Inhibition of LuxS by S-ribosylhomocysteine analogues containing a [4-aza]ribose ring

Article abstract of DOI:10.1016/j.bmc.2011.07.043

LuxS (S-ribosylhomocysteinase) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a small signaling molecule that mediates interspecies bacteria

Full text of DOI:10.1016/j.bmc.2011.07.043

Bioorganic & Medicinal Chemistry 19 (2011) 5507–5519
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Bioorganic & Medicinal Chemistry
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Inhibition of LuxS by S-ribosylhomocysteine analogues containing
a [4-aza]ribose ring
Venkata L.A. Malladi a, Adam J. Sobczak a, , Tiffany M. Meyer b, Dehua Pei b, Stanislaw F. Wnuk a,
⇑
a Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA
b Department of Chemistry and Ohio State Biochemistry program, 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:
Available online 28 July 2011
LuxS (S-ribosylhomocysteinase) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine
(SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a small sig-
naling molecule that mediates interspecies bacterial communication called autoinducer 2 (AI-2). Inhibi-
tors of LuxS should interfere with bacterial interspecies communication and potentially provide a novel
class of antibacterial agents. In this work, SRH analogues containing substitution of a nitrogen atom for
the endocyclic oxygen as well as various deoxyriboses were synthesized and evaluated for LuxS inhibi-
Keywords:
Azahemiacetals
Azasugars
Homocysteine
LuxS
S-Ribosylhomocysteinase
tion. Two of the [4-aza]SRH analogues showed modest competitive inhibition (KI ꢀ40
lM), while most
of the others were inactive. One compound that contains a hemiaminal moiety exhibited time-dependent
inhibition, consistent with enzyme-catalyzed ring opening and conversion into a more potent species
(KI⁄ = 3.5
l
M). The structure–activity relationship of the designed inhibitors highlights the importance
of both the homocysteine and ribose moieties for high-affinity binding to LuxS active site.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
unstable DPD has been accomplished recently by the groups of Jan-
da13 and Semmelhack,14 which allowed the vital complexation
properties of DPD with borate15 to be studied and provided DPD
as a reliable standard for investigation of AI-2 regulated QS
processes.
Quorum sensing (QS) is a type of bacterial cell-to-cell commu-
nication mediated through the production, release and detection
of the small signaling molecules called autoinducers (AIs).1–3 Such
communication allows bacterial control of crucial functions in uni-
ted communities for enhancement of symbiosis, virulence, antibi-
otic production, biofilm formation, and many other processes.4,5
Hence, there have been great interests in the synthesis of small
molecules that can modulate QS pathways.6–8 S-Ribosylhomocy-
steinase (LuxS) is a key enzyme in the biosynthetic pathway of
type II autoinducer, which mediates the interspecies quorum sens-
ing among both Gram-positive and Gram-negative bacteria.
The biosynthesis of AI-2 starts with the dual substrate-specific
microbial enzyme 50-methylthioadenosine/AdoHcy nucleosidase
LuxS is a small metalloenzyme (157 amino acids in the Bacillus
subtilis enzyme) containing Fe2+ coordinated by His-54, His-58,
Cys-126, and a water molecule. The native enzyme is unstable un-
der aerobic conditions, but substitution of Co2+ for Fe2+ gives a
highly stable variant with essentially wild-type catalytic activ-
ity.16–18 In the proposed catalytic mechanism, LuxS catalyzes con-
secutive aldose–ketose (1a ? 1b) and ketose–ketose (1b ? 1c)
isomerization steps and then b-elimination of Hcy from a 3-keto
intermediate (1c ? 1d) to form DPD.11,19 LuxS-catalyzed cleavage
of the C5–S thioether bond in SRH is analogous to that of SAH
hydrolase, which effects cleavage of an equivalent thioether bond
in SAH by first oxidizing the C30 secondary alcohol into a ketone
with an NAD+ cofactor.20,21
(MTAN), which catalyzes the depurination of S-adenosyl-
cysteine (SAH), a byproduct of many S-adenosyl- -methionine-
dependent methyltransferases reactions, to form S-ribosyl- -homo-
cysteine (SRH, Fig. 1).9,10 SRH is subsequently converted to
L-homo-
L
L
Zhou and co-workers designed and synthesized two LuxS sub-
L
-homocysteine and 4,5-dihydroxy-2,3-pentadione (DPD) by the
strate analogues, the S-(anhydroribosyl)-L-homocysteine (2) and
LuxS enzyme.11 DPD undergoes spontaneous cyclization to 1e and
complexation with borate to form a furanosyl borate diester, which
acts as the AI-2 in some bacteria.11,12 Chemical synthesis of the
S-(homoribosyl)-L-cysteine compounds, which blocked initial and
final mechanistic steps, respectively (Fig. 2).22 Pei and co-workers
have prepared a series of stable analogues of the putative enedio-
late intermediate, some of which showed submicromolar inhibi-
tion of the enzyme (e.g., KI = 0.72 l
M for isostere 3).23 Zhang
⇑
Corresponding author. Tel.: +1 305 348 6195; fax: +1 305 348 3772.
et al. found that the brominated furanones 4 covalently modify
and inactivate LuxS.24 Recognizing structural similarities between
substrates of mammalian AdoHcy hydrolase and bacterial
E-mail address: wnuk@fiu.edu (S.F. Wnuk).
On a faculty leave from University of Life Sciences, Department of Chemistry,
Poznan, Poland
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.043

5508
V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
NH2
NH2
A
O
O
MTAN (Pfs Nucleosidase)
Ade
HO2C
S
HO2C
S
OH
HO OH
SRH
HO OH
SAH
LuxS
RS
RS
H
OH
RS
H
OH
OH
H
H
OH
H
H
H
H
H
H
H
H
H
H
O
O
HO
RSH
OH
OH
HO
OH
OH
O
H
(Hcy)
OH
1b
O
HO
O
1d
1c
1a
HO
OH
-
B(OH)4
HO
HO
B
OH
OH
CH3
O
O
H2O
OH
HO
HO
CH3
O
O
2 H2O
HO
O
1e
DPD
AI-2
Figure 1. Biosynthetic pathway to AI-2. Enzymatic conversion of SRH to DPD by LuxS.
2. Results and discussion
NH2
O
O
O
HO
OH
S
2.1. Chemistry
S
OH
OH
O
N
H
HO
Our first target was 1,4-dideoxy-[4-aza]SRH 12 lacking the hy-
droxyl group at C1 (Scheme 1). Compound 12 cannot undergo ring
opening (which will preclude the initial step of the LuxS-catalyzed
reaction) and may act as a competitive inhibitor of LuxS. Synthesis
of 12 started with the protected 1-amino-1,4-anhydro-1-deoxy-
OH
HO OH
NH2
NH2
2
3
Br
D
-ribitol 6, which was readily prepared from the commercially
O
HO
-lactone.37 However, attempted mesyla-
S
available -gulonic acid
D
c
O
O
tion of the N-benzyl protected 6 resulted in the formation of piper-
idine derivative 13 as a mixture of two diastereomers (ꢀ3:1).
Presumably, the mesylated pyrrolidine underwent a rearrange-
ment reaction into the piperidines through an aziridine intermedi-
ates.38,39 We found that replacement of the benzyl protecting
group at ring nitrogen of 6 with a Boc group suppressed the nucle-
ophilicity of the nitrogen and prevented ring expansion, allowing
the formation of stable 5-O-mesyl derivatives. Thus, silylation of
6 with TBDMSCl and subsequent hydrogenation (5% Pd/C) in the
presence of (Boc)2O39,40 yielded 8 (97% from 5). Desilylation of 8
(70%) followed by mesylation gave 10 as a stable compound
(96%). Displacement of the mesylate group with a thiolate, gener-
O
Br
X
OH
4
5 X = H, F, Br, OMe
Figure 2. LuxS inhibitors.
S-ribosylhomocysteine (SRH) hydrolase (LuxS enzyme), we de-
signed and synthesized SRH analogues with 6-(fluoro)vinyl moiety
in place of the C5 and sulfur atoms which acted as weak/moderate
inhibitors of LuxS enzyme.25 The SRH analogues 5 lacking enoliz-
able hydroxyl group at C3 were found to be competitive substrate
of LuxS.26,27 The time dependence inhibition with C3 halogenated
substrates was caused by enzyme-catalyzed elimination of halide
ions.27
Here, we report synthesis of [4-aza]SRH mimics in which the
furanose ring oxygen has been replaced by a nitrogen atom. The
resulting hemiaminals should have different stabilities28 relative
to the O,O-hemiacetals present in SRH and as a result different
rates of metabolic alteration. The higher basicity of the aza ana-
logues is expected to have different binding strengths and rates
for productions of the open chain aldehyde form–necessary for
the first isomerization to occur. Also, the aminosugars can be
protonated at physiological pH and the corresponding positive
charge may have an effect on binding to the enzymatic active site.
Azasugars29 have been found to be potent inhibitors of glycosi-
dases and glycosyltransferases30,31 and have been targeted as
transition-state models.32,33 The 40-azanucleosides34 function as
transition-state inhibitors of MTAN at the femtomolar level.35,36
ated by reduction of properly protected L
-homocystine19 with
water soluble tris(2-carboxyethyl)phosphine hydrochloride,26 gave
thioether 11 (86%). Treatment of 11 with TFA effected simulta-
neous removal of the N-Boc, acetonide and t-butyl ester protection
groups to give the desired [4-aza]SRH analogue 12 in good yields
(66%).
The second target was c-lactam 21, which contains an amide
carbonyl at C1 and nitrogen as a replacement of the ring oxygen
(Scheme 2). It is noteworthy that, as opposed to the [4-aza]SRH
analogue 12 (or 23), the lactam nitrogen cannot be protonated at
physiological pH. Selective oxidation of the 5-O-TBDMS-azasugar
7 at C1 with RuO2/NaIO4 under EtOAc/H2O biphasic conditions41
produced N-benzyl lactam 14a (65%) and a small amount (18%)
of the corresponding N-benzoylpyrrolidinone byproduct, resulted
from oxidation of the benzylic carbon of the N-protecting group.
Desilylation of 14a with TBAF, followed by mesylation and
displacement of the mesylate group with protected Hcy gave

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
5509
Boc
Boc
N
Bn
N
RO
N
RO
MsO
c
b
O
O
O
O
O
O
6 R = H
8 R = TBDMS
9 R = H
10
a
d
7 R = TBDMS
e
c
Boc
N
NHBoc
Bn
NH2
t-BuO
H
N
N
HO
S
f
S
O
O
O
O
Cl
O
O
OH OH
11
12
13
Scheme 1. Reagents and conditions: (a) TBDMSCl/imidazole/DMAP/CH2Cl2/rt; (b) H2/Pd-C/(Boc)2O/Et3N/EtOH/rt; (c) MsCl/Et3N/CH2Cl2/rt; (d) TBAF/THF/rt; (e)
BocNHCH(CH2CH2SH)CO2t-Bu/LDA/DMF; (f) (i) TFA, (ii) TFA/H2O.
R
N
R
N
R
N
MsO
R'O
TBDMSO
a
O
a
O
c
10
O
O
O
O
O
O
16 R = Bn
14a R = Bn, R' = TBDMS
14b R = Bn, R' = H
7 R = Bn
b
17 R = Boc
8 R = Boc
15a R = Boc, R' = TBDMS
15b R = Boc, R' = H
b
d
R
N
NHBoc
R
N
NH2
t-BuO
HO
S
e
O
S
O
O
O
O
O
OH OH
20 R = Bn
21 R = H
18 R = Bn
19a R = H
f
19b R = Boc
g
Boc
N
H
NHBoc
NH2
5
7
6
10
t-BuO
HO
N
1
9
e
S
S
OH
OH
8
4
2
3
O
O
O
O
OH OH
23
22
Scheme 2. Reagents and conditions: (a) NaIO4/RuO2 x H2O/EtOAc/H2O/rt; (b) TBAF/THF/rt; (c) MsCl/Et3N/CH2Cl2/rt; (d) BocNHCH(CH2CH2SH)CO2t-Bu/LDA/DMF; (e) (i) TFA,
(ii) TFA/H2O; (f) (Boc)2O/Et3N/CH2Cl2/rt; (g) LiEt3BH/THF/ꢁ78 °C.
thioether 18. Treatment of 18 with TFA removed all of the acid-la-
bile protection groups to yield N-benzyl protected [4-aza]SRH lac-
tam 20 (48%). However, all attempts to remove the N-benzyl group
from 18 or 20 (to yield 21) were unsuccessful [e.g., H2/Pd-C or
Pd(OH)2-C, Na/NH3(liq.), BCl3]. Our attempt to mesylate the
N-Boc protected 15b (prepared by RuO2-catalyzed oxidation of 8
and desilylation of the resulting 15a) failed to produce 17, yielding
only the starting material 15b. Fortunately, oxidation of the 5-O-
mesyl and N-Boc protected pyrrolidine 10 with RuO2/NaIO4
afforded 17 efficiently (95%). Coupling of 17 with homocysteinate

