Tight-binding inhibition of jack bean α-mannosidase by glycoimidazole clusters

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Takeharu Haino et al.

Solvent-induced emission of organogels based on tris(phenylisoxazolyl)

benzene

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Tight-binding Inhibition of Jack bean -Mannosidase by

Glycoimidazole Clusters

Received 00th January 20xx,

Accepted 00th January 20xx

Maëva M. Pichon, Fabien Stauffert, Anne Bodlenner, and Philippe Compain*

The best multivalent effects observed in glycosidase inhibition have been achieved so far with Jack bean -mannosidase

(JBα-man) using iminosugar clusters based on weakly binding mismatching active-site-directed inhibiting epitopes

(inhitopes) in the D-gluco series. Here, we synthesize and evaluate as JBα-man inhibitors a series of mono- to 14-valent

glycoimidazoles with inhitopes displaying inhibitions values up to the hundreds of nM range to study the impact of

inhitope affinity on the multivalent effect. The most potent inhibitor of the series, a 14-valent mannoimidazole derivative,

inhibits JBα-man with a nanomolar K_ivalue (2 ± 0.5 nM) and binding enhancements observed are, at best, relatively small

(up to 25-fold on a valency-corrected basis). Results of this study support the fact that JBα-man-inhitope affinity and the

strength of the inhibitory multivalent effect evolve in opposite direction. The major impact of the glycoimidazole-based

inhitope is found on the binding scenario; most of the synthesized mannoimidazole clusters as well as a 14-valent

glucoimidazole derivative prove to be tight binding inhibitors of JBα-man.

DOI: 10.1039/x0xx00000x

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affinity enhancements observed were explained by the

Introduction

formation of

a sandwich-type complex in which the

multivalent inhibitor simultaneously bridges the four catalytic

sites of two dimeric JBα-man molecules, resulting in a strong

chelate effect. Other arrangements may be obtained

depending on the structure of the multimeric inhibitors. For

example, the formation of discrete “S”-shaped arrangement

resulting from the interaction of two molecules of the 9-valent

Research in the field of multivalent glycosidase inhibitors has

experienced an impressive growth in the last decade.¹Yet

multivalency has long been neglected as a solid option to

achieve potent and selective glycosidase inhibition. It was the

discovery of the first quantifiable²and large³binding

enhancements achieved with multimeric mannosidase

inhibitors that ended a long lag period. The stakes involved are

high considering the therapeutic potential of glycosidase

inhibitors, exemplified by the antiviral Tamiflu^TMor the

inhibitor

2 with three molecules of JBα-man has been recently

proposed based on TEM study.⁸In addition to the use of

physical methods, structure-activity relationship investigations

have been performed to get further insights into multimeric

binding modes.¹Such studies were motivated also by the

objective of pushing the limits of the inhibitory multivalent

effect and to assess its potential as a tool for modulating

glycosidase activity in cells⁹and in vivo.¹⁰Over the last ten

years, chemists have thus synthesized numerous multimeric

systems from low-valency clusters to nanoparticle,¹¹

polymeric¹²and supramolecular¹⁰assemblies.¹Structure-

activity relationship studies focused on the impact of key

structural features on the multivalent effect. The list includes

the number, the nature and the spatial orientation of the

active-site-directed inhibiting epitopes (i. e. inhitope,^13,14by

analogy with epitope-paratope interaction). Relative inhibition

potency (rp) is estimated by a direct comparison of the

inhibition constant values between the n-valent inhibitors and

the corresponding monovalent control.

antidiabetic Glyset^{TM 4}

Glycosidase-catalyzed carbohydrate

.

hydrolysis is indeed a biologically widespread reaction involved

in a diversity of key processes including cell wall degradation

and the turnover of signalling molecules.⁵Attempts to assess

the structural basis of the inhibitory multivalent effect were

performed using physical methods. Most works focused on

Jack bean

-mannosidase (JBα-man), a dimeric high-

molecular-weight glycosidase which is by far the most

sensitive to multivalent binding known to date.¹Very recently,

an important step has been taken with the disclosing of the

first high-resolution crystal structure⁶of JBα-man in complex

with 1, the multimeric inhibitor displaying the largest binding

enhancements reported so far (Fig. 1).⁷The exceptionally large

^a.Laboratoire d'Innovation Moléculaire et Applications (LIMA)

Univ. de Strasbourg | Univ. de Haute-Alsace | CNRS (UMR 7042)

Equipe de Synthèse Organique et Molécules Bioactives (SYBIO), ECPM

25 Rue Becquerel, 67000 Strasbourg, France

E-mail:philippe.compain@unistra.fr

Electronic Supplementary Information (ESI) available: [details of any

supplementary information available should be included here]. See

DOI: 10.1039/x0xx00000x

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Fig. 1 Potent multivalent inhibitors of JBα-man and their corresponding monovalent controls.

A qualitative evaluation of the multivalent effect is thus nanomolar inhibitor of JBα-man, the selected glycocluster

provided by calculation of rp/n values. To date, the best head groups have to be accessible by a straightforward

multivalent effects were described with inhitopes displaying synthesis. The attachment of a linker to the inhitope in order

modest affinity for the targeted glycosidases.^1,7,8For example, to construct the multivalent systems by Cu(I)-catalyzed azide–

with regards to JBα-man inhibition, the largest rp/n values alkyne cycloaddition (CuAAC) must also be performed without

have been obtained with clusters based on weakly binding negatively affecting binding to JBα-man active site. After an

mismatching inhitopes in the pseudo D

-gluco series (Fig. 1).^7,8,15extensive review of the literature, mannoimidazole derivatives

Multivalent inhibitors based on 1-deoxynojirimycin (DNJ) have of type I were selected (Fig. 2). Related 2-alkyl substituted

been mainly used.¹For such systems, the corresponding imidazoles are indeed potent inhibitors of JBα-man with

monovalent controls typically display inhibition in the inhibition values in the nanomolar range.^16,17

hundreds of

M range. Even if clusters based on inhitopes in

the matching

D-mannno series have already been evaluated,

13,22

the impact of inhitopes in the nM range has never been

studied. Despite the impressive number of multivalent

glycosidase inhibitors synthesized to date,¹a puzzling question

remains unanswered: what would be the impact of high

affinity targeting inhitopes on the multivalent effect? Would

this lead to changes in the binding modes or to improved

relative inhibition potencies? To answer these questions, we

have now designed neoglycoclusters based on glycoimidazole

inhitopes. Here, we describe the full details of our study from

the stereodivergent synthesis of two glycoimidazole-based

inhitopes to the inhibition results obtained with the model

glycosidase JBα-man.

Fig. 2 Devised strategy towards click glycoimidazole clusters.

Our synthetic approach was partly based on the optimization

Results and discussion

Neoglycocluster design. The choice of the inhitope structure

of a strategy developed by Vasella et al. from readily prepared

D

-glucono-δ-thiolactam derivatives (Fig. 2).¹⁸In this

was guided by different criteria. In addition to being a potent

stereodivergent synthesis, both

D-gluco- and D-manno-

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configurated glycoimidazole intermediates could be obtained. The initial conditions described by Vasella et al.¹⁸which led in

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This offers the opportunity to access multivalent JBα-man our hands to a 2:1 mixture of the gluc^Do^O/m^{I: 1}a⁰n^.1n⁰o³-⁹c^/o^Cn^9Ofi^Bg⁰u⁰r⁸a²t⁶e^Hd

inhibitors with inhitopes displaying inhibitions from the low imidazoles



6 in 77% yield were modified to increase the

M range to the nM range.^16,17Concerning the choice of the reaction yield as well as the amount of the manno-imidazole

scaffold, β-cyclodextrin (β-CD) holds many advantages to build product 6b. After a systematic study into the effects of

rapidly multivalent constructs that may display large reaction conditions, it was found that the best yields (up to

multivalent effects with rp/n up to 620-fold (Fig. 1)¹⁵and will 96%) were obtained using dry toluene and higher

allow for direct comparison with previously synthesized concentrations for the annulation step. Under these

clusters with different inhitopes.^15,22Pentaerythritol was conditions, the amount of manno-imidazole product 6b was

selected as well in the present study as a core to access di- to slightly increased (6a

tetravalent systems. The objective was to evaluate if high chromatography of diastereoisomers

affinity inhitopes could compensate for a reduced valency.. continued on both epimers separately. Diiodo derivatives

were obtained by treatment of with an excess of N-iodo

Neoglycocluster synthesis. The first key step of the synthesis is succinimide in DMF at 80°C.^17,20In our hands, the mono-

the annulation of an imidazole ring to -gluconothiolactam

deiodination reaction using Vasella’s protocol¹⁷(EtMgBr at

obtained by treatment with Lawesson’s reagent of the room temperature) afforded the expected iodo derivative

corresponding

-glucono-δ-lactam derivative (Scheme 1).¹⁸modest yield (57%) along with the di-deiodination product

/6b ratio 3:2). After separation by flash

6

, the synthesis was

7

6

D

5

8

6

in

D

The former compound was obtained in three steps on gram 32% yield. Increasing the reaction time or the amount of

scale from commercially available tetra-O-benzyl- EtMgBr did not improve the product yield with possible

D

-

glucopyranose following a robust synthetic route developed decomposition of the Grignard reagent. After various

recently in our group.¹⁹The key tetrahydroimidazopyridine attempts, the best results were obtained with 1.1 equivalent

skeleton was generated by a two-step process: nucleophilic of i-PrMgCl at -15°C. Under these conditions, the desired

addition of aminoacetaldehyde dimethyl acetal to the mono-iodo derivatives

8 could be obtained in yields up to 84%

thiocarbonyl group of

5

followed by acid-mediated cyclization.

on a gram scale. The precursor of the “clickable” linker was

then introduced by way of a Sonogashira coupling.^17,20,21

Optimal reaction conditions using Pd(PPh₃)₂Cl₂(5 mol%) in

DMF in the presence of diisopropylamine (DIPA, 5 equivalents)

and CuI (0.1 equivalent) afforded the 2-alkyl substituted

imidazoles

9 in good yields. One-pot alkyne reduction/O-

debenzylation followed by peracetylation of the corresponding

crude tetrols provided 10 in high yields for the two steps. The

use of acetate protecting groups is critical for the final

challenging deprotection step in the synthesis of the targeted

neoglycoclusters. The displacement of the chloride with

sodium azide in the presence of TBAI was achieved in

quantitative yields in DMF to afford the desired protected

clickable glycoimidazole derivatives 11

. The protected

monovalent controls 13 were then synthesized by treatment

of glycoimidazoles 11 with ethynylcyclopropane in the

presence of CuSO₄.5H₂O and sodium ascorbate under

microwave conditions (Scheme 2).

Scheme 1 Access to azide-armed glycoimidazole 11.

Scheme 2 Synthesis of monovalent models 13.

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The use of cyclopropylated alkyne in the CuAAC reaction glycoimidazole clusters 18, 20 and 22 in 47 to 85% yields

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DOI: 10.1039/C9OB00826H

avoided the formation of side-products arising from prior (Scheme 4).

aerobic copper-catalyzed oxygenation at the propargylic

methylene group in 1-pentyne.^14,22The reaction proceeded in

high yields for the conversion of glucoimidazole 11a. However,

in contrast to the previous steps of the synthesis, the CuAAC

reaction showed a marked difference of reactivity between the

D

-gluco and the D-manno series, mannoimidazole 12b being

obtained in only 65% yield. The monovalent models 13 were

then obtained by saponification of peracetates 12 according to

a convenient method based on the use of anion exchange

Amberlite IRA-400 (OH^-) resin.¹⁵Having in hands the clickable

glycoimidazole derivatives 11, we then focused our attention

on the final steps of the neoglycoclusters synthesis. We first

prepared the pentaerythritol-based scaffolds for the synthesis

of di-, tri- and tetravalent glycoimidazole derivatives (Scheme

3).

Scheme 3 Synthesis of O-propargyl scaffolds 17.

The first step in the synthesis of alkynes 17a,b involved the

monosylation of 14 to give 15.²³Complete propargylation of

triol 15 proved difficult, dipropargyl ether 16a being obtained

as the predominant product with its tripropargyl ether

analogue 16b. Taking advantage of this incomplete reaction,

the two desired pentaerythritol-based scaffolds 17a,b could be

readily synthesized by a divergent approach. After separation

on silica gel, TBAF treatment of silyl ethers 16 provided the

desired alcohols 17 in good to excellent yields. The

tetrapropargyl ether 17c was then prepared in one step from

pentaerythritol (14) following Haag’s protocol.²⁴Microwave-

assisted click conjugation of 11 with di-, tri- and tetrapropargyl

ethers 17 provided the corresponding protected

Scheme 4 Synthesis of glycoimidazole clusters 19, 21 and 23.

