Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform

Article abstract of DOI:10.1016/j.bmc.2009.02.008

cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit Staphylococcus aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents.

Full text of DOI:10.1016/j.bmc.2009.02.008

Bioorganic & Medicinal Chemistry 17 (2009) 2886–2893
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of
mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform
b
b
Konstantin V. Kudryavtsev a, , Matthew L. Bentley , Dewey G. McCafferty
*
a Department of Chemistry, M.V. Lomonosov Moscow State University, 119991, Moscow, Russian Federation
b Department of Chemistry, Duke University, Durham, NC 27708-0317, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were
synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile.
4-Vinylsulfonyl 5-phenyl prolinates inhibit Staphylococcus aureus sortase SrtA irreversibly by modifica-
tion of the enzyme Cys184 and could be used as hits for the development of antibacterials and antiviru-
lence agents.
Received 25 December 2008
Revised 6 February 2009
Accepted 7 February 2009
Available online 13 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Staphylococcus aureus
Sortase
Inhibitor
Vinyl sulfone
1. Introduction
small-molecule inhibitors4–12 are presented in Figure 1. Three
types of small molecules, aryl (b-amino)ethyl ketones,7 trans-3-
Methicillin-resistant Staphylococcus aureus (MRSA) has been
nominated by the Antimicrobial Availability Task Force of the
Infectious Diseases Society of America as one of six high-priority
problematic pathogens.1 This nomination reflects the high inci-
dence of MRSA infections, substantial morbidity, and peculiar vir-
ulence factors circumventing usual antimicrobial therapy. Other
concerns are caused by emerging resistance of S. aureus strains to
modern therapies and a lack of novel drug candidates, especially
those with new mechanism of action. S. aureus, like other Gram-
positive organisms, utilizes surface proteins for adhesion to host
cells and invasion of tissues. The vast majority of surface proteins
involved in these aspects of staphylococcal disease are substrates
of sortases — cysteine transpeptidases which link surface proteins
to peptidoglycan, thus incorporating them into the envelope and
leading to their display on the microbial surface.2 S. aureus strains
in which the srtA gene has been deleted display no defect in sur-
vival and growth but exhibit reduced pathogenicity and virulence,
due to a failure to display MSCRAMMs (microbial surface compo-
nents recognizing adhesive matrix molecules) on the bacterial
surface. The same effect has been demonstrated for wild Gram-po-
sitive organisms treated with sortase inhibitors, implicating sor-
tase enzymes as important targets for the treatment of
staphylococcal disease.2 Different types of sortase inhibitors have
been recently reviewed,3 and the most active and promising
(furan-2-yl) acrylic acid amides8 and aaptamines,12 have been re-
ported since the publication of the review. This work describes
our studies towards the design and synthesis of novel types of S.
aureus SrtA inhibitors, and an understanding of their mechanisms
of action.
S. aureus sortase SrtA is a 206 amino acid cysteine transpepti-
dase with an N-terminal transmembrane anchor. His120, Cys184
and Arg197 conserved side chains constitute the enzyme active
site.2,3 Of the reported small molecule inhibitors of SrtA, only a
few have been subjected to detailed kinetic and mechanistic stud-
ies. Commercial vinyl sulfones4 and aryl (b-amino)ethyl ketones7
irreversibly modify Cys184, preventing acylation of the Thr-Gly
carbonyl, and thus cleavage of the sorting signal. Diarylacrylonitr-
iles demonstrate a competitive character of inhibition,5 although
the strong Michael acceptor moiety of these molecules does not
exclude interaction with different nucleophilic species in vivo.
Synthetic trans-3-hetaryl acrylic acid amides SrtA inhibitors have
been developed from in silico virtual screening of commercial
compound libraries and docking model interactions of inhibitors
with amino acid residues in SrtA active site have been identified
recently.8 All other small-molecule inhibitors of SrtA have been
isolated from natural plant extracts, and only indirect data of
their mechanism of interaction with the enzyme are available.3
Although X-ray structures of both S. aureus SrtA with its substrate
complex13 and Bacillus anthracis SrtB with and without inhibitor7
have been solved, this information is insufficient to allow for
* Corresponding author. Tel.: +7 4959393564; fax: +7 4959328846.