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V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
afforded thioether 19a with concomitant loss of Boc group at ring
nitrogen. Subsequent deprotection with TFA followed by TFA/H2O
gave [4-aza]SRH lactam 21 (58%).
deprotected hemiaminal 25a as a mixture of anomers susceptible
to dehydration at pH higher than 7 to form imine 25c.43 Subse-
quent treatment of 25a with O-benzylhydroxylamine gave ex-
pected oxime 26 as the only product. The formation of oxime 26
indicates that azasugar 25a exists in equilibrium with the open
aldehyde form (25b) and that the equilibrium could be shifted by
subsequent transformations.
Our next target was hemiaminal 23. Since only the open alde-
hyde form of SRH is catalytically active, we were interested in
the effect of the nitrogen substitution on the ring opening. The
existence of azahemiacetals in equilibrium with dehydrated form
(imine) as well as with open aldehyde and dimeric forms was re-
ported for 4-azapentofuranoses.42,43 It is noteworthy that sugar
N,O-acetals were found to be stable enough to undergo coupling
with nucleoside bases,41 or transformation to proline.44 Although
direct reduction of lactam 21 (or 19a) with LiBEt3H failed to yield
hemiaminal 23 (or 22), the protection of the ring nitrogen with a
Boc group facilitated the reduction reaction.44 Thus, treatment of
19a with (Boc)2O/DMAP gave N-Boc protected lactam 19b (93%)
which upon treatment with LiBEt3H produced hemiaminal 22
(92%) as a mixture of two anomers. Deprotection of 22 with TFA
followed by TFA/H2O gave desired [4-aza]SRH (N,O-acetal) ana-
2,3,4-Trideoxy-[4-aza]SRH 38 lacking the enolizable hydroxyl
groups at C2 and C3 was next prepared to examine the importance
of C2 and C3-OH groups for LuxS binding and catalysis. The key
starting material (S)-5-(bromomethyl)-2-pyrrolidone (27) was
conveniently prepared from
placement of the bromide in 27 with the
L
-pyroglutamic acid45 (Scheme 4). Dis-
-homocysteinate affor-
L
ded thioether 33 (79%), which was deprotected with TFA
quantitatively to give 2,3-dideoxy-4-azaSRH analogue 34 as a tri-
fluoroacetate. Displacement with the unprotected D/L-homocyste-
ine produced racemic 36 (75%) as a sodium salt, which upon
treatment with TFA was also converted to its trifluoroacetate salt.
As expected, 1H NMR spectrum of 34 showed only one set of peaks
which are present in the spectrum of racemic 36. Treatment of 33
with (Boc)2O/DMAP gave the N-Boc protected lactam 35, which
was reduced with LiBEt3H to give hemiaminal 37. Subsequent
deprotection with TFA produced 38.
The 5-S-alkyl-2,3-dideoxy-[4-aza]SRH (e.g., 28/29) and the 5-S-
alkyl-[4-aza]SRH analogues with different length of the alkylthio
chain were also prepared.46 These cyclic azahemiacetals and their
ancestor lactams were found to modulate Pseudomonas aeruginosa
logue 23 (72%) as a mixture of a/b anomers. Interestingly, no free
aldehyde or imine proton peaks were visible on 1H NMR spectra.
Compound 23 is stable when kept at 4 °C but decomposes slowly
in solution at ambient temperature especially at basic pH.
To determine whether the cyclic [4-aza]SRH exists in equilib-
rium with the open chain aldehyde form, we carried out a limited
model study. Thus, N-Boc protected lactam 15a44 was reduced
with LiBEt3H to afford protected hemiaminal 24a (Scheme 3). Desi-
lylation with TBAF yielded 24b, which was treated with TFA to give
Boc
N
H
N
Boc
N
RO
H
O
RO
a
c
O
OH
OH
O
O
OH OH
O
O
24a R = TBDMS
24b R = H
25a
15a R = TBDMS
b
(
H2O)
OBn
HO
N
HO
O
NH2
NH2
HO
N
d
H
H
OH OH
OH OH
OH OH
26
25b
25c
Scheme 3. Reagents and conditions: (a) LiEt3BH/THF/-78 °C; (b) TBAF/THF/rt; (c) (i) TFA/0 °C, (ii) TFA/H2O/0 °C; (d) BnONH2/pyr/rt.
NHR'
R
N
H
N
H
N
Br
a-d
e or f
R''OOC
S
O
O
RS
OH
(Ref. 46)
28 R = C3H7
29 R =C6H13
33 R = H, R' = Boc, R'' = t-Bu
34 R = R' = R'' = H
27
d
b
35 R = R' = Boc, R'' = t-Bu
36 R = R' = R'' = H (9R/S)
g
(
H2O)
N
c
OBn
NHR'
R
N
NH2
N
C3H7S
RS
R''OOC
S
OH
H
30 R = C3H7
31 R = C6H13
32
37 R = R' = Boc, R'' = t-Bu
38 R = R' = R'' = H
d
Scheme 4. Reagents and conditions: (a) RSH/NaH/DMF; (b) (Boc)2O/DMAP/CH2Cl2; (c) LiEt3BH/THF/CH2Cl2/ꢁ78 °C; (d) TFA; (e) BocNHCH(CH2CH2SH)CO2t-Bu/LDA/DMF; (f)
-Hcy/NaH/DMF; (g) BnONH2/pyr
D/L

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
5511
QS.46 The alkylthiomethyl azahemiacetal 28/29 existed in solution
O
N
O
N
R'
N
MsO
as an equilibrium mixture of anomers along with the open chain
RO
RO
aldehydes [5–25%, 1H NMR (d 8.90), 13C NMR (d 180.8)] and the
d
b
1
corresponding imines 30/31 [3–30%; H NMR (d 7.63), 13C NMR
(d 167.0)].46 Treatment of 28 with O-benzylhydroxylamine also
O
O
O
O
O
O
produced the expected oxime 32,46 as observed for 25a.
To explore the possibility of the LuxS-mediated addition of
water across carbon–nitrogen double bond, we synthesized an
imine-type analogue 43 (Scheme 5). The precursor 1-methylimino-
cyclitol 39 was prepared by the Moriarty rearrangement47 of the
exo-imino to endo-iminocyclitol, which involves inversion at C4
47
45 R = TBDMS
46 R = H
7 R = TBDMS, R' = Bn
44 R = TBDMS, R' = H
c
a
e
O
N
NHBoc
of the
was mesylated at the primary alcohol to give 40 (85%), which
was coupled with protected -Hcy to give 41 (85%, Scheme 5).
L-lyxo sugar to give the D
-ribo azasugar. The imine 3947
O
N
NH2
t-BuO
HO
S
f
S
L
O
O
Treatment of 41 with TFA for a short time gave only isopropylidene
protected 42. We found that the protons at the C1-methyl group
are exchangeable with deuterium within few hours when com-
pound 42 is dissolved in D2O. Treatment of 42 with aqueous TFA
(9:1) yielded fully deprotected 43 in quantitative yield. Protons
at C1-methyl group of 43 were also exchangeable with deuterium.
These exchange indicate that 1,4-ketimine-SRH analogue 43 might
be expected to undergo enzyme-catalyzed hydrolysis to generate a
[4-aza]SRH analogue with a methyl ketone rather than an aldehyde
at C1. This change might affect the regioselectivity and rate of the
first isomerization step in the LuxS-catalyzed reaction. We also
proved that the methyl group protons in 39 are not susceptible
to exchange even if 39 was dissolved in D2O for several hours.
Additionally, we noticed that observed low rate of exchange in
39 (relatively to 42 and 43) can be enhanced exclusively in the
presence of acid or amino acid (TFA and glycine were used,
respectively). Attempted, one-step deprotection of 41 with BCl3
led to a partial loss of chirality at C9 giving 43 as a mixture of
diastereomers (2:3).
O
48
O
OH OH
49
Scheme 6. Reagents and conditions: (a) H2/Pd-C/EtOH/rt; (b) SeO2/H2O2/Me2CO/
ꢁ4 °C; (c) TBAF/THF; (d) MsCl/Et3N/CH2Cl2/ꢁ4 °C; (e) BocNHCH(CH2CH2SH)CO2t-
Bu/LDA/DMF/ꢁ20 °C; (f) (i) TFA/ꢁ4 °C, (ii) TFA/H2O/ꢁ4 °C.
protected Hcy afforded a nitrone-SRH derivative 48 (43%). Depro-
tection of 48 with TFA produced unstable nitrone derivative 49
(40%).
2.2. Inhibition of LuxS
Compounds 12, 20, 21, 23, 28, 36, 38, and 43 were evaluated as
potential inhibitors of Co(II)-substituted B. subtilis LuxS. Compound
12 inhibited LuxS in a concentration-dependent manner that is
consistent with competitive inhibition (Fig. 3a), with a KI value of
Our attempt to prepare the imine derivative of [4-aza]SRH was
unsuccessful. Thus, debenzylation of 7 and treatment of the
aminoribitol 44 with N-chlorosuccinimide (NCS) followed by
dehydrochlorination of the resulting N-chloroamine with lithium
tetramethylpiperidine gave unstable aldoimine of type 39 (H
instead of CH3), as reported.48 However, couplings of such
aldoimine with Hcy to give the imine SRH analogue failed. Acid-
catalyzed hydrolysis of such imine analogue could serve as an
alternative route to 4-azaSRH 23. Also, enzyme-mediated proton-
ation of the imine nitrogen atom and the addition of water might
generate 23 and/or new species with an ‘amino group’ within the
enzyme active site.
A nitrone analogue of SRH 49 was also targeted. Since nitrones
are more electrophilic than imines such analogue might act as irre-
versible inhibitors by forming a covalent adduct(s) with enzyme. It
is noteworthy that nitrones are overall neutral and cannot be pro-
tonated at physiological pH. Thus, treatment of the aminoribitol 44
with SeO2/H2O2 gave nitrone 4549 (74%; Scheme 6). Desilylation
and subsequent mesylation gave 47 (56%). Coupling of 47 with
48
l
M (Table 1). Similarly, lactam 21 also behaved as a competitive
inhibitor with KI value of 37
lM. As expected, the lactam 20, which
contains a bulky benzyl group at the ring nitrogen, was found to be
inactive, likely due to steric reasons. Compounds 36 and 38 were
both inactive toward LuxS, highlighting the importance of the ri-
bose hydroxyl groups for enzyme binding. The proposed mecha-
nism predicts that the C2 and C3 hydroxyl groups directly
coordinate with the catalytic metal ion during different catalytic
steps (Fig. 1). The lack of activity of compound 43, which contains
a methyl group instead of a hydroxyl group at the C1 position, may
be caused by both loss of favorable interactions with the OH group
and the bulky size of the methyl group. Collectively, these results
suggest that proper interactions between the ribose ring and the
enzyme active site critically contribute to the formation of a pro-
ductive E–S complex and subsequent catalysis.
Unlike the other analogues described above, inhibition of LuxS
by the hemiaminal-containing analogue 23 was time dependent
(Fig. 3b). Its inhibition kinetics can be described by the slow-bind-
KI
E + I
E I
ing equation
where KI is the equilibrium constant
k
KI*
NHR'
E I*
RO
N
N
b
for the formation of the initial EꢂI complex, k is the rate constant
for the conversion of the EꢂI complex to the tighter EꢂI⁄ complex,
and KI⁄ represents the dissociation constant of the EꢂI⁄ complex.
To assess its potency, different concentrations of compound 23
were preincubated with LuxS for 30 min at 4 °C and the residual
enzymatic activity was measured. Plot of the residual activity
against the inhibitor concentration resulted in an IC50 value of
ROOC
S
R''O
OR''
O
O
41 R = t-Bu, R' = Boc, R'',R'' = CMe2
42 R = R' = H, R'',R'' = CMe2
43 R = R' = R'' = H
39 R = H
c
d
a
40 R = Ms
60 l lM was estimated (Table 1).
M, from which a KI⁄ value of 3.5
Unfortunately, the complex inhibition kinetics precluded an accu-
Scheme 5. Reagents and conditions: (a) MsCl/Et3N/CH2Cl2; (b) BocNHCH(CH2-
CH2SH)CO2t-Bu/LDA/DMF; (c) TFA/rt; (d) TFA/H2O.
rate determination of the KI value. While further work is clearly

