Synthesis of peptidoglycan units with UDP at the anomeric position

Article abstract of DOI:10.1135/cccc20051615

A series of UDP-disaccharide peptide compounds were synthesized as synthetic substrate analogues or potential inhibitors of glycosyl transferase. Fluorescent compounds have been prepared with the aim of developing a screening method for selecting transglycosylase inhibitors.

Full text of DOI:10.1135/cccc20051615

Synthesis of Peptidoglycan Units with UDP
1615
SYNTHESIS OF PEPTIDOGLYCAN UNITS WITH UDP
AT THE ANOMERIC POSITION
b1
Thierry LIOUXa1, Roger BUSSONa2, Jef ROZENSKIa3, Martine NGUYEN-DISTÈCHE ,
b2
a4,
Jean-Marie FRÈRE and Piet HERDEWIJN
*
a Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, Minderbroedersstraat 10,
B-3000 Leuven, Belgium; e-mail: tlioux@yahoo.fr, roger.busson@rega.kuleuven.ac.be,
jef.rozenski@rega.kuleuven.ac.be, piet.herdewijn@rega.kuleuven.ac.be
Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, B-4000 Sart
Tilman, Belgium; e-mail: mng.disteche@ulg.ac.be, jmfrere@ulg.ac.be
1
2
3
4
b
1
2
Received March 18, 2005
Accepted Jun e 17, 2005
A series of UDP-disacch aride peptide com poun ds were syn th esized as syn th etic substrate
an alogues or poten tial in h ibitors of glycosyl tran sferase. Fluorescen t com poun ds h ave been
prepared with th e aim of developin g a screen in g m eth od for selectin g tran sglycosylase in -
h ibitors.
Keyw ord s: Peptidoglycan s; UDP-disacch aride; Dan syl; Dipeptides; Oligosacch arides;
Glycopeptides; Am in o sugars; Carboh ydrates; Glycosidation s; Glycosyl tran sferases.
Th e recen t in crease in bacterial resistan ce to active agen ts, like glycopeptide
an tibiotics (van com ycin ) an d β-lactam an tibiotics (pen icillin ) wh ich in h i-
bit th e growth of peptidoglycan , by specific in h ibition of pen icillin -
bin din g-protein s (PBPs), h as reach ed an alarm in g level an d h as begun to
erode th eir on ce reliable clin ical efficacy1.
Th e peptidoglycan (PG), wh ich form s a sacculus aroun d th e bacterial cell,
is an essen tial cell wall polym er protectin g bacteria from lyses un der h igh
osm otic pressures2. Man y of th e safe an d poten t an tibiotics fun ction by in -
h ibitin g peptidoglycan syn th esis an d a lot of research h as been devoted to
isolate an d ch aracterize en zym es in volved in th e syn th esis of peptido-
glycan . All th ese protein s are poten tial targets for design in g n ew an tibiotics.
Peptidoglycan is presen t in prokaryotes an d h as n ever been iden tified in
eukaryote cells. In Gram -positive bacteria as m uch as 90% of th e cell wall
con sists of peptidoglycan wh ereas in Gram -n egative bacteria th is figure is
on ly 5–20% 3. Structurally th e peptidoglycan layer is com posed of lin ear
polysacch aride ch ain s of altern in g N-acetylglucosam in e an d N-acetyl-
m uram ic acid un its, cross-lin ked via sh ort peptides appen ded to m uram ic
Collect. Czech. Chem. Commun. (Vol. 70) (2005)
doi:10.1135/cccc20051615

1616
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
acid4. Th e peptidoglycan biosyn th esis takes place in th ree distin ct stages5.
Th e first occurs in th e cytosol in wh ich UDP-N-acetylglucosam in e is con -
verted in two steps in to UDP-N-acetylm uram ic acid by MurA an d MurB en -
zym es. Th en , MurC, MurD, MurE an d MurF ATP-depen den t ligases catalyse
th e form ation of th e UDP-N-acetylm uram oyl-pen tapeptide by sequen tial
addition of L-alan in e, D-glutam ic acid, L-lysin e (in th e case of Gram -positive
bacteria) or meso-diam in opim elic acid (in th e case of Gram -n egative bacte-
ria) an d th e preform ed D-alan yl-D-alan in e dipeptide. Th e secon d step occurs
at th e cytoplasm ic surface of bacteria m em bran e in wh ich MraY catalyzes a
pyroph osph ate exch an ge reaction in wh ich th e UDP-MurNAc-pen tapeptide
is coupled to a tran sm em bran e carrier, th e C55-isopren oid alcoh ol ph os-
ph ate. Th e product of th is reaction provides th e lipid I in term ediate. Th en
MurG catalyses th e tran sfer of GlcNAc from UDP-GlcNAc to th e C(4)-h y-
droxyl group of lipid I. Th e product of th is reaction provides in turn th e ul-
tim ate m on om eric in term ediate of bacterial cell wall: th e lipid II. Th e th ird
stage occurs in th e bacterial cell wall after th e tran slocation of lipid II to th e
extracellular site of th e cell m em bran e. Th e assem bly of th e lipid II un its
in to th e peptidoglycan is carried out by specialized tran sferases, i.e. glycosyl
tran sferase, wh ich catalyses glycan ch ain elon gation (tran sglycosylation ) by
displacin g th e pyroph osph ate lin ked to C(1) of N-acetylm uram ic acid of
on e disacch aride un it by th e C(4) h ydroxyl group of N-acetylglucosam in e
of an oth er disacch aride un it. Th en an acyl serin e tran sferase workin g as
tran speptidase catalyses peptide cross-lin kin g between glycan stran ds. Th e
rupture of th e D-alan yl-D-alan in e bon d at th e carboxy en d of a
pen tapeptide un it an d th e attack of th e pen ultim ate D-alan yl by th e am in o
group of a meso-diam in opim elic acid or lysin e residue of an oth er peptide
proceeds via th e form ation of a peptidyl en zym e in wh ich th e D-alan yl m oi-
ety is lin ked as an ester to a serin e residue at th e en zym e active site.
Th e path way to peptidoglycan is on e of th e m ost h igh ly con served m eta-
bolic path ways in bacteria an d is a m ajor target for an tibiotics. Un derstan d-
in g h ow peptidoglycan is m ade is essen tial for un derstan din g h ow bacteria
build up th eir cell wall an d it is im portan t in form ation for design in g n ew
an tibiotics. In a recen t con tribution , Kah n e an d co-workers suggest th at th e
an tibacterial effects of som e glycopeptide derivatives are n ot n ecessarily
based on stron g peptide bin din g but rath er on in teraction s with protein s
in volved in th e tran sglycosylation steps of th e cell wall biosyn th esis6. Here
we h ave developed ch em ical tools, syn th etic substrate an alogues an d po-
ten tial in h ibitors th at en able th e study of en zym es in volved in th e fin al
steps of peptidoglycan biosyn th esis an d especially th e glycosyl tran sferase
step for wh ich th e m ech an ism of action is n ot yet kn own . We h ave syn th e-
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1617
sized peptidoglycan m on om er with m odification in th e peptide m oiety an d
with a uridin e diph osph ate at th e an om eric position . We selected th e
uridin e m oiety (in stead of a lipid m oiety) because previous studies h ave
dem on strated th at UDP-disacch aride-pen tapeptide in h ibits th e glycosyl
tran sferase reaction 7. Fluorescen t as well as n on -fluorescen t probes h ave
been obtain ed. Th e fluorescen t com poun d h as been syn th esized with th e
aim of developin g a screen in g m eth od for selectin g tran sglycosylase in h ibi-
tors.
CHEMISTRY AND DISCUSSION
Recen tly, th ree syn th etic sch em es8–10 to obtain n atural lipid II h ave been
publish ed. Th e first describes th e con version of lipid I to lipid II usin g puri-
fied E. coli MurG 8. In th e secon d an d th e th ird on e, th e com poun ds were
obtain ed by total syn th eses9,10. Th ese last on e10 h as been used as a guide for
th e preparation of lipid II an alogues described in th is article.
To in troduce th e desired m odification s, we first syn th esized th e core
disacch aride of th e peptidoglycan un it. Th e key startin g m aterial con sists of
th e fully protected N-acetyl-D-glucosam in e an d N-acetylm uram ic acid. Th e
discon n ection in th e retrosyn th etic sch em e of th is disacch aride leads to
two structurally related glycoside derivatives A (don or) an d B (acceptor)
wh ich could be obtain ed, respectively, from D-glucosam in e (GlcNH2) an d
N-acetyl-D-glucosam in e (GlcNAc) for th e glycosylation step11 (Sch em e 1).
For th e large-scale syn th esis of th e core of th e glycan , we in vestigated th e
best ch oice of th e leavin g group at th e an om eric position of th e glycosyl
don or A, th e best way to liberate th e 4-h ydroxyl group of th e acceptor frag-
m en t B, an d fin ally th e activatin g agen t wh ich is m ost appropriate for th e
couplin g reaction . To obtain don or fragm en ts with differen t reactivity at
th e an om eric cen ter we h ave prepared various exam ples of N-[(2,2,2-tri-
ch loroeth oxy)carbon yl] (N-Troc) acetylated don or A (Sch em e 2). Our in ves-
tigation s cover th ree basic m eth ods for oxazolin e in term ediate form ation
i.e. th e Koen igs–Kn orr glycosylation m eth od with an α-brom o glycoside12
a th ioglycoside activation 13 an d Sch im dt’s trich loroacetim idate m eth od14
,
.
Glycoside bon d form ation with don ors derived from GlcNAc occurs gen er-
ally via n eigh borin g group participation . N-Ph th aloyl (N-Ph th ) protected
don or usually gives th e desired β-glycoside in h igh yield an d stereo-
selectivity, but recen tly, th e N-Troc protectin g group h as resulted in a
40-fold m ore reactive buildin g block th an th e ph th aloyl protectin g con ge-
n ers13. Th erefore we decided to use th e Troc group for protection of th e
2-am in o substituen t of glucosam in e. Protection of th e am in o group with
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1618
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
O
OH
O
O
N
NH
O
O
HO
HO
O
NH
O
O
O
P
O-
O
P
O-
NH
OH
O
O
O
O
O
HN
Peptide
OH OH
O
O
OAc
NH
O
AcO
AcO
O
NH
O
O
O
OAc
O
P
O-
O
NH
O
O-
-O
P
O-
O
N
O
O
O
HN
Peptide
OH OH
O
OAc
NH
O
AcO
AcO
O
O
O
Peptides
O
OAc
O
P
NH
O
O
OBn
HO
OBn
O
OAc
O
NH
O
AcO
AcO
O
OBn
OBn
O
O
O
OAc
N
P
NH
OH
O
O
O
S
OR
O
O
O
HO
NH
O
AcO
AcO
X
NH
OBn
O
OAc
O
O
O
O
B
A
S
OH
NH2
O
O
HO
HO
HO
HO
OH
NH
OH
OH
O
D-Glucosamine (GlcNH2)
N-Acetyl-D-glucosamine (GlcNAc)
SCHEME 1
Retrosyn th etic an alysis
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1619
Troc
Troc
NH2
NH
O
NH
O
(i), (ii)
(v)
HO
HO
AcO
AcO
AcO
AcO
OH
OAc
OH
O
OH
OAc
OAc
D-Glucosamine (GlcNH2)
2
5
(iii)
(iv)
(vi)
NH
Cl
Cl
Br
NH
Troc
O
Troc
Troc
NH
NH
Cl
AcO
AcO
AcO
AcO
AcO
AcO
S
O
OAc
O
O
OAc
OAc
4
6
3
(i) TrocCl, NaHCO3, H2O; (ii) Ac2O, pyridine, 98%; (iii) HBr, 33% in AcOH/CH2Cl2, 97%;
(iv) 4-methylbenzenethiol, BF3·(Et2O)2, CH2Cl2. 85%; (v) NH2NH2, CH3COOH;
(vi) Cl3CCN, DBU, CH2Cl2, 79%
SCHEME 2
Don or fragm en t syn th esis
(2,2,2-trich loroeth oxy)carbon yl ch loride in th e presen ce of aqueous sodium
h ydrogen carbon ate is followed by per-O-acetylation to give 2 (as α, β m ix-
ture) in alm ost quan titative yield12. Th en com poun d 2 was brom in ated us-
in g 33% HBr in AcOH in dich lorom eth an e to give th e first don or 3 in h igh
yield12. In th e secon d exam ple com poun d 2 was treated with p-th iocresol in
presen ce of boron trifluoride dieth yl eth erate as catalyst to provide com -
poun d 4 in 85% yield13. Regioselective h ydrazin olysis of th e an om eric
O-acetyl group of 2 followed by activation of th e an om eric position for
