Université René Descartes – Paris 5
UFR Biomédicale des Saints-Pères
Ecole Doctorale du Médicament
Strategic investigations for the design of a
library of liposidomycins analogs, natural
antibiotics dedicated to the MraY translocase
Maryon GINISTY
Direction : Pr. Yves Le Merrer
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques
Direction : Dr. Daniel Mansuy - UMR 8601 – CNRS
45, rue des Saints-Pères - 75270 Paris Cedex 06- France
ANTIBACTERIAL RESISTANCE :
A MAJOR OBSTACLE FOR ANTIBIOTHERAPY
 1940’s : development of penicillin and appearance of the concept of antibiotics
« Agents with specific antibacterial action and with toxicity selectively
directed against bacteria in low concentrations »
► Bacteriostatic effect (decrease or stop of bacterial growth)
► Bactericid effect (destruction of bacteria)
● Complexity and adaptability of bacterial world
► Therapeutic failure
► Development of a large number of antibiotics classified according to various
criteria : site of action, origin, administation route, structure
⇒ Eight major families : b-lactams, aminosides, macrolides,
sulfamides, poly- et glyco-peptides, cyclins, (fluoro)quinolons…
2
RESISTANT STRAINS AND MECHANISMS
⇒ Resistant strain : strain able to develop in the presence of an antibiotic concentration
antibiotic
antibiotic
antibiotic
« modifying
»
notably higher than
that which
inhibits development of other strains of same species
enzyme
pump
● Two types of resistance :
► Natural resistance (intrinsic property related to the bacterial genetic program)
► Acquired resistance (property resulting from genetic modifications of the
resistance
receptor
modified
modified
bacterial
cells)
antibiotic
receptor
gene
● Five major mechanisms of resistance :
- Overproduction of antibiotic target
- Metabolic bypass of inhibited reaction
- Inactivation of antibiotic by enzymatic modification
- Modification of target eliminating or reducing antibiotic binding to target
- Decrease of cellular permeability to antibiotic
3
SITES OF ACTION OF ANTIBIOTICS AND
POTENTIAL TARGETS
⇒ Four sites of action specific to procaryote bacterial cells
- ribosomes responsible for protein synthesis
- metabolism of nucleic acids
⇒ inhibition of DNA synthesis
⇒ inhibition of DNA transcription into messenger RNA
- oxydoreduction (5-nitro-imidazoles) via formation of superoxides
and nitro radicals responsible of irreversible damage on bacterial DNA
O2N
- cell wall biosynthesis
BACTERIA
Bacterial wall
Gram (+) cell
N
CH3
N
R
mRNA
RNA-polymerase mRNA
DNA
external
membrane
ribosome
5-nitro-imidazoles
lipopolysaccharide
periplasm
cytoplasmic membrane
cytoplasm
DNA-gyrase
aminoacid
peptidoglycan
Gram (-) cell
cytoplasmic membrane
cytoplasm
4
fosfomycin
ANTIBIOTICS
AND BACTERIAL
PEPTIDOGLYCAN
BIOSYNTHESIS
UDP-GlcNAc
MurA
O
tunicamycin
muraymycin
PO32mureidomycin
Fosfomycin
liposidomycin
PEP
UDP-GlcNAc-enolpyruvate
UMP
MurB
MraY
NADPH
UDP-MurNAc
Undecaprenyl-P
MurC
L-Ala
Pi
BacA
D-cycloserin
Lipid
Alr I
D-AlaUDP-GlcNAc
MurG
CYTOPLASM
MEMBRANE
UDP-MurNAc-L-Ala
-
OPO32-bacitracin
O2 C
UDP
MurI
MurD
D-Glu
L-Glu
Undecaprenyl-PP
Lipid II
HN O
UDP-MurNAc-dipeptide
PEP
MurE
Polymer
PBPs
meso-A2pm
Acceptor
D-cycloserin
NH2
PERIPLASM
UDP-MurNAc-tripeptide
penicillin
cephalosporin
pentapeptide
PBPs
MurF
Ddl
moenomycin
D-Ala
vancomycin
D-Ala-D-Ala
Peptidoglycan
UDP-MurNAc-pentapeptide
N-acetylmuramic acid
D-Cycloserin
O-
N-acetylglucosamine
O
H3C
O
CH
tétrapeptide
O
tetrapeptide
C
O
O
NHCOCH3
O
NH3+
O
O
D-Alanine
NHCOCH3
NH
N-acetylmuramic acid
N-acetylglucosamine
5
INHIBITORS AND NATURAL SUBSTRATE OF
MraY TRANSLOCASE
Tunicamycins
A : n=9
B : n=10
C : n=8
D : n=11
HO2C
O
CH2 n
HO
HO
HO
O
OH
OH
HO
O
N
O
N O
H
AcHN
H
N
N
H
O
HN
HO OH
Me
R1
NH
HO
O
O
N
O
AcHN O
pentapeptide-HN
HO
O
O
P
O
P
O
A1: R=COC11H22N(OH)C(NH2)=NH
A3: R=COC11H22NHC(NH2)=NH
C1: R=H
NH
O-
OO
N
O
O
O
HO3SO OH
O
OH
HO OH
NH2
R
CH3
HN
O
O
O HN
O
Muraymycins
-
NH
N
HO OCH3
UDP-Mur-NAc-pentapeptide
O
NH
CO2H
HO OH
O
R2
H2N
N
H
HN
O
H3C
H
N
O
O
NH
O
O
OR
H
N
O
O
HO2C
O
O
N
HN
NH
O
CH3O
N
O
HO2C
N
CH3
O
O
O
O
N
O
OH OH
O
R3
O
A: R=
OH
OH
Mureidomycins
R1=H, glycinyl
R2= CH3S(CH2)2R3= m-hydroxyphenyl
Liposidomycins
B: R=
C: R=
NH
6
STRUCTURE OF LIPOSIDOMYCINS
A: R=
B: R=
C: R=
H O 3S O
OH
NH2
R
O
O
O
HO2C
O
CH3 O
6
S 5
HO2C
S
5'
S 7 1N S
2
4 3
N
H
O
HO
O
O
O
N
4' 1'
3' 2'
NH
O
OH
CH3
7
SYNTHETIC APPROACHES DESCRIBED IN
LITTERATURE
 1,4-diazepan-2-one moiety
OHC
acrolein
CH3
⇖
Z N
O
3
Z CHCH
3
4N N
PhCOO
EtO
EtO2C
5
6
3
7 1 2
Boc
N H
O
O H3CCH3 O
N
HO
O
N-Z-sarcosine
Boc
CH3
Knapp et coll. Tetrahedron Lett. 1992, 33, 5485.