4 | J. Name., 2012, 00, 1-3

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Except in one case (clusters 20), no marked difference of orders of magnitude compared to the monoval_Ve_ien_wt_Am_rtico_led_Oe_nl_lin4_e

DOI: 10.1039/C9OB00826H

reactivity was observed between the azide-armed based on the prevalent DNJ inhitope. We were pleased to see

tetrahydroimidazopyridines in the

series. Quite surprisingly, first attempts to perform O- displayed a K_ivalue in the low

D

-gluco- and the

D

-manno that the

D

-gluco-configurated monovalent model 13a

M range, almost halfway

and mannoimidazole 13b



deacetylation of glycoimidazole clusters 18, 20 and 22 using between DNJ derivative

4

.

anion exchange Amberlite IRA-400 (OH^-) led to low yields of Evaluation of di- to tetravalent tetrahydroimidazopyridines

the desired deprotected clusters and to unidentified side- indicated clearly that the use of inhitopes displaying higher

products. Alternatively, deacetylation of pentaerythritol-based affinity than DNJ motifs did not lead to significant multivalent

clusters 18, 20 and 22 could be performed in good to high effects (Table 1). Moreover, the best rp/n values ranging from

yields by treatment with ammonia in methanol at room 2 to 3 were obtained for the clusters displaying the D-gluco-

temperature to provide the corresponding free-OH products configurated inhitope. The use of the mannoimidazole

19, 21 and 23 (Scheme 4).²⁵CuAAC was also performed with inhitope had either no significant impact or even negative

heptakis(2,6-di-O-propargyl) β-cyclodextrin (24),²⁶leading to impact on the valency-corrected inhibition enhancements.

the desired tetradecavalent glycoimidazoles 26 after Double-reciprocal Lineweaver-Burk plots revealed

a

ammonia-mediated deprotection of the acetate groups in 25 competitive inhibition mode for the whole cluster series with

(Scheme 5a).

the exception of tetravalent glucoimidazole 23a which

displayed mixed-type inhibition mode of JBα-man.

a

Interestingly, clusters 21b and 23b displaying three to four

copies of the mannoimidazole-based inhitope were found to

be competitive tight binding inhibitors of JBα-man. For those

compounds, a similar trend was observed: Dixon curves

showed

a

strong non linearity, and double-reciprocal

pure non-

Lineweaver-Burk plots were suggesting

a

competitive behaviour and the respective secondary curves

(slope as a function of inhibitor concentration) were non-linear

(see figures S92, S93, S97 and S98). Such a behaviour can be

misleading and often hints a tight binding inhibition pattern. ²⁷

In such cases, the classical steady-state kinetic model does not

apply anymore. Enzyme (E) and inhibitor (I) concentrations are

in the same range; inhibitor depletion due to the E.I complex

formation cannot be neglected anymore and has therefore to

be taken into account. Proper experiment design and data

treatment for determination of accurate values of inhibition

constants in the case of fast tight binding inhibition has been

thoroughly discussed in the literature.²⁸Inhibitor

concentration range was chosen in order to have relevant

data^28g,29and the generic Morrison equation^28badapted by

Cha^28dfor data analysis (eq. 1) was fitted to mean values of

triplicate experiments in order to get the apparent inhibition

constant K_i^appand then the real inhibition constant K_ifrom

equation 2 in accordance to a competitive inhibitor type:

2

푎푝푝

푖

푎푝푝

푖

푎푝푝

푖

√

푣 (퐸−퐼−퐾

0

+

(퐸−퐼−퐾

) +4퐸퐾

)

푣_푖=

(eq. 1)

2퐸

푆

퐾_푖^푎푝푝= 퐾_푖(1 +

)

(eq. 2)

퐾_푚

Scheme 5 Synthesis of β-CD-based glycoimidazoles 26 and structure of the

corresponding DMJ analogue 27 and its monovalent counterpart 28.

where v_iis the initial rate in presence of inhibitor and v₀in the

absence of inhibitor, E represents the total concentration of

active sites, I the total inhibitor concentration, S the substrate

concentration, 퐾_푖^푎푝푝the apparent inhibition constant, K_ithe

real inhibition constant and K_mthe Mickaelis-Menten constant

determined independently. The initial fit using the Mickaelis

Menten constant evaluated independently for each

experiment and the calculated enzyme concentration as

constants gave the K_i.

Biological results. As indicated in the introduction, JBα-man

has been widely used as a model probe to study multivalent

glycosidase-inhibitor interactions.¹The largest multivalent

effects in glycosidase inhibition have been observed with this

enzyme.⁷We first evaluated the inhibition potency of

monovalent models 13 (Table 1). As expected,

D-manno-

configurated glycoimidazole 13b has an inhibition constant (K_i)

value in the hundreds of nM range. This is a gain of three

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Table 1 JBα-man inhibitory activities (K_i, μM) for glycoimidazoles 13, 19, 21

and 23.^a

sharp contrast to our results, a study on the pentameric

cholera toxin lectin showed that a strong multivalent effect

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DOI: 10.1039/C9OB00826H

(rp/n ~10000) was conserved by decreasing ligand IC₅₀values

by 3 orders of magnitude.³²An analysis of the inhibition

modality in table 2 points out that tight binding inhibition is

observed only for the glycoimidazole cluster series (entries 3

and 4). The development of tight binding inhibition appeared

to be both dependent of JBα-man-inhitope affinity and cluster

valency. With the exception of divalent derivative 19b, tight

binding inhibition is thus observed for all multimeric

mannoimidazoles, i.e. clusters based on the inhitope displaying

the highest affinity. When the inhibition potency of the

inhitope is reduced by one order of magnitude as in the

glucoimidazole cluster series, tight binding inhibition is

cluster

13a

n

Inhitope

K_i

rp

rp/n

Inhibition

mode

1

Gluco-

2.23 ± 0.13

-

Competitive

Mixed-type

Competitive

imidazole

19a

21a

23a

13b

19b

21b

23b

2

3

4

1

2

3

4

Gluco-

0.49 ± 0.64

0.25 ± 0.03

4.5

9

2.3

3

imidazole

Gluco-

imidazole

Gluco-

imidazole

0.21 ± 0.05

1.00 ± 0.10

10.6

-

2.6

-

Manno-

imidazole

0.11 ± 0.02

observed only for the 14-valent cluster 26a

.

Manno-

imidazole

0.037

± 0.007

3

1.5

1

Conclusions

Manno-

imidazole

0.037

± 0.010

3

Competitive

Tight binding

In conclusion, we have synthesized and evaluated as JBα-man

inhibitors a series of multivalent glycoimidazole in the -gluco

and -manno series. A special attention was paid to clusters

D

Manno-

0.091

1.2

0.3

Competitive

Tight binding

D

imidazole

± 0.017

based on strongly binding mannoimidazole inhitopes. Modest

multivalent effects were observed for multimeric

glycoimidazoles which were nevertheless found to be potent

inhibitors of JBα-man with inhibition values up to 2 nM. A

comparison study with iminosugar clusters based on DNJ or

DMJ heads indicated that JBα-man-inhitope affinity and the

strength of the inhibitory multivalent effect seemed to evolve

in opposite direction. It seems from those results that the

potential improvement of a strong inhibitor by multivalency is

less important than for a loose inhibitor for JBα-man.

Multivalency proved however to remain an interesting tool for

modulation even in such cases. This trend still has to be

confirmed on other systems. Interestingly, we found that

mannoimidazole clusters (with the exception of 19b) and 14-

valent glucoimidazole cluster 26a are tight binding inhibitors of

JBα-man. To our knowledge, this is the first report of

multimeric inhibitors that display tight binding inhibition of a

carbohydrate-processing enzyme. These observations add new

insights into the inhibitory multivalent effect. There is still

space for improvements in finding appropriate arm length,

geometry and rigidity while keeping those inhitopes with the

help of molecular modelling thanks to the recently published

JBα-man X-ray structure.⁶

[a] rp = K_i(monovalent reference)/K_i(glycocluster), n = number of inhitope units.

In our case, since K_i^appwere higher than the enzyme

concentration, the latter can not be evaluated from the first

region of the curve but it has been demonstrated that errors

on active enzyme concentration have no effect on the

inhibition constant in such cases.^28g,fWe then turned our

attention to glycoimidazole-cyclodextrin conjugates 26 (Table

2). The corresponding multivalent systems 3¹⁵(Figure 1) and

27²²(scheme 5b) based on DNJ or 1-deoxymannojirimycin

(DMJ) inhitopes have been included for comparative purposes.

Both 14-valent glycoimidazole clusters 26a and 26b were

found to be competitive, tight binding inhibitors of JBα-man

with K_ivalues of 6 and 2 nM, respectively. The multivalent

effects remained modest (rp/n of 26 and 4 respectively),

although 14-valent glycoimidazole-based clusters 26 showed

inhibition values similar to 36-valent iminosugar 1⁷and a 120-

valent cluster³⁰which were both based on DNJ heads.

Comparison of rp/n values of the 14-valent clusters 3, 26 and

27 and the K_ivalues of the corresponding monovalent models

revealed an interesting trend, showing that JBα-man-inhitope

affinity and the strength of the inhibitory multivalent effect

evolved in opposite direction. An increase of three orders of

magnitude in the inhibition potency of the inhitopes led to a

decrease of two orders of magnitude in the multivalent effect

as judged by the rp/n values (Table 2, entries 1 and 4). A

similar trend had been observed with sp²-iminosugars 1-

amino-5N,6O-oxomethylydenenojirimycin (1N-ONJ) and 1-

amino-5N,6O-oxomethylydenemannojirimycin (1N-OMJ) in a

different inhibitory scale. A shift in the monovalent inhibitory

power from the hundreds of µM to the low µM led to a one-

order magnitude reduction in rp/n values or to a total loss of

the multivalent effect depending on the alkyl chain length.¹³In

6 | J. Name., 2012, 00, 1-3

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Table 2. JBα-man inhibitory activities (Ki, μM) for 14-valent clusters 3, 27 and 26 and their corresponding monovalent models^a

Monovalent derivatives

14-valent derivatives

Inhibition mode

Entry

Monovalent

models

Inhibition mode

14-valent

clusters

Inhitope

K_i

rp/n

rp

K_i

1

2

3

4¹⁵

28²²

13a

Competitive

DNJ

188¹⁵

111²²

2.23

610

115

26

8545

1585

372

0.022¹⁵

0.07²²

Competitive

3¹⁵

27²²

26a

DMJ

Competitive

Tight binding

Competitive

Glucoimidazole

Mannoimidazole

0.006 ±

0.001

4

Competitive

Tight binding

13b

0.11

4

55

0.0020 ±

0.0005

26b

[a] rp = K_i(monovalent reference)/K_i(glycocluster), n = number of inhitope units.

(J) are expressed in Hz, integration value and assignment. The

subscript Ar means aromatic. The superscript III means tertiary

carbon and the superscript IV means quaternary carbon. Carbon

multiplicities were assigned by Distortionless Enhancement by

Experimental Section

Commercially available starting materials were purchased from Polarization Transfer (DEPT) experiments. ¹H and ¹³C signals were

commercial suppliers as Sigma-Aldrich Co., Merck Co., Alfa Aesar assigned by correlation spectroscopy (COSY), Heteronuclear Single

GmbH & Co., Acros Organics, Fluorochem, Carbosynth Limited or Quantum Correlation (HSQC), Heteronuclear Multiple-Bond

VWR and were used without further purification. When specified, Correlation spectroscopy (HMBC) and Nuclear Overhauser Effect

anhydrous solvents were required. Tetrahydrofuran (THF) was Spectroscopy (NOESY) when required. Optical rotations were

distilled

over

sodium/benzophenone

under

argon. measured at 589 nm (sodium lamp) and 20 °C on Anton Paar MCP

Dimethylformamide (DMF) was purchased anhydrous over 200 polarimeter with a path length of 1 dm. The concentration (c) is

molecular sieves. Toluene was distilled over CaH₂, pyridine and indicated in gram per deciliter. Infrared (IR) spectra were recorded

diisopropylamine were distilled over KOH and all of them were neat or in solution on

a Perkin-Elmer Spectrum Two FT-IR

stored over KOH under argon. All reactions were performed in spectrometer. ESI-TOF high resolution mass spectra (HRMS) were

standard glassware or in vials adapted to a Biotage Initiator® carried out on a Bruker MicroTOF® mass spectrometer.

microwave reactor. Reaction monitoring was achieved by Thin

Layer Chromatography (TLC) on aluminum sheets coated with silica 2,3,4,6-Tetra-O-benzyl-D-glucono-δ-thiolactam (5)

gel 60 F254 purchased from Merck KGaA. Crude mixtures were Lawesson's reagent (2.02 eq., 0.789 g, 1.89 mmol) was added to a

purified by flash column chromatography on silica gel 60 (230-400 mixture containing 2,3,4,6-Tetra-O-benzyl-D-glucono-δ-lactam (1

mesh, 0.040-0.063 mm) purchased from Merck KGaA. Proton (¹H) eq., 0.500 g, 0.93 mmol), pyridine (0.038 mL), freshly activated 4 Å

and carbon (¹³C) nuclear Magnetic Resonance (NMR) spectra were molecular sieves and dry toluene (38.0 mL). The reaction mixture

recorded at 298K on either Bruker Avance 300 MHz, Bruker Avance was stirred for 20 h. The mixture was stirred with MeOH (5 ml) for 2

III HD 400 MHz with BBFO probe or Bruker 500 MHz Avance III HD h, filtered and the solvent were removed under reduced pressure.

with Prodigy BBO probe spectrometers. The chemical shifts are The residue was subjected to flash chromatography (1:6

reported as δ values in parts per million (ppm) relative to residual EtOAc/petroleum ether) to afford product 5 (0.408 g, 0.737 mmol,

solvent signals used as internal reference. Data are presented as 99%) as a pale orange solid. Analytical data were in accordance with

followed: chemical shift, multiplicity (s = singlet, br s = broad singlet, those reported in literature.³¹

d = doublet, dd = doublet of doublets, ddd = doublet of doublets of ¹H NMR (400 MHz, CDCl₃) δ (ppm) 3.37 (dd, J = 9.8, 7.6 Hz, 1H),

doublets, dt = doublet of triplets, t = triplet, td = triplet of doublets, 3.56 (dd, J = 9.4, 4.6 Hz, 1H), 3.64 (dd, J = 9.8, 3.3 Hz, 1H), 3.85-

q = quadruplet, qnt = quintuplet, m = multiplet), coupling constants 3.93 (m, 2H), 4.35 (d, J = 11.5 Hz, 1H), 4.43-4.49 (m, 4H), 4.59 (d, J

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= 11.5 Hz, 1H), 4.67 (d, J = 11.5 Hz, 1H), 4.74 (d, J = 11.6 Hz, 1H),

5.01 (d, J = 11.5 z, 1H), 7.11-7.44 (m, 20H), 8.05 (s, 1H).

