Novel S-ribosylhomocysteine analogues as potential inhibitors of LuxS enzyme

Article abstract of DOI:10.1080/15257770701513190

(Chemical Equation presented) Selective cross-coupling of the protected 6-fluoro-6-iodo-α-D-ribo-hex-5-enofuranose with 2 equivalents of 4-ethoxy-4-oxobutylzinc bromide in the presence of Pd[P(Ph)3]4 followed by deprotections gave methyl 5,6,7,8,9-pentadeoxy-6-fluoro-α/β-D-ribo- dec-5(Z)-enofuranuronate; a S-ribosylhomocysteine analogue with the sulfur and carbon-5 atoms replaced by the fluoro(vinyl) unit. Copyright Taylor & Francis Group, LLC.

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Novel S -Ribosylhomocysteine Analogues
as Potential Inhibitors of LuxS Enzyme
Stanislaw F. Wnuk a , Jennifer Lalama a , Jenay Robert a & Craig A.
Garmendia a
a Department of Chemistry & Biochemistry , Florida International
University , Miami, Florida, USA
Published online: 10 Dec 2007.
To cite this article: Stanislaw F. Wnuk , Jennifer Lalama , Jenay Robert & Craig A. Garmendia
(2007) Novel S -Ribosylhomocysteine Analogues as Potential Inhibitors of LuxS Enzyme, Nucleosides,
Nucleotides and Nucleic Acids, 26:8-9, 1051-1055, DOI: 10.1080/15257770701513190
To link to this article: http://dx.doi.org/10.1080/15257770701513190
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1051–1055, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701513190
NOVEL S-RIBOSYLHOMOCYSTEINE ANALOGUES AS POTENTIAL
INHIBITORS OF LUXS ENZYME
Stanislaw F. Wnuk, Jennifer Lalama, Jenay Robert, and Craig A. Garmendia
Department of Chemistry & Biochemistry, Florida International University, Miami,
2
Florida, USA
Selective cross-coupling of the protected 6-fluoro-6-iodo-α-D-ribo-hex-5-enofuranose with 2
2
equivalents of 4-ethoxy-4-oxobutylzinc bromide in the presence of Pd[P(Ph)3]4 followed by de-
protections gave methyl 5,6,7,8,9-pentadeoxy-6-fluoro-α/β-D-ribo-dec-5(Z)-enofuranuronate; a S-
ribosylhomocysteine analogue with the sulfur and carbon-5 atoms replaced by the fluoro(vinyl) unit.
Keywords S-Ribosylhomocysteine; Negishi cross-coupling; vinyl fluorides; luxS enzyme
INTRODUCTION
Hydrolytic cleavage of the S-adenosyl-L-homocysteine (SAH) by SAH
hydrolase produced adenosine (Ado) and L-homocysteine (Hcy).[1] The
cellular levels of SAH are critical because SAH is a potent feedback
inhibitor of crucial transmethylation enzymes. Alternatively, hydrolysis
of SAH by nucleosidase pfs yields adenine and S-ribosyl-L-homocysteine
(SRH). S-Ribosylhomocysteinase (LuxS) enzyme catalyzes the cleavage
of the thioether linkage in SRH to produce Hcy and 4,5-dihydroxy-2,3-
pentanedione (DHPD) (Figure 1).[2] DHPD spontaneously cyclizes to A and
complexes with borate to form a furanosyl borate diester, a small signaling
This investigation was partially supported by NIH/NIGMS S06GM08205. C.A.G and J.R were spon-
sored by MBRS RISE programs (NIH/NIGMS; R25GM61347). We wish to thank Professor Dehua Pei
and Jinge Zhu (The Ohio State University) for testing SRH analogues against LuxS enzyme.
Address correspondence to Stanislaw F. Wnuk, Department of Chemistry & Biochemistry, Florida
International University, Miami, FL 33199, E-mail: wnuk@fiu.edu
1051

1052
S.F. Wnuk et al.
FIGURE 1 Enzymatic conversion of SAH by Pfs nucleosidase and LuxS: Biosynthetic pathway to AI-2.
molecule called autoinducer (AI) of type 2. Autoinducers function in in-
terspecies communications and mediate a quorum sensing process of cell-cell
communication that bacteria use to coordinate gene expression in response
to fluctuation in cell density.[3]
LuxS is a metalloenzyme containing an Fe2+ ion coordinated by His-
54, His-58, and Cys-126 and a water molecule. The native enzyme is unsta-
ble under aerobic conditions however substitution of Co2+ for the native
metal ion produces a highly stable variant with wild-type catalytic activity.
In the proposed catalytic mechanism of LuxS, the metal ion acts as a Lewis
acid, facilitating two consecutive aldose-ketose (C1 → C2) and ketose-ketose
(C2 → C3) isomerization steps and a final β-elimination of Hcy from 3-keto
intermediates.[4]
Two substrate analogues S-anhydroribosyl-L-homocysteine (B) and S-
homoribosyl-L-cysteine (C) which were found to prevent the initial step and
the final step of the mechanism have been recently synthesized (Figure 2).[5]
Pei’s laboratory prepared a series of structural analogues in which the un-
stable enediolate moiety formed during isomerizations was replaced with
a planar hydroxamate group. The stable isostere D showed submicromo-
lar inhibition of the enzyme (KI = 0.72 µM).[6] We report herein synthesis
FIGURE 2 LuxS inhibitors.