Knapp et coll. J. Org. Chem. 2001, 66, 5822.
N-Boc-sarcosine ethyl ester
8
SYNTHETIC APPROACHES DESCRIBED
IN LITTERATURE
 Ribosyl-diazepanone
tBuOOH,
Ti(OiPr)4
OMPM
OH
OMPM
OH
BnO 88%
N
BnO
TBDMSO
O
N
H
N
OCH3
O
O
N
H
Pd/C, H2
O
BnO
OCH
3
O
36% O
O
HO
OMPM
N3
OH
OTBDMSO
O
OH
OMPM
N3
N
H
N3
OHC
O
tBuOOH,
Ti(OiPr)4
OCH3
HO
O
R
L-DET
OBn
DCC / HOBt
R
O
O
N3
NaN3
HO
R
OTBDMS
(S1)
N3
O
OMPM
BnO
50%
OBn
OBn
TBDMSO
NaN3
O
D-DET
OH
OH
Isono et coll. Heterocycles 1992, 34, 1147.
HO
OBn
OH
O
HO2C
R
O
O
78%
(S2)
O
R:
O
OCH3
OCH3
O
9
SYNTHETIC APPROACHES DESCRIBED
IN LITTERATURE
 Nucleosidyl-diazepanone
O
H
Knapp et coll. Org. Lett. 2002, 4, 603.
O
O
O
OH
HO2C
1/ Oxidation
O
O
N3
TBDMSO
2/ NaN3
O
TBDMSO
O
HO
O
HO
OH
H O
O
1/ Ozonolysis
O
H3C
N
EtO2C
OH
H
N CH
3
TBDMSO
2/ Azide
reduction
O
CO2Et O
O
O
O
3/ Reductive
amination
PhCOO
OH
H O
O
N
CH3
NH
N3 2
TBDMSO
EEDQ
CO2Et
O
NHCH3
PhCOO
O
NH
deprotection, acylation
then
glycosylation
O
H 3C
OH
O
N
EtO2C
N CH
3
HO
N
OH
O
10
SYNTHETIC APPROACHES DESCRIBED
IN LITTERATURE
 Nucleosidyl-ribosyl-diazepanone
Angew. Chem. Int. Ed. 2005, 44, 1854.
BocHN
reductive
amination
H22N
N
H
HO
HO
2
glycosylation
4
HO
HO
O
O
HO
HO
NH
NH
O
O
HO
C
HO22C
N
N
Me
Me
3
NH
H2O
C
O
O
N
N
N
N
HO
HO
O
O
TBSO
O
CbzHN
OH
OH
TBDPSO
1 Wittig
H2C
peptide coupling
TBSO
N
O
H
N HO2C
O
O
H
O
O
H
O
O H
F
O
O
OO
O
O
O
Me
Me
O
O
N3
MeO
CbzHN
2C
NHMe
MeO2C
OTBDPS
OH
O
O
NH
O
N
H
O
O
11
O
STRUCTURE-ACTIVITY RELATIONSHIP :
DEVELOPMENT OF A NEW PHARMACOPHORE
O
pentapeptide-HN
O O- O OO
P
P
O
O
O
AcHN
HO O
O
OH
NH
O
HO
N
O
8
P OO
O
O
OH
Undecaprenylphosphate
UDP-N-acetyl-muramoylpentapeptide
OH
O
O
R
HO
HO
OH
HO
HN
AcHN
O
OH
O
OH
O
O
HO
NH
N
O
MraY
R
OH
O
pentapeptide-HN
O O- O OO
P
P
O
O
O
AcHN
HO O
O
OH
Lipid I
O
O
O
HO2C
HO2C
Tunicamycins
O(SO3)H
H 2N
Liposidomycins
O
O
N
NH
O
N
N
O
HO
OH
8
12
O
STRUCTURE-ACTIVITY RELATIONSHIP:
DEVELOPMENT OF A NEW PHARMACOPHORE
OH
HO
HO
NH2
HO
CH3
O
N
O
O
O
O
O
O
N
O
HO2C
NH
O
NH
N
O
O
OH
HO
Structure of natural molecules
OH
Pharmacophore structure
OH
HO
R2
R3O
R4 O
RO
*
*
N
H
Target scaffold
O
N
H3C
HO
NH2
N
O
*
R1
NH22
O
O
O
R2=
N
NH
O
13
SCAFFOLD RETROSYNTHESIS
HO
Y
O
PO
O
HO
H 2N
7
PO
PO
PEPTIDE
COUPLING
1
6
NH
2
5
OH
amino-dihydroxybutane
OH
HO
L-ascorbic acid
N-ALKYLATION
4
HN
1''
3
O
O
4'
H2N 5'
O
⇒
HO2C
NH2
OH
1'
L-Serine
3' 2'
PO
OP
O-GLYCOSYLATION
4
P: protecting groups
X: halogen
H2N
O 1
5
X
HO 5
3 2
PO
4
OP
5-amino-ribose
O 1
OH
3 2
HO
OH
D-ribose
14
STRATEGIES TOWARDS SCAFFOLD SYNTHESIS
amino-dihydroxy- PO
butane
H2N
OH
O
H2N
1
5
3
PO
N-ALKYLATION
X
2
H 2N
PEPTIDE
COUPLING
L-serine
O
Y
NH2
HO
4
HO
B
HO
H2N
O
O
O
4'
A
5'
2'
3'
GLYCOSYLATION
PO
OP
PO
1'
PO
5
7 11
6
NH
B 22
C
4
OP
HN
5-amino-ribose
H2N
PO
aminodihydroxybutane
HO
Y
N-ALKYLATION
PO
PO
7 1
6
4
NH2
HN
H2N
3
4
O
PEPTIDE
COUPLING
OH
L-serine
H2N
5-amino-ribose
5
3
PO
3'
3'
O
A
1
X
GLYCOSYLATION
2
OP
O
O
1'
1'
2'
2'
OP
OP
OH
O
HO
4'
4'
O
O
A
PO
NH
B 2
C
5
5'
1''
1''
33
15
ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »
NHR3
R2O2C
glycosylation
 STEPS AND PRECURSORS
amine
amine
deprotection
