Exploring a glycosylation methodology for the synthesis of hydroxamate-modified alginate building blocks

Article abstract of DOI:10.1039/c9ob02053e

Alginate, an anionic polysaccharide, is an important industrial biomaterial naturally harvested from seaweed. Many of its important physicochemical properties derive from the presence of charged carboxylate groups presented as uronic acids within the poly

Full text of DOI:10.1039/c9ob02053e

Organic &
Biomolecular Chemistry
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Exploring a glycosylation methodology for the
synthesis of hydroxamate-modified alginate
building blocks†
Cite this: Org. Biomol. Chem., 2019,
17, 9321
Eleni Dimitriou and Gavin J. Miller
*
Alginate, an anionic polysaccharide, is an important industrial biomaterial naturally harvested from
seaweed. Many of its important physicochemical properties derive from the presence of charged carbox-
ylate groups presented as uronic acids within the polysaccharide backbone. An ability to modify these
carboxylates with alternate functional groups would enable the design and implementation of new algi-
nate systems possessing different physicochemical properties. We present herein our approach to the
chemical synthesis of alginate disaccharides, modified at the carboxylate C6 position with bioisosteric
hydroxamate residues. The synthesis and utilisation of C6-hydroxamate donor and acceptor building
blocks enables a first access to protected α- and β-linked hydroxamate-containing disaccharides,
additionally equipped with a functional tether at the reducing terminus. The evaluation of these building
blocks for chemical glycosylation demonstrates the incorporation of bioisosteric functional groups into
an alginate disaccharide backbone and highlights the important contribution of both acceptor and donor
reactivity underpinning uronate glycosylations.
Received 20th September 2019,
Accepted 3rd October 2019
DOI: 10.1039/c9ob02053e
rsc.li/obc
important use as an industrial biomaterial, currently sourced
from marine algae, where it is applied as a stabiliser, viscosi-
1. Introduction
Alginate, 1, a heterogenous polysaccharide composed of β-1,4- fier and gelling agent across the food, beverage, paper and
linked D-mannuronic acid (M) and its C5 epimer α-L-guluronic pharmaceutical industries.2–5 Alginate is therefore something
acid (G) (Fig. 1), was first extracted from brown algae of a double-edged sword from the perspective of its deleterious
(Phaeophyceae) in the late nineteenth century and has been role in microbial infections countered by its profound utility
commercially available since the early twentieth century. It is as a biomaterial.
also produced by two genera of bacteria, Pseudomonas and
As part of a program to develop accessing next-generation
Azotobacter, and the study of alginate biochemistry and biosyn- alginate materials, through the provision of modified oligosac-
thesis has, to date, largely focused on the Pseudomonas genera. charide sequences with improved or altered functional group
This is owed to the prevalence of Pseudomonas aeruginosa in properties, we targeted a bottom-up synthetic approach to
causing chronic infections for cystic fibrosis patients, contri- provide structurally defined, modified alginate building
buting to a reduction in lung function and increased mortality blocks. Utilisation of such an approach for both chemical6–9
rates.1
Within alginate sub-structure the relative proportions of M
and automated10 syntheses of poly-M and GM-containing algi-
and G units, their homo- or hetereopolymeric block-groupings
and the possibility for acetylation at the C2 and/or C3 posi-
tions of M residues produces a structurally diverse biopolymer.
This structural microheterogeneity varies depending on the
alginate source and consequently affects the viscosity and gel-
forming capacity of the final polysaccharide material. Such
physicochemical properties mean that alginate has also found
Lennard-Jones Laboratory, School of Chemical and Physical Sciences,
Keele University, Keele, Staffordshire, ST5 5BG, UK. E-mail: g.j.miller@keele.ac.uk
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
C9OB02053E
Fig. 1 Chemical structure of the alginate polysaccharide 1 showing
constituent M and G residues and C2/C3 acetylation for one M residue.
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nate oligosaccharides have been reported, which provides extraction compared to native C6-carboxylic acids.14 We report
essential understanding of the glycosylation chemistry herein our approach to the first examples of hydroxamate
required to assemble such complex materials.11 The assembly modified alginate disaccharide building blocks and discuss
of β-1,4-linked mannuronates is challenging, owing to the the initial data arising from utilising C6-modified mannuro-
required 1,2-cis linkage and the presence of a carboxylate oxi- nates for glycosylation.
dation level at C6. Uronate donors were typically classed as
inferior donors compared to their unoxidised counterparts,
due to the electron withdrawing effect of the C6 group making
them less reactive. Recently Codée’s group have countered this
general classification and demonstrated a highly β-selective
2. Results and discussion
2.1. Synthesis of monosaccharide building blocks
glycosylation methodology for the assembly of alginate We envisaged our synthetic methodology to derive from a
oligosaccharides. They proposed that a C5-carboxylate ester in common precursor that could give access to several different
the mannuronate donor prefers to occupy an axial position in mannuronate building blocks (donors and acceptors), for gly-
the oxocarbenium half-chair intermediate (Fig. 2a), which cosylation and assembly into longer systems. We also wished
undergoes reaction with the acceptor, delivering the to incorporate capability for immobilisation or conjugation
required 1,2-cis linked glycosylation products with excellent through a reducing-end tether, as has been employed success-
diastereoselectivity.11,12
fully for many carbohydrate fragment syntheses.15–17
Inspired by this work we envisaged that incorporating Accordingly, we identified carboxylate 3, reported by Codèe for
changes to the parent monosaccharide component (through automated alginate oligosaccharide synthesis,10 as our starting
modification of the carboxylate residue) could enable the point. Thioglycoside hydroxamate donors 5 and 6 were pre-
assembly of modified alginate oligosaccharides using a glyco- pared on multi-gram scale from 3 (Scheme 1). To install the
sylation approach similar to that seen for the native system. C6-hydroxamate group we investigated coupling of carboxylate
We chose to investigate a bioisosteric carboxylate replacement 3 with O-benzyl hydroxylamine using PyBOP as the activating
(Fig. 2b), with a view to providing C6-modified alginates with reagent. This reaction proceeded smoothly and in good yield
new functional properties; in this case as possible sidero- (81%) and was followed by benzyl protection of the hydroxa-
phores through the known ion-chelating abilities of a hydroxa- mate nitrogen. We evaluated coupling of 3 directly with N,O-
mic acid.13 Hydroxamic acid incorporation at C6 in glycosides dibenzyl hydroxylamine to avoid this subsequent protection
has been harnessed to produce biodegradable surfactants with step, but this reagent, more basic than O-benzyl hydroxyl-
good chelating properties for the removal of contaminant amine, triggered a competing C4–C5 elimination and so was
metals in wastewater; Kovensky and co-workers demonstrated abandoned. Similarly, use of an acetyl group to protect the
that hydroxamic acid derivatives exhibited improved iron hydroxamate nitrogen proved problematic, being readily
Fig. 2 (a) Glycosylation chemistry to access β-mannuronates (b) building blocks required for assembly of modified alginates. R = protecting group
e.g. Bn, Lev.
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Scheme 1 (a) BnO–NH2, PyBOP, DIPEA, DCM, rt, 3 h, 81% (b) K2CO3, BnBr, DMF, RT, 2 h, 42% (c) Ac2O, pyridine DCM, rt, 24 h, 68% (d) Lev2O, pyri-
dine, DCM, rt, 24 h, 88% (e) for R = Ac, HO(CH2)3Br, Ph2SO, TTBP, Tf2O, DCM, −90 °C to −20 °C, 1 h, 89%, for R = Lev, Ph2SO, TTBP, Tf2O, HO
(CH2)3Br, DCM, −90 °C to −20 °C, 1 h, 85% (f) For R = Ac, NaN3, acetone, 76%, for R = Lev, NaN3, acetone, 54% (g) For R = Ac, Na(s), MeOH, RT, 24 h,
61%. (h) For R = Lev, H2NNH2·H2O, pyridine/AcOH (4/1), 30 min, 55%. Anomeric 1JC–H coupling constants shown in blue to support later assignment
of glycosylation stereochemistry.
cleaved under the reaction conditions needed for thioglycoside 2.2. Synthesis of C6-hydroxamate-modified disaccharides
donor activation to access 7 or 8. Following successful coup-
ling and N-Bn protection, O-4 was protected as either a levuli- For comparative purposes we first completed a synthesis of
noyl or acetyl ester to deliver thioglycoside donors 5 and 6 native mannuronate disaccharides 14 and 15,8 glycosylating
(Scheme 1).
native acceptor 13 with either of donors 11 or 12 using NIS/
Both 5 and 6 were additionally manipulated into a reducing TMSOTf activation (Scheme 3). Confirmation of the desired
end acceptor, to allow iteration with appropriate donors. 1,2-cis glycosylation products 14 and 15 was made through
1
Thioglycoside activation using Ph2SO–Tf2O allowed glycosyla- comparison of the anomeric JC–H coupling constants, which
tion with 3-bromopropanol, installing the precursor to a redu- closely matched those previously reported for 15.8 Whilst pro-
cing-end, conjugable linker within 7 and 8. Initial attempts ceeding with O-4-deprotection of disaccharides 14 and 15 we
using NIS or NBS to activate 5 or 6 for reaction with 3-bromo- observed that removal of the O-4 acetyl group from 15 using
propanol failed, with N-Bn deprotection observed, followed by NaOMe delivered a product whose analytical data were not
the formation of an α-linked glycosylation product, possibly consistent with the literature reported for the expected product
through anchimeric assistance of the now deprotected nitro- 16 (which was accessed form an O-4 Lev deprotection using
gen blocking the top face of the intermediate. SN2 displace- hydrazine).8 1H NMR analysis of our material (17) showed H1′
ment of alkyl bromides 7 and 8 was completed using sodium at 5.43 ppm instead of the reported 4.73 ppm. Moreover,
1
1
azide, followed by appropriate O-4 deprotection to deliver coupled HSQC data showed JC1–H1 = 156 Hz and JC1′–H1′
=
hydroxamate acceptor 9. 176 Hz. These data suggested that the reaction conditions had
With C6-modified donor and acceptor building blocks 5, 6 liberated the C4-OH (observed at 2.95 ppm), but also altered
and 9 in hand we also completed the synthesis of native man- the anomeric integrity at the non-reducing end of the disac-
nuronate donor (10–12) and acceptor 13 materials from 3 charide. ESI-MS analysis found the sodium adduct of 17 and
(Scheme 2), delivering a small matrix of appropriate building comparison to a disaccharide containing L-guluronic acid as
blocks to subsequently explore modified alginate disaccharide the non-reducing end monomer ruled out C5 epimerisation
synthesis.
(H1′Gul = 5.24 ppm).8 An nOe experiment using 17 and irradiat-
Scheme 2 (a) MeI, K2CO3, DMF rt, 24 h, 77% (b) Lev2O, pyridine, 18 h, 78% (c) Ac2O, pyridine, 3 h, 78% (d) TBDMSOTf, imidazole, DMAP, DMF, RT,
24 h, 70% (e) for R = Ac, Ph2SO, TTBP, Tf2O, HO(CH2)3Br, DCM, −60 to −90 °C, 1 h, 66% (f) NaN3, acetone, 55 °C, 48 h, 97% (g) Na(s), MeOH, RT,
24 h, 87%. Anomeric 1JC–H coupling constants shown in blue.
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Scheme 3 (a) For 11, NIS, TMSOTf, CH2Cl2, −60 °C, 30 min, 56%, for 12, NIS, TMSOTf, CH2Cl2, −60 °C, 30 min, 61%, (b) NaOMe, MeOH, 2 h, 46% (c)
for 14 AcCl, MeOH, 18 h, 40%. Anomeric 1JC–H coupling constants shown in blue.
ing H1′ (5.43 ppm) showed transfer to H4 (see ESI†), but not to vering β-linked products 7 and 8 in high yields (Scheme 1).
H5′. Comparatively, an nOe experiment for 16 showed transfer This same activation protocol was thus applied in the glycosy-
from H1′ to H5′ but not to H4, supporting a change in the disac- lation of 9 with 5, but unfortunately disaccharide 18 was not
charide linkage stereochemistry from β to α for 17 under these formed (Scheme 4).
reaction conditions. To investigate this unusual observation
Variations to the reaction conditions using Ph2SO/Tf2O
further, we repeated the experiment and stopped the de- were scrutinised, but still did not deliver 18. For this pre-acti-
protection after 1 h, neutralising with Amberlite as before. 13C vation protocol we generally observed acceptor, amounts of
NMR clearly showed a mixture of α- and β-linked products (see hydrolysed donor and formation of a polar, baseline material,
ESI†), suggesting the anomerisation was underway, but not suggesting that donor 5 was undergoing an alternative reac-
complete. We took this material and stirred it overnight in tion. Despite isolating the baseline material, we were unable to
MeOH with Amberlite (observed pH = 5–6), but no further characterise this side-product. Evaluation of several further gly-
change was observed by 13C NMR, suggesting that the reaction cosylation conditions, including BSP/Tf2O, DMTST and inverse
time and/or pH and for 4-O-Ac deprotection were causing this glycosylation, all failed to produce 18.
unwanted reaction. At present we cannot fully explain why this
Based on these observations, we synthesised an
occurred under these conditions beyond being able to report N-phenyltrifluoroacetimidate (N-PhTFA) donor 19 from 5 via
the observed data; an E1CB elimination from the reducing end the hemiacetal (Scheme 4) and used this directly for glycosyla-
uronate, mutarotation of the released hemi-acetal to the tion with 9. tert-Butyldimethylsilyl trifluoromethanesulphonate
α-anomer, followed by re-addition to the bottom face of the elim- (TBDMSOTf) was employed as the activator, as it had been
ination product could deliver 17 from 15. Alternative attempts to used successfully for activation of the native N-PhTFA mannur-
remove the acetate group in 15 with NH3 or triethylamine in onate donor.10 This glycosylation returned mostly unreacted 9
methanol at room temperature and 35 °C were unsuccessful, (87%) and a small amount of the anomeric tert-butyldimethyl-
recovering only starting material. Comparatively, when O-4 TBS silanol adduct of donor 5 (16%). We next attempted pre-acti-
disaccharide 14 was treated with AcCl in MeOH at room temp- vation of donors 5 or 10 prior to the addition of the α-thio accep-
erature the reaction proceeded in 40% yield to deliver 16 whose tors (9 or 13). However, this reaction was only successful when
anomeric integrity was confirmed as expected (Scheme 3).
an acceptor with a primary C6-OH was used, not with 9 or 13.