n ucleoph ilic attack h as been carried out by treatm en t of 5 with
trich loroaceton itrile in th e presen ce of 1,8-diazabicyclo[5.4.0]un dec-7-en e
(DBU) as base to give com poun d 6 in 79% yield14 (Sch em e 2). In parallel,
we h ave prepared th e m uram ic acid acceptors B from com m ercially avail-
able GlcNAc (Sch em e 3). Startin g from th e ben zyl α-D-glycoside 7, wh ich
was obtain ed accordin g to th e Fisch er m eth od15,16, th e 4,6-ben zyliden e de-
rivative 8 was obtain ed in 95% yield. Th erefore, 7 was treated with
ben zaldeh yde an d trieth yl orth oform ate in presen ce of p-toluen esulfon ic
acid in a m ixture of DMF an d dioxan e17. Com poun d 9 was obtain ed by
O-alkylation of th e sodium salt of ben zyl 2-acetam ido-4,6-ben zyliden e-
2-deoxy-α-D-glucopyran oside (gen erated from 8 with sodium h ydride in
dioxan e) usin g L-2-ch loropropion ic acid in 80% yield18,19. Th e carboxylic
fun ction of 9 was protected as 2-(ph en ylsulfon yl)eth yl ester 10 20. Startin g
from th is derivative 10, we h ave studied th e possibilities to rem ove th e pro-
tective group at th e 4-h ydroxyl group for th e glycosylation step. Th e first
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1620
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
OH
O
OH
O
O
O
(ii)
Ph
(i)
HO
HO
HO
HO
O
O
HO
NH
NH
OH
NH
O
OBn
O
OBn
7
8
N-Acetyl-D-Glucosamine (GlcNAc)
(iii)
O
Ph
O
O
Ph
O
O
O
O
O
(iv)
O
NH
OBn
NH
O
O
OBn
O
O
O
O
HO
S
10
Ph
9
(i) PhCH2OH/AcCl, 70 oC, 65%; (ii) PhCHO, (EtO)3CH, TsOH, dioxane/DMF, 95%; (iii) NaH,
L-2-chloro-propionic acid, dioxane, 65 oC, 80%; (iv) PhSO2EtOH, EDCl, DMAP, CH2Cl2, 98%
SCHEME 3
Acceptor fragm en t syn th esis
possibility is to rem ove th e ben zyliden e group in acid m edia an d selectively
m on oacetylate th e 6-h ydroxyl group to gen erate th e glycosyl acceptor
9,21
12
(Sch em e 4). An altern ative way is to obtain th e O-6 ben zyl-protected
in term ediate by reductive open in g of th e ben zyliden e group of com poun d
10. Sah a an d co-workers11 h ave described th e possibility to con vert th e
4,6-O-ben zyliden e-protected m uram ic acid derivatives in th e correspon din g
6-O-ben zyl-4-h ydroxy derivatives 13 usin g trieth ylsilan e (TES) an d tri-
fluoroacetic acid (TFA) in dich lorom eth an e (Sch em e 5). However, un der th e
reported con dition s on ly 17% of th e dideprotected com poun d 11 an d 83%
OH
OAc
O
Ph
O
O
O
O
O
O
(i)
(ii)
HO
O
HO
O
O
O
O
NH
NH
NH
OBn
O
OBn
O
O
OBn
O
O
O
O
O
O
S
O
S
Ph
O
S
Ph
Ph
12
10
11
(i) CH3COOH 80%, 60 oC, 95%; (ii) AcCl, pyridine, CH2Cl2, –30 oC, 87%
SCHEME 4
Acceptor fragm en t syn th esis
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1621
OH
O
O
HO
Y
O
NH
OBn
O
17%
O
11
Ph
O
TFA/TES
O
Y
Y = OCH2CH2SO2Ph
O
NH
CH2Cl2, 0°C
OBn
O
O
OBn
O
HO
O
10
NH
OBn
O
O
Y
61%
13
Y = NH-[L-Ala]CO2CH2CH2SO2Ph
SCHEME 5
of th e startin g m aterial was observed after 16 h reaction (Sch em e 5). A m ore
recen t work described th e reductive rin g open in g of 4,6-O-ben zyliden e ace-
tal in carboh ydrates usin g triflic acid an d trieth ylsilan e as reductive agen ts
in dich lorom eth an e at –78 °C 22. By ch an gin g th e acid from TFA to TfOH
th e reaction took place sm ooth ly an d with excellen t selectivity to give th e
desired product 14 in a yield of 84% (Sch em e 6). Wh en we reacted th e
OBn
O
Ph
O
O
O
HO
O
TfOH/TES
O
O
O
O
NH
CH2Cl2, -78°C
NH
O
OBn
O
OBn
O
O
O
O
O
S
S
10
14
84%
Ph
Ph
SCHEME 6
glycosyl acceptor 12 with th e glycosyl brom ide 3 utilizin g AgOTf as pro-
m oter un der th e sam e con dition s as described by Sch wartz et al.9 in th e first
ch em ical total syn th esis of lipid II, we h ave n ot observed th e form ation of
th e aim ed disacch aride (Sch em e 7). We h ave obtain ed th e sam e n egative re-
sult wh en we com bin ed th e th ioglycoside don or 4 with th e acceptor 12 in
presen ce of a m ixture of N-iodosuccin im ide an d triflic acid as described in
th e syn th esis of lipid II an alogues by Sadam oto et al.21 Th ese results dem -
on strates th e difficulty of th is reaction wh ich could be explain ed in part by
th e poor n ucleoph ilicity of th e 4-h ydroxyl group of th e m uram ic acid resi-
due. However, wh en th e glycoside acceptor 14 with ben zyl protective group
at th e 6 position of th e m uram ic acid residue was reacted with th e glycosyl
brom ide 3 un der Koen igs–Kn orr con dition s (Sch em e 8), th e desired
disacch aride was obtain ed in 55% yield (74% reported in th e literature11).
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1622
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
TROC
Br
NH
OAc
OAc
TROC
AgOTf/MS
AcO
AcO
NH
O
O
O
HO
O
AcO
AcO
O
O
+
O
O
OAc
NH
CH2Cl2, -30°C
NH
OBn
OAc
O
OBn
3
O
O
O
O
O
O
O
O
S
S
12
Ph
Ph
OAc
O
TROC
TROC
NIS/TfOH
CH2Cl2
NH
O
NH
O
OAc
O
O
AcO
AcO
AcO
AcO
O
S
O
O
NH
HO
O
+
OAc
OAc
O
OBn
NH
O
O
4
O
OBn
O
S
O
O
Ph
O
S
12
Ph
SCHEME 7
OBn
O
OBn
O
TROC
TROC
Br
NH
O
O
NH
O
(i)
HO
AcO
AcO
O
AcO
AcO
O
O
+
O
NH
NH
OAc
OBn
OAc
OBn
O
O
O
O
3
O
O
O
O
S
S
Ph
Ph
14
15
TROC
NH
OBn
NH
O
TROC
Cl
Cl
AcO
AcO
OBn
O
NH
O
O
(ii)
O
AcO
AcO
O
Cl
O
O
OAc
HO
O
NH
OBn
O
OAc
6
O
+
NH
O
O
OBn
O
O
S
O
O
O
Ph
S
14
15
Ph
(i) AgOTf, MS 4Å, CH2Cl2, 55%; (ii) TMSOTf, MS 4Å, CH2Cl2, 76%
SCHEME 8
Th e best way to obtain th e desired β-lin ked disacch aride 15 is usin g th e
trich loroacetim idate don or 6 an d th e acceptor 14 in m ild con dition s utiliz-
in g on ly 0.2 equivalen t of TMSOTf un der rigorously an h ydrous con di-
tion s23 (yield 76%). Th e 6-O-ben zyl group of 15 was rem oved (Sch em e 9)
usin g a m ixture of acetic an h ydride, acetic acid (3:1) an d an h ydrous zin c
ch loride an d th e free alcoh ol gen erated in situ was acetylated11. Zin c dust
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1623
O
OAc
O
OBn
TROC
NH
NH
O
O
AcO
AcO
O
AcO
AcO
O
(i), (ii)
O
O
O
OAc
O
O
OAc
NH
NH
OBn
OBn
O
O
O
O
O
O
O
O
S
S
Ph
Ph
16
15
O
OAc
O
(iii)-(v)
NH
O
AcO
AcO
O
O
O
OAc
O
NH
O
P
O
O
OBn
O
O
BnO
S
Ph
17
(i) ZnCl2, AcOH/Ac2O (1/3); (ii) Zn dust, THF/Ac2O/AcOH (3/2/1), 84%; (iii) H2, Pd/C, MeOH;
(iv) dibenzyl-N,N-diethylphosphoramidite/tetrazole, CH3CN; (v) H2O2 45%, THF, –78 oC, 75% (3 steps)
SCHEME 9
was added to th e reaction m ixture to rem ove th e Troc group from th e
glucosam in e m oiety, liberatin g th e am in e fun ction wh ich is acetylated to
afford com poun d 16 in 84% yield11,23. Hydrogen olytic cleavage of th e ben -
zyl protectin g group
followed
by treatm en t
with
diben zyl
N,N-dieth ylph osph oram idite an d tetrazole in dich lorom eth an e an d oxida-
tion with h ydrogen peroxide afforded th e desired α-ph osph ate product 17
in 75% yield10. In parallel we h ave prepared via stan dard peptide syn th esis
protocols several peptide side ch ain s th at m ay be con n ected to th e
carboxylic acid fun ction of th e m uram ic acid m oiety. We h ave ch osen to
syn th esize sh ort peptides (Sch em e 10) in order to fin d out th e m in im al re-
quirem en t for recogn ition by en zym es in volved in th e peptidoglycan syn -
th esis. From peptides 24, 25, an d 26 we h ave prepared com poun ds 29, 30,
an d 31 (Sch em e 11). Rem oval of th e 2-(ph en ylsulfon yl)eth yl ester was
ach ieved by treatm en t of 17 with DBU. Th e in term ediate carboxylic acid 27
was activated th rough con version to th e correspon din g NHS-ester 28 an d
added to a solution of th e peptides (24, 25 or 26) (wh ich were first
deblocked usin g TFA in dich lorom eth an e) in DMF with iPr2NEt providin g
com poun ds 29, 30, an d 31.
Th e exact m ech an ism of th e glycosyl tran sferase is n ot yet kn own 24. Th e
lin ear assem bly of th e glycan ch ain proceeds by repetitive addition eith er of
th e disacch aride peptide un its on th e preform ed peptidoglycan or by addi-
tion of growin g peptidoglycan on th e disacch aride peptide un it. In th e first
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1624
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
(i)
(ii)
BOC-L-Lys(Z)NH2
BOC-L-Lys(Z)
BOC-L-LysNH2
18
19
(iii)
(iv)
BOC-L-Lys(Dansyl)NH2
BOC-L-Lys(Fmoc)NH2
21
20
(v)
BOC-D-Glu
BOC-L-Ala
BOC-D-isoGlnNH2
22
(vi)
BOC-L-AlaNHS
23
(vii), (viii)
(vii), (viii)
BOC-L-Lys(Dansyl)NH2
BOC-L-Ala-L-Lys(Dansyl)NH2
20
24
BOC-L-Lys(Fmoc)NH2
BOC-L-Ala-L-Lys(Fmoc)NH2
25
21
(vii), (viii)
BOC-D-isoGlnNH2
BOC-L-Ala-D-isoGlnNH2
22
26
(i) isobutyl chloroformate/Et3N, THF, NH4OH (28%); (ii) H2, Pd/C, MeOH; (iii) DansylCl/Et3N, CH2Cl2;
(iv) FmocCl, NaHCO3/H2O; (v) 4-nitrophenol/DCC, THF and NH4OH (28%); (vi) NHS/EDCl, DMF;
(vii) CH2Cl2/TFA; (viii) 23, DIEA, DMF
SCHEME 10
O
O
OAc
O
OAc
O
NH
NH
O
O
(i)
AcO
AcO
AcO
AcO
O
O
O
O
O
OAc
O
O
OAc
NH
NH
O
O
O
O
P
P
O
O
OBn
OBn
O
RO
O
BnO
BnO
S
Ph
17
27 (R= H)
28 (R= NHS)
O
OAc
O
NH
O
(ii)-(iv)
AcO
AcO
O
O
O
OAc
NH
O
O
P
O
OBn
HN
Peptide
BnO
29 Peptide =
30 Peptide =
31 Peptide =
L
L
L
-Ala-
-Ala-
-Ala-
L
L
D
-Lys(Dansyl)NH2
-Lys(Fmoc)NH2
-isoGlnNH2
(i) DBU, CH2Cl2; (ii) NHS/EDCl in DMF; (iii) 24, 25, 26, CH2Cl2/TFA (2/1);
(iv) unprotected peptides/ DIEA in DMS
SCHEME 11
Syn th esis of peptide con jugated disacch aride
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1625
O
O
OAc
OAc
NH
O
N
(i), (ii)
NH
O
O
AcO
AcO
O
AcO
AcO
O
O
NH
O
O
O
O
NH
NH
O
O
O
P
OAc
OAc
O
O
O
O
O
P O P O
OH OH
O
O
OBn
HN
HN
Peptide
BnO
Peptide
OH OH
32 Peptide = L-Ala-L-Lys(Dansyl)NH2
33 Peptide = L-Ala-L-Lys(Fmoc)NH2
34 Peptide = L-Ala-D-isoGlnNH2
29 Peptide = L-Ala-L-Lys(Dansyl)NH2
30 Peptide = L-Ala-L-Lys(Fmoc)NH2
31 Peptide = L-Ala-D-isoGlnNH2
O
OH
(iii)
NH
O
O
NH
HO
HO
O
O
NH
O
O
O
OH
O
O
N
O
P O
P O
O
O
HN
Peptide
OH OH
OH OH
35 Peptide = L-Ala-L-Lys(Dansyl)NH2
36 Peptide = L-Ala-L-LysNH2
37 Peptide = L-Ala-D-isoGlnNH2
O
O
OAc
O
OAc
NH
(i), (ii)
NH
O
N
O
O
AcO
AcO
O
AcO
O
O
O
NH
O
OAc
O
AcO
O
OAc
O
NH
NH
O
P
O
O
O
O
O
O
O
P O P O
OH OH
O
OBn
O
O
O
O
BnO
O
S
S
Ph
Ph
17
38
OH OH
O
OH
(iii)
NH
O
N
O
HO
HO
O
O
NH
O
NH
O
O
OH
O
O
O
P O
P O
O
O
HO
OH OH
39
OH OH
(i) H2, Pd/C, MeOH; (ii) Uridine 5'-phosphomorpholidate/tetrazole, CH3CN; (iii) 1M NaOH, DMF
SCHEME 12
UDP couplin g an d total deprotection
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1626
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
case, lipid II is con sidered as th e glycosyl don or substrate an d in th e secon d
case lipid II is fun ction in g as th e acceptor an d th e growin g ch ain is tran s-
ferred to th e 4-OH group of th e GlcNAc of lipid II. Th at m ean s th at, wh en
th e lipid II m on om er is an acceptor, it is n ot n ecessarily boun d to un deca-
pren yl pyroph osph ate7. Th erefore, we ch ose to couple th e disacch aride un it
to uridin e m on oph osph ate in stead of a lipid ch ain . After h ydrogen olytic
cleavage of th e ph osph odiester protectin g groups an d treatm en t with
uridin e 5-m on oph osph om orph olidate un der an h ydrous con dition s20 in
presen ce of tetrazole in DMF, th e correspon din g protected UDP-
disacch aride peptide com poun ds 32, 33, an d 34 are obtain ed (Sch em e 12).
