Inhibition of S-ribosylhomocysteinase (LuxS) by substrate analogues modified at the ribosyl C-3 position

Article abstract of DOI:10.1016/j.bmc.2009.07.057

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond of S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), which is the precursor of type 2 autoinducer for bacterial cell-cell communication. In this work, we have synthesized several SRH analogues modified at the ribose C3 position as potential inhibitors of LuxS. While removal or methylation of the C3-OH resulted in simple competitive inhibitors of moderate potency, inversion of the C3 stereochemistry or substitution of fluorine for C3-OH resulted in slow-binding inhibitors of improved potency. The most potent inhibitor showed a K_I^* value of 0.43 μM.

Full text of DOI:10.1016/j.bmc.2009.07.057

Bioorganic & Medicinal Chemistry 17 (2009) 6699–6706
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of S-ribosylhomocysteinase (LuxS) by substrate analogues modified
at the ribosyl C-3 position
a
Stanislaw F. Wnuk a, , Jenay Robert , Adam J. Sobczak a,à, Brandon P. Meyers a, Venkata L. A. Malladi a,
*
Jinge Zhu b, Bhaskar Gopishetty b, Dehua Pei b,
a Department of Chemistry and Biochemistry Florida International University, Miami, FL 33199, USA
b Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond of S-ribosylhomocysteine
(SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), which is the precursor of
type 2 autoinducer for bacterial cell–cell communication. In this work, we have synthesized several
SRH analogues modified at the ribose C3 position as potential inhibitors of LuxS. While removal or meth-
ylation of the C3–OH resulted in simple competitive inhibitors of moderate potency, inversion of the C3
stereochemistry or substitution of fluorine for C3–OH resulted in slow-binding inhibitors of improved
Received 2 June 2009
Revised 22 July 2009
Accepted 23 July 2009
Available online 26 July 2009
Keywords:
Enzyme inhibition
Fluoro sugars
potency. The most potent inhibitor showed a KꢀI value of 0.43
lM.
Ó 2009 Elsevier Ltd. All rights reserved.
LuxS enzyme
S-Ribosylhomocysteine
1. Introduction
tion.16–19 Therefore, proteins involved in QS including LuxS are
being pursued as targets for designing novel antibacterial agents.
LuxS is a small metalloenzyme containing a Fe2+ ion coordi-
nated by His-54, His-58, Cys-126, and a water molecule. The native
enzyme is unstable under aerobic conditions, but substitution of
Co2+ for Fe2+ gives a highly stable variant with wild-type catalytic
activity.20,21 In the proposed catalytic mechanism of LuxS, the me-
tal ion acts as a Lewis acid and catalyzes two consecutive aldose–
ketose (C1?C2) and ketose–ketose (C2?C3) isomerization steps
and the b-elimination of Hcy from a 3-keto intermediate to give
DPD.22–24 Cleavage of the C5–S thioether bond of SRH by LuxS is
mechanistically distinct from the reversible reaction catalyzed by
SAH hydrolase, which cleaves the equivalent thioether bond in
SAH. The latter process is initiated by oxidation of the C30 alcohol
of SAH with an NAD+ cofactor to give a 30-keto intermediate that
undergoes b-elimination of Hcy.25,26
The bacterial enzyme 50-methylthioadenosine/S-adenosylho-
mocysteine (SAH) nucleosidase (MTAN, EC 3.2.2.9) catalyzes the
depurination of SAH to form adenine and S-ribosyl-L-homocysteine
(SRH) (Fig. 1).1 Encoded by the pfs gene, MTAN is involved in sev-
eral bacterial processes including polyamine biosynthesis, quorum
sensing (QS), methyl-transfer reactions, and adenine and methio-
nine salvage.2 SRH is subsequently converted to
L-homocysteine
and 4,5-dihydroxy-2,3-pentanedione (DPD) by the enzyme LuxS
(or S-ribosylhomocysteinase).3 DPD is in equilibrium with its cyclic
hemiketal A and forms several QS molecules, which function as
bacterial interspecies signaling molecules called autoinducers 2
(AI-2), and are detected by a wide range of bacteria.4–6 The
(2S,4S)-isomer of the hemiketal A undergoes complexation with
borate to form a furanosyl borate diester as a distinct AI-2.7 Recent
chemical synthesis of the unstable DPD and their analogues has
made it possible to assess the complexation properties of DPD with
borate and provided DPD samples for investigation of AI-2-regu-
lated processes.8–15 QS regulates many crucial bacterial functions
such as symbiosis, virulence, antibiotic production, biofilm forma-
Recently, Zhou and co-workers designed two substrate ana-
logues, the S-(anhydroribosyl)-L-homocysteine (B) and S-(homori-
bosyl)-
L
-cysteine (C) compounds (Fig. 2). The initial mechanistic
step was prevented by B, and the final step by C.27 Pei and co-
workers28 prepared a series of structural analogues of the enedio-
late intermediates, in which the unstable enediolate moiety was
replaced with a stable hydroxamate group. Among them, stable
isostere
(KI = 0.72
D
inhibited LuxS at submicromolar concentrations
* Corresponding author. Tel.: +1 305 348 6195; fax: +1 305 348 3772.
E-mail address: wnuk@fiu.edu (S.F. Wnuk).
lM). The ribosyl and xylosylhomocysteines in which
the carbon-5 and sulfur atoms were replaced by a vinyl or (flu-
oro)vinyl unit did not show significant inhibition of LuxS.29 We
now describe the synthesis and evaluation of several carbon-3
Correspondence concerning enzymatic studies please direct to pei.3@osu.edu.
Faculty leave from the Department of Chemistry, Poznan University of Life
à
Sciences, Poland.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.057

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S. F. Wnuk et al. / Bioorg. Med. Chem. 17 (2009) 6699–6706
NH2
NH2
A
O
O
MTAN (Pfs Nucleosidase)
Ade
HO2C
S
HO2C
S
OH
HO OH
SAH
HO OH
SRH
LuxS
Hcy
-
HO
OH
B(OH)4
CH3
OH
B
O
OH
O
O
O
OH
HO
HO
CH3
O
HO
O
O
A
H2O
DPD
AI-2
Figure 1. Enzymatic conversion of SAH by MTAN and LuxS: biosynthetic pathway to AI-2.
modified SRH analogues that lack a hydroxyl group which can be
converted into an enol. These compounds act as mechanism-based
inhibitors of LuxS and could impede the production of autoinduc-
ers of type 2, and disrupt quorum sensing and cell–cell communi-
cation in bacteria.
oxidation of the resulting sulfite with RuCl3 and NaIO4. The cyclic
sulfate 14 was readily opened22 with the protected
L-homocysteine
in the presence of BuLi in DMF to give desired thioether 15, which
was converted into the target compound SXH 16 by treatment with
TFA.
