Total Synthesis of 6-Amino-2,6-dideoxy-α-Kdo from d -Mannose

Total Synthesis of 6 ‑Amino-2,6-dideoxy-α -Kdo from D ‑Mannose

Letter

Oscar Javier Gamboa Marin, Nazar Hussain, Gokulakrishnan Ravicoularamin, Nassima Ameur,

Paul Gormand, Janelle Sauvageau, and Charles Gauthier*

Cite This: https://dx.doi.org/10.1021/acs.orglett.0c01847

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ABSTRACT: 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) bio-

synthetic pathway is a promising target in antibacterial drug

discovery. Herein, we report the total synthesis of 6-amino-2,6-

dideoxy-α-Kdo in 15 steps from D-mannose as a potential inhibitor

of Kdo-processing enzymes. Key steps of the synthetic sequence

involve a Horner−Wadsworth−Emmons reaction for the two-

carbon chain homologation followed by either a 6-exo-trig Pd-catalyzed reductive cyclization or a tandem Staudinger/aza-Wittig

reaction with concomitant α-iminoester reduction, enabling the α-stereoselective formation of the Kdo-like six-membered azacyclic

ring.

-Deoxy-D-manno-oct-2-ulosonic acid (Kdo, Figure 1) is a

Kdo linker, containing alternating β-(1→4) and β-(1→7)

glycosidic linkages, is attached to a (lyso)phosphatidylglycerol

moiety, allowing the CPS to anchor into the outer

polyfunctional eight-carbon ketose found in the surface

membrane. Because of their ubiquitous presence in

encapsulated GNB, KpsC and KpsS are considered as

important targets for developing therapeutics acting via the

“

anti-virulence strategy”. β-Kdo residues have also been

found in the exopolysaccharides (EPS) of pathogenic

Burkholderia spp., including B. pseudomallei, the causative

Figure 1. Structure of Kdo, 2-deoxy-β-Kdo, and 6-amino-2,6-dideoxy-

α-Kdo (1).

agent of melioidosis, and B. cepacia complex, a group of

opportunistic pathogens causing fatal infections in cystic

ﬁbrosis patients. As this Kdo-containing EPS is an important

polysaccharides of Gram-negative bacteria (GNB). Kdo is

virulence factor, the yet unknown Kdo transferases involved

typically present in lipopolysaccharide (LPS) core regions of

virtually all GNBs except for Shewanella spp. Within the LPS

inner core, two α-Kdo units are attached to the lipid A forming

in its biosynthesis could represent potential therapeutic targets

against life-threatening Burkholderia infections.

Azacyclic compoundsthe so-called iminosugars in which

the ring oxygen atom is replaced by a nitrogenhave proved

eﬃcient for inhibiting glycosidases and glycosyltransferases by

mimicking the positive charge of the glycosyl cation

lipid A-Kdo , which is biosynthesized through the action of

several sugar-processing enzymes. The last biosynthetic step

of lipid A-Kdo involves a Kdo transferase (WaaA), which adds

two Kdo units to lipid A using CMP-Kdo as an activated sugar

nucleotide. The biosynthesis of CMP-Kdo is catalyzed by

CMP-Kdo synthase (CKS), adding free Kdo to CTP. As the

intermediate usually involved in the active site of these

20,21

enzymes.

Interestingly, as amino acid like derivatives, Kdo

iminosugars could be actively processed to reach their

enzymatic targets into the cytoplasm via bacterial transport

presence of lipid A-Kdo is essential to the integrity of the

outer membrane as well as for cell viability, WaaA and CKS

systems. Kdo iminosugars, in either α- or β-conﬁgurations,

stand as exquisite targets for the discovery of new antibiotics.

have already been synthesized in 11 steps from Kdo (∼50$/

Since the 1980s, tremendous eﬀorts have been deployed for

mg, Sigma) by Norbeck and Kramer in 1987 and proved to be

the design of Kdo derivatives as inhibitors of CKS. Among

only modest inhibitors of CKS. Despite this, we believe it

the synthesized compounds, 2-deoxy-β-Kdo (Figure 1) was

would be worth reinvestigating the inhibitory potential of 2-

found to be a potent CKS inhibitor. On the other hand, only

cytosine-5′-monophospho-3-deoxy-3-ﬂuoro-Kdo was shown to

Received: June 1, 2020

interfere directly with WaaA, pointing out that this ﬁeld of

research is still in its infancy.