Rapid deprotection with aqueous sodium h ydroxide an d ch rom atograph ic
purification by reverse ph ase HPLC fin ally afforded pure UDP-com poun ds
35, 36, an d 37 in average 17% yield based on 17.
We h ave also prepared th e an alogue lackin g a peptide side ch ain . Th e
com poun d 39 was obtain ed in on e pot from th e com poun d 17 (16% yield).
Th ese com poun ds were tested for th eir poten tial to in h ibit th e glycosyl
tran sferase activity of E. coli pen icillin -bin din g protein type 1b (PBP 1b).
However, we did n ot fin d in h ibitory activity of th e glycosyl tran sferase ac-
tivity at 100 µm ol l–1. Th is activity was m easured in th e presen ce of
[14C]lipid II (1.5 µm ol l–1) an d PBP 1b (18 n m ol l–1) at 30 °C durin g 15 m in .
Th e full biological testin g of all th e syn th esized com poun ds will be th e sub-
ject of a separate publication 7.
EXPERIMENTAL
Gen eral
All reaction s usin g air- or m oisture-sen sitive reagen ts were con ducted in an in ert n itrogen
atm osph ere. Solven ts were dried an d distilled un der argon prior to use or purch ased in an -
h ydrous form from com m ercial sources. Brin e refers to a saturated aqueous solution of NaCl.
TLC was perform ed usin g silica gel F254 precoated alum in um sh eets an d visualized by ultra-
violet ligh t or by an isaldeh yde, 25% H2SO4 in eth an ol, followed by h eatin g. Colum n ch ro-
m atograph y was perform ed usin g silica gel 60, 230–400 m esh . HPLC an alyses an d
purification s were perform ed usin g Alltech ® HS Hyper Prep 100 BDS colum n C-18 8µ
(len gth 250 m m ) with specified solven t an d flow rate. NMR spectra were acquired on a
Varian Un ity 500 MHz spectrom eter. 1H an d 13C NMR spectra were recorded at 500 an d 125
MHz, respectively, in CDCl3 or DMSO-d6 un less oth erwise n oted. Ch em ical sh ifts are given
in ppm (δ-scale) with th e residual solven t peak (1H CHCl3, 7.26; 13C CDCl3, 77) as in tern al
stan dard. Couplin g con stan ts (J) are reported in Hz. Exact m ass spectra were acquired on a
quadrupole tim e-of-fligh t m ass spectrom eter (Q-Tof-2, Microm ass, Man ch ester, U.K.)
equipped with a stan dard electrospray-ion ization (ESI) in terface, sam ples were in fused in
iPrOH/H2O (1:1) at 3 µl m in –1
.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1627
1,3,4,6-Tetra-O-acetyl-2-deoxy-2-[(2,2,2-trich loroeth oxy)carbon ylam in o]-
(α,β)-D-glucopyran oside (2)
(Trich loroeth oxy)carbon yl ch loride (46 m l, 347.7 m m ol) was added dropwise at room tem -
perature to a vigorously stirred solution of D-glucosam in e h ydroch loride (50 g, 231.8 m m ol)
an d NaHCO3 (39 g, 463.7 m m ol) in water (300 m l). Th e m ixture was stirred overn igh t. Th e
precipitate was filtered off an d wash ed with water un til th e un reacted ch loride was rem oved.
Th e wh ite solid product was recrystallized from EtOH to yield 73.2 g (89%) of waxy m ate-
rial 1 (ESI-MS for C9H14Cl3NNaO7 calculated [M + Na]+: 375.9734; foun d: 375.9734). Th e
Troc-am in oglucosam in e 1 (72 g, 203.4 m m ol) was dissolved in dry pyridin e (200 m l), Ac2O
(105.7 m l, 1118.7 m m ol) was added, an d th e m ixture was stirred at room tem perature for
12 h . Th e product was con cen trated an d coevaporated with toluen e. Th e residue was dis-
solved in CH2Cl2 (500 m l) an d th e organ ic layer was wash ed with 1 M HCl, H2O an d brin e,
an d dried with Na2SO4. Th e solven t was rem oved to obtain
a wh ite foam of th e
Troc-am in operacetylglucosam in e 2 wh ich was dried in vacuo (104 g, 98%). 1H NMR
(CDCl3): 6.23 (d, J = 3.66, 1 H, H1), 5.27 (t, J = 9.52, 1 H, H3), 5.19 (t, J = 10.1, 1 H, H4),
4.82 an d 4.63 (AX, J = 11.96, CH2), 4.24 (m , 2 H, H2, H6), 4.05 (m , 2 H, H5, H6′), 2.20 (s, 3 H,
CH3), 2.09 (s, 3 H, CH3), 2.05 (s, 3 H, CH3), 2.04 (s, 3 H, CH3). 13C NMR (CDCl3): 171.14,
170.55, 169.09, 168.53 (4 × C=O), 154.01 (C=O), 95.18 (CCl3), 90.36 (C1), 74.58 (CH2),
70.32 (C3), 69.66 (C5), 67.54 (C4), 61.44 (C6), 53.15 (C2), 20.79, 20.58, 20.55, 20.46 (4 ×
CH3). ESI-MS for C17H22Cl3NNaO11 calculated [M + Na]+: 544.0156; foun d: 544.0156 (ref.2).
3,4,6-Tri-O-acetyl-2-deoxy-2-[(2,2,2-trich loroeth oxy)carbon ylam in o]-α-D-glucopyran osyl
Brom ide (3)
Th e N-Troc-peracetylglucosam in e 2 (8.28 g, 15 m m ol) was dissolved in CH2Cl2 (200 m l) an d
HBr (20.0 m l, 33% in AcOH) was added at 0 °C an d kept at 0 °C for 1 h . Th e reaction was
allowed to warm up to room tem perature durin g 1 h . After 2 h th e reaction was com plete by
TLC an d was worked up. Th e solution was diluted with CH2Cl2 (200 m l) an d wash ed with
ice water, 1% NaHCO3, saturated NaHCO3, brin e an d dried over Na2SO4. Th e sam ple was fil-
tered an d con cen trated to a colorless oil an d recrystallized from Et2O/h exan es to yield com -
poun d 3 (7.83 g, 96%), m .p. 97–98 °C.
p-Tolyl 3,4,6-Tri-O-acetyl-2-deoxy-l-th io-2-[(2,2,2-trich loroeth oxy)carbon ylam in o]-
β-D-glucopyran oside (4)
To a m ixtu re of com p ou n d 2 (6.69 g, 12.8 m m ol) an d 4-m eth ylth iop h en ol (2.18 g,
15.3 m m ol) in CH2Cl2 (30 m l) was added boron trifluoride dieth yl eth erate (48%; 4.87 m l,
38.4 m m ol), an d th e m ixture was stirred overn igh t. Th en th e reaction was diluted with
CH2Cl2 (100 m l) an d wash ed with saturated NaHCO3, water an d brin e. Th e organ ic layer
was dried over Na2SO4, filtered an d con cen trated. Th e residue obtain ed was purified by silica
gel ch rom atograph y with h exan e/EtOAc (3:1) as eluen t to give 4 (6.15 g, 82%). 1H NMR
(CDCl3): 7.41 (d, J = 8.06, 2 H, Harom ), 7.11 (d, J = 7.81, 2 H, Harom ), 5.40 (d, J = 9.03, 1 H,
NH), 5.28 (t, J = 9.76, 1 H, H3), 5.00 (t, J = 9.77, 1 H, H4), 4.81–4.67 (m , 3 H, CH2, H1),
4.15–4.24 (m , 2 H, H6, H6′), 3.72 (m , 1 H, H5), 3.67 (m , 1 H, H2), 2.34 (s, 3 H, CH3), 2.08 (s,
3 H, CH3), 2.00 (s, 3 H, CH3), 1.98 (s, 3 H, CH3). 13C NMR (CDCl3): 170.05, 170.00, 169.41
(3 × C=O), 153.87 (C=O), 138.64 (Carom ), 133.59, 129.68 (4 × CHarom ), 127.93 (Carom ), 95.39
(CCl3), 86.66 (C1), 75.70 (C3), 74.49 (CH2), 73.23 (C5), 68.57 (C4), 62.29 (C6), 54.95 (C2),
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1628
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
21.11 (CH3), 20.67, 20.56, 20.50 (3 × CH3). ESI-MS for C22H26Cl3NNaO9 calculated [M + Na]+:
608.0291; foun d: 608.0312.
3,4,6-Tri-O-acetyl-2-deoxy-2-[(2,2,2-trich loroeth oxy)carbon ylam in o]-α-D-glucopyran osyl
Trich oroacetim idate (6)
A solution of 2 (35.0 g, 67.0 m m ol) an d h ydrazin ium acetate (7.4 g, 80.0 m m ol) in DMF
(250 m l) was stirred at room tem perature for 20 m in , th en diluted with EtOAc (250 m l),
wash ed with water, th en with saturated aqueous NaHCO3, an d again with water, dried with
Na2SO4 an d con cen trated. Th e com poun d 5 obtain ed was dissolved in CH2Cl2 (250 m l),
trich loroaceton itrile (66.0 m l, 670 m m ol) an d DBU (2.0 m l, 13.4 m m ol) were added an d th e
m ixture was stirred at room tem perature for 1 h . Th e solven t was rem oved in vacuo an d th e
residue was purified by flash ch rom atograph y on silica gel usin g h exan e/EtOAc (2:1) con -
tain in g 0.1% of trieth ylam in e as eluen t to afford com poun d 6 as a wh ite foam (35 g, 83%).
1H NMR (CDCl3): 8.82 (s, 1 H, NH), 6.43 (d, J = 3.4, 1 H, H1), 5.35 (t, J = 10.1, 1 H, H3), 5.25
(t, J = 10.1, 1 H, H4), 4.73 an d 4.71 (AX, J = 12.2, 2 H, CH2), 4.20–4.32 (m , 2 H, H2, H6),
4.09–4.16 (m , 2 H, H5, H6′), 2.08 (s, 3 H, CH3), 2.06 (s, 6 H, 2 × CH3). 13C NMR (CDCl3):
171.08, 170.52, 169.21 (3 × COO), 160.34 (OCN)) 154.10 (OCON), 94.48 (C1), 95.17, 91.70
(2 × CCl3), 74.60 (CH2), 70.21 (C3, C5), 67.36 (C4), 61.34 (C6), 53.83 (C2), 20.60 (2 × CH3),
20.50 (CH3). ESI-MS for C17H21Cl6N2NaO10 calculated [M + Na]+: 645.9; foun d: 645.6.
Ben zyl 2-Acetam ido-2-deoxy-α-D-glucopyran oside (7)
Acetyl ch loride (11 m l, 154.7 m m ol) was added to a suspen sion of fin ely powdered
N-acetyl-D-glucosam in e (25.7 g, 116.4 m m ol) in ben zyl alcoh ol (150 m l). Th e m ixture was
stirred at 75 °C for 4 h . Th en , th e excess of ben zyl alcoh ol was rem oved in vacuo, th e result-
in g syrup was suspen ded in diisopropyl eth er (500 m l) an d was kept at 4 °C overn igh t. Th e
precipitate was filtered off an d wash ed with cold diisopropyl eth er an d dieth yl eth er to yield
30.0 g (70%) of com poun d 7. 1H NMR (DMSO-d6): 7.81 (d, J = 8.0, AcNH, 1 H), 7.38–7.24
(m , Ar-Ho,m, 4 H), 7.27 (m , 1 H, Ar-Hp), 5.02 (d, J = 5.6, 1 H, 4-OH), 4.77 (d, J = 5.6, 1 H,
3-OH), 4.73 (d, J = 3.6, 1 H, H1), 4.67 an d 4.18 (AX, J = 12.5, 1 H, CH2), 4.53 (t, J = 6.0, 1 H,
6-OH), 3.69 (ddd, J = 10.7, 8.0, 3.4, 1 H, H2), 3.66 (ddd, J = 11.4, 5.6, 1.8, 1 H, H6), 3.56
(ddd, J = 10.7, 8.5, 5.4, 1 H, H3), 3.51 (dt, J = 11.4, 5.8, 5.8, 1 H, H6′), 3.47 (ddd, J = 10.0,
5.6, 1.9, 1 H, H5), 3.18 (ddd, J = 9.9, 8.5, 5.6, 1 H, H4), 1.84 (s, 3 H, CH3). 13C NMR
(DMSO-d6): 169.55 (C=O), 138.01 (Carom ), 128.21, 127.58 (CHarom ), 127.45 (CHarom ), 96.01
(C1), 73.16 (C5), 70.96 (C4), 70.63 (C3), 67.84 (CH2), 60.95 (C6), 53.87 (C2), 22.62 (CH3).
ESI-MS for C15H22NO6 calculated [M + H]+: 312.1447; foun d: 312.1446.
