo-thio-dimannoside 5d
(OMe), 49.0 (C4′), 41.0 (C5′, C2′, C1′), 29.1 (C6′), 28.6 (C3′), 28.4
(tBu-3 × Me), 23.1 (NHAc), 20.8 (OAc), 20.6 (OAc), 20.7 (OAc);
Thioacetate 1 (78 mg, 0.19 mmol) and aziridine 4d (50 mg,
0.15 mmol) were dissolved in dry DMF (250 μl, 0.6 M) into a
MW-reactor. After addition of Et2NH (30μl, 0.28 mmol), the
reaction was stirred at 60 °C under MW irradiation for 1 h.
After that, the mixture was diluted with EtOAc and washed
with 1 M HCl. The aqueous phase was extracted twice with
EtOAc and finally the organic layers collected were dried over
Na2SO4, filtered and the solvents removed under vacuum.
LC-MS (Rt 16.67 min (β), 17.33 min (α)) calcd for
=
C
29H44N2O14S [M + Na]+ m/z: 699.25; found m/z: 698.75; MS
(HRMS) calcd for C29H44N2O14S [M + Na]+ m/z: 699.2411;
found m/z: 699.2413.
Synthesis of the pseudo-thio-dimannoside 5c
Prepared from peracetylated thio-mannose
1
(46 mg, Compound 5d was isolated through flash chromatography
0.11 mmol) and aziridine 4c (30 mg, 0.090 mmol) according to (1 : 1 Hex : EtOAc) in 68% yield (72 mg, 0.10 mmol). Rf = 0.27
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(Hex/EtOAc 1 : 1); [α]D18 (CHCl3, c 1.55): +69; 1H NMR (400 MHz, Hex : EtOAc) to give 33 in 92% yield (79 mg, 0.086 mmol) as
CDCl3): δ 6.09–5.99 (m, 1H, NH), 5.41 (d, J1-2 = 1.1 Hz, 1H, H1), colourless oil. Rf = 0.25 (Hex/EtOAc 1 : 1); [α]2D3 (CHCl3, c 1.00):
5.33 (dd, J2-3 = 3.3 Hz, J2-1 = 1.1 Hz, 1H, H2), 5.29 (t, J4-3 = J4-5
=
−37; 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.4 Hz, 2H,
10.0 Hz, 1H, H4), 5.20 (dd, J3-4 = 10.0 Hz, J3-2 = 3.3 Hz, 1H, H3), CH-Ar-Fmoc), 7.62 (d, J = 7.5 Hz, 2H, CH-Ar-Fmoc), 7.45–7.35
4.42–4.29 (mult., 2H, H5, H6a), 4.18–4.08 (mult., 2H, H4′, H6b), (m, 2H, CH-Ar-Fmoc), 7.36–7.26 (m, 2H, CH-Ar-Fmoc), 6.97 (d,
3.72 (s, 3H, OMe), 3.71 (s, 3H, OMe), 3.40 (AB system, Japp
15.0 Hz, 2H, CH2N3), 3.29–3.23 (m, 1H, H5′), 3.08 (ddd, J1′-2′
=
=
J = 6.7 Hz, 1H, NH), 5.64 (d, J = 8.0 Hz, 1H, NH), 5.35 (s, 2H,
H1, H2), 5.16 (dd, J3-4 = 10 Hz, J3-2 = 2.9 Hz, 1H, H3), 5.09 (dd,
12.6 Hz, J1′-6′ax = 8.8 Hz, J1′-6′eq = 4.2 Hz, 1H, H1′), 2.90–2.80 (m, J4-3 = J4-5 = 10 Hz, 1H, H4), 4.50–4.40 (mult., 2H, CH2-Fmoc),
1H, H2′), 2.28–2.18 (m, H3′eq), 2.18–2.00 (mult., 2H, H6′eq, H6′ 4.28–4.14 (mult., 4H, CH-Fmoc, H5, CH-Glu, H4′), 3.65 (s, 3H,
ax), 2.15 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.97 COOMe), 3.63 (s, 3H, COOMe), 3.37–3.32 (m, 1H, C5′),
(s, 3H, OAc), 1.92–1.80 (m, 1H, H3′ax), 1.20 (2× s, 6H, 2 × CH3 3.07–2.96 (mult., 2H, C2′, C1′), 2.30–1.82 (mult., 8H, C3′, C6′,
linker); 13C NMR (100 MHz, CDCl3): δ 175.0 (CO), 174.2 (CO), CH2-Glu, CH2-Glu), 2.12 (s, 3H, OAc), 2.06 (s, 3H, OAc), 1.98 (s,
173.5 (CO), 170.7 (CO), 169.9 (CO), 169.8 (CO), 169.7 (CO), 81.8 3H, OAc), 1.47 (s, 9H, tBu), 1.23 (d, JCH3-5 = 6.2 Hz, 3H, CH3-
(C1), 71.0 (C2), 69.5 (C3), 69.5 (C5), 66.3 (C4), 62.6 (C6), 60.0 Rha); 13C NMR (101 MHz, CDCl3) δ 174.7 (CO), 174.3 (CO),
(CH2N3), 52.4 (OMe), 52.3 (OMe), 48.0 (C4′), 43.9 (C5′), 40.2 171.9 (CO), 171.2 (CO), 170.2 (CO), 170.1 (CO), 128.0 (CH-Ar-
(C1′), 40.1 (C2′), 29.5 (C6′), 28.9 (C3′), 23.4 (CH3 linker), 23.4 Fmoc), 127.3 (CH-Ar-Fmoc), 125.3 (CH-Ar-Fmoc), 120.2 (CH-Ar-
(CH3 linker), 20.1 (OAc), 20.7 (OAc), 20.7 (OAc), 20.7 (OAc); MS Fmoc), 83.5 (C1), 71.6 (C2), 71.3 (C4), 69.6 (C3), 67.7 (C5), 67.4
(ESI) calcd for C29H42N4O14 [M + Na]+ m/z: 725.23; found m/z: (CH2-Fmoc), 53.9 (CH-Glu), 52.3 (COOMe), 52.2 (COOMe), 48.7
725.66.