deprotection
NH2
PO
O22C
R
R22O
O
2
OP
OP22
OP
2
O
PP2O
O
R1HN
X
P1O
OP1
functionalization
P1O moiety
OP1
of ribosyl
O
O
HN
RR11HN
HN
R2O2C
N-alkylation
PO
O
1
O
O
1
NHR33
PO
N3
R2O2C
OHO
O
azido-epoxide
NHR3
Functionalized
NHR
L-serinyl-O-ribofuranoside
Functionalized
L-serinyl-O-ribofuranoside
STRATEGY 1
3
OP2
HO
glycosylation
P2O
1
RO
NR2
HO
⇗
OH
HN
O
STRATEGY 2NH
X
R1HN
O
O
O
OH
HO
O P2O
OP2
O
P2O
OP2
PO
PO
glycosylation
functionalization
of ribosyl moiety
N3
R2O2C
O
NH
N-alkylation
O
P1 O
1
PO
O
R1HN
⇘
scaffold
N3
HO2C
prefunctionalized
ribofuranose
R1HN
H2 N
HO
TBDPSO
NH
TBDPSO
HN
O
O
R2O2C
OP
1
PO
R2O2C
P1O
NHR3
O
azido-époxyde
OH
NH2
N3
O
X
R2O2C
1
PO
O
O
1
11O
P
OP
PO
1
PO
OP1
1
OP
amine
deprotection
NHR33
NHR
STRATEGY 1
P11O
R22O2C
R O2C
O
O
O
O
PO
P11O 16 OP11
PO
OP
ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »
 GLYCOSYLATION STEP
X= Br,
X=
Br,Cl,
Cl,F,F SR
= OCOR,
O3SR, OP(OR)2, OPO2-OR'.
Substitution via activation of anomeric position
O
X
O
OH
"donor" sugar
R-OH (acceptor)
H+
O
R-OH
OR
acceptor
Direct acid-catalyzed substitution
17
ELABORATION OF O-GLYCOSYLATION STEP (1)
R
R
O
O
X
Y
Y
+
PP11O
O
HO
OP
OP11
OR
O
O
CO2P2
R-OH
NHP3
CO2P2
NHP3
Y
P1O
R = H : L-serine
R = CH3 : L-threonine
OP1
NHR2
⇒ Tricky step :
NHR2
O
OR1
OR1
O
- for the formation
of O-glycosidic derivatives, less known
than that
O
Y
Basic lability
of N-glycosidic analogs
PO
O
O
OP
O
Y
- in the particular case of threonyl and serinyl acceptors
R2HN H
O
1
O
OR
PO
B
OP
Y
H 2O
O
PO
OP
O
Y
"H"
O
PO
OP
Y
H
O
Acid lability
OH
NHR
2
PO
OR1
O
OP
Y
O
PO
NHR2
OP
OR1
HO
O
18
ELABORATION OF O-GLYCOSYLATION STEP (2)
O
X
Y
+
1
1
PO
CO2tBu
HO
activator
O
O
NHP3
Y
NHP3
P1O
OP
CO2tBu
b
a
P3 = Boc, Cbz, Fmoc
OP1
⇒ Success of the reaction and control of stereochemistry depending on three principal factors :
- nature of glycosylation activator
- nature of activation in anomeric position
-nature of the C-2 substituent of the ribose controling a- or b-selective introduction of
serine (anchimeric assistance)
PO
PO
PO
PO
R'-OH
O
O
O
O
OR'
H
X
PO
O
O
R
H
PO
O
PO
O
O
R
O
PO
ROCO
H
banomer
X
R
19
ACTIVATION IN ANOMERIC POSITION
STRATEGY 1
O
O
OP1
glycosylation
reagent
X
Y
Y
P2O
OP2
O
BnO
Y
P2O
OP2
P2O
NHP3
BnO
DAST, THF, -30°C to RT, 1h.
95%
(b/a= 99/1)
SOCl2, DCM,
0°C to RT
O
RO
OR
R = Ac X = Br, Cl
R = Bz X = Br
R = Bn X = Br
X = Br
TMS-Br, DCM,
-40°C to RT.
Koenigs-Knorr method
R2O
O
OR1
R2O
RO
OBn
OP2
X = Cl
X
NHP3
CO2tBu
HO
O
OCO2tBu
F
O
R
Y
O
OR2
1,1=
R1=RR
Ac
R2H= H
2 = Bz,
R2 =RAc,
Bn Bn
O
R = OH, F
Y = N3, PhtN, NHZ
O
OAc
N3
AcO
OAc
STRATEGY 2
20
FORMATION OF PREFUNCTIONALIZED RIBOFURANOSIDES
O
OH
HO
D-Ribose
HO
OH
a : 1/ H2SO4 (0,1N),
65°C, 4h;
2/ Me2C(OMe)2, CSA, Me2CO, 50°C, 30 min.
Me2CO, MeOH, HCl(g),
RT.
75%
*
2 NaN3 + H2SO4
H2O
O
2 HN3 + Na2SO4
0°C
O
O
HN3* (2,8M), PPh3,
DIAD, THF, 0°C.
1/ DOWEX 50W-H+, MeOH, 65°C, 17h.
2/ Ac
98%
2O, pyridine, RT, 2h.
Pd/C,
Pd/C,
H2, EtOH,
H2, EtOH,
CHCl3,
CHCl
RT,
,
TA,
1h30.
1h30.
3
98%
O
O
OO
OH
OMe
N3
N
3
OO
OMe
O
OO
O
O
AcOH, Ac2O,
H2SO4,
RT, 2h.
35%
OAc
N3
a 25%
PhtNK, HMPT,
120°C, 24h.