Ben zyl 2-Acetam ido-4,6-O-ben zyliden e-2-deoxy-α-D-glucopyran oside (8)
Com poun d 7 was dried by coevaporation with an h ydrous eth an ol an d toluen e in vacuo. To
th e dry com poun d 7 (18.5 g, 59.4 m m ol) was added an h ydrous DMF (150 m l) an d an h y-
drous dioxan e (150 m l), trieth yl orth oform ate (30 m l, 180.4 m m ol), ben zaldeh yde (24 m l,
236.3 m m ol) an d p-toluen esulfon ic acid (5.7 g). Th e m ixture was stirred at room tem pera-
ture for 20 h . Absolute dieth yl eth er was added an d th e m ixture was stirred at 0 °C for 1 h
an d th e precipitate was filtered off. After wash in g with dieth yl eth er th e filtrate was dried in
vacuo to give 8 (22.5 g, 95%). 1H NMR (DMSO-d6): 7.99 (d, J = 8.0, 1 H, AcNH), 7.45 (m , 2
H, Harom ), 7.42–7.34 (m , 7 H, Harom ), 7.30 (m , 1 H, Harom ), 5.62 (s, 1 H, CH), 5.19 (d, J = 5.8,
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1629
1 H, 3-OH), 4.81 (d, J = 3.6, 1 H, H1), 4.70 an d 4.49 (AX, J = 12.5, 2 H, CH2), 4.14 (dd, J =
8.5, 3.7, 1 H, H6), 3.85 (ddd, J = 3.6, 8.0, 10.3, 1 H, H2), 3.75 (m , 2 H, H3, H6′), 3.70 (dt, J =
4.4, 10.2, 1 H, H5), 3.52 (t, J = 10.0, 1 H, H4), 1.85 (s, 3 H, CH3). 13C NMR (DMSO-d6):
169.56 (C=O), 137.80 (Carom ), 128.91, 128.29, 128.07, 127.69, 127.62, 126.45 (CHarom ),
100.94 (CHben zyliden e), 97.02 (C1), 82.13 (C4), 68.68 (CH2), 68.09 (C6), 67.29 (C3), 62.93 (C5),
54.35 (C2), 22.59 (CH3). ESI-MS for C22H26NO6 calculated [M + H]+: 400.1760; foun d:
400.1758.
Ben zyl 2-Acetam ido-4,6-O-ben zyliden e-2-deoxy-3-O-(D-1-carboxyeth yl)-
α-D-glucopyran oside (9)
Com poun d 8 (11.36 g, 28.5 m m ol) was dissolved in an h ydrous dioxan e (250 m l). Th e sus-
pen sion was treated with NaH (18.3 g of a 60% suspen sion in m in eral oil, 457.5 m m ol) in
sm all portion s. Th e m ixture was stirred at 95 °C for 2 h an d cooled down to 65 °C.
(S)-2-Ch loropropion ic acid (4 m l, 46.4 m m ol) in an h ydrous dioxan e (75 m l) was slowly
added an d th e m ixture was stirred at 60 °C for 3 h . Th e reaction was cooled to 10 °C an d di-
luted with a sm all portion of m eth an ol an d water. Th e solution was con cen trated in vacuo,
an d th e residue, after dissolution in dieth yl eth er was vigorously stirred with a saturated
aqueous solution of NaCl. Th e eth er layer was con cen trated in vacuo to 100 m l, an d to th is
solution a 6 M HCl solution was slowly added. Th e precipitate was filtered off, wash ed with
dieth yl eth er an d recrystallized from CHCl3/MeOH (95:5) to obtain 9 (10.79 g, 80%). m .p.
269–270 °C (lit.16 264–265 °C). 1H NMR (DMSO-d6): 7.96 (d, J = 6.1, 1 H, NH), 7.45–7.28 (m ,
10 H, Harom ), 5.69 (s, 1 H, CH), 5.04 (d, J = 3.4, 1 H, H1), 4.69 an d 4.49 (AX, J = 12.4, 2 H,
CH2), 4.29 (q, J = 6.8, 1 H, CH), 4.27 (m , 1 H, H3), 4.15 (dd, J = 9.8, 4.2, 1 H, H6), 3.84–3.70
(m , 5 H, H2, H3, H4, H5, H6′), 1.86 (s, 3 H, CH3), 1.28 (d, J = 6.8, 3 H, CH3). 13C NMR
(DMSO-d6): 175.39 (C=Oacid), 169.64 (C=Oam ide), 137.72 (Carom ), 128.95, 128.41, 128.29,
127.79, 125.97 (CHarom ), 100.42 (CHben zyliden e), 96.94 (C1), 81.67 (C4), 75.21 (C3, CH), 69.14
(CH2), 68.00 (C6), 63.02 (C5), 53.63 (C2), 22.71 (CH3), 18.78 (CH3). ESI-MS for C25H30NO8
calculated [M + H]+: 472.1971; foun d: 472.1974.
Ben zyl 2-Acetam ido-4,6-O-ben zyliden e-2-deoxy-3-O-(D-1-{[2-(ph en ylsulfon yl)eth oxy]-
carbon yl}eth yl)-α-D-glucopyran oside (10)
To a suspen sion of 9 (10.13 g, 21.5 m m ol) in an h ydrous CH2Cl2 (150 m l) was added DMAP
(131 m g, 1.75 m m ol) an d 2-(ph en ylsulfon yl)eth an ol (4.39 g, 23.6 m m ol). Th e m ixture was
diluted with CH2Cl2 an d was wash ed with a 0.5 M HCl. Th e organ ic layer was dried with
Na2SO4, filtered an d con cen trated in vacuo. Th e crude m aterial was purified on silica gel
with CHCl3 as eluen t to obtain pure com poun d 10 (11.55 g, 84%). 1H NMR (CDCl3): 7.83
(br d, 2 H, Harom ), 7.48–7.24 (m , 14 H, Harom , NH), 5.55 (s, 1 H, CH), 5.35 (d, J = 3.6, 1 H,
H1), 4.68 an d 4.53 (AX, J = 12.0, 2 H, CH2), 4.47 (m , 2 H, CH2), 4.22 (dd, J = 10.3, 4.6, 1 H,
H6), 4.17 (q, J = 7.1, 1 H, CH), 3.92 (ddd, J = 9.0, 5.1, 3.6, 1 H, H2), 3.84 (ddd, J = 10.1, 9.2,
4.6, 1 H, H5), 3.74 (t, J = 10.3, 1 H, H6′), 3.70 (t, J = 9.0, 1 H, H3), 3.65 (t, J = 9.0, 1 H, H4),
3.44 (m , 2 H, CH2), 2.01 (s, 3 H, CH3), 1.18 (d, J = 7.0, 3 H, CH3). 13C NMR (CDCl3): 174.28
(C=Oacid), 170.69 (C=Oam ide), 139.11, 137.38, 137.31 (Carom ), 134.17, 129.27, 129.05, 128.37,
128.29, 127.92, 127.88, 127.84, 125.90 (CHarom ), 101.33 (CHben zyliden e), 97.29 (C1), 83.27
(C4), 74.89 (C3), 74.84 (CH), 70.35 (CH2), 68.93 (C6), 62.87 (C5), 58.31 (CH2), 54.91 (CH2),
54.06 (C2), 23.07 (CH3), 18.33 (CH3). ESI-MS for C33H38NO10S calculated [M + H]+:
640.2216; foun d: 640.2220.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1630
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
Ben zyl 2-Acetam ido-2-deoxy-3-O-(D-1-{[2-(ph en ylsulfon yl)eth oxy]carbon yl}eth yl)-
α-D-glucopyran oside (11)
A suspen sion of com poun d 10 (6.07 g, 9.5 m m ol) in 80% acetic acid was stirred at 60 °C un til
after 90 m in th e startin g m aterial disappeared as sh own by TLC an alysis (EtOAc). Th e sol-
ven t was rem oved an d th e resultin g oil was coevaporated with toluen e an d th en purified by
silica gel ch rom atograph y with CHCl3/MeOH (95:5) as eluen t to yield a wh ite foam (4.92 g,
94%). 1H NMR (CDCl3): 7.89 (dt, J = 8.30, 1.95, 2 H, Harom ), 7.65 (tt, J = 7.56, 1.23, 1 H,
H
arom ), 7.54 (t, J = 8.3, 2 H, Harom ), 7.40 (d, J = 5.61, 1 H, NH), 7.30 (m , 5 H, Harom ), 5.23 (d,
J = 3.17, 1 H, H1), 4.66–4.42 (m , 5 H, CH2Bn , CH2, CH), 3.79 (m , 2 H, H2, H6), 3.72 (m , 2 H,
H4, H-6′), 3.60 (m , 2 H, H3, H-5), 3.46 (td, J = 6.11, 1.71, 2 H, CH2-5), 1.99 (s, 3 H, CH3),
1.26 (s, 3 H, CH3). 13C NMR (CDCl3): 175.06, 171.16 (2 × C=O), 138.91, 137.52 (Carom ),
134.19, 129.44, 128.32, 127.97, 127.73, 127.68 (CHarom ), 96.44 (C1), 77.00 (C3), 74.57 (CH),
71.96 (C5), 71.90 (C4), 69.85 (CH2), 61.68 (C6), 58.01 (CH2), 54.83 (CH2), 53.43 (C2), 23.01
(CH3), 18.60 (CH3). ESI-MS for C26H34NO10S calculated [M + H]+: 552.1903; foun d:
552.1904.
Ben zyl 2-Acetam ido-6-O-acetyl-2-deoxy-3-O-(D-1-{[2-(ph en ylsulfon yl)eth oxy]carbon yl}-
eth yl)-α-D-glucopyran oside (12)
Com poun d 11 (1.49 g, 2.7 m m ol) was dissolved in CH2Cl2 (20 m l), th e solution was cooled
to –30 °C, an d pyridin e (0.45 m l, 5.4 m m ol) was added to th e solution . A solution of acetyl
ch loride (0.25 m l, 3.5 m m ol) in CH2Cl2 was added dropwise at –30 °C over a period of 45 m in .
Th e solution was warm ed slowly to 0 °C an d was diluted with CH2Cl2 (50 m l). Th is solution
was wash ed with 0.01 M HCl (50 m l) an d with brin e (50 m l). Th e organ ic layer was dried
with Na2SO4, filtered, an d con cen trated in vacuo. Th e crude m aterial was purified by silica
gel ch rom atograph y with CHCl3 as eluen t to obtain a foam y solid 12 (1.41 g, 87%). 1H NMR
(CDCl3): 7.94 (d, J = 8.30, 2 H, Harom ), 7.68 (t, J = 7.45, 1 H, Harom ), 7.60 (t, J = 8.41, 2 H,
H
arom ), 7.40 (d, J = 5.63, 1 H, NH), 7.30 (m , 5 H, Harom ), 5.30 (d, J = 3.41, 1 H, H1),
4.65–4.49 (m , 5 H, CH2Bn , CH2, CH), 3.98 (m , 2 H, H2, H6), 3.83 (m , 2 H, H4, H6′), 3.68 (m ,
2 H, H3, H5), 3.36 (d, J = 5.62, 2 H, CH2-5), 2.12 (s, 3 H, CH3), 2.00 (s, 3 H, CH3), 1.44 (s,
3 H, CH3). 13C NMR (CDCl3): 177.04, 174.93, 171.46 (3 × C=O), 138.98, 137.20 (Carom ),
133.97, 129.38, 128.22, 127.91, 127.88, 127.78 (CHarom ), 96.59 (C1), 76.88 (C3), 74.87 (CH),
71.62 (C4), 71.09 (C5), 70.62 (CH2), 62.97 (C6), 58.17 (CH2), 56.16 (CH2), 54.84 (C2), 22.83
(CH3), 22.75 (CH3), 18.50 (CH3). ESI-MS for C28H36NO11S calculated [M + H]+: 594.2009;
foun d: 594.2009.
Ben zyl 2-Acetam ido-6-O-ben zyl-2-deoxy-3-O-(D-1-{[2-(ph en ylsulfon yl)eth oxy]carbon yl}-
eth yl)-α-D-glucopyran oside 14
A solution of 10 (3.9 g, 6.1 m m ol) in dich lorom eth an e (50 m l) in presen ce of m olecular
sieves (2.3 g) was stirred at room tem perature for 30 m in an d th en was cooled to –78 °C.
Trieth ylsilan e (2.95 m l, 18.1 m m ol) an d triflic acid (1.1 m l, 12.2 m m ol) were added succes-
sively. After stirrin g for 1 h , NaHCO3 an d MeOH were added. Th e m ixture was warm ed up
to room tem perature, diluted with CH2Cl2 an d wash ed with saturated solution of NaHCO3,
water an d brin e. Th e organ ic layer was dried with Na2SO4, filtered an d con cen trated in
vacuo; th e residue was purified on silica gel colum n with CHCl3/MeOH (0–5%) as eluen t
givin g pure com poun d 14 (3.55 g, 91%). 1H NMR (CDCl3): 7.9 (m , 2 H, Harom ), 7.5 (m , 3 H,
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1631
Harom ), 7.3 (m , 10 H, Harom ), 7.2 (d, J = 5.4, 1 H, NH), 5.3 (d, J = 3.2, 1 H, H1), 4.65 an d 4.49
(AB system , J = 12.0, 2 H, CH2), 4.59 an d 4.52 (AB system , J = 12.0, 2 H, CH2), 4.50–4.42 (m ,
3 H, CH2, CH), 3.80 (ddd, J = 10.9, 5.4, 3.2, 1 H, H2), 3.75–3.70 (m , 2 H, H5, H6), 3.68 (dt, J =
2.3, 9.8, 1 H, H4), 3.60 (m , 2 H, H3, H6′), 3.4 (m , 2 H, CH2), 3.3 (d, J = 2.4, 1 H, 4-OH), 1.9
(s, 3 H, CH3), 1.2 (d, J = 7.0, 3 H, CH3). 13C NMR (CDCl3): 175.02 (C=Oester), 169.61
(C=Oam ide), 139.09, 137.63 (Carom ), 134.15, 129.43, 128.50, 128.33, 128.20, 128.02, 127.94,
127.74 (CHarom ), 96.62 (C1), 77.33 (C3), 75.09 (C4), 74.33 (CH), 73.82 (CH2Bn ), 71.33 (C6),
70.11 (CH2Bn ), 69.24 (C5), 58.09 (CH2), 54.94 (CH2S), 53.03 (C2), 23.10 (CH3), 18.65 (CH3).
ESI-MS for C33H40NO10S calculated [M + H]+: 642.2372; foun d: 642.2371.
