Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-dihydroxypyrrolidines: Rhamnulose iminosugar mimics inhibit α-glucosidase

Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-

dihydroxypyrrolidines: rhamnulose iminosugar mimics inhibit

α

-glucosidase

a,c

b

a

c

d

Zilei Liu, Akihide Yoshihara, Sarah F. Jenkinson, Mark R. Wormald, Ciarán Kelly, John T.

d

a

e

a

f

Heap, Mikkel H. S. Marqvorsen, Ramón J. Estévez, George W. J. Fleet *, Shinpei Nakagawa, Ken

b

g

*f

Izumori, Robert. J. Nash, Atsushi Kato

a

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA,

_bUK

International Institute of Rare Sugar Research and Education, Kagawa University, Miki, Kagawa

61-0795, Japan

7

c

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, OX1

QU, UK

Centre for Synthetic Biology, Department of Life Sciences, Imperial College London, SW7 2AZ, UK

Departamento de Química Orgánica and Centro Singular de Investigación en Química Biolóxica e

3

d

e

Materiais Moleculares, Universidade de Santiago de Compostela, 15782 Santiago de Compostela,

Spain

f

Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan

PhytoQuest Ltd, Aberystwyth, Ceredigion SY23 3EB, UK

g

*

george.fleet@chem.ox.ac.uk; kato@med.u-toyama.ac.jp

Graphical abstract

Abstract: The key step in the syntheses of highly substituted trans-3,4-

dihydroxypyrrolidines is introduction of bromide by stereospecific and regiospecific

Hanessian-Hullar reactions; benzylidene lactones of

L-rhamnonolactone and 6-deoxy-L-

gulonolactone allow introduction of N at C2 with inversion or retention of configuration.

Initially a protecting group, the benzylidene acetal then provides a bromide at C5 to allow

formation of the pyrrolidine ring. With silyl protecting groups, bromide was introduced at C5

with inversion of configuration whereas benzoyl protection gave a mixture of retention and

inversion, indicative of neighbouring group participation in a Hanessian-Hullar reaction. Four

stereoisomeric pyrrolidines - iminosugar mimics of α- and β-L-rhamnulose and α- and β-6-

deoxy- -sorbose were prepared. Only the α- -rhamnulose mimic showed moderate

D

L

inhibition of rhamnosidase but some were good inhibitors of α-glucosidases; none inhibited

rhamnose isomerase and they had a small effect as synthetic inducers of the rhamnose

catabolic operon in E. coli.

1

. Introduction

1

Hundreds of iminosugars, in which the ring oxygen of a monosaccharide has been replaced

by nitrogen, have been isolated as natural products. Although one of the largest families of

glycosidase inhibitors, iminosugars have many other bioactivities as sugar mimics. The

glycosidase inhibition profiles of the 16 piperidine analogues of the 16 aldohexoses are quite

well correlated with the structures of the parent sugars; however, the 10 pyrrolidine

2

analogues of the 8 ketohexoses provide many surprises. This paper describes the synthesis

and preliminary evaluation of the α- 1 and β- 2 iminosugar analogues of rhamnulose and of

their epimers at C5, α- 3 and β- 4, iminosugars analogues of 6-deoxy-

D

-sorbose, four of the

1

sixteen pyrrolidine analogues of 6-deoxy ketohexoses [Figure 1]. An example of

neighbouring group participation was found in the Hanessian-Hullar reaction, a key reaction

in their syntheses.

Figure 1: Iminosugar structures

3

6

-Deoxy-DGDP (6-deoxy-2,5-dideoxy-2,5-imino-

D

-glucitol) 1D and 6-deoxy-DMDP (6-

4

deoxy-2,5-dideoxy-2,5-imino-D-mannitol) 2D, the enantiomers of 1 and 2 respectively, have

been isolated from Angylocalyx pynaertii. A further stereoisomer, a strong inhibitor of α-

fucosidase, was also isolated and ascribed the structure 4; the synthetic 4 prepared in this

5

paper did not inhibit α-L-fucosidase. The natural product is therefore likely to be the

6

enantiomer 4L, a fuculose analogue prepared previously by Wong. The rhamnulose

analogues 2 and 4 may also be considered to be the α- and β-1-C-methyl analogues of LAB

7

(1,4-dideoxy-1,4-imino-

L

-arabinitol) 5, a potent and specific α-glucosidase inhibitor. The

glycosidase inhibition profiles of the new iminosugars are compared with structurally related

iminosugars; their effects on rhamnose isomerase and the rhamnose operon were also

investigated.

2

Results and Discussion

.1 Synthesis

.1.1 Strategy

The key step in the syntheses of the highly substituted trans-3,4-dihydroxypyrrolidines 1-4

was the introduction of bromide by a stereospecific and regiospecific Hanessian-Hullar

8

reaction [Scheme 1]. The benzylidene lactones 6 and 7, epimeric at C5, have only C2 OH

unprotected and allow efficient access to the azidolactones 8-11. In general, displacement of

a triflate at C2 of a lactone by sodium azide in DMF can introduce nitrogen with either

inversion or retention of configuration. Displacement of the 2-O-triflate in a γ-lactone in

which all the ring substituents are cis gives a kinetic azide with a 2,3-trans relationship with

the neighbouring oxygen; base catalysed isomerisation by azide gives the thermodynamically

9

more stable azide in which all the substituents on the lactone ring are cis. The epimeric

1

0

azides are always very different in polarity and easily separated by flash chromatography.

2

Scheme 1: Synthetic strategy for trans-3,4-dihydroxypyrrolidines 1-4

Reduction of the lactones to the corresponding diol, followed by silyl ether protection and

oxidation with N-bromosuccinimide give the bromo-benzoates 12-15 with inversion of

configuration at C5; the benzylidene acetal, initially a protecting group, subsequently allows

a Hanessian-Hullar reaction to introduce the bromide leaving group at C5. When benzoate

was substituted for silyl ether protection, both retained and inverted bromides were formed,

indicating neighbouring group participation; the regiospecificity and stereoselectivity is

discussed fully below.

Reduction of the azides 12-15 to the corresponding amines allows cyclization to the

pyrrolidines with a second inversion at C5. Deprotection of the pyrrolidines give the

diastereomers 1-4 in an efficient and general approach, in which the configuration at C2 of

the original sugar may be inverted or retained, but that at C5 is always retained.

2

.1.2 Synthesis of azidolactones 8, 9, 10 and 11

For the rhamnonolactones 8 and 9, the benzylidene derivative 6 can readily be obtained from

1

12

rhamnose in a one pot sequence on a 50 g scale [Scheme 2]. As previously described, the

stable triflate 16, formed in 95% yield from 6, with sodium azide in DMF at -40 °C formed

the kinetic glucono-azide 8 (75%) but at room temperature gave the thermodynamic

mannono-azide 9 (85%); the initially formed 8 was equilibrated to the more stable all cis-

azide 9.

3

Scheme 2: (i) (CF SO ) O, pyridine, THF, -20ꢀ°C, 95ꢀ%; (ii) NaN , DMF, -40 °C, 75%; (iv) NaN ,

3

2 2

3

DMF, rt, 84%, 42 h [as reported in reference 10].

Scheme 3: (i) PhCHO, conc HCl, 92%; (ii) (CF SO ) O, Pyridine, THF, -20 °C, 98%; (iii) NaN ,

3

2 2

3

DMF, -10 °C, 20h, 77%; (iv) NaN , DMF, rt, 78%, 42 h; (v) CF CO Cs, DMF, 60 °C, 100% 52 h ;

3

2

(

vi) (CF SO ) O, Pyridine, THF, -20 °C; (vii) NaN , DMF, rt, 30 h, 45% (2 steps)

3 2 2 3

For the synthesis of the idono- and gulono-azides 10 and 11, the starting material 6-deoxy-

D-

gulonolactone 18 was prepared from the triacetonide 17 in 61% yield with no

1

3

chromatography required; the route is far more convenient than procedures from

rhamnose or

L-

1

2

14

D-gulonolactone (Scheme 3). Reaction of 18 with benzaldehyde in the

presence of concentrated hydrochloric acid gave the crystalline lactone 7 (92%) in which the

methyl group is equatorial. The stereochemistry of 7 was confirmed by the Nuclear

Overhauser Effect (nOe) NMR analysis; significant 1,3-H nOe effects between the protons on

the benzylidene ring showed the phenyl group to be equatorial in 7 in contrast to the axial

1

5

phenyl substituent in the rhamnono-epimer 6.

Esterification of the alcohol in 7 with triflic anhydride in THF in the presence of pyridine

gave the stable triflate 19 (98%) which, on treatment with sodium azide in DMF at -10 °C for

2

0 h, gave the ido-azide 10 with inversion at C2 (77%); when the reaction was performed at

room temperature for 42 h, the more stable gulo-azide 11 was isolated in 78% yield. The

epimeric azides 10 and 11 were easily separated by chromatography. Both nucleophilic

displacement by azide and the equilibration to the all cis-azide were much slower for the

gulono-triflate 7 than for the analogous reactions of the rhamnono epimer 16. Displacement

1

6

of the triflate 19 by caesium trifluoroacetate in DMF at 60 °C for 52 h gave the idono-

lactone 20 (100%). The triflate 21 was unstable and substantially decomposed during

isolation and treatment with sodium azide, giving 11 in poor yield (45%). The different

behaviour between the mannono- 6 and gulono- 7 lactones may be ascribed to substantially

4

different conformations of the bicyclic acetals where the methyl group is, respectively, either

1

5

axial or equatorial. In both the rhamnono- and gulono-series, efficient syntheses of the

easily separable retained and inverted azides 8-11 can be obtained in good yield.

2

.1.3 6-Deoxy-L-DGDP 1 and 6-deoxy-L-DMDP 2 from rhamnono-azides 8 and 9

Scheme 4: (i) DIBALH, DCM, -78 °C, then NaBH , MeOH, 92% (2 steps); (ii) TBDMSOTf,

4

pyridine, DCM, 98%; (iii) NBS, BaCO , CCl , reflux, 86%; (iv) Pd/C, H , NaOAc, EtOH, 72%; (v)

3

4

2

TFA/water/1,4-dioxane, 50 °C, then NaOH (2 M, aq), 50 °C, 81% (2 steps); (vi) DIBALH, DCM, -78

C, then NaBH , MeOH, 89% (2 steps); (vii) TBDMSOTf, pyridine, DCM, 94%; (viii) NBS, BaCO ,

°

4

3

CCl , reflux, 92%; (ix) Pd/C, H , NaOAc, EtOH, 71%; (x) TFA/water/1,4-dioxane, 50 °C, then NaOH

4

2

(

2 M, aq.), 50 °C, 64% (2 steps);

The epimeric azides 8 and 9 derived from rhamnose were used for the synthesis of the α- 1

and β-2 iminosugar analogues of β-rhamnulose respectively [Scheme 4]. Reduction of 8 with

DIBALH followed by in situ treatment with sodium borohydride gave the diol 22 (92%)

which, with tert-butyldimethylsilyl (TBDMS) triflate in dichloromethane in the presence of

pyridine, afforded the bis-silyl ether 23 (98%). The benzylidene acetal 23 with N-

5

bromosuccinimide (NBS) in the presence of barium carbonate in carbon tetrachloride at

reflux underwent a Hanessian-Hullar reaction to produce the bromide 12 in 1 h with

inversion of configuration at C5 (86%). Hydrogenation of azide 12 in ethanol in the presence

of sodium acetate gave the protected pyrrolidine 24 (72%), formed by closure of the resulting

amine with a second inversion at C5. Removal of the silyl groups in 24 by trifluoroacetic

acid, followed by hydrolysis of the benzoyl ester with aqueous sodium hydroxide and ion

exchange purification, gave the unprotected

conditions for global deprotection of 24 were found. The overall yield of 6-deoxy-

from benzylidene rhamnonolactone 6, with inversion at C2 and overall retention at C5, was

3%. 6-Deoxy- -DMDP 2 was formed in an identical sequence from benzylidene

rhamnonolactone 6, with retention of configuration at both C2 and C5, in 29% overall yield

Scheme 4].

L

-gluco-pyrrolidine 1 (81%); no efficient

L-DGDP 1

3

L

[

2

.1.4 6-Deoxy-

D

-ido-DMDP 3 and 6-deoxy-

D

-gulo-DMDP 4 from

D

-gulono-azides 10

and 11

Ph

TBDMSOH C

N₃

O

N₃

O

2

O

(

i)

(ii)

O

N₃

O

HO

O

HO

TBDMSO

CH₃

10

28

29

(iii)

TBDMSOH C

HO

OH

BzO

OTBDMS

CH₃

2

OBz

Br

(

v)

(iv)

N₃

HOH C

CH₃

TBDMSOH C

2

N

H

N

TBDMSO

H

CH₃

3

30

14

Ph

TBDMSOH C

N₃

O

N₃

O

2

O

(

vi)

(vii)

O

N₃

O

HO

O

HO

TBDMSO

CH₃

11

31

32

(viii)

TBDMSOH C

HO

HOH C

OH

CH₃

BzO

OTBDMS

CH₃

2

OBz

Br

(

x)

(ix)

N₃

TBDMSOH C

2

N

H

N

H

TBDMSO

CH₃

4

33

15

Scheme 5: (i) DIBALH, DCM, -78 °C, then NaBH , MeOH, 66% (2 steps); (ii) TBDMSOTf,

4

pyridine, DCM, 74%; (iii) NBS, BaCO , CCl , reflux, 99%; (iv) Pd/C, H , NaOAc, EtOH; (v)

3

4

2

TFA/water/1,4-dioxane, 50 °C, then NaOH (2M, aq), 50 °C, 74% (3 steps from 14); (vi) LiBH , THF,

4

-

20 °C, 90%; (vii) TBDMSOTf, pyridine, DCM, 84%; (viii) NBS, BaCO , CCl , reflux, 76%; (ix)

3 4

Pd/C, H , NaOAc, EtOH; (x) TFA/water/1,4-dioxane, 50 °C, then NaOH (2M, aq), 50 °C, 51% (3

2

steps from 15)

6

The chemistry developed for the rhamnono-azidolactones proceeded equally reliably for the

-deoxy-gulonono-azidolactones and is summarised in Scheme 5. The overall yield of 6-

deoxy- -ido-DMDP 3 was 36% from 10. Although usually the base-sensitivity of 2-

azidolactones requires a two step reduction, the lactone 11 on treatment with lithium

borohydride in THF gave the diol 31 in 90% yield; 6-deoxy- -gulo-DMDP 4 was obtained in

6

D

2

9% overall yield from 11.

2

.1.4 Benzoate protection: neighbouring group participation (NGP) in the Hanessian-

Hullar Reaction

The Hanessian-Hullar reaction of either the benzylidene lactone 8 or the unprotected diol 22

gave complex mixtures of products. As described above, treatment of the silyl ether 23 with

N-bromosuccinimide (NBS) in carbon tetrachloride in the presence of barium carbonate gave

the bromide 12 in 86% yield [Scheme 6]. There is some uncertainty in the mechanism of the

8

formation of the bromide 34. However, nucleophilic attack at C5 of the cation 35 is less

hindered than attack at C3; additionally there are both β-oxygen and β-nitrogen substituents

which slow S 2 displacement at C3. All the four diastereomeric silyl-protected ethers at C2

N

and C5 - 23, 26, 29 and 32 - undergo exclusive attack by bromide at C5 with inversion of

configuration.