E-mail address: kudr@org.chem.msu.ru (K.V. Kudryavtsev).
construction of
a
pharmaceutically suitable inhibitor and
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.008

K. V. Kudryavtsev et al. / Bioorg. Med. Chem. 17 (2009) 2886–2893
2887
MeO
O
O
N
O
O
Me
Cl
Cl
S
O
N
N
OMe
Me
O
N
H
COOMe
MeO
736 µM [4]
9.2 µM [5]; 2.7 µg/ml [6]
15 µM [7]
58 µM [8]
OH
OSO3-
Br
NH2+
O
O
Br
N+
Me
Cl-
N
N
Me
OMe
OMe
O
O
S
S
N
N
N
N
HO
OH
8.7 µg/ml [6]
39.4 µg/ml [6]
OH
HO
H
HO
O
H
H
O
OH
HO
O
OH
OH
O
OH
OH
18.3 µg/ml [6]; 31.72 µM [9]
37.39 µM [9]
OMe
N
H
O
HO
Me
O
O
N
OMe
OH
MeO
HO
N
N
N
H
N
H
15.67 µg/ml [10]
13.8 µg/ml [11]
3.7 µg/ml [12]
Figure 1. Small-molecule SrtA inhibitors. IC50 values with literature source (in square brackets) are indicated.
cysteine protease inhibitors.15 Similarly, the nitrile group has been
shown to be a key structural element for effective SrtA inhibition.5
We planned to attach vinyl sulfonyl and nitrile groups to a func-
tionalized pyrrolidine scaffold and to synthesize molecules A and
B for subsequent testing of S. aureus SrtA inhibition (Fig. 2). Besides
conformational constraints these molecules have the following
attractive properties for drug discovery: druglikeness, the possibil-
ity of synthesis of a diverse set of analogs (combinatorial ap-
proach), an amino acid motif for possible additional interactions
with the enzyme, and 5-aryl- and R2-substituents as points for
hydrophobic interactions with the enzyme. 1,3-Dipolar cycloaddi-
tion of appropriate dipolarophiles to azomethine ylides was uti-
lized as an effective synthetic strategy16,17 for A and B synthesis.
Target compounds were synthesized from commercial benzalde-
MeO
N
O
O
S
OMe
MeO
N
O
S
O
R2
COOR3
R2
COOR3
N
H
N
H
B
A
R1
R1
hydes, a-amino acid esters, divinyl sulfone (DVS) and acrylonitrile,
using established methods.18,19 Starting imino esters 1 have been
synthesized from substituted benzaldehydes and methyl esters of
glycine, alanine and glutamic acid (Table 1) under dehydrating
conditions (Scheme 1).
Figure 2. Structural design of novel 5-phenyl proline SrtA inhibitors.
additional studies of sortase–small molecules interactions are de-
sired. We have employed conformationally constrained deriva-
tives of cis-5-phenyl prolines with directed functional groups to
evaluate the binding modes of these compounds with S. aureus
SrtA (Fig. 2).
Interaction of imino esters 1 with divinyl sulfone was achieved
through preliminary generation of silver(I)-azomethine ylide
(Scheme 2). The subsequent cycloaddition proceeded stereospecif-
ically as an endo-process, leading to racemic 5-phenyl prolinates 2
with cis-stereochemistry at the phenyl, methoxycarbonyl and
vinylsulfonyl substituents. Structural assignments were confirmed
by X-ray crystallography of 2c20 and 2j.18 In the case of glycine imi-
no esters 1a–c, yields of target pyrrolidinyl vinyl sulfones 2a–c
were low due to the concurrent conjugated addition of divinyl sul-
fone and subsequent tandem transformations.21 Glutamate imino
esters 1j–l formed cycloadducts 2j–l as single products with high
yields and allowed for subsequent ring annulation under acidic
2. Chemistry
Only two types of synthetic compounds were known to inhibit
SrtA at the beginning of this project — vinyl sulfones4 and diary-
lacrylonitriles.5 The utility of vinyl sulfones for different medicinal
chemistry applications has been recently reviewed.14 As effective
Michael acceptors, some vinyl sulfones are in clinical studies as

2888
K. V. Kudryavtsev et al. / Bioorg. Med. Chem. 17 (2009) 2886–2893
Table 1
Substituents in synthesized compounds
Index
a
b
c
d
e
f
g
h
j
k
l
R1
R2
H
H
2-F
H
3-F
H
3-Cl
H
2,3-DiF
H
2-Cl
Me
4-Cl
Me
2-Me
Me
H
3,4-DiMeO
CH2CH2COOMe
3-Pyridyl
CH2CH2COOMe
CH2CH2COOMe
R2
O
H
H
O
2
R
R
2
O
S
O
H
OMe
N
H
OMe
O
H2N
+
O
N
O
H3
R1
1
R
R
x HCl
O
1a-k
OMe
O
H
OMe
OMe
O
Figure 3. NOESY interactions in 3j.
OMe
N
O
H2N
+
O
N
O
N
3. Inhibition studies with SrtA
D
N24
x HCl
1l
Scheme 1. Imino esters synthesis. Reaction conditions: MgSO4, Et3N, CH2Cl2, rt.