PhtN
PhtN
OO
OMe
OAc
AcO
OO
O
OMe
OH
84%
TsO
O
O
ZCl, DIEA,
CH2Cl2, RT, 100 %
2h. a 10%
TsCl, Et3N,
DMAP, CH2Cl2,
0°C to RT.
N3
O
O
ZHN
ZHN
50%
(b/a= 3,4)
a 40%
OMe
ClH3N
OMe
HO
HO
AcO
74%
O
OH
O
OMe
O
O
O
O 21
OAc
SYNTHESIS OF L-SERINYL ACCEPTORS
CO2H
HO
HO
CO2tBu
NHR
NH2
R= Fmoc,
Cbz
Boc,
Boc Cbz,
CbzFmoc
N-carbamoyl-L-serine
tert-butyl ester
L-serine
NH
Cl3C
CO2H
BnO
OtBu
CO2tBu
BnO
BF3.OEt2,
cyclohexane, CH2Cl2,
TA, 17 h.
NHBoc
NHBoc
H2, Pd(OH)2/ C,
EtOH abs., AcOH,
TA, 72 h.
96%
HO
CO2tBu
NHBoc
99%
tBu-Br, BnEt3NCl,
K2CO3, CH3CN,
R= Cbz
HO
HO
50°C, 24h.
NHZ
88%
CO2H
CO2tBu
NH
NHR
R= Fmoc
Cl3C
OtBu
HO
AcOEt,cyclohexane,
20°C, 24h.
84%
CO2tBu
NHFmoc
22
SELECTION OF ACTIVATORS AND OPTIMIZATION OF
GLYCOSYLATION CONDITIONS
 STRATEGY
1 (riboses not functionalized
)
OP2
X
O
O
glycosylation
activation
Y
Y
OP1
O
OAc
P1O
OP1
P1O
O
X
RO
RO
RO
RO
OR
P1
P2
Y
Ac
Ac
OAc
CO2tBu
HO activator
NHP3
OR
HO
Activation X = Br, Cl, X
F
P3
Br
Z
TMSBr,
DCM, -40°C à TA
Ac OBz
Brriboses
Z )
 STRATEGIE
2 (prefunctionalized
Bz
Bn
Ac
CMe2
Y
CMe2
Bn
Y
OBn
H O N3OH
H
O
H
N3
O
OBn
Br
DAST, THF,
-30°C à TA, 1h.
NHP
P1O
O
F
O
F
OP1
O
O
RO
OR
Glycosylation
SnCl2/ AgClO4
AgOTf, DCM, -15°C, 15 h.
Boc
Fmoc
HO
Boc
CO2tBu
NHP
RO
O F F Boc activateur
DAST, THF, -30°CYà TA,
2h.
CO2tBu
 Hg(CN)2
 AgClO4
 TMS-OTf
CO2tBu
O
 BFO3.OEt2
NHP3
 AgOTf
 SnCl2/ AgClO4
O
O
SnCl2, AgClO4, Y
-15°C à TA,
48 à 72 h. O
CO2tBu
O
Ratio
(b/a)
Yield
(%)
b: 100%
32
b: 100%
92
0,3
69
CO2tBu
2,15
NHP
1,7
100
1,2
44
NHP
Ginisty M., Gravier-Pelletier C., Le Merrer Y., Tetrahedron: Asymmetry 2004, 15, 189-193.
Ginisty M., Gravier-Pelletier C., Le Merrer Y., Tetrahedron: Asymmetry 2006, 17, 142-150 .
64
23
ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »
NHR
O
O
PO
glycosylation
R2O2C
azido-epoxide
NHR3
3
O
O
PO
amine
deprotection
NH2
N3
R 2O 2 C
O
PO
1
R HN
P 2O
OP2
OP1
P1 O
functionalization of
ribosyl moiety
O
R1HN
O
P2 O
P1O
R2O2C
O
X
1
NHR
OP1
STRATEGY 1
OP2
functionalized
L-serinyl-O-ribofuranoside
NHR3
R2O2C
HO
OH
glycosylation
TBDPSO
NH
O
N3
HO2C
O
X
STRATEGY 2
1
R HN
P2O
OP2
O
prefunctionalized
ribofuranose
R1HN
P1O
OH
1
3
⇗
R2O2C
OP1
O
PO
STRATEGY 1
azido-epoxide
⇘
NH2
N3
NHR3
amine
deprotection
R2O2C
2
R O 2C
O
O
P1O
O
PO
OP
1
R2O2C
OH
glycosylation
O
1
PO
O
X
1
PO
1
1
NHR3
PO
1
OP
P1O
24
OP1
FUNCTIONALIZATION OF RIBOSYL MOIETY
NHR3
1
R2O2C
4'
1
PO
O
3
2
1'
O
functionalization
3
NHR3
at C-2’ and C-3’ NHR
1C-3’
positionsR2O2C1 2 3 C-2’R2and
O2C 2 3
protection
deprotection
4'
5'
3'
P1O
HO
2'
3'
OP1
O
1'
O
5'
HO
2'
4'
HO
1'
NHR3
1
R2O2C
O
4'
1
5'
3'
P 2O
OH
O
substitution
substitution
of the
of the
5’-OH
5’-OH function
function
R HN
2'
O
O
O
OP2
OH
BzO
BzO
NHZ
NHR NHZ
BocHN
NHZ
K22CO33,
1
Pd(OH)
1 2
MeOH/
H
O,H2,Et3N, CH2BzO
1
11
TsCl, DMAP,
Cl2, 1
2/C,
2
3
2
1 22
BzO
OBz tBuO C1 2 3
22
R = Z 82%
33
3OBz
2C
tBuO
C
tBuO
C
tBuO
C
tBuO
C
2
ZHN
tBuO
3
CH
C(OMe)
,
MeOH,
APTS,
2
2
23
3
tBuO222C
3HTA,
2 3CO
EtOH
abs.,1h.
CH
H, 6h.tBuO2C 2
2OMe
0°C
to TA,
PhtNK,
DMF,
+
O
O
O
Boc O
O
O +1'O
(CH
O
O
O
O
3)2CO.
TA,
24h.
OtBu
4'
5'
O
5' O 4' 5' O 4'1'
4'
5'5'
160°C,
12h.