Whitﬁeld and co-workers

−11

have recently characterized a

new family of retaining Kdo transferases (KpsC and KpsS),

which biosynthesize a poly-Kdo linker found at the reducing

end of capsular polysaccharides (CPS) from GNB. This poly-

XXXX American Chemical Society

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

deoxy-imino-Kdo scaﬀold would be built upon a 6-exo-trig

Letter

deoxy-imino-Kdo and their derivatives toward WaaA and the

recently disclosed retaining Kdo transferases. As a ﬁrst step

toward this goal, we herein report an innovative and

stereoselective total synthesis of 6-amino-2,6-dideoxy-α-Kdo

subjected to a Pﬁtzner−Moﬀatt oxidation/NaBH -mediated

reduction sequence providing (S)-alcohol 3 in 80% yield (ratio

2:3 = 1:9). Upon its conversion into mesylate 4 (or crude

triﬂate 5), several conditions were tested for the S 2 reaction

(

1, Figure 1) in 15 steps starting from aﬀordable D-mannose

∼120$/100 g, Sigma).

including type of azido salts (NaN and TBAN ), solvents

(DMF, DMSO, ACN, and H O), as well as additives (18-

As depicted in our retrosynthetic analysis (Figure 2), the 2-

crown-6 and 4 Å MS). Unfortunately, all these attempts led to

the formation of elimination byproducts without any trace of

azido derivative 6, which we postulate was due to steric

crowding at C5.

In parallel, we tried to epimerize the C6 position of α,β-

unsaturated ester 7. Attempted oxidation−reduction con-

ditions failed to provide inverted alcohol 8. Furthermore,

alkene 3, either used as a free alcohol (3) or TBS-protected

derivative (9), was subjected to cross metathesis with ethyl

or methyl) acrylate in the presence of Grubbs ﬁrst- and

(

second-generation catalysts. Unfortunately, this reaction

sequence was not successful either.

These failed synthetic routes from acyclic derivatives 2 and 7

prompted us to study an alternative sequence in which the

azido group would be instead appended on the cyclic mannose

scaﬀold prior to the two-carbon chain elongation (Scheme

). Therefore, conveniently protected mannose derivative 11

Scheme 2. Synthesis of Hemiketal 14 and Attempt to

Synthesize Azide Derivative 15

Figure 2. Retrosynthetic analysis of 6-amino-2,6-dideoxy-Kdo. P =

protecting groups.

intramolecular cyclization of either α-halogenated ester I or α-

keto ester II, both bearing a preinstalled azido group at the C6

24,25

position. Tandem Staudinger/aza-Wittig

condensation of

α-keto ester II would be alternatively used for the formation of

the 2-deoxy-imino-Kdo. Both esters I and II would be obtained

from protected 4-azido-4-deoxy-D-mannose III via Wittig or

Horner−Wadsworth−Emmons (HWE) reactions, respec-

tively. Hemiketal III would be synthesized in few steps from

commercially available D-mannose following protecting group

manipulation, C4 epimerization, and azidation.