We next attempted to apply the established glycosylation As we had evaluated several unsuccessful approaches to effect
methodology using hydroxamate donor 5 and hydroxamate glycosylation towards a hydroxamate disaccharide, we next inves-
acceptor 9 (Scheme 4). We had previously confirmed that tigated the reactivity of modified C6 donor 5 and acceptor 9
donor pre-activation with Ph2SO/Tf2O was successful in deli- systems with native mannuronate building blocks 10 and 13.
Scheme 4 (a) 5, Ph2SO/Tf2O or BSP/Tf2O or Me2S2/Tf2O or NIS/TMSOTf, see Table 1 in ESI† for details of reaction conditions attempted (b) (i) NIS,
AgOTf, DCM/H2O, 75% (ii) N-PhTFA-Cl, K2CO3, H2O, acetone, 68%. Anomeric 1JC–H coupling constants shown in blue.
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2.3. Synthesis of mixed C6-hydroxamate disaccharides
α-linkage had formed as the major product (9/1 as adjudged
by 1H NMR). This glycosylation was repeated using donors 5
The coupling of hydroxamate acceptor 9 with mannuronate and 6 at different temperatures and for different periods of
donor 10 using an NIS/TMSOTf promoter system produced time (1 h at −40 °C, 2 h at −25 °C, 3 h at −20 °C and 30 min at
β-linked disaccharide 20 in 55% yield, as indicated by the −10 °C). The yields obtained were however only slightly
1
observed JC–H coupling constants (Scheme 5). This result improved with 22 isolated in a maximum 30%, alongside
suggested the balance of both donor and acceptor reactivity recovered 13 (14%). Subsequent O-4-deprotection of 22 or 23
during glycosylation was improved, relative to forming 18, was effected using hydrazine or sodium methoxide giving 24
possibly through the known reactivity of mannuronate donor in acceptable yields (76% from 22 and 54% from 23) and
10.8,10 C6-modified alginate disaccharide 20 could be con- noting that the anomerisation issues observed for deprotecting
veniently 4-O-deprotected, using hydrazine hydrate, to regener- 15 were not repeated. Here we generally found 4-O-Lev de-
ate acceptor capability in the form of 21 in good yield (81%).
protection to be better yielding at disaccharide level, compared
We also attempted the reverse reaction, using donors 5 or 6 to 4-O-Ac.
with mannuronate acceptor 13 (Scheme 6). Using donor 6,
In isolating α-linked disaccharides 22 and 23, we propose
TLC analysis indicated the formation of a complex mixture that the inclusion of a protected hydroxamate group enabled
after 30 min at 0 °C. Purification yielded 23 in low yield (12%). its participation in the reaction through blocking the top face
1
A coupled HSQC spectrum showed JC1–H1 = 156 Hz for the of the donor. This may proceed through a bicyclic glycosyl
reducing end (C1, 13C δ 99.5 ppm) and 1JC1′–H1′ = 176 Hz for the cation of type 25, illustrated in Fig. 3, or through coordination
non-reducing end linkage (C1′, 13C δ 102.0 ppm), suggesting an of the hydroxamate oxygen to the anomeric carbon (not
1
Scheme 5 (a) 10, NIS, TMSOTf, CH2Cl2, −40 °C, 60 min, 55% (b) H2NNH2·H2O, pyridine/AcOH (4/1), 30 min, 81%. Anomeric JC–H coupling con-
stants shown in blue.
Scheme 6 (a) 6, NIS, TMSOTf, CH2Cl2, 0 °C, 30 min, 12% or 5, NIS, TMSOTf, CH2Cl2, −40 °C to −10 °C, 6.5 h, 30% (b) for R = Ac, Na(s), MeOH, RT,
16 h, 54%, For 22, H2NNH2·H2O, pyridine/AcOH (4/1), 30 min, 76%. Anomeric 1JC–H coupling constants shown in blue.
Fig. 3 (a) A possible glycosyl intermediate 25 formed during glycosylation to 22, blocking the top face of the donor (b) cyclic amide 26. A 1C4 con-
formation was indicated through analysis of the 1H coupling constants e.g. H4 δ 5.06 (app. t, J = 1.9 Hz).
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shown). As the yields for this reaction were generally poor and Sigma Aldrich. All reactions in non-aqueous solvents were con-
coincided with recovered acceptor, we hypothesised that an ducted in oven dried glassware under a nitrogen atmosphere
alternative pathway may exist for the reaction, giving rise to a with a magnetic stirring device. Solvents were purified by
cyclic product with loss of N-Bn (Fig. 3, red dotted pathway). passing through activated alumina columns and used directly
This arose from earlier experiments using an N-Ac protected from a Pure Solv-MD solvent purification system and were
hydroxamate donor (attempting to install the anomeric linker transferred under nitrogen. Reactions requiring low tempera-
group) where we isolated and characterised bicyclic N-linked tures used the following cooling baths: −78 °C (dry ice/
hydroxamate 26. We were however unable to isolate any of 26 acetone), −30 °C (dry ice/acetone), −15 °C (NaCl/ice/water) and
from the reaction to form 22 and also confirmed through 0 °C (ice/water). Infra-red spectra were recorded neat on a
HRMS and HMBC analyses of 22 that the N-Bn remained PerkinElmer Spectrum 100 FT-IR spectrometer; selected
intact. Additionally, we did not detect N- to O-4 Bn transfer absorbtion frequencies (νmax) are reported in cm−1
between 6 and 13 and at this juncture are unable to fully spectra were recorded at 400 MHz and GATED-13C spectra at
explain the lower than average yield for these glycosylations.
100 MHz respectively using a Bruker AVIII400 spectrometer. 1H
.
1H NMR
The results of these experiments using C6-hydroxamate NMR signals were assigned with the aid of gDQCOSY. 13C
modified glycosyl donors and acceptors indicate there is a deli- NMR signals were assigned with the aid of gHSQCAD.
cate balance of reactivity contributed from both reaction com- Coupling constants are reported in Hertz. Chemical shifts
ponents. From this initial data we conclude that modified accep- (δ, in ppm) are standardised against the deuterated solvent
tor 9 can be used effectively with native mannuronate donors to peak. NMR data were analysed using Nucleomatica iNMR soft-
1
deliver β-1,4-linked 20 in acceptable yields. A subsequent small ware. H NMR splitting patterns were assigned as follows: br s
scale iteration attempt to the trisaccharide using modified (broad singlet), s (singlet), d (doublet), app. t (apparent
donor 6 and acceptor 21 switched the linkage stereochemistry to triplet), t (triplet), dd (doublet of doublets), ddd (doublet of
α and proceeded in poor yield (14%). This implies a redundancy doublet of doublets), or m (multiplet and/or multiple reso-
for the formation of multiple β-1,4-linkages. Similarly, use of nances). Reactions were followed by thin layer chromatography
native acceptor 13 with modified donor 6 gave α-linked products (TLC) using Merck silica gel 60F254 analytical plates (alu-
and in low yield, but glycosylation of 5 or 6 with a more reactive minium support) and were developed using standard visualis-
primary alcohol acceptor was successful. We are currently evalu- ing agents: short wave UV radiation (245 nm) and 5% sulfuric
ating approaches to alternative C6-hydroxamate building blocks acid in methanol/Δ. Purification via flash column chromato-
that will deliver capability for β-linked elongation beyond disac- graphy was conducted using silica gel 60 (0.043–0.063 mm).
charide level and will report on this in due course.
Melting points were recorded using open glass capillaries on a
Gallenkamp melting point apparatus and are uncorrected.
Optical activities were recorded on automatic polarimeter
Rudolph autopol I or Bellingham and Stanley ADP430 (concen-
tration in g per 100 mL). MS and HRMS (ESI) were obtained on
4. Conclusion
Elucidating new chemical methodologies to modify carbo- Waters (Xevo, G2-XS TOF) or Waters Micromass LCT spec-
hydrate monomer building blocks is underpinning to their trometers using a methanol mobile phase. High resolution
glycosylation to create longer oligo- and polysaccharides that (ESI) spectra were obtained on a Xevo, G2-XS TOF mass
contain non-native functional groups. This will contribute spectrometer. HRMS was obtained using a lock-mass to adjust
to the development of next-generation carbohydrate-based the calibrated mass.
materials with improved or altered physicochemical properties
O-Benzyl (phenyl 2,3-di-O-benzyl-1-thio-α-D-mannopyrano-
and will enable the essential study of polysaccharide architec- side) hydroxamate. To a mixture of 312 (700 mg, 1.50 mmol,
tures in more detail.18 Targeting the provision of modified 1.0 equiv.) and O-benzylhydroxylamine hydrochloride (260 mg,
alginate oligosaccharides, we have described our approach to 1.65 mmol, 1.1 equiv.) in CH2Cl2 (5 mL) was added succes-
access C6-hydroxamate derivatives of native mannuronic acid. sively under N2 atmosphere, PyBOP (860 mg, 1.65 mmol,
Bioisosteric hydroxamate donor and acceptor monosacchar- 1.1 equiv.) and DIPEA (653 µL, d = 0.742, 3.75 mmol,
ides were successfully synthesised and enabled the first prep- 2.5 equiv.). The reaction mixture was left stirring at room
arations of related disaccharide species (in protected form). Of temperature for 3 h. Upon completion of the reaction, the
note is an observed switch between the ability to deliver α- or solvent was removed under reduced pressure and the crude
β-linked glycosylation products, depending on donor/acceptor was purified using silica gel flash column chromatography,
pairing when using these non-native building blocks.
eluting with EtOAc/hexane (30/70, 40/60, 50/50, 90/10) to
afford the title compound as a colourless oil (770 mg,
1.35 mmol, 90%). Rf 0.30 (EtOAc/hexane, 1/2); [α]2D2 +120.0
(c. 1.0, CHCl3); H NMR (400 MHz; CDCl3) δ 8.84 (1 H, br. s,
C(O)NHOBn), 7.39–7.21 (20H, m, Ar–H), 5.38 (1 H, d, J =
1.4 Hz, H1), 4.87 (1 H, d, J = 11.1 Hz, CH2Ph-attached to C3),
1
5. Experimental section
5.1. General methods and materials
All reagents and solvents which were available commercially 4.84 (1 H, d, J = 10.7 Hz, C(O)NHOCH2Ph), 4.83 (1 H, d, J =
were purchased from Acros, Alfa Aesar, Fisher Scientific, or 11.2 Hz, CH2Ph-attached to C3), 4.66 (1 H, d, J = 12.0 Hz,
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CH2Ph-attached to C2), 4.64 (1 H, d, J = 12.0 Hz, C(O) column chromatography, eluting with a gradient of diethyl
NHOCH2Ph), 4.58 (1 H, d, J = 12.0 Hz, CH2Ph-attached to C2), ether/petroleum ether (30%–90% diethyl ether) to afford 5 as a
4.54 (1 H, d, J = 9.7 Hz, H5), 4.30 (1 H, app. t, J = 9.5 Hz, H4), colourless oil (90 mg, 0.12 mmol, 91%). Rf 0.58 (EtOAc/hexane,
1
3.90 (1 H, dd, J = 2.8, 1.8 Hz, H2), 3.72 (1 H, dd, J = 9.2, 3.0 Hz, 1/2); [α]2D2 +51.50 (c. 1.00, CHCl3); H NMR (400 MHz; CDCl3)
H3); 13C NMR (101 MHz; CDCl3) δ 168.4 (C(O)NHOBn), 138.5, δ 7.38–7.22 (25 H, m, Ar–H), 5.74 (1 H, app. t, J = 9.6 Hz, H4),
137.9, 134.9, 133.1, 132.2, 129.5, 129.4, 128.9, 128.7, 128.5, 5.54 (1 H, d, J = 2.0 Hz, H1), 5.39 (1 H, d, J = 12.0 Hz, C(O)N
128.5, 128.5, 128.2, 128.1, 128.0, 127.95, 127.9, 127.8 (18 C, Ar– (CH2Ph)OBn), 5.32 (1 H, d, J = 12.0 Hz, C(O)N(CH2Ph)OBn),
C), 86.7 (C1), 78.4 (C(O)NHOCH2Ph), 77.9 (C3), 76.7 (C2), 73.2 4.99 (2 H, s, C(O)N(Bn)OCH2Ph), 4.67 (1 H, d, J = 12.5 Hz,
(CH2Ph-attached to C3), 73.0 (CH2Ph-attached to C2), 71.2 CH2Ph-attached to C2), 4.64 (1 H, d, J = 12.9 Hz, CH2Ph-
(C5), 69.8 (C4); HRMS (ES+) m/z [found: (M + H)+ 572.2111 attached to C2), 4.64 (1 H, d, J = 9.8 Hz, H5), 4.58 (1 H, d, J =
C33H33NO6S requires (MH)+, 572.2107]; IR νmax/cm−1 3313 (m, 12.2 Hz, CH2Ph-attached to C3), 4.53 (1 H, d, J = 12.2 Hz,
N–H), 1659 (s, CvO), 1071 (s, C–Oether).