View Article Online

DOI: 10.1039/C9OB00826H

Compound 7a

A solution of 6a (1 eq., 0.48 g, 0.855 mmol) in DMF (9.3 mL) was

Gluco- and manno-imidazole (6a and 6b)

treated with NIS (10 eq., 2.02 g, 8.55 mmol) and stirred under argon

Thiolactam 5 (1 eq., 517 mg, 0.935 mmol) was dissolved in atmosphere at 80°C for 14 h. The brown mixture was cooled,

NH₂CH₂CH(OMe)₂(15 eq., 1.52 mL, 14 mmol) and stirred under diluted with Et₂O, and washed with a 10% Na₂S₂O₃solution

argon atmosphere for 20h. At the beginning, the reaction was (120mL). The combined H₂O layers were extracted with Et₂O. The

green-yellowish and turned progressively to orange and light brown combined organic layers were washed with H₂O and brine, dried

solution. The reaction was diluted with Et₂O and water. The organic over Na₂SO₄, filtered and concentrated. Flash chromatography on

and aqueous phases were separated. Aqueous phase was extracted silica gel (petroleum ether/EtOAc 7:1) gave product 7a (515 mg,

three times with Et₂O. Combined organic layers were washed with 0.634 mmol, 75%) as an oil. Analytical data were in accordance with

water and then with brine. The organic fraction was dried over those reported in literature.²⁰

Na₂SO₄, filtered and concentrated under reduced pressure. The ¹H NMR (400 MHz, CDCl₃) δ (ppm) 3.64 (dd, J = 9.5, 4.4 Hz, 1H, H6^a),

crude was used for the next step without purification.

3.72 (t, J = 9.2 Hz, 1H, H6^b), 4.08 (t, J = 4.0 Hz, 1H, H3), 4.34 (dd, J =

4.3, 2.1 Hz, 1H, H4), 4.42 (s, 2H, OCH₂Ph), 4.46 (m, 1H, H5), 4.48 (d, J

TsOH. H₂O (1.87 eq., 333 mg, 0.312 mL, 1.75 mmol) was added to a = 11.7 Hz, 1H, OCH₂Ph), 4.57 (dd, J = 11.9, 4.8 Hz, 2H, OCH₂Ph),

solution of crude amidine (1 eq., 586 mg, 0.939 mmol) in dry 4.65-4.71 (m, 2H, OCH₂Ph and H2), 4.82 (d, J = 11.9 Hz, 1H,

toluene (13.4 mL). The solution was stirred at 60 °C for 63h under OCH₂Ph), 5.1 (d, J = 12.0 Hz, 1H, OCH₂Ph), 7.16-7.45 (m, 20H, H_Ar).

air. The cooler system was equipped with a drierite drying tube to

Compound 7b

avoid water in the medium. The solution was diluted with DCM and

sat. NaHCO₃. Aqueous and organic phase were separated. Aqueous

phase was extracted with DCM. Organic fractions were gathered

and washed with water, brine, dried over Na₂SO₄and concentrated

under reduced pressure. The crude residue was purified by flash

column chromatography on silica gel (4:6 EtOAc/petroleum ether).

Glucoimidazole 6a (205 mg, 0.367 mmol, 39%) and mannoimidazole

6b (291 mg, 0.52 mmol, 55%) were obtained as oils. Analytical data

were in accordance with those reported in the literature.¹⁸

A solution of 6b (1 eq., 1.33 g, 2.37 mmol) in DMF (20 mL) was

treated with NIS (10 eq., 5.62 g, 23.7 mmol) and stirred under argon

atmosphere at 80°C for 20h. The brown mixture was cooled, diluted

with Et₂O, and washed with sat. Na₂S₂O₃(x3). The combined H₂O

layers were extracted with Et₂O (x3). The combined organic layers

were washed with H₂O and brine, dried over Na₂SO₄, filtered and

concentrated. The crude was purified by flash column

chromatography on silica gel (7:1 petroleum ether/EtOAc) and

product 7b (1.43 g, 1.76 mmol, 74%) was obtained as an orange oil.

Analytical data were in accordance with those reported in

literature.¹⁷

6a: R_f0.35 (petroleum ether / EtOAc 1:1), ¹H NMR (400 MHz,

CDCl₃) δ (ppm) 3.75 (dd, J = 10.4, 5.3 Hz, 1H, H6^a), 3.83-3.90 (m, 2H,

H6^b, H3), 4.10 (dd, J = 7.6, 5.7 Hz, 1H, H4), 4.19 (ddd, J = 7.9, 5.7, 3.0

Hz, 1H, H5), 4.46 (d, J = 2.3 Hz, 2H, OCH₂Ph), 4.51 (d, J = 11.2 Hz, 1H,

OCH₂Ph), 4.70 (d, J = 11.2 Hz, 1H, OCH₂Ph), 4.75 (d, J = 5.7 Hz, 1H,

H2), 4.83 (d, J = 10.4 Hz, 1H, OCH₂Ph), 4.85 (d, J = 11.2 Hz, 1H,

OCH₂Ph), 4.88 (d, J = 11.7 Hz, 1H, OCH₂Ph), 5.18(d, J = 11.7 Hz, 1H,

OCH₂Ph), 7.05 (d, J = 1.1 Hz, 1H, H7), 7.12 (d, J = 1.1 Hz, 1H, H8),

7.17-7.46 (m, 20H, H_Ar), ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 58.2,

68.5, 72.9, 73.4, 74.2, 74.4, 76.2, 82.3, 117.4, 127.7 (C7), 128.0,

128.02, 128.09, 128.1, 128.2, 128.3, 128.5, 128.51, 128.57, 128.60,

128.7 (C_Ar), 129.5 (C8), 137.4, 137.7, 138.0, 138.8 (C^IV_Ar), 144.1 (C1).

¹H NMR (400 MHz, CDCl₃) δ (ppm) 3.61 (dd, J = 9.4, 4.9 Hz, 1H), 3.71

(dd, J = 9.3, 8.7 Hz, 1H), 3.74 (dd, J = 7.6, 3.1 Hz, 1H), 3.76 (m, 2H),

4.35 (ddd, J = 8.7, 4.8, 2.4 Hz, 1H), 4.43 (d, J = 7.6 Hz, 1H), 4.45 (dd, J

= 7.7, 2.4 Hz, 1H), 4.47-4.52 (m, 2H), 4.54 (d, J = 11.6 Hz, 1H), 4.57

(d, J = 11.6 Hz, 1H), 4.60 (d, J = 11.2 Hz, 1H), 4.73-4.78 (m, 3H), 7.20-

7.39 (m, 20H).

Compound 8a

Isopropyl magnesium chloride (1.11 eq., 1.99 M, 0.82 mL, 1.63

mmol) in solution in THF was added dropwise for 15 min to a stirred

solution of 7a in freshly distilled THF (14 mL) at -15°C. Reaction was

stopped 2h after the beginning of the addition. The mixture was

treated with sat. NH₄Cl and diluted with Et₂O. The layers were

separated, and the organic layer was washed with sat. NH₄Cl twice.

The combined H₂O layers were extracted with Et₂O (x3). The

combined organic layers were washed with H₂O twice and brine,

dried over Na₂SO₄, filtered, and evaporated. The crude was purified

by flash column chromatography on silica gel (Pentane/ EtOAc 9:1

to 8:2). Product 8a (843 mg, 1.23 mmol, 84%) was isolated as an oil.

Analytical data were in accordance with those reported in

literature.²⁰

6b: R_f0.21 (petroleum ether / EtOAc 1:1), ¹H NMR (400 MHz, CDCl₃)

δ (ppm) 3.63 (dd, J = 9.9, 7.1 Hz, 1H, H6^a), 3.77 (dd, J = 10.1, 3.1 Hz,

1H, H6^b), 3.87 (dd, J = 9.4, 3.2 Hz, 1H, H3), 4.14 (td, J = 7.0, 2.9

Hz,1H, H5), 4.29 (dd, J = 9.6, 7.3 Hz, 1H, H4), 4.41-4.52 (m, 2H,

OCH₂Ph), 4.58 (d, J = 12.0 Hz, 1H, OCH₂Ph), 4.63 (d, J = 11.2 Hz, 1H,

OCH₂Ph), 4.67 (d, J = 12.8 Hz, 2H, OCH₂Ph), 4.74 (d, J = 12.2 Hz, 1H,

OCH₂Ph), 4.82 (d, J = 2.9 Hz, 1H, H2), 5.01 (d, J = 11.1 Hz, 1H,

OCH₂Ph), 7.07 (s ,1H, H7), 7.17 (s, 1H, H8), 7.23-7.43 (m, 20H, H^Ar),

¹³C NMR (100 MHz, CDCl₃) δ (ppm) 56.0 (C5), 68.3 (C2), 70.6

(OCH₂Ph), 71.2 (C6), 71.8, 73.3 (OCH₂Ph), 74.3 (C4), 75.0 (OCH₂Ph),

80.3 (C3), 119.5 (C7), 127.6, 127.8, 127.9, 127.9, 128.0, 128.3,

128.34, 128.4, 128.53, 128.54, 128.56, 128.6 (C^III_Ar), 129.3 (C8),

137.7, 138.0, 138.2, 138.3 (C^IV_Ar), 143.0 (C1).

¹H NMR (400 MHz, CDCl₃) δ (ppm) 3.69 (dd, J = 10.5, 5.6 Hz, 1H,

H6a), 3.78 (dd, J = 10.3, 3.0 Hz, 1H, H6b), 3.80 (t, J = 7.5 Hz, 1H, H4),

8 | J. Name., 2012, 00, 1-3

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4.07 (dd, J = 7.1, 5.1 Hz, 1H, H3), 4.17 (ddd, J = 8.1, 5.1, 2.9 Hz, 1H, 128.59 (C^III_Ar), 137.4, 137.6, 137.9, 138.3 (4xC^IV_Ar), 143.8 (C1), HRMS

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+

DOI: 10.1039/C9OB00826H

H5), 4.40-4.51 (m, 3H, OCH₂Ph), 4.63 (d, J = 11.4 Hz, OCH₂Ph), 4.70 (ESI) m/z 675.2971 ([M + H] calcd. For C₄₂H₄₄ClN₂O₄: 678.2984).

(d, J = 5.1 Hz, 1H, H2), 4.74-4.82 (m, 3H, OCH₂Ph), 5.11 (d, J = 11.6

Hz, OCH₂Ph), 7.09 (s, 1H, H7), 7.13-7.43 (m, 20 H, H_Ar).

Compound 9b

A degassed suspension of DMF (4.3 mL) and CuI (0.1 eq., 4.8 mg,

0.0252 mmol) was added to 8b (1 eq., 175 mg, 0.255 mmol) under

Compound 8b

Isopropyl magnesium chloride (1.11 eq., 1.99 M, 0.065 mL, 0.123 argon. Then 6-chloro-1-hexyne (4.12 eq., 0.13 mL, 1.05 mmol) and

mmol) in solution in THF was added dropwise for 15 min to a stirred Pd(PPh₃)₂Cl₂(4.98%, 9 mg, 0.0127 mmol) were added under argon.

solution of 7b in freshly distilled THF (0.9 mL) at -15°C. Reaction was DIPA (5 eq., 0.18 mL, 1.27 mmol) was finally added and the reaction

stopped 30 min after the beginning of the addition. The mixture mixture was stirred at 40°C for 16 h. The solution was cooled to

was treated with sat. NH₄Cl and diluted with Et₂O. The layers were room temperature, diluted with Et₂O and washed with NH₄Cl (x3).

separated, and the organic layer was washed with sat. NH₄Cl twice. The combined organic layers were extracted with Et₂O (x3). The

The combined H₂O layers were extracted with Et₂O (x3). The combined organic layers were washed with H₂O and brine, dried

combined organic layers were washed with H₂O twice and brine, over Na₂SO₄, filtered and evaporated. The crude was purified by

dried over Na₂SO₄, filtered, and evaporated. The crude was purified flash column chromatography on silica gel (2:8 EtOAc/pentane).

by flash column chromatography on silica gel (Pentane/ EtOAc 9:1). Product 9b (131 mg, 0.194 mmol, 76%) was obtained as an oil.

R_f0.3 (EtOAc/petroleum ether 2:8), [α]²_D⁰= -31 (c 1,0, CHCl₃), IR

(neat) 3063, 3030, 2925, 2865, 1497, 1454, 1365, 1028, 1114, 1027,

913, 737, 698 cm^-1, ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.74 (qnt, J =

7.4 Hz, 2H, H12), 1.95 (qnt, J = 7.2 Hz, 2H, H13), 2.45 (t, J = 7.0 Hz,

2H, H11), 3.54-3.61 (m, 3H, H14 and H6a), 3.71 (dd, J = 10.0, 2.9 Hz,

1H, H6b), 3.83 (dd, J = 9.3, 3.0 Hz, 1H, H3), 4.08 (td, J = 7.1, 2.7 Hz,

1H, H5), 4.25 (dd, J = 9.3, 7.4 Hz, 1H, H4), 4.44 (s, 2H, OCH₂Ph), 4.54-

4.68 (m, 4H, OCH₂Ph), 4.71-4.76 (m, 2H, OCH₂Ph and H2), 4.98 (d, J

= 11.2 Hz, 1H, OCH₂Ph), 7.19-7.43 (m, 20H, H_Ar),¹³C NMR (100 MHz,

CDCl₃) δ (ppm) 18.8 (C11), 25.8 (C12), 31.6 (C13), 44.7 (C14), 60.1

(C5), 68.4 (C2), 70.7 (OCH₂Ph), 70.9 (C6), 71.8, 73.2 (2 x OCH₂Ph),

73.9 (C4), 74.6 (C9), 75.0 (OCH₂Ph), 80.0 (C3), 89.4 (C10), 122.6 (C8),

127.6-128.5 (C^III_Ar), 137.4, 137.8, 138.0, 138.1, (4 x C^IV_Ar), 142.8 (C1),

HRMS (ESI) m/z 675.2988 ([M + H]⁺calcd. For C₄₂H₄₄ClN₂O₄:

675.2984).

Product 8b (61.2 mg, 0.089 mmol, 80%) was obtained as an oil.