As a first test, the compounds were assayed for the ability to in-
hibit SrtA activity in an HPLC-based assay at three separate con-
centrations: 39.1 lM, 625 lM, and 5 mM. Compounds 2a–c
conditions, and synthesis of pyrrolizidinyl vinyl sulfones 3j–l
(Scheme 2). Compounds 2k,l and 3j–l are novel and were exhaus-
tively characterized by NMR and elemental analysis. Pyrrolidines
2k,l differ from the X-ray defined 2j only by the type of aryl substi-
tuent and all three of these analogs were characterized by well-
correlated signals in 1H NMR spectra. The stereochemistry of pyrr-
olizidinones 3 was confirmed by cross-peaks between H2/H3 and
phenyl/methoxy hydrogens in NOESY experiments (Fig. 3).
displayed complete inhibition at a concentration of 5 mM, while
the remainder showed more modest inhibition. However, only
the vinyl sulfone compounds were potent inhibitors at mid-micro-
molar concentrations. The carbonitriles 4–6 and 9 failed to com-
pletely inhibit SrtA even at a concentration of 5 mM and were
not evaluated further.
In order to more accurately rank the potencies of our inhibitory
compounds, we performed dose–response studies to determine
IC50 values for each of the compounds. Compounds 2a–c, k, l,
Cycloadditions of acrylonitrile to azomethines 1a–h were con-
ducted using the published protocol of Tsuge and co-workers.22
Lithium bromide was used as a Lewis acid for metallodipole gener-
ation, and previously subsequent stereospecific endo-cycloaddition
with tert-butyl acrylate was applied for the synthesis of diverse
proline-glutamate chimeras.19 In the case of the acrylonitrile dipol-
arophile, the stereoselectivity of the cycloaddition is poor and two
products are formed (Scheme 3). Preliminary structural assign-
ments of cycloaddition products have been made using published
spectral data for isomeric 4a and 5a,22 that differs from that shown
in Scheme 3 in the stereochemistry of the 4-cyano substituents.
Following transformation of major isomer 4a into thymine-
substituted pyrrolidine 9 and X-ray structural analysis of 9 re-
vealed cis-stereochemistry of the cyano group with respect to the
phenyl and (N1-thyminyl)methyl substituents of the final product
(Scheme 4).23 This places the cyano group of the pyrrolidine ring
of isomer 4a and intermediates 6–8 with a cis orientation with re-
spect to the phenyl substituent. Thus, we believe the position of
the 4-cyano group in pyrrolidines 4a and 5a was likely assigned
incorrectly,22 and the major cycloaddition products 4 correspond
to an endo-process as for the majority of other dipolarophiles. Min-
or products 5 correspond to an exo-interaction of syn,syn-azome-
thine ylide and dipolarophilic acrylonitrile.
and 3j–l were incubated at various concentrations with 1 lM SrtA
and assayed as described above. For each of the compounds, the
fractional activity remaining after treatment was measured and
plotted versus compound concentration using GRAFIT v.4.03 (Eritha-
cus Software), and fit as described in materials and methods. Com-
pounds 2a–c exhibited modest activity, and had IC50 values
ranging from 850 lM to 1.32 mM (Table 2). The more highly-
substituted compounds 2l and 3j–l were less potent and had IC50
values of 1.86–2.68 mM (Table 2). Compound 2k showed signifi-
cantly lower inhibition, with an IC50 value of greater than 5 mM.
For compounds 2a–2c, fitting the resultant curves to a single-expo-
nential function allowed the calculation of the observed rate con-
stants for inactivation (kobs), which were then plotted as an
inverse function of inhibitor concentration to determine
kainpapct=KaI pp values, the apparent second-order rate constant for inac-
tivation (Table 2). Because of the nature of the reverse protonation
mechanism of SrtA and the markedly enhanced rate of vinyl sulf-
ones to undergo conjugate addition with a Cys thiolates over a
Cys thiol, true kinact=KI values were determined from the apparent
values by correcting for the fraction of enzyme in the enzymati-
cally active thiolate-imidazolium form (determined previously to
be 0.06%).24
O
MeO
O
R2
O
S
O
S
O
ii)
i)
R2
OMe
OMe
SO2
N
N
+
N
O
O
H
O
R1
R1
R1
1a-c, j-l
( )-2a-c, j-l
( )-3j-l
Scheme 2. Reaction conditions: (i) AgOAc, Et3N, toluene, rt; (ii) AcOH, toluene, 110 °C.

K. V. Kudryavtsev et al. / Bioorg. Med. Chem. 17 (2009) 2886–2893
2889
N
N
R2
N
R2
R2
OMe
OMe
OMe
+
N
+
N
N
H
O
H
O
O
R1
R1
R1
1a-h
( )-4a-h
( )-5a-h
Scheme 3. Reaction conditions: LiBr, Et3N, THF, rt.