CH2Ph-attached to C3), 3.98–3.94 (1 H, m, H2), 3.75 (1 H, dd,
O-Benzyl, N-benzyl (phenyl-2,3-di-O-benzyl-1-thio-α-D-man- J = 9.4, 2.9 Hz, H3), 2.66–2.38 (3 H, m, CH2 Lev), 2.31–2.19 (1
nopyranoside) hydroxamate 4. To a stirred solution of O-benzyl H, m, CH2 Lev), 2.12 (3 H, s, CH3 Lev); 13C NMR (101 MHz;
(phenyl 2,3-di-O-benzyl-1-thio-α-D-mannopyranoside) hydroxa- CDCl3) δ 206.2 (CvO Lev ketone), 171.1 (CvO Lev), 150.6
mate (180 mg, 0.31 mmol, 1.0 equiv.) and K2CO3 (65.3 mg, (C(O)N(Bn)OBn), 137.9, 137.9, 137.8, 137.1, 131.6, 129.1, 128.4,
0.47 mmol, 1.5 equiv.) in DMF (3.5 mL) was added benzyl 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.7, 127.6,
bromide (41.2 µL, d = 1.438, 0.35 mmol, 1.1 equiv.) at room 127.5, 86.1 (C1), 77.2 (C3), 76.4 (C(O)N(Bn)OCH2Ph), 76.1 (C2),
temperature. The reaction mixture stirred for 4 h, diluted with 73.1 (C(O)N(CH2Ph)OBn), 72.4 (CH2Ph-attached to C2), 72.1
EtOAc (60 mL), washed with H2O (50 mL) and brine (50 mL), (CH2Ph-attached to C3), 71.1 (C5), 68.6 (C4), 38.0 (CH2 Lev),
dried over MgSO4, filtered and concentrated under reduced 29.8 (CH3 Lev), 28.0 (CH2 Lev); 13C-GATED (101 MHz; CDCl3):
pressure. Purification of the crude product by Reveleris® auto- 86.1 (1JC1–H1 = 168 Hz, C1); HRMS (ES+) m/z [found: (M + H)+
mated silica gel flash column chromatography (liquid injec- 760.2955 C45H45NO8S requires (MH)+, 760.2939].
tion onto column), eluting with EtOAc/hexane (0/100, 5/95, 20/
O-Benzyl, N-benzyl (phenyl 4-O-acetyl-2,3-di-O-benzyl-1-thio-
80 and 90/10) afforded 4 as a colourless oil (90 mg, 0.13 mmol, α-D-mannopyranoside) hydroxamate 6. To a stirred solution of
44%). Rf 0.75 (EtOAc/hexane, 1/2); [α]2D2 +55.7 (c. 0.22, CHCl3); 4 (900 mg, 1.47 mmol, 1.0 equiv.) and K2CO3 (240 mg,
1H NMR (400 MHz; CDCl3) δ 7.40–7.14 (25 H, m, Ar–H), 5.59 1.76 mmol, 1.2 equiv.) in DMF (11 mL) was added benzyl
(1 H, d, J = 1.3 Hz, H1), 5.22 (1 H, d, J = 12.2 Hz, C(O)N(CH2Ph) bromide (0.2 mL, d = 1.438, mmol, 1.1 equiv.) at room temp-
OBn), 5.16 (1 H, d, J = 12.2 Hz, C(O)N(CH2Ph)OBn), 5.06 (1 H, erature. The reaction mixture stirred for 15 h, diluted with
d, J = 12.3 Hz, C(O)N(Bn)OCH2Ph), 5.00 (1 H, d, J = 12.3 Hz, EtOAc (60 mL), washed with H2O (50 mL) and brine (50 mL),
C(O)N(Bn)OCH2Ph), 4.71 (1 H, d, J = 11.9 Hz, CH2Ph), 4.69 dried over MgSO4, filtered and concentrated under reduced
(1 H, d, J = 12.2 Hz, CH2Ph), 4.64 (1 H, d, J = 10.7 Hz, CH2Ph), pressure. Purification of the crude product by Reveleris® auto-
4.62 (2 H, d, J = 8.8 Hz, H5), 4.59 (1 H, d, J = 11.6 Hz, CH2Ph), mated silica gel flash column chromatography (liquid injec-
4.43 (1 H, td, J = 9.6, 2.9 Hz, H4), 3.96 (1 H, dd, J = 2.9, 1.6 Hz, tion onto column), eluting with EtOAc/hexane (0/100, 5/95, 20/
H2), 3.66 (1 H, dd, J = 9.8, 3.0 Hz, H3), 2.57 (1 H, d, J = 2.9 Hz, 80 and 90/10) afforded 6 as a colourless oil (450 mg,
C4-OH); 13C NMR (101 MHz; CDCl3) δ 151.8 (C(O)N(Bn)OBn), 0.64 mmol, 42%). Rf 0.80 (EtOAc/hexane, 1/2); [α]2D2 +46.7
1
138.2, 137.9, 137.7, 136.7, 134.0, 130.8, 129.2, 128.5, 128.4, (c. 1.17, CHCl3); H NMR (400 MHz; CDCl3) δ 7.40–7.20 (25 H,
128.4, 128.2, 128.0, 127.94, 127.9, 127.8, 127.7, 127.4, 86.3 m, Ar–H), 5.73 (1 H, t, J = 9.6 Hz, H4), 5.55 (1 H, d, J = 2.0 Hz,
(C1), 78.2 (C3), 77.2 (C2), 76.5 (C(O)N(Bn)OCH2Ph), 72.8 H1), 5.42 (1 H, d, J = 11.9 Hz, C(O)N(CH2Ph)OBn), 5.35 (1 H, d,
(CH2Ph), 72.6 (CH2Ph), 72.3 (C(O)N(CH2Ph)OBn), 71.8 (C5), J = 12.0 Hz, C(O)N(CH2Ph)OBn), 5.00 (1 H, d, J = 12.5 Hz, C(O)
67.8 (C4); 13C-GATED (101 MHz; CDCl3): 86.3 (1JC1–H1 = 168 Hz, N(Bn)OCH2Ph), 4.97 (1 H, d, J = 12.5 Hz, C(O)N(Bn)OCH2Ph),
C1); HRMS (ES+) m/z [found: (M + H)+ 662.2589 C40H39NO6S 4.65 (2 H, s, CH2Ph-attached to C2), 4.63 (1 H, d, J = 9.8 Hz,
requires (MH)+, 662.2571]; IR νmax/cm−1 1640 (m, CvOamide), H5), 4.57 (1 H, d, J = 12.2 Hz, CH2Ph-attached to C3), 4.49 (1 H,
1454 (m, CvCaromatic), 1223 (s, C–Oester), 1024 (s, C–Oether).
d, J = 12.2 Hz, CH2Ph-attached to C3), 3.98 (1 H, app. t, J =
O-Benzyl, N-benzyl (phenyl 4-O-levulinoyl-2,3-di-O-benzyl-1- 2.5 Hz, H2), 3.74 (1 H, dd, J = 9.4, 2.9 Hz, H3), 1.80 (3 H, s,
thio-α-D-mannopyranoside) hydroxamate 5. To a mixture of 4 C(O)CH3); 13C NMR (101 MHz; CDCl3) δ 169.3 (C(O)CH3), 150.7
(90 mg, 0.13 mmol, 1.0 equiv.) and levulinoyl anhydride (C(O)N(Bn)OBn), 137.9, 137.9, 137.8, 137.2, 133.7, 131.7, 129.1,
(43 mL, 0.27 mmol, 2.0 equiv.) in CH2Cl2 (1 mL) was added 128.4, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7, 127.7, 127.7,
pyridine (44 mL, 0.54 mmol, 4.0 equiv.) under N2 atmosphere. 127.7, 127.6, 127.6, 86.2 (C1), 76.8 (C3), 76.4 (C(O)N(Bn)
The reaction mixture was left stirring at room temperature for OCH2Ph), 76.0 (C2), 73.2 (C(O)N(CH2Ph)OBn), 72.4 (CH2Ph-
18 h. Upon completion of the reaction, the mixture was attached to C2), 72.0 (CH2Ph-attached to C3), 71.3 (C5), 68.3
diluted with CH2Cl2 (15 mL), and the organic layer was washed (C4), 20.7 (C(O)CH3); 13C-GATED (101 MHz; CDCl3): 86.2
successively with 1 M HCl (2 × 10 mL) and sat. aq. NaHCO3 (1JC1–H1 = 168 Hz, C1); HRMS (ES+) m/z [found: (M + H)+
solution (2 × 10 mL). The organic layer was dried over MgSO4, 704.2681 C42H41NO7S requires (MH)+, 704.2676]; IR νmax/cm−1
filtered and concentrated under reduced pressure furnishing a 1751 (m, CvOester), 1639 (m, CvOamide), 1496, 1454
colourless oil. The crude was purified using silica gel flash (CvCaromatic), 1223 (s, C–Oester), 1024 (s, C–Oether).
This journal is © The Royal Society of Chemistry 2019
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Organic & Biomolecular Chemistry
3-Bromopropyl O-benzyl, N-benzyl (4-O-levulinoyl-2,3-di-O- (c. 2.00, CHCl3); 1H NMR (400 MHz; CDCl3) 7.41–7.23 (20 H,
benzyl-β-D-mannopyranoside) hydroxamate 7. A solution of 5 m, Ar–H), 5.65 (1 H, app. t, J = 9.9 Hz, H4), 5.48 (1 H, d, J =
(100 mg, 0.13 mmol, 1.0 equiv.), diphenyl sulphoxide (34 mg, 12.0 Hz, C(O)N(CH2Ph)OBn), 5.42 (1 H, d, J = 12.0 Hz, C(O)N
0.187 mmol, 1.3 equiv.) and tri-tert-butylpyrimidine (81 mg, (CH2Ph)OBn), 4.98 (1 H, d, J = 12.4 Hz, C(O)N(Bn)OCH2Ph),
0.33 mmol, 2.5 equiv.) in CH2Cl2 (2.5 mL) was stirred over acti- 4.95 (1 H, d, J = 12.5 Hz, C(O)N(Bn)OCH2Ph), 4.88 (1 H, d, J =
vated 4ÅMS for 40 min. The mixture was cooled to −60 °C and 12.3 Hz, CH2Ph-attached to C2), 4.81 (1 H, d, J = 12.3 Hz,
triflic anhydride (28 µL, d = 1.720, 0.17 mmol, 1.3 equiv.) was CH2Ph-attached to C2), 4.52 (1 H, d, J = 12.3 Hz, CH2Ph-
then added. The mixture was stirred for 5 min followed by attached to C3), 4.43 (1 H, d, J = 12.3 Hz, CH2Ph-attached to
cooling to −90 °C, upon 3-bromopropanol (18 µL, d = 1.537, C3), 4.37 (1 H, s, H1), 3.90–3.84 (1H, m, OCH2CH2CH2N3) 3.88
0.20 mmol, 1.5 equiv.) was added. The reaction mixture was (1 H, d, J = 3.0 Hz, H2), 3.82 (1 H, d, J = 10.0 Hz, H5), 3.48 (1 H,
allowed to warm up to −20 °C, and stirring was continued for ddd, J = 6.6, 6.0, 3.6 Hz, OCH2CH2CH2N3), 3.44 (1 H, dd, J =
1 h. At that temperature triethylamine was added until pH = 7, 9.8, 2.8 Hz, H3), 3.30 (2 H, ddd, J 13.9, 9.9, 4.0 Hz,
the organic layer was washed with H2O (10 mL), dried over OCH2CH2CH2N3), 2.63–2.37 (3 H, m, CH2 Lev), 2.24 (1 H, ddd,
MgSO4, filtered and concentrated under reduced pressure. J = 10.8, 8.8, 4.0 Hz, CH2 Lev), 2.10 (3 H, s, CH3 Lev), 1.81 (2 H,
Purification using silica gel flash column chromatography, ddt, J = 24.6, 12.3, 6.3 Hz, OCH2CH2CH2N3); 13C NMR
eluting with diethyl ether/petroleum ether (10/90, 20/80, 30/ (101 MHz; CDCl3) δ 206.2 (CvO Lev ketone), 171.2 (CvO Lev),
170) afforded 7 as a colourless oil (80 mg, 0.10 mmol, 78%). Rf 150.7 (C(O)N(Bn)OBn), 138.5, 137.8 (2 C), 137.3, 128.4, 128.3,
1
0.46 (EtOAc/hexane, 1/2); [α]2D2 +39.5 (c. 0.84, CHCl3); H NMR 128.1, 128.1, 128.1, 128.0, 127.7, 127.7, 127.6, 127.6, 127.4,
(400 MHz; CDCl3) δ 7.40–7.24 (20 H, m, Ar–H), 5.65 (1 H, app. 101.7 (C1), 78.7 (C3), 76.3 (C(O)N(Bn)OCH2Ph), 74.0, 73.9, 73.9
t, J = 9.9 Hz, H4), 5.48 (1 H, d, J = 12.0 Hz, C(O)N(CH2Ph)OBn), (3C, C2, C5, CH2Ph-attached to C2), 73.1 (C(O)N(CH2Ph)OBn),
5.42 (1 H, d, J = 12.0 Hz, C(O)N(CH2Ph)OBn), 4.98 (1 H, d, J = 71.5 (CH2Ph-attached to C2), 68.3 (C4), 66.6 (OCH2CH2CH2N3),
12.5 Hz, C(O)N(Bn)OCH2Ph), 4.95 (1 H, d, J = 12.4 Hz, 48.3 (OCH2CH2CH2N3), 37.9 (CH2 Lev), 29.8 (CH3 Lev), 29.1
C(O)N(Bn)OCH2Ph), 4.88 (1 H, d, J = 12.3 Hz, CH2Ph-attached (OCH2CH2CH2N3), 27.8 (CH2 Lev); 13C-GATED (101 MHz;
to C2), 4.81 (1 H, d, J = 12.3 Hz, CH2Ph-attached to C2), 4.52 CDCl3): 101.7 (1JC1–H1 = 156 Hz, C1); HRMS (ES+) m/z [found:
(1 H, d, J = 12.3 Hz, CH2Ph-attached to C3), 4.43 (1 H, d, J = (M + H)+ 751.3342 C42H46N4O9 requires (MH)+, 751.3338].