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V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
necessary to determine the exact mechanism of inhibition by 23,
we propose a working hypothesis to explain the observed time
dependence (Fig. 4). Since compounds 12 and 21, which are struc-
turally similar to 23, did not exhibit time-dependent inhibition and
our model study shows that the hemiaminal 25a exists in equilib-
rium with the free aldehyde form (25b), we propose that hemiami-
nal 23 may undergo ring opening to form aldehyde 23a. Due to its
structural similarity to catalytic intermediate 1a (Fig. 1), 23a may
undergo the aldose-ketose isomerization reaction to form 2-ketone
23b, which presumably binds to the LuxS active site with higher
affinity than the ribose analogue 23. This behavior is very similar
to that of a class of halogenated SRH analogues (e.g., [3-F]SRH
and [3-Br]SRH), which have been shown to undergo LuxS-
catalyzed ring opening to form open-chain species that are more
potent LuxS inhibitors than the initial ribose analogues.27
The remaining compound 28 and its ancestor lactam showed no
significant inhibition of LuxS.
3. Conclusions
We have synthesized [4-aza] S-ribosylhomocysteine analogues
in which the furanose ring oxygen has been substituted by a
nitrogen atom having also the additional modifications at
anomeric carbon. Coupling of the protected 4-amino-5-O-meth-
anesulfonyl-4-deoxy-D-ribono-1,4-lactam with homocysteinate
and subsequent deprotection with TFA gave [4-aza]SRH with an
amide carbonyl at anomeric carbon. Reduction of the N-Boc
protected lactam with LiBEt3H and acid catalyzed deprotection
produced [4-aza]SRH hemiaminal analogue. The [4-aza]SRH
analogue lacking the hydroxyl group at C1 and the corresponding
lactam derivative showed modest competitive inhibition (KI
ꢀ40–50
l
M) of LuxS. The hemiaminal analogue exhibited time-
dependent inhibition (KI⁄ = 3.5
lM), consistent with the enzyme-
catalyzed ring opening and generation of 2- and/or 3-ketone
intermediates, which presumably bind to the LuxS active site with
higher affinity than the ribose natural substrate.
4. Experimental procedure
The 1H (400 or 600 MHz) and 13C (100 MHz) NMR spectra were
determined with solutions in CDCl3 unless otherwise noted. Mass
spectra (MS) were obtained with atmospheric pressure chemical
ionization (APCI) technique and HRMS in AP-ESI or TOF-ESI mode.
TLC was performed with Merck kieselgel 60-F254 sheets products
were detected with 254 nm light or by visualization with
Ce(SO4)2/(NH4)6Mo7O24ꢂ4H2O/H2SO4/H2O reagent. Merck kieselgel
60 (230–400 mesh) was used for column chromatography. HPLC
purifications were performed using XTerraÒ preparative RP18
Figure 3. Inhibition of LuxS by compounds 12 and 23. (A) Reaction progress curves
in the presence of increasing concentrations of inhibitor 12 (0, 200, 400, 800, 1600,
and 3200 lM). The last two curves were control reactions in the absence of LuxS.
Inset, plot of remaining LuxS activity as a function of inhibitor 12 concentration. (B)
Reaction progress curves of LuxS in the presence of increasing concentrations of
OBDTM column (5
l
m 19 ꢃ 150 mm) with gradient program using
inhibitor 23 (0, 20, 40, and 50
lM) (without preincubation). Inset, plot of remaining
CH3CN/H2O as a mobile phase. Reagent grade chemicals were used,
and solvents were dried by reflux over and distillation from CaH2
(except for THF/potassium) under argon.
LuxS activity as
preincubation).
a function of inhibitor 12 concentration (after 30 min
4.1. 1-Amino-1,4-anhydro-N-benzyl-5-O-tert-
Table 1
Inhibition constants of [4-aza]SRH analogues against B. subtilis LuxS
butyldimethylsilyl-1-deoxy-2,3-O-isopropylidene-D-ribitol (7)
Compound
KI or KI⁄
(lM)
To a stirred solution of 637 (150 mg, 0.57 mmol) in anhydrous
CH2Cl2 (5 mL) at rt under Ar atmosphere were added DMAP
(7 mg, 0.05 mmol) and imidazole (93 mg, 1.36 mmol) followed by
TBDMSCl (103 mg, 0.68 mmol). The mixture was then stirred for
12
21
23
48
37
3.5
RS
RS
RS
RS
HO
H
H
N
NH2 HO
NH2
NH2
OH
23b
Figure 4. Proposed mechanism for the time-dependent inhibition of LuxS by [4-aza]SRH hemiaminal 23.
?
O
OH
M2+
O
O
OH
23a
OH
OH
23c
OH
23
OH
M2+
M2+
M2+

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
5513
10 h and partitioned (CH2Cl2//ꢂNaHCO3/H2O). The organic layer
4.5. S-(1-Amino-1,4-anhydro-N-tert-butoxycarbonyl-1,5-
dideoxy-2,3-O-isopropylidene- -ribitol-5-yl)-N-tert-
butoxycarbonyl- -homocysteine tert-butyl ester (11)
was washed (brine), dried (MgSO4) and evaporated. The residue
was purified by column chromatography (15% EtOAc/hexane) to
give 750 (204 mg, 98%) as a colorless oil. 1H NMR d ꢁ0.13 (s, 3,
CH3), 0.00 (s, 3, CH3), 0.83 (s, 9, t-Bu), 1.26 (s, 3, CH3), 1.49 (s, 3,
CH3), 2.65 (dd, J = 2.7, 10.3 Hz, 1, H1), 2.94 (‘q’, J = , 2.2 Hz, 1, H4),
3.04 (dd, J = 5.5, 10.3 Hz, 1, H10), 3.57 (dd, J = 4.1, 10.6 Hz, 1, H5),
3.64 (d, J = 13.4 Hz, 1, Bn), 3.71 (dd, J = 4.3, 10.6 Hz, 1, H50), 3.94
(d, J = 13.4 Hz, 1, Bn), 4.49 (dd, J = 2.0, 6.5 Hz, 1, H3), 4.58 (‘dt’,
J = 2.7, 6.2 Hz, 1, H2), 7.13–7.23 (m, 5, Bn); 13C NMR d ꢁ5.5,
(CH3), ꢁ5.4, (CH3), 18.2 (t-Bu), 25.2 (CMe2), 25.7 (CH3), 25.9
(CH3), 27.2 (CMe2), 56.9 (Bn), 59.3 (C1), 63.2 (C5), 68.9 (C4), 79.4
(C2), 83.3 (C3), 111.9 (CMe2), 128.2, 128.5, 126.8, 139.4 (Bn); MS
(APCI) m/z 378 (100, MH+).
D
L
Procedure B. Step a. H2O (0.4 mL) and tris(2-carboxy-
ethyl)phosphine hydrochloride (140 mg, 0.5 mmol) were added
to a stirred solution of N,N’-di(tert-butoxycarbonyl)-L-homocys-
tine di(tert-butyl) ester19 (250 mg, 0.4 mmol) in anhydrous
DMF (4 mL) at ambient temperature under Ar atmosphere. After
24 h, the reaction mixture [TLC (EtOAc/hexane, 2:8) showed con-
version of disulfide (Rf 0.55) into thiol (Rf 0.65)] was partitioned
between EtOAc and saturated NaHCO3/H2O. Aqueous layer was
extracted with EtOAc, and the combined organic layer was
washed with brine, dried (MgSO4) and concentrated to give N-
tert-butoxycarbonyl-
99%) as colorless oil of sufficient purity to be directly used in
next step. Step b. LDA (85 L, 2.0 M/THF and heptane,
L
-homocysteine tert-butyl ester19 (240 mg,
4.2. 1-Amino-1,4-anhydro-N-tert-butoxycarbonyl-5-O-tert-
butyldimethylsilyl-1-deoxy-2,3-O-isopropylidene-
D
-ribitol (8)
l
0.17 mmol) was added dropwise (10 min) to a stirred solution
of freshly prepared thiol from step a (200 mg, 0.6 mmol) in
anhydrous DMF (5 mL) under a vigorous stream of argon at
0 °C (ice-bath). After an additional 10 min, 10 (100 mg,
0.2 mmol) in anhydrous DMF (5 mL) was added via a syringe.
After 15 min ice-bath was removed and the reaction mixture
was stirred for 24 h at ambient temperature. 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 (brine), dried (MgSO4) and was evaporated. The res-
idue was column chromatographed (40 ? 50% EtOAc/hexane) to
give 11 (130 mg, 86%) as a mixture of rotamers (ꢀ1:1): 1H NMR
d 1.29 (s, 3, CH3), 1.41 (s, 12H, t-Bu, CH3), 1.44 (s, 18H, t-Bu),
1.79–1.92 (m, 2, H8,8’), 2.39–2.80 (m, 4, H5,5’,7,7’), 3.37 (dd,
J = 4.2, 11.7 Hz, 0.5, H1), 3.43 (dd, J = 4.5, 11.7 Hz, 0.5, H1), 3.70
(d, J = 12.6 Hz, 0.5, H1’), 3.84 (d, J = 12.8 Hz, 0.5, H1’), 3.99–4.05
(m, 0.5, H4), 4.11–4.17 (m, 0.5, H4), 4.18–4.29 (m, 1, H9), 4.56
(dd, J = 5.6, 10.4 Hz, 0.5, H3), 4.60 (dd, J = 5.6, 10.4 Hz, 0.5, H3),
4.69 (d, J = 4.8 Hz, 0.5, H2), 4.71 (d, J = 4.8 Hz, 0.5, H2), 5.06 (br
d, J = 7.3 Hz, 0.5, NH), 5.29 (br d, J = 6.1 Hz, 0.5, NH); 13C NMR
d 25.0 (CMe2), 26.9 (CMe2), 27.9 (C7), 28.0 (t-Bu), 28.3 (t-Bu),
28.4 (t-Bu), 32.2 (C5), 32.6 (C8), 32.9 (C8), 33.2 (C5), 51.7 (C1),
52.4 (C1), 53.5 (C9), 62.8 (C4), 63.2 (C4), 78.5 (C2), 78.5 (t-Bu),
79.2 (C2), 79.2 (t-Bu), 80.0 (t-Bu), 80.1 (t-Bu), 83.5 (C3), 84.1
(C3), 111.9 (CMe2), 154.1 (CO), 154.9 (CO), 155.4 (CO), 171.2
(C10), 171.5 (C10); MS (APCI) m/z 547 (100, MH+); HRMS
A solution of 7 (145 mg, 0.38 mmol), triethylamine (0.105 mL,
0.76 mmol), di-tert-butyldicarbonate (126 mg, 0.57 mmol) and
Pd/C (5%, 300 mg) in ethanol (6 mL) was stirred under an atmo-
sphere of hydrogen at room temperature for 6 h. The reaction mix-
ture was filtered through Celite to remove the catalyst. The Celite
was washed with ethanol (5 mL) and washings and the filtrate
were combined and evaporated. The residue was partitioned
(EtOAc//ꢂNaHCO3/H2O). The organic layer was washed (brine),
dried (MgSO4) and evaporated. The residue was column chromato-
graphed (20 ? 30% EtOAc/hexane) to give 8 (147 mg, 99%) with
spectral properties as reported.44
4.3. 1-Amino-1,4-anhydro-N-tert-butoxycarbonyl-1-deoxy-2,3-
O-isopropylidene-D-ribitol (9)
TBAF (1 M/THF; 0.25 mL, 0.25 mmol) was added to a stirred
solution of 8 (66 mg, 0.17 mmol) in THF (5 mL) at ambient temper-
ature. After stirring for 30 min, the reaction mixture was parti-
tioned (EtOAc//ꢂNaHCO3/H2O). The organic layer was washed
(brine), dried (MgSO4) and evaporated. The residue was column
chromatographed (50 ? 60% EtOAc/hexane) to give 9 (32 mg,
70%) with spectral properties as reported.51
4.4. 1-Amino-1,4-anhydro-N-tert-butoxycarbonyl-1-deoxy-2,3-
O-isopropylidene-5-O-methanesulfonyl-D-ribitol (10)
Procedure A. Triethylamine (99
l
L, 0.71 mmol) and MsCl
(AP-ESI) m/z calcd for C
26H47N2O8S [MH]+ 547.3048; found
(25 L, 0.33 mmol) were added dropwise to stirred solution of 9
l
547.3042.
(60 mg, 0.22 mmole) in anhydrous CH2Cl2 (6 mL) at 0 °C (ice-bath).
After 5 min, ice-bath was removed and the reaction mixture was
allowed to stir at ambient temperature for 30 min. The reaction
mixture was quenched with saturated NaHCO3/H2O and was ex-
tracted with CH2Cl2. The organic layer was washed (brine), dried
(MgSO4) and evaporated to give 10 (73 mg, 96%) as a mixture
(ꢀ3:2) of two rotamers of sufficient purity to be directly used in
next step: 1H NMR d 1.28 (s, 3, CH3), 1.42 (s, 12H, t-Bu, CH3),
2.96 (s, 1.2, Ms), 2.98 (s, 1.8, Ms), 3.39 (dd, J = 4.8, 12.5 Hz, 0.4,
H1), 3.46 (dd, J = 4.8, 12.5 Hz, 0.6, H1), 3.69 (d, J = 12.5 Hz, 0.6,
H1’), 3.82 (d, J = 12.5 Hz, 0.4, H1’), 4.10–4.14 (m, 0.4, H4), 4.22–
4.30 (m, 0.6, H4), 4.22–4.29 (m, 1.4, H5,5’), 4.45 (dd, J = 4.1,
10.1 Hz, 0.6, H5), 4.65 (‘d’, J = 5.9 Hz, 1, H3); 4.72 (‘t’, J = 5.3 Hz, 1,
H2); 13C NMR (major rotamer) d 24.9 (CMe2), 26.9 (CMe2), 29.6
(t-Bu), 37.1 (Ms), 52.5 (C1), 62.4 (C4), 68.9 (C5), 79.2 (C2), 80.4
(t-Bu), 81.7 (C3), 112.1 (CMe2),154.2 (NHCO); 13C NMR (minor rot-
amer) d 24.9 (CMe2), 26.9 (CMe2), 29.6 (t-Bu), 37.5 (Ms), 53.1 (C1),
62.6 (C4), 68.6 (C5), 78.5 (C2), 80.6 (t-Bu), 82.5 (C3), 112.1 (CMe2),
153.6 (NHCO); MS (APCI) m/z 352 (10, MH+), 252 (100, [MH2-
Boc]+).
4.6. S-(1-Amino-1,4-anhydro-1,5-dideoxy-D-ribitol-5-yl)-L-
homocysteine (12)
Procedure C. Step a. Compound 11 (39 mg, 0.07 mmol) dis-
solved in TFA (4.0 mL) was stirred at 0 °C for 3 h. Volatiles were
coevaporated with toluene to give an oily residue, which was used
directly in next step. Step b. Product from step a was treated with
TFA/H2O (9:1, 4.0 mL) for 1 h at 0 °C. Volatiles were evaporated and
the crude product was purified on RP-HPLC (5% CH3CN/H2O at
2.5 mL/min; tR = 12 min) to give 12 (12 mg, 66%) as a colorless
oil: 1H NMR (D2O) d 2.10–2.25 (m, 2, H8,8’), 2.71–2.81 (m, 2,
H7,7’), 2.87 (dd, J = 10.4, 14.5 Hz, 1, H5), 3.15 (dd, J = 4.3, 14.5 Hz,
1, H5’), 3.34 (dd, J = 1.9, 13.0 Hz, 1, H1), 3.53 (dd, J = 4.0, 13.0 Hz,
1, H1’), 3.69 (ddd, J = 4.3, 8.7, 10.4 Hz, 1, H4), 3.86 (t, J = 6.2 Hz, 1,
H9), 4.14 (dd, J = 4.1, 8.7 Hz, 1, H3), 4.39 (ddd, J = 1.9, 4.0, 4.1 Hz,
1, H2); 13C NMR (D2O) d 26.8 (C8), 30.1 (C7), 30.6 (C5), 49.2 (C1),
53.6 (C9), 59.5 (C4), 69.5 (C2), 74.3 (C3), 174.0 (C10); MS m/z
251 (100, MH+); HRMS (TOF MS-ESI) m/z calcd for C9H19N2O4S
[M+H]+ 251.1060; found 251.1063