(C4′), 47.4 (CH-Fmoc), 45.1 (C5′), 40.1 (C1′), 39.8 (C2′), 33.0
Product 5d (31 mg, 0.044 mmol) was deacetylated according (CH2-Glu), 32.1 (CH2-Glu), 29.9 (C3′, C6′), 28.2 (tBu-3 × Me),
to the general procedure for deacetylation (reaction time 3 h). 21.1 (OAc), 21.0 (OAc), 20.8 (OAc), 17.3 (CH3-Rha); MS (HRMS)
Deacetylated product was obtained in 68% (16 mg, calcd for C46H58N2O16S [M + Na]+ m/z: 949.3405; found m/z:
0.030 mmol) yield after purification. Rf = 0.21 (CH2Cl2/MeOH 949.3400.
1
9 : 1); [α]1D6 (MeOH, c 0.55): +105; H NMR (400 MHz, CD3OD):
δ 5.34 (d, J1-2 = 1.2 Hz, 1H, H1), 4.1 (ddd, J4′-3′ax = 8.1 Hz, J4′-3′eq
= 7.7 Hz, J4′-5′ = 4.1 Hz, 1H, H4′), 3.93–3.82 (mult., 3H, H5, H2, Conflicts of interest
H6a), 3.78–3.67 (m, 1H, H6b), 3.72 (s, 3H, OMe), 3.72 (s, 3H,
There are no conflicts to declare.
OMe), 3.67–3.57 (mult., 2H, H4, H3), 3.44 (AB system, Japp
=
15.7 Hz, 2H, CH2N3), 3.22–3.08 (mult., 3H, H5′, H1′, H2′), 2.34
(ddd, J6′eq- 6′ax = 14.3 Hz, J6′eq-1′ = 7.7 Hz, J6′eq-5′ = 3.8 Hz, 1H,
Acknowledgements
H
6′eq), 2.15 (ddd, J3′eq-3′ax = 14 Hz, J3′eq-2′ = 7 Hz, J3′eq-4′ = 4 Hz,
1H, H3′eq), 2.02 (ddd, J6′ax-6′eq = 14.3 Hz, J6′ax-5′ = 7.9 Hz, J6′ax-1′
=
This research was funded by the European Union’s Horizon
2020 research and innovation program under the Marie
Skłodowska-Curie grant agreement no. 765581 (PhD4GlycoDrug).
4.4 Hz, 1H, H6′ax), 1.88 (ddd, J3′ax-3′eq = 14.1 Hz, J3′ax-4′ = 7.8 Hz,
J3′ax-2′ = 4.6 Hz, 1H, H3′ax), 1.21 (s, 6H, 2× CH3 linker); 13C NMR
(100 MHz, CD3OD): δ 177.9 (CO), 175.6 (CO), 175.6 (CO), 85.3
(C1), 75.5 (C5), 73.9 (C2), 73.3 (C3), 68.9 (C4), 63.0 (C6), 61.1
(CH2N3), 52.7 (2 × OMe), 49.5 (C4′), 45.2 (C5′), 42.0 (C1′), 41.6
(C2′), 31.4 (C6′), 30.5 (C3′), 23.8 (CH3 linker), 23.5 (CH3 linker);
HR-MS (ESI) calcd for C21H34N4O10S [M + Na] + m/z: 557.1893;
found: 557.1899.
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crude was
dissolved
in
CH3CN
(1.9
ml)
and
N-methylmorpholine (0.010 ml, 0.093 mmol), amino acid 32
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0.093 mmol) were added. Reaction mixture was kept stirring
overnight. Then the reaction mixture was concentrated, dis-
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