S-(3-Deoxy-3-fluoroxylosyl)homocysteine 21a and the corre-
sponding glycoside 21b were prepared from the readily available
diacetone 3-deoxy-3-fluoroglucose 17.34 Thus, sequential treat-
2. Results and discussion
2.1. Chemistry
31
ment of 17 with H5IO6 and NaBH4 gave 3-deoxy-3-fluoro-l,2-O-
isopropylidene-
converted into 5-O-mesylate 19b (Scheme 3). Displacement of
the mesylate group with thiolate generated from N-Boc- –Hcy–
a-D-xylofuranose (18, 60%), which was then
We initially targeted 3-deoxy-SRH analogue 10 because it can-
not undergo the second enolization step to produce the 3-keto
intermediate.3,22 Treatment of diacetone 3-deoxyglucose30 with
periodic acid selectively removed the 5,6-O-isopropylidene group
and oxidatively cleaved the resulting vicinal 5,6-diol31 to give the
corresponding 5-aldehyde, which was reduced with NaBH4 to give
3-deoxy furanoside 1 (Scheme 1). Mesylation at C5, displacement
L
CO2t-Bu (LDA/DMF) gave thioether 20b in 92% yield. It is notewor-
thy that the use of water soluble tris(2-carboxyethyl)phosphine for
the generation of the protected homocysteine free thiol from N,N0-
di(tert-butoxycarbonyl)-L-homocystine di(tert-butyl) ester not
only improved the reaction yield but also simplified the purifica-
tion process, as compared to the more commonly used tributyl-22
and triethylphoshines27 for the reduction of homocystine ana-
logues. Deprotection (TFA followed by TFA/H2O) of 20b gave
S-(3-deoxy-3-fluoroxylosyl)homo-cysteine 21a, which showed
two sets of peaks at the 19F NMR spectrum at d ꢁ200.55 (ddd,
J = 12.6, 28.0, 50.6 Hz, 0.6F; b) and ꢁ202.41 (ddd, J = 18.7, 25.4,
with thiolate derived from
aqueous trifluoroacetic acid gave the desired S-(3-deoxyribo-
syl)homocysteine 10 ( /b, 1:3; 25% from 1).
D/L
-homocysteine,32 and treatment with
a
Next, the 3-O-methyl xylo- (11) and ribofuranosyl analogues
(12) were prepared as potential LuxS inhibitors, because the 3-
methoxy group was expected to prevent the second enolization
step as well. We were also interested to see whether the LuxS ac-
tive site can tolerate both R and S epimers at the C3 position. Thus,
52.1 Hz, 0.4F;
a
). Treatment of thioether 20b with BCl3 at ꢁ50 °C
followed by quenching of the reaction mixture with MeOH re-
sulted in the removal of all protection groups and the formation
mesylation of l,2-O-isopropylidene-3-O-methyl-
2 afforded ester 5. Nucleophilic displacement with the thiolate
group of -homocysteine afforded thioether 8. Deprotection
(TFA/H2O) and purification by reversed-phase HPLC gave S-(3-O-
methylxylosyl)homocysteine 11 as a mixture of two anomers (
b, 1:1; 50%). S-(3-O-Methylribosyl)homocysteine 12 ( /b, 3:7)
a-D-xylofuranose
of methyl glycoside 21b as a 1:1.1 mixture of
as well as small amounts of 21a (7%). Displacement of the tosylate
group from 19a with -homocysteinate thiolate produced 20a
(9R/S, 1:1) and subsequent acid-catalyzed removal of the acetonide
group afforded 21a (9R/S, 1:1; /b, 2:3), which showed four sets of
peaks at the 19F NMR spectrum.
a/b anomers (29%)
D/L
D/L
a/
a
a
was similarly prepared from the corresponding ribose 3, except
that the C5–OH group was activated by conversion into 5-O-tosyl-
ate 6 (instead of 5-O-mesylate) because treatment of the latter
with Hcy/NaOH led to the hydrolysis of the mesylate and recovery
of alcohol 3.
Synthesis of 3-deoxy-3-fluoro-SRH 22 and 3-bromo-3-deoxy-
SRH analogue 23 have recently been described.35
2.2. Inhibition of LuxS
S-Xylosylhomocysteine (SXH, compound 16) was synthesized
from 1,2-isopropylidene-a-D
-xylofuranose 1333 (Scheme 2). The
The SRH analogues 10–12, 16, 21a, and 21b were evaluated as
potential inhibitors of Bacillus subtilis LuxS. Compound 10 acted as
a simple, competitive inhibitor of moderate potency, with a KI
diol of 13 was converted into cyclic sulfate 14 by treatment with
thionyl chloride in the presence of Et3N in DCM and followed by
NH2
NH2
O
O
OH
O
HO
O
HO
S
S
OH
S
OH
O
N
O
HO
H
OH
NH2
HO OH
HO OH
B
C
D
Figure 2. LuxS inhibitors.

S. F. Wnuk et al. / Bioorg. Med. Chem. 17 (2009) 6699–6706
6701
HO
RO
O
X
O
X
a
O
O
O
O
Y
Y
4 X = Y = H, R = Ms
5 X = OMe, Y = H, R = Ms
6 X = H, Y = OMe, R = Ts
1 X = Y = H,
2 X = OMe, Y = H
3 X = H, Y = OMe
b
NH2
NH2
O
X
O
HO
c
HO
S
S
X
OH
O
O
O
O
Y
Y
OH
7 X = Y = H,
8 X = OMe, Y = H
9 X = H, Y = OMe
10 X = Y = H,
11 X = OMe, Y = H
12 X = H, Y = OMe
Scheme 1. Reagents: (a) MsCl or TsCl/Et3N; (b) Hcy/NaOH/MeOH/H2O; (c) TFA/H2O.
O
O
HO
c
a, b
O
S
O
O
HO
O
O
O
O
O
13
14
NH2
NHBoc
O
O
HO
tBuO
OH
OH
d
S
S
O
O
O
HO
HO
O
16
15
Scheme 2. Reagents and conditions: (a) SOCl2/Et3N/DCM/ꢁ78 °C to rt; (b) RuCl3/NaIO4/CCl4/CH3CN/H2O/0 °C to rt; (c) (i) BocNH–CH(CH2CH2SH)–CO2t-Bu/BuLi/DMF/0 °C to
rt, (ii) THF/H2SO4/H2O/0 °C; (d) TFA/H2O/0 °C to rt.
O
RO
O
O
F
O
F
a, b
O
O
O
O
17
18 R = H
19a
19b
R = Ts
R = Ms
c or d
NHR'
NH2
O
X
HO
f or g
O
F
RO
S
OR
9
S
O
O
O
O
Y
OH
21a X = F, Y = H, R = H
21b
20a R = R' = H
20b
e
R = t-Bu, R' = Boc
X = F, Y = H, R = Me
22 X = H, Y = F, R = H
23
X = H, Y = Br, R = H
Scheme 3. Reagents: (a) (i) H5IO6/EtOAc, (ii) NaBH4/EtOH; (b) TsCl/C5H5N or MsCl/Et3N/CH2Cl2; (c) Hcy/NaOH/MeOH/H2O; (d) BocNH–CH(CHCHSH)–CO2t-Bu/LDA/DMF; (e)
TFA; (f) TFA/H2O; (g) BCl3/CH2Cl2/MeOH.
value of 55
6
l
M (Table 1 and Fig. 3). We also tested whether
spectrum of an overnight reaction mixture of 10 and LuxS failed
to show any signal in the d 180–230 region, indicating that no sig-
nificant amount of the 2-keto intermediate was generated under
the equilibrium conditions. Unfortunately, this result does not re-
veal whether removal of the C3–OH prevented the formation of
the 2-ketone intermediate or simply shifted the equilibrium posi-
tion between the substrate and the 2-ketone intermediate. Like
compound 10 can act as a substrate of LuxS. In particular, we were
interested in whether it could be converted into the corresponding
2-ketone intermediate in the LuxS reaction. Since the intermediate
cannot be converted into DPD product due to its lack of the 3-hy-
droxyl group, this would provide an alternative substrate to study
the detailed mechanism of the first catalytic step. The 13C NMR

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S. F. Wnuk et al. / Bioorg. Med. Chem. 17 (2009) 6699–6706
Table 1
0.05
Inhibition constants of C3-modified SRH analogues against Bacillus subtilis LuxSa
0.2 mM
0.6 mM
1.0 mM
2.0 mM
4.0 mM
KIꢀ
(lM)
0.04
0.03
0.02
0.01
0
Compound
KI (
l
M)
10
11
12
16
21a
21b
22
55
66 10
42
4.2 1.2
7.7 1.3
42
6
—
—
—
8
0.43 0.04
2.8 0.3
8.8 1.6
0.70 0.26
1.4 0.4
1
10.6 2.4
7.9 1.6
23
a
Inhibition constants for compounds 10–12 were determined using an epimeric
mixture (9R/S), while for the remaining compounds the pure 9S isomers were used.