5514
V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
4.7. 1-Benzyl-5-chloro-3,4-dihydroxy-3,4-O-
isopropylidenepiperidine [13(3S,4S,5R/S)]
4.08 (d, J = 15.2 Hz, 1, Bn), 4.64 (d, J = 5.6, Hz, 1, H3), 4.77 (d,
J = 5.2 Hz, 1, H2), 5.04 (d, J = 15.2 Hz, 1, Bn), 7.29–7.37 (m, 5, Bn);
MS (APCI) m/z 278 (100, MH+).
Treatment of
6 (50 mg, 0.19 mmol) with MsCl (21.9 lL,
0.28 mmol) by Procedure A [column chromatography (20 ? 30%
EtOAc/hexane)] gave 13 (25 mg, 46%) as a 3:1 mixture of diastereo-
mers: The major isomer had: 1H NMR d 1.30 (s, 3, CH3), 1.50 (s, 3,
CH3), 2.13–2.19 (m, 1, H2), 2.49 (dd, J = 3.7, 13.3 Hz, 1, H6), 2.87
(dq, J = 1.7, 12.0 Hz, 1, H2’), 3.05 (‘dt’, J = 2.1, 13.3 Hz, 1, H6’), 3.50
(d, J = 13.4 Hz, 1, Bn), 3.61 (d, J = 13.4 Hz, 1, Bn), 3.93–3.99 (m, 2,
H3,5), 4.19 (dd, J = 3.7, 7.5 Hz, 1, H4), 7.20–7.25 (m, 5, Bn); 13C
NMR d 26.2 (CMe2), 28.4 (CMe2), 53.5 (C6), 56.5 (C2), 58.5 (C5),
61.5 (Bn), 73.8 (C4), 79.5 (C3), 112.8 (CMe2), 127.3, 128.4, 128.9,
4.10. 4-Amino-N-tert-butoxycarbonyl-5-O-tert-butyldimethyl-
silyl-4-deoxy-2,3-O-isopropylidene-
D-ribono-1,4-lactam (15a)
Oxidation of (90 mg, 0.23 mmol) with NaIO4 (126 mg,
8
0.7 mmol) and RuO2ꢂxH2O (8 mg, 0.05 mmol) by Procedure D [col-
umn chromatography (50 ? 60% EtOAc/hexane)] gave 15a (56 mg,
60%) as a colorless oil with data as reported.44
4.11. 4-Amino-N-tert-butoxycarbonyl-4-deoxy-2,3-O-
isopropylidene-D-ribono-1,4-lactam (15b)
137.2 (Bn); MS m/z 282 (100, MH+[35Cl]), 284 (40, MH+
[
37Cl]);
+
HRMS (TOF MS-ESI) m/z calcd for
C
15H2035ClNO2 [M+H]
282.1255; found 282.1259.
Desilylation of 15a (50 mg, 0.12 mmol) with TBAF (1 M/THF,
0.14 mL, 0.14 mmol), as described for 14b, [column chromatogra-
phy (70 ? 80% EtOAc/hexane)] gave 15b (27 mg, 77%) as a color-
less oil: 1H NMR d 1.36 (s, 3, CH3), 1.46 (s, 12, t-Bu, CH3), 3.88
(‘dt’, J = 3.9, 5.3 Hz, 1, H4), 4.03 (dd, J = 4.1, 11.4 Hz, 1, H5), 4.19
(dd, J = 3.8, 11.4 Hz, 1, H5’), 4.57 (dd, J = 5.3, 5.7 Hz, 1, H3), 4.63
(d, J = 5.7 Hz, 1, H2); 13C NMR d 25.7 (CMe2), 27.0 (CMe2), 27.6 (t-
Bu), 56.7 (C4), 66.6 (C5), 76.7 (C2), 77.0 (C3), 83.3 (t-Bu), 112.4
(CMe2), 153.0 (CO), 173.7 (C1); MS (APCI) m/z 288 (100, MH+);
HRMS (AP-ESI) m/z calcd for C13H22NO6 [MH]+ 288.1442; found
288.1437.
4.8. 4-Amino-N-benzyl-5-O-tert-butyldimethylsilyl-4-deoxy-
2,3-O-isopropylidene- -ribono-1,4-lactam (14a)
D
Procedure D. RuO2ꢂꢃH2O (4.3 mg, 0.032 mmol) was added to a
stirred solution of NaIO4 (83 mg, 0.39 mmol) in H2O (1 mL) at
ambient temperature. After 5 min,
a solution of 7 (50 mg,
0.13 mmol) in EtOAc (1 mL) was added dropwise and the reaction
mixture was continued to stir for 12 h. H2O (10 mL) and EtOAc
(10 mL) were added and the separated aqueous layer was further-
more extracted with EtOAc (2 ꢃ 10 mL). The combined organic lay-
ers were washed (brine), dried (MgSO4) and evaporated. The
residue was column chromatographed (50 ? 60% EtOAc/hexane)
to give 14a (33 mg, 65%) and N-benzoylated byproduct (10 mg,
18%). Compound 14a had: 1H NMR d 0.01 (s, 6, 2ꢃ CH3), 0.84 (s,
9, t-Bu), 1.35 (s, 3, CH3), 1.42 (s, 3, CH3), 3.51 (t, J = 2.1 Hz, 1, H4),
3.62 (dd, J = 2.0, 10.9 Hz, 1, H5), 3.69 (dd, J = 2.3, 10.9 Hz, 1, H5’),
3.93 (d, J = 15.2 Hz, 1, Bn), 4.50 (d, J = 5.6 Hz, 1, H3), 4.69 (d,
J = 5.6 Hz, 1, H2), 5.00 (d, J = 15.2 Hz, 1, Bn), 7.22–7.32 (m, 5, Bn);
13C NMR d ꢁ5.7, (CH3), ꢁ5.6, (CH3), 18.1 (t-Bu), 25.8 (t-Bu), 25.8
(CMe2), 27.3 (CMe2), 44.2 (Bn), 60.2 (C5), 62.0 (C4), 76.7 (C3),
78.0 (C2), 111.7 (CMe2), 127.7, 128.2, 128.7, 135.6 (Bn), 171.9
(C1); MS (APCI) m/z 392 (100, MH+). The 4-amino-N-benzoyl-5-
4.12. 4-Amino-N-benzyl-4-deoxy-2,3-O-isopropylidene-5-O-
methanesulfonyl-D-ribono-1,4-lactam (16)
Mesylation of 14b (67 mg, 0.24 mmol) with MsCl (27
l
L,
0.36 mmol) by Procedure A gave 16 (83 mg, 97%) as a colorless
oil of sufficient purity to be used directly in next step: 1H NMR
d 1.30 (s, 3, CH3), 1.36 (s, 3, CH3), 3.07 (s, 3, Ms), 3.65 (t,
J = 2.7 Hz, 1, H4), 4.06 (d, J = 15.2 Hz, 1, Bn), 4.16 (dd, J = 2.2,
11.0 Hz, 1, H5), 4.22 (dd, J = 3.0, 11.0 Hz, 1, H5’), 4.51 (d,
J = 5.6 Hz, 1, H3), 4.69 (d, J = 5.6 Hz, 1, H2), 4.92 (d, J = 15.2 Hz, 1,
Bn), 7.18–7.30 (m, 5, Bn); MS (APCI) m/z 388 (100, [MH+MeOH]+),
356 (40, MH+).
O-tert-butyldimethylsilyl-4-deoxy-2,3-O-isopropylidene-D-ribono-
1,4-lactam byproduct had: 1H NMR d 0.01 (s, 3, CH3), 0.05 (s, 3,
CH3), 0.87 (s, 9, t-Bu), 1.40 (s, 3, CH3), 1.51 (s, 3, CH3), 3.83 (dd,
J = 1.5, 10.7 Hz, 1, H5), 4.19 (dd, J = 2.2, 10.7 Hz, 1, H5’), 4.58 (‘t’,
J = 1.8 Hz, 1, H4), 4.65 (d, J = 5.5 Hz, 1, H3), 4.76 (d, J = 5.5 Hz, 1,
H2), 7.38–7.43 (m, 2, Bn), 7.51 (‘dt’, J = 1.3, 6.7 Hz, 2, Bn), 7.54–
7.58 (m, 1, Bn); 13C NMR d ꢁ5.7, (CH3), ꢁ5.6, (CH3), 18.2 (t-Bu),
25.3 (CMe2), 25.8 (t-Bu), 27.2 (CMe2), 61.7 (C4), 62.1 (C5), 76.3
(C3), 78.7 (C2), 112.1 (CMe2), 127.9, 128.7, 132.2, 134.1 (Bn)
170.6 (CO), 171.7 (C1); MS (APCI) m/z 406 (100, MH+).
4.13. 4-Amino-N-tert-butoxycarbonyl-5-O-methanesulfonyl-4-
deoxy-2,3-O-isopropylidene-D-ribono-1,4-lactam (17)
Oxidation of 10 (80 mg, 0.32 mmol) with NaIO4 (172 mg,
0.96 mmol) and RuO2ꢂxH2O (8.5 mg, 0.064 mmol) by procedure D
[column chromatography (EtOAc)] gave 17 (78 mg, 95%) as a color-
less oil: 1H NMR d 1.37 (s, 3, CH3), 1.44 (s, 3, CH3), 1.54 (s, 9H, t-Bu)
3.01 (s, 3, Ms), 4.39–4.43 (m, 2, H4,5), 4.58 (d, J = 5.5 Hz, 1, H3),
4.64 (dd, J = 3.1, 11.2 Hz, 1, H5’), 4.70 (d, J = 5.5 Hz, 1, H2); 13C
NMR d 25.6 (CMe2), 27.0 (CMe2), 28.0 (t-Bu), 37.7 (Ms), 59.2 (C4),
67.0 (C5), 74.5 (C3), 77.5 (C2), 84.7 (t-Bu), 112.8 (CMe2), 149.7
(CO), 170.2 (C1); MS (APCI) m/z 366 (5, MH+), 297 (100, [MH2-
Boc+MeOH]+).
4.9. 4-Amino-N-benzyl-4-deoxy-2,3-O-isopropylidene-D-ribono-
1,4-lactam (14b)
TBAF (1 M/THF; 0.18 mL, 0.18 mmol) was added dropwise to a
stirred solution of 14a (49 mg, 0.12 mmol) in THF (10 mL) at 0 °C.
After 5 min, the ice-bath was removed and reaction mixture was
allowed to stir at ambient temperature for 2 h. The reaction mix-
ture was quenched with water and volatiles were evaporated.
The residue was partitioned (EtOAc//ꢂNaHCO3/H2O) and the organ-
ic layer was washed (brine), dried (MgSO4) and evaporated. The
residue was column chromatographed (80 ? 90% EtOAc/hexane)
to give 14b52as a white solid (33 mg, 97%): 1H NMR d 1.37 (s, 3,
CH3), 1.47 (s, 3, CH3), 3.35 (s, 1, OH), 3.54 (‘t’, J = 1.8 Hz, 1, H4),
3.64 (d of m, J = 11.8 Hz, 1, H5), 3.85 (br d, J = 12.0 Hz, 1, H5’),
4.14. S-(4-Amino-N-benzyl-4,5-dideoxy-2,3-O-isopropylidene-D-
ribono-1,4-lactam-5-yl)-N-tert-butoxycarbonyl-L-homocysteine
tert-butyl ester (18)
Treatment of 16 (85 mg, 0.24 mmol) with lithium homocystei-
nate (104 mg, 0.36 mmol) by Procedure B [column chromatogra-
phy (60 ? 70% EtOAc/hexane)] gave 18 (94 mg, 70%) as
a
colorless oil. 1H NMR d 1.28 (s, 3, CH3), 1.37 (s, 12, t-Bu, CH3),
1.39 (s, 9, t-Bu), 1.74–1.86 (m, 1, H8), 1.95–2.17 (m, 1, H8’),
2.46–2.57 (m, 2, H7,7’), 2.69–2.73 (m, 2, H5,5’), 3.59–3.71 (m, 1,