Analytical data were in accordance with those reported in

literature.¹⁷

¹H NMR (400 MHz, CDCl₃) δ (ppm) 3.57 (dd, J = 10.0, 7.2 Hz, 1H,

H6a), 3.71 (dd, J = 10.1, 2.7 Hz, 1H, H6b), 3.84 (dd, J = 9.3, 2.8 Hz,

1H, H3), 4.11 (td, J = 7.1, 2.9 Hz, 1H, H5), 4.26 (dd, J = 8.9, 7.4 Hz,

1H, H4), 4.46 (s, 2H, OCH₂Ph), 4.57-4.70 (m, 4H, OCH₂Ph), 4.76 (d, J

= 13.2 Hz, 1H, OCH₂Ph), 4.78 (d, J = 2.8 Hz, 1H, H2), 4.99 (d, J = 11.2

Hz, 1H, OCH₂Ph), 7.21-7.45 (m, 20H, H_Ar).

Compound 9a

A degassed suspension of CuI (0.1 eq., 23.3 mg, 0.122 mmol) in

DMF (20.4 mL) was added to 8a (1 eq., 839 mg, 1.22 mmol) under

argon. 6-chloro-1-hexyne (4 eq., 0.61 mL, 4.93 mmol) and

Pd(PPh₃)₂Cl₂(5.02%, 43.5 mg, 0.0614 mmol) were then added under

argon. DIPA (5 eq., 0.87 mL, 6.16 mmol) was finally added and the

reaction mixture was stirred at 40°C for 17 h. The solution was

cooled to room temperature, diluted with Et₂O and washed with

NH₄Cl (x3). The combined organic layers were extracted with Et₂O

(x3). The combined organic layers were washed with H₂O and brine,

dried over Na₂SO₄, filtered and evaporated. The crude was purified

by flash column chromatography on silica gel (15:85 EtOAc/pentane

to 2:8 EtOAc/pentane) affording 9a (0.65 g, 0.97 mmol, 79%) as an

oil.

Compound 10a

Pd(OH)₂/C (4,55 eq., 620 mg, 4.42 mmol) was placed in a round

bottom flask. Three cycles vacuum/argon were done, followed by a

vacuum/H₂cycle. A solution of 9a (1 eq., 653 mg, 0.968 mmol) in a

3:1:1 EtOAc/MeOH/H₂O mixture (29 mL) and AcOH (17.6 mL) were

added on the activated catalyst, then degassed with H₂and the

reaction mixture was stirred under H₂for 3 days. The solution was

R_f0.2 (EtOAc/petroleum ether 2:8), [α]²_D⁰= + 29 (c 1.0, CHCl₃), IR filtered over a pad of Silica with 85:15 DCM/MeOH (300 mL). The

(neat) 3031, 2923, 2867, 1497, 1454, 1362, 1338, 1095, 1067, 1028, crude of was used without further purification for the next step.

1

737, 698 cm^-1, H NMR (400 MHz, CDCl₃) δ (ppm) 1.76 (qnt, J = 7.4 Ac₂O (12.8 mL) and dry pyridine (12.5 mL) were added to the crude

Hz, 2H, H12), 1.97 (qnt, J = 7.1 Hz, H13), 2.47 (t, J = 6,9 Hz, 2H, H11), and the solution was stirred for 20 h under argon atmosphere.

3.60 (t, J = 6,5 Hz, 2H, H14), 3.71 (dd, J = 10.3, 5.6 Hz, 1H, H6^a), 3.79- Water was slowly added at 0 °C. The aqueous phase was extracted

3.84 (m, 2H, H4 and H6^b), 4.08 (dd, J = 7.3, 5.3 Hz, 1H, H3), 4.17 (m, with CH₂Cl₂(x3). The combined organic layers were then washed

1H, H5), 4.42-4.51 (m, 3H, OCH₂Ph), 4.64 (d, J = 11.3 Hz, 1H, with an aqueous solution of 2M HCl (x3), sat. NaHCO₃solution,

OCH₂Ph), 4.69 (d, J = 5.2 Hz, 1H, H2), 4.75-4.84 (m, 3H, OCH₂Ph), water (x3) and dried over Na₂SO₄, filtered and concentrated. Crude

5.15 (d, J = 11.7 Hz, 1H, OCH₂Ph), 7.15 (s, 1H, H7), 7.23-7.44 (m, residue was purified by flash column chromatography on silica gel

20H, H_Ar), ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 19.0 (C11), 25.9 (C12), (6:4 EtOAc/pentane). Product 10a (419 mg, 0.862 mmol, 89%) was

31.8 (C13), 44.8 (C14), 58.3 (C5), 68.5 (C6), 72.8, 73.4 (2x OCH₂Ph), obtained as an oil.

R_f0.4 (EtOAc/petroleum ether 6:4), [α]²_D⁰= +25 (c 1,1, CHCl₃), IR

73.9 (C2), 74.0, 74.2 (OCH₂Ph), 76.1 (C4), 81.8 (C3), 121.0 (C10),

127.7 (C9), 128.0, 128.08, 128.09, 128.2, 128.3, 128.4, 128.57,

1

(neat) 2934, 2858, 1749, 1455, 1370, 1219, 1035, 644, 602 cm^-1, H

NMR (400 MHz, CDCl₃) δ (ppm) 1.35-1.42 (m, 2H, H11), 1.42-1.51

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(m, 2H, H12), 1.64 (m, 2H, H10), 1.77 (qnt, J = 7.0 Hz, 2H, H13), 2.08 (283 mg, 0.575 mmol, 100%) was obtained as a pure product

View Article Online

DOI: 10.1039/C9OB00826H

(s, 3H, C(O)CH₃), 2.09 (s, 3H, C(O)CH₃), 2.10 (s, 3H, C(O)CH₃), 2.11 (s, without purification.

[α]²_D⁰= +23 (c 1,1, CHCl₃), IR (neat) 2934, 2858, 1749, 1455, 1370,

1219, 1035, 644, 602 cm^-1, ¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.34-

1.41 (m, 4H, H11 and H12), 1.54-1.65 (m, 4H, H10 and H13), 2.06 (s,

3H, C(O)CH₃), 2.07 (s, 3H, C(O)CH₃), 2.08 (s, 3H, C(O)CH₃), 2.09 (s,

3H, C(O)CH₃), 2.55 (t, J = 7.8 Hz, 2H, H9), 3.23 (t, J = 6.9 Hz, 2H,

H14), 4.31 (dd, J = 10.9, 4.3 Hz, 1H, H6^a), 4.35-4.44 (m, 2H, H5 and

H6^b), 5.38 (dd, J = 7.2, 5.4 Hz, 1H, H4), 5.48 (dd, J = 7.2, 5.4 Hz, 1H,

H3), 5.98 (d, J = 5.2 Hz, 1H, H2), 6.71 (s, 1H, H7), ¹³C NMR (125 MHz,

CDCl₃) δ (ppm) 20.70, 20.75, 20.8, 21.0 (4x C(O)CH₃), 26.6 (C12),

28.5 (C9), 28.8 (C13), 29.0 (C10), 29.2 (C11), 51.5 (C14), 56.3 (C5),

62.2 (C6), 66.2 (C2), 66.4 (C4), 70.3 (C3), 113.7 (C7), 139.2 (C1),

144.9 (C8), 169.2, 169.4, 169.8, 170.4 (4x C(O)CH₃), HRMS (ESI) m/z

494.2242 ([M + H]⁺calcd. For C₂₂H₃₂N₅O₈: 494.2245).

3H, C(O)CH₃), 2.57 (t, J = 7.8 Hz, 2H, H9), 3.52 (t, J = 6.7 Hz, 2H,

H14), 4.30-4.46 (m, 3H, H6, H5), 5.39 (dd, J = 7.1, 5.3 Hz, 1H, H4),

5.50 (dd, J = 7.2, 5.3 Hz, 1H, H3), 6.0 (d, J = 5.2 Hz, 1H, H2), 6.72 (s,

1H, H7), ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 20.7, 20.8, 20.8, 21.1 (4

C(O)CH₃), 26.8 (C12), 28.5 (C9), 28.7 (C11), 29.2 (C10), 32.6 (C13),

45.2 (C14), 56.3 (C5), 62.2 (C6), 66.3 (C2), 66.5 (C4), 70.3 (C3), 113.5

(C7), 139.2 (C1), 144.9 (C8), 169.2, 169.5, 169.8, 170.4 (4 C(O)CH₃),

HRMS (ESI) m/z 487.1838 ([M + H]⁺calcd. For C₂₂H₃₂ClN₂O₈:

487.1842).

Compound 10b

Pd(OH)₂/C (454%, 667 mg, 4.75 mmol) was placed in a round

bottom flask. Three cycles vacuum/argon were done, followed by a

vacuum/H₂cycle. A solution of 9b (1 eq., 706 mg, 1.05 mmol) in a

3:1:1 EtOAc/MeOH/H₂O mixture (31.3 mL) and AcOH (19 mL) were

added on the activated catalyst, then degassed with H₂and the

reaction mixture was stirred under H₂for 3 days. The reaction was

filtered over a pad of silica gel with 84:16 DCM/MeOH (250mL). The

filtrate was evaporated to dryness and the crude was used for the

next step without purification.

Ac₂O (13.8 mL) and dry pyridine (13.5 mL) were added to the crude.

And the solution was stirred for 22 h under argon atmosphere.

Water was slowly added at 0 °C. The aqueous phase was extracted

with DCM (x3). The combined organic layers were then washed with

2M HCl (2 x 4 mL), sat. NaHCO₃solution, water and dried over

Na₂SO₄, filtered and concentrated. Crude residue was purified by

flash column chromatography on silica gel (6:4 EtOAc/pentane).

Product 10b (421 mg, 0.865 mmol, 83%) was obtained as an oil.

R_f0.3 (EtOAc/petroleum ether 6:4), [α]²_D⁰= -39 (c 0.7, CHCl₃), IR

(neat) 2932, 2857, 1749, 1455, 1370, 1221, 1054, 950 cm^-1, ¹H NMR

(500 MHz, CDCl₃) δ (ppm) 1.36 (qnt, J = 7.4 Hz, 2H, H11), 1.45 (qnt, J

= 7.4 Hz, 2H, H12), 1.59-1.66 (m, 2H, H10), 1.76 (qnt, J = 7.1 Hz, 2H,

H13), 2.03 (s, 3H, C(O)CH₃), 2.10 (s, 3H, C(O)CH₃), 2.11 (s, 6H,

C(O)CH₃), 2.55 (td, J = 7.8, 2.4 Hz, 2H, H9), 3.51 (t, J = 6.7 Hz, 2H,

H14), 4.24-4.33 (m, 2H, H5 and H6a), 4.53 (dd, J = 11.7, 4.0 Hz, 1H,

H6b), 5.41 (dd, J = 8.9, 3.8 Hz, 1H, H3), 5.63 (dd, J = 8.9, 6.1 Hz, 1H,

H4), 6.33 (d, J = 3.8 Hz, 1H, H2), 6.76 (s, 1H, H7), ¹³C NMR (125 MHz,

CDCl₃) δ (ppm) 20.7, 20.78, 20.81, 20.9 (4 x C(O)CH₃), 26.7 (C12),

28.4 (C9), 28.7 (C11), 29.2 (C10), 32.6 (C13), 45.2 (C14), 57.3 (C5),

63.4 (C6), 63.6 (C2), 65.8 (C4), 68.9 (C3), 114.4 (C7), 139.1 (C1),

144.9 (C8), 169.6, 169.66, 169.67, 170.3 (4 x C(O)CH₃), HRMS (ESI)

m/z 487.1845 ([M + H]⁺calcd. For C₂₂H₃₂ClN₂O₈: 487.1842).

Compound 11b

Substrate 10b (1 eq., 421 mg, 0.865 mmol) was dissolved in DMF (5

mL), TBAI (0.198 eq., 63.4 mg, 0.172 mmol) and NaN₃(5.88 eq., 334

mg, 5.09 mmol) were added. Reaction mixture was heated at 60°C

for 18h. Reaction was diluted with EtOAc and water. The aqueous

phase was extracted with EtOAc (x3). The gathered organic phase

was washed with water (x5) and then brine. Organic phase was

then dried over Na₂SO₄, filtered and evaporated under reduced

pressure. Compound 11b (427 mg, 0.865 mmol, 100%) was

obtained as a pure product without purification.

[α]²_D⁰= -41 (c 1.1, CHCl₃), IR (neat) 2935, 2859, 2097, 1749, 1456,

1370, 1221, 1054, 950 cm^-1, ¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.33-

1.43 (m, 4H, H11 and H12), 1.56-1.67 (m, 4H, H10 and H13), 2.04 (s,

3H, C(O)CH₃), 2.107 (s, 3H, C(O)CH₃), 2.114 (s, 3H, C(O)CH₃), 2.116

(s, 3H, C(O)CH₃), 2.56 (td, J = 7.8, 2.6 Hz, 2H, H9), 3.25 (t, J = 6.9 Hz,

2H, H14), 4.25-4.34 (m, 2H, H5 and H6^a), 4.54 (dd, J = 11.8, 4.1 Hz,

1H, H6^b), 5.42 (dd, J = 8.9, 3.7 Hz, 1H, H3), 5.64 (dd, J = 8.9, 6.1 Hz,

1H, H4), 6.34 (d, J = 3.7 Hz, 1H, H2), 6.76 (s, 1H, H7), ¹³C NMR (125

MHz, CDCl₃) δ (ppm) 20.7, 20.81, 20.85, 21.0 (4x C(O)CH₃), 26.6

(C12), 28.5 (C9), 28.8 (C13), 29.0 (C10), 29.2 (C11), 51.5 (C14), 57.3

(C5), 63.4 (C6), 63.6 (C2), 65.8 (C4), 69.0 (C3), 114.4 (C7), 139.2 (C1),

145.0 (C8), 169.6, 169.7, 170.3 (4x C(O)CH₃), HRMS (ESI) m/z

494.2250 ([M + H]⁺calcd. For C₂₂H₃₂N₅O₈: 494.2245).