We ﬁrst planned to install the azido group from known

acyclic precursor 2 once the hydroxyl group is epimerized

Scheme 1). Following optimization, (R)-alcohol 2 was

(

Scheme 1. Attempts to Synthesize Acyclic Azide Derivatives

bearing a free OH at C4 was eﬃciently and stereoselectively

converted into compound 12 (90%) featuring the D-talo

conﬁguration using Dess−Martin periodinane and NaBH as

the oxidizing and reducing agents, respectively. Pleasingly,

conversion of alcohol 12 into a triﬂate derivative followed by

S 2 displacement with NaN in DMF in the presence of 4 Å

MS proceeded with complete inversion of conﬁguration to

provide azide 13 (71%). The anomeric allyl group was then

removed in two steps yielding hemiketal 14 (86%, α/β = 7:1)

ready for subsequent Wittig or HWE homologation.

Wittig oleﬁnation of hemiketal 14 using (carboethoxy-

methylene)triphenylphosphorane was then attempted. How-

ever, we were surprised to ﬁnd that, instead of providing target

azide derivative 15, the reaction led to triazole 16 (22%) along

with α-diazo ester 17 (60%) (see the SI for analytical and

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

Letter

physical details). The formation of these compounds was

Scheme 3. Synthesis of 8-Amino-2,6-dideoxy-Kdo (1) from

α-Keto Ester 20

rationalized by the in situ intramolecular Michael addition of

the C6 azido group at the α,β-unsaturated ester followed by

subsequent rearrangement of resulting triazole 16 into diazo

compound 17. Such chemical behavior was already reported by

Buchanan and co-workers using structurally similar azido-

containing six-carbon derivatives.

Inspired by Chai and co-workers who recently reported a

convenient procedure for the large-scale synthesis of Kdo, we

then studied the HWE reaction with our newly developed

azido-containing hemiketal 14. As shown in Table 1, several

Table 1. Optimization of the HWE Reaction

entry

base

solvent

toluene

DMF

1,4-dioxane

TBME

THF

yield (%)

tBuOLi

tBuOK

NaH

the cyclization step, we tried, without success, to acetylate the

C7 free hydroxyl group in compound 19 (see Table S1). These

results showed that, as this position seemed to be sterically

hindered, it would not be an issue to leave it unprotected for

subsequent steps.

With α-keto ester 20 in hand, we were able to explore its 6-

exo-trig intramolecular cyclization following two alternative

THF

synthetic pathways, namely reductive cyclization and tandem

LDA

nBuLi

LiHMDS

EtOLi

24,25

Staudinger/aza-Wittig condensation

(Table 2). We ﬁrst

screened several conditions known to promote cyclization

through azide reduction (entries 1−11). While no reaction

THF

40,41

occurred for indium(III) chloride or NaI-mediated

The reaction was performed in anhydrous solvents at 50 °C for 2 h

using 3.6 equiv of base and 5.1 equiv of HWE reagent 18 unless

Table 2. Screening of Conditions for the 6-Exo-Trig

Cyclization via Azide Reduction or Tandem Staudinger/

Aza-Wittig Condensation

otherwise stated. Isolated yields. Only traces of enol 19 were

detected by TLC. 1.2 equiv of base and 1.7 equiv of HWE reagent

9 were used. Room temperature for 6 h. Reﬂuxing conditions.

conditions were investigated to increase the reaction yield. We

were pleased to ﬁnd that using an excess of phosphate ester 18

(

5.1 equiv) in the presence of tBuOLi (3.6 equiv) in heated

toluene (50 °C) provided target compound 19 in moderate

yield (41%, entry 1). Switching the solvent for THF allowed

increasing the reaction yield to 45% (entries 2−5). However,

varying the number of equivalents (entry 6), temperature

entry

reductive conditions

yield (%)

PhSeH, Et N, 60 °C

HS(CH ) SH, Et N, DMF, 60 °C

InCl , Et SiH, MeCN, rt

NaI, FeCl , MeCN, rt

NaI, TMSCl, MeCN, rt

(

entries 7 and 8), and type of base (entries 9−14) did not

allow us to further improve the HWE reaction yield. The

moderate yield was ascribed to the unavoidable formation of

diazo byproduct S7, which was generated via the intra-

molecular Michael addition of azide 19 followed by in situ

decarboxylation and subsequent triazole rearrangement as for

compound 17 (see Scheme S4 for details).