Novel S-Ribosylhomocysteine Analogues
1053
of the SRH analogues with the sulfur and carbon-5 atoms replaced by a
fluoro(vinyl) unit (e.g., 6), which are not capable to undergo the final elim-
ination step. These ribosyl (depurinated) analogues of SAH were also de-
signed as probes to evaluate similarities between SAH hydrolase and SRHase
(LuxS).
RESULTS AND DISCUSSION
Treatment of the diacetone 3-O-benzoylallose with periodic acid ef-
fected the regioselective removal of 5,6-O-isopropylidene group, and se-
quential oxidative cleavage of the exposed vicinal diol[7] gave the ribose 5-
aldehyde 1 (Scheme 1). Wittig-Horner treatment of 1 with sulfonyl-stabilized
fluorophosphonate[8] gave 6-(fluoro)homovinyl sulphones 2. Stannyldesul-
fonylation followed by iododestannylation[9] of the resulting vinyl stan-
nanes 3 afforded the protected 5,6-dideoxy-6-fluoro-6-iodo-α-D-ribo-hex-5-
enofuranose 4 (E/Z, 3:2). Trans selective Negishi cross-coupling[10] of 4
using 2 equivalents of 4-ethoxy-4-oxobutylzinc bromide gave fluoro(vinyl)
SRH analogue 5 (Z, 54%; 90% based on the conversion of the E iso-
mer only).[11] Treatment of 5 with NH3/MeOH affected debenzoylation
and transesterification and subsequent deacetonization with aqueous triflu-
oroacetic acid (TFA) gave methyl 5,6,7,8,9-pentadeoxy-6-fluoro-α/β-D-ribo-
dec-5(Z)-enofuranuronate 6 (40%; α/β, 3:7). To investigate whether the
stereochemistry of the 3-OH group in the SRH analogues can effect a sec-
ond enolization step,[4a] the xylo analogue of 6 was prepared similarly from
diacetone 3-O-benzoylglucose. Moreover, debenzoylation of 3 (E/Z, 7:3)
with NH3/MeOH and subsequent concomitant protiodestannylation and
deacetonization with TFA/H2O afforded 5,6-dideoxy-6-fluoro-D-ribo-hex-5-
enofuranose (E/Z, ∼3:1; α/β ∼1:6).
SCHEME 1