Peracetylated monovalent 12a

To a solution of substrate 11a (1 eq., 91.4 mg, 0.185 mmol) and

cyclopropylacetylene (5 eq., 0.081 mL, 0.926 mmol) in DMF (4.92

mL) was added a solution of CuSO₄.5H₂O (6.6 mg, 0.0264 mmol) and

sodium ascorbate (10.4 mg, 0.0525 mmol) in H₂O (1.3 mL). The

resulting solution was heated under microwave irradiation

conditions at 80 °C for 30 min. Water was added, and the aqueous

phase was extracted with EtOAc (3x). The organic phases were

combined, washed with water (3x) and brine, dried over Na₂SO₄,

filtered and concentrated under reduced pressure. Traces of copper

salts were removed by filtration through a short pad of silica gel

eluting with CH₃CN/H₂O/NH₄OH (10:1:1). Crude residue was

purified by flash column chromatography on silica gel (100 EtOAc).

Product 12a (88.4 mg, 0.158 mmol, 85%) was obtained as an oil.

Compound 11a

Substrate 10a (1 eq., 276 mg, 0.567 mmol) was dissolved in DMF

(3.2 mL). TBAI (0.201 eq., 42.1 mg, 0.114 mmol) and NaN₃(6.08 eq.,

224 mg, 3.45 mmol) were added. Reaction mixture was heated at

60°C for 20 h. The mixture was diluted with EtOAc and water. The

aqueous phase was extracted with EtOAc (x3). Gathered organic

phases were washed with water (x5) and brine, dried over Na₂SO₄,

filtered and concentrated under reduced pressure. Compound 11a

10 | J. Name., 2012, 00, 1-3

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Organic & Biomolecular Chemistry

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ARTICLE

R_f0.3 (DCM/MeOH 97:3), [α]_D²⁰= +19 (c 0.9, CHCl₃), IR (neat) 2931,

2857, 1749, 1370, 1219, 1040 cm^-1, ¹H NMR (500 MHz, CDCl₃) δ

concentrated under reduced pressure. Product 13a (15,6 mg, 0.040

DOI: 10.1039/C9OB00826H

mmol, 97%) was obtained pure without purification.

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(ppm) 0.86 (dd, J =11.5, 4.5 Hz, 2H, H18), 0.95-1.00 (m, 2H, H18), [α]²_D⁰= -13 (c 0.4, MeOH), IR (neat) 3338, 3142, 3096, 3004, 2928,

1.35-1.47 (m, 4H, H11 and H12), 1.61-1.69 (m, 2H, H10), 1.91 (qnt, J 2857, 1563, 1459, 1106, 1023, 666 cm^-1, ¹H NMR (400 MHz, CD₃OD)

= 7.3 Hz, 2H, H13), 1.98 (m, 1H, H17), 2.12 (s, 3H, C(O)CH₃), 2.131 δ (ppm) 0.72-0.79 (m, 2H, H18), 0.92-0.98 (m, 2H, H18), 1.26-1.43

(s, 3H, C(O)CH₃), 2.135 (s, 3H, C(O)CH₃), 2.15 (s, 3H, C(O)CH₃), 2.59 (m, 4H, H11 and H12), 1.61 (qnt, J = 7.4 Hz, 2H, H10), 1.87 (qnt, J =

(t, J = 7.9 Hz, 2H, H9), 4.31 (t, J = 7.2 Hz, 2H, H14), 4.38 (dd, J = 11.3, 7.3 Hz, 2H, H13), 1.92-1.99 (m, 1H, H17), 2.50 (t, J = 7.5 Hz, 2H,

4.3 Hz, 1H H6^a), 4.43-4.51 (m, 2H, H5 and H6^b), 5.44 (dd, J = 7.2, 5.4 H9), 3.67 (t, J = 8.3 Hz, 1H, H3), 3.80 (dd, J = 17.1, 8.3 Hz, 1H, H4),

Hz, 1H, H4), 5.54 (dd, J = 7.3, 5.3 Hz, 1H, H3), 6.04 (d, J = 5.2 Hz, 1H, 3.84 (dd, J = 8.4, 2.9 Hz, 1H, H5), 3.93 (dd, J = 11.9, 3.9 Hz, 1H, H6^a),

H2), 6.78 (s, 1H, H7), 7.34 (s, 1H, H15), ¹³C NMR (125 MHz, CDCl₃) δ 4.16 (dd, J = 12.0, 2.2 Hz, 1H, H6^b), 4.31 (t, J = 7.1 Hz, 2H, H14), 4.46

(ppm) 6.7 (C17), 7.7 (C18 and C18’), 20.6, 20.67, 20.73, 21.0 (4x (d, J = 7.9 Hz, 1H, H2), 6.98 (s, 1H, H7), 7.66 (s, 1H, H15), ¹³C NMR

C(O)CH₃), 26.3 (C12), 28.3 (C9), 28.7 (C11), 29.0 (C10), 30.2 (C13), (100 MHz, CD₃OD) δ (ppm) 7.3 (C17), 8.2 (C18 and C18’), 27.2 (C12),

50.1 (C14), 56.3 (C5), 62.1 (C6), 66.1 (C2), 66.3 (C4), 70.2 (C3), 113.8 28.9 (C9), 29.6 (C11), 30.2 (C10), 31.1 (C13), 51.2 (C14), 61.5 (C6),

(C7), 119.5 (C15), 139.1(C1), 144.5 (C8), 150.2 (C16), 169.1, 169.3, 62.6 (C5), 69.3 (C4), 69.8 (C2), 76.7 (C3), 114.5 (C7), 121.8 (C15),

169.7, 170.3 (4x C(O)CH₃), HRMS (ESI) m/z 560.2720 ([M + H]⁺calcd. 143.5 (C16), 146.9 (C1), 151.4 (C8), HRMS (ESI) m/z 392.2290 ([M +

For C₂₇H₃₈N₅O₈: 560.2715).

H]⁺calcd. For C₁₉H₃₀N₅O₄: 392.2292).

Peracetylated monovalent 12b

Monovalent 13b

To a solution of substrate 11b (1 eq., 40.7 mg, 0.0825 mmol) and Substrate 12b (1 eq., 29.5 mg, 0.0527 mmol) was dissolved in

cyclopropylacetylene (5 eq., 0.036 mL, 0.412 mmol) in DMF (2.2 mL) MeOH (5.9 mL) and freshly prepared Amberlite IRA-400 (OH^-) (1 g)

was added a solution of CuSO₄.5H₂O (4 mg, 0.016 mmol) and was added. The suspension was smoothly stirred for 18 h. The resin

sodium ascorbate (6.5 mg, 0.0328 mmol) in H₂O (0.6 mL). The was filtered and rinsed with MeOH and H₂O. The filtrate was

resulting solution was heated under microwave irradiation concentrated under reduced pressure. Product 13b (17.9 mg, 0.046

conditions at 80 °C for 30 min. Water was added, and the aqueous mmol, 87%) was obtained pure without purification as a solid.

[α]²_D⁰= -18 (c 0.3, MeOH), m.p. 93 °C IR (neat) 3335, 3142, 3090,

phase was extracted with EtOAc (3x). The organic phases were

combined, washed with water (3x) and brine, dried over Na₂SO₄,

filtered and concentrated under reduced pressure. Traces of copper

salts were removed by filtration through a short pad of silica gel

eluting with CH₃CN/H₂O/NH₄OH (10:1:1). Crude residue was

purified by flash column chromatography on silica gel (97:3

DCM/MeOH to 9:1). Product 12b (30 mg, 0.054 mmol, 65%) was

obtained as a beige oil.

1

2926, 2855, 1685, 1563, 1465, 1097, 901, 825 cm^-1, H NMR (400

MHz, CD₃OD) δ (ppm) 0.72-0.79 (m, 2H, H18), 0.92-0.98 (m, 2H,

H18), 1.26-1.44 (m, 4H, H11, H12), 1.61 (qnt, J = 7.3 Hz, 2H, H10),

1.87 (qnt, J = 7,3 Hz, 2H, H13), 1.95 (m, 1H, H17), 2.5 (t, J = 7.6 Hz,

2H, H9), 3.76 (dd, J = 9.4, 3.7 Hz, 1H, H3), 3.80 (ddd, J = 7.9, 5.4, 2.7

Hz, 1H, H5), 3.89 (dd, J = 11.9, 5.5 Hz, 1H, H6^a), 4.09 (dd, J = 9.4, 7.8

Hz, 1H, H4), 4.16 (dd, J = 11.7, 2.8 Hz, 1H, H6^b), 4.31 (t, J = 7.1 Hz,

R_f0.3 (DCM/MeOH 97:3), [α]²_D⁰= -34 (c 0.7, CHCl₃), IR (neat) 2934, 2H, H14), 4.78 (d, J = 3.7 Hz, 1H, H2), 7.04 (s, 1H, H7), 7.66 (s, 1H,

1

2857, 1749, 1460, 1433, 1370, 1221, 1052, 949 cm^-1, H NMR (500 H15), ¹³C NMR (100 MHz, CD₃OD) δ (ppm) 7.3 (C17), 8.2 (C18, C18’),

MHz, CDCl₃) δ (ppm) 0.78-0.83 (m, 2H, H18), 0.89-0.95 (m, 2H, H18), 27.2 (C12), 28.8 (C9), 29.6 (C11), 30.3 (C10), 31.1 (C13), 51.2 (C14),

1.29-1.41 (m, 4H, H11 and H12), 1.6 (qnt, J = 7.2 Hz, 2H, H10), 1.86 63.0 (C6), 63.5 (C5), 65.7 (C2), 67.1 (C4), 73.2 (C3), 115.5 (C7), 121.8

(qnt, J = 7.1 Hz, 2H, H13), 1.90-1.95 (m, 1H, H17), 2.03 (s, 3H, (C15), 143.5 (C8), 145.9 (C1), 151.4 (C16), HRMS (ESI) m/z 392.2291

C(O)CH₃), 2.10 (s, 3H, C(O)CH₃), 2.11 (s, 6H, C(O)CH₃), 2.53 (td, J = ([M + H]⁺calcd. For C₁₉H₃₀N₅O₄: 392.2292).

7.7, 3.4 Hz, 2H, H9), 4.22-4.33 (m, 4H, H14, H5, H6^a), 4.54 (dd, J =

11.4, 3.6 Hz, 1H, H6^b), 5.41 (dd, J = 9.0, 3.6 Hz, 1H, H3), 5.63 (dd, J = Compounds 16a and 16b

9.0, 6.0 Hz, 1H, H4), 6.33 (d, J = 3.7 Hz, 1H, H2), 6.75 (s, 1H, H7), A solution of 15 (1 eq., 3.2 g, 12.8 mmol) in dry THF was cooled to 0

7.19 (s, 1H, H15), ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 6.8 (C17), 7.8 °C under argon atmosphere. NaH (4.0 eq., 2.05 g, 51.3 mmol) was

(C18, C18’), 20.7, 20.81, 20.82, 21.0 (4x C(O)CH₃), 26.4 (C11 ou C12), added portion-wise and after stirring for 30 min, propargyl bromide

28.4 (C9), 28.8 (C11 ou C12), 29.1 (C10), 30.3 (C13), 50.2 (C14), 57.3 (6.04 eq., 8.6 mL, 77.2 mmol) was added dropwise over 45 min. The

(C5), 63.4 (C6), 63.6 (C2), 65.8 (C4), 69.0 (C3), 114.4 (C7), 119.6 reaction was allowed to warm to room temperature and was stirred

(C15), 139.2 (C1), 144.8 (C8), 150.3 (C16), 169.6, 169.7, 170.3 (4x for 16 h. After quenching the excess of NaH with NH₄Cl, the solvent

C(O)CH₃), HRMS (ESI) m/z 280.6399 ([M

C₂₇H₃₉N₅O₈: 280.6394).

+

2H]⁺calcd. For was evaporated under reduced pressure. The residue was diluted

with Et₂O and washed with water. The aqueous phase was

extracted with Et₂O (2x). The combined organic layers were washed

with water and brine, dried over Na₂SO₄, and concentrated under

Monovalent 13a

Substrate 12a (1 eq., 23 mg, 0.0411 mmol) was dissolved in MeOH reduced pressure. The crude residue was purified by flash column

(4.6 mL) and freshly prepared Amberlite IRA-400 (OH^-) (1000 mg) chromatography on silica gel (100:0 to 8:2 petroleum ether/EtOAc)

was added. The suspension was smoothly stirred for 14 h. The resin to give 16a (2,92 g, 8.94 mmol, 70%) as a yellow oil and 16b (0.9 g,

was filtered and rinsed with MeOH and H₂O. The filtrate was

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J. Name., 2013, 00, 1-3 | 11

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Page 12 of 18

ARTICLE

Journal Name

2.47 mmol, 19%) as a yellow oil. Analytical data of 16b were in was stirred and heated under microwave irradiation at 80°C for 30-

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accordance with those reported in the literature.²⁶

40 min. Water was added, and the aqueous phase was extracted

DOI: 10.1039/C9OB00826H

16a: R_f0.35 (DCM/MeOH 9:1), IR (neat) 3372 cm^-1, H NMR (400 with EtOAc (3x). The organic phases were combined, washed with

MHz, CDCl₃) δ (ppm) 0.05 (s, 6H, CH₃), 0.89 (s, 9H, t-Bu), 2.41 (t, J = water (3x) and brine, dried over Na₂SO₄, filtered and concentrated

2.4 Hz, 2H, C-H), 2.66 (t, J =5.9 Hz, 1H, OH), 3.54 (m, 4H, CH₂-O), under reduced pressure. Traces of copper salts were removed by

3.64 (s, 2H, CH₂-OSi), 3.70 (d, J =5.9 Hz, 2H, CH₂-OH), 4.12 (d, J = 2.4 filtration through a short pad of silica gel eluting with a 10:1:1

Hz, 4H, CH₂), ¹³C NMR (100 MHz, CDCl₃) δ (ppm) -5.6, 18.3, 25.9, CH₃CN/H₂O/NH₄OH solution. The crude residue was purified by

45.2, 58.8, 63.5, 65.5, 70.1, 74.5, 79.8, HRMS (ESI) m/z 349.180 ([M flash column chromatography on silica gel (97:3 to 9:1

1

+ Na]⁺calcd. For C₁₇H₃₀O₄SiNa: 349.181).