Ben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-[(2,2,2-trich loroeth oxy)carbon ylam in o]-
β-D-glucopyran osyl-(1→4)-2-acetam ido-6-O-ben zyl-2-deoxy-3-O-(D-1-{[2-(ph en ylsulfon yl)-
eth oxy]carbon yl}eth yl)-α-D-glucopyran oside (15)
To a solution of 14 (4.0 g, 6.2 m m ol) in CH2Cl2 (10 m l) were added 4Å m olecular sieves
(7 g) an d TMSOTf (0.11 m l, 1.2 m m ol). To th is m ixture was added dropwise a solution of
com poun d 6 (11.7 g, 18.7 m m ol) in CH2Cl2 (10 m l). Th en th e solution was stirred for 24 h .
Th e reaction m ixture was filtered th rough Celite an d wash ed with CH2Cl2. Th e organ ic layer
was wash ed with NaHCO3 solution , water an d brin e, th en dried over Na2SO4 an d con cen -
trated in vacuo. Th e residue was purified on silica gel utilizin g a gradien t of MeOH in
CH2Cl2 (0–5%) as eluen t an d yieldin g th e disacch aride 15 as a wh ite foam (4.9 g, 72%).
1H NMR (CDCl3): 7.93 (br d, 2 H, Harom ), 7.70 (br t, 1 H, Harom ), 7.25–7.62 (overlapped m ,
14 H, 12 Harom , 2 NH), 5.36 (d, J = 3.4, 1 H, H1m ), 4.95 (t, J = 9.7, 1 H, H4g), 4.88 an d 4.34
(d, J = 12.0, 2 H, CH2Bn ), 4.76 (t, J = 10.0, 1 H, H3g), 4.72 an d 4.62 (d, J = 12.0, 2 H,
CH2Troc), 4.58 an d 4.49 (d, J = 12.0, 2 H, CH2Bn ), 4.50 an d 4.42 (m , 2 H, CH2), 4.38 (m , 1 H,
CHO), 4.34 (overlapped m , H6g), 4.22 (d, J = 8.3, 1 H, H1g), 3.99 (d, J = 12.0, 1 H, H6′g), 3.91
(t, J = 9.3, 1 H, H3m ), 3.78 (m , 1 H, H2m ), 3.64 (d, J = 10.8, 1 H, H6m ), 3.56 (d, J = 10.0, H5m ),
3.51 (t, J = 9.3, 1 H, H4m ), 3.48 (m , 1 H, H2g), 3.45 (m , 2 H, CH2S), 3.41 (m , 1 H, H5g), 3.39
(d, J = 10.8, 1 H, H6′m ), 2.05 (s, 3 H, CH3), 2.03 (s, 3 H, CH3), 2.00 (s, 3 H, CH3), 1.97 (s, 3 H,
CH3), 1.18 (d, J = 6.8, CH3). 13C NMR (CDCl3): 174.60 (COO), 170.58, 170.36, 170.15 (3 ×
Ac), 169.53 (CON), 153.89 (OCON), 139.11, 137.20 (Carom ), 134.14 (CHarom ) 129.47 (4 ×
CHarom ), 129.14 (2 × CHarom ), 129.17 (CHarom ), 128.34, 128.09 (4 × CHarom ), 127.75 (2 ×
CHarom ) 127.79 (CHarom ), 100.12 (C1g), 96.59 (C1m ), 92 (CCl3), 77.48 (C3m ), 75.25 (C4m ),
74.63 (CHO), 74.42 (CH2Troc), 73.81 (CH2Bn ), 72.51 (C3g), 71.32 (C5g), 70.39 (CH2Bn ), 69.90
(C5m ), 68.33 (C4g), 67.06 (C6m ), 61.49 (C6g), 58.14 (CH2O), 56.19 (C2g), 54.88 (CH2S), 54.15
(C2m ), 23.01, 20.58 (4 × CH3), 18.05 (CH3). ESI-MS for C48H58Cl3N2O19S calculated [M + H]+:
1103.2421; foun d: 1103.2411.
Ben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-acetam ido-β-D-glucopyran osyl-(1→4)-2-acetam ido-
6-O-acetyl-2-deoxy-3-O-(D-1-{[2-(ph en ylsulfon yl)eth oxy]carbon yl}eth yl)-
α-D-glucopyran oside (16)
To a solution of 15 (3 g, 2.7 m m ol) in Ac2O/AcOH (2:1, 30 m l) was added Zn Cl2 (3.7 g, 27.2
m m ol). Th e m ixture was stirred overn igh t. Th en th e solven t was rem oved an d th e residual
oil was dissolved in a m ixture of THF/Ac2O/AcOH (3:2:1, 30 m l). Zn dust (7.0 g, 108.8
m m ol) was added an d th e m ixture was stirred overn igh t. Th e reaction m ixture was filtered
th rough Celite, wash ed with CH2Cl2 an d th en con cen trated un der reduced pressure. Th e res-
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1632
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
idue was coevaporated with toluen e th ree tim es an d th en dissolved in CH2Cl2. Th e organ ic
layer was wash ed with saturated aqueous NaHCO3, H2O an d brin e, dried with Na2SO4, fil-
tered an d con cen trated in vacuo. Th e residue was purified on a colum n of silica gel an d
eluted with 2% MeOH in CH2Cl2 affordin g th e desired com poun d 16 (2.1 g, 85%). 1H NMR
(CDCl3): 7.95 (d, J = 7.3, 2 H, Harom ), 7.73 (t, J = 7.4, 1 H, Harom ), 7.63 (t, J = 7.3, 2 H,
H
arom ), 7.63 (obs, 1 H, NHm ), 7.35–7.27 (m , 5 H, Harom ), 6.17 (d, J = 9.0, 1 H, NHg), 5.28 (d,
J = 3.4, 1 H, H1m ), 5.11 (t, J = 9.5, 1 H, H3g), 5.09 (t, J = 9.3, 1 H, H4g), 4.61 an d 4.51 (AX, J =
12.2, 2 H, CH2Bn ), 4.57–4.43 (overlapped m , 2 H, CH2O), 4.50 (q, J = 6.9, 1 H, CH), 4.39 (d,
J = 8.6, 1 H, H1g), 4.37 (dd, J = 12.4, 4.6, 1 H, H6g), 4.24 (dd, J = 12.0, 2.2, 1 H, H6m ), 4.19
(d, J = 11.8, 1 H, H6′m ), 4.05 (dd, J = 12.4, 2.0, 1 H, H6′g), 4.05 (overlapped m , 1 H, H2g),
3.78 (m , 1 H, H2m ), 3.73–3.66 (m , 2 H, H3m , H5m ), 3.65–3.57 (overlapped m , 2 H, H5g, H4m ),
3.57–3.47 (m , 2 H, CH2S), 2.13 (s, 3 H, CH3), 2.05 (s, 3 H, CH3), 2.03 (s, 3 H, CH3), 2.02 (s,
3 H, CH3), 1.98 (s, 3 H, CH3), 1.93 (s, 3 H, CH3), 1.21 (d, J = 6.8, 3 H, CH3). 13C NMR
(CDCl3): 175.28 (COO), 171.16 (Ac), 170.70 (2 Ac), 170.69 (Ac), 170.32 (Ac, CON), 169.32
(CON), 139.04 (Carom ), 137.36 (Carom ), 134.16 (CHarom ), 129.50 (2 × CHarom ), 128.30 (2 ×
CHarom ), 128.06 (2 × CHarom ), 127.78 (CHarom ), 127.67 (2 × CHarom ), 101.06 (C1g), 96.31
(C1m ), 78.17 (C3m ), 74.86 (C4m ), 74.68 (CH), 72.87 (C3g), 71.89 (C5g), 70.32 (CH2Bn ), 69.37
(C5m ), 68.07 (C4g), 61.92 (C6m ), 61.59 (C6g), 58.17 (CH2O), 54.79 (CH2S), 54.27 (C2g), 53.89
(C2m ), 23.01, (CH3), 22.97 (CH3), 20.51 (4 × CH3), 18.05 (CH3). ESI-MS for C42H55N2O19
calculated [M + H]+: 923.3120; foun d: 923.3114.
S
Diben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-acetam ido-β-D-glucopyran osyl-(1→4)-2-acetam ido-
6-O-acetyl-2-deoxy-3-O-(D-1-{[2-(ph en ylsulfon yl)eth oxy]carbon yl}eth yl)-α-D-glucopyran osyl
Ph osph ate (17)
Com poun d 16 (1.8 g, 2.0 m m ol) was dissolved in THF/MeOH (4:1, 20 m l), th en 10% Pd/C
(500 m g) was added an d th e suspen sion was stirred an d cooled in ice-bath . Th e suspen sion
was warm ed to room tem perature an d h ydrogen ated at room tem perature for 2 h . Th e cata-
lyst was collected by filtration on Celite an d th e filtrate was con cen trated in vacuo to afford
a wh ite solid wh ich was triturated with dieth yl eth er/h exan e. Th e resultin g solid (1.5 g,
92%) was collected an d dried un der h igh vacuum for 16 h (ESI-MS for C35H49N2O19S calcu-
lated [M + H]+: 833.2650; foun d 833.2653). Th e wh ite powder (1.5 g, 1.83 m m ol) was dis-
solved in 0.5
M solution of tetrazole in aceton itrile (20 m l, 9.2 m m ol) an d diben zyl
N,N-dieth ylph osph oram idite (1.6 m l, 5.5 m m ol). Th e m ixture was stirred at room tem pera-
ture for 4 h . Th en th e m ixture was diluted with dich lorom eth an e (20 m l) an d wash ed with
saturated aqueous solution of NaHCO3, water an d brin e. Th e organ ic layer was dried with
Na2SO4, filtered an d con cen trated in vacuo. Th e residue obtain ed was dried overn igh t over
P2O5 in h igh vacuum . Th e oil was dissolved in THF (30 m l), cooled to –80 °C, an d th en h y-
drogen peroxide (45%, 3.5 m l) was added dropwise. Th e m ixture was allowed to warm to
room tem perature over 2 h . Th e solution was cooled in an ice-bath , an d diluted with eth yl
acetate followed by a saturated solution of Na2S2O3 (10 m l), with stirrin g for a few m in utes.
Th e organ ic layer was wash ed with water an d brin e an d dried with Na2SO4, filtered an d con -
cen trated in vacuo to provide a colorless oil, wh ich was triturated with Et2O/h exan e. Th e
solid was filtered an d dried over P2O5 in h igh vacuum to give com poun d 17 (1.5 g, 77%).
1H NMR (CDCl3): 7.96 (d, J = 7.3, 2 H, Harom ), 7.74 (m , 1 H, 2 H, Harom an d NHm ), 7.65 (t, J =
7.7, 2 H, Harom ), 7.38–7.27 (overlapped m , 10 H, 2 × Bn ), 6.21 (m , 1 H, NHg), 6.05 (dd, JH/H
=
3.2, JH/P = 5.6, 1 H, H1m ), 5.13 (t, J = 9.5, 1 H, H3g), 5.09 (t, J = 9.5, 1 H, H4g), 5.05 an d 5.00
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1633
(2 × d, JH/P = 7.5, 7.8, 2 × 2 H, 2 × CH2Bn ), 4.57 (m , 1 H, O-CH2A), 4.52 (q, J = 6.8, O-CH),
4.43 (m , 1 H, O-CH2B), 4.43 (d, J = 9.7, 1 H, H1g), 4.38 (dd, J = 12.5, 4.5, 1 H, H6g),
4.22–4.16 (m , 2 H, H6m an d H6′m ), 4.08–4.01 (m , 2 H, H6′g an d H2g), 3.88 (dm , J = 9.7, 1 H,
H
H
5m ), 3.82 (m , 1 H, H2m ), 3.77 (t, J = 9.8, 1 H, H3m ), 3.64 (m , 1 H, H5g), 3.56 (t, J = 9.8,
4m ), 3.58–3.46 (m , 2 H, CH2S), 2.05 (s, 3 H, CH3), 2.03 (s, 3 H, CH3), 2.03 (s, 3 H, CH3),
2.02 (s, 3 H, CH3), 1.92 (s, 3 H, CH3), 1.85 (s, 3 H, CH3), 1.23 (d, J = 6.8, 3 H, CH3). 13C NMR
(CDCl3): 175.32 (COO), 171.08 (Ac), 170.95 (Ac), 170.63 (Ac), 170.35 (Ac), 170.32 (CON),
169.33 (CON), 139.08 (Carom ), 135.50 (d, JC/P = 6.8, 2 × Carom ), 134.19 (CHarom ), 129.55 (2 ×
CHarom ), 128.55 (6 × CHarom ), 128.06 (2 × CHarom ), 127.87 (2 × CHarom ), 127.83 (2 ×
CHarom ), 100.89 (C1g), 95.25 (C1m ), 77.13 (C3m ), 74.89 (CH), 74.04 (C4m ), 72.77 (C3g), 71.94
(C5g), 71.05 (C5m ), 69.46 (d, JC/P = 6.0, CH2Bn ), 69.38 (d, JC/P = 6.0, CH2Bn ), 68.08 (C4g),
61.59 (C6m ), 61.45 (C6g), 58.28 (CH2O), 54.77 (CH2S), 54.31 (C2g), 53.82 (d, JC/P = 8.0, C2m ),
23.03 (CH3, Ac), 22.72 (CH3, Ac), 20.76 (CH3, Ac), 20.53 (CH3, Ac), 20.52 (2 × CH3, Ac),
17.99 (CH3). 31P NMR (CDCl3, 202 MHz): –2.553. ESI-MS for C49H61N2NaO22PS calculated
[M + Na]+: 1115.3071; foun d: 1115.3105.
Nε-(Ben zyloxycarbon yl)-Nα-(tert-butoxycarbon yl)-L-lysin am ide (18)
Boc-Lys(CBz) (4.75 g, 12.5 m m ol) was dissolved in dry THF (25 m l) an d cooled to –10 °C.