N
N
N
i)
ii)
iii)
R2
R2
OMe
OH
OMe
N
N
Me
N
Me
H
O
O
R1
R1
( )-4a,b,e,f,h
( )-6a,b,e,f,h
( )-7
Me
O
Me
O
N
O
N
N
iv)
NH
O
N
N
O
N
Me
N
Me
( )-8
( )-9
ORTEP of ( )-9
Scheme 4. Reaction conditions: (i) MeI, K2CO3, DMF, rt; (ii) LiBH4, THF; (iii) DEAD, Ph3P, THF; (iv) NH3, MeOH.
Table 2
Inhibition of S. aureus SrtA in vitro by pyrrolidinyl vinyl sulfones 2 and 3
kainpapct=KIapp (Mꢁ1 minꢁ1
)
kinact=KI (Mꢁ1 minꢁ1
)
a
Compound
IC50 (mM)
Hill coefficient
2a
2b
2c
2k
2l
3j
3k
3l
0.85 0.05
1.32 0.27
1.04 0.27
> 5.0
1.86 0.42
2.20 0.17
2.68 0.59
2.47 0.27
1.23 0.07
1.32 0.27
1.04 0.12
n.d.
1.40 0.33
1.90 0.23
1.39 0.28
1.42 0.14
12.73 0.71
9.18 1.55
10.67 1.28
n.d.
n.d.
n.d.
(2.12 0.12) ꢀ 104
(1.53 0.26) ꢀ 104
(1.78 0.21) ꢀ 104
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d. = Not determined.
a
Corrected for the fraction of active enzyme in the reverse protonation state (0.06%).
SrtA treated with vinylsulfones depicted in Table 2 exhibited
irreversible time-dependent inhibition with values for kinact=KI,
ranging from 1.5 to 2.2 ꢀ 104 Mꢁ1 minꢁ1 (Table 2). Of the inhibitors
examined, compound 2a proved most potent. Further inspection
revealed complicated inhibition kinetics suggestive of a multistep
inactivation mechanism for compounds 2a–2c.
The rest of the vinyl sulfone compounds 2 and 3 are confirmed
to act in the same inhibitory manner (data not shown).
4. Discussion
Inspecting known S. aureus SrtA inhibitors structures (Fig. 1),
it is interesting to note that many are two-dimensional mole-
cules with limited possibility for further modification. In this re-
gard it seems attractive to investigate the three-dimensional
space of enzyme–ligand interactions within the SrtA active site
and to explore a set of related structural analogs. We selected
cis-5-phenyl proline scaffold for construction of potential inhibi-
tors (Fig. 2) since efficient straightforward synthetic procedures
and diversification methods are developed for these compounds
and due to druglikeness of this compound class. An electrophilic
moiety attached to this scaffold could form a reversible or an
irreversible covalent bond with the enzyme cysteine residue
and therefore be envisaged as a route to mechanism-based SrtA
inhibitors (Scheme 5). The inhibition kinetics and mechanism of
To unequivocally establish that the compounds inhibited SrtA
via covalent modification of the active-site nucleophile Cys184,
we used mass spectrometry to ascertain if compound 2c formed
a covalent inhibitor-SrtA adduct with SrtA. SrtA was incubated
with 2c then run on a 4–12% gradient SDS–PAGE gel. The band cor-
responding to SrtA was excised, trypsin digested, and analyzed by
ESI-MS. A peptide was found whose mass (2256.1 before modifica-
tion, 2569.4 after modification) corresponds to the fragment
178QLTLITCDDYNEKTGVWEK196
, containing a 313.3 mass unit
modification, equivalent to the mass of 2c. Sequencing of the pep-
tide by ESI-MS/MS confirmed the sequence, and localized the mod-
ification to Cys184 (data not shown). This result confirms that 2c
inactivates SrtA via formation of a covalent adduct with Cys184.

2890
K. V. Kudryavtsev et al. / Bioorg. Med. Chem. 17 (2009) 2886–2893
O
O
S
S
R2
S
O
R2
O
OMe
OMe
OMe
R1
SrtA
SrtA
N
R1
N
H
H
O
O
NH
S
N
R2
S. aureus
R2
1
sortase SrtA
RR
N
R3
OMe
+
O
H
1
RR
N
N
R3
O
Cys184
O
HS
O
H
N
N
O
H
N
N
O
Me
O
Me
N
N
1
RR
S
N
R2
1
RR
N
R2
SrtA
Scheme 5. Proposed modes of action of synthesized compounds.