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
5515
H4), 3.86 (d, J = 15.0 Hz, 1, Bn), 4.17–4.18 (m, 1, H9), 4.43 (d,
J = 5.7 Hz, 1, H3), 4.79 (d, J = 5.5 Hz, 1, H2), 4.97 (d, J = 15.0 Hz, 1,
Bn), 5.05 (br d, J = 7.1 Hz, 1, NH), 7.17–7.27 (m, 5, Bn); 13C NMR d
25.6 (CMe2), 27.0 (CMe2), 28.0 (t-Bu), 28.3 (t-Bu), 29.0 (C7), 33.1
(C5), 33.2 (C8), 44.4 (Bn), 53.1 (C9), 60.3 (C4), 77.2 (C3), 77.5
(C2), 79.9 (t-Bu) 82.4 (t-Bu), 112.1 CMe2, 127.9, 128.2, 128.8,
135.2 (Bn), 153.0 (CO), 171.1 (C10), 171.4 (C1); MS m/z 551 (100,
MH+). HRMS (AP-ESI) m/z calcd for C28H43N2O7S [MH]+ 551.2785;
found 551.2792.
(AP-ESI) m/z calcd for C16H22N2NaO5S [M+Na]+ 377.1142; found
377.1156.
4.18. S-(4-Amino-4,5-dideoxy-D-ribono-1,4-lactam-5-yl)-L-
homocysteine (21)
Treatment of 19a (30 mg, 0.065 mmol) with TFA by Procedure C
(step a, 3 h) gave crude 21 as colorless oil. RP-HPLC purification (5%
CH3CN/H2O at 2.5 mL/min; tR = 16 min) gave 21 (10 mg, 58%): 1H
NMR (D2O) d 2.10–2.28 (m, 2, H8,8’), 2.75–2.77 (m, 3, H5’,7,7’),
2.83–2.84 (m, 1, H5), 3.73 (‘t’, J = 6.5 Hz, 1, H4), 3.95 (‘t’,
J = 5.8 Hz, 1, H9), 4.29 (‘d’, J = 5.2 Hz, 1, H3), 4.57 (d, J = 5.2 Hz, 1,
H2); 13C NMR (D2O) dd 26.2 (C7), 28.5 (C8), 32.7 (C5), 58.8 (C4),
51.1 (C9), 69.7 (C2), 70.5 (C3), 170.9 (C1), 176.1 (C10); MS (ESI)
m/z 264 (100, M+); HRMS (AP-ESI) m/z calcd for C9H17N2O5S
[MH]+ 265.0853; found 265.0859.
4.15. S-(4-Amino-4,5-dideoxy-2,3-O-isopropylidene-
D-ribono-
1,4-lactam-5-yl)-N-tert-butoxycarbonyl-
L-homocysteine tert-
butyl ester (19a)
Treatment of 17 (70 mg, 0.19 mmol) with lithium homocystei-
nate (83 mg, 0.28 mmol) by Procedure B [column chromatography
(70 ? 80% EtOAc/hexane)] gave 19a (40 mg, 45%) as a light yellow
oil. 1H NMR d 1.36 (s, 3, CH3), 1.43 (s, 9, t-Bu), 1.45 (s, 12, t-Bu, CH3),
1.82–1.90 (m, 1, H8), 1.97–2.06 (m, 1, H8’), 2.58 (‘t’, J = 7.4 Hz, 2,
H7,7’), 2.64–2.72 (m, 2, H5,5’), 3.83 (‘t’, J = 5.8 Hz 1, H4), 4.23–
4.24 (m, 1, H9), 4.48 (d, J = 4.4 Hz, 1, H3), 4.69 (d, J = 4.6 Hz, 1,
H2), 5.21 (br d, J = 7.9 Hz, 1, NH); 13C NMR d 25.6 (CMe2), 27.0
(CMe2), 28.0 (t-Bu), 28.4 (t-Bu), 29.2 (C7), 32.9 (C8), 33.8 (C5),
53.2 (C9), 60.4 (C4), 75.9 (C3), 77.5 (C2), 80.0 (t-Bu) 82.4 (t-Bu),
112.4 CMe2 , 155.4 (CO), 170.4 (C1), 171.2 (C10); MS (APCI) m/z
461 (100, MH+).
4.19. S-(4-Amino-N-tert-butoxycarbonyl-4,5-dideoxy-2,3-
isopropylidene-a/b-D-ribofuranos-5-yl)-N-tert-butoxycarbonyl-
L-homocysteine tert-butyl ester (22)
Procedure F. LiEt3BH (1 M/THF; 125 lL, 0.125 mmol) was
added to a stirred solution of 19b (28 mg, 0.05 mmol) in anhydrous
THF (1 mL) at ꢁ78 °C under N2 atmosphere. After 30 min, the solu-
tion was quenched with water and volatiles were evaporated. The
residue was partitioned (EtOAc//ꢂNaHCO3/H2O), washed (brine)
and dried (MgSO4). The resulting oil was chromatographed (40%
EtOAc/hexane) to give 22 [26 mg, 92%; mixture of anomers (3:2)
which appear as a set of rotamers as colorless oil: 1H NMR d 1.30
(s, 3, CH3), 1.43 (s, 12, t-Bu, CH3), 1.46 (s, 9, t-Bu), 1.48 (s, 9, t-
Bu), 1.83–1.94 (m, 1, H8), 2.03–2.10 (m, 1, H8’), 2.51–2.62 (m, 3,
H7,7’,5), 2.83 (dd, J = 3.7, 13.5 Hz, 0.6, H5’), 2.92 (dd, J = 3.5,
13.7 Hz, 0.4, H5’), 3.45 (dd, J = 3.5, 10.6 Hz, 0.3, H4), 3.99–4.28
(m, 1.7, H4,9), 4.57 (d, J =5.8 Hz, 0.6, H3), 4.59 (d, J = 5.9 Hz, 0.4,
H3), 4.66 (d, J = 6.7 Hz, 0.4, H2), 4.72 (d, J = 5.8 Hz, 0.6, H2), 5.09
(br d, J = 7.2 Hz, 0.6, NH), 5.32 (br d, J = 7.8 Hz, 0.4, NH), 5.39 (s,
0.4, H1), 5.50 (s, 0.6, H1); 13C NMR d (major isomer) 24.8 (CMe2),
26.7 (CMe2), 28.0 (t-Bu), 28.3 (t-Bu), 28.4 (t-Bu), 29.7 (C7), 32.9
(C8), 34.7 (C5), 53.2 (C9), 63.9 (C4), 81.2 (2 ꢃ t-Bu), 82.3 (t-Bu),
82.8 (C2), 84.4 (C3), 87.1 (C1), 112.7 (CMe2), 154.3 (CO), 155.4
(CO), 171.3 (C10); MS (ESI) m/z 585 (100, [M+Na]+).
4.16. S-(4-Amino-N-tert-butoxycarbonyl-4,5-dideoxy-2,3-O-
isopropylidene-D-ribono-1,4-lactam-5-yl)-N-tert-butoxy-
carbonyl- -homocysteine tert-butyl ester (19b). Procedure E
L
DMAP (18.8 mg, 0.15 mmol) and (Boc)2O (46.4 mg, 0.21 mmol)
were added to a stirred solution of compound 19a (27 mg,
0.06 mmol) in CH2Cl2 (2 mL) at ambient temperature under Ar
atmosphere. After 48 h, the reaction mixture was quenched with
H2O (5 mL) and partitioned between CH2Cl2//NaHCO3/H2O. The or-
ganic layer was washed (brine), dried (MgSO4) and evaporated. The
residue was column chromatographed (30 ? 40% EtOAc/hexane)
to give 19b (30 mg, 93%) as a colorless oil: 1H NMR dd 1.36 (s, 3,
CH3), 1.43 (s, 9, t-Bu), 1.45 (s, 3, CH3), 1.46 (s, 9, t-Bu), 1.54 (s, 9,
t-Bu), 1.82–1.87 (m, 1, H8), 2.02–2.04 (m, 1, H8’), 2.49–2.55 (m,
1, H7), 2.58–2.62 (m, 1, H7’), 2.70 (dd, J = 7.1, 14.2 Hz, 1, H5),
2.95 (dd, J = 2.5, 14.2 Hz, 1, H5’), 4.22–4.24 (m, 1, H9), 4.23 (dd,
J = 2.5, 7.1 Hz, 1, H4), 4.46 (d, J = 5.5 Hz, 1, H3), 4.80 (d, J = 5.5 Hz,
1, H2), 5.08 (br d, J = 7.9 Hz, 1, NH); 13C NMR d 25.6 (CMe2), 26.9
(CMe2), 28.0 (t-Bu), 28.3 (t-Bu), 28.4 (t-Bu), 28.9 (C7), 33.2 (C8),
36.6 (C5), 53.2 (C9), 58.0 (C4), 76.7 (C3), 79.1 (C2), 82.3 (2ꢃ t-
Bu), 84.1 (t-Bu), 112.7 CMe2 , 149.9 (CO), 155.5 (CO), 171.2 (C10),
174.0 (C1); MS (ESI) m/z 583 (100, [M+Na]+).
4.20. S-(4-Amino-4,5-dideoxy-a/b-D-ribofuranos-5-yl)-L-
homocysteine (23)
Treatment of 22 (24 mg, 0.04 mmol) with TFA by Procedure C
(step a, 1 h; step b, 10 h at 0 °C) gave crude 23. RP-HPLC (5%
CH3CN/H2O at 2.5 mL/min; tR = 14 min) yielded 23 (8 mg, 72%) as
a light yellow oil of a mixture of anomers (3:2): 1H NMR (D2O) d
2.15–2.24 (m, 1, H8), 2.26–2.37 (m, 1, H8’), 2.75–2.82 (m, 2,
H7,7’), 2.84–2.98 (m, 1, H5,5’), 3.09–3.17 (m, 1, H5,5’), 3.55–3.69
(m, 0.6, H4), 3.76–3.83 (m, 0.4, H4), 4.15–4.21 (m, 2, H2,3,9),
4.23–4.25 (m, 0.4, H2), 4.34 (dd, J = 4.8, 6.3 Hz, 0.6, H3), 5.27 (d,
J = 2.6 Hz, 0.6, H1), 5.41 (d, J = 2.2 Hz, 0.4, H1); 13C NMR d (major
isomer) 25.8 (C7), 28.7 (C8), 30.7 (C5), 51.3 (C9), 58.8 (C4), 72.0
(C3), 73.0 (C2), 86.5 (C1), 171.2 (C10); MS (ESI) m/z 267 (50,
MH+), 249 (100, [M-17]+).
4.17. S-(4-Amino-N-benzyl-4,5-dideoxy-D-ribono-1,4-lactam-5-
yl)- -homocysteine (20)
L
Treatment of 18 (40 mg, 0.07 mmol) with TFA by Procedure C
(step a, 3 h) gave crude 20 as colorless oil. RP-HPLC purification
(5% CH3CN/H2O for 30 min followed by gradient 5 ? 90% CH3CN/
H2O for 30 min at 2.5 mL/min; tR = 45 min) gave 20 (12 mg,
48%): 1H NMR (D2O) d 1.98–2.11 (m, 2, H8,8’), 2.57–2.61 (m, 2,
H7,7’), 2.71 (dd, J = 8.4, 14.0 Hz, 1, H5), 2.82 (dd, J = 3.9, 14.0 Hz,
1, H5’), 3.57 (dd, J = 3.6, 8.3 Hz, 1, H4), 3.78 (‘t’, J = 5.8 Hz, 1, H9),
4.36 (d, J = 5.3 Hz, 1, H3), 4.37 (d, J = 14.7 Hz, 1, Bn), 4.72 (d,
J = 5.2 Hz, 1, H2), 4.78 (d, J = 14.7 Hz, 1, Bn), 7.33–7.46 (m, 5, Bn);
13C NMR (D2O) d 27.7 (C7), 30.3 (C8), 30.8 (C5), 45.2 (Bn), 53.6
(C9), 63.9 (C4), 70.0 (C3), 70.1 (C2), 128.0, 128.1, 128.0, 135.1
(Bn), 173.9 (C1), 174.7 (C10); MS (APCI) m/z 355 (100, MH+); HRMS
4.21. 4-Amino-N-tert-butoxycarbonyl-5-O-tert-butyldimethyl-
silyl-4-deoxy-2,3-O-isopropylidene-a/b-D-ribofuranose (24a)
Reduction of 15a44 (68 mg, 0.17 mmol) with LiEt3BH (1 M/THF;
0.43 mL, 0.43 mmol) in anhydrous THF (2 mL) at -78 °C by the pro-
cedure F gave 24a44 (68 mg, 100%) as a colorless oil with data as
reported.