Th en trieth ylam in e (2.15 m l, 15 m m ol) an d isobutyl ch loroform ate (2 m l, 15 m m ol) were
added. Th e m ixture was stirred in in ert atm osph ere at –10 °C for 2 h . Th en aqueous am m o-
n ia solution (25%, 40 m l) was added, th e solution was stirred at –10 °C for 30 m in an d was
allowed to warm to room tem perature overn igh t. Th en th e solven ts were rem oved in vacuo
an d th e residue was redissolved in dich lorom eth an e an d wash ed with 1 M NaHCO3 an d wa-
ter. Th e organ ic layer was dried with Na2SO4, filtered an d con cen trated. Th e crude m aterial
obtain ed was recrystallized from CH2Cl2/MeOH to provide com poun d 18 (4.14 g, 87%), m .p.
148–149 °C (lit.24 142 °C). 1H NMR (DMSO-d6): 7.38–7.28 (m , 5 H, Harom ), 7.20 (br, 2 H,
2 NH), 6.91 (br, 1 H, NH), 6.66 (d, J = 8.0, NH), 5.00 (s, 2 H, CH2O), 3.81 (m , 1 H, CH), 2.97
(q, J = 6.2, 2 H, CH2ε), 1.56 an d 1.47 (m , 2 H, CH2β), 1.38 (m , 2 H, CH2S), 1.37 (s, 9 H, t-Bu),
1.25 (m , 2 H, CH2γ). 13C NMR (DMSO-d6): 174.35 (C=Oam ide), 156.16 (COOCBz), 155.40
(COOBoc), 137.36 (Carom ), 128.47 (CHm), 127.78 (CHo+p), 77.96 (C-O), 65.18 (CH2), 54.19
(CHα), 40.19 (CH2ε), 31.73 (CH2β), 29.19 (CH2δ), 28.27 (CH3Boc), 22.87 (CH2γ). ESI-MS for
C
19H29N3NaO5 calculated [M + Na]+: 402.2005; foun d: 402.1994.
Nα-(tert-Butoxycarbon yl)-Nε-dan syl-L-lysin am ide (20)
Am in o acid 18 (930 m g, 2.1 m m ol) was added to a suspen sion of 10% palladium on carbon
(100 m g) in m eth an ol (15 m l). Th e m ixture was cooled an d degassed, warm ed to room tem -
perature an d h ydrogen ated at atm osph eric pressure for 1.5 h . Th e catalyst was collected by
filtration on Celite an d th e filtrate was con cen trated in vacuo to an off-wh ite solid wh ich
was dried over P2O5 in h igh vacuum overn igh t to obtain 650 m g of crude com poun d 19
([M + H]+: 317.1). Th e off-wh ite solid (500 m g, 2.0 m m ol) was dissolved in dry dich loro-
m eth an e, an d dan syl ch loride (604 m g, 2.2 m m ol) an d trieth ylam in e (0.57 m l, 4.1 m m ol)
were added. Th e m ixture was stirred at room tem perature for 3 h , th en diluted with di-
ch lorom eth an e an d wash ed with 0.5 M HCl, water an d brin e. Th e organ ic layer was dried
with Na2SO4, filtered an d con cen trated. Th e residue was eluted from a colum n of silica gel
with CH2Cl2/MeOH (9:1) to give com poun d 20 (780 m g, 80%). 1H NMR (DMSO-d6): 8.45 (d,
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1634
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
J = 8.5, 1 H, CHarom ), 8.31 (d, J = 8.6, 1 H, CH), 8.09 (d, J = 7.3, 1 H, CH), 7.85 (t, J = 5.7,
NH), 7.61 (dd, J = 7.3, 8.3, 1 H, CH), 7.58 (dd, J = 7.6, 8.5, 1 H, CH), 7.25 (d, J = 7.5, 1 H,
CH), 7.15 an d 6.88 (s, 2 × 1 H, NH2), 6.60 (d, J = 8.0, 1 H, NH), 3.74 (m , 1 H, CH), 2.83 (s, 6
H, N(CH3)2), 2.74 (q, J = 6.6, 2 H, CH2ε), 1.44 an d 1.35 (m , 2 H, CH2β), 1.36 (s, 9 H, t-Bu),
1.34 (m , 2 H, CH2δ), 1.23–1.12 (m , 2 H, CH2γ). 13C NMR (DMSO-d6): 174.23 (C=Oam ide),
155.33 (C=OBoc), 151.25, 136.24 (Carom ), 129.30 (CHarom ), 129.17, 129.09 (Carom ), 128.20,
127.81, 123.65, 119.32, 115.22 (CHarom ), 77.95 (C-O), 54.06 (CHα), 45.14 (2 × NCH3), 42.38
(CH2ε), 31.49 (CH2β), 29.03 (CH2δ), 28.24 (CH3Boc), 22.65 (CH2γ). ESI-MS for C23H35N4O5S
calculated [M + H]+: 479.2328; foun d: 479.2329.
Nα-(tert-Butoxycarbon yl)-Nε-[(fluoren -9-ylm eth oxy)carbon yl]-L-lysin am ide (21)
In th e sam e m an n er as for com poun d 20, th e crude com poun d 19 was obtain ed by h ydroge-
n ation of com poun d 18. Th e off-wh ite solid (500 m g, 2.0 m m ol) was dissolved in saturated
aqueous solution of NaHCO3 an d Fm oc ch loride (633 m g, 2.4 m m ol) was added. A precipi-
tate appeared an d stirrin g was kept overn igh t. Th en th e suspen sion was diluted with di-
ch lorom eth an e an d wash ed with water an d brin e. Th e organ ic layer was dried with Na2SO4,
filtered an d con cen trated. Th e residue was eluted from
a colum n of silica gel with
CH2Cl2/MeOH (9:1) to give com poun d 21 (744 m g, 81%). 1H NMR (DMSO-d6): 7.88 (d, J =
7.3, 2 H, Harom ), 7.68 (d, J = 7.4, 2 H, Harom ), 7.41 (t, J = 7.4, 2 H, Harom ), 7.33 (t, J = 7.4, 2 H,
H
arom ), 7.25 (t, J = 5.5, 1 H, NH), 7.21 an d 6.91 (s, 2 × 1 H, NH2), 6.66 (d, J = 8.1, 1 H, NH),
4.29 (d, J = 6.8, 2 H, CH2), 4.20 (t, J = 6.8, 1 H, CH), 3.83 (m , 1 H, CHα), 2.96 (q, J = 6.4,
2 H, CH2ε), 1.57 an d 1.48 (m , 2 H, CH2β), 1.37 (s, 9 H, t-Bu), 1.36 (m , 2 H, CH2S), 1.26 (m ,
2 H, CH2γ). 13C NMR (DMSO-d6): 174.34 (C=Oam ide), 156.14 (C=OFm oc), 155.39 (COOBoc),
144.01, 140.80 (Carom ), 127.64, 127.10, 125.20, 120.15 (CHarom ), 77.95 (C-O), 65.26 (CH2-O),
54.20 (CHα), 46.85 (CH), 40.13 (CH2ε), 31.74 (CH2β), 29.16 (CH2δ), 28.25 (CH3Boc), 22.86
(CH2γ). ESI-MS for C26H33N3NaO5 calculated [M + Na]+: 490.2318; foun d: 490.2330.
N-(tert-Butoxycarbon yl)-D-glutam ic Diam ide (22)
Com m ercial Boc-D-Glu (2.5 g, 10.1 m m ol) an d 4–n itroph en ol (2.8 g, 20.2 m m ol) were dis-
solved in THF (40 m l). DCC (4.4 g, 21.2 m m ol) was added at 0 °C an d th e solution was
stirred at room tem perature overn igh t. Th e resultin g N,N′-dicycloh exylurea was filtered off
an d NH4OH (25%, 20 m l) was added to th e filtrate. Th e solution was stirred at room tem -
perature for 18 h . Th en th e solution was con cen trated in vacuo an d th e residue was dis-
solved in H2O (50 m l). Th e solution was n eutralized with 10% aqueous form ic acid, th e
aqueous solution was extracted with eth yl acetate (3 × 50 m l) an d lyoph ilized. Th e residue
was recrystallized from EtOH to give com poun d 22 (1.5 g, 62%), m .p. 125–126 °C (lit.26
132–133 °C). 1H NMR (DMSO-d6): 7.30 (br, 2 H, NH2), 6.96 (s, 1 H, NH), 6.73 (s, 2 H, NH),
3.81 (m , 1 H, CH), 2.08 (m , 2 H, CH2γ), 1.81 an d 1.68 (m , 2 H, CH2β), 1.36 (s, 9 H, t-Bu).
13C NMR (DMSO-d6): 174.07 (C=Oam ide), 173.95 (C=O), 155.34 (C=OBoc), 78.06 (C-O), 54.04
(CHα), 31.08 (CH2γ), 28.28 (CH3Boc), 27.94 (CH2β). ESI-MS for C10H19N3NaO4 calculated [M +
Na]+: 268.1273; foun d: 268.1270.
N-(tert-Butoxycarbon yl)-L-alan yl N-Hydroxysuccin im idyl Ester (23)
1-[3-(Dim eth ylam in o)propyl]-3-eth ylcarbodiim ide h ydroch loride (5.6 g, 29.2 m m ol) an d
N-h ydroxysuccin im ide (3.1 g, 26.9 m m ol) were added to a solution of Boc-L-Ala (5.08 g, 26.8
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1635
m m ol) in an h ydrous dioxan e (20 m l). Th e m ixture was stirred at room tem perature over-
n igh t. Th en th e solution was con cen trated in vacuo an d redissolved in eth yl acetate (50 m l)
an d wash ed with water an d brin e. Th e organ ic layer was dried with Na2SO4, filtered an d
con cen trated to provide a foam wh ich was recrystallized from EtOH to provide com poun d
23 (6.9 g, 89%), m .p. 154–155 °C. 1H NMR (DMSO-d6): 7.61 (d, J = 7, 1 H, NH), 4.41 (m ,
1 H, CH), 2.80 (s, 4 H, 2 × CH2), 1.41 (d, J = 7.3, CH3), 1.39 (s, 9 H, t-Bu). 13C NMR
(DMSO-d6): 170.01 (C=Osuccin im ide), 169.49 (C=Oacid), 155.11 (C=OBoc), 78.75 (C-O), 47.47
(CH), 28.18 (CH3Boc), 25.53 (2 × CH2), 17.01 (CH3). ESI-MS for C12H18N2NaO6 calculated
[M + Na]+: 309.1062; foun d: 309.1049.
N-(tert-Butoxycarbon yl)-L-alan yl-Nε-dan syl-L-lysin am ide (24)
Trifluoroacetic acid (5 m l) was added to a solution of com poun d 20 (932 m g, 1.9 m m ol) in
dich lorom eth an e (5 m l) an d th e m ixture was stirred at room tem perature for 2 h . Th e solu-
tion was con cen trated in vacuo an d dried un der h igh vacuum to provide th e trifluoroacetate
salt as an oil. Th e salt th us obtain ed was added to a solution of 23 (588 m g, 1.95 m m ol) in
an h ydrous DMF (10 m l) an d N,N-diisopropyleth ylam in e (0.61 m l, 5.9 m m ol) was added.
Th e m ixture was stirred at room tem perature overn igh t. Th en th e solven t was rem oved an d
th e residue was redissolved in dich lorom eth an e (20 m l) an d was wash ed with 0.5 M HCl, wa-
ter an d brin e. Th e organ ic layer was dried with Na2SO4, filtered an d con cen trated. Th e resi-
due was purified on silica gel with CH2Cl2/MeOH (9.5:0.5) to give com poun d 24 (749 m g,
70%). 1H NMR (DMSO-d6): 8.45 (d, J = 8.3, 1 H, Hdan syl), 8.31 (d, J = 8.6, 1 H, Hdan syl), 8.09
(d, J = 7.3, 1 H, Hdan syl), 7.84 (t, J = 5.7, 1 H, NH), 7.61 (dd, J = 8.6, 7.1, 1 H, Hdan syl), 7.59
(d, J = 8.3, NH), 7.58 (dd, J = 8.5, 7.5, 1 H, Hdan syl), 7.23 (d, J = 7.3, 1 H, Hdan syl), 7.26 an d
7.00 (brs, 2 H, NH2), 6.97 (d, J = 7.0, 1 H, NH), 4.12 (m , 1 H, CHLys), 3.92 (m , 1 H, CHAla),
2.81 (s, 6 H, (CH3)2), 2.73 (q, J = 6.6, 2 H, CH2ε), 1.53 an d 1.40 (m , 2 H, CH2β), 1.33 (m , 2 H,
CH2δ), 1.33 (s, 9 H, t-Bu), 1.18 (m , 2 H, CH2γ), 1.15 (d, J = 7.0, 3 H, CH3). 13C NMR
(DMSO-d6): 173.50 (C=Oam id e), 172.50 (C=O), 155.25 (C=OBoc), 151.41, 136.18 (Carom ),
129.38 (CHarom ), 129.22, 129.17 (Carom ), 128.26, 127.85, 123.63, 119.25, 115.17 (CHarom ),
78.23 (C-O), 51.95 (CHLys), 50.07 (CHAla), 45.13 (NMe2), 42.47 (CH2ε), 31.73 (CH2β), 29.08
(CH2δ), 28.22 (CH3Boc), 22.28 (CH2γ), 17.90 (CH3). ESI-MS for C26H40N5O6S calculated [M +
H]+: 550.2699; foun d: 550.2673.
N-(tert-Butoxycarbon yl)-L-alan yl-Nε-[(fluoren -9-ylm eth oxy)carbon yl]-L-lysin am ide (25)
Trifluoroacetic acid (5 m l) was added to a solution of com poun d 21 (500 m g, 1.1 m m ol) in
dich lorom eth an e (5 m l) an d th e m ixture was stirred at room tem perature for 2 h . Th e solu-
tion was con cen trated in vacuo an d dried un der h igh vacuum to provide th e trifluoroacetate
salt as an oil. Th e salt th us obtain ed was added to a solution of 23 (315 m g, 1.1 m m ol) in
an h ydrous DMF (10 m l) an d N,N-diisopropyleth ylam in e (0.57 m l, 3.3 m m ol) was added.