covalent labeling of SrtA is consistent with the proposed mech-
anism of thiolate mediate conjugate addition. Vinyl sulfonyl pyr-
rolidines 2 and pyrrolizidines 3 readily available in 2–3 synthetic
steps from commercial starting materials have been equal with
our expectations and inhibit S. aureus sortase A at concentrations
similar to phenyl vinyl sulfone (PVS). The mechanism of SrtA
inhibition by heterocyclic vinyl sulfones 2 and 3 includes cova-
lent modification of the enzyme active site Cys184 analogously
to PVS.4 These compounds were more potent than simple alkyl
vinyl sulfones4 indirectly indicating an importance of hydropho-
bic interactions between ligands and the enzyme. It should be
also concluded that substituents at second position or annulation
of five-membered cycle to positions 1 and 2 of proline ring de-
crease affinity of vinyl sulfonyl prolinates 2 to SrtA while the
character of substituents in aryl fragment at fifth position
slightly influences activity (Table 2). It is remained intriguing
to test both enantiopure forms of 2 and 3 and find if chiral
three-dimensional environment is critical for enzyme–ligand
interactions. Development of asymmetric version of dipolar
cycloaddition of DVS to azomethine ylides is under active inves-
tigation in our labs to evaluate this issue. It should be also noted
that vinyl sulfonyl prolinates 2a–c are crystalline compounds
stable during two years storage at +4 °C notwithstanding that
the molecules contain both effective Michael acceptor (vinyl sul-
fonyl) and Michael donor (secondary amine) parts.
that is confirmed by X-ray.26 To test pyrimidine nucleobases as po-
tential Michael acceptors in the sortase environment we modified
5-phenyl proline scaffold with the thymine residue (Scheme 4)
with the aim to increase inhibitory properties of carbonitriles 4
and 6 (Scheme 5). Unfortunately compound 9 is practically inactive
toward SrtA (%I = 6 at 9 concentration 1 mM).
5. Conclusions
In conclusion we have developed an effective synthetic ap-
proach to a diverse set of cis-5-phenyl prolinates functionalized
by electrophilic groups. We have also tested these synthesized
compounds on the in vitro inhibitory activity of the S. aureus sor-
tase SrtA transpeptidase. Racemic vinyl sulfonyl 5-phenyl proli-
nates 2 inhibit S. aureus sortase SrtA irreversibly by modification
of the enzyme Cys184. Modifications of most active compounds
including asymmetric synthetic approaches are under develop-
ment for increasing inhibitory properties of ligands based on cis-
5-phenyl proline scaffold. Present work is centered on elucidating
structure–activity relationships with these compound classes and
on evaluating of the specificity profile of these agents against sor-
tase activity in S. aureus and other Gram-positive microorganisms
in vivo.
6. Experimental
6.1. Chemistry
Nitrile-containing compounds are another chemotype for inhib-
iting enzymes with activity dependent on cysteine thiol nucleo-
philes.25
A possible mechanism of inhibition could involve
formation of thioimidate ester (Scheme 5). This mechanism could
be also applied to discovered diarylacrylonitrile SrtA inhibitors5
for explanation of their activity. Six of our tested pyrrolidine carbo-
nitriles 4–6, namely compounds 4b, 4d, 5a, 5b, 6b and 6f, inhibited
SrtA with percent inhibition (%I) value 20–30% at 5 mM of inhibitor
concentration. %I ꢂ 20 was the criterion for primary selection of
sortase inhibitors hits.7 All other nitriles were less active summa-
rizing a poor efficacy of 4-cyano-5-phenyl prolinates 4–6 towards
sortase. Assuming the same or similar orientation of 5-phenyl pro-
linate fragment in the SrtA active site, weaker inhibitory properties
of carbonitriles 4–6 compared to vinyl sulfones 2 could be ac-
counted for by decreasing of electrophilic properties of the former
compounds.
Reagents were obtained from Alfa Aesar and used without fur-
ther purification unless otherwise stated. Solvents were dried
using standard procedures. Reactions were monitored by thin layer
chromatography (TLC) on precoated silica gel plates (Sorbfil) with a
UV indicator. Column chromatography was performed with Alfa
Aesar Silica Gel 60 (0.040–0.063 mm). Melting points were deter-
mined in open capillary and are uncorrected.
NMR spectra were recorded with a Bruker Avance 400 MHz spec-
trometer. The chemical shifts (d) are reported in parts per million
upfield using residual signals of solvents as internal standards.
Coupling constants (J values) were measured in Hertz (Hz). Com-
bustion analyses were performed with a Carlo Erba CHN analyzer.