0.0
0.50
1.0
Time (h)
1.5
2.0
Figure 4. Time-dependent inhibition of VhLuxS by compound 21a. Co-VhLuxS
(96 M) was preincubated with the indicated concentrations of compound 21a
(0.2ꢁ4.0 mM) at room temperature. At varying time points (0ꢁ2 h), 10- L aliquots
were withdrawn and rapidly diluted into a 1.0-mL solution containing 20 M SRH
and the rate of homocysteine formation was monitored with DTNB (absorbance
change at 412 nm).
l
the 3-deoxy analogue 10, the 3-methoxy analogues 11 and 12 also
acted as competitive inhibitors, with KI values of 66 and 42 lM,
respectively, against BsLuxS.
l
l
On the other hand, compounds 16, 21a, and 21b all exhibited
time-dependent inhibition of LuxS. Figure 4 shows the example
of inhibition of Vibrio harveyi LuxS (VhLuxS) by compound 21a.
Preincubation of LuxS with the inhibitor resulted in gradual loss
of LuxS activity with time; the activity decrease continued for
ꢂ60 min, when there was no further change in the residual activ-
ity. The compounds were also incubated with BsLuxS and the reac-
tion mixtures were analyzed by electrospray ionization mass
spectrometry to detect any covalent adduct between the enzyme
and the inhibitors. We only observed signals that correspond to
the unmodified BsLuxS protein, indicating that compounds 16,
21a, and 21b do not covalently modify the LuxS protein. Thus,
these compounds act as slow-binding inhibitors of LuxS and their
inhibition kinetics may be described by equation
the addition of SRH (KM = 2.4 lM) as the last component. The rate
constant k could not be determined accurately, because the rela-
*
tively fast EꢃI to EꢃI conversion makes the KI measurement less reli-
able. Among the three compounds, SXH (16) was most potent,
having KI and Kꢀ values of 4.2 and 0.43
l
M, respectively (Table 1).
M,
and
M), probably because the methyl group at the C1 position
creates steric clashes with the active-site residues.
I
The 3-fluorinated 21a had KI and KꢀI values of 7.7 and 2.8
l
respectively. The glycoside was least potent (KI = 42
lM
KꢀI = 8.8
l
The observed slow-binding behavior of compounds 16, 21a, and
21b is very similar to that of [3-F]SRH (22) and [3-Br]SRH (23),
both of which exhibited time-dependent inhibition of LuxS and
their time dependency was caused by enzyme-catalyzed release
of halide ions.35 Because compounds 16 and 21a are structurally
similar to 22 and 23, we hypothesize that compounds 16 and
21a may undergo similar structural changes in the LuxS active site
(e.g., the formation of 2-ketone intermediate-like species). The in-
verted stereochemistry at the C3 position in SXH would prevent
the conversion of the intermediate into products. Unfortunately,
all of our attempts to isolate/detect the altered inhibitor species
KI
E + I
E I
k
KI*
E I*
*
where EꢃI represents the initial enzyme–inhibitor complex, EꢃI is
the final, tighter enzyme–inhibitor complex, KI is the equilibrium
constant for the formation of the initial EꢃI complex, k is the rate
*
(I ) failed. Thus, we cannot rule out the possibility that the ob-
served slow-binding inhibition was caused by the conversion of
*
*
EꢃI into E ꢃI, where E is LuxS in an alternative conformation in-
duced by inhibitor binding. The origin of time dependency of gly-
coside 21b is less clear, as it cannot undergo ring opening and
therefore any catalyzed transformation.
*
*
constant for the conversion of the EꢃI complex to the tighter EꢃI
complex, and KꢀI represents the dissociation constant of the EꢃI
complex. The KI values were determined by initiating the enzymatic
reactions with the LuxS enzyme (no preincubation), whereas the KIꢀ
values were obtained by incubating the enzyme with the inhibitors
for 30 min at room temperature prior to initiating the reactions by
2.3. Conclusion
In this work, we have synthesized several SRH analogues that
lack an enolizable hydroxyl group at the carbon-3 position. Their
evaluation against LuxS showed that removal or methylation of
the C3–OH resulted in simple competitive inhibitors of moderate
potency, while inversion of the C3 stereochemistry or substitution
of fluorine for C3–OH resulted in slow-binding inhibition with the
improved potency.
[I], μM
1500
1000
1000
500
300
150
50
500
3. Experimental section
0
1H (Me4Si) NMR spectra were determined at 400 or 600 MHz,
13C (Me4Si) at 100.6 MHz and 19F (CFCl3) at 376.5 MHz with solu-
tion in CDCl3 unless otherwise noted. Mass spectra (MS) were
obtained by atmospheric pressure chemical ionization (APCI) and
HRMS by electron impact techniques unless otherwise noted.
Reagent grade chemicals were used and solvents were dried by
reflux over and distillation from CaH2 under an argon atmosphere
-500
-0.1 -0.05
0
0.05 0.1 0.15 0.2
1/[S] (μM-1)
Figure 3. Lineweaver–Burk plot showing competitive inhibition of 3-deoxy-SRH 10
against Co2+-substituted BsLuxS.

S. F. Wnuk et al. / Bioorg. Med. Chem. 17 (2009) 6699–6706
6703
except THF (K/benzophenone). TLC was performed on Merck kie-
selgel 60-F254 precoated aluminium sheets, and products were de-
tected by visualization with Ce(SO4)2/(NH4)6Mo7O24ꢃ4H2O/H2SO4/
H2O reagent or with 254 nm light. Merck kieselgel 60 (230–400
mesh) was used for column chromatography. HPLC purifications
were performed using XTerraÒ preparative RP18 OBDTM column
dissolved in EtOAC and washed with NaHCO3 and NaCl. The organ-
ic layer was evaporated and column chromatographed (hexane/
EtOAc, 1:1) to give 640 (117 mg, 72%) of sufficient purity to be used
in the next step: 1H NMR d 1.34 (s, 3H), 1.52 (s, 3H), 2.44 (s, 3H),
3.44 (s, 3H), 3.64 (dd, J = 4.2, 9.0 Hz, 1H), 4.07 (dt, J = 2.4, 9.1 Hz,
1H), 4.18 (dd, J = 3.3, 11.2 Hz, 1H), 4.27 (dd, J = 2.1, 11.2 Hz, 1H),
4.64 (t, J = 3.9 Hz, 1H), 5.66 (d, J = 3.6 Hz, 1H), 7.34 (d, J = 8.4 Hz,
2H), 7.79 (d, J = 8.4 Hz, 2H).
(5
l
m 19 ꢄ 150 mm) with program using CH3CN/H2O as a mobile
phase.
3.1. 3-Deoxy-1,2-O-isopropylidene-
a
-D
-erythro-pentofuranose
3.5. S-(3,5-Dideoxy-l,2-O-isopropylidene-a-D-erythro-
(1)
pentofuranos-5-yl)homocysteine (7)
H5IO6 (922 mg, 4.1 mmol) was added to a stirred solution of
diacetone 3-deoxyglucose30 (500 mg, 2.05 mmol) in dried EtOAc
(40 mL) at ambient temperature. A precipitate appeared within
the first 5 min and after 15 min, the somehow unstable 3-deoxy-
Compound 4 (45.6 mg, 0.18 mmol) was added to 1 M NaOH/
H2O (5 mL) and degassed with N2 for 30 min. -Homocysteine
D/L
(36.5 mg, 0.27 mmol) was then added, and the suspension was
heated to 60 °C. After 12 h, the reaction mixture was cooled to
room temperature, neutralized with dilute HCl to pH ꢂ 7 and
washed with EtOAc (3ꢄ). The water layer was evaporated and
purified by HPLC (25% CH3CN/H2O for 35 min at 2.5 mL/min;
tR = 12.6 min) to give 7 (16 mg, 31%): 1H NMR (D2O) d 1.39 (s,
3H), 1.55 (s, 3H), 1.83 (ddd, J = 4.7, 10.9, 14.0 Hz, 1H), 2.08–2.21
(m, 2H), 2.24 (dd, J = 4.3, 14.0, 1H), 2.72–2.78 (m, 2H), 2.81 (dd,
J = 6.8, 13.9 Hz, 1H), 2.93 (dd, J = 4.5, 13.8 Hz, 1H), 3.88 (t,
J = 6.3 Hz, 1H), 4.46 (dddd, J = 4.3, 4.5, 6.8, 10.9 Hz, 1H), 4.98 (t,
J = 4.1 Hz, 1H), 5.94 (d, J = 3.7 Hz, 1H); MS m/z 292 (100%, MH+).