5516
V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
4.22. 4-Amino-N-tert-butoxycarbonyl-4-deoxy-2,3-O-
isopropylidene- /b- -ribofuranose (24b)
13.3 Hz, 1, H5), 2.57–2.65 (m, 2, H7,7’), 2.72 (dd, J = 5.1, 13.2 Hz,
1, H5’), 3.80 (‘quin’, J = 6.4 Hz, 1, H4), 4.28 (br ‘d’, J = 3.7 Hz, H9),
5.20 (br d, J = 7.1 Hz, 1, NHBoc), 6.45 (br s, 1, CONH); 13C NMR d
26.7 (C3), 28.0 (t-Bu), 28.3 (t-Bu), 28.3 (C7), 30.1 (C2), 33.2 (C8),
38.7 (C5), 53.3 (C9), 53.7 (C4), 79.9 (t-Bu), 82.3 (t-Bu), 155.4 (CO),
171.2 (C10), 177.7 (C1); HRMS (AP-ESI) m/z calcd for C18H33N2O5S
[MH]+ 389.2105; found 389.2110.
a
D
Desilylation of 24a (55 mg, 0.13 mmol) with TBAF (1 M/THF,
0.19 mL, 0.19 mmol), as described for 14b, [column chromatogra-
phy (50 ? 60% EtOAc/hexane)] gave 24b (39 mg, 96%) as a color-
less oil of a mixture of anomers (3:2): 1H NMR d 1.30 (s, 3, CH3),
1.40 (s, 3, CH3), 1.46 (s, 5.4, t-Bu), 1.49 (s, 3.6, t-Bu), 2.87 (br s,
0.4, OH), 3.15 (br s, 0.6, OH), 3.60–3.77 (m, 1.4, H5,5’), 3.84–3.90
(m, 0.8, H5,OH), 4.10 (br s, 0.6, H4), 4.27 (br s, 0.4, H4), 4.30 (br
s, 0.6, OH), 4.55 (d, J = 5.9 Hz, 1, H3), 4.73 (d, J = 5.9 Hz, 0.4, H2),
4.77 (d, J = 5.9 Hz, 0.6, H2), 5.36 (d, J = 5.7 Hz, 0.4, H1), 5.51 (s,
0.6, H1); 13C NMR (major anomer) d 24.6 (CMe2), 26.6 (CMe2),
28.3 (t-Bu), 62.6 (C5), 65.8 (C4), 81.9 (C2), 81.3 (t-Bu), 85.3 (C3),
86.4 (C1), 111.4 (CMe2), 154.3 (CO); 13C NMR (minor anomer) d
24.7 (CMe2), 26.7 (CMe2), 28.3 (t-Bu), 62.6 (C5), 65.8 (C4), 81.2
(C2), 81.3 (t-Bu), 86.4 (C3), 86.5 (C1), 111.4 (CMe2), 153.5 (CO);
MS (APCI) m/z 272 (50, [M-OH+]), 213 [100, [MH-Boc-
OH+CH3CN]+).
4.26. S-(4-Amino-2,3,4,5-tetradeoxy-
lactam-5-yl)- -homocysteine (34)
D-glycero-pentono-1,4-
L
Compound 33 (9 mg, 0.02 mmol) was dissolved in TFA (0.7 mL),
and the resulting mixture was stirred at ambient temperature for
60 min. The reaction mixture was evaporated, and coevaporated
with toluene to give a trifluoroacetate of 34 (7.5 mg, 95%) as a col-
orless oil: 1H NMR (D2O) d 1.84–1.94 (m, 1, H3), 2.12–2.23 (m, 1,
H8), 2.23–2.31 (m, 1, H8’), 2.31–2.37 (m, 1, H3’,), 2.37–2.52 (m,
2, H2,2’), 2.73 (dd, J = 6.5, 13.6 Hz, 1, H5), 2.76 (t, J = 7.6 Hz, 2,
H7,7’), 2.82 (dd, J = 5.4, 13.6 Hz, 1, H5’), 3.99 (‘quin’, J = 6.3 Hz, 1,
H4), 4.17 (t, J = 6.4 Hz, 1, H9); 13C NMR (D2O) d 25.3 (C3), 27.1
(C7), 29.7 (C8), 29.9 (C2), 36.8 (C5), 51.9 (C9), 54.3 (C4), 172.0
(C10), 181.5 (C1); HRMS (AP-ESI) m/z calcd for C9H17N2O3S [MH]+
233.0954; found 233.0957.
4.23. 4-Amino-4-deoxy-a/b-D-ribofuranose (25a)
Treatment of 24b (27 mg, 0.09 mmol) with TFA by Procedure C
(step a, 5 h; step b, 6 h at 0 °C) gave crude 25a43 (13 mg, 92%) as a
light yellow oil of a mixture of anomers (
a
/b, 0.65:0.35): 1H NMR
4.27. S-(4-Amino-N-tert-butoxycarbonyl-2,3,4,5-tetradeoxy-D-
(D2O; pD = 5–6) 3.52–3.53 (m, 0.35, H4), 3.56–3.59 (m, 0.65, H4),
3.76–3.78 (m, 0.65, H3), 3.85 (dd, J = 6.0, 13.1 Hz, 0.35, H5), 3.86
(dd, J = 2.1, 12.8 Hz, 0.65, H5), 3.97–4.00 (m, 0.35, H3), 4.06 (dd,
J = 3.0, 4.0 Hz, 0.35, H2), 4.10 (dd, J = 2.8, 13.5 Hz, 0.35, H5’), 4.16
(dd, J = 3.0, 12.8 Hz, 0.65, H5’), 4.20 (‘t’, J = 3.4 Hz, 0.65, H2), 4.84
(d, J = 1.3 Hz, 0.35, H1), 5.12 (d, J = 4.0 Hz, 0.65, H1); 13C NMR (ma-
jor anomer) d 50.1 (C4), 58.9 (C5), 65.6 (C2), 69.8 (C3), 94.1 (C1);
13C NMR (minor anomers) d 49.9 (C4), 61.5 (C5), 63.6 (C2), 69.8
(C3), 94.1 (C1); MS (APCI) m/z 150 (100, MH+).
glycero-pentono-1,4-lactam-5-yl)-N-tert-butoxycarbonyl-
homocysteine tert-butyl ester (35)
L-
Treatment of 33 (40 mg, 0.1 mmol) in CH2Cl2 (2 mL) with DMAP
(20 mg, 0.16 mmol), and (Boc)2O (63 mg, 0.29 mmol) by procedure
E [column chromatography (30 ? 35% EtOAc/hexane)] gave 35
(42 mg, 83%) as a colorless oil: 1H NMR (isomers ratio ꢀ3:2) d
1.45 (s, 9H), 1.47 (s, 9H), 1.55 (s, 9H), 1.82–1.94 (m, 1, H8), 1.99–
2.22 (m, 3, H3,3’,8’), 2.44 & 2.45 (2ꢃ ddd, J = 2.5, 9.6, 17.9 Hz, 1,
H2), 2.54–2.70 (m, 4, H2’,5,7,7’), 2.94 (‘dt’, J = 3.5, 13.4 Hz, 1, H5’),
4.22–4.32 (m, 2, H4,9), 5.11 & 5.16 (2ꢃ br d, J = 7.9 Hz, 1, NHBoc);
13C NMR d 22.0 (C3), 28.0 (t-Bu), 28.1 (t-Bu), 28.3 (t-Bu), 28.8 (C7),
31.1 & 31.2 (C2), 33.0 & 33.2 (C8), 35.1 & 35.5 (C5), 53.2 & 53.3 (C9),
57.2 & 57.5 (C4), 79.8 (t-Bu), 82.1 & 82.2 (t-Bu), 83.2 & 83.3 (t-Bu),
149.9 & 150.0 (CO), 155.5 (CO), 171.3 (C10), 173.7 & 173.8 (C1); MS
(APCI) m/z 489 (30, MH+), 389 (100, [MH2-Boc]+).
The 1H NMR (D2O) at pD = 11 showed a singlet at 7.76 ppm
which suggested formation of imine 25c.
4.24. 4-Amino-4-deoxy-a/b-D-ribose O-benzyloxime (26)
A solution of the crude 25a (13 mg. 0.09 mmol) and O-ben-
zylhydroxylamine hydrochloride (43 mg, 0.27 mmol) in anhydrous
pyridine (4 mL) was stirred under an atmosphere of nitrogen at
room temperature for 12 h. Pyridine was evaporated to afford 26
of sufficient purity (ꢀ95%) for spectroscopic characterization to-
gether with the excess of BnONH2 used: 1H NMR (MeOH-d4) d
3.52 (‘dt’, J = 4.1, 8.4 Hz, 1, H4), 3.79 (dd, J = 3.8, 11.5 Hz, 1, H5),
3.85 (dd, J = 4.4, 8.7 Hz, 1, H3), 3.94 (dd, J = 8.4, 11.5 Hz, 1, H5’),
4.13 (dd, J = 6.8, 8.7 Hz, 1, H2), 4.92–5.16 (2H, Bn; signals for ben-
zylic protons were within the envelope of the solvent peak but
cross peaks between them were observed in COSY), 7.41 (1, H1;
signal for H1 was within the envelope of protons from benzyl
group but cross peaks of H1 to H2 were observed in COSY), 7.35–
7.47 (m, 5H, Bn); 13C NMR d 56.2 (C4), 58.8 (C5), 71.2 (C3), 71.6
(C2), 77.0 (Bn, confirmed by HETCOR), 128.9, 129.3, 129.5, 139.1
(Bn), 151.9 (C1); MS (ESI) m/z 255 (100, MH+).
4.28. S-(4-Amino-2,3,4,5-tetradeoxy-D-glycero-pentono-1,4-
lactam-5-yl)-D/ -homocysteine (36)
L
Treatment of 2745 (62 mg, 0.35 mmol) with
D/L-Hcy (52 mg,
0.385 mmol)/NaH (44 mg, 1.1 mmol; 60%/ mineral oil) by proce-
dure G gave crude 36. RP-HPLC purification (5% CH3CN/H2O at
2.5 mL/min; tR = 14 min) gave 36 (60.5 mg, 75%) as a Na salt: 1H
NMR (D2O) d 1.83–1.95 (m, 2, H3,8), 1.95–2.04 (m, 1, H8’), 2.29–
2.38 (m, 1, H3’), 2.38–2.53 (m, 2, H2,2’), 2.65 (t, J = 7.7 Hz, 2,
H7,7’), 2.72 (dd, J = 6.5, 13.6 Hz, 1, H5), 2.81 (dd, J = 5.6, 13.6 Hz,
1, H5’), 3.47 (br s, 1, H9), 3.99 (‘quin’, J = 6.3 Hz, 1, H4); 13C
NMR (D2O) d 25.4 (C3), 27.9 & 28.0 (C7), 29.9 & 29.9 (C2), 33.5
& 33.6 (C8), 36.8 (C5), 54.4 (C4), 54.8 (br, C9), 180.4 (br, C10),
180.5 (C1); MS (APCI) m/z 233 (100, MH+); (ESI) m/z 233 (100,
MH+).
4.25. S-(4-Amino-2,3,4,5-tetradeoxy-
D
-glycero-pentono-1,4-
lactam-5-yl)-N-tert-butoxycarbonyl-
ester (33)
L
-homocysteine tert-butyl
Stirring sodium salt of 36 (10 mg) in TFA (1 mL) for 1 h at ambi-
ent temperature and evaporation of volatiles gave trifluoroacetate
of 36 as a mixture of diastereomers (9R/S, ꢀ1:1): 1H NMR (D2O) d
1.79–1.89 (m, 1, H3), 2.09–2.19 (m, 1, H8), 2.21–2.28 (m, 1, H8’),
2.28–2.33 (m, 1, H3’,), 2.34–2.48 (m, 2, H2,2’), 2.678 (dd, J = 6.5,
13.6 Hz, 0.5, H5, 9R), 2.683 (dd, J = 6.5, 13.6 Hz, 0.5, H5, 9S), 2.72
(t, J = 7.6 Hz, 2, H7,7’), 2.776 (dd, J = 5.4, 13.6 Hz, 0.5, H5’, 9R),
2.781 (dd, J = 5.4, 13.6 Hz, 0.5, H5’, 9S), 3.94 (‘quin’, J = 6.3 Hz, 1,
H4), 4.181 (t, J = 6.4 Hz, 1, H9R), 4.186 (t, J = 6.4 Hz, 1, H9S).
Treatment of 2745 (5S; 18 mg, 0.1 mmol) with protected
L-Hcy
(35 mg, 0.12 mmol) by Procedure B (step a and b, 48 h) gave
50.5 mg of the yellow oil. This material was column chromato-
graphed (EtOAc/MeOH, 19:1) to give 33 (31 mg, 79%) as a colorless
oil: 1H NMR d 1.45 (s, 9), 1.48 (s, 9), 1.77–1.92 (m, 2, H3,8), 2.02–
2.14 (m, 1, H8’), 2.27–2.47 (m, 3, H2,2’,3’), 2.51–2.57 (dd, J = 8.1,