Compounds 1, 2a–c, 2j, and 4–6 have been synthesized as previ-
ously reported.18,20 3-Benzoylthymine was obtained from thymine
according to literature protocol.27
1H NMR and 13C
A stable covalent complex is formed by Escherichia coli thymid-
ilate synthase (TS) and deoxyuridine monophosphate (dUMP) by
Michael addition of reactive thiolate of TS Cys198 to C-6 of dUMP

K. V. Kudryavtsev et al. / Bioorg. Med. Chem. 17 (2009) 2886–2893
2891
*
*
*
6.2. General procedure for the synthesis of pyrrolidinyl vinyl
sulfones (2k) and (2l)
6.7. Methyl (2S ,3S ,7aS )-3-(3,4-dimethoxyphenyl)-2-
ethenesulfonyl-5-oxotetrahydropyrrolizine-7a-carboxylate
(3k)
Iminoester 1k or 1l (8.4 mmol) was dissolved in 20 ml of tolu-
ene under argon atmosphere and divinyl sulfone (0.89 ml,
8.4 mmol) and silver(I) acetate (1.55 g, 9.3 mmol) were added in
one portions. A solution of Et3N (1.4 ml, 10 mmol) in 40 ml of tol-
uene was then added to the resulting suspension under stirring.
The mixture was stirred for 24 h at room temperature under argon
atmosphere with protection from light. The precipitate was filtered
off and washed with 10 ml of toluene, the organic phase was con-
centrated under reduced pressure, and the residue was purified by
chromatography on silica gel using hexane/AcOEt as an eluent.
Yield 87%, beige solid, mp 95–97 °C. 1H NMR (400 MHz; DMSO-
d6): d 6.94 (s, 1H, Ar), 6.83 (s, 2H, Ar), 6.06 (dd, J 16.8, 10.0, 1H,
CH@CH2), 5.78 (d, J 10.0, 1H, CH@CH2), 5.68 (d, J 16.8, 1H, CH@CH2),
5.22 (d, J 8.0, 1H, H-3), 4.51–4.46 (m, 1H, H-2), 3.75 (s, 3H, OCH3),
3.73 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.07 (dd, J 14.0, 5.6, 1H, H-1),
2.75–2.66 (m, 1H, H-7), 2.56–2.50 (m, 1H, H-1), 2.36–2.32 (m, 2H,
H-6), 2.26–2.21 (m, 1H, H-7). 13C NMR (100 MHz; CDCl3): d 177.62,
173.20, 149.22, 148.66, 134.69, 130.09, 127.46, 121.08, 112.31,
110.56, 72.52, 69.63, 61.15, 56.00 (2C), 53.14, 36.49, 35.92, 33.66.
Anal. Calcd for C19H23NO7S: C, 55.73; H, 5.66; N, 3.42. Found: C,
55.35; H, 5.45; N, 3.20.
*
*
*
6.3. Methyl (2S ,4S ,5S )-5-(3,4-dimethoxyphenyl)-4-
ethenesulfonyl-2-(2-methoxycarbonylethyl)pyrrolidine-2-
carboxylate (2k)
*
*
*
6.8. Methyl (2S ,3S ,7aS )-2-ethenesulfonyl-5-oxo-3-pyridin-3-
yl-tetrahydropyrrolizine-7a-carboxylate (3l)
Yield 67%, colorless viscous oil. 1H NMR (400 MHz; DMSO-d6): d
6.92 (d, J 2.0, 1H, Ar), 6.87 (d, J 8.1, 1H, Ar), 6.82 (dd, J 8.1, 2.0, 1H,
Ar), 5.93 (dd, J 16.4, 9.6, 1H, CH@CH2), 5.74–5.63 (m, 2H, CH@CH2),
4.54 (dd, J 10.0, 6.0, 1H, H-5), 4.05 (m, 1H, H-4), 3.72 (s, 3H, OCH3),
3.71 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.51 (s, 3H, OCH3), 3.29 (d, J