1,2-O-isopropylidene-a-D-erythro-pentodialdo-1,4-furanose alde-
hyde can be detected on TLC and 1H NMR spectra (d 9.75 (d, J5–
4 = 4.8 Hz, ꢂ0.9, H5). The white precipitate was filtered off and
was washed with EtOAc (3 ꢄ 5 mL). The combined organic layer
was evaporated, and the residue was quickly dissolved in EtOH
(30 mL). Sodium borohydride (115 mg, 3.0 mmol) and water
(10 mL) was added until all of the sodium borohydride was dis-
solved. After 45 min, the reaction mixture was evaporated, and
the resulting residue was dissolved in EtOAc and washed with
NaHCO3. The organic layer was dried (Na2SO4), evaporated and
flash column chromatographed (40?60% EtOAc/hexane) to give
136 (217 mg, 61%).
3.6. S-(5-Deoxy-l,2-O-isopropylidene-3-O-methyl-a-D-
xylofuranos-5-yl)homocysteine (8)
3.2. 3-Deoxy-l,2-O-isopropylidene-5-O-methylsulfonyl-
erythro-pentofuranose (4)
a
-
D
-
Treatment of 5 (35 mg, 0.12 mmol) with D/L-homocysteine
(25 mg, 0.19 mmol), as described for 7, and purification by HPLC
(5% CH3CN/H2O for 45 min at 2.5 mL/min; tR = 30 min) gave 8
(11 mg, 28%): 1H NMR (D2O) d 1.36 (s, 3H), 1.53 (s, 3H), 1.90–
2.10 (m, 2H), 2.65–2.73 (m, 2H), 2.82 (dd, J = 7.5, 13.6 Hz, 1H),
2.87 (dd, J = 6.6, 13.6 Hz, 1H), 3.46 (s, 3H), 3.57 (‘t’, J = 6.9 Hz,
1H), 3.91 (d, J = 2.9 Hz, 1H), 4.38 (dt, J = 2.9, 7.2 Hz, 1H), 4.88 (d,
J = 3.9 Hz, 1H), 5.98 (d, J = 3.9 Hz, 1H); 13C NMR (D2O) d 24.9,
25.4, 27.9, 28.0, 28.42, 32.71, 54.54, 57.42, 79.8, 79.9, 81.0, 83.3,
104.3, 113.8, 174.0; HRMS (AP-ESI) m/z calcd for C13H24NO6S
[M+H]+ 322.1325, found 322.1321.
Triethylamine (125
lL, 90.9 mg, 0.9 mmol) and CH3SO2Cl
(35 L, 51.3 mg, 0.45 mmol) were added to a stirred solution of 1
l
(52 mg, 0.3 mmol) in anhydrous CH2Cl2 (10 mL) under N2 at 0 °C.
After 10 min, the reaction mixture was partitioned (NaHCO3/
H2O//CHCl3), and the organic layer was washed (brine), dried
(Na2SO4) and evaporated to yield 437 (73 mg, 97%) of sufficient pur-
ity to be used in the next step: 1H NMR d 1.34 (s, 3H), 1.53 (s, 3H),
1.80 (ddd, J = 4.7, 10.7, 13.5 Hz, 1H), 2.14 (dd, J = 4.2, 13.2 Hz, 1H),
3.08 (s, 3H), 4.27 (dd, J = 4.9, 11.4 Hz, 1H), 4.45 (dd, J = 2.8, 11.2 Hz,
1H), 4.44–4.53 (m, 1H), 4.79 (t, J = 4.2 Hz, 1H), 5.85 (d, J = 3.6 Hz,
1H).
3.7. S-(5-Deoxy-l,2-O-isopropylidene-3-O-methyl-a-D-
ribofuranos-5-yl)homocysteine (9)
3.3. l,2-O-Isopropylidene-3-O-methyl-5-O-methylsulfonyl-
a-
D-
Treatment of 6 (48 mg, 0.14 mmol) with D/L-homocysteine
xylofuranose (5)
(28 mg, 0.06 mmol), as described for 7, and purification by HPLC
(5% CH3CN/H2O for 45 min at 2.5 mL/min; tR = 35.0 min) gave 9
(13 mg, 30%): 1H NMR (D2O) d 1.40 (s, 3H), 1.56 (s, 3H), 2.03–
2.23 (m, 2H), 2.72 (t, J = 7.6 Hz, 2H), 2.79 (dd, J = 7.1, 14.3 Hz,
1H), 3.03 (dd, J = 3.2, 14.3 Hz, 1H), 3.48 (s, 3H), 3.80 (dd, J = 4.1,
9.0 Hz, 1H), 3.78–3.83 (m, 1H), 4.12 (ddd, J = 3.1, 7.1, 9.3 Hz, 1H),
4.97 (t, J = 3.9 Hz, 1H), 5.90 (d, J = 3.7, 1H); MS m/z 322 (100%,
MH+).
Treatment of 238 (50 mg, 0.25 mmol; prepared in 77% yield by
dehomologation31 of diacetone 3-O-methylglucose39 as described
for 1) with triethylamine (104
lL, 76 mg, 0.75 mmol) and
CH3SO2Cl (30
lL, 43 mg, 0.38 mmol), as described for 4, gave 5
(68 mg, 96%) of sufficient purity to be used in the next step: 1H
NMR d 1.35 (s, 3H), 1.55 (s, 3H), 3.08 (s, 3H), 3.42 (s, 3H), 3.81 (d,
J = 3.2 Hz, 1H), 4.37–4.51 (m, 3H), 4.62 (d, J = 3.8 Hz, 1H), 5.94 (d,
J = 3.7 Hz, 1H); HRMS (ESI) m/z calcd for C10H19O7S [M+H]+
283.0851, found 283.0844.
3.8. S-(3,5-Dideoxy-D-erythro-pentofuranos-5-yl)homocysteine
(10)
3.4. l,2-O-Isopropylidene-3-O-methyl-5-O-p-toluenesulfonyl-
a
-
A solution of 7 (9 mg, 0.03 mmol) in TFA/H2O (9:1, 3 mL) was
stirred for 45 min. at 0 °C (ice-bath). The reaction mixture was
evaporated and coevaporated [toluene (3ꢄ), CH3CN (3ꢄ)] and the
residue was purified by HPLC (5% CH3CN/H2O for 25 min at
D
-ribofuranose (6)
TsCl (93 mg, 0.49 mmol) was added to a stirred solution of
40(90 mg, 0.45 mmol; prepared in 67% yield by dehomologation31
3
2.5 mL/min) to yield 7 (7 mg, 90%,
a
/b, ꢂ1:3; tR = 12.6 min): 1H
of diacetone 3-O-methylallose40 as described for 1) in pyridine
(1 mL) at 0 °C. After 12 h, an additional TsCl (93 mg, 0.49 mmol)
was added and the stirring was continued for 8 h. Pyridine was
evaporated/coevaporated with toluene (3ꢄ), and the residue was
NMR (D2O) d 2.02 (ddd, J = 4.7, 9.4, 14.0 Hz, 1H), 2.06–2.25 (m,
3H), 2.69–2.78 (m, 2H), 2.78–2.91 (m, 2H), 3.87 (‘t’, J = 6.1 Hz,
1H), 4.26 (d, J = 4.6 Hz, 0.75H), 4.35 (‘dt’, J = 4.1, 6.8 Hz, 0.25H),
4.44–4.53 (m, 1H), 5.26 (s, 0.75H), 5.35 (d, J = 4.0 Hz, 0.25H); HRMS

6704
S. F. Wnuk et al. / Bioorg. Med. Chem. 17 (2009) 6699–6706
(LCT-ESI) m/z calcd for C9H17O5NSNa [M+Na]+ 274.0725, found
274.0725.