Scheme 6: Neighbouring group participation (NGP) in Hanessian-Hullar reaction

Benzoate was studied as an alternative protecting group. NBS oxidation of the dibenzoate 36

[

obtained by benzolation of the diol 22 (86%)] gave a mixture of the

D-ido 39 and L-gluco-

4

0 bromides in a 5:1 ratio and 91% yield. The cation 37 is thus mainly trapped by bromide in

an S 2 inversion at C5 to give the ido-bromide 39. Competitive NGP by the benzoate at C4

N

in 37 formed the cation 38 which undergoes attack by bromide to give a second inversion at

C5 to form the gluco-bromide 40 with overall retention at C5; this may be the first report of

7

NGP in a Hanessian-Hullar reaction. Both cations 37 and 38 undergo regiospecific

nucleophilic displacement at C5.

Scheme 7: (i) BzCl, Pyridine, DCM, 86%; (ii) NBS, BaCO , CCl , reflux, 39:40 5:1, 91%;

3

4

(

iii) Pd/C, H , NaOAc, EtOH, 41:42 4:1, 82%; (iv) NaOH (aq., 2M), 50 °C, 1:3 5:1, 73%. (v)

2

BzCl, Pyridine, DCM, 97%; (vi) NBS, BaCO , CCl , reflux, 43:44 3:1, 94%; (vii) Pd/C, H ,

3

4

2

NaOAc, EtOH, 45:46 10:7, 89%; (viii) NaOH (aq., 2M), 50 °C, 2:4 5:3, 70%.

The inseparable bromides 39 and 40 [5:1] (91% from 36) on hydrogenation in the presence of

palladium on charcoal in ethanol gave an inseparable epimeric mixture of the pyrrolidine

tribenzoates 41 and 42 [4:1] (82%) [Scheme 7]. Removal of the benzoate protecting groups

1

gave 1 and 3 (73%) in a ratio of 5:1, determined by comparison with H NMR of pure

samples of 1 and 3 prepared as above.

Benzoylation of the epimeric azidodiol 25 afforded the dibenzoate 26 (97%) which on

treatment with NBS gave a mixture of the epimeric bromides 43 : 44, 3:1 (94%) which on

hydrogenation gave the tribenzoates 45 : 46 (94%). Deprotection with aqueous sodium

hydroxide gave a mixture of 2 : 4, 5:3 (94%). Again, none of the intermediate mixtures were

separable. The use of silyl protection in Hanessian-Hullar reactions ensures there is no NGP,

whereas other protecting groups may give epimeric bromides as products.

3

. Biological assays

Compounds that affect enzymes in the biosynthesis and metabolism of

that has no role in mammalian metabolism, have potential to affect pathogens and to

L

-rhamnose, a sugar

1

7

8

1

8

provide novel antibiotics. The effects of the rhamnulose mimics 1-4 on glycosidases,

rhamnose isomerase and the rhamnose operon were investigated.

3

.1 Glycosidase Inhibition Profile

Figure 2: Iminosugar structural relationships

PLEASE KEEP CLOSE TO TABLE BELOW

2,19

The inhibition of glycosidases by iminosugar furanose analogues is difficult to predict.

The four trans-3,4-dihydroxypyrrolidines 1-4 prepared in this paper were compared with the

corresponding iminosugars 47-49 in which the methyl group is replaced by a hydroxymethyl

9

2

0

group [

glycosidases : α- and β-glucosidases (from various sources), α- and β-galactosidase, α- and

β-mannosidase, α- -fucosidase, α,α-trehalase, amyloglucosidase, α- -rhamnosidase, and β-

glucuronidase (Table 1). -DGDP 47 and -DMDP 48 are the enantiomers of the natural

products DGDP 47D [isolated from the Thai traditional medicine Non tai yak and a weak

D-guloDMDP is identical by rotation to L-DGDP 47] as inhibitors of the following

2

1

L

2

3

inhibitor of α-glucosidases] and DMDP 48D [a most abundant natural iminosugar and a

potent inhibitor of β-galactosidase

DMDP 48 are both specific and potent inhibitors of α-glucosidases; there are many cases in

which enantiomers of iminosugars are more potent inhibitors of the same enzyme than the

2

,24

]. In contrast, their enantiomers L-DGDP 47 and L-

2

5

natural product. As 1 and 2 are the iminosugar mimics of α- and β-

L-rhamnulose,

L-DGDP

4

7 and

L-DMDP 48 are the corresponding mimics of α- and β-D-fructose.

1

. Only the α-rhamnulose mimic, 6-deoxy-L-DGDP, 1 showed moderate inhibition of α-

rhamnosidase. None of the other iminosugar hexose analogues – including the β-rhamnulose

mimic 2 – showed any rhamnosidase inhibition. 6-Deoxy- -DGDP 1 was also a moderate

inhibitor of α-glucosidases; the substitution of Me in 1 for the CH OH group in -DGDP 47

-ido-DMDP 3, in

L

2

considerably reduced glucosidase and galactosidase inhibition. 6-deoxy-

D

which the methyl group is epimeric to that in 1, did not show significant inhibition of any

glycosidase.

2

. 6-Deoxy-

Comparison of 6-deoxy-

OH group slightly increased the inhibition of rice and rat intestinal sucrase. Furthermore, 6-

deoxy- -DMDP 2 lost inhibition of porcine kidney α,α-trehalase, showing more selectivity.

L

-DMDP 2 was a potent and highly specific inhibitor of α-glucosidases.

L

-DMDP 2 and -DMDP 48 revealed that deoxygenation of C6-

L

3

. 6-Deoxy-L-DMDP 2 was also compared to the potent and α-glucosidase inhibitor LAB 5.

2

6

Its enantiomer DAB 5D, isolated from Angylocalyx boutiqueanus is a good inhibitor of α-

2

7

glucosidase and glycogen phosphorylase as a potential drug for the treatment of diabetes.

Although LAB 5 was 10-times better as an inhibitor of rat intestinal maltase than 6-deoxy-

DMDP 2, LAB 5 also showed broad inhibition toward α,α-trehalase and α- -rhamnosidase.

L-

L

4

. 6-Deoxy-D-gulo-DMDP 4, with its methyl group epimeric to that in 2, was a moderate

[

and highly specific] inhibitor of α-glucosidase. 2 and 4 may be viewed as the α- and β-1-C-

methyl-LAB derivatives. α-1-C-methyl-LAB 2 is a significantly more potent glucosidase

inhibitor than its β-anomer 4. Broussonetines, a large family of natural glucosidase and

2

8

galactosidase inhibitors, are α-1-C-alkyl-DAB derivatives.

3

.2 Rhamnose Isomerase

The only previous report of iminosugar inhibition of aldose isomerases is a crystallographic

study of mimics of the substrates and products in the xylose isomerase catalyzed

isomerization of glucose to fructose; DGDP 47D (an α-fructose mimic) was a weak inhibitor

2

9

whereas DMDP 48D (β-fructose mimic) showed no inhibition. There are no aldose

isomerases in mammalian metabolism; no inhibitors of rhamnose isomerase have been

3

0

described.

L

-Rhamnose isomerase activity was measured by the conversion of

L

-rhamnose

3

1

as substrate to

L-rhamnulose as previously described. None of the four pyrrolidines 1-4 had

any significant effect on the activity of rhamnose isomerase (for details, see Supplementary

Material).

1

0

3

.3 Rhamnose Operon

In synthetic biology, a precisely-controlled gene expression system based on the regulatory

elements (transcriptional promoter and activator) of the -rhamnose catabolism gene operon

in E. coli, rhaBAD, has been frequently used for the production of various proteins. Free

L

3

2

L-

rhamnose is an inducer of rhaBAD but has the significant disadvantage of being metabolized

to rhamnulose, which causes induction of gene expression to cease. The performance of this

promoter system would therefore be enhanced by the development of a non-metabolizable

rhamnose substitute; some 35 rare sugars have been studied as potential alternative inducers

3

to rhamnose.

The four iminosugars 1-4, tested for their ability to induce expression of the rhaBAD

3

promoter in Escherichia coli cells using a reporter assay system showed much weaker

induction than the nature inducer, -rhamnose; the rhamnulose 1, 2 and 6-deoxy- -sorbose 3

mimics had low but statistically significant induction of the rhaBAD promoter system (P <

.05) relative to no inducer (see supplementary material). -Rhamnose is easily metabolized

L

0

L

in E. coli, causing gene expression to cease and a much reduced protein production. To

overcome this problem, a non-metabolizable inducer is of both commercial and academic

interest. The rhamnulose iminosugar analogues 1, 2 and 3, with no 6-hydroxyl group, are

unlikely to be metabolized by rhamnose metabolic pathways and thus might be suitable to

induce sustainable gene expression.

4

. Conclusion

The high yield syntheses of iminosugar mimics of

L-rhamnulose and 6-deoxy-D-sorbose

depended on (i) azide substitution of 2-O-triflates of lactones with either retention or

inversion in consistent yields of >80%; and (ii) efficient Hanessian Hullar NBS oxidation of

benzylidene acetals – silyl ether protection ensured bromide was introduced at C5 of the

sugar with complete regio- and stereo-specificity whereas using benzoate as an alternative

protecting group lead to a mixture arising from NGP. Only the α-L-rhamnulose mimic 1

showed moderate inhibition of rhamnosidase whereas 2 and 4 were good inhibitors of α-

glucosidases; 2 and 4 may be viewed as α- and β-1-C-methyl analogues of LAB 5, a potent

α-glucosidase inhibitor. None inhibited rhamnose isomerase and they had only a small effect

as synthetic inducers of the rhamnose catabolic operon in E. coli.

5

. Experimental

5

.1 General Experimental

All commercial reagents were used as supplied. Solvents were used as supplied (Analytical or HPLC

grade), without prior purification. Thin layer chromatography (TLC) was performed on aluminium

sheets coated with 60 F₂₅₄silica. Plates were visualised using a 0.2% w/v cerium (IV) sulfate and 5%

ammonium molybdate solution in 2 M sulfuric acid. Melting points were recorded on a Kofler hot

block and are uncorrected. Optical rotations of the protected sugars were recorded on a Perkin-Elmer

-

1

2

41 polarimeter with a path length of 1 dm; concentrations are quoted in g 100 mL . Optical rotations

+

were recorded on a Jasco R1030 polarimeter, Na lamp, (Jasco, Tokyo, Japan) at 20 °C, polarimeter

with a path length of 1 dm. Infrared spectra were recorded on a Perkin-Elmer 1750 IR Fourier

Transform spectrophotometer using thin films on a diamond ATR surface (thin film). Only the

characteristic peaks are quoted. Nuclear magnetic resonance (NMR) spectra were recorded on a

Bruker AMX 500 ( H: 500 MHz and C: 125.7 MHz) or Bruker AVIII 400 HD nanobay and Bruker

DQX 400 ( H: 400 MHz, C: 100.6 MHz and F: 375 MHz) spectrometers in the deuterated solvent

1

13

1

13

19

1

13

stated. H and C NMR spectra were assigned by utilizing 2D COSY, HSQC and HMBC spectra. All

chemical shifts (δ) are quoted in ppm and coupling constants (J) in Hz. Residual signals from the

solvents were used as an internal reference. For solutions in D O acetonitrile was used as an internal

2

reference. HRMS measurements were made using a microTOF mass analyzer using electrospray

ionization (ESI) or an HP 5988A mass spectrometer using chemical ionization (CI).

5

.2 Azidogulonolactones 10 and 11

(

R)-3,5-O-Benzylidene-6-deoxy-

D-gulono-1,4-lactone 7

Concentrated hydrochloride acid (37%, 4 mL) was added to a suspension of 6-deoxy-

D

-gulono-1,4-

12

lactone 18 (887 mg, 5.5 mmol) in benzaldehyde (15 mL). The reaction mixture was stirred at room

temperature for 15 h during which time a solid product separated. The solid was collected by filtration

and washed with diethyl ether (20 mL) to yield the benzylidene acetal 7 (1.2 g, 92%) as white

crystals.

+

HRMS (ESI+ve): found 273.0733 [M + Na] ; C H O Na requires 273.0733; m.p. 226 - 230 °C;

[

1

3

14

5

25

α]_D-54 (c 0.8, MeCN); v_m_a_x(thin film): 1785 (s, C=O); δ (CD CN, 500 MHz): 1.36 (3H, d, H6,

H 3

^J⁶,5 6.7), 3.80 (1H, br-d, OH, J_O_H_,₂8.9), 4.19 (1H, t, H4, J₄_,₃= J₄_,₅2.0), 4.25 (1H, dq, H5, J₅_,₄1.9, J₅_,₆

6

7

.6), 4.58 (1H, dd, H2, J₂_,₃4.1, J₂_,_O_H8.2), 4.68 (1H, dd, H3, J₃_,₄2.0, J₃_,₂4.1), 5.68 (1H, s, H7), 7.38 -

.48 (5H, m, -Ar); δ (CD CN, 100 MHz): 15.8 (C6), 71.4 (C2), 72.8 (C5), 72.1 (C4), 74.5 (C3), 98.9

C

3

+

(

C7), 126.3, 128.2, 129.1, 137.9 (-Ar), 175.4 (C=O); m/z (ESI+ve): 273 ([M + Na] , 100%).

(

R)-3,5-O-Benzylidene-6-deoxy-2-O-trifluoromethanesulfonyl- -gulono-1,4-lactone 19

D

Anhydrous pyridine (0.3 mL, 0.36 mmol) and triflic anhydride (0.5 mL, 0.31 mmol) were sequentially

added dropwise to a solution of the benzylidene lactone 7 (600 mg, 2.40 mmol) in anhydrous THF (10

mL) at -20 °C. After 3 h, TLC (cyclohexane/ethyl acetate, 1:1) indicated the formation of one product

(

R 0.57). The reaction mixture was diluted with dichloromethane (10 mL) and washed with HCl (2

f

M, aq, 2 x 10 mL). The organic layer was dried (MgSO ); the solvent was removed in vacuo to give a

4

residue that was purified by flash chromatography (ethyl acetate/cyclohexane, 1:5 to 1:2) to obtain

pure triflate 19 (898 mg, 98%) as a white solid.

+

HRMS (ESI+ve): Found 405.2226 [M + Na] ; C H F O SNa requires 405.2226; m.p. 198 - 200 °C

1

4

13

3

7

2

5

(

decomp); [α]_D-64 (c 0.79, MeCN); v_m_a_x(thin film): 1795 (s, C=O); δ (CD CN, 400 MHz): 1.44

H 3

3H, d, H6, J₆_,₅6.6), 4.36 (1H, dq, H5, J₅_,₄1.4, J₅_,₆6.6), 4.44 (1H, br-t, H4, J = J 1.5), 5.12 (1H,

5,4

3,4

dd, H3, J₃_,₄1.9, J₃_,₂4.0), 5.77 (1H, s, H7), 5.91 (1H, d, H2, J₂_,₃4.0), 7.44 - 7.46 (5H, m, -Ar); δ_C

CD CN, 100 MHz): 15.6 (C6), 71.4 (C5), 72.7, 72.8 (C3, C4), 80.2 (C2), 98.9 (C7), 126.1, 128.3,

(

1

3

+

29.3, 137.1 (-Ar), 168.2 (C1); m/z (ESI+ve): 405 ([M + Na] , 100%).

2

-Azido-(R)-3,5-O-benzylidene-2,6-dideoxy- -idono-1,4-lactone 10

D

Sodium azide (168 mg, 2.58 mmol) was added to a solution of triflate 19 (898 mg, 2.35 mmol) in

anhydrous DMF (10 mL). The resulting solution was stirred at -10 °C for 20 h until TLC

(

cyclohexane/ethyl acetate, 1:1) indicated the formation of a major product (R 0.70). After dilution

f

with ethyl acetate (10 mL), the reaction mixture was washed with half saturated brine (20 mL). The

organic phase was dried (MgSO ), filtered and the solvent was removed in vacuo to obtain a residue

4

that was purified by flash chromatography (ethyl acetate/cyclohexane, 1:7 to 1:5) to yield the idono-

azide 10 (500 mg, 77%) as a white solid.