10.0, 1H, NH), 2.76 (dd, J 15.0, 4.2, 1H, H-3), 2.38–2.26 (m, 2H, H-3,
CH2COOMe), 2.23–2.15 (m, 1H, CH2COOMe), 2.00 (t, J 7.6, 2H,
CH2CH2COOMe). Anal. Calcd for C20H27NO8S: C, 54.41; H, 6.16; N,
3.17. Found: C, 54.48; H, 6.14; N, 3.25.
Yield 69%, viscous oil. 1H NMR (400 MHz, DMSO-d6): d 8.55 (s, 1H,
Py), 8.43 (d, J 4.0, 1H, Py), 7.77 (d, J 8.0, 1H, Py), 7.30 (dd, J 8.0, 4.0, 1H,
Py), 6.46 (dd, J 16.8, 10.0, 1H, CH@CH2), 5.82 (d, J 10.0, 1H, CH@CH2),
5.60 (d, J 16.8, 1H, CH@CH2), 5.38 (d, J 8.0, 1H, H-3), 4.64–4.59 (m, 1H,
H-2), 3.76 (s, 3H, OCH3), 3.15 (dd, J 14.4, 3.2, 1H, H-1), 2.78–2.69 (m, 1
H, H-7), 2.62(dd, J 14.4, 8.8, 1H, H-1), 2.39–2.26 (m, 3H, H-6, H-7). 13C
NMR (100 MHz; CDCl3): d 177.31, 172.94, 149.75, 149.57, 136.73,
134.78, 131.34, 130.81, 123.36, 72.57, 68.80, 58.95, 53.25, 36.43,
36.32, 33.77. Anal. Calcd for C16H18N2O5S: C, 54.85; H, 5.18; N,
7.99. Found: C, 55.03; H, 5.15; N, 8.09.
*
*
*
6.4. Methyl (2S ,4S ,5S )-4-ethenesulfonyl-2-(2-methoxycarbon-
ylethyl)-5-pyridin-3-yl-pyrrolidine-2-carboxylate (2l)
*
*
*
6.9. (2R ,3S ,5S )-5-Hydroxymethyl-1-methyl-2-
phenylpyrrolidine-3-carbonitrile (7)
Yield 72%, beige solid, mp 118–120 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.54 (d, J 2.0, 1H, Py), 8.44 (dd, J 4.8, 1.6, 1H, Py),
7.72 (dt, J 8.0, 1.6, 1H, Py), 7.33 (dd, J 8.0, 4.8, 1H, Py), 6.40 (dd, J
16.4, 9.6, 1H, CH@CH2), 5.80 (d, J 9.6, 1H, CH@CH2), 5.63 (d, J
16.4, 1H, CH@CH2), 4.74 (dd, J 9.2, 6.8, 1H, H-5), 4.22–4.17 (m,
1H, H-4), 3.72 (s, 3H, OCH3), 3.54 (s, 3H, OCH3), 3.47 (d, J 9.2, 1H,
NH), 2.75 (dd, J 14.8, 5.2, 1H, H-3), 2.40–2.32 (m, 2H, H-3,
CH2COOMe), 2.28–2.20 (m, 1H, CH2COOMe), 2.04 (t, J 8.0, 2H,
CH2CH2COOMe). Anal. Calcd for C17H22N2O6S: C, 53.39; H, 5.80;
N, 7.32. Found: C, 53.33; H, 5.74; N, 7.32.
LiBH4 (0.200 g, 11 mmol) was added to the solution of N-methyl
prolinate 6a (2.690 g, 11 mmol) in 50 ml of dry THF under stirring at
room temperature. After 24 h additional LiBH4 (0.200 g, 11 mmol)
was added and the mixture was heated at 40 °C during 18 h. After
cooling to room temperature obtained suspension was filtered and
concentrated at vacuum. Methanol (120 ml) was added by portions
to the residue under external cooling to 0 °C and solution was kept at
room temperature during 2 h. Concentration and chromatography
on silica gel using hexane/AcOEt as an eluent led to 2.120 g of amino
alcohol 7 as white crystals. Yield 89%, mp 98–99 °C. 1H NMR
(400 MHz; CDCl3): d 7.46–7.40 (m, 4H, Ar), 7.39–7.34 (m, 1H, Ar),
3.87 (dd, J 11.4, 2.6, 1H, CH2O), 3.66 (d, J 6.4, 1H, H-2), 3.64–3.58
(m, 1H, CH2O), 3.24–3.19 (m, 1H, H-3), 2.75 (br, 1H, OH), 2.48–2.34
(m, 3H, H-4, H-5), 2.25 (s, 3H, NCH3). 13C NMR (100 MHz; CDCl3): d
128.71, 128.68 (2C), 127.98 (2C), 120.15, 72.60, 65.39, 60.94,
38.24, 35.67, 30.81. Anal. Calcd for C13H16N2O: C, 72.19; H, 7.46; N,
12.95. Found: C, 72.20; H, 7.55; N, 12.89.
6.5. General procedure for the synthesis of pyrrolizidinones (3)
Pyrrolidine 2 (1.4 mmol) was dissolved in 20 ml of toluene, then
2 ml of glacial acetic acid was added and the reaction mixture was
heated at 110 °C under stirring and argon atmosphere during 2 h.
After cooling to room temperature the mixture was concentrated
under reduced pressure and residue was purified by chromatogra-
phy on silica gel using hexane/AcOEt as an eluent.