DCM/MeOH).

1

16b: H NMR (400 MHz, CDCl₃) δ (ppm) 0.03 (s, 6H, CH₃), 0.88 (s,

9H, t-Bu), 2.38 (t, J = 2.4 Hz, 3H, C-H), 3.49 (s, 6H, CH₂), 3.56 (s, 2H, Peracetylated divalent cluster 18a

CH₂-OSi), 4.11 (d, J = 2.3 Hz, 6H, CH₂).

Product 18a (67.8 mg, 0.0565 mmol, 59%) was obtained following

general procedure A starting from 11a (111 mg, 0.225 mmol) and

17a (20.5 mg, 0.0966 mmol).

Compound 17a³³

R_f0.25 (DCM/MeOH 95:5), [α]²_D⁰= +19 (c 0.8, CHCl₃), m.p. 67-70 °C

To a solution of 16a (1 eq., 197 mg, 0.603 mmol) in dry THF (10 mL)

at 0 °C was added dropwise a 1 M solution of TBAF (5 eq., 3.02 mL,

3.02 mmol) in THF over a period of 20 min. The reaction mixture

was allowed to warm to room temperature and stirred for 5 h

under argon atmosphere. The solvent was evaporated under

reduced pressure. The residue was diluted with EtOAc and washed

with water (2x) and brine. The organic layers were dried over

Na₂SO₄, and concentrated under reduced pressure. The crude

residue was purified by flash column chromatography on silica gel

(55:45 EtOAc/pentane) to afford 17a (122 mg, 0.579 mmol, 96%) as

a colorless oil.

1

IR (neat) 2932, 2860, 1750, 1462, 1371, 1222, 1048 cm^-1, H NMR

(500 MHz, CDCl₃) δ (ppm) 1.26-1.42 (m, 8H, H11 and H12), 1.54-

1.64 (m, 4H, H10), 1.85-1.94 (m, 4H, H13), 2.02-2.14 (m, 24H,

C(O)CH₃), 2.51 (t, J = 7.6 Hz, 4H, H9), 3.54 (s, 4H, H18), 3.62 (s, 4H,

H18’), 4.28-4.35 (m, 6H, H14 and H6^a), 4.60 (s, 4H, H17), 5.35-5.40

(m, 2H, H4), 5.44-5.50 (m, 2H, H3), 5.98 (d, J = 5.0 Hz, 2H, H2), 6.71

(s, 2H, H7), 7.52 (s, 2H, H15), ¹³C NMR (125 MHz, CDCl₃) δ (ppm)

20.7, 20.73, 20.8, 21.0 (8x C(O)CH₃), 26.2 (C12), 28.3 (C9), 28.7

(C11), 29.0 (C10), 30.2 (C13), 45.2 (C19), 50.3 (C14), 56.3 (C5), 62.1

(C6), 64.4 (C18’), 65.0 (C17), 66.1 (C2), 66.4 (C4), 70.3 (C3), 71.0

(C18), 113.8 (C7), 122.3 (C15), 139.2 (C1), 144.6 (C8), 145.1 (C16),

169.2, 169.4, 169.8, 170.4 (8x C(O)CH₃), HRMS (ESI) m/z 1199.5470

([M + H]⁺calcd. For C₅₅H₇₉N₁₀O₂₀: 1199.5467).

IR (neat) 3373 cm^-1, H NMR (400 MHz, CDCl₃) δ (ppm) 2.34 (br s,

1

2H, OH), 2.45 (t, J = 2.4 Hz, 2H, H5), 3.60 (s, 4H, H2’), 3.68 (s, 4H,

H2), 4.15 (d, J = 2.4 Hz, 4H, H3). ¹³C-NMR (75.5 MHz, CDCl₃) δ (ppm)

45.0, 58.9, 64.3, 71.0, 74.9, 79.6, HRMS (ESI) m/z 235.0969 ([M +

Na]⁺calcd. For C₁₁H₁₆O₄Na: 235.0941).

Peracetylated divalent cluster 18b

Product 18b (57.1 mg, 0.0476 mmol, 64%) was obtained following

general procedure A starting from 11b (82.2 mg, 0.167 mmol) and

17a (15.9 mg, 0.0749 mmol).

Compound 17b

To a solution of 16b (1 eq., 54.3 mg, 0.149 mmol) in dry THF (2.9

mL) at 0 °C was added dropwise a solution of TBAF (5 eq., 1 M,

0.745 mL, 0.745 mmol) in THF over a period of 30 min. Solution

turned orange directly after TBAF addition. The reaction mixture

was allowed to warm to room temperature and was stirred for 5 h

under argon atmosphere. The solvent was evaporated under

reduced pressure. The residue was diluted with EtOAc and washed

with water (2x) and brine. The organic layers were dried over

Na₂SO₄and concentrated under reduced pressure. The crude

residue was purified by flash column chromatography on silica gel

(8:2 to 7:3 petroleum ether/EtOAc) to afford 17b (27 mg, 0.11

mmol, 72%) as a colorless oil. Analytical data were in accordance

with those reported in literature.²³

R_f0.2 (DCM/MeOH 95:5), [α]²_D⁰= -35 (c 0.8, CHCl₃), m.p. 60 °C IR

(neat) 2928, 2860, 1749, 1461, 1370, 1222, 1052, 950 cm^-1, ¹H NMR

(400 MHz, CDCl₃) δ (ppm) 1.25-1.40 (m, 8H, H11 and H12), 1.60

(qnt, J = 7.3 Hz, 4H, H10), 1.90 (qnt, J = 7.2 Hz, 4H, H13), 2.01 (s, 6H,

C(O)CH₃), 2.08 (s, 6H, C(O)CH₃), 2.088 (s, 6H, C(O)CH₃), 2.09 (s, 6H,

C(O)CH₃), 2.51 (td, J = 7.6, 2.6 Hz, 4H, H9), 3.53 (s, 4H, H18), 3.61 (s,

4H, H18’), 4.25-4.35 (m, 8H, H14, H5 and H6^a), 4.50-4.57 (m, 2H,

H6^b), 4.59 (s, 4H, H17), 5.41 (dd, J = 9.0, 3.7 Hz, 2H, H3), 5.64 (dd, J =

9.0, 6.1 Hz, 2H, H4), 6.33 (d, J = 3.7 Hz, 2H, H2), 6.75 (s, 2H, H7),

7.52 (s, 2H, H15), ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 20.6, 20.7,

20.8, 20.9 (8x C(O)CH₃), 26.2 (C12), 28.2 (C9), 28.6 (C11), 29.0 (C10),

30.1 (C13), 45.2 (C19), 50.3 (C14), 57.2 (C5), 63.2 (C6), 63.5 (C2),

64.2 (C18’), 64.9 (C17), 65.7 (C4), 68.9 (C3), 70.9 (C18), 114.4 (C7),

122.2 (C15), 139.1 (C1), 144.6 (C8), 145.0 (C16), 169.5, 169.62,

169.65, 170.3 (8x C(O)CH₃), HRMS (ESI) m/z 1221.5290 ([M + Na]⁺

calcd. For C₅₅H₇₈N₁₀NaO₂₀: 1221.5286).

¹H NMR (400 MHz, CDCl₃) δ (ppm) 2.42 (t, J = 2.4 Hz, 3H), 3.56 (s,

6H), 3.69 (s, 2H), 4.13 (d, J = 2.4 Hz, 6H).

General procedure A

Azide substrate 11 (1.1 eq by alkyne) and platform 17 or 24 (1 eq.)

were dissolved in DMF (20 mL/mmol of alkyne) in a microwave vial.

Solution was stirred till the solution was homogeneous. A solution

of sodium ascorbate (0.2 eq. by alkyne) and CuSO₄.5H₂O (0.1 eq. by

alkyne) in H₂O (0.25 mL per mL of DMF) was added. The mixture

Peracetylated trivalent cluster 20a

Product 20a (71.4 mg, 0.0413 mmol, 85%) was obtained following

general procedure A starting from 11a (81 mg, 0.164 mmol) and

17b (12.1 mg, 0.0483 mmol).

12 | J. Name., 2012, 00, 1-3

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Organic & Biomolecular Chemistry

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Journal Name

ARTICLE

R_f0.45 (DCM/MeOH 94:6), [α]²_D⁰= +21 (c 0.5, CHCl₃), m.p. 65-67 °C

(C3), 113.8 (C7), 122.5 (C15), 139.1 (C1), 144.6 (C8 )_V,_ie1_w4_A5_r._ti3_cle(C_O1_nl6_in)_e,

1

IR (neat) 2930, 2859, 1749, 1461, 1370, 1220, 1049, 756 cm^-1, H

DOI: 10.1039/C9OB00826H

169.1, 169.4, 169.7, 170.3 (16x C(O)CH₃), HRMS (ESI) m/z 754.6768

([M + 3H]³⁺calcd. For C₁₀₅H₁₄₇N₂₀O₃₆: 754.6757).

NMR (500 MHz, CDCl₃) δ (ppm) 1.29-1.43 (m, 12H, H11 and H12),

1.56-1.65 (m, 6H, H10), 1.89 (qnt, J = 7.3 Hz, 6H, H13), 2.06 (s, 9H,

C(O)CH₃), 2.075 (s, 9H, C(O)CH₃), 2.077 (s, 9H, C(O)CH₃), 2.10 (s, 9H,

C(O)CH₃), 2.53 (t, J = 7.7 Hz, 6H, H9), 3.50 (s, 6H, H18), 3.61 (s, 2H,

H18’), 4.29-4.35 (m, 9H, H14 and H6^a), 4.37-4.45 (m, 6H, H5 and

H6^b), 4.56 (s, 6H, H17), 5.38 (dd, J = 7.2, 5.6 Hz, 3H, H4), 5.48 (dd, J =

Peracetylated tetravalent cluster 22b

Product 22b (70.0 mg, 0.0309 mmol, 76%) was obtained as a solid

following general procedure A starting from 11b (91.1 mg, 0.180

mmol) and 17c (11.8 mg, 0.0409 mmol).

7.3, 5.3 Hz, 3H, H3), 5.99 (d, J = 5.3 Hz, 3H, H2), 6.77 (s, 3H, H7), R_f0.3 (DCM/MeOH 94:6), [α]²_D⁰= -31 (c 0.8, CHCl₃), m.p. 75 °C IR

7.53 (s, 3H, H15), ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 20.7, 20.75, (neat) 2931, 2859, 1748, 1460, 1370, 1221, 1051, 950, 755 cm^-1, H

1

20.8, 21.1 (12x C(O)CH₃), 26.3 (C12), 28.4 (C9), 28.8 (C11), 29.1 NMR (500 MHz, CDCl₃) δ (ppm) 1.28-1.42 (m, 16H, H11 and H12),

(C10), 30.3 (C13), 45.3 (C19), 50.4 (C14), 56.3 (C5), 62.2 (C6), 64.5 1.60 (qnt, J = 7.2 Hz, 8H, H10), 1.89 (qnt, J = 6.9 Hz, 8H, H13), 2.02

(C18’), 65.1 (C17), 66.2 (C2), 66.4 (C4), 70.3 (C3), 70.4 (C18), 113.8 (s, 12H, C(O)CH₃), 2.09 (s, 12H, C(O)CH₃), 2.096 (s, 12H, C(O)CH₃),

(C7), 122.4 (C15), 139.2 (C1), 144.6 (C8), 145.2 (C16), 169.2, 169.4, 2.10 (s, 12H, C(O)CH₃), 2.51 (td, J = 7.6, 3.2 Hz, 8H, H9), 3.44 (s, 8H,

169.8, 170.4 (12x C(O)CH₃), HRMS (ESI) m/z 865.8943 ([M + 2H]²⁺H18), 4.25-4.34 (m, 16H, H5, H14 and H6^a), 4.51-4.57 (m, 12H, H17

calcd. For C₈₀H₁₁₃N₁₅O₂₈: 865.8934).

and H6^b), 5.39 (dd, J = 9.2, 3.7 Hz, 4H, H3), 5.63 (dd, J = 9.1, 6.2 Hz,

4H, H4), 6.32 (s, J = 3.7 Hz, 4H, H2), 6.76 (s, 4H, H7), 7.54 (s, 4H,

H15), ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 20.7, 20.8, 20.9 (16x

Peracetylated trivalent cluster 20b

Product 20b (38.0 mg, 0.0219 mmol, 47%) was obtained as a white C(O)CH₃), 26.4 (C12), 28.4 (C9), 28.8 (C11), 29.2 (C10), 30.4 (C13),

solid following general procedure A starting from 11b (79.9 mg, 45.3 (C19), 50.3 (C14), 57.2 (C5), 63.3 (C6), 63.6 (C2), 65.2 (C17),

0.157 mmol) and 17b (11.8 mg, 0.0471 mmol).

65.7 (C4), 69.0 (C3), 69.2 (C18), 144.4 (C7), 122.5 (C15), 139.1 (C1),

144.8 (C8), 145.4 (C16), 169.6, 169.7, 170.3 (16x C(O)CH₃), HRMS

(ESI) m/z 754.6767 ([M + 3H]³⁺calcd. For C₁₀₅H₁₄₇N₂₀O₃₆: 754.6757).