+

25

D

HRMS (ESI+ve): found 276.0980 [M + H] ; C H O N requires 276.0979; m.p.: 68 - 69 °C; [α]

-

1

3

14

4

3

8

9 (c 0.88, CHCl ); v (thin film): 2112 (s, N ), 1780 (s, C=O); δ (CDCl , 400 MHz): 1.51 (3H, d,

3 max 3 H 3

H6, J₆_,₅6.6), 4.20 (1H, dq, H5, J₅_,₄1.8, J₅_,₆6.6), 4.21 (1H, br-s, H4), 4.35 (1H, dd, H3, J₃_,₄1.9, J₃_,₂

.2), 4.44 (1H, d, H2, J₂_,₃2.2), 5.65 (1H, s, H7), 7.37 - 7.45 (5H, m, -Ar); δ (CDCl , 100 MHz): 17.5

2

C

3

(

C6), 63.3 (C5), 72.4 (C4), 76.1, 76.2 (C2, C3), 100.1 (C7), 126.5, 128.8, 129.9, 136.9 (-Ar), 171.4

C1); m/z (ESI+ve): 298 ([M + Na] , 100%).

+

(

R)-3,5-O-Benzylidene-6-deoxy-D-idono-1,4-lactone 20

Caesium trifluoroacetate (48 mg, 0.19 mmol) was added to a solution of 10 (50 mg, 0.13 mmol) in

anhydrous DMF (1.5 mL). The solution was then stirred at 60 °C for 50 h until TLC (ethyl

1

2

acetate/cyclohexane, 2:1) indicated the disappearance of the starting material (R 0.68) and the

f

formation of one product (R 0.62). After dilution with ethyl acetate (5 mL), the reaction mixture was

f

washed with half saturated brine (10 mL). The organic phase was dried (MgSO ), filtered and the

4

solvent was removed in vacuo to give a residue that was purified by flash chromatography (ethyl

acetate/cyclohexane, 1:5 to 1:3) to obtain 20 (32.5 mg, 100%) as a white solid.

+

25

D

HRMS (ESI+ve): found 273.0734 [M + Na] ; C H O Na requires 273.0733; m.p. 82 - 84 °C; [α]

1

3

14

5

-39 (c 0.78, CH CN); ν (thin film): 3220 (br, OH), 1776 (s, C=O); δ (CD CN, 400 MHz): 1.39

3 max H 3

(

5

3H, d, H6, J₆_,₅6.6), 4.10 (1H, br-s, H3), 4.29 (1H, dq, H5, J₅_,₄1.7, J₅_,₆6.6), 4.51 – 4.53 (1H, m, H4),

.68 (1H, s, H7), 5.87 (1H, d, H2, J₂_,₃4.0), 7.37 - 7.44 (5H, m, -Ar); δ (CD CN, 100 MHz): 17.3

C

3

(

C6), 72.7 (C5), 73.7 (C3), 76.9 (C4), 78.1 (C2), 99.8 (C7), 127.0, 129.1, 130.0, 138.8 (-Ar), 175.7

C1); m/z (ESI+ve): 273 ([M + Na] , 100%).

+

2

-Azido-(R)-3,5-O-benzylidene-2,6-dideoxy-

D-gulono-1,4-lactone 11

Method 1: from gulono-triflate 19

Sodium azide (228 mg, 3.36 mmol) was added to a solution of the gulono-triflate 19 (838 mg, 2.19

mmol) in anhydrous DMF (10 mL). The mixture was stirred at rt for 42 h until TLC (ethyl

acetate/cyclohexane, 1:1) indicated the formation of major product (R 0.20). After being diluted with

f

ethyl acetate (10 mL), the mixture was washed with half saturated brine (20 mL). The organic phase

was dried (MgSO ), filtered and the solvent removed in vacuo to obtain a residue that was purified by

4

flash chromatography (ethyl acetate/cyclohexane, 1:5 to 1:1) to yield the gulono-azide 11 (470 mg,

7

8%) as a white solid, identical to material obtained by Method 2 below.

Method 2: from idono-lactone 20

Anhydrous pyridine (0.03 mL, 0.36 mmol) and triflic anhydride (0.05 mL, 0.31 mmol) were

sequentially added dropwise to a solution of the idono-lactone 20 (60 mg, 0.24 mmol) in anhydrous

THF (3 mL) at -20 °C. After 4 h, TLC (ethyl acetate/cyclohexane, 1:1) indicated the formation of the

only product (R 0.83). The reaction mixture was diluted with dichloromethane (5 mL) and washed

f

with HCl (2 M, aq, 2 x 5 mL). The organic layer was dried (MgSO ) and the solvent was removed in

4

vacuo to give the residue (~100 mg) that was used without further purification; in contrast to gulono-

triflate 21, this triflate was not stable.

Sodium azide (23 mg, 0.36 mmol) was added to a solution of the crude triflate (~100 mg) in

anhydrous DMF (2 mL). The mixture was stirred at rt for 30 h until TLC (ethyl acetate/cyclohexane,

1

:1) indicated the formation of the product (R 0.20). After being diluted with ethyl acetate (5 mL),

f

the mixture was washed with half saturated brine (10 mL). The organic phase was dried (MgSO₄),

filtered and the solvent was removed in vacuo to obtain a crude that was purified by flash

chromatography (ethyl acetate/cyclohexane 1:5 to 1:1) to yield the azido lactone 11 as a white solid

(

30 mg, 45%).

+

°

HRMS (ESI+ve): Found 298.0799 [M + Na] ; C H O N Na requires 298.0798; m.p. 144 - 148 C;

[

(

1

3

13

4

3

25

α]_D-76 (c 0.70, CHCl ); v (thin film): 2108 (s, N ), 1775 (s, C=O); δ (CDCl , 400 MHz): 1.49

3 max 3 H 3

3H, d, H6, J₆_,₅6.6), 4.04 (1H, d, H2, J₂_,₃3.8), 4.14 (1H, br-s, H4), 4.22 (1H, dq, H5, J₅_,₄1.2, J₅_,₆6.6),

4

1

.78 (1H, dd, H3, J₃_,₄1.8, J₃_,₂3.8), 5.61 (1H, s, H7), 7.37 - 7.49 (5H, m, -Ar); δ (CDCl , 100 MHz):

C 3

7.0 (C6), 62.1 (C2), 72.5 (C5), 73.7 (C4), 75.2 (C3), 100.1 (C7), 126.5, 128.8, 129.8, 136.8 (-Ar),

+

70.9 (C1); m/z (ESI+ve): 298 ([M + Na] , 100%).

5

.3 6-Deoxy-

L-DGDP 1 and 6-deoxy-

L-DMDP 2 from rhamnono-azides 8 and 9

2

-Azido-(R)-3,5-O-benzylidene-2,6-dideoxy-

L

-glucitol 22

Diisobutylaluminum hydride (25% w/v in toluene, 0.31 ml, 0.54 mmol) was added dropwise to a

solution of the lactone 8 (100 mg, 0.36 mmol) in anhydrous dichloromethane (3.6 mL) at -78 °C. The

reaction mixture was stirred at -78 °C for 1 h until mass spectrometry showed the formation of desired

+

product peak ([M + Na] 300) and disappearance of the starting material peak ([M + Na] 298). The

reaction mixture was diluted with ethyl acetate (15 mL) and potassium sodium tartrate (sat. aq., ~1

mL) was added. The reaction mixture was stirred for 8 h, then diluted with water (15 mL) and

extracted with ethyl acetate (3 x 15 mL). The organic phase was dried (MgSO ) and the solvent was

4

removed in vacuo; the residue was dissolved in methanol (3.6 mL) and then sodium borohydride (14

mg, 0.36 mmol) was added. After stirring the reaction mixture at rt for one h, TLC (ethyl

1

3

acetate/cyclohexane, 2:3) showed the formation of a single spot (R 0.18). Acetic acid was added to

f

neutralize the reaction mixture. The solvent was removed in vacuo and the residue was purified by

flash column chromatography (ethyl acetate/cyclohexane, 3:7 to 1:1) to afford the diol 22 (93.6 mg,

9

2 %) as a white solid.

+

HRMS (ESI+ve): found 302.1111 [M + Na] ; C H N O Na requires 302.1111; m.p. 105 - 106 °C;

1

3

17

3

4

25

[

(

3

α]_D-5.2 (c 1.0, CH CN); v (thin film): 3395 (br, OH), 2102 (s, N ); δ (CD CN, 400 MHz): 1.49

3 max 3 H 3

3H, d, H6, J₅_,₆7.2), 3.42 (1H, br-t, OH, J_O_H_,₁= J_O_H_,₁_’5.5), 3.39 (1H, dt, H4, J₄_,₃= J₄_,₅1.2, J₄_,_O_H9.5),

.53 (1H, d, OH, J_O_H_,₄9.5), 3.57 - 3.66 (1H, m, H1), 3.68 - 3.78 (2H, m, H1’, H3), 4.17 - 4.27 (2H, m,

H2, H5), 5.93 (1H, s, H7), 7.26 - 7.54 (5H, m, Ar); δ (CD CN, 100 MHz): 15.4 (C6), 61.2 (C1), 65.5

C

3

(

C3), 67.2 (C4), 76.2 (C2), 76.8 (C5), 94.9 (C7), 127.3, 129.1, 129.7, 139.8 (-Ar); m/z (ESI+ve): 302

[M + Na] , 100%).

+

2

-Azido-(R)-3,5-O-benzylidene-1,4-bis-(O-tert-butyldimethylsilyl)-2,6-dideoxy-L-glucitol 23

tert-Butyldimethylsilyl triflate (0.51 mL, 2.2 mmol) and anhydrous pyridine (0.22 mL, 2.7 mmol)

were added to a solution of diol 22 (250 mg, 0.90 mmol) in anhydrous dichloromethane (6 mL) at

0

°C. After stirring at 0 °C for 1 h, the mixture was stirred at rt for another 3 h. TLC (ethyl

acetate/cyclohexane, 1:19) showed the formation of one spot (R 0.27). The solvent was removed in

f

vacuo; the residue was dissolved in cyclohexane (25 mL) and washed with water (2 x 25 mL).

Organic phase was dried (MgSO ), filtered and the solvent removed to obtain a residue that was

4

purified by flash column chromatography (ethyl acetate/cyclohexane, 0:10 to 1:24) to afford the silyl

ether 23 (444 mg, 98 %) as a white solid.

+

HRMS (ESI+ve): found 530.2843 [M + Na] ; C H N O Si Na requires 530.2841; m.p. 61 - 62 °C;

2

5

45

3

4

2

25

[

α]_D-33.2 (c 1.0, MeCN); v_m_a_x(thin film): 2098 (s, N ); δ (CD CN, 400 MHz): 0.13 (3H, s, CH ),

3 H 3 3

0

1

.14 (3H, s, CH ), 0.15 (3H, s, CH ), 0.18 (3H, s, CH ), 0.95 (9H, s, 3 x CH ), 0.96 (9H, s, 3 x CH ),

3 3 3 3 3

.44 (3H, d, H6, J₆_,₅7.2), 3.60 (1H, s, H4), 3.66 (1H, a-dt, H2, J₂_,₁_’= J₂_,₁4.5, J₂_,₃8.8), 3.79 (1H, dd,

H1, J₁_,₂4.5, J_g_e_m11.1), 3.87 (1H, dd, H1’, J₁_’_,₂4.5, J_g_e_m11.1), 4.26 - 4.34 (2H, m, H3, H5), 5.94 (1H,

s, H7), 7.35 - 7.56 (5H, m, Ar); δ (CD CN, 100 MHz): -4.9 (CH ), -5.0 (CH ), -3.9 (CH ), -3.2

C

3

(

CH ), 16.1 (C6), 19.2 (C(CH ) ), 19.3 (C(CH ) ), 26.6 (C(CH ) ), 26.7 (C(CH ) ), 63.4 (C1), 65.1

3 3 3 3 3 3 3 3 3

C2), 69.1 (C4), 75.6 (C3), 76.3 (C5), 95.3 (C7), 127.8, 129.5, 130.2, 140.6 (-Ar); m/z (ESI+ve): 530

+

[M + Na] , 100%).

2

-Azido-3-O-benzoyl-5-bromo-1,4-bis-(O-tert-butyldimethylsilyl)-2,5,6-trideoxy-D-iditol 12

N-Bromosuccinimide (185 mg, 1.0 mmol) and barium carbonate (256 mg, 1.3 mmol) were added to a

solution of 23 (440 mg, 0.87 mmol) in carbon tetrachloride (5.8 mL). The stirred reaction mixture was

refluxed for 1 h until TLC (ethyl acetate/cyclohexane, 1:19) indicated the disappearance of starting

material (R 0.27) and the formation of one major product (R 0.52). After removal of the solvent in

f

vacuo, the residue was purified by flash column chromatography (ethyl acetate/hexane, 0:10 to 1:24)

to give the bromide 12 (437 mg, 86%) as a colorless oil.

+

HRMS (ESI+ve): found 586.2127, 588.2107 [M + H] ; C H BrN O Si requires 586.2127,

2

5

45

3

4

2

5

88.2106; [α]_D- 17.5 (c 1.0, MeCN); v_m_a_x(thin film): 1728 (s, C=O), 2102 (s, N ); δ (400 MHz,

3 H

CD CN): -0.08 (3H, s, CH ), 0.02 (3H, s, CH ), 0.04 (3H, s, CH ), 0.13 (3H, s, CH ), 0.85 (9H, s,

3

CH ), 0.88 (9H, s, CH ), 1.76 (3H, d, H6, J 6.9), 3.77 - 3.86 (2H, m, H1, H2), 3.88 - 3.96 (1H, m,

3

6,5

H1’), 4.08 (1H, dd, H4, J₄_,₅3.0, J₄_,₃6.6), 4.42 (1H, dq, H5, J₅_,₄3.0, J₅_,₆6.9), 5.54 (1H, dd, H3, J₃_,₂2.9,

J₃_,₄6.6), 7.53 (2H, t, Ar, J 7.7), 7.67 (1H, t, Ar, J 7.4), 8.03 - 8.10 (2H, m, Ar); δ (100 MHz,

C

CD CN): -5.0 (CH Si), -4.9 (CH Si), -3.2 (CH Si), -3.1 (CH Si), 19.1 (C(CH ) ), 19.2 (C(CH ) ), 23.9

3

3 3

(

1

C6), 26.5 (C(CH ) ), 26.6 (C(CH ) ), 53.3 (C5), 63.4 (C2), 64.8 (C1), 75.3 (C4), 75.8 (C3), 130.0,

31.1, 131.2, 134.8 (Ar), 166.7 (C=O); m/z (ESI+ve): 586, 588 ([M + H] , 100%, 93%).

3 3 3 3

+

3

-O-Benzoyl-1,4-bis-(O-tert-butyldimethylsilyl)-2,5-imino-2,5,6-trideoxy- -glucitol 24

L

Palladium on charcoal (10 % wt., 72 mg) and sodium acetate (84 mg, 1.0 mmol) were added to a

solution of 12 (399 mg, 0.68 mmol) in ethanol (20 mL). The reaction mixture was flushed

sequentially with nitrogen, argon and hydrogen gas and stirred vigorously overnight at rt under a

hydrogen atmosphere until TLC (ethyl acetate/cyclohexane, 3:7) showed the formation of one major

product (R 0.32). After filtration, the solvent was removed in vacuo and the residue purified by flash

f

1

4

column chromatography (triethylamine/ethyl acetate/cyclohexane, 0.01:3:7) to afford 24 (236 mg,

2%) as a yellow oil.