V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
5517
Chemical shifts observed for TFA salt of 36(9R/S) were different
from its sodium salt but parallel the signals for the trifluoroacetate
of 34(9S) derived from Hcy.
2.22–2.31 (m, 1, H8’), 2.57 (br s, 3 ? 0, exch. with deuterium with-
in few hours, N@CMe), 2.75 (t, J = 7.3 Hz, 2, H7,7’), 2.99 (d,
J = 6.2 Hz, 2, H5,5’), 4.17 (t, J = 6.4 Hz, 1, H9), 4.73 (‘t’, J = 6.3 Hz, 1,
H4), 4.91 (d, J = 5.4, 1, H2), 5.64 (d, J = 5.3, 1, H3); 1H NMR (DMSO)
d 1.28 (s, 3), 1.30 (s, 3), 1.95–2.07 (m, 2, H8,8’), 2.08 (s, 3, N@CMe),
2.60 (dd, J = 7.4, 13.6 Hz, 1, H5) 2.65 (‘t’, J = 7.9 Hz, 2, H7,7’), 2.82
(dd, J = 5.2, 13.7 Hz, 1, H5’), 4.01 (br s, 1, H9), 4.21 (‘t’, J = 5.8 Hz,
1, H4), 4.51 (d, J = 5.5, 1, H2), 5.10 (d, J = 5.5, 1, H3), 7.28 (br s, 3,
+NH3); 13C NMR (D2O) d 15.5 (‘quin’, J = 20 Hz, N@CCD3), 24.0
(CMe2), 25.4 (CMe2) 27.4 (C7), 29.5 (C8), 31.6 (C5), 51.4 (C9), 71.2
(C4), 79.0 (C2), 84.0 (C3), 114.2 (CMe2), 171.5 (C10), 191.7
(N@CCD3); MS (APCI) m/z 303 (100, MH+).
4.29. S-(4-Amino-N-tert-butoxycarbonyl-2,3,4,5-tetradeoxy-
-glycero-pentofuranos-5-yl)-N-tert-butoxycarbonyl- -homo-
cysteine tert-butyl ester (37)
a/b-
D
L
Treatment of 35 (42 mg, 0.086 mmol) in CH2Cl2 (2 mL) with
LiEt3BH (1 M/THF, 0.22 mL, 0.22 mmol), by procedure F [quenched
with MeOH (3 mL) at low temp., column chromatography
(30 ? 40% EtOAc/hexane)] gave 37 (31 mg, 73%; colorless oil) as
a complex mixture of isomers (1H, and 13C NMR): MS (APCI) m/z
473 (100, [M-17]+), 373 (55, [M-Boc-18]+); (ESI) m/z 473 (50, [M-
17]+), 373 (100, [M-Boc-18]+).
4.34. S-(1-Amino-1,4-anhydro-5-deoxy-1,N-didehydro-1-
methyl-D-ribitol-5-yl)-L-homocysteine (43)
4.30. S-(4-Amino-2,3,4,5-tetradeoxy-
a
/b-
D
-
D
-glycero-pento-
Treatment of 42 (41 mg, 0.136 mmol) with TFA/H2O by Proce-
dure C (step b, TFA/H2O 4:1, ambient temperature, 12 h) gave
homogenous 43 (36 mg, 98%) as a colorless oil: 1H NMR (D2O) d
2.05–2.16 (m, 1, H8), 2.17–2.27 (m, 1, H8’), 2.45 (br s, 3 ? 0, exch.
with deuterium within few hours, N@CMe), 2.73 (dt, J = 3.3, 7.3 Hz,
2, H7,7’), 2.78 (dd, J = 8.3, 14.4 Hz, 1, H5), 2.92 (dd, J = 6.0, 14.3 Hz,
1, H5’) 4.14 (t, J = 6.4 Hz, 1, H9), 4.35 (‘t’, J = 7.1 Hz, 1, H4), 4.37 (d,
J = 5.3, 1, H2), 5.12 (d, J = 5.5, 1, H3); 13C NMR (D2O) d 15.4 (‘quin’,
J = 21 Hz, N@CCD3), 27.0 (C7), 29.3 (C8), 30.7 (C5), 51.3 (C9), 71.6
(C2), 71.9 (C4), 76.8 (C3), 171.4 (C10), 195.8 (N@CCD3); MS (APCI)
m/z 263 (100, MH+); HRMS (AP-ESI) m/z calcd for C10H19N2O4S
[MH]+ 263.1060; found 263.1065.
furanos-5-yl)- -homocysteine (38)
L
Treatment of 37 (20 mg, 0.04 mmol) with an excess of TFA
(1 mL) by Procedure C (step a, 2 h at ambient temperature) gave
trifluoroacetate of 38 (13 mg, 95%; light yellow oil) as a complex
mixture of isomers accompanied (ꢀ10%) by the open aldehyde
form [1H NMR d 8.89 (s)]: MS (APCI) m/z 217 (100, [M-17]+).
4.31. 1-Amino-1,4-anhydro-1,N-didehydro-2,3-O-isopropyl-
idene-5-O-methanesulfonyl-1-methyl-D-ribitol (40)
Treatment of 3947 (48.5 mg, 0.26 mmol) with MsCl (0.031 mL,
45 mg, 0.39 mmol) in the presence of Et3N (0.11 mL, 80 mg,
4.35. 1-Amino-1,4-anhydro-5-O-tert-butyldimethylsilyl-1-
0.79 mmol) by Procedure
A
[3 h; column chromatography
deoxy-2,3-O-isopropylidene-D-ribitol (44)
(EtOAc ? 10% MeOH/EtOAc)] gave 40 (59 mg, 85%) as a colorless
oil: 1H NMR d 1.38 (s, 3), 1.39 (s, 3), 2.16 (d, J = 1.0 Hz, 3, N@CCH3),
3.00 (s, 3, Ms), 4.38 (s, 1, H4), 4.39 (dd, 1, J = 3.6, 11.2 Hz, 1, H5),
4.53 (dd, J = 4.2, 11.3 Hz, 1, H5’), 4.64 (d, J = 5.8 Hz, 1, H3), 4.95
(‘quin’, J = 5.6 Hz, 1, H2); 13C NMR d 17.1 (N@CMe), 25.7 (CMe2),
26.8 (CMe2), 37.4 (Ms), 69.7 (C5), 75.0 (C4), 79.8 (C3), 87.4 (C2),
112.4 CMe2, 177.0 (C=N); MS (APCI) m/z 264 (100, MH+).
To a solution of 7 (109 mg, 0.28 mmol) in EtOH (6 mL) was
added 5% Pd/C (300 mg) and stirred under an atmosphere of H2
at room temperature for 6 h. The mixture was filtered through Cel-
ite to remove the catalyst. The Celite bed was washed with ethanol
(5 mL) and the filtrate and washings were combined and evapo-
rated. The residue was column chromatographed (30% EtOAc/hex-
ane) to give 4450 as a colorless oil (70 mg, 80%). 1H NMR d 0.04 (s, 3,
CH3), 0.04 (s, 3, CH3), 0.87 (s, 9, t-Bu), 1.32 (s, 3, CH3), 1.46 (s, 3,
CH3), 2.33 (s, 1, NH), 2.98 (‘d’, J = 2.6 Hz, 2, H1,1’), 3.20 (‘dt’,
J = 0.6, 5.8 Hz, 1, H4), 3.52 (dd, J = 5.9, 10.3 Hz, 1, H5), 3.62 (dd,
J = 5.1, 10.3 Hz, 1, H5’), 4.63 (dd, J = 0.8, 5.8 Hz, 1, H3), 4.68 (‘dt’,
J = 2.6, 5.8 Hz, 1, H2); MS (APCI) m/z 288 (100, MH+).
4.32. S-(1-Amino-1,4-anhydro-5-deoxy-1,N-Didehydro-2,3-O-
isopropylidene-1-methyl-D-ribitol-5-yl)-N-tert-butoxycarbonyl-
L
-homocysteine tert-butyl ester (41)
Treatment of 40 (49.5 mg, 0.19 mmol) with protected L-Hcy
(82 mg, 0.284 mmol) by Procedure B (step a and b, 36 h) gave
124 mg of yellow oil. Crude product was chromatographed
(50 ? 60% EtOAc/hexane) to give 41 as an colorless oil (73 mg,
85%): 1H NMR d 1.35 (s, 3), 1.35 (s, 3),1.43 (s, 9), 1.46 (s, 9), 1.84
(‘sx’, J = 7.3 Hz, 1, C8), 1.98–2.07 (m, 1, H8’), 2.11 (d, J = 1.1 Hz, 3,
N@CMe), 2.54 (‘t’, J = 7.6 Hz, 2, H7,7’), 2.65 (dd, J = 6.4, 13.5 Hz, 1,
H5), 2.87 (dd, J = 4.4, 13.5 Hz, 1, H5’), 4.17–4.27 (m, 1, H9), 4.33
(br ‘t’, J = 4.6 Hz, 1, H4), 4.48 (d, J = 5.6 Hz, 1, H2) 4.96 (d,
J = 5.6 Hz, 1, H3) 5.13 (br d, J = 7.7 Hz, 1, NH); 13C NMR d 17.0
(N@CMe), 25.7 (CMe2), 26.8 (CMe2), 28.0 (t-Bu), 28.3 (t-Bu), 29.2
(C7), 33.1 (C8), 35.3 (C5), 53.3 (C9), 76.5 (C4), 79.8 (t-Bu), 81.8
(C2), 82.1 (t-Bu), 87.3 (C3), 111.9 (CMe2), 155.3 (CO), 171.2 (C10),
174.7 (N@C); MS (APCI) m/z 459 (100, MH+); HRMS (TOF MS-ESI)
m/z calcd for C22H39N2O6S [MH]+ 459.2523; found 459.2523.
4.36. 1-Amino-1,4-anhydro-5-O-tert-butyldimethylsilyl-1,N-
didehydro-2,3-O-isopropylidene-D-ribitol N–oxide (45)
A
stirred solution of 44 (70 mg, 0.24 mmol) and SeO2
(0.01 mmol, 1.1 mg) in acetone (3 mL) was cooled to ꢁ4 °C under
N2 atmosphere and H2O2 (25%) was added slowly (3–4 h) until
the reaction was completed (as judged by TLC). Volatiles are evap-
orated and the residue was partitioned (EtOAc//NaHCO3/H2O).The
organic layer was collected, washed (brine) and dried (MgSO4).
The resulting solid was chromatographed (50% EtOAc/hexane) to
give 45 (54 mg, 73%) as a white solid with data as reported.49
4.37. 1-Amino-1,4-anhydro-1,N-didehydro-2,3-O-isopropyl-
idene-D-ribitol N-oxide (46)
4.33. S-(1-Amino-1,4-anhydro-5-deoxy-1,N-didehydro-2,3-O-
isopropylidene-1-methyl-
D
-ribitol-5-yl)-
L
-homocysteine (42)
Desilylation of 45 (155 mg, 0.51 mmol) with TBAF (1 M/THF,
0.77 mL, 0.77 mmol) at ꢁ4 °C as described for 14b [column chro-
matography (10 ? 20% MeOH/CHCl3)] gave 46 (40 mg, 42%) as a
white solid: 1H NMR d 1.34 (s, 3, CH3), 1.41 (s, 3, CH3), 3.87 (dd,
J = 2.6, 11.9 Hz, 1, H5), 4.02–4.03 (m, 1, H4), 4.14 (dd, J = 2.3,
Treatment of 41 (62 mg, 0.136 mmol) with TFA by Procedure C
(step a, ambient temperature) gave 42 (41 mg, 99%) as a colorless
oil: 1H NMR (D2O) d 1.38 (s, 3), 1.41 (s, 3), 2.10–2.20 (m, 1, H8),