Th e m ixture was stirred at room tem perature overn igh t. Th en th e solven t was rem oved an d
th e residue was redissolved in dich lorom eth an e (20 m l) an d wash ed with 0.5 M HCl, water
an d brin e. Th e organ ic layer was dried with Na2SO4, filtered an d con cen trated. Th e residue
was eluted from silica gel with CH2Cl2/MeOH (9:1) to give com poun d 25 (455 m g, 77%). 1H
NMR (DMSO-d6): 7.88 (d, J = 7.6, 2 H, Harom ), 7.68 (d, J = 7.1, 2 H, Harom ), 7.65 (d, J = 8.1,
1 H, NH), 7.41 (t, J = 7.7, 2 H, Harom ), 7.33 (t, J = 8.5, 2 H, Harom ), 7.31 an d 7.02 (br, 2 H,
NH2), 7.24 (t, J = 5.3, 1 H, NH), 7.01 (d, J = 6.3, NH), 4.28 (d, J = 6.8, 2 H, CH2O), 4.20 (m ,
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1636
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
1 H, CHLys), 4.20 (m , 1 H, CH), 3.95 (m , 1 H, CHAla), 2.96 (q, J = 6.2, 2 H, CH2ε), 1.66 an d
1.52 (m , 2 H, CH2β), 1.38 (m , 2 H, CH2δ), 1.37 (s, 9 H, t-Bu), 1.24 (m , 2 H, CH2γ), 1.18 (d,
J = 7.1, 3 H, CH3). 13C NMR (DMSO-d6): 173.57 (C=Oam ide), 172.52 (C=O), 156.13 (C=OFm oc),
155.26 (C=OBoc), 144.01, 140.75 (Carom ), 127.15, 127.11, 125.29, 120.16 (CHarom ), 78.26
(C-O), 65.28 (CH2-O), 52.09 (CHLys), 50.17 (CHAla), 46.85 (CH), 40.23 (CH2ε), 31.95 (CH2β),
29.19 (CH2δ), 28.23 (CH3Boc), 22.47 (CH2γ), 17.97 (CH3). ESI-MS for C29H39N4O6 calculated
[M + H]+: 539.2870; foun d: 539.2869.
[N-(tert-Butoxycarbon yl)-L-alan yl]glutam ic Diam ide (26)
Trifluoroacetic acid (5 m l) was added to a solution of com poun d 22 (1.1 g, 4.6 m m ol) in di-
ch lorom eth an e (5 m l) an d th e m ixture was stirred at room tem perature for 2 h . Th e solution
was con cen trated in vacuo an d dried un der h igh vacuum to provide th e trifluoroacetate salt
as an oil. Th e salt th us obtain ed was added to a solution of 23 (1.3 m g, 4.6 m m ol) in an h y-
drous DMF (15 m l) an d N,N-diisopropyleth ylam in e (1 m l, 9.3 m m ol) was added. Th e m ix-
ture was stirred at room tem perature overn igh t. Th en th e solven t was rem oved an d th e resi-
due was redissolved in dich lorom eth an e (40 m l), an d wash ed with 0.5 M HCl, water an d
brin e. Th e organ ic layer was dried with Na2SO4, filtered an d con cen trated. Th e residue was
elu ted from silica gel with CH2Cl2/ MeOH (8:2) to give com p ou n d 26 (843 m g, 58%).
1H NMR (DMSO-d6): 7.87 (d, J = 7.6, 1 H, NH), 7.30 an d 6.93 (s, 2 H, NH2), 7.27 an d 7.10 (s,
2 H, NH2), 7.04 (d, J = 6.1, 1 H, NH), 4.12 (m , 1 H, CHGlu), 3.95 (m , 1 H, CHAla), 2.135 (t,
J = 7.8, 2 H, CH2γ), 1.97 an d 1.70 (m , 2 H, CH2β), 1.37 (s, 9 H, t-Bu), 1.18 (d, J = 7.1, 3 H,
CH3). 13C NMR (DMSO-d6): 173.90 (C=Oam ide), 173.40 (C=Oam ide), 171.64 (C=O), 155.44
(C=OBoc), 78.33 (C-O), 52.66 (CHGlu), 50.07 (CHAla), 31.79 (CH2γ), 28.27 (CH3Boc), 27.97
(CH2β), 17.82 (CH3). ESI-MS for C13H24N4NaO5 calculated [M + Na]+: 339.1644; foun d:
339.1648.
Diben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-acetam ido-β-D-glucopyran osyl-(1→4)-2-acetam ido-
6-O-acetyl-2-deoxy-3-O-{D-1-[(N-h ydroxysuccin im idyloxy)carbon yl]eth yl}-
α-D-glucopyran osyl Ph osph ate (28)
To a solution of com poun d 17 (1.2 g, 1.1 m m ol) in dich lorom eth an e (20 m l) was added
dropwise at 0 °C DBU (0.175 m l, 1.1 m m ol). After 30 m in th e solution was diluted with di-
ch lorom eth an e an d wash ed with 1 M HCl, water an d brin e. Th e organ ic layer was dried with
Na2SO4, filtered, con cen trated an d triturated with Et2O. After filtration , th e crude com -
poun d 27 was dried over P2O5 in h igh vacuum . Th e acid (1 g, 1.1 m m ol) was dissolved in
an h ydrous DMF (20 m l) an d N-h ydroxysuccin im ide (187 m g, 1.6 m m ol) an d 1-[3-(dim eth yl-
am in o)propyl]-3-eth ylcarbodiim ide h ydroch loride (311 m g, 1.62 m m ol) were added. Th e
m ixture was stirred at room tem perature overn igh t. Th en th e solution was con cen trated in
vacuo, th e residue was redissolved in eth yl acetate (30 m l) an d wash ed with water an d
brin e. Th e organ ic layer was dried with Na2SO4, filtered an d con cen trated to provide a color-
less foam wh ich was triturated with Et2O to give th e NHS-ester 28 (977 m g, 87%). ESI-MS
for C45H56N3NaO22P calculated [M + Na]+: 1044.2991; foun d: 1044.2988.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1637
Diben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-acetam ido-β-D-glucopyran osyl-(1→4)-2-acetam ido-
6-O-acetyl-2-deoxy-3-O-(D-2-propion yl-L-alan yl-Nε-dan syl-L-lysin am ide)-α-D-glucopyran osyl
Ph osph ate (29)
Th e dipeptide 24 (364.0 m g, 0.66 m m ol) was treated with trifluoroacetic acid (5 m l) in di-
ch lorom eth an e (5 m l) for 2 h , th en th e solven t was rem oved in vacuo an d th e residue was
coevaporated with toluen e to provide th e trifluoroacetate salt as an oil. Th e salt th us ob-
tain ed was added to a solution of th e NHS-ester 28 (517 m g, 0.51 m m ol) an d N,N-diiso-
propyleth ylam in e (0.283 m l, 1.6 m m ol) in DMF (3 m l), an d stirred at room tem perature
un der in ert atm osph ere for 24 h . Th en th e solven t was rem oved un der reduce pressure an d
th e residue was redissolved in dich lorom eth an e, wash ed with 0.5 M HCl solution , water an d
brin e. Th e organ ic layer was dried with Na2SO4, filtered an d con cen trated. Th e residue was
triturated with dieth yl eth er to provide th e com poun d 29 (634 m g, 92%). 1H NMR (CDCl3):
8.52 (d, J = 8.5, 1 H, Hdan syl), 8.34 (d, J = 8.8, 1 H, Hdan syl), 8.18 (d, J = 7.2, 1 H, Hdan syl),
8.03 (m , 1 H, NHm ), 7.52 (d, J = 8.7, 1 H, Hdan syl), 7.50 (d, J = 7.6, 1 H, Hdan syl), 7.16 (d, J =
7.6, 1 H, Hdan syl), 7.46 (m , 1 H, NHαAla), 7.19 (m , 1 H, NHαLys), 6.84 (m , 1 H, NHg), 6.63
(brs, 1 H, NH2/A), 6.37 (m , 1 H, NHεLys), 6.33 (brs, 1 H, NH2/B), 5.99 (dd, J = 3.2, 5.8, 1 H,
H
1m ), 5.26 (t, J = 10.1, 1 H, H3g), 5.08 (t, J = 9.8, 1 H, H4g), 5.08–5.02 (dAB, JH/P = 7.3, 2 H,
CH2Bn ), 5.01–4.93 (dAB, JH/P = 6.4, 2 H, CH2Bn ), 4.72 (q, J = 6.6, 1 H, CHO), 4.67 (d, J =
8.3, 1 H, H1g), 4.45 (p, J = 7.1, 1 H, CHAla), 4.40–4.37 (m , 2 H, CHLys, H6g), 4.31 (d, J = 12.3,
H
6m ), 4.16 (dd, J = 3.4, 12.2, 1 H, H6′m ), 4.08 (d, J = 10.5, 1 H, H6′g), 4.03 (m , 2 H, H5m , H2g),
3.90 (m , 2 H, H2m , H4m ), 3.68 (brd, J = 8.3, 1 H, H5g), 3.61 (t, J = 9.9, 1 H, H3m ), 2.86 (s, 6 H,
N(CH3)2), 2.84 (m , 2 H, CH2ε), 2.03 (s, 3 H, CH3), 2.01 (s, 3 H, CH3), 2.00 (s, 3 H, CH3), 1.97
(s, 3 H, CH3), 1.93 (s, 3 H, CH3), 1.79 (s, 3 H, CH3), 1.73 an d 1.63 (br, 2 H, CH2β), 1.50 an d
1.40 (obs, 4 H, CH2γ, CH2δ), 1.47 (d, J = 7.1, CH3Ala), 1.43 (d, J = 6.6, 3 H, CH3). 13C NMR
(CDCl3): 175.28 (CONH), 174.08 (COam ide), 172.60 (CONH), 171.41, 171.34, 170.83, 170.68
(4 × OAc), 170.57 (NHAc), 169.46 (NHAc), 151.78 (Cdan syl), 135.39 (d, JC/P = 7.5, C, Bn ),
135.29 (d, JC/P = 7.5, C, Bn ), 134.98 (Cdan syl), 130.20 (CHdan syl), 129.82, 129.60 (Cdan syl),
129.15 (CHdan syl), 128.57 (6 × CHarom ), 128.16 (CHdan syl), 127.94 (4 × CHarom ), 123.16
(CHdan syl), 118.93 (CHdan syl), 115.17 (CHdan syl), 99.59 (C1g), 95.80 (d, J = 7.0, C1m ), 76.64
(C3m ), 75.15 (CHO), 74.64 (C4m ), 72.30 (C3g), 71.97 (C5g), 70.97 (C5m ), 69.68 (d, J = 5.6,
CH2Bn ), 69.61 (d, J = 5.6, CH2Bn ), 68.63 (C4g), 62.05 (C6m ), 61.82 (C6g), 54.80 (C2g), 53.97
(C2m ), 52.36 (CHα, Lys), 49.99 (CHα, Ala), 45.32 (2 × N-CH3), 42.34 (CH2ε, Lys), 31.13 (CH2β
,
Lys), 28.25 (CH2δ, Lys), 23.18 (CH3, NHAc), 22.70 (CH3, NHAc), 21.45 (CH2γ, Lys), 20.74
(CH3, OAc), 20.63 (CH3, OAc), 20.54 (2 × CH3, OAc), 19.24 (CH3Ala), 17.42 (CH3Ala).31
P
NMR (CDCl3, 202 MHz): –3.085. ESI-MS for C62H83N7O23PS calculated [M + H]+: 1356.4998;
foun d: 1356.4943.
Diben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-acetam ido-β-D-glucopyran osyl-(1→4)-2-acetam ido-
6-O-acetyl-2-deoxy-3-O-{D-2-propion yl-L-alan yl-Nε-(fluoren -9-ylm eth oxy)carbon yl-
L-lysin am ide}-α-D-glucopyran osyl Ph osph ate (30)
Com poun d 30 was prepared from com poun d 28 (200.0 m g, 0.196 m m ol) an d th e
deprotected dipeptide 25 (161.0 m g, 0.300 m m ol) as described for th e preparation of com -
poun d 29, yieldin g 160.68 m g (61%). 31P NMR (DMSO-d6, 202 MHz): –2.048. ESI-MS for
C
65H82N6O23P calculated [M + H]+: 1345.5168; foun d: 1345.5248.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1638
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
Diben zyl 3,4,6-Tri-O-acetyl-2-deoxy-2-acetam ido-β-D-glucopyran osyl-(1→4)-2-acetam ido-
6-O-acetyl-2-deoxy-3-O-(D-2-propion yl-L-alan yl-D-glutam ic diam ide)-
α-D-glucopyran osyl Ph osph ate (31)
Com poun d 31 was prepared from com poun d 28 (200.0 m g, 0.196 m m ol) an d th e depro-
tected dipeptide 26 (100 m g, 0.294 m m ol) as described for th e preparation of com poun d 29,
yieldin g 120.95 m g (55%). 31P NMR (DMSO-d6, 202 MHz): –1.951. ESI-MS for C49H68N6O22
calculated [M + H]+: 1123.4125; foun d: 1123.4156.
P
P1-Uridin -5′-yl P2-[(2-Acetam ido-2-deoxy-β-D-glucopyran osyl)-(1→4)-2-acetam ido-2-deoxy-
3-O-(D-2-propion yl-L-alan yl-Nε-dan syl-L-lysin am ide)-α-D-glucopyran osyl] Diph osph ate (35)
Th e disacch aridyl dipeptide 29 (118.30 m g, 0.09 m m ol) was dissolved in m eth an ol (4 m l)
an d 10% palladium on carbon (100 m g) was added. Th e suspen sion was cooled to 0 °C an d
th e reaction m ixture was degassed. Th en th e m ixture was warm ed to room tem perature an d
h ydrogen ated at atm osph eric pressure for 2 h . Th e catalyst was collected by filtration on
Celite an d th e filtrate was treated with pyridin e (0.5 m l) an d con cen trated in vacu o to
afford a wh ite solid wh ich was triturated with dieth yl eth er. Th e resultin g solid (99.6 m g,
95%) was collected an d dried un der h igh vacuum for 16 h . Th en th e dried solid was reacted
with uridin e 5′-m on oph osph om orph olidate 4-m orph olin e-N,N′-dicycloh exylcarboxam idin e
salt (92.7 m g, 0.135 m m ol) an d 0.5 M tetrazole in aceton itrile (0.6 m l) in pyridin e (1 m l).