HRMS (ESI+ve): found 480.2957 [M + H] ; C H NO Si requires 480.2960; [α] +17.4 (c 1.0,

7

+

25

2

5

45

4

2

D

MeCN); v_m_a_x(thin film): 1722 (s, C=O); δ (CD CN, 400 MHz): -0.09 (3H, s, CH ), -0.05 (3H, s,

H

3

CH ), 0.05 (3H, s, CH ), 0.09 (3H, s, CH ), 0.82 (9H, s, CH ), 0.88 (9H, s, 3 x CH ), 1.18 (3H, d, H6,

3

J₆_,₅6.4), 2.98 (1H, quintet, H5, J 6.3), 3.45-3.51 (1H, m, H2), 3.60 (1H, dd, H1, J 4.9, J_g_e_m10.1),

5,4

1,2

3

.68 (1H, dd, H1’, J₁_’_,₂6.6, J_g_e_m10.1), 3.88 (1H, dd, H4, J₄_,₃4.2, J₄_,₅6.3), 5.27 (1H, dd, H3, J₃_,₄4.2,

J₃_,₂6.2), 7.50-7.56 (2H, m, Ar), 7.59 - 7.64 (1H, m, Ar), 8.05 - 8.07 (2H, m, Ar); δ (CD CN, 100

C

3

MHz): -5.1 (CH ), -5.0 (CH ), -4.1 (CH ), -3.9 (CH ), 18.9 C(CH ) , 19.1 (C(CH ) ), 20.0 (C6), 26.5

3

3 3

(

1

C(CH ) ), 26.6 (C(CH ) ), 60.6 (C5), 61.3 (C2), 63.5 (C1), 82.3 (C3), 84.0 (C4), 129.9, 130.8, 131.6,

34.6 (Ar), 167.0 (C=O); m/z (ESI+ve): 480 ([M + H] , 100%).

3 3 3 3

+

2

,5-Imino-2,5,6-trideoxy-L-glucitol (6-Deoxy-L-DGDP) 1

2

4 (236 mg, 0.49 mmol) was dissolved in a mixture of trifluoroacetic acid/water/1,4-dioxane (1:1:1, 6

mL). The solution was stirred at 50 °C for 24 h until mass spectrometry showed the deprotection of

+

the silyl groups ([M + H] 252). The solvent was removed in vacuo; the residue was dissolved in

aqueous sodium hydroxide (aq., 2 M, 10 mL) and stirred at 50 °C for 24 h until mass spectrometry

+

showed the formation of desired product ([M + H] 148). After removal of the solvent in vacuo, the

residue was dissolved in ethanol (5 mL), filtered and then loaded with water (~1 mL) onto a short

®

column of DOWEX 50WX8-200 resin (pre-washed with water, 1,4-dioxane and water sequentially

until the eluent was neutral). The ion exchange column was then washed with water, 1,4-dioxane and

then water and the pure product was then eluted with aqueous ammonia (2 M). Removal of the

solvent in vacuo gave the unprotected pyrrolidine 1 as a yellow oil (58 mg, 81%).

+

25

HRMS (ESI+ve): found 148.0967 [M + H] ; C H NO requires 148.0968; [α]_D–12.1 (c 0.45,

6

14

3

4

29

water) [lit. for 1D,the enantiomer of 1: [α]_D+5.2 (c 1.0, MeOH)]; v_m_a_x(thin film): 3300 (br, NH,

OH); δ (D O, 400 MHz): 1.25 (3H, d, H6, J 6.6), 2.92 (1H, quintet, H5, J₅_,₄= J₅_,₆6.6), 3.05 (1H, q,

H

2

6,5

H2, J₂_,₁= J₂_,₁_’= J₂_,₃6.0), 3.64 (1H, dd, H4, J₄_,₃3.5, J₄_,₅6.6), 3.67 (1H, dd, H1, J₁_,₂6.0, J_g_e_m11.4), 3.79

(

5

1H, dd, H1’, J₁_’_,₂6.0, J_g_e_m11.4), 4.12 (1H, dd, H3, J₃_,₄3.5, J₃_,₂6.0); δ (D O, 100 MHz): 17.9 (C6),

9.2 (C5), 60.6 (C1), 61.2 (C2), 78.3 (C3), 84.4 (C4); m/z (ESI+ve): 148 ([M + H] , 100%).

C 2

+

2

-Azido-(R)-3,5-O-benzylidene-2,6-dideoxy- -mannitol 25

L

Diisobutylaluminium hydride (25% w/v in toluene, 2.33 ml, 4.05 mmol) was added dropwise to a

solution of 9 (500 mg, 1.80 mmol) in anhydrous dichloromethane (18 mL) at -78 °C. The solution

was stirred at -78°C for 1.5 h until mass spectrometry showed the formation of desired product peak

+

(

[M + Na] 300) and disappearance of starting material peak ([M+Na] 298). Then the mixture was

diluted with ethyl acetate (25 mL) and potassium sodium tartrate (sat, aq., ~5 mL) was added and

allowed to stand at rt for 8h. The reaction mixture was diluted with water (50 mL) and extracted with

ethyl acetate (3 x 50 mL). Organic phase was dried (MgSO ), filtered and solvent was removed in

4

vacuo to obtain a residue that was dissolved in methanol (18 mL) and sodium borohydride (69 mg,

1

.80 mmol) was added. After stirring at rt for one h, TLC (ethyl acetate/cyclohexane, 2:3) showed the

formation of a single spot (R 0.32). Acetic acid was added to neutralize the reaction mixture. After

f

removing the solvent in vacuo, the crude product was purified by flash column chromatography (ethyl

acetate/cyclohexane, 3:7 to 1:1) to afford the diol 25 (452 mg, 89 %) as a white solid.

+

HRMS (ESI+ve): found 302.1111 [M + Na] ; C H N O Na requires 302.1111; m.p. 108 - 109 °C;

1

3

17

3

4

25

[

α]_D+19.8 (c 1.0, MeCN); v_m_a_x(thin film): 3406 (br, OH), 2102 (s, N ); δ (CD CN, 400 MHz):

3 H 3

1

.44 (3H, d, H6, J₆_,₅7.2), 3.11 (1H, br-t, OH, J_O_H_,₁= J_O_H_,₁_’5.7), 3.43 - 3.51 (2H, m, H4, OH), 3.65

(

1H, dt, H1, J₁_,₂5.9, J_g_e_m11.8), 3.73 (1H, ddd, H2, J₂_,₁_’2.6, J₂_,₁5.9, J₂_,₃9.1), 3.88 (1H, ddd, H1’, J₁_’_,₂

2

7

.6, J₁_’_,_O_H5.7, J_g_e_m11.8), 4.01 (1H, d, H3, J₃_,₂9.1), 4.26 (1H, q, H5, J₅_,₆7.2), 5.85 (1H, s, H7), 7.33 -

.53 (5H, m, Ar); δ (CD CN, 100 MHz): 15.2 (C6), 62.4 (C1), 63.4 (C2), 66.8 (C4), 73.9 (C3), 76.9

C

3

+

(

C5), 94.7 (H7), 127.2, 129.1, 129.7, 139.8 (Ar); m/z (ESI+ve): 302 ([M + Na] , 100%).

2

-Azido-(R)-3,5-O-benzylidene-1,4-bis-(O-tert-butyldimethylsilyl)-2,6-dideoxy- -mannitol 26

L

tert-Butyldimethylsilyl triflate (1.0 mL, 4.50 mmol) and anhydrous pyridine (0.45 mL, 5.4 mmol)

were added to a solution of 25 (500 mg, 1.80 mmol) in anhydrous dichloromethane (12 mL) at 0 °C.

After stirring at 10 °C for 3 h, TLC (ethyl acetate/cyclohexane, 1:4) showed the formation of a new

1

5

spot (R 0.73) but reaction was not complete. Further tert-butyldimethylsilyl triflate (0.33 mL, 1.49

f

mmol) and anhydrous pyridine (0.15 mL, 1.80 mmol) were added to the reaction mixture which was

stirred at -18 °C for 16 h until TLC indicated the completion of reaction. After removing the solvent

in vacuo, the residue was dissolved in ethyl acetate (50 mL) and washed with half saturated brine (50

mL) and then brine (50 mL). The organic phase was dried (MgSO ), filtered and solvent was removed

4

to obtain a residue that was purified by flash column chromatography (ethyl acetate/cyclohexane, 0:10

to 1:4) to afford 26 (852 mg, 94 %) as a colorless oil.

+

25

HRMS (ESI+ve): found 530.2838 [M + Na] ; C H N O Si Na requires 530.2841; [α] +7.6 (c 1.0,

2

5

45

3

4

2

D

MeCN); v_m_a_x(thin film): 2099 (s, N ); δ (CD CN, 400 MHz): 0.08 (6H, s, CH ), 0.150 (3H, s, CH ),

3

H

3

0

.153 (3H, s, CH ), 0.92 (9H, s, 3 x CH ), 0.95 (9H, s, 3 x CH ), 1.41 (3H, d, H6, J 7.2), 3.63 (1H,

3 3 3 6,5

br-s, H4), 3.66 (1H, ddd, H2, J₂_,₁_’1.9, J₂_,₁5.0, J₂_,₃7.5), 3.87 (1H, dd, H1, J₁_,₂5.0, J_g_e_m10.9), 4.01 (1H,

dd, H3, J₃_,₄1.1, J₃_,₂7.5), 4.06 (1H, dd, H1’, J₁_’_,₂1.9, J_g_e_m10.9), 4.25 (1H, q, H5, J₅_,₆7.2), 5.80 (1H, s,

H7), 7.32 - 7.47 (5H, m, Ar); δ (CD CN, 100 MHz): -5.3 (CH Si), -5.3 (CH Si), -4.7 (CH Si), -4.2

C

3

(

CH Si), 15.4 (C6), 18.79 (C(CH ) ), 18.84 (C(CH ) ), 26.1 (C(CH ) ), 26.2 (C(CH ) ), 62.1 (C2),

3 3 3 3 3 3 3 3 3

6

5

4.1 (C1), 67.8 (C4), 73.3 (C3), 76.2 (C5), 94.5 (C7), 127.2, 129.0, 129.7, 140.1 (Ar); m/z (ESI+ve):

30 ([M + Na] , 100%).

+

2

-Azido-3-O-benzoyl-5-bromo-1,4-bis-(O-tert-butyldimethylsilyl)-2,5,6-trideoxy-D-gulitol 13

N-Bromosuccinimide (358 mg, 2.0 mmol) and barium carbonate (496 mg, 2.5 mmol) were added to a

solution of 26 (851 mg, 1.70 mmol) in carbon tetrachloride (11.2 mL). The mixture was refluxed with

stirring for 50 min until TLC (ethyl acetate/cyclohexane, 1:19) indicated the disappearance of starting

material (R 0.50) and the formation of one major product (R 0.62). After removal of the solvent in

f

vacuo, the residue was purified by flash column chromatography (ethyl acetate/hexane 0:10 to 1:24)

to afford 13 (909 mg, 92%) as a colorless oil.

+

HRMS (ESI+ve): found 608.1944, 610.1924 [M + Na] ; C H BrN O Si Na requires 608.1946,

6

2

5

44

3

4

2

5

08.1926; [α]_D-22.2 (c 1.0, MeCN); v_m_a_x(thin film): 2098 (s, N ), 1728 (s, C=O); δ (CD CN, 400

3 H 3

MHz): 0.05 (3H, s, CH ), 0.06 (3H, s, CH ), 0.08 (3H, s, CH ), 0.16 (3H, s, CH ), 0.91 (9H, s, 3 x

3

CH ), 0.93 (9H, s, 3 x CH ), 1.66 (3H, d, H6, J 6.9), 3.79 - 3.91 (2H, m, H1, H2), 3.96 (1H, dd, H1’,

3

6,5

J₁_’_,₂3.7, J_g_e_m10.0), 4.21 (1H, t, H4, J₄_,₃= J₄_,₅4.0), 4.34 (1H, dq, H5, J₅_,₄4.0, J₅_,₆6.9), 5.60 (1H, dd,

H3, J₃_,₄4.0, J₃_,₂6.3), 7.49 - 7.57 (2H, m, -Ar), 7.63 - 7.70 (1H, m, -Ar), 8.01 - 8.09 (2H, m, -Ar); δ_C

(

CD CN, 100 MHz): -5.1 (CH Si), -5.0 (CH Si), -3.5 (CH Si), -3.3 (CH Si), 19.1 (C(CH ) ), 22.5

3 3 3 3 3 3 3

(

1

C6), 26.4 (C(CH ) ), 26.5 (C(CH ) ), 52.9 (C5), 63.5 (C2), 64.0 (C1), 73.8 (C3), 74.6 (C4), 130.0,

3 3 3 3

30.9, 131.2, 134.8 (-Ar), 166.4 (C=O); m/z (ESI+ve): 586, 588 ([M + H] , 100%, 97%).

+

3

-O-Benzoyl-1,4-bis-(O-tert-butyldimethylsilyl)-2,5-imino-2,5,6-trideoxy- -mannitol 27

L

Palladium on charcoal (10 % wt., 165 mg) and sodium acetate (191 mg, 2.3 mmol) were added to a

solution of 13 (909 mg, 1.5 mmol) in ethanol (45 mL). The reaction mixture was flushed with

nitrogen, argon and hydrogen gas sequentially. Then the reaction mixture was stirred vigorously

overnight at rt under a hydrogen atmosphere for 12 h until TLC (ethyl acetate/cyclohexane, 1:4)

showed the formation of one major product (R 0.17). Filtration and evaporation in vacuo gave a

f

crude bromide salt (~1.2 g). Part of the salty residue (120 mg) was purified by flash column

chromatography (triethylamine/ethyl acetate/cyclohexane, 0.01:3:7) to give a pure sample of 27 (64

mg, 71%) as a yellow oil for full characterization. The crude salt was used in the next step.

+

25

HRMS (ESI+ve): found 480.2959 [M + H] ; C H NO Si requires 480.2960; [α] +10.6 (c 1.0,

2

5

45

4

2

D

MeCN); v_m_a_x(thin film): 3350 (br, NH), 1722 (s, C=O); δ (CD OD, 400 MHz): 0.03 (3H, s, CH ),

H

3

0

1

.05 (3H, s, CH ), 0.12 (3H, s, CH ), 0.16 (3H, s, CH ), 0.87 (9H, s, 3 x CH ), 0.94 (9H, s, 3 x CH ),

.32 (3H, d, H6, J₆_,₅6.6), 3.35 - 3.49 (2H, m, H2, H5), 3.84 (1H, dd, H1, J₁_,₂3.9, J_g_e_m10.6), 4.03 (1H,

3 3 3 3 3

dd, H1’, J₁_’_,₂4.4, J_g_e_m10.6), 4.16 (1H, dd, H4, J₄_,₃5.2, J₄_,₅7.3), 5.32 (1H, t, H3, J₃_,₂= J₃_,₄4.6), 7.51

(

2H, t, -Ar, J 7.7), 7.64 (1H, t, -Ar, J 7.4), 8.07 (2H, d, -Ar, J 7.4); δ (CD OD, 100 MHz): -5.3

C 3

(

CH Si), -5.2 (CH Si), -4.6 (CH Si), -4.2 (CH Si), 17.1 (C6), 18.7 (C(CH ) ), 19.1 (C(CH ) ), 26.2

3 3 3 3 3 3 3 3

(

1

C(CH ) ), 26.4 (C(CH ) ), 60.2 (C5), 64.0 (C2), 64.7 (C1), 82.8 (C3), 83.3 (C4), 129.7, 130.7, 130.8,

34.6 (Ar), 167.4 (C=O); m/z (ESI+ve): 480 ([M + H] , 100%).