R_f0.4 (DCM/MeOH 94:6), [α]_D²⁰= -39 (c 1.8, CHCl₃), m.p. 83-85 °C IR

(neat) 2934, 2860, 1747, 1461, 1370, 1220, 1051, 950, 753 cm^-1, H

1

NMR (500 MHz, CDCl₃) δ (ppm) 1.29-1.41 (m, 12H, H11 and H12),

1.60 (qnt, J = 7.2 Hz, 6H, H10), 1.89 (qnt, J = 7.2 Hz, 6H, H13), 2.02

(s, 9H, C(O)CH₃), 2.09 (s, 9H, C(O)CH₃), 2.098 (s, 9H, C(O)CH₃), 2.10

(s, 9H, C(O)CH₃), 2.51 (td, J = 7.6, 3.1 Hz, 6H, H9), 3.50 (s, 6H, H18),

3.61 (s, 2H, H18’), 4.25-4.34 (m, 12H, H5, H14 and H6^a), 4.51-4.58

(m, 9H, H17 and H6^b), 5.40 (dd, J = 9.1, 3.7 Hz, 3H, H3), 5.64 (dd, J =

9.1, 6.3 Hz, 3H, H4), 6.32 (d, J = 3.7 Hz, 3H, H2), 6.75 (s, 3H, H7),

7.53 (s, 3H, H15), ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 20.7, 20.8,

21.0 (12x C(O)CH₃), 26.3 (C12), 28.4 (C9), 28.8 (C11), 29.1 (C10),

30.3 (C13), 45.2 (C19), 50.3 (C14), 57.2 (C5), 63.3 (C6), 63.6 (C2),

64.5 (C18’), 65.1 (C17), 65.7 (C4), 69.1 (C3), 70.4 (C18), 144.4 (C7),

122.4 (C15), 139.2 (C1), 144.8 (C8), 145.2 (C16), 169.6, 169.7, 170.3

(12x C(O)CH₃), HRMS (ESI) m/z 865.8928 ([M + 2H]²⁺calcd. For

C₈₀H₁₁₃N₁₅O₂₈: 865.8934).

Peracetylated tetradecavalent cluster 25a

Product 25a (22.9 mg, 0.0027 mmol, 57%) was obtained as an oil

following general procedure A starting from 11a (36.8 mg, 0.074

mmol) and 24 (7.8 mg, 0.0047 mmol).

R_f0.65 (DCM/MeOH 9:1), [α]²_D⁰= +27 (c 1.15, MeOH), IR (neat)

3452, 3139, 2929, 2857, 1750, 1462, 1440, 1371, 1223, 1047, 604

cm^-1, ¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.28-1.44 (m, 56H, H11 and

H12), 1.53-1.66 (m, 28H, H10), 1.82-1.95 (m, 28H, H13), 2.03-2.12

(m, 168H, CH₃(O)C), 2.52 (t, J = 7.0 Hz, 28H, H9), 3.34-3.44 (m, 7H,

H23), 3.44-3.50 (m, 7H, H21), 3.58-3.75 (m, 21H, H19 and H18), 3.92

(t, J = 8.9 Hz, 7H, H22), 4.26-4.47 (m, 70H, H5, H6 and H14), 4.53 (m,

14H, H17), 4.76 (br s, 7H, H20), 4.86-5.03 (m, 14 H, H17’), 5.36-5.42

(m, 14H, H4), 5.45-5.51 (m, 14H, H3), 5.99 (d, J = 5.4 Hz, 14H, H2),

6.75 (br s, 14H, H7), 7.66 (s, 7H, H15’), 7.74 (s, 7H, H15), ¹³C NMR

(125 MHz, CDCl₃) δ (ppm) 20.7, 20.78, 20.83, 21.1 (CH₃(O)C), 26.4,

Peracetylated tetravalent cluster 22a

Product 22a (77.2 mg, 0.0341 mmol, 79%) was obtained following 26.5 (C12 and C12’), 28.6 (C9), 28.9, 29.0 (C11 and C11’), 29.2 (C10),

general procedure A starting from 11a (97.2 mg, 0.191 mmol) and 30.5 (C13), 50.3, 50.4 (C14 and C14’), 56.3 (C5), 62.1 (C6), 64.9

17c (12.4 mg, 0.043 mmol).

(C17), 65.2 (C17’), 66.3 (C2), 66.4 (C4), 68.8 (C18), 70.4 (C3), 70.6

(C19), 73.4 (C22), 79.1 (C21), 83.1 (C23), 101.7 (C20), 113.8 (C7),

123.0 (C15’), 123.7 (C15), 139.2 (C1), 144.1 (C16), 144.7 (C8), 169.2,

169.4, 169.8, 170.4 (CH₃(O)C), HRMS (ESI) m/z 1072.4607 ([M +

8H]⁸⁺calcd. For C₃₉₂H₅₄₀N₇₀O₁₄₇: 1072.4611).

R_f0.35 (DCM/MeOH 94:6), [α]²_D⁰= +20 (c 1.0, CHCl₃), m.p. 82-85 °C

IR (neat) 2933, 2859, 1747, 1461, 1370, 1217, 1048, 750 cm^-1, H

1

NMR (500 MHz, CDCl₃) δ (ppm) 1.30-1.41 (m, 16H, H11 and H12),

1.54-1.63 (m, 8H, H10), 1.88 (qnt, J = 7.2 Hz, H13), 2.05 (s, 12H,

C(O)CH₃), 2.06 (s, 24H, C(O)CH₃), 2.08 (s, 12H, C(O)CH₃), 2.52 (t, J =

7.8 Hz, 8H, H9), 3.44 (s, 8H, H18), 4.27-4.34 (m, 12H, H14 and H6^a),

4.36-4.44 (m, 8H, H5 and H6^b), 4.52 (s, 8H, H17), 5.37 (dd, J = 7.2,

5.6 Hz, 4H, H4), 5.47 (dd, J = 7.3, 5.3 Hz, 4H, H3), 5.98 (d, J = 5.3Hz,

4H, H2), 6.72 (s, 4H, H7), 7.54 (s, 4H, H15), ¹³C NMR (125 MHz,

Peracetylated tetradecavalent cluster 25b

Product 25b (29.0 mg, 0.0034 mmol, 55%) was obtained as an oil

following general procedure A starting from 11b (49.2 mg, 0.0987

mmol) and 24 (10.3 mg, 0.0062 mmol).

CDCl₃) δ (ppm) 20.67, 20.71, 20.8, 21.0 (16x C(O)CH₃), 26.4 (C12), R_f0.5 (DCM/MeOH 9:1), [α]²_D⁰= -169 (c 0.5, CHCl₃), IR (neat) 3417,

28.4 (C9), 28.8 (C11), 29.1 (C10), 30.3 (C13), 45.3 (C19), 50.3 (C14), 2930, 2860, 1749, 1457, 1370, 1222, 1051, 949, 755, 600 cm^-1, H

1

56.3 (C5), 62.1 (C6), 65.1 (C17), 66.1 (C2), 66.4 (C4), 69.2 (C18), 70.3 NMR (500 MHz, CDCl₃) δ (ppm) 1.27-1.43 (m, 56 H, H11 and H12),

J. Name., 2013, 00, 1-3 | 13

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Organic & Biomolecular Chemistry

Page 14 of 18

ARTICLE

Journal Name

1.53-1.66 (m, 28H, H10), 1.80-1.95 (m, 28H, H13), 2.00-2.13 (m (t, J = 7.0 Hz, 4H, H14), 4.55 (s, 4H, H17), 4.76 (d, J = 3_V.7_iewH_Az_r,_ti2_clH_e,_OH_nl2_in)_e,

13

DOI: 10.1039/C9OB00826H

168H, CH₃(O)C), 2.45-2.55 (m, 28H, H9), 3.33-3.43 (m, 7H, H21), 7.04 (s, 2H, H7), 7.93 (s, 2H, H15), C NMR (125 MHz, CD₃OD) δ

3.43-3.57 (m, 14H, H19 and H23), 3.60-3.74 (m, 14H, H18), 3.86- (ppm) 27.2 (C12), 28.7 (C9), 29.6 (C11), 30.2 (C10), 31.1 (C13), 46.9

3.96 (m, 7H, H22), 4.23-4.40 (m, 56H, H14, H5 and H6a), 4.46-4.60 (C19), 51.3 (C14), 62.6 (C18’), 62.9 (C6), 63.5 (C5), 65.3 (C17), 65.7

(m, 21H, H6b and H17), 4.76 (s, 7H, H20), 4.83-5.05 (m, 14H, H17’), (C2), 67.1 (C4), 70.4 (C18), 73.2 (C3), 115.6 (C7), 124.8 (C15), 143.3

5.35-5.45 (m, 14H, H3), 5.59-5.68 (m, 14H,H4), 6.32 (s, 14H, H2), (C8), 145.9 (C1), 146.1 (C16), HRMS (ESI) m/z 863.4633 ([M + H]⁺

6.80 (s, 14H, H7), 7.67 (s, 7H, H15’), 7.75 (s, 7H, H15), ¹³C NMR (125 calcd. For C₃₉H₆₃N₁₀O₁₂: 863.4621).

MHz, CDCl₃) δ (ppm) 20.7, 20.8, 20.9 (CH₃(O)C), 26.4 (C12), 28.3

(C9), 28.9 (C11), 29.2 (C10), 30.4 (C13), 50.3, 50.4 (C14 and C14’), Trivalent cluster 21a

57.3 (C5), 63.2 (C6), 63.5 (C2), 64.9, 65.2 (C17 and C17’), 65.7 (C4), Product 21a (13.8 mg, 0.0113 mmol, 100%) was obtained as a white

68.9 (C3), 70.4, 70.6 (C18 and C18’), 73.0 (C22), 75.5 (C19), 79.1 solid following general procedure B, starting from 20a (19.5 mg,

(C21), 83.0 (C23), 101.7 (C20), 114.6 (C7), 123.1 (C15’), 123.8 (C15), 0.0113 mmol).

[α]²_D⁰= -24 (c 1.0, MeOH), m.p. 125 °C, IR (neat) 3325, 3142, 2926,

139.1 (C1), 144.0 (C8), 144.6 (C16), 169.6, 169.7, 170.3 (CH₃(O)C),

HRMS (ESI) m/z 1225.5263 ([M + 7H]⁷⁺calcd. For C₃₉₂H₅₃₉N₇₀O₁₄₇

:

1

2858, 1567, 1461, 1092, 1059, 1024, 778, 649 cm^-1, H NMR (500

1225.5259).

MHz, CD₃OD) δ (ppm) 1.27-1.41 (m, 12H, H11 and H12), 1.60 (qnt, J

= 7.5 Hz, 6H, H10), 1.89 (qnt, J = 7.2 Hz, 6H, H13), 2.49 (t, J = 7.7 Hz,

6H, H9), 3.43 (s, 6H, H18), 3.52 (s, 2H, H18’), 3.67 (dd, J = 8.7, 8.2

Hz, 3H, H3), 3.76-3.85 (m, 6H, H4 and H5), 3.92 (dd, J = 12.0, 3.9Hz,

3H, H6^a), 4.15 (dd, J = 12.1, 2.5 Hz, 3H, H6^b), 4.37 (t, J = 7.0 Hz, 6H,

H14), 4.46 (d, J = 7.9Hz, 3H, H2), 6.97 (s, 3H, H7), 7.92 (s, 3H, H15),

¹³C NMR (125 MHz, CD₃OD) δ (ppm) 27.2 (C12), 28.8 (C9), 29.6

(C11), 30.2 (C10), 31.2 (C13), 46.2 (C19), 51.3 (C14), 61.5 (C6), 62.3

(C18’), 62.6 (C5), 65.3 (C17), 69.3 (C4), 69.8 (C2), 70.1 (C18), 76.7

(C3), 114.5 (C7), 124.9 (C15), 143.5 (C8), 146.1 (C16), 146.9 (C1),

HRMS (ESI) m/z 409.5577 ([M + 3H]³⁺calcd. For C₅₆H₉₀N₁₅O₁₆:

409.5558).

General procedure B

To the peracetylated multivalent cluster (1 eq.) dissolved in MeOH

(55.5 mL/mmol), a 2M ammonia solution in MeOH (110 eq.) was

added. The reaction was stirred for 16-20h under argon atmosphere

and then concentrated. Pure deprotected cluster was obtained

after acetamide removal by co-evaporation with toluene and then

MeOH.

Divalent cluster 19a

Product 19a (11.6 mg, 0.0135 mmol, 100%) was obtained as an oil

following general procedure B, starting from 18a (16.2 mg, 0.0135

mmol).

Trivalent cluster 21b

[α]²_D⁰= -14 (c 0.6, MeOH), IR (neat) 3326, 3142, 2926, 2857, 1664, Product 21b (19.3 mg, 0.0157 mmol, 75%) was obtained as a white

1461, 1094, 1056, 1031, 778, 640 cm^-1, H NMR (500 MHz, CD₃OD) solid following general procedure B, starting from 20b (36.5 mg,

1

δ (ppm) 1.28-1.42 (m, 8H, H11 and H12), 1.61 (qnt, J = 7.4 Hz, 4H, 0.0211 mmol).

[α]²_D⁰= -18 (c 1.3 MeOH), m.p. 82-85 °C, IR (neat) 3325, 3142, 2926,

2857, 1659, 1462, 1088, 1060, 900 cm^-1, ¹H NMR (500 MHz, CD₃OD)

δ (ppm) 1.26-1.43 (m, 12H, H11 and H12), 1.60 (qnt, J = 7.4 Hz, 6H,

H10), 1.89 (qnt, J = 7.3 Hz, 6H, H13), 2.49 (t, J = 7.5 Hz, 6H, H9), 3.43

(s, 6H, H18), 3.52 (s, 2H, H18’), 3.76 (dd, J = 9.4, 3.7 Hz, 3H, H3),

3.79 (ddd, J = 8.0, 5.1, 2.6 Hz, 3H, H5), 3.89 (dd, J = 11.8, 5.4 Hz, 3H,

H6^a), 4.09 (dd, J = 9.4, 8.0 Hz, 3H, H4), 4.16 (dd, J = 11.8, 2.8 Hz, 3H,

H6^b), 4.37 (t, J = 7.1 Hz, 6H, H14), 4.51 (s, 6H, H17), 4.78 (d, J = 3.8

Hz, 3H, H2), 7.03 (s, 3H, H7), 7.92 (s, 3H, H15), ¹³C NMR (125 MHz,

CD₃OD) δ (ppm) 27.2 (C12), 28.8 (C9), 29.6 (C11), 30.3 (C10), 31.2

(C13), 46.7 (C19), 51.3 (C14), 62.3 (C18’), 62.9 (C6), 63.5 (C5), 65.4

(C17), 65.7 (C2), 67.1 (C4), 70.2 (C18), 73.2 (C3), 115.5 (C7), 124.9

(C15), 143.5 (C8), 145.9 (C1), 146.1 (C16), HRMS (ESI) m/z 409.5547

([M + 3H]³⁺calcd. For C₅₆H₉₀N₁₅O₁₆: 409.5558).