20% Pd(OH) /C, H , DCE, rt

Pd black, H , DCE, rt

10% Pd/C, H , DCE, rt

Pd Lindlar, H , DCE, 40 °C

PdO, H , MeOH, rt

Chemoselective cleavage of the TBS group in compound 19

under buﬀered TBAF conditions (AcOH) provided α-keto

ester 20 in 87% yield (Scheme 3). 1D and 2D NMR analyses

revealed a complex mixture of chemical species with α-keto

ester 20 as the major compound. The other species were

tentatively assigned as the α- and β-seven-membered sugars

10% Pd/C, H-Cube (20 bar), 40 °C

Staudinger then NaBH , MeOH, −10 °C

Staudinger then NaBH , MeOH, −78 °C

Staudinger then NaBH , CeCl ·7H O, THF, −78 °C

Staudinger then NaBH(OAc) , AcOH, DCE, 0 °C

Staudinger then NaBH CN, EtOH, 0 °C

(

septanoses) coming from the intramolecular attack of the

C7-alcohol at the C2-ketone position. To prevent the

formation of septanoses that could potentially interfere in

Isolated yields from α-keto ester 20. Degradation. No reaction.

PPh , THF, 60 °C, 5 h.

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

The Supporting Information is available free of charge at

Letter

reductions (entries 3−5), using benzeneselenol or 1,3-

ASSOCIATED CONTENT

sı Supporting Information

■

propanedithiol as the reducing agent led to degradation of

the starting material (entries 1 and 2). Gratifyingly, Pd-

catalyzed reductive cyclization cleanly converted α-keto ester

0 into azacyclic 2-deoxy-α-Kdo derivative 21 (entries 6−8).

The reaction was fully stereoselective, and the yield was

optimal (73%) when 10% Pd/C was used as the catalyst (entry

https://pubs.acs.org/doi/10.1021/acs.orglett.0c01847.

Experimental details and NMR spectra (PDF)

). Interestingly, the reaction was very speciﬁc to the type of

AUTHOR INFORMATION

■

Pd source as only Pearlman’s catalyst, Pd black, and 10% Pd/C

provided clean cyclization products (entries 6−8) as compared

to Pd Lindlar and PdO (entries 9 and 10). We also tried to

perform the cyclization using a microﬂuidic continuous ﬂow

Corresponding Author

Charles Gauthier − Centre Armand-Frappier Sante

Biotechnologie, Institut National de la Recherche Scienti ﬁ que

INRS), Laval, Quebec, Canada H7V 1B7; orcid.org/

000-0002-2475-2050 ; Email: charles.gauthier@iaf.inrs.ca

Complete contact information is available at:

reactor (H-Cube); however, degradation mainly occurred

entry 11). The α-selectivity of the cyclization was

(

Authors

subsequently conﬁrmed by H NMR following cleavage of

the 4,5-O-isopropylidene group (see Scheme 3), which allowed

Oscar Javier Gamboa Marin − Centre Armand-Frappier Sante

Biotechnologie, Institut National de la Recherche Scientiﬁque

the six-membered ring to retrieve the usual C conformation.

(

INRS), Laval, Queb

Nazar Hussain − Centre Armand-Frappier Sante

Institut National de la Recherche Scientiﬁque (INRS), Laval,

Quebec, Canada H7V 1B7

Gokulakrishnan Ravicoularamin − Centre Armand-Frappier

Sante Biotechnologie, Institut National de la Recherche

Scientiﬁque (INRS), Laval, Quebec, Canada H7V 1B7

Nassima Ameur − Centre Armand-Frappier Sante

Biotechnologie, Institut National de la Recherche Scientiﬁque

(INRS), Laval, Quebec, Canada H7V 1B7

Paul Gormand − Centre Armand-Frappier Sante

Institut National de la Recherche Scientiﬁque (INRS), Laval,

ec, Canada H7V 1B7

As a plausible mechanism for this diastereoselective one-pot,

38,45

Biotechnologie,

three-step sequence

(Scheme 3), we propose that upon

azido group reduction the resulting primary amine underwent

an intramolecular 6-exo-trig cyclization to give a hemiaminal.