3 3 3 3

+

2

,5-Imino-2,5,6-trideoxy- -mannitol (6-Deoxy- -DMDP) 2

L

1

6

The crude product 27 (617 mg) was dissolved in a solution of trifluoroacetic acid/water/1,4-dioxane

2:1:1, 20 mL). The solution was stirred at 50 °C for 18 h until mass spectrometry showed the

(

+

deprotection of the silyl groups ([M + H] 252). After the solvent was removed in vacuo, the residue

was dissolved into sodium hydroxide solution (aq, 2M, 5 mL) and stirred at 50 °C for 24 h until mass

+

spectrometry showed the formation of desired product ([M + H] 148). The mixture was neutralized

with HCl (2M, aq) and the solvent removed in vacuo. The residue was dissolved in ethanol (5 mL)

and filtered (glass microfiber) to afford a crude salt that was then loaded with water (~1 mL) onto a

®

short column of DOWEX 50WX8-200 resin. The ion exchange column was then washed with water,

1

,4-dioxane and then water; the pure product was then eluted with aqueous ammonia (2 M). Removal

of solvent in vacuo gave a residue which was subjected to a further purification cycle by resin column

to yield 6-deoxy- -DMDP 2 as a yellow oil (121 mg, 64% from crude salt of 27).

HRMS (ESI+ve): found 148.0968 [M + H] ; C H NO requires 148.0968; [α] -42 (c 1.0, MeOH)

L

+

25

6

13

3

D

3

5

25

[

lit. [α] -33 (c 1.2, MeOH)]; v_m_a_x(thin film): 3277 (br, NH, OH); δ (D O, 400 MHz): 1.19 (3H,

D

H

2

d, H6, J₆_,₅6.4), 2.96 (1H, dq, H5, J₅_,₆6.4, J₅_,₄7.3), 3.05 (1H, br-q, H2, J 6.5), 3.58 – 3.64 (2H, m, H4,

H1), 3.68 (1H, dd, H1’, J₁_’_,₂4.6, J_g_e_m11.4), 3.81 (1H, t, H3, J₃_,₄= J₃_,₂6.8); δ (D O, 100 MHz): 17.8

C

2

+

(

C6), 58.2 (C5), 62.0, 63.0 (C1, C2), 78.7 (C3), 83.3 (C4); m/z (ESI+ve): 148 ([M + H] , 100%); m/z

ESI+ve): 148 ([M + H] , 100%).

+

5

.4 6-Deoxy-

D-ido-DMDP 3 and 6-deoxy-

D-gulo-DMDP 4 from

D-gulono-azides 10 and 11

2

-Azido-(R)-3,5-O-benzylidene-2,6-dideoxy-

D-iditol 28

Diisobutylaluminium hydride (25% w/v in toluene, 4.83 ml, 8.50 mmol) was added dropwise to a

solution of 10 (467 mg, 1.70 mmol) in anhydrous dichloromethane (15 mL) at -78 °C. The reaction

mixture was stirred at -78 °C for 3 h until mass spectrometry showed the formation of desired product

+

peak ([M+Na] 300) and disappearance of starting material peak ([M + Na] 298). Then the mixture

was diluted with ethyl acetate (20 mL) and potassium sodium tartrate (sat, aq., ~2 mL) was added.

After stirring for 12 h, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (3

x 20 mL). Organic phase was dried (MgSO ), filtered and solvent was removed in vacuo to obtain a

4

residue that was dissolved in methanol (15 mL) and sodium borohydride (84 mg, 2.20 mmol) was

added. After stirring at rt for one h, TLC (ethyl acetate/cyclohexane, 1:1) showed the formation of a

new spot (R 0.32). Acetic acid was added to neutralize the reaction mixture. The solvent was

f

removed in vacuo, the resulting crude was purified by flash column chromatography (ethyl

acetate/cyclohexane, 1:4 to 1:1) to afford the ido-diol 28 (312 mg, 66 %) as a pale syrup.

+

25

HRMS (ESI+ve): Found 302.1111 [M + Na] ; C H O N Na requires 302.1111; [α] +5.2 (c 0.78,

1

3

17

4

3

D

CHCl ); v (thin film): 3400 (br, OH), 2108 (s, N ); δ (CDCl , 400MHz): 1.37 (3H, d, H6, J 6.4),

3

max

3

H

3

6,5

2

3

.24 (1H, br-s, OH), 3.48 (1H, br-s, H4), 3.76 (1H, dd, H1, J₁_,₂4.2, J_g_e_m12.0), 3.82 (1H, dd, H1’, J₁_’_,₂

.1, J_g_e_m12.0), 3.90 (1H, ddd, H2, J₂_,₁_’3.1, J₂_,₁4.2, J₂_,₃8.5), 4.06 (1H, dq, H5, J₅_,₄0.6, J₅_,₆6.5), 4.35

(

1H, dd, H3, J₃_,₄0.6, J₃_,₂8.5), 5.69 (1H, s, H7), 7.36 - 7.56 (5H, m, -Ar); δ (CDCl , 100MHz): 17.2

C 3

C6), 61.2 (C1), 64.3 (C2), 66.6 (C4), 76.6 (C5), 81.5 (C3), 101.4 (C7), 126.1, 128.6, 129.3, 137.6 (-

+

Ar); m/z (ESI+ve): 302 ([M + Na] , 100%).

2

-Azido-(R)-3,5-O-benzylidene-1,4-bis-(O-tert-butyldimethylsilyl)-2,6-dideoxy-D-iditol 29

tert-Butyldimethylsilyl triflate (1.03 mL, 4.5 mmol) and anhydrous pyridine (0.59 mL, 7.2 mmol)

were added to a solution of 28 (250 mg, 0.90 mmol) in anhydrous dichloromethane (6 mL) at 0 °C.

Then the mixture was stirred at rt for 18 h until TLC (ethyl acetate/cyclohexane, 1:4) showed the

formation of a major spot (R 0.78). Removal of the solvent in vacuo gave a residue that was

f

dissolved in cyclohexane (25 mL) and washed with water (2 x 25 mL). The organic phase was dried

(

MgSO ), filtered and the solvent removed to obtain a residue that was purified by flash column

4

chromatography (ethyl acetate/cyclohexane, 1:9) to afford the fully protected 29 (334 mg, 74 %) as a

colorless oil.

+

25

HRMS (ESI+ve): found 530.2840 [M + Na] ; C H N O Si Na requires 530.2841; [α]

-35

c 0.81, CHCl ); v (thin film): 2100 (s, N ); δ (CDCl , 400 MHz): 0.10 (3H, s, CH ), 0.11 (6H, s, 2

25

45

3

4

2

D

(

3

max

3

H

3

x CH ), 0.15 (3H, s, CH ), 0.92 (9H, s, 3 x CH ), 0.97 (9H, s, 3 x CH ), 1.31 (3H, d, H6, J 6.4), 3.56

3

6,5

(

1H, br-s, H4), 3.60 (1H, ddd, H2, J₂_,₁_’3.2, J₂_,₁4.6, J₂_,₃9.0), 3.75 (1H, dd, H1, J₁_,₂4.6, J_g_e_m10.7), 3.82

1H, dd, H1’, J₁_’_,₂3.1, J_g_e_m10.7), 3.92 (1H, q, H5, J₅_,₆6.5), 4.00 (1H, d, H3, J₃_,₂9.0), 5.62 (1H, s, H7),

1

7

.33 - 7.55 (5H, m, -Ar); δ (CDCl , 100 MHz): -5.2 (CH Si), -5.1 (CH Si), -2.8 (2 x CH Si), 18.6 (2

C 3 3 3 3

x (C(CH ) )), 19.1 (C6), 26.1 (C(CH ) ), 26.5 (C(CH ) ), 62.4, 62.5 (C1, C2), 68.4 (C4), 76.9 (C5),

3

3 3

+

7

9.8 (C3), 102.2 (C7), 126.8, 128.6, 129.2, 138.6 (-Ar); m/z (ESI+ve): 530 ([M + Na] , 100%).

2

-Azido-3-O-benzoyl-5-bromo-1,4-bis-(O-tert-butyldimethylsilyl)-2,5,6-trideoxy- -glucitol 14

L

N-Bromosuccinimide (146 mg, 0.83 mmol) and barium carbonate (205 mg, 1.04 mmol) were added

into a solution of 29 (350 mg, 0.69 mmol) in carbon tetrachloride (5 mL). The reaction mixture was

refluxed with stirring for 1 h until TLC (ethyl acetate/cyclohexane, 1:19) indicated the disappearance

of the starting material (R 0.52) and the formation of one major product (R 0.71). After removing the

f

solvent in vacuo, the residue was purified by flash column chromatography (ethyl acetate/hexane, 1:9)

to afford the bromide 14 (400 mg, 99%) as a yellow oil.

+

HRMS (ESI+ve): found 586.2127, 588.2105 [M + H] ; C H BrN O Si requires 586.2127,

5

2

5

45

3

4

2

5

88.2106; [α]_D-11 (c 0.66, CHCl ); v (thin film): 2098 (s, N ), 1728 (s, C=O); δ (400 MHz,

3 max 3 H

CDCl ): 0.03 (3H, s, CH ), 0.04 (3H, s, CH ), 0.08 (3H, s, CH ), 0.18 (3H, s, CH ), 0.86 (9H, s, 3 x

3

CH ), 0.87 (9H, s, 3 x CH ), 1.74 (3H, d, H6, J 6.9), 3.69 – 3.73 (1H, m, H2), 3.79 (1H, dd, H1, J₁_,₂

3

6,5

7

.0, J_g_e_m10.3), 3.87 (1H, dd, H1’, J₁_’_,₂4.9, J_g_e_m10.2), 4.26 (1H, t, H4, J₄_,₃= J₄_,₅5.0), 4.41 (1H, dq,

H5, J₅_,₄4.8, J₅_,₆6.7), 5.28 (1H, dd, H3, J₃_,₂3.7, J₃_,₄5.2), 7.46 (2H, t, -Ar, J 7.7), 7.60 (1H, t, -Ar, J

7

.3), 8.08 – 8.10 (2H, m, -Ar); δ (100 MHz, CDCl ): -5.3 (2 x CH Si), -3.9 (CH Si), -3.8 (CH Si),

C 3 3 3 3

1

8.5 (C(CH ) ), 18.6 (C(CH ) ), 22.1 (C6), 26.1 (C(CH ) ), 26.2 (C(CH ) ), 49.5 (C5), 62.2 (C2), 63.8

3

3 3

(

C1), 73.8 (C4), 75.9 (C3), 128.6, 129.9, 130.4, 133.7 (-Ar), 165.9 (C=O); m/z (ESI+ve): 586, 588

[M + H] , 100%, 98%).

+

3

-O-Benzoyl-1,4-bis-(O-tert-butyldimethylsilyl)-2,5-imino-2,5,6-trideoxy-L-glucitol 30

Palladium on charcoal (10 % wt., 60 mg) and sodium acetate (82 mg, 0.99 mmol) were added to a

solution of the bromide 14 (385 mg, 0.66 mmol) in ethanol (20 mL). The solution was flushed with

nitrogen, argon and hydrogen gas sequentially, and then stirred vigorously at rt under hydrogen

atmosphere for 2 h until TLC (ethyl acetate/cyclohexane, 1:4) showed the formation of one major

product (R 0.30). The reaction mixture was filtered, the solvent removed and the resultant crude

f

product 30 (400 mg) was used for the next step without further purification. A small portion of the

crude material was purified for characterization by flash column chromatography (triethylamine/ethyl

acetate/cyclohexane, 0.01:3:7) to afford 30 as a yellow oil.

+

25

HRMS (ESI+ve): found 480.2955 [M + H] ; C H BrNO Si requires 480.2960; [α] -13 (c 1.45,

2

5

45

4

2

D

MeOH); v_m_a_x(thin film): 1723 (s, C=O); δ (CD OD, 400 MHz): -0.09 (3H, s, CH ), 0.01 (3H, s,

H

3

CH ), 0.17 (3H, s, CH ), 0.26 (3H, s, CH ), 0.82 (9H, s, 3 x CH ), 0.99 (9H, s, 3 x CH ), 1.20 (3H, d,

3

H6, J₆_,₅6.6), 3.44 (1H, dq, H5, J₅_,₄3.5, J₅_,₆6.6), 3.74 – 3.85 (3H, m, H1, H1’, H2), 4.10 (1H, br-d, H4,

J 2.0), 5.39 (1H, br-d, H3, J 2.1), 7.52 (2H, t, -Ar, J 7.7), 7.65 (1H, t, -Ar, J 7.4), 8.08 (2H, d, -Ar, J

7

.3); δ (CD OD, 100 MHz): -4.6 (CH Si), -4.5 (CH Si), -4.1 (CH Si), -3.6 (CH Si), 15.5 (C6), 19.8

C 3 3 3 3 3

(

C(CH ) ), 19.7 (C(CH ) ), 27.1 (C(CH ) ), 27.2 (C(CH ) ), 58.3 (C5), 62.1 (C2), 63.3 (C1), 79.6

3 3 3 3 3 3 3 3

+

C4), 81.5 (C3), 130.5, 131.5, 132.2, 135.3 (-Ar), 167.7 (C=O); m/z (ESI+ve): 480 ([M + H] , 100%).

,5-Imino-2,5,6-trideoxy- -iditol 3

2

D

The crude protected pyrrolidine 30 (400 mg) was dissolved in a solution of trifluoroacetic

acid/water/1,4-dioxane (2:1:1, 10 mL). The solution was stirred at 50 °C for 24 h until mass

+

spectrometry showed the deprotection of the silyl groups ([M + H] 252). The solvent was removed

and the residue dissolved in sodium hydroxide solution (aq., 2M, 8 mL); the resulting solution was

then stirred at 50 °C for 24 h until mass spectrometry showed the formation of the desired product

+

(

[M + H] 148). The reaction mixture was neutralized with HCl (2M, aq) and the solvent removed in

vacuo. The residue was dissolved in ethanol (5 mL) and filtered (glass microfiber) to afford a crude

®

salt that was then loaded with water (~1 mL) onto a short column of DOWEX 50WX8-200 resin.

The ion exchange column was then washed with water, 1,4-dioxane and then water; the pure product

was then eluted with aqueous ammonia (2 M). Removal of solvent in vacuo gave a residue which was

subjected to a further purification cycle by resin column to give the title compound 3 (72 mg, 74% 3

steps from 14) as a yellow oil.

+

25

HRMS (ESI+ve): found 148.0968 [M + H] ; C H N O requires 148.0968; [α]_D+2.4 (c 0.17,

6

14

3

water); v_m_a_x(thin film): 3300 (br, NH, OH); δ (D O, 400 MHz): 1.15 (3H, d, H6, J 6.7), 3.43 (1H,

H

2

6,5

1

8

dq, H5, J₅_,₄3.7, J₅_,₆6.7), 3.05 (1H, ddd, H2, J₂_,₃4.9, J ₂_,₁_’6.6, J₂_,₁7.2), 3.65 (1H, dd, H1, J₁_,₂7.2, J_g_e_m

1

1.4), 3.77 (1H, dd, H1’, J₁_’_,₂6.6, J_g_e_m11.4), 3.98 (1H, dd, H4, J₄_,₃1.5, J₄_,₅3.6), 4.24 (1H, dd, H3, J₃_,₄

.4, J₃_,₂4.9); δ (D O, 100 MHz): 13.2 (C6), 55.5 (C5), 60.7, 60.8 (C1, C2), 77.2 (C3), 78.9 (C4); m/z

C

2

+

(

ESI+ve): 148 ([M + H] , 100%).

