Synthesis of isomeric analogues of S-ribosylhomocysteine analogues with homocysteine unit attached to C2 of ribose

Article abstract of DOI:10.1016/j.bmcl.2017.03.004

LuxS (S-ribosylhomocysteinase; EC 4.4.1.21) is an enzyme that catalyzes the cleavage of the thioether linkage in the catalytic pathway of S-ribosylhomocysteine (SRH) which produces homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). DPD is the precursor of the signaling molecules known as autoinducer 2 (AI-2) responsible for the bacterial quorum sensing (QS) identified as cell to cell communication. Inhibitors of LuxS should be able to interfere with its catalytic pathway thus preventing the formation of the autoinducer molecules. In this work, the synthesis of 2-deoxy-2-bromo-SRH analogues was attempted by the coupling of the corresponding 2-bromo-2-deoxypentafuranosyl sugars with the homocysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the mesylate group from C5 was observed leading to a novel isomeric analogue of SRH in which Hcy moiety is attached to a ribose ring via C2-sulfur bond.

Full text of DOI:10.1016/j.bmcl.2017.03.004

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of isomeric analogues of S-ribosylhomocysteine analogues
with homocysteine unit attached to C2 of ribose
Christiane Chbib
Department of Pharmaceutical Sciences, Larkin College of Pharmacy, 18301 North Miami Ave, Miami, FL 33169, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
LuxS (S-ribosylhomocysteinase; EC 4.4.1.21) is an enzyme that catalyzes the cleavage of the thioether
linkage in the catalytic pathway of S-ribosylhomocysteine (SRH) which produces homocysteine and
4,5-dihydroxy-2,3-pentanedione (DPD). DPD is the precursor of the signaling molecules known as autoin-
ducer 2 (AI-2) responsible for the bacterial quorum sensing (QS) identified as cell to cell communication.
Inhibitors of LuxS should be able to interfere with its catalytic pathway thus preventing the formation of
the autoinducer molecules. In this work, the synthesis of 2-deoxy-2-bromo-SRH analogues was
attempted by the coupling of the corresponding 2-bromo-2-deoxypentafuranosyl sugars with the homo-
cysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the
mesylate group from C5 was observed leading to a novel isomeric analogue of SRH in which Hcy moiety is
attached to a ribose ring via C2-sulfur bond.
Received 25 January 2017
Revised 1 March 2017
Accepted 2 March 2017
Available online xxxx
Ó 2017 Elsevier Ltd. All rights reserved.
Bacteria are known to release a large variety of small molecules.
They also produce and respond to diffusible signal molecules
(termed autoinducers or pheromones). One of the cell to cell
communication subtypes is related to population density in
bacteria known as quorum sensing (QS) (Fig. 1). It is shown to be
accomplished through the exchange of extracellular signaling
molecules called autoinducers.1–4 LuxS is a small metalloenzyme
containing Fe2+ ion tetrahedrally coordinated by His-54 and
to prevent the final mechanistic step of LuxS catalytic cycle.
Moreover, brominated furanone derivatives were found to modify
LuxS selectively leading to the covalent inhibition.17
His-58, Cys-126 and
a water molecule. A highly conserved
Cys-84 is located 4.86 Å from the metal ion. It is widely preserved
among Gram negative and Gram positive bacteria.5–9 Pei and
coworkers in 2003 proposed the pathway for the catalytic mecha-
nism of S-ribosylhomocysteine presented in Scheme 1.
Various S-ribosylhomocysteine [SRH] analogues have been
designed as mechanistic probes and/or inhibitors of the LuxS
enzyme.10 The most important one among them is the family of
SRH analogues that targets mechanistic steps of LuxS catalytic
cycle by effecting the initial ring opening step e.g., 1-deoxy-SRH
analogue11 and [4-aza]12 or 4-[thio]-SRH analogues13 or one of
the tautomerization/isomerization steps. These included
substrates lacking enolizable hydroxyl group at C3 (e.g., OMe),14
including mechanistically significant C3 halogenated [3-Br or F]-
SRH analogues.15 Zhou and coworkers synthesized substrate
analogue S-homoribosyl-L-cysteine11 and Chbib and coworkers
prepared 4-C-alkyl/aryl-SRH derivatives16 which were designed
Fig. 1. X-ray crystallography of LuxS enzyme (Resolution: 2.2 Å, R-Value Free:
E-mail address: cchbib@ularkin.org
0.242, R-Value Work: 0.174).