The mixture was stirred for 1 h at 0 °C and then partitioned be-
tween EtOAc (100 mL) and 5% NaHCO3 solution (40 mL). The or-
ganic layer was collected, washed with H2O (20 mL) and brine
(20 mL), dried over MgSO4 and concentrated under vacuum. The
crude product was purified by silica gel chromatography (EtOAc/
hexane, 2:3) to afford alcohol 15 (0.47 g, 67%) as a white solid:
1H NMR d 1.29 (s, 3H), 1.43 (s, 9H), 1.45 (s, 9H), 1.48 (s, 3H), 1.87
(m, 1H), 2.09 (m, 1H), 2.50 (d, J = 5.6 Hz, 1H), 2.56–2.69 (m, 2H),
2.81 (dd, J = 8.4, 13.2 Hz, 1H), 2.87 (dd, J = 5.2, 13.2 Hz, 1H), 4.21–
4.30 (m, 3H), 4.51 (d, J = 3.6 Hz, 1H), 5.10 (d, J = 5.6 Hz, 1H), 5.90
(d, J = 3.6 Hz, 1H); 13C NMR d 26.2, 26.7, 28.0, 28.3, 28.6, 29.2,
33.0, 53.2, 74.9, 79.7, 80.0, 82.4, 85.2, 104.7, 111.6, 155.5, 171.4;
HRMS (ESI) m/z calcd for C21H37NO8SNa+ [M+Na+] 486.2138, found
486.2141.
3.9. S-(5-Deoxy-3-O-methyl-D-xylofuranos-5-yl)homocysteine
(11)
Treatment of 8 (22 mg, 0.07 mmol) with TFA/H2O, as described
for 10, and purification by HPLC (5% CH3CN/H2O for 45 min at
2.5 mL/min; tR = 20 min) gave 11 (12 mg, 60%;
a
/b, ꢂ1:1): 1H
NMR (D2O) d 2.06–2.24 (m, 2H), 2.68–2.76 (m, 2H), 2.78–2.85
(m, 1.5H), 2.91 (dd, J = 5.9, 13.7 Hz, 0.5H), 3.41 (s, 1.5H), 3.44 (s,
1.5H), 3.80 (dd, J = 1.7, 4.7 Hz, 0.5H), 3.83–3.89 (m, 1H), 3.92 (dd,
J = 4.0, 4.8 Hz, 0.5H), 4.20 (s, 0.5H), 4.24 (t, J = 4.1 Hz, 0.5H), 4.38–
4.47 (m, 1H), 5.20 (s, 0.5H), 5.39 (d, J = 4.4 Hz, 0.5H); HRMS (LCT-
ESI) m/z calcd for C10H19O6NSNa [M+Na]+ 304.0831, found
304.0829.
3.13. S-(5-Deoxy-D-xylofuranos-5-yl)-L-homocysteine (16)
3.10. S-(5-Deoxy-3-O-methyl-D-ribofuranos-5-yl)homocysteine
(12)
Alcohol 15 (40 mg, 0.09 mmol) was treated with TFA containing
10% anisole and 10% water (5 mL) at room temperature for 6 h. The
volatile solvents were removed by evaporation, and the residue
was dissolved in H2O (3 mL) and washed with CH2Cl2 (3 ꢄ 2 mL).
The aqueous solution was lyophilized to give the desired SXH 16
(20 mg, 61%) as a trifluoroacetate salt: 1H NMR (250 MHz, D2O) d
1.85–2.10 (m, 2H), 2.35–2.70 (m, 4H), 3.60–4.40 (m, 3H), 4.96 (s,
1H), 5.20 (d, J = 4.3 Hz, 1H); HRMS (ESI) m/z calcd for C9H17NO6S-
Na+ [M+Na+] 290.0674, found 290.0684.
Treatment of 9 (10 mg, 0.03 mmol) with TFA/H2O, as described
for 10, and purification by HPLC (5% CH3CN/H2O for 45 min at
2.5 mL/min; tR = 20 min) gave 12 (5 mg, 59%;
a
/b, ꢂ3:7): 1H NMR
(D2O) d 2.07–2.26 (m, 2H), 2.70–2.79 (m, 2H), 2.79–2.99 (m, 2H),
3.44 (s, 0.9H), 3.47 (s, 2.1H), 3.78 (t, J = 5.0 Hz, 0.3H), 3.84 (t,
J = 5.5 Hz, 0.3H), 3.86 (dd, J = 5.5, 7.0 Hz, 0.7H), 3.94 (dd, J = 4.5,
6.5 Hz, 0.7H), 4.07–4.14 (m, 1H), 4.23 (dd, J = 1.3, 4.3 Hz, 0.7H),
4.29 (‘t’ J = 4.6 Hz, 0.3H) 5.26 (s, 0.7H), 5.37 (d, J = 4.0 Hz, 0.3H);
HRMS (LCT-ESI) m/z calcd for C10H19O6NSNa [M+Na]+ 304.0831,
found 304.0827.
3.14. 3-Deoxy-3-fluoro-l,2-O-isopropylidene-a-D-xylofuranose
(18)
Treatment of 1734 (75 mg, 0.29 mmol) with H5IO6 (77 mg,
0.34 mmol) and NaBH4 (16.5 mg, 0.43 mmol), as described for 1,
followed by flash column chromatography (EtOAc/hexane, 1:1)
3.11. 1,2-O-Isopropylidene-3,5-O-sulfonyl-a-D-xylofuranose
(14)
gave 18 (39 mg, 71%) with data as reported:41 19F NMR
ꢁ208.66 (ddd, J = 11.2, 30.1, 50.4 Hz).
d
To a stirred solution of diol 1333 (0.4 g, 2.11 mmol) in CH2Cl2
(12 mL) were added TEA (1.2 mL, 8.42 mmol) and SOCl2 (2 M/
DCM; 2.1 mL, 4.22 mmol) at ꢁ78 °C under argon atmosphere. The
mixture was allowed to warm to 0 °C and stirred until the reaction
was completed (1 h). The reaction mixture was diluted in DCM
(50 mL), washed with H2O (20 mL), dried over MgSO4, and concen-
trated to dryness. The residue was dissolved in CCl4 (6 mL) and
CH3CN (6 mL). To the resulting solution was added H2O (9 mL) fol-
lowed by RuCl3 hydrate (15 mg) and NaIO4 (0.9 g, 4.22 mmol) at
0 °C. The mixture was stirred for 2 h, diluted in EtOAc (50 mL),
washed with H2O (20 mL), dried over MgSO4, and concentrated
in reduced pressure. The crude product was purified by silica gel
chromatography (EtOAc/hexane, 1:2) to afford the cyclic sulfate
14 (0.39 g, 74%) as a white solid: 1H NMR d 1.33 (s, 3H), 1.49 (s,
3H), 4.24 (m, 1H), 4.70 (d, J = 3.6 Hz, 1H), 4.81 (d, J = 12.8 Hz, 1H),
4.95 (dd, J = 2.0, 12.8 Hz, 1H), 5.16 (d, J = 2.0 Hz, 1H), 6.03 (d,
J = 4.0 Hz, 1H); 13C NMR d 26.1, 26.5, 69.3, 72.4, 82.8, 86.5, 104.7,
113.1; HRMS (ESI) m/z calcd for C8H12O7SNa+ [M+Na+] 275.0201,
found 275.0212.