4'
5'
5'
4'
1'
5'
1'
4'
1'
5'
1'
1'1'
4'
1'
TsO
MeOH
PhtN
HO
HO
HO
AcO
HO
BnO
BzO
3' 2'
3' 2'
3' 2'
3' 2'
3'
CO2tBu
3'
2'
3' 2'
81% HO 3' NHZ
2'
2'
3'
MeO
O70%
100%
O
O
O
OH
HO
AcO
OAc
HO HO OHOOtBu
BzO
OBz
O
O
OBn
OH O O
BnO
NHZ
NHR
NHBoc
NHZ
NHZ
X
O
MeO
O
2'
3'
P1 = Ac, Bz, Bn
P2 = C(CH3)2
O
1'
3
5'
P 2O
OP2
2
major product
25
ACCESS TO THE SCAFFOLD BY « CHAIN EXTENSION »
NHR3
NHR3
R2O2C
azido-epoxide
O
PO
glycosylation
O
R2O2C
OH
O
P1O
N3
NH2
R 2O 2 C
O
NHR
amine
deprotection
P1O
R2O2C
O
PO
OP1
P1 O
⇗
P2O
P2 O
OP2
OP2
NHR3
functionalized
L-serinyl-O-ribofuranoside
R2O2C
HO
OH
glycosylation
TBDPSO
O
STRATEGY 2
NH
X
1
R HN
N3
HO2C
O
OP1
STRATEGY 1
1
R HN
X
1
functionalization
of the ribosyl moiety
O
R1HN
O
O
3
P2O
OP2
prefunctionalized
ribofuranose
O
R1HN
P1O
O
PO
OP1
⇘
STRATEGY 1
azido-epoxide
N3
NH2
NHR3
amine
deprotection
R2O2C
2
R O 2C
O
O
P1O
O
PO
OP
1
R2O2C
OH
glycosylation
O
1
PO
O
X
1
PO
1
1
NHR3
PO
1
OP
P1O
26
OP1
N3
AMINE DEPROTECTION
 STRATEGY 1
NHZ
1
tBuO2C
2
3
5'
RO
4'
1'
RO
O
3
2
O
O
5'
1'
4'
O
1
O
tBuO2C
azido
HCO
2NH4
reduction
Pd/C 10%
MeOH, TA.
3' 2'
O
4'
1'
3' 2'
OR
HO
N3NH
NHFmoc
2
X
3
PO
NHFmoc
tBuO2C
5'
RO
OR
R = Ac, Bz
 STRATEGY 2
2
O
3' 2'
RO
1
1
tBuO2C
X
O
O
NH2
H2, Pd(OH)2/ C, CH3CO2H, EtOH abs., RT, 24h.
H2, Pd(OH)2/ C, CH3CO2H, EtOH abs., RT, 48h.
H2, Pd black, CH3CO2H, RT, 48h.
22
O
H222N
N
H
5' 4'
5'
5'
4'
4'
O
O
3'
2'
3' N
2'3
2'
3'
PONHFmoc
NH2
protection
tBuO2C of
C5'-amino group
33
O
O
O
tBuO2C
Y
O
Y= PhtN-, ZHN-
3
2
5'
4'
O
N3 deprotection
of
C2-amino
group
tBuO
2C
O
O
O
1'
1'
1'
O
O O
1
1'
3' 2'
O
H2N
NH
O
1
tBuO2C
O
O
Y
N3
O
O
2
5' 4'
O
27
1'
3' 2'
O
ACCES TO THE SCAFFOLD BY « CHAIN EXTENSION » :
CONCLUSION AND PERSPECTIVES
 Powerful glycosylation conditions for the diastereoselective formation of serinyl-5’-amino-b-Dribofuranoside derivatives
⇒ unfinished strategy because of difficult functionalization of the ribosyl moiety and amine
deprotection.
 Perspective :
⇒ strategy 2 : glycosylation of 2,3-O-isopropyliden-D-ribofuranoside derivatives differently
N-protected, whose synthesis was already carried out.
.
HO
1) glycosylation
functionalization
of the
OH ribosyl moiety
O
5 4
HO
3 2
1
OH
O
N3
5 4
O
3 2
O
OMe
1
Y
O
5 4
3 2
O
2) amine
deprotection
X
1
tBuO2C
O
Y
O
5'
4'
3'
A
HO
3
2
NH2
1 2
CO2tBu
NHFmoc
28
3
O
1'
2'
O
O
1
Y = PhtN-, ZHNX = activated group
B
ACCESS TO THE SCAFFOLD BY DIRECT COUPLING
amino-dihydroxy- PO
butane
NH2
H2N
PO
H2 N
HO
OH
O
1'
5'
3'
N-ALKYLATION
PEPTIDE
COUPLING
L-serine
4'
Y
NH2
HO
O
HO
X
H 2N
GLYCOSYLATION
OP
O
O
O
4'
A
5'
1'
PO
2'
3'
2'
PO
PO
7 1
6
5
NH
B 2
C
4
OP
HN
5'-amino-ribose
4'
H2N
PO
aminodihydroxybutane
PO
HO
Y
PO
7 1
6
4
HN
NH2
HO
4'
OH
L-sérine
H 2N
5'-amino-ribose
O
1'
5'
3'
PO
X GLYCOSYLATION
2'
OP
1'
OP
OH
O
O
2'
3'
PO
3
H2N
O
5'
O
O
A
NH
B 2
C
5
1''
3
29
ACCESS TO THE SCAFFOLD BY DIRECT COUPLING
CAG STRATEGY
Y
1
PO
N-alkylation
2
peptide
coupling
H 2N
6
PO
HO
7
3
5
O
O
HO
4
N
H
O
OH
OH
HO
L-ascorbic acid
Y
44
PO
PO
NH
C B 3
55
6
7
11 N 2
H
PO
H2N
PO
O
OH
OH
NH2
amino-butanol
1,4-diazépan-2-one
1,4-diazepan-2-one
HO
O
L-serine
peptide
coupling
PO
NH
3
6
1
2
PO
7
ACG
STRATEGY
4
5
NH2CO2H OH
N-alkylation
30
FORMATION OF N1-C2 LINKAGE BY PEPTIDE COUPLING
- FIRST STEP OF THE CAG STRATEGY  SYNTHESIS
OF AMINO-BUTANOL PRECURSORS
OTBDPS
O
OTBDPS
O
NH2
amino-epoxide
N3
azido-epoxide
HO
OTBDPS
O
O
NH2
amino-acetonide
EtO2C
TBDPSO
O
O
OTBDPS
NH2
amino-ester
O
CO2OTBDPS
Et
O
N
OH
3
HO
azido-acetonide
3,4-O-methylethylidenL-threonine ethyl ester
TBDPSO
CO2Et
N3
OTBDPS
azido-ester
O
HO
OH
L-ascorbic acid
CO2Et
O
O
OTBDPS
31
FORMATION OF N1-C2 LINKAGE BY PEPTIDE COUPLING
- FIRST STEP OF THE CAG STRATEGY  SYNTHESIS
OF AMINO-BUTANOL PRECURSORS
TBDPSO
Introduction
of an electrophilic group
in C1 position
CO2Et
R
PO
4
O
1
3
2
N3
introduction in
C3 position
of the azido group
N3
OTBDPS
PO 4
HO
O
HO
HO
OH
Me2C(OMe)2, Me2CO, PO
HCl(g), RT, 12h.