2

-Azido-(R)-3,5-O-benzylidene-2,6-dideoxy-D-gulitol 31

Lithium borohydride (2M in THF, 0.74 mL, 1.47 mmol) was added to a solution of lactone 11 (270

mg, 0.98 mmol) in anhydrous THF at -20 °C. The reaction mixture was stirred at -20 °C for 2 h when

TLC (ethyl acetate/cyclohexane, 2:1) showed the consumption of starting material (R 0.51) and the

f

formation of a new spot (R 0.71). After addition of acetic acid (0.4 mL) to the mixture at -20 °C with

f

stirring, the solvent was removed in vacuo to obtain a residue that was re-dissolved in ethyl acetate

(

20 mL) and washed with water (20 mL). The aqueous layer was back extracted with ethyl acetate (2

x 15 mL). The organic phase was combined, dried (MgSO ) and the solvent was removed in vacuo to

4

yield a residue that was purified by flash column chromatography (ethyl acetate/cyclohexane, 1:2 to

2

:1) to afford the diol 31 (246 mg, 90%) as a white solid.

+

HRMS (ESI+ve): Found 302.1111 [M + Na] ; C H N O Na requires 302.1113; m.p.: 100 – 102 °C;

1

3

17

3

4

25

[

α]_D+19 (c 0.69, CHCl ); v (thin film): 2102 (s, N ); δ (CDCl , 400 MHz): 1.40 (3H, d, H6, J

3 max 3 H 3 6,5

6

.4), 3.58 (1H, s, H4), 3.82 (1H, dd, H1, J₁_,₂4.9, J_g_e_m11.4), 3.85 (1H, d, H3, J₃_,₂9.1), 3.88 – 3.92 (1H,

m, H4), 3.98 (1H, dd, H1’, J₁_’_,₂3.2, J_g_e_m12.0), 3.90 (1H, ddd, H2, J₂_,₁_’3.1, J₂_,₁4.2, J₂_,₃8.5), 4.07 (1H,

q, H5, J₅_,₆6.4), 5.60 (1H, s, H7), 7.37 - 7.48 (5H, m, -Ar); δ (CDCl , 100 MHz): 17.5 (C6), 62.4,

C

3

6

2.5 (C1, C2), 66.2 (C4), 76.9 (C5), 79.1 (C3), 101.5 (C7), 126.2, 128.7, 129.5, 137.6 (-Ar); m/z

+

(

ESI+ve): 302 ([M + Na] , 100%).

2

-Azido-(R)-3,5-O-benzylidene-1,4-bis-(O-tert-butyldimethylsilyl)-2,6-dideoxy-D-gulitol 32

tert-Butyldimethylsilyl triflate (0.27 mL, 1.18 mmol) and anhydrous pyridine (0.11 mL, 1.41 mmol)

were added to a solution of 31 (130 mg, 0.47 mmol) in anhydrous dichloromethane (6 mL) at 0 °C.

The reaction mixture was stirred at rt for 15 h until TLC (ethyl acetate/cyclohexane, 1:9) showed the

formation of a major spot (R 0.73). Removal of solvent in vacuo gave a residue that was dissolved in

f

cyclohexane (20 mL) and washed with water (2 x 15 mL). The organic phase was dried (MgSO₄),

filtered and the solvent was removed to obtain a residue that was purified by flash column

chromatography (ethyl acetate/cyclohexane, 1:9) to afford the silyl ether 32 (200 mg, 84%) as a

colorless oil.

+

25

HRMS (ESI+ve): found 530.2840 [M + Na] ; C H N O Si Na requires 530.2841; [α]

+6.6

c 0.80, CHCl ); v (thin film): 2103 (s, N ); δ (400 MHz, CDCl ): 0.04 (3H, s, CH ), 0.05 (3H, s,

2

5

45

3

4

2

D

(

3

max

3

H

3

CH ), 0.14 (3H, s, CH ), 0.17 (3H, s, CH ), 0.90 (9H, s, 3 x CH ), 0.99 (9H, s, 3 x CH ), 1.30 (3H, d,

3

H6, J₆_,₅6.4), 3.53 (1H, ddd, H2, J₂_,₁_’2.3, J₂_,₁4.9, J₂_,₃10.0), 3.66 (1H, s, H4), 3.76 (1H, d, H3, J₃_,₂

1

7

0.0), 3.89 – 3.95 (2H, m, H1, H5), 4.01 (1H, dd, H1’, J₁_’_,₂2.4, J_g_e_m10.7), 5.51 (1H, s, H7), 7.32 -

.47 (5H, m, -Ar); δ (100 MHz, CDCl ): -5.3 (CH Si), -5.2 (CH Si), -3.6 (CH Si), -3.4 (CH Si), 18.6

C

3

(

C(CH ) ), 18.8 (C(CH ) ), 22.3 (C6), 19.0 (C(CH ) ), 26.1 (C(CH ) ), 26.6 (C(CH ) ), 59.9 (C2),

3 3 3 3 3 3 3 3 3 3

6

3.2 (C1), 66.7 (C4), 76.9 (C5), 78.1 (C3), 102.1 (C7), 126.9, 128.6, 129.3, 138.8 (-Ar); m/z

+

(

ESI+ve): 530 ([M + Na] , 100%).

2

-Azido-3-O-benzoyl-5-bromo-1,4-bis-(O-tert-butyldimethylsilyl)-2,5,6-trideoxy-L-mannitol 15

N-Bromosuccinimide (55 mg, 0.47 mmol) and barium carbonate (115 mg, 0.59 mmol) were added to

a solution of 32 (200 mg, 0.39 mmol) in carbon tetrachloride (6 mL). The reaction mixture was

refluxed for 1 h until TLC (ethyl acetate/cyclohexane, 1:19) indicated the disappearance of starting

material (R 0.70) and the formation of one major product (R 0.62). After removal of the solvent in

f

vacuo, the residue was purified by flash column chromatography (ethyl acetate/hexane, 1:9) to give

the manno-bromide 15 (173 mg, 76%) as a yellow oil.

+

HRMS (ESI+ve): found 586.2128, 588.2107 [M + H] ; C H BrN O Si requires 586.2127,

5

2

5

45

3

4

2

5

88.2106; [α]_D-8.1 (c 0.93, CHCl ); v (thin film): 2103 (s, N ), 1730 (s, C=O); δ (CDCl , 400

3 max 3 H 3

MHz): -0.01 (3H, s, CH ), 0.01 (3H, s, CH ), 0.21 (3H, s, CH ), 0.22 (3H, s, CH ), 0.87 (9H, s, 3 x

3

CH ), 0.98 (9H, s, 3 x CH ), 1.69 (3H, d, H6, J 6.9), 3.70 – 3.81 (2H, m, H1, H2), 3.86 (1H, dd,

3

6,5

H1’, J₁_’_,₂2.7, J_g_e_m10.4), 4.16 (1H, dq, H5, J₅_,₄5.7, J₅_,₆6.7), 4.29 (1H, dd, H4, J₄_,₃1.7, J₄_,₅5.7), 5.41

1H, dd, H3, J₃_,₄1.7, J₃_,₂9.2), 7.46 (2H, t, -Ar, J 7.6), 7.59 (1H, t, -Ar, J 7.5), 8.05 (2H, d, -Ar, J 7.3);

(

1

9

δ (CDCl , 100 MHz): -5.3 (2 x CH Si), -3.9 (CH Si), -3.8 (2 x CH Si), 18.5 (C(CH ) ), 18.8

C

3

3 3

(

C(CH ) ), 22.3 (C6), 26.1 (C(CH ) ), 26.4 (C(CH ) ), 50.3 (C5), 61.9 (C2), 64.0 (C1), 71.9 (C3), 75.4

C4), 128.9, 130.0, 130.3, 133.7 (-Ar), 165.7 (C=O); m/z (ESI+ve): 586, 588 ([M + H] , 100%, 98%).

3 3 3 3 3 3

+

3

-O-Benzoyl-1,4-bis-(O-tert-butyldimethylsilyl)-2,5-imino-2,5,6-trideoxy- -gulitol 33

D

Palladium on charcoal (10 % wt., 40 mg) and sodium acetate (49 mg, 0.60 mmol) were added to a

solution of 15 (233 mg, 0.40 mmol) in ethanol (15 mL). The reaction mixture was flushed with

nitrogen, argon and hydrogen gas sequentially. Then the reaction mixture was stirred vigorously at rt

under hydrogen atmosphere for 5 h until TLC (ethyl acetate/cyclohexane, 1:4) showed the formation

of one major product (R 0.14). Filtration and evaporation to dryness in vacuo gave the crude

f

protected pyrrolidine 33 (170 mg) which was used for the next step without further purification. A

small portion was purified for characterization by flash column chromatography (triethylamine/ethyl

acetate/cyclohexane, 0.01:2:1) to produce a pure sample of 33 as a yellow oil.

+

25

HRMS (ESI+ve): found 480.2958 [M + H] ; C H BrNO Si requires 480.2960; [α] -23 (c 0.90,

2

5

45

4

2

D

MeOH); v_m_a_x(thin film): 1721 (s, C=O); δ (CD OD, 400 MHz): 0.13 (6H, s, 2 x CH ), 0.15 (3H, s,

H

3

CH ), 0.23 (3H, s, CH ), 0.93 (9H, s, 3 x CH ), 0.98 (9H, s, 3 x CH ), 1.23 (3H, d, H6, J 6.6), 3.25

3

6,5

(

1H, dt, H2, J₂_,₃3.2, J₂_,₁= J₂_,₁_’6.0), 3.31 (1H, dq, H5, J₅_,₄3.5, J₅_,₆6.6), 3.85 (1H, dd, H1, J₁_,₂6.4, J_g_e_m

1

2

4

0.1), 3.92 3.85 (1H, dd, H1’, J₁_’_,₂6.0, J_g_e_m10.1), 4.15 (1H, br-d, H4, J₄_,₅3.2), 5.19 (1H, br-d, H3, J₃_,₂

.8), 7.52 (2H, t, -Ar, J 7.6), 7.65 (1H, t, -Ar, J 7.3), 8.06 (2H, d, -Ar, J 7.2); δ (CD OD, 100 MHz): -

C

3

.4 (CH Si), -4.3 (CH Si), -4.1 (CH Si), -3.5 (CH Si), 15.0 (C6), 19.9 (C(CH ) ), 20.0 (C(CH ) ), 27.2

3

3 3

(

1

C(CH ) ), 27.3 (C(CH ) ), 59.9 (C5), 65.3 (C2), 67.7 (C1), 80.9 (C4), 84.7 (C3), 130.5, 131.5, 132.0,

35.3 (-Ar), 167.9 (C=O); m/z (ESI+ve): 480 ([M + H] , 100%).

3 3 3 3

+

2

,5-Imino-2,5,6-trideoxy- -gulitol [2,5-Imino-1,2,5-trideoxy-D-glucitol] 4

D

A solution of the protected pyrrolidine 33 (170 mg) in trifluoroacetic acid/water/1,4-dioxane (2:1:1, 4

mL) was stirred at 50 °C for 24 h until mass spectrometry showed the deprotection of the silyl groups

+

(

[M + H] 252). The solvent was removed and the residue dissolved in sodium hydroxide solution (aq,

2

M, 3 mL) and stirred at 50 °C for 24 h until mass spectrometry showed the formation of desired

+

product ([M + H] 148). The mixture was neutralized with HCl (2M, aq) and the solvent removed in

vacuo. The residue was dissolved in ethanol (5 mL) and filtered (glass microfiber) to afford a crude

salt that was then loaded with water (~1 mL) onto a short column of DOWEX 50WX8-200 resin.

®

The ion exchange column was then washed with water, 1,4-dioxane and then water; the pure product

was then eluted with aqueous ammonia (2 M). Removal of solvent in vacuo gave a residue which was

subjected to a further purification by ion exchange column chromatography to yield the gulo-

pyrrolidine 4 (30 mg, 51% 3 steps from 15) as a light yellow gum.

+

25

HRMS (ESI+ve): found 148.0968 [M + H] ; C H N O requires 148.0968; [α] -8.9 (c 0.57, water)

6

14

3

D

3

6

24

[

(

lit. for 4L the enantiomer of 4: [α]_D+17 (c 0.50, water)]; v_m_a_x(thin film): 3290 (br, NH, OH); δ_H

D O, 400 MHz): 1.21 (3H, d, H6, J₆_,₅6.7), 3.13 (1H, ddd, H2, J₂_,₃4.4, J ₂_,₁_’4.9, J₂_,₁6.7), 3.45 (1H,

2

dq, H5, J₅_,₄4.3, J₅_,₆6.7), 3.71 (1H, dd, H1, J₁_,₂7.0, J_g_e_m11.8), 3.80 (1H, dd, H1’, J₁_’_,₂4.9, J_g_e_m11.8),

3

5

.93 (1H, dd, H3, J₃_,₄1.8, J₃_,₂4.4), 3.99 (1H, dd, H4, J₄_,₃1.8, J₄_,₅4.3); δ (D O, 100 MHz): 12.6 (C6),

7.1 (C5), 61.9 (C1), 66.7 (C2), 78.9 (C3), 79.4 (C4); m/z (ESI+ve): 148 ([M + H] , 100%).

C 2

+

5

2

.5 Benzoyl protection: NGP in the Hanessian-Hullar reaction

-Azido-(R)-3,5-O-benzylidene-1,4-di-O-benzoyl-2,6-dideoxy-L-glucitol 36

Benzoyl chloride (0.24 mL, 2.2 mmol) and pyridine (0.24 mL, 2.8 mmol) were added to a solution of

the diol 22 (200 mg, 0.72 mmol) in anhydrous dichloromethane (7 mL). The reaction mixture was

stirred at rt for 16 h until TLC (ethyl acetate/cyclohexane, 1:4) showed the formation of one major

product (R 0.41). The solvent was removed in vacuo and the residue purified by flash column

f

chromatography (ethyl acetate/cyclohexane, 1:19 to 1:4) to form the dibenzoate 36 (300 mg, 86%) as

a clear oil; trituration of the oil with acetonitrile gave a white crystalline solid.

+

HRMS (ESI+ve): found 510.1634 [M + Na] ; C H N O Na requires 510.1636; m.p. 111 - 114 °C;

2

7

25

3

6

25

[

(

α]_D-29.6 (c 1.0, MeCN); v_m_a_x(thin film): 1716 (s, C=O), 2104 (s, N ); δ (CD CN, 400 MHz): 1.58

3H, d, H6, J₆_,₅7.2), 4.14 - 4.21 (1H, m, H2), 4.35 (1H, dd, H1, J₁_,₂5.8, J_g_e_m12.1), 4.46 (1H, br-q, H5,

3 H 3

J₅_,₆7.2), 4.50 (1H, dd, H1’, J₁_’_,₂3.3, J_g_e_m12.1), 4.68 (1H, dd, H3, J₃_,₄1.4, J₃_,₂7.9), 5.02 (1H, br-s,

2

0

H4), 6.11 (1H, s, H7), 7.37 - 7.68 (10H, m, Ar x 2), 7.95 - 8.01 (2H, m, Ar), 8.09 - 8.15 (2H, m, Ar);

δ (CD CN, 100 MHz): 15.4 (C6), 60.8 (C2), 64.0 (C1), 69.8 (C4), 73.8 (C5), 74.5 (C3), 95.1 (C7),

C

3

1

27.3, 129.2, 129.6, 129.7, 129.9, 130.4, 130.6, 130.9, 134.4, 134.5, 139.5 (-Ar), 166.7 (C=O), 166.8

+

(

C=O); m/z (ESI+ve): 510 ([M + Na] , 100%).