H10), 1.90 (qnt, J = 7.3 Hz, 4H, H13), 2.50 (t, J = 7.5 Hz, 4H, H9), 3.45

(s, 4H, H18), 3.54 (s, 4H, H18’), 3.67 (t, J = 8.5 Hz, 2H, H3), 3.79 (t, J =

8.8 Hz, 2H, H4), 3.84 (dt, J = 11.0, 3.0 Hz, 2H, H5), 3.93 (dd, J = 12.1,

4.0 Hz, 2H, H6^a), 4.15 (dd, J = 12.0, 2.3 Hz, 2H, H6^b), 4.38 (t, J = 7.1

Hz, 4H, H14), 4.46 (d, J = 7.9 Hz, 2H, H2), 4.55 (s, 4H, H17), 6.97 (s,

2H, H7), 7.93 (s, 2H, H15), ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 27.2

(C12), 28.8 (C14), 29.6 (C11), 30.2 (C10), 31.1 (C13), 46.9 (C19), 51.3

(C14), 61.4 (C6), 62.6 (C18’), 62.7 (C5), 65.3 (C17), 69.3 (C4), 69.8

(C2), 70.4 (C18), 76.7 (C3), 114.6 (C7), 124.8 (C15), 143.4 (C8), 146.1

(C16), 146.9 (C1), HRMS (ESI) m/z 863.4621 ([M + H]⁺calcd. For

C₃₉H₆₃N₁₀O₁₂: 863.4621).

Divalent cluster 19b

Product 19b (30.1 mg, 0.035 mmol, 100%) was obtained as a beige

solid, following general procedure B, starting from 18b (41.8 mg,

0.035 mmol).

Tetravalent cluster 23a

[α]²_D⁰= -16 (c 1.6, MeOH), m.p. 63-65 °C, IR (neat) 3308, 3142, 2926,

Product 23a (42 mg, 0.0264 mmol, 100%) was obtained as a white

solid following general procedure B, starting from 22a (58.4 mg,

0.0258 mmol).

1

2858, 1658, 1462, 1090, 1059, 901, 773 cm^-1, H NMR (500 MHz,

CD₃OD) δ (ppm) 1.27-1.43 (m, 8H, H11 and H12), 1.61 (qnt, J = 7.6

Hz, 4H, H10), 1.90 (qnt, J = 7.2 Hz, 4H, H13), 2.50 (t, J = 7.6 Hz, 4H,

H9), 3.45 (s, 4H, H18), 3.54 (s, 4H, H18’), 3.77 (dd, J = 9.3, 3.7 Hz,

2H, H3), 3.81 (m, 2H, H5), 3.89 (dd, J = 11.8, 5.4 Hz, 2H, H6^a), 4.09

(dd, J = 9.2, 7.8 Hz, 2H, H4), 4.16 (dd, J = 11.9, 2.8 Hz, 2H, H6^b), 4.38

[α]²_D⁰= -32 (c 0.4, MeOH/H₂O 2:1), m.p. 122-125 °C, IR (neat) 3307,

1

2925, 2857, 1565, 1455, 1083, 1021, 775, 638 cm^-1, H NMR (500

MHz, CD₃OD) δ (ppm) 1.27-1.41 (m, 16H, H11 and H12), 1.59 (qnt, J

= 7.6 Hz, 8H, H10), 1.88 (qnt, J = 7.3 Hz, 8H, H13), 2.48 (t, J = 7.6 Hz,

14 | J. Name., 2012, 00, 1-3

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8H, H9), 3.42 (s, 8H, H18), 3.67 (dd, J = 8.7, 7.9 Hz, 4H, H3), 3.78 (dd, Product 26b (11.4 mg, 0.0018 mmol, 89%) was obtained as an

View Article Online

DOI: 10.1039/C9OB00826H

J = 8.8, 8.5 Hz, 4H, H4), 3.82 (m, 4H, H5), 3.92 (dd, J = 12.1, 3.9 Hz, orange solid following general procedure B, starting from 25b (17.7

4H, H6^a), 4.15 (dd, J = 12.0, 2.3 Hz, 4H, H6^b), 4.36 (t, J = 7.1 Hz, 8H, mg, 0.0021 mmol).

H14), 4.46 (d, J = 7.9 Hz, 4H, H2), 4.48 (s, 8H, H17), 6.97 (s, 4H, H7), m.p. 170 °C (decomp), IR (neat) 3342, 2927, 2857, 1735, 1460,

7.90 (s,4H, H15), ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 27.2 (C12), 1370, 1225, 1085, 1044, 732 cm^-1, H NMR (500 MHz, DMSO-d₆) δ

1

28.9 (C9), 29.6 (C11), 30.2 (C10), 31.2 (C13), 46.0 (C19), 51.3 (C14), (ppm) 1.15-1.31 (m, 56H, H11 and H12), 1.43-1.52 (m, 28H, H10),

61.5 (C6), 62.6 (C5), 65.4 (C17), 69.3 (C4), 69.8 (C2), 69.9 (C18), 76.7 1.69-1.80 (m, 28H, H13), 2.31-2.41 (m, 28H, H9), 3.26-3.52 (m, 63H,

(C3), 144.5 (C7), 124.9 (C15), 145.5 (C8), 146.1 (C16), 146.9 (C1), H21, H23, H17, H17’,H19 and H18), 3.62-3.71 (m, 42H, H3, H5 and

HRMS (ESI) m/z 530.6219 ([M + 3H]³⁺calcd. For C₇₃H₁₁₅N₂₀O₂₀: H6a), 3.90-4.00 (m, 28H, H4 and H6b), 4.19-4.26 (m, 14H, H14),

530.6193).

4.27-4.34 (m, 14H, H14’), 4.36-4.43 (m, 7H, H22), 4.63 (s, 14H, H2),

4.83-4.92 (m, 7H, H20), 6.98 (s, 14H H7), 8.02 (s, 7H, H15’), 8.10 (s,

7H, H15), ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm) 25.7, 25.8 (C12

and C12’), 27.4, 27.5 (C9 and C9’), 28.1, 28.3 (C11 and C11’), 28.6,

28.7 (C10 and C10’), 29.7 (C13), 49.2, 49.3 (C14 and C14’), 61.6 (C6),

62.3 (C5), 63.6 (C22), 64.0 (C2), 65.7 (C4), 69.8 (C18), 71.3 (C3),

72.3, 72.4 (C17 and C17’), 74.6 (C19), 79.1 (C21), 82.4 (C23), 100.2

(C20), 113.9 (C7), 123.6, 124.0 (C15 and C15’), 140.3 (C8), 143.3,

143.8 (C16 and C16’), 144.7 (C1), HRMS (ESI) m/z 622.9111 ([M +

10H]¹⁰⁺calcd. For C₂₈₀H₄₃₀N₇₀O₉₁: 622.9112).

Tetravalent cluster 23b

Product 23b (15.4 mg, 0.0097 mmol, 88%) was obtained as a beige

solid following general procedure B, starting from 22b (24.9 mg,

0.011 mmol).

[α]²_D⁰= -19 (c 0.7, MeOH), m.p. 128 °C (decomp) IR (neat) 3338,

3142, 2926, 2858, 1462, 1087 cm^-1, H NMR (500 MHz, CD₃OD) δ

1

(ppm) 1.26-1.41 (m, 16H, H12 and H11), 1.59 (qnt, J = 7.2 Hz, 8H,

H10), 1.88 (qnt, J = 7.2 Hz, 8H, H13), 2.49 (t, J = 7.5 HZ, 8H, H9), 3.42

(s, 8H, H18), 3.77 (dd, J = 9.3, 3.7 Hz, 4H, H3), 3.80 (ddd, J = 7.8, 5.5,

2.8 Hz, 4H, H5), 3.89 (dd, J = 11.8, 5.5 Hz, 4H, H6^a), 4.09 (dd, J = 9.2,

7.8 Hz, 4H, H4), 4.15 (dd, J = 11.8, 2.8 Hz, 4H, H6^b), 4.36 (t, J = 7.0

Inhibition assay on α-mannosidase of Jack-bean

p-nitrophenyl-α-D-mannopyranoside and α-mannosidase (EC

Hz, 8H, H14), 4.45 (s, 8H, H17), 4.79 (d, J = 3.7 Hz, 4H, H2), 7.04 (s, 3.2.1.24, from Jack bean) were purchased from Sigma Aldrich.

4H, H7), 7.90 (s, 4H, H15), ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 27.2

Inhibition constants were determined by spectrophotometrically

measuring the residual hydrolytic activities of the mannosidase

against p-nitrophenyl-α-D-mannopyranoside in the presence and

absence of inhibitor. Each well was filled with a total volume of 100

µL containing 0.2 M acetate buffer pH 5, inhibitor, substrate and

enzyme. All kinetics are performed between 25 and 27 °C and

started by enzyme addition. After 30-40 min incubation, the

reaction was quenched by addition of 100 µL of 1M Na₂CO₃. The

absorbance of the resulting solution was determined at 405 nm. K_i

values were determined in duplicate or triplicate, using the Dixon

and Lineweaver Burk graphical method with Microsoft Excel, or

using non-linear regression with GraphPad Prism Software. When

inhibitors were only partially soluble in water, stock solutions in 1:1

DMSO/buffer were prepared. The final DMSO content was under

2.5 %. The stability of the enzyme in presence of the same

concentrations of DMSO was controlled and the enzyme activity

was unaffected.

(C12), 28.8 (C9), 29.6 (C11), 30.2 (C10), 31.2 (C13), 46.4 (C19), 51.3

(C14), 63.0 (C6), 63.5 (C5), 65.4 (C17), 65.7 (C2), 67.2 (C4), 69.9

(C18), 73.1 (C3), 115.6 (C7), 124.9 (C15), 143.3 (C8), 145.9 (C1),

146.1 (C16), HRMS (ESI) m/z 530.6181 ([M + 3H]³⁺calcd. For

C₇₃H₁₁₅N₂₀O₂₀: 530.6193).

Tetradecavalent cluster 26a

Product 26a (5 mg, 0.0008 mmol, 98%) was obtained as a white

solid following general procedure B, starting from 25a (7 mg,

0.00082 mmol).

[α]²_D⁰= +7 (c 0.07, DMSO), m.p. 205 °C (decomp), IR (neat) 3343,

2925, 2860, 1733, 1456, 1369, 1227, 1084, 1044, 731 cm^-1, ¹H NMR

(500 MHz, DMSO-d₆) δ (ppm) 1.13-1.30 (m, 56H, H11 and H12),

1.41-1.51 (m, 28H, H10), 1.67-1.80 (m, 28H, H13), 2.29-2.39 (m,

28H, H9), 3.45-3.52 (m, 14H, H3), 3.52-3.59 (m, 14H, H4), 3.62-3.73

(m, 28H, H5 and H6a), 3.95-4.03 (m, 14H, H6b), 4.18-4.33 (m, 42H,

H14 and H2), 4.33-4.44 (m, 7H, H19), 4.68-4.79 (m, 14H, H23 and

H18a), 4.80-4.92 (m, 14H, H22 and H18b), 5.00-5.10 (m, 7H, H21), Conflicts of interest

5.33-5.45 (m, 28H, H17 and H17’), 5.76-5.88 (m, 7H, H20), 6.91 (s,

14H, H7), 8.02 (s, 7H, H15’), 8.10 (s, 7H, H15), ¹³C NMR (125 MHz,

There are no conflicts to declare.

DMSO-d₆) δ (ppm) 25.7 (C2), 27.9 (C9), 28.2 (C11), 28.8 (C10), 29.7

(C13), 49.3 (C14), 60.0 (C6), 60.9 (C5), 63.6 (C17), 64,8 (C17’), 67.6

(C4), 68.2 (C2), 69.9 (C22), 73.0 (C19), 75.1 (C3), 79.2 (C21), 82.3

(C23), 100.6 (C20),112.6 (C7), 123.6 (C15’), 124.1 (C15), 141.3 (C8),

Acknowledgements

The authors are grateful to financial supports from the CNRS (UMR

143.4 (C16’), 143.8 (C16), 145.8 (C1) carbon C18 is not visible, HRMS

7042), the University of Strasbourg, and the International Centre for

Frontier Research in Chemistry (icFRC). M.M.P thank the French

Department of Research for a doctoral fellowship. The authors

express their gratitude to Robin Henches and Alexis Taponard for

assistance with synthetic work and to Dr Camille Decroocq for the

preparation of compound 24.

(ESI) m/z 1037.5147 ([M

+

3H]⁶⁺calcd. For C₂₈₀H₄₂₆N₇₀O₉₁:

1037.5138).

Tetradecavalent cluster 26b

J. Name., 2013, 00, 1-3 | 15

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Journal Name

12 (a) Y. Brissonnet, S. Ladevèze, D. Tezé, E. Fabre, D. Deniaud,

Notes and references

F. Daligault, C. Tellier, S. Šesták, M. Remau^Vd^ie-S^wim^Arte^ico^len^O,^nliG^ne.

DOI: 10.1039/C9OB00826H

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33 M. Ortega-Muñoz, F. Perez-Balderas, J. Morales-Sanfrutos, F.

Hernandez-Mateo, J. Isac-García and F. Santoyo-Gonzalez,

Eur. J. Org. Chem., 2009, 2454–2473.

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DOI: 10.1039/C9OB00826H

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DOI: 10.1039/C9OB00826H

HO

*

O

OH

O

N

O

HO

N

O

OH

O

HO

O

high-affinity

inhitopes

O

OH

O

HO

O

OH

O

OH

O

TIGHT-BINDING INHIBITORS

Examples of multimeric inhibitors displaying tight binding inhibition of a carbohydrate-processing

enzyme are presented.

Article Doi

Tight-binding inhibition of jack bean α-mannosidase by glycoimidazole clusters

DOI: 10.1039/c9ob00826h

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c9ob00826h

Full text of DOI:10.1039/c9ob00826h

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