O
O3
PO
94%
L-ascorbic acid
introduction
of the azido group in
C2 position
OOP O
180°
1 rotation
4
2
HO
OP
OTBDPS
O
O
1) H2O2, H2O, K2CO3,
0°C, 2h.
2) EtI, CH3CN,
85°C, 12h.
PO
O
PO
N3
OP
1
-
Y
N3
2
OP
OH
OH
OP
4
3 2
O
introduction
of an electrophilic group
in C4 position
+
4
3 2
1 CO
180°
Et
CO22rotation
Et
O
4 3 2 1
X
4
3
78%
OH
SN en C3
X

O
1 OP
3 2
PO
SN en C2
OP
1 OP
N3
P : protecting group
R = OEt, H
32
FORMATION OF N1-C2 LINKAGE BY PEPTIDE COUPLING
- FIRST STEP OF THE CAG STRATEGY  SYNTHESIS
O
CO2Et
O
OF AMINO-BUTANOL PRECURSORS
TBDPSCl, ImH,
DMF,
0°C to RT, 12h.
O
CO2Et
TBDPSO
4
3
OTBDPS
N3
O
OH
TFA,
H2O,
1) MeC(OMe)
, PPTS, CH2ClCO
TBDPSCl, ImH, TBDPSO
3HO
2 Et
2
CO0°C,
EtAcBr,
4
22)
Et
THF,
3h.
DMF, 0°C to RT.
3N, CH2Cl2
1
3) K2CO3, MeOH.
2
Sharpless epoxidation
HO
OTBDPS
HO
OTBDPS
100 %
74%
PPh3,
H2O, THF,
RT, 72h.
TBDPSO
87%
3 2
H2N
O
CO2Et
CO2Et
O
LiAlH4, THF,
0°C
OH
98%
O
O
O
N3
OTBDPS
3
OTBDPS
1
CO2Et
4
TFA, 1)Tf
THF,2O,
H22,6-lutidine,
O,
CH
0°C, 2h2. Cl2, -78°C.
NH2 HO
O
98%
N3
N3
OTBDPS
2) NaN3, DMF,
55%
0°C to RT, 12h.
PPh3, DIAD,
130°C,
0,1 mmHg
1
ImH,
4 TBDPSCl,
OTBDPS
OH
DMF,
2 0°C to RT.
OHO
OTBDPS
3 2
Mitsunobu reaction
O
2Et
74%
HCO2NH4, Pd/ C 10%,
HOOTBDPS
OTBDPS
MeOH, RT, 2h.
TBDPSO
1
4
87%
1CO
3 2
N3
88%
1)Tf2O, 2,6-lutidine,
OTBDPS CH2Cl2, -78°C.
78%
O
O
O
2) NaN3, DMF,
0°C to RT, 12h.
OH
80%
OTBDPS
O
N3
90%
O
OTBDPS
O
NH2
PPh3, H2O, THF,
RT, 72h.
78%
33
PEPTIDE COUPLING : SUBSTRATES AND PRODUCTS
"AMINO" PRECURSORS
PEPTIDE
COUPLING
COUPLING
PRODUCTS
CO2Et
TBDPSO
YIELD
CO2Et
NHFmoc
TBDPSO
100 %
a
NH2
N
H
TBDPSO
TBDPSO
amino-ester
OBn
O
O
O
P1HN
HO2C
NH2
O
a
O
NHFmoc
OP
TBDPSO
N
H
L-SERINE
amino-acetonide
TBDPSO
O
O
4'
a or b
1'
TBDPSO
O
P2
2
2'
NH2
OBn
P1
NHP1
3'
TBDPSO
100 %
2
3
1
N
H
OP2
Fmoc
Coupling
reagent
PyBOP
78 %
HBTU *
49 %
80 %
Bn
O
amino-epoxide
a : PyBOP, DIEA, CH2Cl2
b : HBTU, DIEA, DMF
Z
tBu
PyBOP
Boc
Bn
PyBOP
* 25 % of epimerization in C2 position
99 %
34
ACCESS TO THE SCAFFOLD BY DIRECT COUPLING
CAG STRATEGY
Y
1
PO
2
N-alkylation
peptide
coupling
H 2N
6
PO
7
3
4
N
H
5
O
OH
Y
4
PO
PO
NH
C B 3
5
6
7
1N 2
H
PO
H2N
PO
O
OH
OH
NH2
amino-butanol
1,4-diazepan-2-one
HO
O
L-serine
peptide
coupling
PO
4
NH
5
3
6
1
2
PO
7
NH2CO2H OH
N-alkylation
ACG STRATEGY
35
ACCESS TO THE SCAFFOLD BY DIRECT COUPLING
CAG STRATEGY
INTRAMOLECULAR PATHWAY
Y
1
PO
2
N-alkylation
peptide
coupling
H 2N
6
PO
7
3
4
N
H
5
O
OH
Y
PO
PO
NH
C B 3
5
6
7
4
1N
H
2
O
PO
H2N
PO
OH
OH
NH2
amino-butanol
1,4-diazepan-2-one
HO
O
L-sérine
peptide
coupling
PO
NH
3
6
1
2
PO
7
ACG STRATEGY
4
5
NH2CO2H OH
N-alkylation
INTERMOLECULAR PATHWAY
36
FORMATION OF N4-C5 LINKAGE BY N-ALKYLATION
INTRAMOLECULAR PATHWAY (CAG Strategy)
Y
O
R
PO
H2N
PO
H2N
OP
N
H
O
OP
N
H
PO
PO
O
PO
Y= Br, OTs
R= O, O-(SO2)-O
H2N
NHP PO
PO
PO
PO
OP
O
N
H
OP
NHP PO
INTERMOLECULAR PATHWAY (ACG Strategy)
O
CHO
NH2
H2N
PO
OP
Reductive amination
Y
O
R
CHO
NH2
O
NHP PO
PO
OP
O
37
FORMATION OF N4-C5 LINKAGE BY N-ALKYLATION :
NUCLEOPHILIC ATTACK OF AN ACTIVATED PRIMARY CARBON
 INTERMOLECULAR PATHWAY
NH
HO
O
H 2N
2
1
tBuO
PO
P
O 2C
NHP
N
3
PO
TBDPSO
BnO
CO2H
HO
OP
OBn
OP2
TBDPSO
O
HCl 3M/AcOEt, RT, 1h.