Because of its instability, the hemiaminal spontaneously

dehydrated into an α-iminoester. Hydrogenation of the latter

occurred from the less hindered (bottom) face of the six-

membered piperidine ring, stereoselectivity leading to 6-

amino-2,6-dideoxy-Kdo derivative 21 featuring the α-conﬁg-

uration.

Biotechnologie,

Alternatively, an intramolecular tandem Staudinger/aza-

4,25

Quebec, Canada H7V 1B7

Janelle Sauvageau − Centre Armand-Frappier Sante

Wittig

reaction sequence was studied to access Kdo

iminosugar derivative 21. The Staudinger reaction was best

Biotechnologie, Institut National de la Recherche Scientiﬁque

performed using PPh in heated THF (60 °C) resulting in the

(

INRS), Laval, Quebec, Canada H7V 1B7; National Research

in situ formation of the α-iminoester, which appeared to be

instable upon isolation. Thereafter, several conditions were

screened for α-iminoester reduction (Table 2, entries 12−16).

Once again, these reductions were fully stereoselective,

exclusively providing α-anomer 21. The yield was optimal

Council Canada (NRC), Ottawa, Ontario, Canada K1A 0R6

https://pubs.acs.org/10.1021/acs.orglett.0c01847

Notes

when NaBH CN was used as the reductive agent at 0 °C in

The authors declare no competing ﬁnancial interest.

EtOH (65% over two steps from α-keto ester 20).

To complete the total synthesis (Scheme 3), fully

deprotected 6-amino-2,6-deoxy-α-Kdo (1) was readily ob-

tained in three nearly quantitative steps from derivative 21

following TBAF-mediated cleavage of the TBDPS group,

ACKNOWLEDGMENTS

■

This work was supported by grants from the Natural Sciences

and Engineering Research Council of Canada (NSERC) under

Award No. RGPIN-2016-04950, the Fonds de recherche du

acidic (TFA/THF/H O) removal of the 4,5-O-isopropylidene,

Queb

FNRS), and the Res

medicaments (RQRM). C.G. holds a Fonds de recherche du

Quebec − Sante (FRQS) Research Scholars Junior 2 Career

Award. O.J.G.M. thanks NSERC, Fonds de recherche du

Queb

ec (FRQ) − Fonds de la recherche scientiﬁque (F.R.S./

and saponiﬁcation (LiOH/THF/H O).

eau quebecois de la recherche sur les

In summary, we describe the total synthesis of 6-amino-2,6-

deoxy-α-Kdo (1) from D-mannose through an intramolecular

-exo-trig reductive cyclization of an azido-containing α-keto

ester. Although our synthetic route toward Kdo iminosugar 1 is

less eﬃcient in terms of number of synthetic steps and overall

ec − Nature et technologies (FRQNT), Centre

d’excellence en recherche sur les maladies orphelines −

Fondation Courtois (CERMO-FC), and Desjardins for MSc

scholarships.

yield than Norbeck and Kramer’s route, it starts from a more

available and aﬀordable compound, i.e., D-mannose instead of

Kdo. Moreover, we believe our synthetic route could be readily

applied for the late-stage diversiﬁcation of the Kdo scaﬀold via,

for instance, functionalization of the in situ formed α-

iminoester intermediate. Importantly, Kdo mimic 1 will have

to be assessed for inhibitory potential against Kdo-processing

enzymes to prove our starting hypothesis. Work toward this

goal is currently in progress in our laboratory.

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