12.3 Hz, CH2Ph-attached to C3), 4.39 (1 H, s, H1), 3.94–3.88
3-Bromopropyl O-benzyl, N-benzyl (4-O-acetyl-2,3-di-O-
(1 H, m, OCH2CH2CH2Br), 3.88 (1 H, d, J = 2.6 Hz, H2), 3.83 benzyl-β-D-mannopyranoside) hydroxamate 8. A solution of 6
(1 H, d, J = 10.0 Hz, H5), 3.56 (1 H, ddd, J = 9.8, 7.7, 4.8 Hz, (100 mg, 0.14 mmol, 1.0 equiv.), diphenyl sulphoxide (37 mg,
OCH2CH2CH2Br), 3.44 (1 H, dd, J = 9.8, 2.9 Hz, H3), 3.46–3.38 0.18 mmol, 1.3 equiv.) and tri-tert-butylpyrimidine (88 mg,
(2 H, m, OCH2CH2CH2Br), 2.64–2.37 (3 H, m, CH2 Lev), 0.35 mmol, 2.5 equiv.) in CH2Cl2 (3 mL) was stirred over acti-
2.27–2.15 (1 H, m, CH2 Lev), 2.11 (3 H, s, CH3 Lev), 2.10–1.97 vated MS4Å for 40 min. The mixture was cooled to −60 °C and
(2 H, m, OCH2CH2CH2Br); 13C NMR (101 MHz; CDCl3) δ 206.3 triflic anhydride (30 μL, d = 1.720, 0.18 mmol, 1.3 equiv.) was
(CvO Lev ketone), 171.2 (CvO Lev), 150.7 (C(O)N(Bn)OBn), then added. The mixture was stirred for 5 min followed by
138.5, 137.9 (2 C), 137.3, 128.4, 128.3, 128.2, 128.1, 128.1, cooling to −80 °C, whereupon 3-bromopropanol (19 μL, d =
128.1, 127.7, 127.7, 127.6, 127.6, 127.4, 101.7 (C1), 78.8 (C3), 1.537, 0.21 mmol, 1.5 equiv.) was added. The reaction mixture
76.3 (C(O)N(Bn)OCH2Ph), 74.0, 73.9, 73.8 (3C, C2, C5, CH2Ph- was allowed to warm up to −20 °C, and stirring was continued
attached to C2), 73.1 (C(O)N(CH2Ph)OBn), 71.5 (CH2Ph- for 1 h. At that temperature triethylamine was added until pH
attached to C3), 68.3 (C4), 67.4 (OCH2CH2CH2Br), 37.9 (CH2 = 7, the organic layer was washed with H2O (10 mL), dried over
Lev), 32.7 (OCH2CH2CH2Br), 30.3 (OCH2CH2CH2Br), 29.8 (CH3 MgSO4, filtered and concentrated under reduced pressure.
Lev), 27.8 (CH2 Lev); 13C-GATED (101 MHz; CDCl3): 101.7 Purification using silica gel flash column chromatography,
(1JC1–H1 = 156 Hz, C1); HRMS (ES+) m/z [found: (M + H)+ eluting with diethyl ether/petroleum ether (10/90, 20/80, 30/
788.2465 C42H46BrNO9 requires (MH)+, 788.2429].
3-Azidopropyl O-benzyl, N-benzyl (4-O-levulinoyl-2,3-di-O- 0.58 (EtOAc/hexane, 1/2); [α]2D2 +12.81 (c. 0.50, CHCl3); 1H NMR
benzyl-β-D-mannopyranoside) hydroxamate. Compound (400 MHz; CDCl3) δ 7.42–7.23 (25 H, m, Ar–H), 5.63 (1 H, t, J =
170) afforded 8 as a colourless oil (91 mg, 0.12 mmol, 89%). Rf
7
(90 mg, 1.14 mmol, 1.0 equiv.) and NaN3 (37 mg, 5.70 mmol, 9.9 Hz, H4), 5.50 (1 H, d, J = 12.0 Hz, C(O)N(CH2Ph)OBn), 5.45
5.0 equiv.) and tetrabutylammonium iodide (2.11 g, (1 H, d, J = 12.0 Hz, C(O)N(CH2Ph)OBn), 4.99 (1 H, d, J = 12.6
5.70 mmol, 5.0 equiv.) were dissolved in acetone (12 mL) and Hz, C(O)N(Bn)OCH2Ph), 4.94 (1 H, d, J = 12.6 Hz, C(O)N(Bn)
the reaction mixture was stirred for 24 h at 55 °C. Upon com- OCH2Ph), 4.89 (1 H, d, J = 12.3 Hz, CH2Ph-attached to C2), 4.81
pletion, the reaction mixture was cooled to room temperature (1 H, d, J = 12.3 Hz, CH2Ph-attached to C2), 4.52 (1 H, d, J =
and EtOAc (25 mL) was added. The organic layer was washed 12.3 Hz, CH2Ph-attached to C3), 4.39 (1 H, s, H1) 4.38 (1 H, d,
with H2O (20 mL), brine (20 mL), dried over MgSO4, filtered J = 11.3 Hz, CH2Ph-attached to C3), 3.95–3.88 (1 H, m,
and concentrated under reduced pressure to afford the crude OCH2CH2CH2Br), 3.89 (1 H, d, J = 2.6 Hz, H2), 3.81 (1 H, d, J =
product. Purification using silica gel flash column chromato- 10.0 Hz, H5), 3.61–3.52 (1 H, m, OCH2CH2CH2Br), 3.43 (1 H,
graphy, eluting with EtOAc/hexane (20/80, 40/60, 50/50) dd, J = 9.7, 3.1 Hz, H3), 3.46–3.39 (2 H, m, OCH2CH2CH2Br),
afforded the title compound as a colourless oil (62 mg, 2.18–2.10 (2 H, m, OCH2CH2CH2Br), 1.77 (3 H, s, C(O)CH3);
0.83 mmol, 73%). Rf 0.38 (EtOAc/hexane, 1/2); [α]2D2 −41.5 13C NMR (101 MHz; CDCl3)
δ 169.3 (C(O)CH3), 150.9
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(C(O)N(Bn)OBn), 138.5, 137.9, 137.9, 137.3, 128.4, 128.3, 128.2, 24 h, then neutralised with ion exchange Amberlite 120 (H+)
128.1, 128.1, 128.0, 127.7, 127.7, 127.7, 127.6, 127.4, 127.4, resin (approximately 0.2 g, 5 min), filtered, and concentrated
101.8 (C1), 78.9 (C3), 76.3 (C(O)N(Bn)OCH2Ph), 74.1 (2 C, C5 under reduced pressure. Flash column chromatography,
and CH2Ph-attached to C2), 73.9 (C2), 73.1 (C(O)N(CH2Ph)OBn), eluting with EtOAc/hexane (20/80, 50/50, 90/10) afforded 9 as a
71.4 (CH2Ph-attached to C3), 68.1 (C4), 67.4 (OCH2CH2CH2Br), colourless oil (200 mg, 0.3 mmol, 76%).
32.8 (OCH2CH2CH2Br), 30.2 (OCH2CH2CH2Br), 20.7 (C(O)CH3);
From: 3-azidopropyl (O-benzyl, N-benzyl (phenyl 4-O-levuli-
13C-GATED (101 MHz; CDCl3): 101.8 (1JC1–H1 = 152 Hz, C1); noyl-2,3-di-O-benzyl-1-thio-α-D-mannopyranoside) hydroxamate):
HRMS (ES+) m/z [found: (M + H)+ 732.2188 C39H42BrNO8 starting material (1.87 g, 2.49 mmol, 1.0 equiv.) was dissolved
requires (M + H)+, 732.2167]; IR νmax/cm−1 1745 (m, CvO ester), in a mixture of pyridine/AcOH (4/1 v/v, 30 mL), after which
1637 (w, CvO amide), 1051 (m, C–O ester).
hydrazine acetate (1.14 g, 12.45 mmol, 5.0 equiv.) was added.
3-Azidopropyl O-benzyl, N-benzyl (4-O-acetyl-2,3-di-O-benzyl- The mixture was stirred for 1 h at room temperature and was
1-β-D-mannopyranoside) hydroxamate. Compound 8 (20 mg, diluted with EtOAc (150 mL). The organic layer was washed
0.03 mmol, 1.0 equiv.) and NaN3 (10 mg, 0.02 mmol, 6.5 with 1 M HCl (2 × 80 mL), sat. aq. NaHCO3 solution (2 ×
equiv.) were dissolved in acetone (1.5 mL) and the reaction 80 mL) and brine (80 mL). The combined organic layers were
mixture was stirred for 2 days at 55 °C. Upon completion, the dried over MgSO4, filtered and concentrated under reduced
reaction mixture was cooled to room temperature and EtOAc pressure to furnish a yellow oil. Purification by silica gel flash
(10 mL) was added. The organic layer was washed with H2O column chromatography, eluting with EtOAc/hexane (20/80,
(10 mL), dried over MgSO4, filtered and concentrated under 50/50, 90/10) afforded 9 as a colourless oil (1.50 g, 2.29 mmol,
reduced pressure to afford the crude product. Purification 92%).
using silica gel flash column chromatography, eluting with
1
Rf 0.56 (EtOAc/hexane, 1/2); [α]2D2 −31.5 (c. 0.65, CHCl3); H
EtOAc/hexane (10/90, 20/80, 40/60) afforded the title com- NMR (400 MHz; CDCl3) δ 7.50–7.25 (20 H, m, Ar–H), 5.41 (1 H,
pound as a colourless oil (16 mg, 0.02 mmol, 76%). Rf 0.60 d, J = 12.1 Hz, C(O)N(CH2Ph)OBn), 5.32 (1 H, d, J = 12.2 Hz,
(EtOAc/hexane, 1/2); [α]2D2 −50.8 (c. 0.93, CHCl3); 1H NMR C(O)N(CH2Ph)OBn), 5.05 (1 H, d, J = 12.1 Hz, C(O)N(Bn)
(400 MHz; CDCl3) 7.43–7.19 (20 H, m), 5.63 (1 H, t, J = 9.9 Hz, OCH2Ph), 4.99 (1 H, d, J = 12.2 Hz, C(O)N(Bn)OCH2Ph), 4.93
H4), 5.50 (1 H, d, J = 12.0 Hz, C(O)N(CH2Ph)OBn), 5.45 (1 H, d, (1 H, d, J = 12.4 Hz, CH2Ph-attached to C2), 4.78 (1 H, d, J =
J = 12.0 Hz, C(O)N(CH2Ph)OBn), 4.99 (1 H, d, J = 12.6 Hz, C(O) 12.4 Hz, CH2Ph-attached to C2), 4.58 (1 H, d, J = 12.4 Hz,
N(Bn)OCH2Ph), 4.94 (1 H, d, J = 12.6 Hz, C(O)N(Bn)OCH2Ph), CH2Ph-attached to C3), 4.54 (1 H, d, J = 12.3 Hz, CH2Ph-
4.89 (1 H, d, J = 12.3 Hz, CH2Ph-attached to C2), 4.81 (1 H, d, attached to C3), 4.37 (1 H, s, H1), 4.32 (1 H, dd, J = 9.5, 2.8 Hz,
J = 12.3 Hz, CH2Ph-attached to C2), 4.52 (1 H, d, J = 12.3 Hz, H4), 3.97–3.88 (1 H, m, OCH2CH2CH2N3), 3.86 (1 H, d, J =
CH2Ph-attached to C3), 4.38 (1 H, d, J = 12.2 Hz, CH2Ph- 2.9 Hz, H2), 3.71 (1 H, dd, J = 9.4, 2.1, H5), 3.52–3.42 (1 H, m,
attached to C3), 4.37 (1 H, s, H1), 3.94–3.85 (1 H, m, OCH2CH2CH2N3), 3.36–3.30 (3 H, m, H3, OCH2CH2CH2N3),
OCH2CH2CH2N3), 3.89 (1 H, d, J = 2.8 Hz, H2), 3.81 (1 H, d, J = 2.53 (1 H, d, J = 2.9 Hz, C4-OH), 1.93–1.76 (2 H, m,
10.0 Hz, H5), 3.48 (1 H, ddd,
J =
9.8, 7.6, 5.1 Hz, OCH2CH2CH2N3); 13C NMR (101 MHz; CDCl3) δ 151.4 (C(O)
OCH2CH2CH2N3), 3.43 (1 H, dd, J = 9.7, 2.8 Hz, H3), 3.38–3.25 N(Bn)OBn), 138.6, 138.1, 137.5, 136.9, 128.5, 128.4, 128.3,
(2 H, m, OCH2CH2CH2N3), 1.85 (2 H, ddd, J = 18.1, 11.5, 128.3, 128.2, 128.1, 128.1, 128.0, 127.7, 127.5, 127.4, 102.1
4.8 Hz, OCH2CH2CH2N3), 1.77 (3 H, s, C(O)CH3); 13C NMR (C1), 80.1 (C3), 76.6 (C(O)N(Bn)OCH2Ph), 74.8 (C5), 74.5 (C2),
(101 MHz; CDCl3) δ 169.3 (C(O)CH3), 150.8 (C(O)N(Bn)OBn), 74.3 (CH2Ph-attached to C2), 72.8 (C(O)N(CH2Ph)OBn), 72.2
138.5, 137.9, 137.8, 137.3, 128.4, 128.3, 128.2, 128.1, (CH2Ph-attached to C3), 67.5 (C4), 66.6 (OCH2CH2CH2N3),
128.0, 127.7, 127.7, 127.7, 127.6, 127.5, 127.4, 124.8, 101.7 48.3 (OCH2CH2CH2N3), 29.2 (OCH2CH2CH2N3); 13C-GATED
(C1), 78.8 (C3), 76.3 (C(O)N(Bn)OCH2Ph), 74.1 (2 C, C5, CH2Ph- (101 MHz; CDCl3): 102.1 (1JC1–H1 = 152 Hz, C1); HRMS (ES+)
attached to C2), 74.0 (C2), 73.1 (C(O)N(CH2Ph)OBn), m/z [found: (M + H)+ 653.2971 C37H40N4O7 requires (MH)+,
71.4 (CH2Ph-attached to C3), 68.1 (C4), 66.6 (OCH2CH2CH2N3), 653.2970].