5518
V. L. A. Malladi et al. / Bioorg. Med. Chem. 19 (2011) 5507–5519
11.9 Hz, 1, H5’), 4.96 (d, J = 6.2 Hz, 1, H3), 5.21 (‘dt’, J = 1.4, 6.2 Hz,
1, H2), 6.90 (s, 1, H1); 13C NMR d 25.7 (CMe2), 27.2 (CMe2), 58.9
(C5), 77.2 (C3), 78.9 (C4), 80.8 (C2), 111.5 (CMe2), 134.6 (C1); MS
(ESI) m/z 186 (100, M+).
For compounds that showed time dependent inhibition, the inhib-
itor and LuxS (1.6 M) were preincubated for 30 min at 4 °C and
the reaction was then initiated by addition of SRH.
l
Acknowledgment
4.38. 1-Amino-1,4-anhydro-1,N-didehydro-2,3-O-
We thank NIH (SC1CA138176, AI62901, and DE019667) and
FIU’s Doctoral Evidence Acquisition Fellowship (V.L.A.M) for their
support.
isopropylidene-5-O-methanesulfonyl-D-ribitol N–oxide (47)
Treatment of 46 (40 mg, 0.21 mmol) at ꢁ4 °C with MsCl (26
lL,
0.32 mmol) by Procedure A [column chromatography (80% ? 90%
EtOAc/hexane)] gave 47 (24 mg, 56%) as a colorless oil: 1H NMR d
1.37 (s, 3, CH3), 1.46 (s, 3, CH3), 3.04 (s, 3, Ms), 4.23–4.24 (m, 1,
H4), 4.55 (dd, J = 1.8, 11.1 Hz, 1, H5), 4.83 (dd, J = 2.4, 11.1 Hz, 1,
H5’), 4.91 (dd, J = 1.1, 6.3 Hz, 1, H3), 5.25 (‘dt’, J = 1.5, 6.4 Hz, 1,
H2), 6.99 (s, 1, H1); 13C NMR d 25.6 (CMe2), 27.1 (CMe2), 37.4
(Ms), 65.5 (C5), 75.8 (C3), 77.8 (C4), 78.4 (C2), 112.6 (CMe2),
134.1 (C1); MS (APSI) m/z 266 (100, MH+); HRMS (AP-ESI) m/z calcd
for C9H16NO6S [MH]+ 266.0693; found 266.0682.
References and notes
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9. Lee, J. E.; Cornell, K. A.; Riscoe, M. K.; Howell, P. L. Structure 2001, 9, 941.
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8761.
11. Pei, D.; Zhu, J. Curr. Opin. Chem. Biol. 2004, 8, 492.
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14. Semmelhack, M. F.; Campagna, S. R.; Federle, M. J.; Bassler, B. L. Org. Lett. 2005,
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15. Semmelhack, M. F.; Campagna, S. R.; Hwa, C.; Federle, M. J.; Bassler, B. L. Org.
Lett. 2004, 6, 2635.
4.39. S-(1-Amino-1,4-anhydro-5-deoxy-1,N-didehydro-2,3-O-
isopropylidene-D-ribitol-5-yl N–oxide)-N-tert-butoxycarbonyl-
L
-homocysteine tert-butyl ester (48)
Treatment of 47 (24 mg, 0.09 mmol) with
L-homocysteine
(38 mg, 0.13 mmol) by Procedure B (step a and b, 8 h at -20 °C)
and purification by column chromatography (80% ? 90% EtOAc/
hexane) gave 48 as a colorless oil (24 mg, 43%): 1H NMR d 1.37
(s, 3, CH3), 1.44 (s, 9, t-Bu), 1.45 (s, 3, CH3), 1.46 (s, 9, t-Bu),
1.81–1.90 (m, 1, H8), 1.96–2.09 (m, 1, H8’), 2.52–2.70 (m, 2,
H7,H7’), 3.07 (dd, J = 3.5, 14.4 Hz, 1, H5), 3.14 (dd, J = 5.2, 14.4 Hz,
1, H5’), 4.20–4.31 (m, 2, H4,9), 4.71 (d, J = 6.2 Hz, 1, H3), 5.06 (m,
1, NH), 5.31 (‘dt’, J = 1.4, 6.4 Hz, 1, H2), 6.97 (s, 1, H1); 13C NMR d
25.6 (CMe2), 26.1 (CMe2), 28.0 (t-Bu), 28.3 (t-Bu), 29.1 (C7), 32.0
(C5), 32.8 (C8), 53.1 (C9), 77.9 (C3), 79.0 (C2), 79.1 (C4), 79.8 (t-
Bu), 82.2 (t-Bu), 112.0 (CMe2), 133.4 (C1), 155.4 (CO), 171.1
(C10); MS (APCI) m/z 461 (100, MH+).
16. Zhu, J.; Dizin, E.; Hu, X.; Wavreille, A.-S.; Park, J.; Pei, D. Biochemistry 2003, 42,
4717.
17. Zhu, J.; Patel, R.; Pei, D. Biochemistry 2004, 43, 10166.
18. Rajan, R.; Zhu, J.; Hu, X.; Pei, D.; Bell, C. E. Biochemistry 2005, 44, 3745.
19. Zhu, J.; Hu, X.; Dizin, E.; Pei, D. J. Am. Chem. Soc. 2003, 125, 13379.
20. Turner, M. A.; Yang, X.; Yin, D.; Kuczera, K.; Borchardt, R. T.; Howell, P. L. Cell
Biochem. Biophys. 2000, 33, 101.
21. Yuan, C.-S.; Liu, S.; Wnuk, S. F.; Robins, M. J.; Borchardt, R. T. Biochemistry 1994,
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24. Zhang, T.; Lee, B. W. K.; Cannon, L. M.; Ritter, K. A.; Dai, S.; Ren, D.; Wood, T. K.;
Zhou, Z. S. Bioorg. Med. Chem. Lett. 2009, 19, 6200.
4.40. S-(1-Amino-1,4-anhydro-5-deoxy-1,N-didehydro-
5-yl N–Oxide)- -homocysteine (49)
D-ribitol-
L
25. Wnuk, S. F.; Lalama, J.; Garmendia, C. A.; Robert, J.; Zhu, J.; Pei, D. Bioorg. Med.
Chem. 2008, 16, 5090.
26. Wnuk, S. F.; Robert, J.; Sobczak, A. J.; Meyers, B. P.; Malladi, V. L. A.; Zhu, J.;
Gopishetty, B.; Pei, D. Bioorg. Med. Chem. 2009, 17, 6699.
27. Gopishetty, B.; Zhu, J.; Rajan, R.; Sobczak, A. J.; Wnuk, S. F.; Bell, C. E.; Pei, D. J.
Am. Chem. Soc. 2009, 131, 1243.
28. Jencks, W. P. Chem. Rev. 1972, 72, 705.
29. Pearson, M. S. M.; Mathé-Allainmat, M.; Fargeas, V.; Lebreton, J. Eur. J. Org.
Chem. 2005, 2159.
30. Kajimoto, T.; Liu, K. K. C.; Pederson, R. L.; Zhong, Z.; Ichikawa, Y.; Porco, J. A.;
Wong, C. H. J. Am. Chem. Soc. 1991, 113, 6187.
31. Wong, C.-H.; Provencher, L.; Porco, J. A.; Jung, S.-H.; Wang, Y.-F.; Chen, L.;
Wang, R.; Steensma, D. H. J. Org. Chem. 1995, 60, 1492.
32. Schramm, V. L. Acc. Chem. Res. 2003, 36, 588.
33. Schramm, V. L. Arch. Biochem. Biophys. 2005, 433, 13.
34. Yokoyama, M.; Momotake, A. Synthesis 1999, 1541.
35. Lee, J. E.; Singh, V.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Cornell, K. A.;
Riscoe, M. K.; Schramm, V. L.; Howell, P. L. J. Biol. Chem. 2005, 280, 18274.
36. Singh, V.; Evans, G. B.; Lenz, D. H.; Mason, J. M.; Clinch, K.; Mee, S.; Painter, G.
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Chem. 2005, 280, 18265.
37. Fleet, G. W. J.; Son, J. C. Tetrahedron 1988, 44, 2637.
38. Calvez, O.; Chiaroni, A.; Langlois, N. Tetrahedron Lett. 1998, 39, 9447.
39. Lee, J.; Hoang, T.; Lewis, S.; Weissman, S. A.; Askin, D.; Volante, R. P.; Reider, P. J.
Tetrahedron Lett. 2001, 42, 6223.
Treatment of 48 (72 mg, 0.15 mmol) with TFA by Procedure C
(step a, 5 h; step b, 6 h at 0 °C) gave crude 49. Purification on HPLC
(5% CH3CN/H2O at 2.5 mL/min; tR = 10–14 min) afforded 49
(16 mg, 40%) as a white solid: 1H NMR (D2O) d 2.01–2.17 (m, 2,
H8,8’), 2.83–2.89 (m, 2, H7,7’), 2.97 (dd, J = 6.3, 14.4 Hz, 1,H5),
3.06 (dd, J = 3.8, 14.4 Hz, 1, H5’), 3.73–3.77 (m, 1, H9), 4.08–4.22
(m, 1, H4), 4.40 (dd, J = 3.2, 6.0 Hz, 1, H3), 4.89–4.96 (m, 1, H2),
7.25 (s, 1, H1); 13C NMR d 27.6 (C7), 29.9 (C5), 30.5 (C8), 53.7
(C9), 70.4 (C3), 78.3 (C4), 80.7 (C2), 141.8 (C1), 173.9 (C10); MS
(APCI) m/z 265 (100, MH+); HRMS (TOF MS-ESI) m/z calcd for
C9H16N2O5SNa [M+Na]+ 287.0672; found 287.0664.
4.41. LuxS Inhibition Assay
SRH was prepared by incubating SAH (typically 10 mM) with
nucleosidase Pfs (2 lM) overnight at 4 °C and the completion of
the reaction was monitored spectrophotometrically by the absorp-
tion difference between SAH and adenine (
D
e
276 = ꢁ1.4 mMꢁ1
cmꢁ1). A typical LuxS reaction (total volume = 1.0 mL) contained
40. Haidle, A. M.; Myers, A. G. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12048.
41. Qiu, X. L.; Qing, F. L. J. Org. Chem. 2005, 70, 3826.
42. Kim, Y. J.; Kitahara, T. Tetrahedron Lett. 1997, 38, 3423.
43. Witte, J. F.; McClard, R. W. Tetrahedron Lett. 1991, 32, 3927.
44. Zanardi, F.; Battistini, L.; Nespi, M.; Rassu, G.; Spanu, P.; Cornia, M.; Casiraghi,
G. Tetrahedron: Asymmetry 1996, 1167, 7.
50 mM HEPES (pH 7.0), 150 mM NaCl, 17.8
5,5’-dithiobis(2-nitrobenzoic acid) (DTNB). The reaction was
initiated by the addition of LuxS (final concentration 0.8 M) and
monitored continuously at 412 nm (
= 14 000 Mꢁ1 cmꢁ1) in a
Perkin-Elmer k20 UV–vis spectrophotometer at room temperature.
lM SRH, and 150 lM
l
e
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