Th e m ixture was stirred at room tem perature for 2 days. Th e m ixture was th en con cen trated
in vacuo an d dissolved in dioxan e (1 m l) followed by 1 M NaOH (0.5 m l). Th e m ixture was
stirred at room tem perature for 1 h . Th e solution was con cen trated in vacuo at 30 °C an d
th e residue was dissolved in water (1 m l), an d th e solution was filtered an d purified by re-
verse ph ase HPLC on a Alltech ® HS Hyper Prep 100 BDS colum n C-18 8µ (len gth 250 m m )
em ployin g a gradien t elution of A/B (100:0) to A/B (80:20) over 15 m in at a flow rate of 3 m l
m in –1 wh ere A = 0.05 M aqueous HCOONH4 an d B = MeCN. Lyoph ilisation of th e colum n
fraction s afforded pure 35 (21.2 m g, 18%) as a yellow powder. 1H NMR (DMSO-d6): 8.44 (d,
J = 8.3, Hdan syl), 8.30 (d, J = 8.6, Hdan syl), 8.09 (d, J = 7.3, Hdan syl), 7.89 (d, J = 8.1, 1 H, H6u),
7.61 (dd, J = 7.3, 8.6, Hdan syl), 7.58 (dd, J = 7.3, 8.6, Hdan syl), 7.23 (d, J = 7.3, Hdan syl), 5.80 (d,
J = 5.1, H1′u), 5.64 (d, J = 8.1, H5u), 5.36 (d, J = 5.4, H1m ), 4.21 (m , 1 H, CHO), 4.15 (m , 1 H,
CHαAla), 4.06 (m , 3 H, H2′u, H5m , CHαLys), 3.93 (m , 1 H, H4′u), 3.90 (m , 3 H, H5′u, H5′′u),
3.88 (m , 1 H, H2g), 3.68 (m , 2 H, H3g, H5g), 3.68 an d 3.40 (m , 2 H, H6g, H6′g), 3.66 an d 3.58
(m , 2 H, H6m , H6′m ), 3.40 (m , 3 H, H2m , H4m , H4g), 3.05 (m , 1 H, H3m ), 3.01 (m , 1 H, H3′u),
2.83 (m , 6 H, NMe2), 2.72 (m , 2 H, CH2ε Lys), 2.80 (m , 2 H, CH2β Lys), 1.81 (s, 6 H, 2 × Ac),
1.34 (m , 4 H, CH2δ, CH2γ Lys), 1.24 (s, 6 H, 2 × CH3). 13C NMR (DMSO-d6): 173.69, 173.63,
172.33, 170.29, 169.42, 163.36, 150.99, 140.98, 102.07, 100.58, 93.79, 87.58, 83.59, 76.55,
76.46, 76.30, 75.14, 74.00, 73.44, 72.58, 71.39, 70.02, 64.61, 61.77, 59.60, 55.90, 53.38, 52.40,
48.86, 45.14, 42.30, 29.51, 29.00, 23.07, 22.67, 21.87, 18.79, 17.87. 31P NMR (DMSO-d6, 202
MHz): –9.540 (d, J = 19.5), –12.56 (d, J = 19.8). ESI-MS for C48H71N9O27P2S calculated [M +
H]+: 1299.3655; foun d: 1299.3656.
P1-Uridin -5′-yl P2-[(2-Acetam ido-2-deoxy-β-D-glucopyran osyl)-(1→4)-2-acetam ido-2-deoxy-
3-O-(D-2-propion yl-L-alan yl-L-lysin am ide)-α-D-glucopyran osyl] Diph osph ate (36)
Th e disacch aridyl dipeptide 30 (145 m g, 0.108 m m ol) was dissolved in m eth an ol (6 m l) an d
10% palladium on carbon (150 m g) was added. Th e suspen sion was cooled to 0 °C an d th e
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1639
reaction m ixture was degassed. Th en th e m ixture was warm ed to room tem perature an d h y-
drogen ated at atm osph eric pressure for 2 h . Th e catalyst was collected by filtration on Celite
an d th e filtrate was treated with pyridin e (0.5 m l) an d con cen trated in vacuo to afford a
wh ite solid wh ich was triturated with dieth yl eth er. Th e resultin g solid (100 m g, 79%) was
collected an d dried un der h igh vacuum for 16 h . Th en th e dried solid was reacted with
uridin e 5′-m on oph osph om orph olidate 4-m orph olin e-N,N′-dicycloh exylcarboxam idin e salt
(88.5 m g, 0.129 m m ol) an d 0.5 M tetrazole in aceton itrile (1.72 m l, 0.86 m m ol). Th e m ixture
was stirred at room tem perature for 2 days. Th e m ixture was th en con cen trated in vacuo
an d dissolved in dioxan e (1 m l) followed by 1 M NaOH (0.5 m l). Th e m ixture was stirred at
room tem perature for 1 h . Th e com poun d 36 was purified as described for 35, yieldin g
12.3 m g (13%). 31P NMR (DMSO-d6, 202 MHz): –10.930 (d, J = 20.7), –12.710 (d, J = 21.4).
ESI-MS for C37H63N8O25P2 calculated [M + H]+: 1081.3379; foun d: 1081.3412.
P1-Uridin -5′-yl P2-[(2-Acetam ido-2-deoxy-β-D-glucopyran osyl)-(1→4)-2-acetam ido-2-deoxy-
3-O-(D-2-propion yl-L-alan yl-D-glutam ic diam ide)-α-D-glucopyran osyl] Diph osph ate (37)
Th e disacch aridyl dipeptide 31 (100 m g, 0.089 m m ol) was dissolved in m eth an ol (3 m l) an d
10% palladium on carbon (80 m g) was added. Th e suspen sion was cooled to 0 °C an d th e
reaction m ixture was degassed. Th en th e m ixture was warm ed to room tem perature an d h y-
drogen ated at atm osph eric pressure for 2 h . Th e catalyst was collected by filtration on Celite
an d th e filtrate was treated with pyridin e (0.5 m l) an d con cen trated in vacuo to afford a
wh ite solid wh ich was triturated with dieth yl eth er. Th e resultin g solid (60.48 m g, 72%) was
collected an d dried un der h igh vacuum for 16 h . Th en th e dried solid was reacted with
uridin e 5′-m on oph osph om orph olidate 4-m orph olin e-N,N′-dicycloh exylcarboxam idin e salt
(66.13 m g, 0.09 m m ol) an d 0.5 M tetrazole in aceton itrile (1.28 m l, 0.64 m m ol). Th e m ixture
was stirred at room tem perature for 2 days. Th e m ixture was th en con cen trated in vacuo
an d dissolved in dioxan e (1 m l) followed by 1 M NaOH (0.5 m l). Th e m ixture was stirred at
room tem perature for 1 h . Th e com poun d 37 was purified as described for 35, yieldin g
13.8 m g (20%). 31P NMR (DMSO-d6, 202 MHz): –11.45 (d, JP,P = 20.8), –13.63 (d, JP,P = 22.6).
ESI-MS for C36H59N8O26P2 calculated [M + H]+: 1081.3015; foun d: 1081.3020.
P1-Uridin -5′-yl P2-[(2-Acetam ido-2-deoxy-β-D-glucopyran osyl)-(1→4)-2-acetam ido-2-deoxy-
3-O-(D-1-carboxyeth yl)-α-D-glucopyran osyl] Diph osph ate (39)
Th e disacch aride 17 (150 m g, 0.137 m m ol) was dissolved in m eth an ol (3 m l) an d 10% palla-
dium on carbon (150 m g) was added. Th e suspen sion was cooled to 0 °C to aid in degassin g
th e reaction m ixture. Th en , th e m ixture was warm ed to room tem perature an d h ydroge-
n ated at atm osph eric pressure for 2 h . Th e catalyst was collected by filtration on Celite an d
th e filtrate was treated with pyridin e (0.5 m l) an d con cen trated in vacuo to afford a wh ite
solid wh ich was triturated with dieth yl eth er. Th e resultin g solid (90 m g, 72%) was collected
an d dried un der h igh vacuum for 16 h . Th en th e dried solid was com bin ed with uridin e
5′-m on oph osph om orph olidate 4-m orph olin e-N,N′-dicycloh exylcarboxam idin e salt (100 m g,
0.147 m m ol) an d 0.5 M tetrazole in aceton itrile (2 m l, 0.986 m m ol). Th e m ixture was stirred
at room tem perature for 2 days. Th e m ixture was th en con cen trated in vacuo an d dissolved
in dioxan e (1 m l) followed by 1 M NaOH (0.5 m l). Th e m ixture was stirred at room tem pera-
ture for 1 h . Th e com poun d 39 was purified as described for 35, yieldin g 19.10 m g (16%).
31P NMR (DMSO-d6, 202 MHz): –10.297 (d, JP,P = 14.0), –11.576 (d, JP,P = 13.9). ESI-MS for
C
28H43N4Na2O24P2 calculated [(M – H) + 2 Na]+: 927.1537; foun d: 927.1539.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

1640
Lioux, Busson, Rozenski, Nguyen-Disteche, Frere, Herdewijn:
Authors are indebted to the Belgian Program on Interuniversity Poles of Attraction initiated by the
Belgian State, Prime Minister’s Office, Science Policy Programming (IAP P5/33).
REFERENCES
1. Russell A. D., Chopra I.: Understanding Antibacterial Action and Resistance, 2nd ed. Ellis
Horwood, London 1996.
2. Rogers H. J., Perkins H. R., Ward J. B.: Microbial Cell Walls and Membranes. Chapman &
Hall, London 1980.
3. Brock T. D., Madigan M. T.: Biology of Microorganisms, p. 72. Prentice Hall, Englewood
Cliffs (NJ) 1988.
4. Ghuysen J. M., Goffin C.: Antimicrob. Agents Chemother. 1999, 43, 2339.
5. Ha S., Chang E., Lo M.-C., Men H., Park P., Ge M., Walker S.: J. Am. Chem. Soc. 1999,
121, 8415.
6. Ge M., Chen Z., Onishi H. R., Kholer J., Silver L. L., Kerns R., Fukuzawa S., Thompson C.,
Kahne D.: Science 1999, 284, 507.
7. Fraipont C., Sapunarich F., Lioux T., Zervosen A., Auger G., Devreese B., Blaot D.,
Mengin-Lecreulx D., Herdewijn P., Frère J.-M., Nguyen-Distèch e M.: Biochemistry 2005,
accepted.
8. Ye X.-Y., Lo M.-C., Brunner L., Walker D., Kahne D., Walker S.: J. Am. Chem. Soc. 2001,
123, 3155.
9. Schwartz B., Markwalder J. A., Wang Y.: J. Am. Chem. Soc. 2001, 123, 11638.
10. Van Nieuwenhze M. S., Mauldin S. C., Zia-Ebrahimi M., Winger B. E., Hornback W. J.,
Saha S. L., Aikins J. A., Blaszczak L. C.: J. Am. Chem. Soc. 2002, 124, 3656.
11. Saha S. L., Van Nieuwenhze M. S., Hornback W. J., Aikins J. A., Blaszczak L. C.: Org. Lett.
2001, 3, 3575.
12. Mitchell S. A., Pratt M. R., Hruby V. J., Polt R.: J. Org. Chem. 2001, 66, 2327.
13. Zhang Z., Ollmann I. R., Ye X.-S., Wischnat R., Baasov T., Wong C.-H.: J. Am. Chem. Soc.
1999, 121, 734.
14. Dullenkopf W., Castro-Palomino J. C., Manzoni L., Schmidt R. R.: Carbohydr. Res. 1996,
1, 135.
15. Fischer E.: Ber. Dtsch. Chem. Ges. 1893, 26, 2400.
16. Pertel S. S., Kadun A. L., Kakayan E. S., Chirva V. Y., Krivorutchenko Y. L., Krivoshein Y. S.,
Bakova A. A., Andronovskaya I. B.: Russ. J. Bioorg. Chem. 1999, 25, 628.
17. Gross P. H., Rimpler M.: Liebigs Ann. Chem. 1986, 37.
18. Jeanloz R. W., Walker E., Sina P.: Carbohydr. Res. 1968, 6, 184.
19. Wang Z.-F., Xu J.-C.: Tetrahedron 1998, 54, 12597.
20. Hitchcock S. A., Eid C. N., Aikins J. A., Zia-Ebrahimi M., Blaszczak L. C.: J. Am. Chem.
Soc. 1998, 120, 1916.
21. Sadamoto R., Niikura K., Sears P. S., Liu H., Wong C.-H.: J. Am. Chem. Soc. 2002, 124,
9018.
22. DeNinno M. P., Etienne J. B., Duplantier K. C.: Tetrahedron Lett. 1995, 36, 669.
23. Ellervik U., Magnusson G.: Carbohydr. Res. 1996, 280, 251.
24. Lazar K., Walker S.: Curr. Opin. Chem. Biol. 2002, 6, 786.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Synthesis of Peptidoglycan Units with UDP
1641
25. Garrouste P., Pawlowski M., Tonnaire T., Sicsic S., Dumy P., de Rosny E., Reboud-Ravaux M.,
Fulcrand P., Martinez J.: Eur. J. Med. Chem. 1998, 33, 423.
26. Poulos C., Stavropoulos G. B., Brown J. R., Jordan C. C.: J. Med. Chem. 1987, 30, 1512.
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