http://dx.doi.org/10.1016/j.bmcl.2017.03.004
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chbib C. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.03.004

2
C. Chbib / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
O
The initial objective of this research was the design and synthe-
O
S
S
sis of novel LuxS inhibitors in which the hydroxyl group at C2 in
the ribofuranose ring of the S-ribosylhomocysteine would be sub-
stituted by halogens such as bromide. The two main targets of the
C2-substituted analogues were 2-deoxy-2-bromo-d-ribosylhomo-
cysteine (2-[Br]-SRH, 10) and its arabino-epimer 11 (Fig. 2). The
targeted 2-halo-SRH analogues were envisioned to interact with
the LuxS protein differently than the natural substrate S-ribosylho-
mocysteine does, upon binding to the LuxS enzyme. Thus, 2-halo-
SRH analogues, lacking the 2-hydroxyl group, should prevent the
formation of the 2-keto intermediates (e.g., complex 2, Scheme 1)
during the interaction with LuxS. Herein we reported synthesis of a
novel isomeric analogues of SRH in which Hcy moiety is attached
to a ribose ring via C2-sulfur bond.
The displacement of the bromide by cysteinate thiol could occur
at either closed or opened form of the ribose ring. However,
because of the different stereochemical requirements of the bromo
substituent in ribo 10 and arabino 11 substrate, inhibition might
also shed some light on the leniency of the active site for the stere-
ochemical regimen of the substituent at the C2 of the ribose ring.
For example, the bromo substituent at the b face in the arabino
substrate 11 might prevent SN2 displacement which requires a
nucleophilic attack by Cys84 from the opposite site of the leaving
group (bromide). The fact that targeted 2-halo-2-deoxy-SRH
analogues lack the 2-hydroxyl group, the first tautomerization step
and the generation of the 2-keto-SRH intermediate, which is
critical for the enzymatic activity of LuxS might be prevented. I
expect that direct replacement of bromine by Cys-84 via SN2 mech-
anism might lead to the covalent inhibition by the attack of Cys-84
at C2. The attack might happen at the hemiacetal (path 1,
Scheme 2) or opened ring form of the bromo-substrate (path 2,
Scheme 2). The formation of enzyme-inhibitor complexes can be
detected using mass spectroscopy.
O
O
HO
HO
OH
OH
Br
NH2
NH2
Br
HO
HO
10
11
2-[Br]-ara-SRH
2-[Br]-SRH
Fig. 2. Targeted 2-deoxy-2-bromo S-ribosylhomocysteine analogues.
Synthesis of 2-bromo-2-deoxy-ribono/arabinonolactones
The synthesis of the 2-bromo-2-deoxy-SRH 10 was divided into
two steps. In the first step the 2-bromo-2-deoxy precursor 21 was
prepared (Scheme 3), while the second step was envisioned as the
coupling between 21 and the homocysteine thiolate 25. The
bromo-sugar precursor 21 was prepared according to the literature
report.18 Thus, oxidation of the 2-deoxyribose 16 with Br2 /H2O
yielded 2-deoxyribonolactone 17. Treatment of the resulting 17
with TBDMSCl produced a disilylated ribonolactone 18 with 80%
yield. Direct bromination of 18 with NBS, following the procedure
developed by Sauve,18 led to the formation of the bromo-lactone
19 as a mixture of arabino/ribo epimers in 2:1 ratio. The formation
of 19A and 19B was consistent with the bromination of the inter-
mediate enolate. However, the obtained mixture of the arabino and
ribo-epimers differed from the reporting in literature (arabino/ribo,
1:1.4)18 ratio. The arabino/ribo epimers of 19 were successfully sep-
arated using column chromatography. Compound 19 was consid-
ered as a precursor for the synthesis of the mesylated derivative