48.3 (OCH2CH2CH2N3), 29.1 (OCH2CH2CH2N3), 20.7
Methyl (phenyl 4-O-tert-butyl dimethylsilyl-2,3-di-O-benzyl-
(C(O)CH3); 13C-GATED (101 MHz; CDCl3): 101.7 (1JC1–H1 = 152 1-thio-α-D-mannopyranoside) uronate 12. To a mixture of
Hz, C1); HRMS (ES+) m/z [found: (M
+
H)+ 695.3097 methyl (phenyl 2,3-di-O-benzyl-1-thio-α-D-mannopyranoside)
C39H42N4O8 requires (MH)+, 695.3075]; IR νmax/cm−1 2095 (m, uronate10 (100 mg, 0.21 mmol, 1.0 equiv.), imidazole (42 mg,
NvNvN), 1746 (m, CvOester), 1637 (w, CvOamide), 1050 (s, 0.62 mmol, 3.0 equiv.) and 4-dimethylaminopyridine (42.5 mg,
C–Oester).
0.62 mmol, 0.5 equiv.) in DMF (2 mL) was added tert-butyldi-
3-Azidopropyl O-benzyl, N-benzyl (2,3-di-O-benzyl-β-D-man- methylsilyl trifluoromethanesulphonate (144 µL, d = 1.151,
nopyranoside) hydroxamate 9. From 3-azidopropyl (O-benzyl, 0.62 mmol, 3.0 equiv.). The reaction mixture was left stirring
N-benzyl (phenyl 4-O-acetyl-2,3-di-O-benzyl-1-thio-α-D-manno- overnight at room temperature and was quenched with H2O
pyranoside) hydroxamate): to a stirred solution of the starting (1 mL). The mixture was concentrated under reduced pressure
material (280 g, 0.40 mmol, 1.0 equiv.) in anhydrous MeOH and the remaining crude was reconstituted in CH2Cl2 (25 mL)
(3.5 mL), Na (0.93 mg, 0.04 mmol, 0.1 equiv.) dissolved in and H2O (20 mL). The organic layer was washed with H2O (2 ×
anhydrous MeOH (0.5 mL) was added dropwise at room temp- 20 mL), separated, dried over MgSO4, filtered and concentrated
erature under a N2 atmosphere. The mixture was stirred for under reduced pressure to furnish a colourless oil. Purification
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by silica gel flash column chromatography, eluting with m/z [found: (M + NH4)+ 973.4639 C51H65N3O13SiNH4 requires
EtOAc/hexane (0/100, 5/95, 10/90) afforded 12, as a colourless (MNH4)+, 973.4625].
oil (120 mg, 0.20 mmol, 96%). Rf 0.77 (EtOAc/hexane, 1/2);
3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(methyl (4-O-
[α]D22 +22.3 (c. 4.65, CHCl3); 1H NMR (400 MHz; CDCl3) acetyl-2,3-di-O-benzyl-β-D-mannopyranosyl) uronate)-β-D-man-
δ 7.62–7.20 (15 H, m, Ar–H), 5.68 (1 H, d, J = 2.6 Hz, H1), 4.58 nopyranoside) uronate 15. A solution of 1110 (160 mg,
(1 H, d, J = 11.8 Hz, CH2Ph), 4.55 (1 H, d, J = 11.5 Hz, CH2Ph), 0.30 mmol, 1.0 equiv.) and 1310 (220 mg, 0.46 mmol,
4.50 (1 H, d, J = 11.9 Hz, CH2Ph), 4.42 (1 H, d, J = 12.0 Hz, 1.5 equiv.) in CH2Cl2 (5 mL) was stirred over activated MS4Å
CH2Ph), 4.40–4.35 (2 H, m, H4, H5), 3.81 (1 H, dd, J = 7.5, for 30 min before N-iodosuccinimide (90 mg 0.40 mmol,
2.6 Hz, H2), 3.60 (3 H, s, CO2CH3), 3.54 (1 H, dd, J = 5.6, 1.3 equiv.) was added. The mixture was cooled to −60 °C
2.7 Hz, H3), 0.80 (9 H, s, SiC(CH3)3), 0.00 (3 H, s, Si(CH3)2), before trimethylsilyl trifluoromethanesulfonate (5.6 µL, d =
−0.08 (3 H, s, Si(CH3)2); 13C NMR (101 MHz; CDCl3) δ 169.7 1.225, 0.03 mmol, 0.1 equiv.) was added. The reaction was left
(CO2CH3), 138.0, 137.9, 134.0, 131.3, 128.8, 128.4, 128.3, 128.1, stirring for 30 min at room temperature, and upon com-
127.9, 127.8, 127.7, 127.0, 82.9 (C1), 77.3 (C3), 76.4 (C4), 73.7 pletion, triethylamine was added until pH = 7. The reaction
(C2), 72.5 (CH2Ph), 72.5 (CH2Ph), 69.7 (C5), 52.0 (CO2CH3), mixture was filtered through Celite® and concentrated under
25.7 (SiC(CH3)3), 18.0 (SiC(CH3)3), −4.7 (Si(CH3)2), −5.2 reduced pressure. Purification by Reveleris® automated silica
(Si(CH3)2); 13C-GATED (101 MHz; CDCl3): 82.9 (1JC1–H1 = 168 gel flash column chromatography (liquid injection onto
Hz, C1); HRMS (ES+) m/z [found: (M + NH4)+ 612.2832 column), eluting with EtOAc/toluene (0/100, 5/95 and 10/90)
C33H42O6SSiNH4 requires (MNH4)+, 612.2810].
afforded 15 as a colourless oil (150 mg, 0.17 mmol, 56%).
3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(methyl (4-O-tert- Rf 0.42 (EtOAc/toluene, 3/7); [α]2D2 −35.3 (c. 3.65, CHCl3); 1H
butyl dimethylsilyl-2,3-di-O-benzyl-β-D-mannopyranosyl) uronate)- NMR (500 MHz; CDCl3) δ 7.50–7.21 (20 H, m, Ar–H), 5.44 (1 H,
β-D-mannopyranoside) uronate 14. A solution of 12 (110 mg, app. t, J = 9.8 Hz, H4′), 4.88 (1H, d, J = 12.3 Hz, CH2Ph), 4.84
0.18 mmol, 1.0 equiv.) and 131 (96 mg, 0.20 mmol, 1.1 equiv.) (1 H, d, J = 12.2 Hz, CH2Ph), 4.79 (1 H, d, J = 12.3 Hz, CH2Ph),
in CH2Cl2 (3.5 mL) was stirred over activated MS4Å for 30 min 4.78 (2 H, d, J = 12.1 Hz, CH2Ph), 4.76 (1 H, d, J = 12.2 Hz,
before N-iodosuccinimide (54 mg, 0.24 mmol, 1.3 equiv.) was CH2Ph), 4.72 (1 H, d, J = 11.9 Hz, CH2Ph), 4.60 (1 H, d, J =
added. The mixture was cooled to −10 °C before trimethylsilyl 12.2 Hz, CH2Ph), 4.54 (1 H, d, J = 12.3 Hz, CH2Ph), 4.52 (1 H,
trifluoromethanesulfonate (6.7 µL, d = 1.225, 0.04 mmol, d, J = 0.9 Hz, H1′), 4.46 (1 H, s, H1), 4.43 (1 H, app. t, J = 8.8 Hz,
0.2 equiv.) was added. The reaction was left stirring for 30 min H4), 4.03 (1 H, dt, J = 9.7, 5.7 Hz, OCH2CH2CH2N3), 3.93 (1 H,
at room temperature, and upon completion, triethylamine was d, J = 8.8 Hz, H5), 3.89–3.88 (2 H, m, H2′, H2), 3.71 (1 H, d, J =
added until pH = 7. The reaction mixture was filtered through 9.8 Hz, H5′), 3.67 (3 H, s, CO2CH3), 3.66 (1 H, dd, J = 8.8,
Celite® and concentrated under reduced pressure. Purification 3.0 Hz, H3), 3.58 (3 H, s, CO2CH3), 3.55 (1 H, m,
by silica gel flash column chromatography, eluting with OCH2CH2CH2N3), 3.49 (1 H, dd, J = 9.8, 2.8 Hz, H3′), 3.37 (2 H,
EtOAc/toluene (0/100, 5/95, 10/90) afforded 14, as a colourless t, J = 6.8 Hz, OCH2CH2CH2N3), 2.02 (3 H, s, C(O)CH3),
oil (105 mg, 0.11 mmol, 61%). Rf 0.76 (EtOAc/toluene, 3/7); 1.95–1.84 (2 H, m, OCH2CH2CH2N3); 13C NMR (126 MHz;
[α]2D2 +8.0 (c. 0.21, CHCl3); 1H NMR (400 MHz; CDCl3) CDCl3) δ 169.8 (C(O)CH3), 168.7 (CO2CH3), 167.8 (CO2CH3),
δ 7.41–7.15 (20 H, m, Ar–H), 4.82 (1 H, d, J = 12.3 Hz, CH2Ph), 138.7 (Cq Bn), 138.6 (Cq Bn), 138.4 (Cq Bn), 137.8 (Cq Bn),
4.80 (1 H, d, J = 12.5 Hz, CH2Ph), 4.74 (1 H, d, J = 12.7 Hz, 128.4, 128.3, 128.2, 128.1, 128.1, 127.9, 127.7, 127.5, 127.5,
CH2Ph), 4.71 (1 H, s, H1′), 4.70 (1 H, d, J = 12.9 Hz, CH2Ph), 127.4, 127.3, 127.2, 102.4 (C1′), 101.9 (C1), 79.4 (C3), 78.5 (C3′),
4.67 (1 H, d, J = 12.3 Hz, CH2Ph), 4.56 (1 H, d, J = 11.8 Hz, 77.6 (C4), 75.0 (C2′ or C2), 74.5 (C5), 74.4 (CH2Ph), 74.0 (C2′ or
CH2Ph), 4.55 (1 H, d, J = 12.2 Hz, CH2Ph), 4.47 (1 H, d, J = C2, CH2Ph), 73.4 (C5′), 72.2 (CH2Ph), 71.7 (CH2Ph), 68.7 (C4′),
11.4 Hz, CH2Ph), 4.45 (1 H, s, H1), 4.41 (1 H, app. t, J = 8.6 Hz, 66.9 (OCH2CH2CH2N3), 52.4 (CO2CH3), 52.4 (CO2CH3), 48.3
H4), 4.30 (1 H, app. t, J = 9.2 Hz, H4′), 4.08–3.99 (1 H, m, (OCH2CH2CH2N3), 29.1 (OCH2CH2CH2N3), 20.8 (C(O)CH3);
OCH2CH2CH2N3), 3.86 (1 H, d, J = 8.6 Hz, H5), 3.83 (1 H, d, J = 13C-GATED (126 MHz; CDCl3): 102.4 (1JC1′–H1′ = 155 Hz, C1′),
2.5 Hz, H2′), 3.80 (1 H, d, J = 2.2 Hz, H2), 3.74 (1 H, d, J = 101.9 (1JC1–H1 = 156 Hz, C1); HRMS (ES+) m/z [found: (M +
9.2 Hz, H5′), 3.62 (6 H, s, CO2CH3), 3.62–3.56 (1 H, m, H3), 3.52 NH4)+ 901.3877 C47H53N3O14NH4 requires (MNH4)+, 901.3866].
(1 H, ddd, J = 9.6, 7.6, 5.2 Hz, OCH2CH2CH2N3), 3.35 (2 H, t,
3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(methyl (2,3-di-O-
J = 6.7 Hz, OCH2CH2CH2N3), 3.28 (1 H, dd, J = 9.2, 2.7 Hz, H3′), benzyl-β-D-mannopyranosyl) uronate)-β-D-mannopyranoside)
1.92–1.82 (2 H, m, OCH2CH2CH2N3), 0.81 (9 H, s, SiC(CH3)3), uronate 16. To a stirred solution of 14 (70 mg, 0.07 mmol,
0.00 (6 H, s, Si(CH3)2); 13C NMR (101 MHz; CDCl3) δ 168.7 1.0 equiv.) in anhydrous MeOH (0.7 mL) at 0 °C, was added
(CO2CH3), 168.7 (CO2CH3), 139.2, 138.8, 138.5, 137.9, 128.4, AcCl (1.6 µL, d = 1.104, 0.02 mmol, 0.3 equiv.) and the reaction
128.2, 128.1, 128.1, 128.0, 127.6, 127.5, 127.5, 127.5, 127.4, was left stirring at room temperature for 24 h. The mixture was
127.3, 127.2, 102.6 (C1′), 101.7 (C1), 81.8 (C3′), 79.1 (C3), 77.5 then neutralised and diluted with sat. aq. NaHCO3 (20 mL).