2

-Azido-5-bromo-1,3,4-tri-O-benzoyl-2,5,6-trideoxy-

D-iditol 39 and 2-Azido-5-bromo-1,3,4-tri-

O-benzoyl-2,5,6-trideoxy- -glucitol 40

L

N-Bromosuccinimide (116 mg, 0.65 mmol) and barium carbonate (160 mg, 0.81 mmol) were added to

a solution of 36A/B (265 mg, 0.54 mmol) in carbon tetrachloride (3.5 mL). The stirred reaction

mixture was refluxed for 1 h until TLC (ethyl acetate/cyclohexane, 1:4) indicated the disappearance

of starting material (R 0.39) and the formation of two products (R 0.45, 0.5). After removal of the

f

solvent, the residue was purified by flash column chromatography (ethyl acetate/hexane, 0:10 to 1:4)

to give a mixture of the inseparable epimeric bromides 39 and 40 (5:1, 280 mg, 91%) as a light yellow

syrup.

3

4

7

9: δ (CD CN, 400 MHz): 1.76 (3H, d, H6, J 6.8), 4.29 (1H, ddd, H2, J₂_,₃3.0, J₂_,₁_’5.2, J₂_,₁6.6),

H 3 6,5

.35 (1H, dd, H1, J₁_,₂6.6, J_g_e_m11.4), 4.63 - 4.71 (2H, m, H1’, H5,), 5.72 (1H, dd, H4, J₄_,₅3.1, J₄_,₃

.3), 5.95 (1H, dd, H3, J₃_,₂3.1, J₃_,₄7.3), 7.39 - 7.72 (15H, m, -Ar x 3), δ (CD CN, 100 MHz): 24.8

C

3

(

C6), 50.4 (C5), 60.9 (C2), 66.0 (C1), 75.4 (C3), 76.5 (C4), 130.9 – 136.1 (-Ar x 3), 167.7 (C=O),

1

4

7

67.8 (C=O), 167.9 (C=O);

0: δ (CD CN, 400 MHz): 1.79 (3H, d, H6, J 6.7), 4.20 (1H, dt, H2, J₂_,₃= J₂_,₁_’4.2, J₂_,₁6.2), 4.54 -

H

3

6,5

.71 (3H, m, H1, H1’, H5,), 5.89 (1H, t, H4, J₄_,₅= J₄_,₃5.4), 5.98 (1H, dd, H3, J₃_,₂4.5, J₃_,₄5.4), 7.39 -

.72 (15H, m, -Ar x 3), δ (CD CN, 100 MHz): 22.9 (C6), 48.1 (C5), 62.3 (C2), 66.3 (C1), 73.4 (C3),

C

3

7.4 (C4), 131.1 – 136.4 (-Ar x 3);

1

,3,4-Tri-O-benzoyl-2,5-imino-2,5,6-trideoxy-L-glucitol 41 and 1,3,4-Tri-O-benzoyl-2,5-imino-

2

,5,6-trideoxy-D-iditol 42

Palladium on charcoal (10 % wt., 30 mg) and sodium acetate (49 mg, 0.6 mmol) were added to a

solution of the mixture of 39 and 40 (260 mg, 0.46 mmol) in ethanol (15 mL). The reaction mixture

was flushed sequentially with nitrogen, argon and hydrogen and then stirred overnight at rt under a

hydrogen atmosphere for 12 h when TLC (ethyl acetate/cyclohexane, 1:1) showed the formation of

two products (R 0.14, 0.17). The solvent was removed and the residue purified by flash column

f

chromatography (triethylamine/ethyl acetate/cyclohexane, 0.01:1:5 to 0.01:2:1) to afford a mixture of

5

-epimers 41 and 42 (4:1, 173 mg, 82%) as a white solid. The mixture was not clean enough for

assignment of the tribenzoates by NMR; this crude product was directly used in the deprotection step.

2

,5-Imino-2,5,6-trideoxy-L-glucitol 1 and 2,5-Imino-2,5,6-trideoxy-D-iditol 3

The mixture of 41 and 42 (100 mg, 0.22 mmol) was dissolved in sodium hydroxide (aq, 2M, 10 mL)

and stirred at 50 °C for 48 h until mass spectrometry showed the formation of desired product

+

(

[M+H] 148). The solvent was removed in vacuo; the residue was dissolved in ethanol (5 mL) and

passed through glass fiber to afford a crude salt that was then loaded with water (~1 mL) onto a short

®

column of DOWEX 50WX8-200 resin. The ion exchange column was then washed with water, 1,4-

dioxane and then water; the pure product was then eluted with aqueous ammonia (2 M). Removal of

solvent in vacuo gave a mixture of the unprotected pyrrolidines 1 and 3 as a yellow oil (5 :1 according

1

to H spectrum, 23 mg, 73%).

2

-Azido-(R)-3,5-O-benzylidene-1,4-di-O-benzoyl-2,6-dideoxy-L-mannitol 26

Benzoyl chloride (0.12 mL, 1.1 mmol) and pyridine (0.12 mL, 1.4 mmol) were added into a solution

of diol 25 (100 mg, 0.36 mmol) in anhydrous dichloromethane (7 mL). The mixture was stirred at rt

for 18 h until TLC (ethyl acetate/cyclohexane, 1:4) showed the formation of one major product (R_f

0

.42). The solvent was in vacuo and the residue was purified by flash column chromatography (ethyl

acetate/cyclohexane, 1:8 to 1:4) to afford the dibenzoate 26 (196 mg, 97%) as a white solid.

+

HRMS (ESI+ve): found 510.1633 [M + Na] ; C H N O Na requires 510.1636; m.p. 79 - 85 °C;

[

2

7

25

3

6

25

α]_D+37.9 (c 1.0, MeCN); v_m_a_x(thin film): 2103 (s, N ), 1719 (s, C=O); δ (CD CN, 400 MHz):

3 H 3

1

9

.59 (3H, d, H6, J₆_,₅7.2), 4.23 (1H, ddd, H2, J₂_,₁_’2.7, J₂_,₁7.4, J₂_,₃9.7), 4.43 (1H, dd, H3, J₃_,₄1.5, J₃_,₂

.7), 4.50 (1H, dd, H1, J₁_,₂7.4, J_g_e_m11.7), 4.51 (1H, dq, H5, J₅_,₄1.5, J₅_,₆7.2), 4.80 (1H, dd, H1’, J₁_’_,₂

2

1

2

8

.7, J_g_e_m11.7), 5.05 (1H, t, H4, J₄_,₃= J₄_,₅1.5), 6.01 (1H, s, H7), 7.34 - 7.69 (10H, m, -Ar x 2), 8.02 -

.18 (4H, m, -Ar); δ (CD CN, 100 MHz): 15.4 (C6), 60.7 (C2), 65.2 (C1), 69.3 (C4), 72.8 (C3), 73.7

C

3

(

C5), 94.7 (C7), 127.2, 129.2, 129.6, 129.7, 129.9, 130.47, 130.52, 130.7, 130.9, 134.3, 134.5, 139.4

-Ar x 2), 166.6, 166.8 (C=O); m/z (ESI+ve): 510 ([M + Na] , 100%).

+

2

-Azido-1,3,4-tri-O-benzoyl-5-bromo-2,5,6-trideoxy-

D-gulitol 43 and 2-Azido-1,3,4-tri-O-

benzoyl-5-bromo-2,5,6-trideoxy- -mannitol 44

L

N-Bromosuccinimide (156 mg, 0.88 mmol) and barium carbonate (217 mg, 1.1 mmol) were added

into a solution of 26 (357 mg, 0.73 mmol) in carbon tetrachloride (5.0 mL). The mixture was refluxed

+

with stirring for 1 h until mass spectrometry indicated the completion of reaction ([M + Na] 588,

5

90) The solvent was removed in vacuo and the residue purified by flash column chromatography

ethyl acetate/hexane, 1:9 to 3:7) to give a mixture of the bromide epimers 43 and 44 (4:1, 388 mg,

4%) as a colorless oil. Separation of the epimers 43 and 44 by flash column chromatography was not

efficient.

HRMS (ESI+ve): found 588.0740, 590.0719; C H BrN O Na requires 588.0741, 590.0720; v

(

9

+

2

7

24

3

6

max

+

(

thin film): 2108 (s, N ), 1722 (s, C=O); m/z (ESI+ve): 588, 590 ([M + Na] , 100%, 95%)

3

4

3: δ (CD CN, 400 MHz): 1.76 (3H, d, H6, J 6.8), 4.31 (1H, dt, H2, J₂_,₁_’3.7, J₂_,₁= J₂_,₃6.7), 4.46

H 3 6,5

(

1H, dd, H1, J₂_,₁6.7, J_g_e_m12.0), 4.55 - 4.63 (1H, m, H5), 4.70 (1H, dd, H1’, J₁_’_,₂3.7, J_g_e_m12.0), 5.72

1H, t, H4, J₄_,₃= J₄_,₅5.0), 5.78 (1H, dd, H3, J₄_,₃5.0, J₃_,₂6.7), 7.41 - 7.71 (9H, m, -Ar), 7.95 - 7.17 (6H,

m, -Ar); δ (CD CN, 100 MHz): 23.1 (C6), 49.4 (C5), 61.0 (C2), 64.3 (C1), 72.4 (C3), 75.1 (C4),

C

3

1

4

5

29.6-130.8, 134.5, 134.7, 134.8 (-Ar), 166.1 (C=O), 166.4 (C=O), 166.7 (C=O).

4: δ (CD CN, 400 MHz): 1.72 (3H, d, H6, J 6.7), 4.25 (1H, ddd, H2, J₂_,₁_’3.7, J₂_,₁6.7, J₂_,₃7.8),

H

3

6,5

.42 (1H, dd, H1, J₁_,₂6.7, J_g_e_m11.9), 4.41 - 4.49 (1H, m, H5), 4.63 (1H, dd, H1’, J₂_,₁_’3.7, J_g_e_m11.9),

.82 (1H, dd, H4, J₃_,₄2.7, J₄_,₅7.7), 5.87 (1H, dd, H3), 7.41 - 7.71 (9H, m, -Ar), 7.95 - 7.17 (6H, m, -

Ar); δ (CD CN, 100 MHz): 22.1 (C6), 46.8 (C5), 61.1 (C2), 64.4 (C1), 71.2 (C3), 75.5 (C4), 129.6 -

C

3

1

30.8, 134.4, 134.87, 134.92 (-Ar), 166.1 (C=O), 166.4 (C=O), 166.6 (C=O).

1

2

,3,4-Tri-O-benzoyl-2,5-imino-2,5,6-trideoxy-

,5,6-trideoxy- -gulitol 46

L-mannitol 45 and 1,3,4-Tri-O-benzoyl-2,5-imino-

D

Palladium on charcoal (10 % wt., 73 mg) and sodium acetate (84 mg, 0.6 mmol) were added to a

solution of 43 and 44 (388 mg, 0.69 mmol) in ethanol (15 mL). The reaction mixture was flushed

sequentially with nitrogen, argon and hydrogen, stirred at rt under hydrogen atmosphere for 16 h until

TLC (ethyl acetate/cyclohexane, 3:7) showed the formation of two products (R 0.12, 0.14). The

f

solvents were removed in vacuo and the residue was purified by flash column chromatography

(

triethylamine/ethyl acetate/ petroleum ether, 0.01:1:8 to 0.01:2:1) to obtain a mixture of 5-epimers 45

and 46 (10:7, 280 mg, 89%) as a yellow oil. Attempted separation of 45 and 46 by flash column

chromatography was not successful.

4

5: δ (CD CN, 400 MHz): 1.34 (3H, d, H6, J 6.6), 3.57 (1H, br-dq, H5, J₅_,₄4.2, J₅_,₆6.6), 3.82 (1H,

H 3 6,5

dt, H2, J₂_,₃4.2, J₂_,₁= J₂_,₁_’5.6), 4.50 (1H, dd, H1, J₁_,₂5.6, J_g_e_m10.9), 4.53 - 4.60 (1H, m, H1’), 5.72

(

1H, dd, H4, J₄_,₃3.7, J₄_,₅4.2), 5.58 (1H, dd, H3, J₃_,₄3.7, J₃_,₂4.1), 7.34 – 7.78 (15H, -Ar x 3); δ_C

CD CN, 100 MHz): 20.3 (C6), 60.2 (C5), 63.4 (C2), 67.7 (C1), 83.4 (C3), 86.9 (C4), 120.7 – 135.8

3

-Ar x 3), 168.0 (C=O), 168.1 (C=O), 168.4 (C=O).

4

6: δ (CD CN, 400 MHz): 1.21 (3H, d, H6, J 6.3), 3.61 – 3.68 (2H, m, H2, H5), 4.45 – 4.60 (2H,

H 3 6,5

m, H1, H1’), 5.42 (1H, dd, H4, J 1.9, J 4.9), 4.48 (1H, dd, H3, J 1.9, J 4.5), 7.34 – 8.08 (15H, m, -Ar

x 3); δ (CD CN, 100 MHz): 16.0 (C6), 58.6 (C5), 64.8 (C2), 67.6 (C1), 82.8 (C3), 83.3 (C4), 120.7 –

C

3

1

35.8 (-Ar x 3), 167.8 (C=O), 167.9 (C=O), 168.3 (C=O).

2

,5-Imino-2,5,6-trideoxy- -mannitol 2 and 2,5-Imino-2,5,6-trideoxy-D-gulitol 4

L

The epimeric mixture of the tribenzoates 45 and 46 (200 mg, 0.44 mmol) was dissolved into sodium

hydroxide solution (aq., 2 M, 15 mL) and stirred at 50 °C for 48 h until mass spectrometry showed the

+

formation of desired product ([M + H] 148). The solvent was removed in vacuo, the residue was

dissolved in ethanol (5 mL) and filtered (glass fiber) to afford a crude salt that was then loaded with

®

water (~1 mL) onto a short column of DOWEX 50WX8-200 resin. The ion exchange column was

then washed with water, 1,4-dioxane and then water; the pure product was then eluted with aqueous

2

ammonia (2 M). Removal of solvent in vacuo yielded a mixture of 2 and 4 as a yellow oil (5:3

1

according to H spectrum, 46 mg, 70%).

6

. Acknowledgements This work was supported in part by: a Grant-in-Aid for Scientific

Research (C) from the Japanese Society for the Promotion of Science (JSPS KAKENHI

Grant Number JP17K08362) (AK); Biotechnology and Biological Sciences Research Council

(BB/M011321/1) (JTH); University of Kagawa (KI, AY); Xunta de Galicia (Centro Singular

de Investigación de Galicia, accreditation 2016–2019, ED431B 2018/13; Project

CN2011/037 and Project GRC2014/040), and the European Union (European Regional

Development Fund-ERDF).

7

. Conflict of interest

None of the authors have any conflict of interest arising from this work.

References

1

(a) Winchester, B. G.; Fleet, G. W. J. Glycobiology, 2 (1992), 199-210; (b) Watson, A. A.; Fleet, G. W .J.;

Asano, N.; Molyneux, R. J.; Nash, R. J. Phytochemistry, 56 (2001), 265-295; (c) Nash, R. J.; Kato, A.; Yu, C.