3.15. 3-Deoxy-3-fluoro-l,2-O-isopropylidene-5-O-p-toluenesul-
fonyl- -xylofuranose (19a)
a
-D
Treatment of 18 (66 mg, 0.34 mmol) with TsCl (130 mg,
0.68 mmol) in pyridine (1 mL), as described for 6, followed by flash
column chromatography (EtOAc/hexane, 25:75) gave 19a42
(72 mg, 62%): 1H NMR d 1.32 (s, 3H), 1.48 (s, 3H), 2.47 (s, 3H),
4.17–4.28 (m, 2H), 4.41 (dtd, J = 2.2, 6.3, 28.2 Hz, 1H), 4.67 (dd,
J = 3.8, 10.6 Hz, 1H), 4.96 (dd, J = 2.2, 50.1 Hz, 1H), 5.93 (d,
J = 3.6 Hz, 1H), 7.38 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H); 19F
NMR d ꢁ208.85 (ddd, J = 10.1, 27.7, 49.7 Hz).
3.16. 3-Deoxy-3-fluoro-1,2-O-isopropylidene-5-O-methanesul-
fonyl-a-D-xylofuranose (19b)
Et3N (0.115 mL, 84 mg, 0.83 mmol) and MsCl (0.032 mL, 47 mg,
0.41 mmol) were added dropwise to a stirred solution of 18
(53 mg, 0.27 mmol) in anhydrous CH2Cl2 (5 mL) at ambient tem-
perature. After 15 min, the reaction mixture was quenched with
saturated NaHCO3/H2O, and extracted with CHCl3. The combined
organic layer was washed with brine, dried (Na2SO4) and was
evaporated and column chromatographed (20?40% EtOAc/hex-
ane) to give 19b (54 mg, 75%) as a white solid: 1H NMR d 1.36 (s,
3H), 1.52 (s, 3H), 3.10 (s, 3H), 4.42–4.48 (m, 2H), 4.52 (dddd,
J = 2.3, 5.5, 7.1, 28.5 Hz, 1H), 4.74 (dd, J = 3.7, 10.8 Hz, 1H), 5.04
(dd, J = 1.8, 50.0 Hz, 1H), 6.03 (d, J = 3.7 Hz, 1H); 13C NMR d 26.2,
26.7, 37.6, 65.9 (d, J = 11.0 Hz) 77.4 (d, J = 18.7 Hz), 82.4 (d,
3.12. N-tert-Butoxycarbonyl-S-(5-deoxy-1,2-O-isopropylidene-
a-
D-xylofuranos-5-yl)-L-homocysteine tert-Butyl ester (15)
To solution of BocNH–CH(CH2CH2SH)–CO2tBu (1.1 g,
a
3.77 mmol) in DMF (15 mL) was added BuLi (2.5 M/hexane;
0.9 mL, 2.26 mmol) at 0 °C under argon atmosphere. After stirring
for 5 min, a solution of cyclic sulfate 14 (0.38 g, 1.51 mmol) dis-
solved in DMF (10 mL) was added. The mixture was stirred for
4 h, diluted with EtOAc (75 mL), washed with H2O (25 mL) and
brine (25 mL). The organic layer was dried over MgSO4 and concen-
trated to dryness. The residue was dissolved in THF (3 mL) and
J = 31.7 Hz), 93.7 (d, J = 184.8 Hz), 105.1, 112.8; 19F NMR
ꢁ208.7 (ddd, J = 10.6, 27.7, 50.2 Hz); MS m/z 271 (MH+).
d
concentrated H2SO4 (2 lL) and H2O (2 lL) were added at 0 °C.

S. F. Wnuk et al. / Bioorg. Med. Chem. 17 (2009) 6699–6706
6705
3.17. S-(3,5-Dideoxy-3-fluoro-l,2-O-isopropylidene-
xylofuranos-5-yl)homocysteine [20a(9R/S)]
a
-
D
-
tR = 16.5 min) gave 21a (3 mg, 55%; 9R/S, ꢂ1:1,
a
/b, ꢂ2:3): 19F
NMR (D2O) d ꢁ202.41 (ddd, J = 18.7, 25.4, 52.1 Hz, 0.2F;
a, 21a-
9S), ꢁ202.38 (ddd, J = 18.7, 25.4, 52.1 Hz, 0.2F;
a, 21a-9R),
A solution of 19a (18.5 mg, 0.05 mmol) in 2 M NaOH/H2O–EtOH
solution (4 mL, 1:1) was degassed with N2 for 30 min. -Homo-
ꢁ200.55 (ddd, J = 12.6, 28.0, 50.6 Hz, 0.3F; b, 21a-9S), ꢁ200.52
(ddd, J = 12.6, 28.0, 50.6 Hz, 0.3F; b, 21a-9R); 1H NMR spectra as re-
ported below for 21a-9S; MS (APCI) m/z 270 (MH+); HRMS (LCT-
ESI) m/z calcd for C9H16FNO5S [M+H]+ 270.0811; found 270.0798.
D/L
cysteine (11 mg, 0.08 mmol) was added, and the solution was
heated to 60 °C. After 36 h, the reaction mixture was cooled to
room temperature, neutralized with concd HCl and washed with
EtOAc (3ꢄ). The water layer was evaporated and purified by HPLC
(5% CH3CN/H2O for 60 min at 2.5 mL/min; tR = 30 min) to give 20a
(5 mg, 32%; 9R/S, ꢂ1:1): 1H NMR (D2O) d 1.37 (s, 3H), 1.52 (s, 3H),
2.05–2.26 (m, 2H), 2.72–2.78 (m, 2H), 2.86–2.97 (m, 2H), 3.83 (dd,
J = 5.7, 7.0 Hz, 1H), 4.45 (dtd, J = 2.0, 6.6, 28.7 Hz, 1H), 4.94 (dd,
J = 3.9, 10.8 Hz, 1H), 5.12 (dd, J = 2.0, 49.1 Hz, 1H), 6.10 (d,
J = 4.0 Hz, 1H); 19F NMR (D2O) d ꢁ208.45 (ddd, J = 10.7, 28.9,
49.1 Hz, 0.5F; 20a-9S), ꢁ208.43 (ddd, J = 10.7, 28.9, 49.1 Hz, 0.5F;
20a-9R); MS (APCI) m/z 310 (MH+).
3.20. S-(3,5-Dideoxy-3-fluoro-D-xylofuranos-5-yl)-L-
homocysteine (21a)
Compound 20b (17 mg, 0.037 mmol) was dissolved in TFA
(1 mL) and the resulting mixture was stirred at ambient tempera-
ture for 4 h and was evaporated and coevaporated with toluene.