(C5′), 77.2 (C4), 75.0 (C2′), 74.6 (C5), 74.5 (CH2Ph), 74.4 (C2), The aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The
73.9 (CH2Ph), 72.6 (CH2Ph), 71.4 (CH2Ph), 68.9 (C4′), 66.8 combined organic layers were dried over Na2SO4, filtered and
(OCH2CH2CH2N3), 52.3 (CO2CH3), 52.0 (CO2CH3), 48.3 concentrated under reduced pressure to furnish a colourless
(OCH2CH2CH2N3), 29.1 (OCH2CH2CH2N3), 25.8 (SiC(CH3)3), oil. Purification by silica gel flash column chromatography,
18.0 (SiC(CH3)3), −3.9 (Si(CH3)2), −5.3 (Si(CH3)2); HRMS (ES+) eluting with diethyl ether/toluene (10/90, 20/80, 25/75)
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afforded 16 as a colourless oil (20 mg, 0.02 mmol, 32%). (C1), 99.8 (C1′), 81.5 (C3), 78.2 (C2′), 75.8 (C5), 75.0 (C3′), 74.7
Rf 0.30 (EtOAc/hexane, 1/2); 1H NMR (400 MHz; CDCl3) (C4), 74.1 (CH2Ph), 72.9 (C2), 72.4 (2 C, CH2Ph), 72.4 (C5′),
δ 7.38–7.22 (20 H, m), 4.84 (1 H, d, J = 12.3 Hz, CH2Ph), 4.79 71.0 (CH2Ph), 68.4 (C4′), 67.0 (OCH2CH2CH2N3), 52.6
(1 H, d, J = 12.1 Hz, CH2Ph), 4.76 (1 H, d, J = 12.1 Hz, CH2Ph), (CO2CH3), 52.5 (CO2CH3), 48.3 (OCH2CH2CH2N3), 29.1
4.75 (1 H, d, J = 12.3 Hz, CH2Ph), 4.72 (1 H, s, H1′), 4.67 (1 H, (OCH2CH2CH2N3); 13C-GATED (101 MHz; CDCl3): 102.0
d, J = 12.2 Hz, CH2Ph), 4.60 (1 H, d, J = 12.3 Hz, CH2Ph), 4.57 (1JC1–H1 = 156 Hz, C1), 99.8 (1JC1′–H1′ = 176 Hz, C1′); HRMS (ES+)
(1 H, d, J = 12.0 Hz, CH2Ph), 4.55 (1 H, d, J = 12.3 Hz, CH2Ph), m/z [found: (M + Na)+ 864.3343 C47H53N3O14Na requires
4.48 (1 H, d, J = 0.7 Hz, H1), 4.45 (1 H, app. t, J = 8.6 Hz, H4), (MNa)+, 864.3314].
4.19 (1 H, app. t, J = 9.6 Hz, H4′), 4.07–3.99 (1 H, m,
O-Benzyl, N-benzyl (phenyl 4-O-levulinoyl-2,3-di-O-benzyl-1-
OCH2CH2CH2N3), 3.90 (1 H, d, J = 8.7 Hz, H5), 3.85 (1 H, d, J = hydroxyl-α/β-D-mannopyranoside) hydroxamate. A solution of
2.3 Hz, H2), 3.83 (1 H, d, J = 2.5 Hz, H2′), 3.70 (1 H, dd, J = 9.0, 5 (100 mg, 0.13 mmol, 1.0 equiv.) in CH2Cl2/H2O (10/1 v/v,
4.4 Hz, H3), 3.64 (3 H, s, CO2CH3), 3.62 (3 H, s, CO2CH3), 3.59 1.4 mL in total) was cooled to 0 °C followed by the addition of
(1 H, d, J = 9.6 Hz, H5′), 3.57–3.51(1 H, m, OCH2CH2CH2N3), N-iodosuccinimide (30 mg 0.13 mmol, 1.0 equiv.) and a cata-
3.36 (2 H, t, J = 6.7 Hz, OCH2CH2CH2N3), 3.32 (1 H, dd, J = 9.5, lytic amount of silver trifluoromethanesulfonate (6.8 mg
2.8 Hz, H3′), 2.93 (1 H, br. s, C4-OH), 1.94–1.81 (2 H, m, 0.03 mmol, 0.2 equiv.). The reaction was left stirring at 0 °C for
OCH2CH2CH2N3); 13C NMR (101 MHz; CDCl3)
δ 169.9 4 h before it was quenched by the addition of 10% aq. Na2S2O3
(CO2CH3), 168.6 (CO2CH3), 138.8, 138.7, 138.4, 138.0, 128.5, solution (5 mL) and diluted with CH2Cl2 (15 mL). The organic
128.3, 128.2, 128.1, 128.1, 127.8, 127.8, 127.7, 127.5, 127.4, layer was subsequently washed with sat. aq. NaHCO3 solution
127.4, 127.1, 102.5 (C1′), 101.8 (C1), 80.4 (C3′), 79.4 (C5′), 77.2 (10 mL) and brine (10 mL), dried over Na2SO4, filtered and
(C4), 75.2 (C2′), 74.8 (C3), 74.6 (CH2Ph), 74.5 (C5), 73.9 (C2, concentrated under reduced pressure. The crude product was
CH2Ph), 72.1 (CH2Ph), 71.9 (CH2Ph), 68.2 (C4′), 66.9 purified using silica gel flash column chromatography, eluting
(OCH2CH2CH2N3), 52.5 (CO2CH3), 52.4 (CO2CH3), 48.3 with diethyl ether/toluene (5/95, 10/90, 20/80) to furnish the
(OCH2CH2CH2N3),
29.1
(OCH2CH2CH2N3);
13C-GATED title compound as a yellow oil (0.056 mg, 0.10 mmol, 78%).
(101 MHz; CDCl3): 102.5 (1JC1–H1 = 156 Hz, C1′), 101.8 (1JC1′–H1′ Rf 0.50 (EtOAc/tolene, 3/7); the NMR data reported refer to the
= 156 Hz, C1); HRMS (ES+) m/z [found: (M + NH4)+ 859.3781 major α-anomer (α/β = 87/13): 1H NMR (400 MHz; CDCl3)
C45H51N3O13NH4 requires (MNH4)+, 859.3760]; these data were δ 7.41–7.20 (20 H, m, Ar–H), 5.70 (1 H, app. t, J = 9.7 Hz, H4),
consistent with literature values.10
5.46 (1 H, d, J = 12.0 Hz, C(O)N(CH2Ph)OBn), 5.42 (1 H, d, J =
3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(methyl (2,3-di-O- 12.0 Hz, C(O)N(CH2Ph)OBn), 5.20 (1 H, dd, J = 3.5, 2.2 Hz, H1),
benzyl-β-D-mannopyranosyl) uronate)-α-D-mannopyranoside) 4.97 (1 H, d, J = 12.7 Hz, C(O)N(Bn)OCH2Ph), 4.93 (2 H, d, J =
uronate 17. To a stirred solution of 15 (50 mg, 0.06 mmol, 13.1 Hz, C(O)N(Bn)OCH2Ph), 4.76 (1 H, d, J = 12.2 Hz, CH2Ph-
1.0 equiv.) in anhydrous MeOH (1 mL), Na (0.06 mg, attached to C2), 4.64 (1 H, d, J = 12.1 Hz, CH2Ph-attached to
0.003 mmol, 0.05 equiv.) dissolved in anhydrous MeOH C2), 4.59 (1 H, d, J = 12.1 Hz, CH2Ph-attached to C3), 4.54 (1 H,
(30 µL) was added at room temperature under a N2 atmo- d, J = 12.2 Hz, CH2Ph-attached to C3), 4.33 (1 H, d, J = 9.9 Hz,
sphere. The mixture was stirred for 2 h, then neutralised with H5), 3.90 (1 H, dd, J = 9.5, 2.9 Hz, H3), 3.77–3.74 (1 H, app. t,
ion exchange Amberlite 120 (H+) resin (approximately 0.1 g, J = 2.6 Hz, H2), 3.38 (1 H, d, J = 3.7 Hz, OH), 2.63–2.35 (3 H, m,
5 min), filtered, and concentrated under reduced pressure. CH2 Lev), 2.30–2.21 (1 H, m, CH2 Lev), 2.09 (3 H, s, CH3 Lev);
Flash column chromatography, eluting with EtOAc/hexane (20/ 13C NMR (101 MHz; CDCl3) δ 206.4 (CvO Lev ketone), 171.4
80, 50/50, 70/30, 90/10) afforded 17 as a colourless oil (15 mg, (CvO Lev), 151.5 (C(O)N(Bn)OBn), 138.2, 137.7, 137.2, 128.3,
0.02 mmol, 46% based on recovered starting material, 16 mg). 128.3, 128.3, 128.2, 128.1, 127.7, 127.7, 127.6, 127.5, 127.5,
Rf 0.42 (EtOAc/toluene, 3/7); [α]2D2 −17.8 (c. 0.74, CHCl3); 1H 93.0 (C1), 76.2 (2C, C3, (C(O)N(Bn)OCH2Ph)), 74.8 (C2), 73.3
NMR (400 MHz; CDCl3) δ 7.65–6.80 (20 H, m, Ar–H), 5.42 (1 H, (C(O)N(CH2Ph)OBn), 72.9 (CH2Ph-attached to C2), 72.2
s, H1′), 4.94 (1 H, d, J = 12.5 Hz, CH2Ph), 4.73 (1 H, d, J = (CH2Ph-attached to C3), 70.5 (C5), 68.7 (C4), 37.9 (CH2 Lev),
12.5 Hz, CH2Ph), 4.63 (1 H, d, J = 11.9 Hz, CH2Ph), 4.55 (1 H, 29.8 (CH3 Lev), 27.9 (CH2 Lev); 13C-GATED (101 MHz; CDCl3):
d, J = 11.9 Hz, CH2Ph), 4.45 (1 H, app. t, J = 9.3, H4), 4.43 (1 H, α-anomer: 93.0 (1JC1–H1 = 176 Hz, C1), β-anomer: 93.7 (1JC1–H1
s, H1), 4.40 (1 H, d, J = 11.6 Hz, CH2Ph), 4.32 (1 H, d, J = = 164 Hz, C1); HRMS (ES+) m/z [found: (M + H)+ 668.2860
12.2 Hz, CH2Ph), 4.23 (1 H, d, J = 12.3 Hz, CH2Ph), 4.22 (1 H, C39H41NO9 requires (MH)+, 668.2854].
d, J = 9.3 Hz, H4′), 4.19 (1 H, d, J = 11.9 Hz, CH2Ph), 4.07–3.98
O-Benzyl, N-benzyl (phenyl 4-O-levulinoyl-2,3-di-O-benzyl-1-
(1 H, m, OCH2CH2CH2N3), 4.01 (1 H, d, J = 9.7 Hz, H5′), 3.91 O-phenyl-N-trifluoroacetimidate-α-D-mannopyranoside) hydro-
(1 H, d, J = 2.6 Hz, H2), 3.84 (1 H, d, J = 9.4 Hz, H5), 3.80 (3 H, xamate 19. O-Benzyl, N-benzyl (phenyl 4-O-levulinoyl-2,3-di-
s, CO2CH3), 3.76 (3 H, s, CO2CH3), 3.67–3.62 (2 H, m, H2′, H3′), O-benzyl-1-hydroxyl-α/β-D-mannopyranoside)
hydroxamate
3.57–3.49 (1 H, m, OCH2CH2CH2N3), 3.43–3.36 (2 H, m, (70 mg, 0.10 mmol, 1.0 equiv.) was dissolved in acetone/H2O
OCH2CH2CH2N3), 3.40 (1 H, dd, J = 9.3, 2.7 Hz, H3), 2.95 (1 H, (20/1 v/v, 1.1 mL in total) and the solution was cooled to 0 °C.
s, C4-OH), 2.01–1.78 (2 H, m, OCH2CH2CH2N3); 13C NMR N-Phenyl trifluoroacetimidoyl chloride (25 µL, d = 1.31,
(101 MHz; CDCl3) δ 170.9 (CO2CH3), 168.3 (CO2CH3), 138.5 (Cq 0.16 mmol, 1.5 equiv.) and K2CO3 (17 mg, 0.12 mmol,
Bn), 138.3 (Cq Bn), 138.3 (Cq Bn), 137.4 (Cq Bn), 128.5, 128.4, 1.2 equiv.) were added and the resulting suspension was
128.2, 128.1, 128.0, 127.6, 127.6, 127.5, 127.4, 127.4, 102.0 stirred for 20 h at room temperature. The reaction mixture was
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diluted with EtOAc (10 mL) and H2O (10 mL), the organic layer OCH2CH2CH2N3), 3.31 (2 H, dd,
J
=
9.9, 3.9 Hz,
was then washed with brine (10 mL), collected dried over OCH2CH2CH2N3), 3.21 (1 H, dd, J = 9.8, 2.8 Hz, H3′), 2.69–2.64
Na2SO4, filtered and concentrated under reduced pressure. (2 H, m, CH2 Lev), 2.55–2.46 (2 H, m, CH2 Lev), 2.14 (3 H, s,
The crude product was purified using silica gel flash column CH3 Lev), 1.88–1.76 (2 H, m, OCH2CH2CH2N3); 13C NMR
chromatography, eluting with diethyl ether/toluene (5/95, 10/ (101 MHz; CDCl3) δ 206.2 (CvO Lev ketone), 171.5 (CvO Lev),
90, 20/80) to furnish 19 as a colourless oil which was used 167.9 (CvO CO2CH3), 151.8 (C(O)N(Bn)OBn), 139.0 (Cq), 138.8
immediately (0.06 mg, 0.07 mmol, 66%). Rf 0.68 (EtOAc/tolene, (Cq), 138.7 (Cq), 138.0 (Cq), 137.1 (Cq), 128.5, 128.4, 128.3,
1
3/7); H NMR (400 MHz; CDCl3) δ 7.38–7.22 (25 H, m, Ar–H), 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 127.6, 127.6, 127.4,
7.11 (1 H, t, J = 7.5 Hz, CH NPh), 6.69 (2 H, d, J = 7.7 Hz, CH 127.4, 127.3, 127.2, 102.3 (C1′), 101.8 (C1), 79.9 (C3), 78.4 (C3′),
NPh), 6.26 (1 H, br. s, H1), 5.76 (1 H, t, J = 9.7 Hz, H4), 5.44 77.3 (C4), 76.6 (CH2Ph), 75.1 (C2), 74.9 (C2′), 74.6 (C5, CH2Ph),
(1 H, d, J = 11.9 Hz, C(O)N(CH2Ph)OBn), 5.38 (1 H, d, J = 11.9 74.3 (CH2Ph), 73.3 (C5′), 73.3 (CH2Ph), 72.8 (CH2Ph), 71.2
Hz, C(O)N(CH2Ph)OBn), 5.02 (1 H, d, J = 12.4 Hz, C(O)N(Bn) (CH2Ph), 68.9 (C4′), 66.6 (OCH2CH2CH2N3), 52.3 (CO2CH3),
OCH2Ph), 4.99 (1 H, d, J = 12.5 Hz, C(O)N(Bn)OCH2Ph), 4.72 48.3 (OCH2CH2CH2N3), 37.9 (CH2 Lev), 29.8 (CH3 Lev), 29.2
(1 H, d, J = 10.2 Hz, CH2Ph), 4.63 (1 H, d, J = 10.3 Hz, CH2Ph), (OCH2CH2CH2N3), 27.9 (CH2 Lev); 13C-GATED (101 MHz;
4.61 (1 H, d, J = 12.1 Hz, CH2Ph), 4.54 (1 H, d, J = 12.1 Hz, CDCl3): 102.3 (1JC1–H1 = 156 Hz, C1′), 101.8 (1JC1′–H1′ = 156 Hz,
CH2Ph), 4.27 (1 H, d, J = 9.9 Hz, H5), 3.82 (1 H, dd, J = 9.5, C1); HRMS (ES+) m/z [found: (M + H)+ 1121.4755 C63H68N4O15