21 intended to be coupled with the homocysteine thiolate to afford
the desired final product 26. Thus, deprotection of 19 with TFA/
H2O (9:1) effected selective removal of TBDMS group from the
RS
RS
H
RS
H
:S-C84
OH
:S-C84
HS-C84
HS-C84
OH
OH
OH
RS
H
H
H
H
5
H2O
O
H
1
4
H
O
H
O
H
O
H
3
2
HO
O
HO
O
HO
O
H
M2+
M2+
O
OH2
M2+
H
O
H
M2+
57E
HO
OH
57E
O
O
H
2
1
3
RS
RS
H
RS
H
RS
H
:S-C84
H
HS-C84
57E
HS-C84
H
H
:S-C84
OH
OH
OH
OH
H
H
H
H
H
H
RSH
OH
OH
OH
OH
O-
O
O
H
O
O
O
O
O
OH
O
H
O
O
O
O
O
H
M2+
M2+
57E
57E
E57
M2+
M2+
O
4
5
:S-C84
RSH
:S-C84
OH
RS
OH
OH
H
H
H
H
OH
2
O
4
O
H
O
OH
OH
H2O
O
3
5
O
OH
O
OH
1
O
OH
O
M2+
M2+
E57
DPD
HO
7
8
9
6
Scheme 1. Proposed mechanism of LuxS-catalyzed reaction.5,6
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C. Chbib / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Cys-84
S
Cys-84
-S
RS
RS
RS
O
O
O
Path 1
OH
OH
OH
Br
HO
RS
HO
Br
HO
12
10
Cys-84
-S
Cys-84
S
H
H
RS
H
RS
OH
OH
OH
Path 2
O
O
O
M2+
HO
Br
M2+
HO
HO
Br
M2+
14
15
13
Scheme 2. A plausible inhibition of LuxS by 2-[Br]-SRH via SN2 mechanism.
Br2/H2O
5 days
TBDMSCl
O
O
O
TBDMSO
TBDMSO
HO
HO
O
OH
O
Imidazole
DMF
HO
HO
17
18
16
1. TMSOTf/Et3N
2. NBS/CH2Cl2
Br
Br
O
HO
TBDMSO
20
TFA/H2O
9/1
O
MsCl,Et3N
CH2Cl2
O
x
TBDMSO
TBDMSO
MsO
TBDMSO
21
O
O
O
y
19A X= H Y=Br
19B X= Br Y=H
Scheme 3. Synthesis of the 2-bromo-2-deoxy-5-O-mesyl-ribono/arabinono lactone derivatives.
primary 5-hydroxyl group of 19 to afford 20 in 90% yield. Treat-
ment of 20 with mesyl chloride in pyridine produced the desired
5-O-mesyl derivative 21 in 60% yield (Scheme 3).
than organic soluble trialkylphosphines19,20 simplified the prepa-
ration of the protected homocysteine substrate 25.
Attempted couplings
Synthesis of protected homocysteine
Two methods for the displacement of the mesylate group in the
ribonolactone 21 with thiolate anion generated from homocysteine
25 were attempted. In the first approach, LDA was used as a base
and DMF as a solvent. Thus, coupling of 25 and 21 in DMF resulted
in the formation of the complex reaction mixture. Careful purifica-
tion of the crude reaction mixture on the column chromatography
resulted in the isolation of few products in low yields which
showed no presence of bromine in their mass spectra. In a second
approach K2CO3 was used as a base and dry acetone as solvent. The
resulting complex reaction mixture was purified on column chro-
matography but desired product 27 was not isolated (Scheme 5).
Proton NMR of the crude reaction mixture showed however the
The protected homocysteine precursor 25 was prepared start-
ing from the commercially available l-homocystine 22 following
the literature protocol19 (Scheme 4). Thus, treatment of the homo-
cystine 22 with di-t-butyl dicarbonate gave N-Boc protected homo-
cysteine 23. Next, the carboxylic group in 23 was protected as tert-
butyl ester upon treatment with diisopropylcarbodiimide (DIC)
and t-butanol. The reduction of the disulfide bond in 24 with
triscarboxyethylphosphine hydrochloride (TCEP) followed by
aqueous workup afforded the homocysteine 25 of the appropriate
purity for direct coupling with the sugar precursors. It is notewor-
thy that employing TCEP14 as water soluble reducing agent rather
t-BuOCOCH(NHBoc)CH2CH2SH
O
MsO
TBDMSO
26
O
MsO
O
25
O
O
O
(t-BuOCO)2O
Na2CO3/Dioxane
S
S
K2CO3/dry Acetone/DMF
S
Br
HO
TBDMSO
HO
2
2
NH2
21
NHBoc
BocHN
22
23
O
t-butanol/DIC
CuCl2/CH2Cl2
O
NBoc
O
O
O
TCEP/H2O
DMF
O
S
SH
S
O
O
O
2
O
NHBoc
25
NHBoc
24
Br
TBDMSO
27
Scheme 5. Coupling between the 5-O-mesyl-2-bromopentafuranose lactone and
homocysteine thiolate.