The crude mixture [19F NMR (D2O) d ꢁ208.45 (0.82F, 20a-9S),
ꢁ202.41 (0.07F; , 21a-9S), ꢁ200.55 (0.11F; b, 21a-9S)] was trea-
a
ted (2 h) with TFA/H2O (9:1, 2 mL), as described for 10, and purified
on HPLC (preparative RP-C18 column, 5% CH3CN/H2O for 45 min at
3.18. N-(tert-Butoxycarbonyl)-S-(3,5-dideoxy-3-fluoro-1,2-O-
2.5 mL/min; tR = 16.5 min) to give 21a (4.7 mg, 46% overall; a/b,
isopropylidene-
a
-D
-xylofuranos-5-yl)-
L
-homocysteine tert-
ꢂ2:3): 1H NMR (D2O) d 2.10–2.35 (m, 2H), 2.72–2.82 (m, 2H),
2.82–3.01 (m, 2H), 4.01 (‘t’ , J = 6.1 Hz, 1H), 4.33 (d, J = 12.6 Hz,
0.6H), 4.40 (ddd, J = 2.4, 4.3, 8.6 Hz, 0.4H), 4.39–4.55 (m, 1H),
5.03 (dd, J = 3.3, 50.6 Hz, 0.6H), 5.10 (ddd, J = 2.5, 3.6, 52.1 Hz,
0.4H), 5.30 (s, 0.6H), 5.53 (dd, J = 1.0, 4.3 Hz, 0.4H); 19F NMR
Butyl ester (20b)
Step a: H2O (0.24 mL) and tris(2-carboxyethyl)phosphine
hydrochloride (88 mg, 0.31 mmol) were added to a stirred solution
of N,N0-di(tert-butoxycarbonyl)-
L-homocystine di(tert-butyl) ester
(D2O) d ꢁ202.41 (ddd, J = 18.7, 25.4, 52.1 Hz, 0.4F;
a), ꢁ200.55
(160 mg, 0.28 mmol) in anhydrous DMF (2.4 mL) at ambient tem-
perature under Ar atmosphere. After 32 h, the reaction mixture
[TLC (EtOAc/hexane, 2:8) showed conversion of disulfide (Rf 0.55)
into thiol (Rf 0.65)] was partitioned between EtOAc, and saturated
NaHCO3/H2O. After separation, the aqueous layer was extracted
with EtOAc, and the combined organic layer was washed with
brine, dried (Na2SO4), and concentrated to give N-tert-butoxycar-
(ddd, J = 12.6, 28.0, 50.6 Hz, 0.6F; b); MS (APCI) m/z 270 (MH+).
HRMS (TOF MS-ESI) m/z calcd for C9H16FNNaO5S [M+Na]+
292.0631; found 292.0611.
3.21. S-(3,5-Dideoxy-3-fluoro-1-O-methyl-D-xylofuranos-5-yl)-
L-homocysteine (21b)
bonyl-
L
-homocysteine tert-butyl ester22 (159 mg, 99%) as colorless
BCl3 solution (1.0 M/CH2Cl2; 0.2 mL) was added dropwise to a
oil of sufficient purity to be directly used in next step. Step b: A
freshly prepared homocysteine (80 mg, 0.275 mmol) was dissolved
in anhydrous DMF (0.5 mL), and was stirred under a vigorous
stream of argon for 10 min at 0 °C (ice-bath). Next, a solution of
stirred solution of 20b (25 mg, 0.054 mmol) in CH2Cl2 (3 mL) at
ꢁ50 °C, under Ar atmosphere. The reaction mixture was stirred
at same temperature for 20 min, and reaction was quenched with
MeOH (2 mL). The volatiles were evaporated and resulting crude
product was purified on RP-HPLC (as described for 21a) to give
LDA (138 lL, 2.0 M/THF–heptane) was added dropwise and after
an additional 10 min 19b (31 mg, 0.115 mmol) in anhydrous
DMF (0.5 mL) was added via syringe. After 15 min, ice-bath was re-
moved and the reaction mixture was stirred for 28 h at ambient
temperature (TLC showed consumption of 19b). Ice-cold saturated
NH4Cl/H2O was added and the resulting suspension was diluted
with EtOAc. The organic layer was separated and the aqueous layer
was extracted with EtOAc. The combined organic layer was washed
with brine, dried (Na2SO4) and was evaporated to give 94 mg of
yellowish oily residue. This crude product was column chromato-
graphed on silica gel (hexane/EtOAc, 4:1) to give 20b as an color-
less oil (49 mg, 92%): 1H NMR d 1.33 (s, 3H), 1.45 (s, 9H), 1.48 (s,
9H), 1.51 (s, 3H), 1.84–1.95 (m, 1H), 2.05–2.16 (m, 1H), 2.63 (t,
J = 7.7 Hz, 2H), 2.78 (dd, J = 8.4, 13.7 Hz, 1H), 2.86 (ddd, J = 1.4,
6.2, 13.7 Hz, 1H), 4.23–4.32 (m, 1H), 4.31 (dm, J = 28.2 Hz, 1H),
4.70 (dd, J = 3.8, 10.7 Hz, 1H), 4.98 (dd, J = 1.9, 49.9 Hz, 1H), 5.12
(br. d, J = 6.7 Hz, 1H), 5.96 (d, J = 3.9 Hz, 1H); 13C NMR d 26.2,
26.7, 28.0, 28.3, 28.6, 28.7, 33.2, 53.3, 79.8, 80.1 (d, J = 19.4 Hz),
82.2, 82.41 (d, J = 32.7 Hz), 93.78 (d, J = 185.0 Hz), 104.8, 112.2,
155.3, 171.3; 19F NMR d ꢁ208.16 (ddd, J = 10.4, 28.4, 49.8 Hz);
HRMS (AP-ESI) m/z calcd for C21H37FNO7S [M+H]+ 466.2269; found
466.2265.
21a (1 mg, 7%; a/b 1:3; tR = 16.5 min), and 21b (4.5 mg, 29%; a/b
45:55; tR = 24.5 min). Compound 21b had: 1H NMR (D2O) d 2.07–
2.28 (m, 2H), 2.76 (q, J = 7.1 Hz, 2H), 2.80–3.00 (m, 2H), 3.43 (s,
0.55H), 3.46 (s, 0.45H), 3.89 (‘t’, J = 6.1 Hz, 1H), 4.30–4.75 (m,
1H), 4.38 (d, J = 12.2 Hz, 0.55H), 4.47 (ddd, J = 2.9, 4.5, 23.2 Hz,
0.45H), 4.97 (s, 0.55H), 5.05 (dd, J = 4.0, 51.2 Hz, 0.55H), 5.09
(ddd, J = 3.0, 4.6, 53.5 Hz, 0.45H), 5.12 (d, J = 4.6 Hz, 0.45H); 13C
NMR (D2O) d 27.3, 27.4, 29.2, 29.3, 30.2, 30.2, 53.6, 55.4, 55.6,
75.6 (d, J = 27.1 Hz), 76.9 (d, J = 19.7 Hz), 77.3 (d, J = 27.1 Hz), 81.2
(d, J = 19.5 Hz), 94.8 (d, J = 185.2 Hz), 96.9 (d, J = 184.2 Hz), 102.1
(d, J = 6.0 Hz), 108.7, 173.9; 19F NMR (D2O) d ꢁ201.21 (dt,
J = 22.8, 53.6 Hz, 0.45F;
a
), ꢁ199.51 (ddd, J = 11.9, 27.1, 50.8 Hz,
0.55F; b); MS (AP-CI) m/z 284 (MH)+. HRMS (TOF MS-ESI) m/z calcd
for C10H18FNNaO5S [M+Na]+ 306.0787; found 306.0775.
3.22. LuxS assay
Inhibition assays were performed in a buffer containing 50 mM
HEPES (pH 7.0), 150 mM NaCl, 150
zoic acid) (DTNB) and various concentrations of SRH (0–55
and inhibitors (0–1 mM). The reactions were initiated by the addi-
tion of Co-BsLuxS (final concentration 0.4–0.5 M) and monitored
continuously at 412 nm (
= 14150 Mꢁ1 cmꢁ1) in a Perkin–Elmer
l
M 5,50-dithio-bis-(2-nitroben-
lM)
l
3.19. S-(3,5-Dideoxy-3-fluoro-
D
-xylofuranos-5-yl)homocysteine
e
[21a(9R/S)]
k25 UV–vis spectrophotometer at room temperature (23 °C). The
initial rates recorded from the early regions of the progress curves
were fitted into the Lineweaver–Burk equation 1/V = K0M/(kcat
[E]0) ꢄ 1/[S] + 1/(kcat [E]0) and the Michaelis–Menten equation
V = kcat [E]0 [S]/(K0M + [S]) using KaleidaGraph 3.5 to determine
Treatment of 20a (6 mg, 0.02 mmol; 9R/S, ꢂ1:1) with TFA/H2O
(9:1, 5 mL), as described for 10, and purification by HPLC (prepara-
tive RP-C18 column, 5% CH3CN/H2O for 45 min at 2.5 mL/min;

6706
S. F. Wnuk et al. / Bioorg. Med. Chem. 17 (2009) 6699–6706
14. Frezza, M.; Balestrino, D.; Soulère, L.; Reverchon, S.; Queneau, Y.; Forestier, C.;
Doutheau, A. Eur. J. Org. Chem. 2006, 4731.