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S.F. Wnuk et al.
The 5,6,7,8,9-pentadeoxy-6-fluoro-D-ribo-dec-5(Z)-enofuranuronate 6
and 5,6-dideoxy-6-fluoro-D-ribo-hex-5-enofuranose and their xylo epimers
were evaluated as potential inhibitors of Bacillus subtilis S-ribosylhomocys-
teinase (LuxS) using the inhibition assays as described previuosly.[6] None
of the compounds showed significant activity. These results might indicate
that LuxS has more rigid requirements for binding than SAH hydrolase
and that the intact Hcy unit in substrate/inhibitor is required for proper
binding.
REFERENCES
1. a) Yuan, C.-S.; Liu, S.; Wnuk, S.F.; Robins, M.J.; Borchardt, R.T. In Advances in Antiviral Drug De-
sign; De Clercq, E., Ed.; JAI Press: Greenwich, 1996; vol. 2, pp. 41–88; b) Wnuk, S.F. Targeting
“hydrolytic” activity of the S-adenosyl-L-homocysteine hydrolase. Mini-Rev. Med. Chem. 2001, 1, 307–
316.
2. a) Chen, X.; Schauder, S.; Potier, N.; Van Dorsselaer, A.; Pelczer, I.; Bassler, B.L.; Hughson, F.M.
Structural identification of a bacterial quorum-sensing signal containing boron. Nature 2002, 415,
545–549; b) Pei, D.; Zhu, J. Mechanism of action of S-ribosylhomocysteinase (LuxS). Curr. Opin.
Chem. Biol. 2004, 8, 492–497.
3. Miller, M.B.; Bassler, B.L. Quorum sensing in bacteria. Annu. Rev. Microbiol. 2001, 55, 165–
199.
4. a) Zhu, J.; Hu, X.; Dizin, E.; Pei, D. Catalytic mechanism of S-ribosylhomocysteinase (LuxS): Direct
observation of ketone intermediates by 13C NMR spectroscopy. J. Am. Chem. Soc. 2003, 125, 13379–
13381; b) Rajan, R.; Zhu, J.; Hu, X.; Pei, D.; Bell, C.E. Crystal structure of S-ribosylhomocysteinase
(LuxS) in complex with
3753.
a catalytic 2-ketone intermediate. Biochemistry 2005, 44, 3745–
5. Alfaro, J.F.; Zhang, T.; Wynn, D.P.; Karschner, E.L. Zhou, Z.S. Synthesis of LuxS inhibitors targeting
bacterial cell-cell communication. Org. Lett. 2004, 6, 3043–3046.
6. Shen, G.; Rajan, R.; Zhu, J.; Bell, C.E.; Pei, D. Design and synthesis of substrate and intermediate
analogue inhibitors of S-ribosylhomocysteinase. J. Med. Chem. 2006, 49, 3003–3011.
7. Xie, M.; Berges, D.A.; Robins, M.J. Efficient “dehomologation” of di-O-isopropylidenehexofuranose
derivatives to give O-isopropylidenepentofuranoses by sequential treatment with periodic acid in
ethyl acetate and sodium borohydride. J. Org. Chem. 1996, 61, 5178–5179.
8. Wnuk, S.F.; Bergolla, L.A.; Garcia, P.I., Jr. Studies toward the synthesis of α-fluorinated phosphonates
via tin-mediated cleavage of α-fluoro-α-(pyrimidin-2-ylsulfonyl)alkylphosphonates. Intramolecular
cyclization of the α-phosphonyl radicals. J. Org. Chem. 2002, 67, 3065–3071.
9. Wnuk, S.F.; Yuan, C.-S.; Borchardt, R.T.; Balzarini, J.; De Clercq, E.; Robins, M.J. Nucleic acid-related
compounds. 84. Synthesis of 6ꢁ-(E and Z)-halohomovinyl derivatives of adenosine, inactivation of
S-adenosyl-L-homocysteine hydrolase, and correlation of anticancer and antiviral potencies with
enzyme inhibition. J. Med. Chem. 1994, 37, 3579–3587.
10. For recent developments on the selective monoalkylation of vinyl dihalides with the alkylzinc halides
(Csp2−Csp3 cross-couplings) see: a) Andrei, D.; Wnuk S.F. Synthesis of the multisubstituted halo-
genated olefins via cross-coupling of dihaloalkenes with alkylzinc bromides. J. Org. Chem. 2006, 71,
405–408; b) Tan, Z.; Negishi, E.-I. Widely applicable Pd-catalyzed trans-selective monoalkylation of
unactivated 1,1-dichloro-1-alkenes and Pd-catalyzed second substitution for the selective synthesis
of E or Z trisubstituted alkenes. Angew. Chem. Int. Ed. 2006, 45, 762–765.
11. Typical Procedure: Pd[P(Ph)3]4 (22 mg, 0.01 mmol) was added to a stirred solution of 4 (42 mg,
0.097 mmol; E/Z, 3:2) in anhydrous benzene (4 mL) under N2 at ambient temperature. After
2 minutes, 4-ethoxy-4-oxobutylzinc bromide (0.5M/THF; 0.30 mmol, 0.60 mL) was added and the
resulting mixture was heated at 55◦C for 5 hours. EtOAc (30 mL) and NaHCO3/H2O (10 mL) were
added and the separated organic layer was washed with H2O (10 mL), NaCl/H2O (10 mL), dried
(Na2SO4), and then was evaporated. Column chromatography (10 → 30% EtOAc/hexanes) gave
5(Z) (22 mg, 54%; 90% based on the conversion of E-isomer): 1H NMR δ 1.24 (t, J = 7.1 Hz, 3,

Novel S-Ribosylhomocysteine Analogues
1055
CH3), 1.37 & 1.60 (2 × s, 2 × 3, 2 × CH3), 1.84 (“quint”, J8-7/7 /9/9 = 7.4 Hz, 2, H8/8ꢁ), 2.25 (dt,
ꢁ
ꢁ
J7-F = 17.6 Hz, J7-8/8 = 7.5 Hz, 2, H7/7ꢁ), 2.32 (t, J9-8/8 = 7.4 Hz, 2, H9/9ꢁ), 4.09 (q, J = 7.1 Hz,
2, CH2), 4.72 (dd, J3-4 = 9.2 Hz, J3-2 = 4.7 Hz, 1, H3), 4.75 (dd, J5-F = 35.0 Hz, J5-4 = 8.9 Hz, 1,
H5), 4.95 (t, J2-1/3 = 4.3 Hz, 1, H2), 5.19 (t, J4-3/5 = 9.1 Hz, 1, H4), 5.89 (d, J1-2 = 3.8 Hz, 1, H1),
ꢁ
ꢁ
7.48–8.09 (m, 5, Ar); 19F NMR δ −102.14 (dt, JF −H5 = 34.1 Hz, JF -H7/7 = 17.6 Hz); HRMS (AP-ESI)
ꢁ
m/z: calcd for C22H28FO7 (MH+) 423.1814; found 423.1815.