Scheme 4. Synthesis of homocysteine-thiolate.
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C. Chbib / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
O
analogue of SRH in which Hcy moiety is attached to a ribose ring
O
MsO
MsO
CH3CH2CH2SH
LDA/DMF
O
O
via C2-sulfur bond. Model reaction with propanethiol confirmed
that the displacement of the bromide from C2 takes precedence
over the substitution of the mesylate from the C5 leading to the
formation 2-S-propyl substituted 2-thioribonolactone. The interac-
tion of LuxS with novel isosteric isomer of SRH with Hcy unit
attached to ribose at C2 position rather than C5 in my finding,
could interact with LuxS via ring opening.
S
Br
TBDMSO
TBDMSO
21
28
Scheme 6. Model reaction of bromo-lactones with propylthiol
Inhibition of LuxS enzyme by the
a-keto substituent
analogue 29 might occur by the nucleophilic displacement of R0
(R0 = S-CH2-CH2-CH3). Based on the previous studies with
3-halo-SRH analogues,15 it is reasonable to expect that after ring
Cys-84
-S
Cys-84
S
RS
H
RS
opening the reactive
be a substrate for nucleophilic substitution by the active site
Cys-84 leading to a covalent inhibition as depicted in Scheme 7.
a-keto substituent intermediate 30 might
RS
OH
O
O
OH
O
OH
M2+
HO
HO
HO
R'
R'
29
31
30
Notes
Scheme 7. Plausible inhibition of LuxS by 2-deoxy-2-propylthiol-S-
ribosylhomocysteine.
LuxS PDB ID code 1IE0.
Acknowledgment
characteristic pattern for the protons of homocysteine and sugar
moieties. However, the mass spectra analysis of the major fraction,
showed no presence of the typical pattern of bromine isotopes
(M/M+2). The unexpected displacement reactions at C2 may be
I would like to thank Dr. Stanislaw F. Wnuk from Florida Inter-
national University for providing his laboratory facility and a help-
ful discussion during the preparation of this manuscript.
Department of Chemistry and Biochemistry Florida Interna-
tional University, Miami, FL 33199, United States. Tel.: +1 (305)
348 6195. E-mail: wnuk@fiu.edu.
attributed to the fact that the bromine at the C2 is
a to an ester
carbonyl in the ribonolactone precursor 21 which is also a good
leaving group and might be involved in nucleophilic substitution
reaction with homocysteinate salts leading to 26. To prove this
hypothesis, I carried out a reaction between 21 with model
alkylthiol.
A. Supplementary data
In order to optimize conditions for the reactions of bromo-lac-
tone 21 with thiols, I carried out reactions between 21 and propy-
lthiol instead of homocysteine under different conditions. Thus,
treatment of 21 (R/S, 60:40) with 1 equiv. of the propylthiolate
generated from propylthiol/LDA in DMF at 0 °C resulted in the for-
mation of new product and disappearance of substrate 21. Purifica-
tion of the crude reaction mixture on the silica column
chromatography gave 1:1 mixture of the 2-S-propyl substituted
2-thiolactone 28 (as 1:1 mixture of the arabino/ribo epimers) in
overall yield of 61% (Scheme 6). Structure for 28 was established
based on spectroscopic data. Thus, 1H NMR spectra shows the sin-
glet at 3.06 ppm indicative of the presence of the mesyl group in
addition to the characteristic peaks for the propylthiol moiety.
On 13C NMR, peaks at 171.47/172.74 ppm indicate the presence
of the carbonyl groups in the lactone 28 for the arabino-ribo epi-
mers, while the lack of the bromine patterns (M/M+2) on mass
spectra analysis suggested the substitution at C2 and the loss of
the bromine substituent. These results indicate that the secondary
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.03.
004.
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