15. Frezza, M.; Soulere, L.; Balestrino, D.; Gohar, M.; Deshayes, C.; Queneau, Y.;
Forestier, C.; Doutheau, A. Bioorg. Med. Chem. Lett. 2007, 17, 1428.
16. Waters, C. M.; Bassler, B. L. Annu. Rev. Cell Dev. Biol. 2005, 21, 319.
17. Bassler, B. L.; Losick, R. Cell 2006, 125, 237.
the inhibition pattern. KI values were calculated from the equation
(K0M = KM ꢄ (1 + [I]/KI), where KM = 2.2
lM.
Acknowledgments
18. Camilli, A.; Bassler, B. L. Science 2006, 311, 1113.
19. Lebeer, S.; De Keersmaecker, S. C. J.; Verhoeven, T. L. A.; Fadda, A. A.; Marchal,
K.; Vanderleyden, J. J. Bacteriol. 2007, 189, 860.
20. Zhu, J.; Dizin, E.; Hu, X.; Wavreille, A.-S.; Park, J.; Pei, D. Biochemistry 2003, 42,
4717.
This work was supported by grants from the NIH (SC1CA138176
and AI062901). J.R. was sponsored by MBRS RISE program (NIH/
NIGMS; R25GM61347).
21. Rajan, R.; Zhu, J.; Hu, X.; Pei, D.; Bell, C. E. Biochemistry 2005, 44, 3745.
22. Zhu, J.; Hu, X.; Dizin, E.; Pei, D. J. Am. Chem. Soc. 2003, 125, 13379.
23. Zhu, J.; Patel, R.; Pei, D. Biochemistry 2004, 43, 10166.
24. Zhu, J.; Knottenbelt, S.; Kirk, M. L.; Pei, D. Biochemistry 2006, 45, 12195.
25. Turner, M. A.; Yang, X.; Yin, D.; Kuczera, K.; Borchardt, R. T.; Howell, P. L. Cell
Biochem. Biophys. 2000, 33, 101.
26. Wnuk, S. F. Mini-Rev. Med. Chem. 2001, 1, 307.
27. Alfaro, J. F.; Zhang, T.; Wynn, D. P.; Karschner, E. L.; Zhou, Z. S. Org. Lett. 2004, 6,
3043.
28. Shen, G.; Rajan, R.; Zhu, J.; Bell, C. E.; Pei, D. J. Med. Chem. 2006, 49, 3003.
29. Wnuk, S. F.; Lalama, J.; Garmendia, C. A.; Robert, J.; Zhu, J.; Pei, D. Bioorg. Med.
Chem. 2008, 16, 5090.
30. Robins, M. J.; Wilson, J. S.; Hansske, F. J. Am. Chem. Soc. 1983, 105, 4059.
31. Xie, M.; Berges, D. A.; Robins, M. J. J. Org. Chem. 1996, 61, 5178.
32. Miles, R. W.; Nielsen, L. P. C.; Ewing, G. J.; Yin, D.; Borchardt, R. T.; Robins, M. J. J.
Org. Chem. 2002, 67, 8258.
33. Moravcová, J.; Capková, J.; Stanek, J. Carbohydr. Res. 1994, 263, 61.
34. Tewson, T. J.; Welch, M. J. J. Org. Chem. 1978, 43, 1090.
35. Gopishetty, B.; Zhu, J.; Rajan, R.; Sobczak, A. J.; Wnuk, S. F.; Bell, C. E.; Pei, D. J.
Am. Chem. Soc. 2009, 131, 1243.
36. Shiozaki, M.; Deguchi, N.; Mochizuki, T.; Wakabayashi, T.; Ishikawa, T.;
Haruyama, H.; Kawai, Y.; Nishijima, M. Tetrahedron 1998, 54, 11861.
37. Bentley, N.; Dowdeswell, C. S.; Singh, G. Heterocycles 1995, 41, 2499.
38. Yadav, J. S.; Subba Reddy, B. V. Carbohydr. Res. 2000, 329, 885.
39. Yan, S.; Klemm, D. Tetrahedron 2002, 58, 10065.
References and notes
1. Lee, J. E.; Cornell, K. A.; Riscoe, M. K.; Howell, P. L. Structure 2001, 9, 941.
2. Singh, V.; Shi, W.; Almo, S. C.; Evans, G. B.; Furneaux, R. H.; Tyler, P. C.; Painter,
G. F.; Lenz, D. H.; Mee, S.; Zheng, R.; Schramm, V. L. Biochemistry 2006, 45,
12929.
3. Pei, D.; Zhu, J. Curr. Opin. Chem. Biol. 2004, 8, 492.
4. Schauder, S.; Shokat, K.; Surette, M. G.; Bassler, B. L. Mol. Microbiol. 2001, 41,
463.
5. Winzer, K.; Hardie, K. R.; Burgess, N.; Doherty, N.; Kirke, D.; Holden, M. T. G.;
Linforth, R.; Cornell, K. A.; Taylor, A. J.; Hill, P. J.; Williams, P. Microbiology 2002,
148, 909.
6. Miller, S. T.; Xavier, K. B.; Campagna, S. R.; Taga, M. E.; Semmelhack, M. F.;
Bassler, B. L.; Hughson, F. M. Mol. Cell 2004, 15, 677.
7. Chen, X.; Schauder, S.; Potier, N.; Van Dorsselaer, A.; Pelczer, I.; Bassler, B. L.;
Hughson, F. M. Nature 2002, 415, 545.
8. Meijler, M. M.; Hom, L. G.; Kaufmann, G. F.; McKenzie, K. M.; Sun, C.; Moss, J. A.;
Matsushita, M.; Janda, K. D. Angew. Chem., Int. Ed. 2004, 43, 2106.
9. Lowery, C. A.; Park, J.; Kaufmann, G. F.; Janda, K. D. J. Am. Chem. Soc. 2008, 130,
9200.
10. Semmelhack, M. F.; Campagna, S. R.; Federle, M. J.; Bassler, B. L. Org. Lett. 2005,
7, 569.
11. Semmelhack, M. F.; Campagna, S. R.; Hwa, C.; Federle, M. J.; Bassler, B. L. Org.
Lett. 2004, 6, 2635.
12. De Keersmaecker, S. C. J.; Varszegi, C.; van Boxel, N.; Habel, L. W.; Metzger, K.;
Daniels, R.; Marchal, K.; De Vos, D.; Vanderleyden, J. J. Biol. Chem. 2005, 280,
19563.
40. Haga, M.; Takano, M.; Tejima, S. Carbohydr. Res. 1972, 21, 440.
41. Jeong, L. S.; Lim, B. B.; Marquez, V. E. Carbohydr. Res. 1994, 262, 103.
42. Tsoukala, E.; Agelis, G.; Dolinsek, J.; Botic, T.; Cencic, A.; Komiotis, D. Bioorg.
Med. Chem. 2007, 15, 3241.
13. Frezza, M.; Soulere, L.; Queneau, Y.; Doutheau, A. Tetrahedron Lett. 2005, 46,
6495.