Y.; Fleet, G. W. J. Future Med. Chem. 3 (2011), 1513-1521.

2

Ayers, B. J.; Ngo, N.; Jenkinson, S. F.; Martínez, R. F.; Shimada, Y.; Adachi, I.; Weymouth-Wilson, A. C.;

Kato, A.; Fleet, G. W. J. J. Org. Chem. 77 (2012), 7777-7792.

Molyneux, R. J.; Pan, Y. T.; Tropea, J. E.; Elbein, A. D.; Lawyer, C. H.; Hughes, D. J.; Fleet, G. W. J. J. Nat.

Prod. 56 (1993), 1356-1364.

Merino, P.; Delso, I.; Tejero, T.; Cardona, F.; Marradi, M.; Faggi, E.; Parmeggiani, C.; Goti, A. Eur. J. Org.

Chem. 2008, 2929-2947.

(a) Asano, N.; Yasuda, K.; Kizu, H.; Kato, A.; Fan, J.-Q.; Nash, R. J.; Fleet, G. W. J.; Molyneux, R. J. Eur. J.

3

4

5

Biochem. 268 (2001), 35-41. (b) Yasuda, K.; Kizu, H.; Yamashita, T.; Kameda, Y.; Kato, A.; Nash, R. J.; Fleet,

G. W. J.; Molyneux, R. J.; Asano, N. J. Nat. Prod. 65 (2002), 198-202.

6

(a) Wang, Y. F.; Dumas, D. P.; Wong, C. H. Tetrahedron Lett. 34 (1993), 403-406. (b) Liu, K. K. C.;

Kajimoto, T.; Chen, L.; Zhong, Z.; Ichikawa, Y.; Wong, C. H. J. Org. Chem. 56 (1991), 6280-6289.

(a) Fleet, G. W. J.; Nicholas, S. J.; Smith, P. W.; Evans, S. V.; Fellows, L. E.; Nash, R. J Tetrahedron Lett. 26

1985), 3127-3131. (b) Fan, A.; Chuah, G. K. ; Jaenicke, S. Carbohydr. Res. 472 (2019), 103-114.

7

(

8

9

Crich, D.; Yao. D. J.; Bowers, A. A. Carbohydr. Res. 341 (2006), 1748-1752.

Krulle, T. M.; Davis, B.; Ardron, H.; Long, D. D.; Hindle, N. A.; Smith, C.; Brown, D.; Lane, A. L.; Watkin,

D. J.; Marquess, D. G.; Fleet, G. W. J. J. Chem. Soc., Chem. Commun. 1996, 1271-1272.

1

0 Dho, J. C.; G. W. J. Fleet, G. W. J ; Peach, J. M.; Prout, K.; Smith, P. W. J. Chem. Soc., Perkin Trans.

, 1987, 1785-1791,

1 Johnson, S. W,; Jenkinson, S. F.; Angus, D.; Taillefumier, C.; Jones, J. H.; Fleet, G. W. J. Tetrahedron:

Asymmetry, 15 (2004), 2681-2686.

2 Liu, Z.; Yoshihara, A.; Kelly, C.; Heap, J.; Marqvorsen, M. H. S.; Jenkinson, S. F.; Wormald, M. R; Estevez,

A.; Kato, A.; Fleet, G. W. J.; Estevez, R. J.; Izumori, K. Chem.- Eur. J. 22 (2016), 12557-12565.

3 Liu, Z.; Yoshihara, A.; Jenkinson, S. F.; Wormald, M. R.; Estevez, R. J.; Fleet, G. W. J.; Izumori, K. Org.

Lett. 18 (2016), 4112-4115.

4 Davis, B. G.; Hull, A.; Smith, C.; Nash, R. J.; Watson, A. A.; Winkler, D. A.; Griffiths, R. C.; Fleet, G. W. J.

Tetrahedron Asymmetry, 9 (1998), 2947-2960.

5 Johnson, S. W.; Angus, D.; Taillefumier, C.; Jones, J. H.; Watkin, D. J.; Floyd. E.; Buchanan, J. G.; Fleet, G.

W. J. Tetrahedron: Asymmetry, 11 (2000), 4113-4125.

1

6 Bell, A. A.; Pickering, L.; Finn, M.; de la Fuente. C.; Krulle, T. M.; Davis, B. G.; Fleet, G. W. J. Synlett 1997

077-1078.

7 Giraud, M. F.; Naismith, J. H. Curr. Opin. Struct. Biol. 10 (2000), 687-96.

8 (a) Dharuman, S.; Wang, Y.; Crich, D. Carbohydr. Res., 419 (2016), 29-32. (b) Shilvock, J. P.; Wheatley, J.

R.; Nash, R. J.; Watson, A. A.; Griffiths, R. C.; Butters, T. D.; Muller, M.; Watkin, D. J.; Winkler, D. A.; Fleet,

G. W. J. J. Chem. Soc., Perkin Trans. 1 1999, 2735-2745. (c) Alphey, M. S.; Pirrie, L.; Torrie, L. S.;

Boulkeroua, W. A.; Gardiner, M.; Sarkar, A.; Maringer, M.; Oehlmann, W.; Brenk, R.; Scherman, M. S.;

McNeil, M.; Rejzek, M.; Field, R. A.; Singh, M.; Gray, D.; Westwood, N. J.; Naismith, J. H. ACS Chem. Biol. 8

2

3

(

2013), 387-396. (d) Garnier, P.; Wang, X. T.; Robinson, M. A.; van Kasteren, S.; Perkins, A. C; Frier, M.;

Fairbanks, A. J.; Davis, B. G. J. Drug Target. 18 (2010), 794-802.

9 (a) Minami, Y.; Kuriyama, C.; Ikeda, K.; Kato, A.; Takebayashi, K.; Adachi, I.; Fleet, G. W. J.; Kettawan,

1

A.; Okamoto, T.; Asano, N. Bioorg. Med. Chem. 16 (2008), 2734-2740. (b) Jenkinson, S. F.; Best, D.; Saville,

A. W.; Mui, J.; Martinez, R. F.; Nakagawa, S.; Kunimatsu, T.; Alonzi, D. S.; Butters, T. D.; Norez, C.; Becq, F.;

Bleriot, Y.; Wilson, F. X.; Weymouth-Wilson, A. C.; Kato, A.; Fleet, G. W. J. J. Org. Chem. 78 (2013), 7380-

7

2

397.

0 (a) Kato, A.; Zhang, Z-L.; Wang, H-Y.; Jia, Y-M,; Yu, C-Y.; Kinami, K.; Hirokami, Y.; Tsuji, Y.; Adachi, I.;

Nash, R. J.; Fleet, G. W. J.; Koseki, J.; Nakagome, I.; Hirono, S. J. Org. Chem., 80 (2015), 4501-4515; (b) Kato,

A.; Harashi, E.; Miyauchi, S.; Adachi, I.; Imahori, T.; Natori, Y.; Yoshimura, Y.; Nash, R. J.; Shimaoka, H.;

Nakagome, I.; Koseki, J.; Takahata, H. J. Med. Chem. 55 (2012), 10347-10362; (c) Li, Y-X.; Huang, M-H.;

Yamashita, Y.; Kato, A.; Jia, Y-M.; Wang, W-B.; Fleet, G. W. J.; Nash, R. J.; Yu, C-Y. Org. Biomol. Chem. 9

(

2

2011), 3405-3414.

1 For details of assays, see: Glawar, A. F. G.; Best, D.; Ayers, B.; Miyauchi, S.; Nakagawa, S.; Aguilar-

Moncayo, M.; García Fernańdez, J. M.; Mellet, C. O.; Crabtree, E. V.; Butters, T. D.; Wilson, F. X.; Kato, A.;

Fleet, G. W. J. Chem. Eur. J., 18 (2012), 9341−9359 and references cited therein.

2

2 Asano, N.; Yamauchi, T.; Kagamifuchi, K.; Shimizu, N.; Takahashi, S.; Takatsuka, H.; Ikeda, K.; Kizu, H.;

Chuakul, W.; Kettawan, A.; Okamoto, T. J. Nat. Prod. 68 (2005), 1238−1242.

2

3 Wrodnigg, T. M. Monatsh. Chem. 133 (2002), 393−426.

4 Wrodnigg, T. M.; Stuetz, A. E.; Tarling, C. A.; Withers, S. G. Carbohydr. Res., 341 (2006), 1717−1722.

5 (a) Best, D.; Wang, C.; Weymouth-Wilson, A. C.; Clarkson, R. A.; Wilson, F. X.; Nash, R. J.; Miyauchi, S.;

Kato, A.; Fleet, G. W. J., Tetrahedron: Asymmetry, 21 (2010), 311-319; (b) Yu, C.-Y.; Asano, N.; Ikeda, K.;

Wang, M.-X.; Butters, T. D.; Wormald, M. R.; Dwek, R. A.; Winters, A. L.; Nash, R. J.; Fleet, G. W. J., Chem.

Commun., 17 (2004), 1936-1937. (c) D’Alonzo, D.; Guaragna, A.; Palumbo, G. Curr. Med. Chem., 16 (2009),

4

2

73−505.

6 Nash, R. J.; Bell, E. A.; Williams, J. M. Phytochemistry, 24 (1985), 1620-1622.

7 (a) Fosgerau, K.; Westergaard, N.; Quistorff, B.; Grunnet, N.; Kristiansen, M., Lundgren, K. Arch. Biochem.

Biophys. 380 (2000), 274–284. (b) Andersen, B.; Rassov,A.; Westergaard, N.; Lundgren, K. Biochem. J. 342

1999), 545–550. (c) Minami, Y., Kuriyama, C., Ikeda, K., Kato, A., Takebayashi, K., Adachi, I., Fleet, G.W. J.,

Kettawan,A., Okamoto,T., Asano, N. Bioorg. Med. Chem. 16 (2008), 2734–2740.

(

2

5

8 (a) Song, Y. -Y.; Kinami, K.; Kato, A.; Li, Y.-X.; Fleet, G. W. J.; Yu, C.-Y. Org. Biomol. Chem., 14 (2016),

157-5174. (b) Zhao, H.;, Kato, A.; Sato, K.; Jia, Y. M.; Yu, C.-Y. J. Org. Chem., 78 (2013), 7896-902. (c) Wu,

Q.-K.; Kinami, K.; Kato A.; Li. Y.-X.; Jia, Y-M.; Fleet, G. W. J.; Yu, C.-Y. Molecules 24 (2019), 3712.

9 Collyer, C. A.; Goldberg, J. D.; Viehmann, H.; Blow, D. M.; Ramsden, N. G.; Fleet, G. W. J.; Montgomery,

F. J.; Grice, P. Biochemistry, 31 (1992), 12211-12218.

2

3

0 Xu, W.; Zhang, W.; Zhang, T.; Jiang, B.; Mu, W. Appl. Microbiol. Biotechnol., 100 (2016), 2985-2992.

1 (a) Dische, A.; Borenfreund. E., J. Biol. Chem., 192 (1951), 583-587. (b) Leang, K.; Takada, G.; Ishimura,

A.; Okita, M.; Izumori, K. App. Environmental Microbiol., 70 (2004), 3298–3304. (c) Bhuiyan, S. H.; Itami, Y.;

Rokui, Y.; Katayama, T.; Izumori, K. J. Ferment. Bioeng., 85 (1998), 539-541.

3

2 (a) Beuttler, H.; Hoffmann, J.; Jeske, M.; Hauer, B.; Schmid, R. D.; Altenbuchner, J.; Urlacher, V. B. Appl.

Microbiol. Biotechnol., 89 (2011), 1137– 1147. (b) Wilms, B.; Hauck, A.; Reuss, M.; Syldatk, C.; Mattes, R.;

Siemann, M.; Altenbuchner, J. Biotechnol. Bioeng., 73 (2001), 95−103. (c) Giacalone, M.; Gentile, A.; Lovitt,

B.; Berkley, N.; Gunderson, C. Surber, M. BioTechniques, 40 (2006), 355−364. (e) Haldimann, A.; Daniels, L.

L.; Wanner, B. L. J. Bacteriol. 180 (1998), 1277−1286.

3

3 Kelly, C. L.; Liu, Z.; Yoshihara, A.; Jenkinson, S. F.; Wormald, M. R.; Otero, J.; Estévez, A.; Kato, A.;

Marqvorsen, M. H. S.; Fleet, G. W. J; Estévez, R. J.; Izumori, K.; Heap, J. T. ACS Synth. Biol., 5 (2016), 1136-

1

3

4

3

145.

4 Aravind, A.; Kumar, P. S.; Sankar, M. G.; Baskaran, S. Eur. J. Org. Chem. 2011, 6980-6988.

5 Espelt, L.; Parella, T.; Bujons, J.; Solans, C.; Joglar, J.; Delgado, A.; Clapes, P. Chem. - Eur. J., 9 (2003),

887-4899.

6 Izquierdo, I.; Plaza, M. T.; Tamayo, J. A.; Lo Re, D.; Sanchez-Cantalejo, F., Synthesis, 2008, 1373-1378.

2

4

Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-

dihydroxypyrrolidines: rhamnulose iminosugar mimics inhibit

α

-glucosidase

a,c

b

a

c

d

Zilei Liu, Akihide Yoshihara, Sarah F. Jenkinson, Mark R. Wormald, Ciarán Kelly, John

d

a

e

a

T. Heap, Mikkel H. S. Marqvorsen, Ramón J. Estévez, George W. J. Fleet *, Shinpei

f

b

g

*f

Nakagawa, Ken Izumori, Robert. J. Nash, Atsushi Kato

HIGHLIGHTS:

•

Iminosugar mimics of

isomerase

Benzylidene protection then allows a stereospecific and regiospecific

activation

Benzoate causes neighbouring group participation in Hanessian-Hullar

reactions

L-rhamnulose inhibit α-glucosidases but not rhamnose

•

Easy efficient synthesis of highly functionalised pyrrolidines

Search for control of rhamnulose and rhamnose metabolising enzymes

1

STATEMENT OF NO CONFLICT OF INTEREST:

Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-

dihydroxypyrrolidines: rhamnulose iminosugar mimics inhibit

α

-glucosidase

a,c

b

a

c

d

Zilei Liu, Akihide Yoshihara, Sarah F. Jenkinson, Mark R. Wormald, Ciarán Kelly, John T.

d

a

e

a

f

Heap, Mikkel H. S. Marqvorsen, Ramón J. Estévez, George W. J. Fleet *, Shinpei Nakagawa, Ken

b

g

*f

Izumori, Robert. J. Nash, Atsushi Kato

a

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA,

_bUK

International Institute of Rare Sugar Research and Education, Kagawa University, Miki, Kagawa

61-0795, Japan

7

c

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, OX1

QU, UK

Centre for Synthetic Biology, Department of Life Sciences, Imperial College London, SW7 2AZ, UK

Departamento de Química Orgánica and Centro Singular de Investigación en Química Biolóxica e

3

d

e

Materiais Moleculares, Universidade de Santiago de Compostela, 15782 Santiago de Compostela,

Spain

f

Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan

PhytoQuest Ltd, Aberystwyth, Ceredigion SY23 3EB, UK

g

None of the authors have any conflict of interest in the work described in this paper.

This is clearly stated in the paper which has been reviewed by all of the authors

1

Article Doi

Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-dihydroxypyrrolidines: Rhamnulose iminosugar mimics inhibit α-glucosidase

DOI: 10.1016/j.tet.2019.130758

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.tet.2019.130758

Full text of DOI:10.1016/j.tet.2019.130758

Products guided by the article

R&D Labs maybe for 322726-62-5

Relevant to this article

Hot Product