P1
P2
100 %
NHBoc
O2Bn
CO
OBn
P1 OP
N3 CO2tBu
TBDMS-Cl,
ImH, DMF,
H
H.HCl
0°C to RT, 15h.
H2N
CO2H
BnO
Opening conditions
tBu100 % Bn
H
H.HCl
Bn
TBDMS
tBu
Bn
NH3Cl
tBuOH, NaH, 100°C
Cs2CO3, DMF, 65°C
CO2Bn
TBDMSO
Cs2CO3, DMF, 110°C+
-
CO2Bn
TBDMSO
NH2100°C
tBuOH, NaH,
OP
70%110°C
Cs2CO3, DMF,
N
TBDMS
H ClOBn
PO
3C-C(NH)-OtBu,
MeOH, Et3N, 60°C
cyclohexane,
CH
Cl
,
2
2
CO2H
2tBu
Yb(OTf)3CO
, (Et
DCM,
TART, 2h.
THF,
50°C, 3h.
BnO
3N), DBU,
NHFmoc
P2
Yield
NH3Cl
BnO
P1
NHFmoc
%
Yb(OTf)3, DCM, RT, 7100
days
NHCHO
-
25%
BnO
65 %
CO2tBu
NH2
38
FORMATION OF N4-C5 LINKAGE BY N-ALKYLATION :
NUCLEOPHILIC ATTACK OF AN ACTIVATED PRIMARY CARBON
 INTRAMOLECULAR PATHWAY
O
NHFmoc
TBDPSO
N
H
OBn
O
piperidine,
deprotection
DMF, RT
X
65%
TBDPSO
epoxide
opening
O
H2N
N
H
HO
NH
X
OBn
O
TBDPSO
N
H
OBn
O
Acid conditions :
Yb(OTf)3, (Et3N), CH2Cl2, RT, 6 days.
LiNTf2, CH2Cl2, RT, 48h.
Basic conditions :
Cs2CO3, DMF, RT to 110°C, 20h.
tBuOH, NaH, 100°C.
« Neutral » conditions :
MeOH, RT, 15 days.
iPrOH, RT, 18h.
iPrOH, 50°C, 4 days.
39
MOLECULAR MODELING OF AMINO-EPOXIDE
Amine function
involved in
epoxide ring opening
Primary carbon atom
of epoxide ring
H NH2
O
H
N
O
O
O
Si
« -stacking » interactions
« -stacking »
interactions
40
N-ALKYLATION BY REDUCTIVE AMINATION
 SYNTHESIS OF PRECURSORS INVOLVED IN REDUCTIVE AMINATION
PO
1/ Aldehyde derivatives
PO
PO
O
PO
peptide
PO
LiBH4, MeOH,
coupling
O
Et O, 0°C, 4h.
NH
N
OH
CO2Et
OP
O
OTBDPS
2
1
83%
target diazepanone
2
H
4
OTBDPS
OP
CO2P
CHO
CHO
NHP PO
DIBAL-H (1M
POin toluene),
CH2Cl2, -78°C, 2h.
96%
PO
OPO
44
CHO
PO
2
1
3
O
4
93%
acetonide
NH2-aldehyde
OP
O
acetonide
-aldehyde
functionalization
of the diol moiety
11
CHO
CHO
22
33
N33
N
H 2N
PO
PO
azido-aldehyde
azido-aldehyde
OP
L-serine
1
(ClCO)2, DMSO, Et3N,
CH2Cl32, -78°C, 2h.
Reductive amination
O
4
CO2P OP
NH
O
O 2 OTBDPS
PO
H 2N
O
2
reductive
amination
N3
OP
reductive
amination
PO
3
CO2P
O
O
HO
NH
PO
OTBDPS
CO2Et
2
CHO 1
NH2
functionalization
of the diol moiety N
PO
NH
4
3
L-serine
CO2P
OP
P= TBDPS
41
N-ALKYLATION BY REDUCTIVE
AMINATION
FUNCTIONALIZATION
OF THE DIOL
MOIETY
TBDPSCl,
R’ = H
ImH,
DMF,
 SYNTHESIS OF PRECURSORS
0°C to RT, 15h.
R’ = TBDPS
TBDPSO
INVOLVED IN REDUCTIVE AMINATION
TFA, H2O,
THF,
0°C, 3h.
NZ
2/ Serinyl derivatives
OH
1/ Tf2O, 2,6-lutidine,
Cl3C-C(NH)-OtBu,
R'O
CH2Cl2, -78°C,
2h.
cyclohexane,
CH2Cl2,
CO2H
BnO
50°C, 3h.
2/ NaN3, DMF,
0°C to RT, 15h.