2.7 Hz, H3), 3.77 (1 H, s, H2), 2.68–2.41 (3 H, m, CH2 Lev), requires (MH)+, 1121.4754].
2.30–2.20 (1 H, m, CH2 Lev), 2.13 (3 H, s, CH3 Lev); 13C NMR
3-Azidopropyl (O-benzyl-N-benzyl-(methyl 2,3-di-O-benzyl-4-
(101 MHz; CDCl3) δ 206.2 (CvO Lev ketone), 171.2 (CvO Lev), O-(2,3-di-O-benzyl-β-D-mannopyranosyl)uronate)-β-D-mannopyr-
150.2 (C(O)N(Bn)OBn), 143.1 (Cq NPh), 142.3 (q, J = 36.0 Hz, anoside)hydroxamate 21. Disaccharide 20 (40 mg, 0.03 mmol,
CvNPh), 137.7, 137.5, 137.00, 128.7, 128.4, 128.3, 128.3, 1.0 equiv.) was dissolved in a mixture of pyridine/AcOH
128.2, 128.1, 128.1, 127.9, 127.8, 127.7, 127.7, 127.5, 124.5, (4/1 v/v, 0.5 mL in total), after which hydrazine acetate (16 mg,
119.4 (CH NPh), 115.8 (d, J = 287.3 Hz, CF3), 94.9 (C1), 75.4, 0.18 mmol, 5.0 equiv.) was added. The mixture was stirred for
73.5 (C3), 73.0 (CH2Ph), 72.8 (2C, C2, C5), 72.6 (CH2Ph), 67.7 30 min and then was diluted with EtOAc (5 mL), washed with
(C4), 37.9 (CH2 Lev), 29.8 (CH3 Lev), 27.8 (CH2 Lev); 1 M HCl (5 mL), sat. aq. NaHCO3 solution (5 mL) and brine
13C-GATED (101 MHz; CDCl3): 94.9 (1JC1–H1 = 172 Hz, C1); (5 mL). The organic layer was then dried over MgSO4 filtered
HRMS (ES+) m/z [found: (M + Na)+ 861.2972 C47H45F3N2O9Na and concentrated under reduced pressure to furnish a yellow
requires (MNa)+, 861.2969].
oil. Purification using silica gel flash column chromatography,
3-Azidopropyl (O-benzyl-N-benzyl-(methyl 2,3-di-O-benzyl- eluting with diethyl ether/toluene (0/100, 30/70, 40/60, 90/10)
4-O-(4-O-levulinoyl-2,3-di-O-benzyl-β-D-mannopyranosyl) uronate)- afforded 21 as a colourless oil (25 mg, 0.02 mmol, 81%).
β-D-mannopyranoside) hydroxamate 20. A solution of donor 1010 Rf 0.60 (EtOAc/toluene, 3/7); [α]D22 −44.5 (c. 0.25, CHCl3); 1H
(120 mg, 0.21 mmol, 1.2 equiv.) and acceptor 9 (110 mg, NMR (400 MHz; CDCl3) δ 7.43–7.20 (30 H, m, Ar–H), 5.46 (1 H,
0.17 mmol, 1.0 equiv.) and in CH2Cl2 (4 mL) was stirred over d, J = 12.2 Hz, CH2Ph), 5.33 (1 H, d, J = 12.2 Hz, CH2Ph), 4.93
activated MS4Å for 30 min before N-iodosuccinimide (60 mg (1 H, d, J = 11.8 Hz, CH2Ph), 4.89 (1 H, d, J = 12.2 Hz, CH2Ph),
0.27 mmol, 1.3 equiv.-based on donor) was added. The 4.88 (1 H, d, J = 10.6 Hz, CH2Ph), 4.86 (1 H, d, J = 11.0 Hz,
mixture was cooled to −40 °C before trimethylsilyl trifluoro- CH2Ph), 4.85 (1 H, d, J = 12.5 Hz, CH2Ph), 4.81 (1 H, d, J =
methanesulfonate (7.5 µL, d = 1.225, 0.04 mmol, 0.2 equiv.) 12.1 Hz, CH2Ph), 4.63 (1 H, d, J = 12.2 Hz, CH2Ph), 4.56 (1 H,
was added. The reaction was allowed to warm at 0 °C within d, J = 12.2 Hz, CH2Ph), 4.52 (1 H, s, H1′), 4.43 (1 H, d, J =
45 min, and upon completion, triethylamine was added until 12.6 Hz, CH2Ph), 4.42 (1 H, t, J = 9.2 Hz, H4), 4.41 (1 H, s, H1),
pH = 7. The reaction mixture was filtered through Celite® and 4.36 (1 H, d, J = 12.0 Hz, CH2Ph), 4.12 (1 H, t, J = 9.6 Hz, H4′),
concentrated under reduced pressure. Flash column chromato- 3.96–3.90 (1 H, m, OCH2CH2CH2N3), 3.88 (1 H, d, J = 9.7 Hz,
graphy, eluting with diethyl ether/toluene (0/100, 5/95 and H5), 3.84 (1 H, d, J = 2.9 Hz, H2), 3.76 (1 H, d, J = 2.2 Hz, H2′),
10/90) afforded 20 as a colourless oil (94 mg, 0.09 mmol, 55%). 3.59 (3 H, s, CO2CH3), 3.58–3.54 (1 H, m, OCH2CH2CH2N3),
Rf 0.38 (EtOAc/toluene, 3/7); [α]2D2 −29.6 (c. 0.5, CHCl3); 1H 3.50 (1 H, dd, J = 9.3, 2.7 Hz, H3), 3.46 (1 H, d, J = 9.6 Hz, H5′),
NMR (400 MHz; CDCl3) δ 7.44–7.16 (30 H, m), 5.46 (1 H, d, J = 3.32 (2 H, td, J = 6.7, 1.5 Hz, OCH2CH2CH2N3), 3.12 (1 H, dd,
12.2 Hz, CH2Ph), 5.35 (1 H, app. t, J = 9.8 Hz, H4′), 5.31 (1 H, d, J = 9.5, 2.8 Hz, H3′), 2.85 (1 H, d, J = 1.7 Hz, C4-OH), 1.90–1.77
J = 12.2 Hz, CH2Ph), 4.92 (1 H, d, J = 11.8 Hz, CH2Ph), 4.88 (2 H, m, OCH2CH2CH2N3); 13C NMR (101 MHz; CDCl3) δ 170.0
(1 H, d, J = 10.0 Hz, CH2Ph), 4.85 (1 H, s, J = 13.9 Hz, CH2Ph), (CvO CO2CH3), 151.8 (C(O)N(Bn)OBn), 139.1, 139.0, 138.8,
4.85 (2 H, s, CH2Ph), 4.83 (4 H, d, J = 14.2 Hz, CH2Ph), 4.67 138.0, 137.1, 137.0, 128.5, 128.4, 128.3, 128.2, 128.2, 128.1,
(1 H, d, J = 12.3 Hz, CH2Ph), 4.57 (1 H, d, J = 12.4 Hz, CH2Ph), 128.1, 128.1, 128.0, 127.7, 127.6, 127.5, 127.4, 127.3, 127.3,
4.44 (1 H, s, H1′), 4.39 (1 H, s, H1), 4.36 (1 H, d, J = 11.5 Hz, 102.6 (C1′), 101.7 (C1), 80.3 (C3′), 80.2 (C3), 77.2 (C4), 76.6
CH2Ph), 4.35 (1 H, d, J = 9.4 Hz, H4), 4.23 (1 H, d, J = 12.4 Hz, (CH2Ph), 75.3 (C2), 75.2 (C2′), 74.9 (C5), 74.8 (CH2Ph), 74.7
CH2Ph), 3.95–3.88 (1 H, m, OCH2CH2CH2N3), 3.85 (1 H, d, J = (C5′), 74.3 (CH2Ph), 73.3 (CH2Ph), 72.6 (CH2Ph), 71.3 (CH2Ph),
9.7 Hz, H5), 3.82 (1 H, d, J = 2.9 Hz, H2), 3.76 (1 H, d, J = 68.0 (C4′), 66.6 (OCH2CH2CH2N3), 52.3 (CO2CH3), 48.3
2.6 Hz, H2′), 3.53 (3 H, s, CO2CH3), 3.50 (1 H, d, J = 9.0 Hz, (OCH2CH2CH2N3), 32.8, 30.3, 29.2 (OCH2CH2CH2N3);
H5′), 3.46 (1 H, dd, J = 9.2, 2.9 Hz, H3), 3.48–3.39 (1 H, m, 13C-GATED (101 MHz; CDCl3): 102.6 (1JC1–H1 = 156 Hz, C1′),
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101.7 (1JC1′–H1′ = 156 Hz, C1); HRMS (ES+) m/z [found: (M + H)+ 0 °C before trimethylsilyl trifluoromethanesulfonate (4.2 µL, d