NHFmoc
TBDPSO
4
1
2
BnBr, K2CO3,
O
CHO
DMF, RT, 3h Bn
BnO
1
N3 CO2R OBn
O
100 %
TBDPSO
BnO
OP
CO2H
NHBoc
R1
PO
reductive
amination
CHO
EtI,
OP3,
N3 Cs2CO
CH3CN,
reflux, 1h30
100 %
1/ Step 1
2/ NaBH3CN, EtOH abs., 18 h.
tBu
tBu
CO2R1 OBn
O
H 2N
O
BnO
DBU, THF, RT, 2h.reductive
2/ NaBH3CN,
EtOH abs., 18 h.
amination
NHFmoc
Aldehyde
derivative
3
R=Z
1/ tBu
Step 1
CO
2
BnO
ZCl, K2CO3,
DMF, TA, 1h.
NR
CO2R1 OBn
100 %
NH
TBDPSO
R=H
R 1O 2C
OBn
NH2
100 %
Step 1Y: reductive
ZC
YAR
YTFA YTBDPS
YAR YN3
amination
l
TFA, CH2Cl2,
DIEA, DCM,
4Ả
molecular
CO84
min.
2Bn
84 sieves,
76 RT, 3094
84 85
RT, 15h.
100 %
NHBoc
Ti(OiPr)495
, DCM, RT,
58
83 3h 90 58
27
CO2tBu
L-serine
CHO
TBDPSO
2
-
3
CO2Bn
BnO
4
acetonide
-aldehyde
27
47
TFA, CH2Cl47
2,
RT, 30 min.
CO2Et
BnO
1
TBDPSO
H2N
100 %
NHBoc
4
1
R O 2C
OBn
L-serine
TBDPSO
O
O 2H
NH2.CF3CO
Ti(OiPr)4, DCM, RT, 3h
Et
1
CO2Et
BnO
CHO
2
3
NH2.CF3CO2H
N3
azido-aldehyde
42
ACCESS TO THE SCAFFOLD BY DIRECT COUPLING
CAG STRATEGY
Y
1
PO
2
N-alkylation
peptide
coupling
H 2N
6
PO
7
3
4
N
H
5
O
OH
Y
4
PO
PO
NH
C B 3
5
6
7
1N 2
H
O
PO
H2N
PO
OH
OH
NH2
amino-butanol
1,4-diazepan-2-one
HO
O
L-serine
peptide
coupling
PO
4
NH
5
3
6
1
2
PO
7
NH2CO2H OH
N-alkylation
ACG STRATEGY
43
FORMATION OF N1-C2 LINKAGE BY PEPTIDE COUPLING
NR2
R3O
azido
1
reduction
1 OBn
N3 CO2R
TBDPSO
Formation
Bn
Z
TBDPS
Reductive amination
Et
H
TBDPS
Reductive amination
tBu
H
H
Epoxide ring opening
tBu
H
TBDPS
Reductive amination
R3
Bn
Z
TBDPS
Et
H
TBDPS
Azido reduction
NH
NH
NR2
deprotection
R2 = H
R3
R33O
2
X
AND
TBDPSO Bu P
deprotection TBDPSO
3
R2
R2
2
NR22
CO22R
R111 OBn
OBn
H
OBn
NH22CO
NH
2R
N
R1
R1
3
R33O
TBDPSO
TBDPSO
R33O
O
R
NH
peptide
coupling
OBn
OBn
N
N
H
H
X
O
O
TBDPSO
TBDPSO
Target diazepanone
diazepanone
Target
Yield
Deprotection
Peptide
Coupling
Yield
DCC, HOBt, DCM, RT
-
Yield
H2, Pd/C 10 %, MeOH, AcOEt, RT, 24h.
1 nBu3P, toluene, RT (3h) to reflux (5h)
OBn
CO22H
H OBn
OBn
NH22CO
NH
N
H
O
2
TFA, THF, H2O, RT, 15h.
-

tBu
H
H
51
HCO2NH4, Pd/C, MeOH,
RT, 20 min
tBu
H
TBDPS
DCC/ HOBt, DIEA,
DCM/ DMF, RT, 24h.
 PyBOP, DIEA, DCM,
RT, 24h
100
TFA, DCM, RT, 20h.
-

71
100
EDCI/ HOBt, DIEA,
DCM/ DMF, RT, 24h.
 DCC, DIEA, DCM, RT. 44
-
MOLECULAR MODELING OF THE
« COMPLEX AMINO-ACIDS »
-stacking
interaction
-stacking
interaction
O
H O
H 2N
N
H
O
H
O
O
Si
Si
-stacking
interaction
« hydrophobic
site »
amine function involved
in peptide coupling
bis-O-silylated compound
O
H
O
NH2
H
N
H
O
acid function involved in
peptide coupling
HO
hydrophobic
interactions
O
Si
Mono-O-silylated compound
45
CONCLUSION AND PERSPECTIVES
OP
⇒ RING CLOSURE ?
PO
OP
NP
PO
N 2
NH
R
PO
CO2H
⇒ HOAt
O
OH
HO
R2
O
O
X
NHP
O
PO
O
N-ALKYLATION
R
O
PHN
N3
OP
6
7
O
5 4N
3
1N
H
2
NH2
O
O-GLYCOSYLATION
O
PO
N
H
O
PEPTIDE COUPLING
46
TOWARDS A NEW FAMILY OF POTENTIAL
ANTIBIOTICS
2
O
1
NR
NHH
2N
RHO
O
3
RPO
O
PO
OOBn O NH
NHP
2 NHP
OBn F
OP
CO2tBu
H O
N
N
NH
33 2 tBuO
O
H
O
OH
O
O
HO
O
O
R1, R2 , R3 =
HO A
OH
O
(CH2)n CH3
OH
(CH2)n CO2H
O
N-Alkylation of
L-serine
tert-butyl ester
+ Intramolecular peptide Coupling
+ O-Glycosylation of diazepanone heterocycle
NH
N
O
A = OH, NH2, NHAc...
Ribosyl-diazepanone scaffold
+
R 1 / R 2/ R 3
Family of powerful MraY inhibitors
⇒ Biologic evaluation (Laboratoire des Enveloppes Bactériennes et
47
Antibiotiques – Dr D. Blanot – Dr. D. Mengin-Lecreulx)