1023.4412 C58H62N4O13 requires (MH)+, 1023.4386].
= 1.225, 0.02 mmol, 0.1 equiv.) was added. The reaction was
3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(O-benzyl-N-benzyl- left stirring for 30 min at 0 °C, and upon completion, triethyl-
(4-O-levulinoy-2,3-di-O-benzyl-α-D-mannopyranosyl) hydroxa- amine was added until pH = 7. The reaction mixture was fil-
mate)-β-D-mannopyranoside) uronate 22. A solution of accep- tered through Celite® and concentrated under reduced
tor 5 (200 mg, 0.26 mmol, 1.0 equiv.) and donor 13 (140 mg, pressure. Flash column chromatography, eluting with EtOAc/
0.29 mmol, 1.1 equiv.) and in CH2Cl2 (5 mL) was stirred over toluene (0/100, 5/95 and 10/90) afforded 23 as a colourless oil
activated MS4Å for 1 h before N-iodosuccinimide (90 mg (30 mg, 0.03 mmol, 12%, α/β = 9/1). Rf 0.53 (EtOAc/toluene, 3/
0.39 mmol, 1.5 equiv.) was added. The mixture was cooled to 7); [α]2D2 +0.8 (c. 0.63, CHCl3); 1H NMR (400 MHz; CDCl3) δ
−40 °C before trimethylsilyl trifluoromethanesulfonate (4.8 µL, 7.44–7.09 (30 H, m, Ar–H), 5.63 (1 H, app. t, J = 9.9 Hz, H4′),
d = 1.225, 0.02 mmol, 0.1 equiv.) was added. The reaction was 5.44 (2 H, s, C(O)N(CH2Ph)OBn), 5.39 (1 H, d, J = 1.8 Hz, H1′),
left stirring for 1 h at −40 °C, 2 h at −25 °C, 3 h at −20 °C and 4.95 (2 H, s, C(O)N(Bn)OCH2Ph), 4.92 (1 H, d, J = 12.6 Hz,
30 min at −10 °C, and quenched with triethylamine until pH = CH2Ph-attached to C2), 4.73 (1 H, d, J = 12.5 Hz, CH2Ph-
7. The reaction mixture was filtered through Celite® and con- attached to C2), 4.53 (1 H, d, J = 12.7 Hz, CH2Ph-attached to
centrated under reduced pressure. Flash column chromato- C3′), 4.50 (1 H, d, J = 12.5 Hz, CH2Ph-attached to C3′) 4.42 (1
graphy, eluting with diethyl ether/toluene (0/100, 10/90, 20/80) H, s, H1), 4.40 (1 H, d, J = 10.3 Hz, H4), 4.38 (1 H, d, J = 11.2
afforded 22 as a colourless oil (90 mg, 0.08 mmol, 30%, α/β = Hz, CH2Ph-attached to C3), 4.34 (1 H, d, J = 12.1 Hz, CH2Ph-
90/10). Rf 0.30 (EtOAc/toluene, 3/7); [α]2D2 −1.8 (c. 1.95, CHCl3); attached to C2′), 4.23 (1 H, d, J = 12.0 Hz, CH2Ph-attached to
1H NMR (400 MHz; CDCl3) δ 7.39–7.10 (30 H, m, Ar–H), 5.64 C2′), 4.18 (1 H, d, J = 11.5 Hz, CH2Ph-attached to C3), 4.06–3.99
(1 H, app. t, J = 9.9 Hz, H4′), 5.43 (1 H, d, J = 12.6 Hz, CH2Ph), (1 H, m, OCH2CH2CH2N3), 3.95 (1 H, d, J = 10.0 Hz, H5′), 3.90
5.40 (1 H, d, J = 12.1 Hz, CH2Ph), 5.38 (1 H, d, J = 2.0 Hz, H1′), (1 H, d, J = 2.6 Hz, H2), 3.81 (1 H, d, J = 9.4 Hz, H5), 3.78 (1 H,
4.97 (1 H, d, J = 12.6 Hz, CH2Ph), 4.94 (1 H, d, J = 12.7 Hz, dd, J = 9.9, 2.7 Hz, H3′), 3.65 (1 H, app. t, J = 2.3 Hz, H2′), 3.59
CH2Ph), 4.92 (1 H, d, J = 12.5 Hz, CH2Ph), 4.73 (1 H, d, J = (3 H, s, CO2CH3), 3.53 (1 H, ddd, J = 9.5, 8.0, 5.1 Hz,
12.5 Hz, CH2Ph), 4.56 (1 H, d, J = 12.2 Hz, CH2Ph), 4.52 (1 H, OCH2CH2CH2N3), 3.38 (2 H, ddd, J = 11.2, 8.6, 2.2 Hz,
d, J = 12.1 Hz, CH2Ph), 4.52 (1 H, d, J = 12.1 Hz, CH2Ph), 4.42 OCH2CH2CH2N3), 3.36 (1 H, dd, J = 9.2, 2.3 Hz, H3) 1.97–1.82
(1 H, s, H1), 4.37 (1 H, d, J = 11.3 Hz, CH2Ph), 4.36 (1 H, app. t, (2 H, m, OCH2CH2CH2N3), 1.79 (3 H, s C(O)CH3); 13C NMR
J = 10.5 Hz, H4), 4.34 (1 H, d, J = 11.9 Hz, CH2Ph), 4.22 (1 H, d, (101 MHz; CDCl3) δ 169.6 (C(O)CH3), 168.1 (CO2CH3), 151.2
J = 12.0 Hz, CH2Ph), 4.18 (1 H, d, J = 11.6 Hz, CH2Ph), 4.03 (C(O)N(Bn)OBn), 138.4, 138.3, 138.2, 137.4, 137.3, 128.5, 128.3,
(1 H, dt, J = 9.7, 5.6 Hz, OCH2CH2CH2N3), 3.96 (1 H, d, J = 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9,
10.0 Hz, H5′), 3.90 (1 H, d, J = 2.7 Hz, H2), 3.81 (1 H, d, J = 127.6, 127.6, 127.5, 127.5, 127.4, 127.4, 127.2, 127.2, 102.0
9.4 Hz, H5), 3.79 (1 H, dd, J = 10.4, 2.1 Hz, H3′), 3.64 (1 H, app. (C1), 99.4 (C1′), 81.5 (C3), 76.1 (C3′), 76.1 (C(O)N(Bn)OCH2Ph),
t, J = 2.1 Hz, H2′), 3.58 (3 H, s, CO2CH3), 3.53 (1 H, ddd, J = 9.5, 75.8 (C5), 75.4 (C2′), 74.5 (C4), 74.0 (CH2Ph-attached to C2),
8.0, 5.1 Hz, OCH2CH2CH2N3), 3.42–3.36 (2 H, m, 73.4 (C(O)N(CH2Ph)OBn), 72.8 (C2), 72.3 (CH2Ph-attached to
OCH2CH2CH2N3), 3.36 (1 H, dd, J = 9.3, 2.7 Hz, H3′), 2.65–2.38 C2′), 72.1 (CH2Ph-attached to C3′), 71.7 (C5′), 71.0 (CH2Ph-
(3 H, m, CH2 Lev), 2.31–2.20 (1 H, m, CH2 Lev), 1.99–1.81 (3 H, attached to C3), 68.5 (C4′), 67.0 (OCH2CH2CH2N3), 52.9
m, CH3 Lev), 1.99–1.81 (2 H, m, OCH2CH2CH2N3); 13C NMR (CO2CH3), 48.3 (OCH2CH2CH2N3), 29.1 (OCH2CH2CH2N3), 20.7
(101 MHz; CDCl3) δ 206.4 (CvO Lev ketone), 171.5 (CvO Lev), (C(O)CH3); 13C-GATED (101 MHz; CDCl3): 102.0 (1JC1–H1 = 156
168.1 (CvO CO2CH3), 151.1 (C(O)N(Bn)OBn), 138.4 (Cq), 138.2 Hz, C1), 99.4 (1JC1′–H1′ = 176 Hz, C1′); HRMS (ES+) m/z [found:
(Cq), 138.2 (Cq), 138.1 (Cq), 137.3 (Cq), 137.3 (Cq), 128.5, 128.3, (M + H)+ 1065.4525 C60H64N4O14 requires (MH)+, 1065.4492];
128.2, 128.2, 128.1, 128.1, 1280, 127.9, 127.6, 127.5, 127.5, IR νmax/cm−1 2095 (m, NvNvN), 1746 (m, CvOester), 1638 (w,
127.4, 127.2, 127.2, 101.9 (C1), 99.4 (C1′), 81.5 (C3), 76.1, 76.0 CvOamide), 1229 (m, C–Oester), 1084 (s, C–Oether), 1024 (C–Oester).
(2 C, C3′ CH2Ph), 75.8 (C5), 75.4 (C2′), 74.5 (C4), 74.0 (CH2Ph),
3-Azidopropyl
(methyl
2,3-di-O-benzyl-4-O-(O-benzyl-N-
73.4 (CH2Ph), 72.7 (C2), 72.3 (CH2Ph), 72.2 (CH2Ph), 71.5 (C5′), benzyl-(2,3-di-O-benzyl-α-D-mannopyranosyl) hydroxamate)-β-
70.9 (CH2Ph), 68.7 (C4′), 67.0 (OCH2CH2CH2N3), 52.9 D-mannopyranoside) uronate 24
(CO2CH3), 48.3 (OCH2CH2CH2N3), 37.9 (CH2 Lev), 29.8 (CH3
From 23 (OAc). To a stirred solution of 23 (10 mg,
Lev), 29.0 (OCH2CH2CH2N3), 27.9 (CH2 Lev); 13C-GATED 0.009 mmol, 1.0 equiv.) in anhydrous MeOH (0.15 mL), Na
(101 MHz; CDCl3): 101.9 (1JC1–H1 = 156 Hz, C1), 99.41 (1JC1′–H1′ (0.01 mg, 0.0004 mmol, 0.05 equiv.) dissolved in anhydrous
= 176 Hz, C1′); HRMS (ES+) m/z [found: (M + H)+ 1121.4770 MeOH (10 µL) was added at room temperature under a N2
C63H68N4O15 requires (MH)+, 1121.4754].
3-Azidopropyl (methyl
benzyl-(4-O-acetyl-2,3-di-O-benzyl-α-D-mannopyranosyl) hydro- 0.05 g, 3 min), filtered through Celite®, and concentrated
xamate)-β-D-mannopyranoside) uronate 23. solution of under reduced pressure. Flash column chromatography,
atmosphere. The mixture was stirred for 16 h, then neutralised
2,3-di-O-benzyl-4-O-(O-benzyl-N- with ion exchange Amberlite 120 (H+) resin (approximately
A
donor 6 (180 mg, 0.26 mmol, 1.1 equiv.) and 13 (110 mg, eluting with diethyl ether/petroleum ether (20/80, 50/50, 90/10)
0.23 mmol, 1.0 equiv.) and in CH2Cl2 (5 mL) was stirred over afforded 24 as a colourless oil (5 mg, 0.05 mmol, 54%).
activated MS4Å for 1 h before N-iodosuccinimide (80 mg,
From 22 (OLev). 22 (100 mg, 0.09 mmol, 1.0 equiv.) was dis-
0.35 mmol, 1.5 equiv.) was added. The mixture was cooled to solved in a mixture of pyridine/AcOH (4/1 v/v, 1.5 mL), after
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which hydrazine acetate (41 mg, 0.44 mmol, 5.0 equiv.) was 50/50, 90/10) to afford compound 26 as a colourless oil (50 mg,
added. The mixture was stirred for 1 h at room temperature 0.1 mmol, 62%). Rf 0.28 (EtOAc/hexane, 1/2); [α]2D2 +21.0
1
and was diluted with EtOAc (20 mL). The organic layer was (c. 1.10, CHCl3); H NMR (300 MHz; CDCl3) δ 7.71–7.21 (15 H,
washed with 1 M HCl (2 × 15 mL), sat. aq. NaHCO3 solution m, Ar–H), 5.80 (1 H, app. t, J = 1.2 Hz, H1), 5.06 (1 H, t, J =
(2 × 10 mL) and brine (15 mL). The combined organic layers 1.9 Hz, H4), 4.94 (2 H, s, C(O)N(C1)OCH2Ph), 4.74 (1 H, app. t,
were dried over MgSO4, filtered and concentrated under J = 2.0 Hz, H5), 4.71 (1 H, d, J = 12.2 Hz, CH2Ph-attached to
reduced pressure to furnish a yellow oil. Purification by silica C3), 4.56 (1 H, d, J = 12.2 Hz, CH2Ph-attached to C3), 4.54 (1 H,
gel flash column chromatography, eluting with EtOAc/hexane d, J = 12.2, Hz, CH2Ph-attached to C2), 4.39 (1 H, d, J = 12.2,
(20/80, 50/50, 90/10) afforded 24 as a colourless oil (65 mg, Hz, CH2Ph-attached to C2), 3.85 (1 H, dd, J = 5.0, 1.6 Hz, H3),
0.63 mmol, 70%).
3.65 (1 H, dd, J = 5.0, 1.6 Hz, H2), 2.09 (3 H, s, C(O)CH3); 13C
1
Rf 0.58 (EtOAc/toluene, 3/7); [α]2D2 −3.2 (c. 0.40, CHCl3); H NMR (101 MHz; CDCl3) δ 169.6 (C(O)CH3), 150.4 (C(O)N(C1)),
NMR (400 MHz; CDCl3) δ 7.38–7.17 (30 H, m, Ar–H), 5.39 (1 H, 145.7, 137.8 (Cq Bn), 137.5 (Cq Bn), 137.3 (Cq Bn), 131.1, 129.3,
s, H1′), 5.39 (1 H, d, J = 11.7 Hz, CH2Ph), 5.33 (1 H, d, J = 128.5, 128.3, 128.2, 128.2, 128.0, 127.8, 127.7, 127.7, 124.8,
12.0 Hz, CH2Ph), 5.05 (1 H, d, J = 12.2 Hz, CH2Ph), 4.99 (1 H, 104.6 (C1), 76.4 (C(O)N(C1)OCH2Ph), 73.7 (C3), 73.0 (C5), 72.4
d, J = 12.2 Hz, CH2Ph), 4.90 (1 H, d, J = 12.3 Hz, CH2Ph), 4.71 (CH2Ph-attached to C3), 72.2 (C2), 71.1 (CH2Ph-attached to
(1 H, d, J = 12.4 Hz, CH2Ph), 4.62 (1 H, d, J = 11.7 Hz, CH2Ph), C2), 69.1 (C4), 20.9 (C(O)CH3); HRMS (ES+) m/z [found:
4.54 (1 H, d, J = 11.8 Hz, CH2Ph), 4.43 (1 H, s, H1), 4.42 (1 H, d, (M + H)+ 504.2009 C29H29NO7 requires (MH)+, 504.2017]; IR
J = 13.7 Hz, CH2Ph), 4.42 (1 H, app. t, J = 8.9 Hz, H4) 4.30 (2 H, νmax/cm−1 1740 (s, CvOester), 1701 (m, CvOamide), 1223 (s,
s, CH2Ph), 4.23 (1 H, app. t, J = 9.6 Hz, H4′), 4.23 (1 H, d, J = C–Oester) 1065 (m, C–Oether).
11.5 Hz, CH2Ph), 4.06–4.00 (1 H, m, OCH2CH2CH2N3), 3.98
(1 H, d, J = 9.6 Hz, H5′), 3.90 (1 H, d, J = 1.9 Hz, H2) 3.82 (1 H,
d, J = 9.1 Hz, H5), 3.67 (1 H, d, J = 2.3 Hz, H2′), 3.66 (1 H, dd, Conflicts of interest
J = 9.6, 2.3 Hz, H3′), 3.58 (3 H, s, CO2CH3), 3.53 (1 H, dd, J =
13.7, 8.6 Hz, OCH2CH2CH2N3), 3.43–3.35 (3 H, m, H3,
There are no conflicts to declare.
OCH2CH2CH2N3), 2.27 (1 H, d, J = 2.2 Hz, C4-OH), 1.90 (2 H,
ddd, J = 28.6, 14.0, 7.4 Hz, OCH2CH2CH2N3); 13C NMR
(101 MHz; CDCl3) δ 168.2 (CO2CH3), 152.4 (C(O)N(Bn)OBn),
Acknowledgements
138.5, 138.4, 138.3, 137.7, 137.4, 137.1, 128.5, 128.4, 128.3,
Keele University are thanked for a studentship to E. D. We also
128.2, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.6,
thank the EPSRC UK National Mass Spectrometry Facility
127.5, 127.4, 127.4, 127.3, 101.9 (C1), 99.4 (C1′), 81.5 (C3), 78.1
(NMSF) at Swansea University.
(C3′), 76.4 (CH2Ph), 75.6 (C5), 75.3 (C2′), 74.0 (CH2Ph and C4),
73.3 (CH2Ph), 72.9 (C5′), 72.8 (C2), 72.3 (CH2Ph), 72.3 (CH2Ph),
70.9 (CH2Ph), 67.3 (C4′), 66.9 (OCH2CH2CH2N3), 52.9
References
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99.4 (1JC1′–H1′ = 176 Hz, C1′); HRMS (ES+) m/z [found: (M + H)+
1023.4412 C58H62N4O13 requires (MH)+, 1023.4386].
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