*
*
*
6.10. (2R ,3S ,5S )-5-(3-Benzoyl-5-methyl-2,4-dioxo-3,4-dihydro-
2H-pyrimidin-1-ylmethyl)-1-methyl-2-phenylpyrrolidine-3-
carbonitrile (8)
*
*
*
6.6. Methyl (2S ,3S ,7aS )-2-ethenesulfonyl-5-oxo-3-
phenyltetrahydropyrrolizine-7a-carboxylate (3j)
Yield 99%, white crystals, mp 69–70 °C. 1H NMR (400 MHz;
DMSO-d6): d 7.34–7.31 (m, 2H, Ar), 7.26–7.23 (m, 3H, Ar), 6.05
(dd, J 16.8, 10.0, 1H, CH@CH2), 5.73 (d, J 10.0, 1H, CH@CH2), 5.62
(d, J 16.8, 1H, CH@CH2), 5.27 (d, 1H, J 8.0, H-3), 4.52 (ddd, J 13.6,
8.0, 4.6, 1H, H-2), 3.74 (s, 3H, OCH3), 3.12 (dd, J 13.6, 4.6, 1H, H-
1), 2.75–2.66 (m, 1H, H-7), 2.54 (dd, J 13.6, 8.0, 1H, H-1), 2.35–
2.31 (m, 2H, H-6), 2.26–2.20 (m, 1H, H-7). 13C NMR (100 MHz;
DMSO-d6): d 177.73, 173.71, 136.76, 135.93, 129.47, 129.23(2C),
128.22, 127.95(2C), 72.69, 67.70, 61.38, 52.13, 35.54, 35.07,
33.74. Anal. Calcd for C17H19NO5S: C, 58.44; H, 5.48; N, 4.01.
Found: C, 58.26; H, 5.36; N, 4.14.
Amino alcohol 7 (0.865 g, 4.0 mmol), PPh3 (2.100 g, 8.0 mmol)
and 3-benzoylthymine (1.842 g, 8.0 mmol) were suspended in
50 ml of anhydrous THF under argon atmosphere. A solution of
diethyl azodicarboxylate (1.400 g, 8.0 mmol) in 60 ml of anhydrous
THF was added within 5 h. After stirring 48 h at room temperature
all volatiles were removed under vacuum. The residue was purified
by column chromatography. Yield 0.240 g (14%), white crystals, mp
224–226 °C. 1H NMR (400 MHz; DMSO-d6): d 8.00 (d, J 7.6, 2H, Ar),
7.79–7.75 (m, 2H, Ar, CH@), 7.56 (t, 4H, J 7.6, 2H, Ar), 7.38–7.30 (m,
5H, Ar), 4.12 (dd, J 14.4, 2.8, 1H, CH2N), 3.84 (dd, J 14.4, 4.4, 1H,
CH2N), 3.73 (d, J 6.0, 1H, H-2), 3.54–3.50 (m, 1H, H-3), 3.00–2.93

2892
K. V. Kudryavtsev et al. / Bioorg. Med. Chem. 17 (2009) 2886–2893
(m, 1H, H-5), 2.50–2.41 (m, 1H, H-4), 2.23 (s, 3H, NCH3), 1.99–1.93
(m, 1H, H-4), 1.93 (s, 3H, CH3). Anal. Calcd for C25H24N4O3: C, 70.08;
H, 5.65; N, 13.08. Found: C, 70.04; H, 5.68; N, 13.12.
8. Solid-phase synthesis of Abz-LPETG-Dap(DNP)-NH2
The peptide Abz-LPETG-Dap(DNP)-NH2 was synthesized by the
Fmoc/piperidine strategy on PAL resin on a 0.25 mmol scale. Cleav-
age from the resin was achieved via incubation with a 95:2.5:2.5
TFA/water/triisopropylsilane (TIPS) mixture for 2.5 h. The peptide
was precipitated using cold diethyl ether following the removal
of excess TFA via rotary evaporation. After filtration, the precipitate
was dissolved in a 50:50 water/acetonitrile mixture and lyophi-
lized to yield crude peptide. Crude peptide was purified by HPLC
*
*
*
6.11. (2R ,3S ,5S )-1-Methyl-5-(5-methyl-2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-ylmethyl)-2-phenylpyrrolidine-3-
carbonitrile (9)
Methanol (25 ml) was saturated with gaseous ammonia. Com-
pound 8 (0.214 g, 0.5 mmol) was added to this solution and stirred
48 h at room temperature. All volatiles were removed under re-
duced pressure. The residue was purified by column chromatogra-
phy. Yield 0.128 g (79%), white crystals, mp 209–211 °C. 1H NMR
(400 MHz; DMSO-d6): d 11.40 (br, 1H, NH), 7.49 (s, 1H, CH@),
7.38–7.36 (m, 4H, Ar), 7.35–7.28 (m, 1H, Ar), 4.06 (dd, J 14.2, 3.4,
1H, CH2N), 3.72–3.68 (m, 2H, CH2N, H-2), 3.50–3.45 (m, 1H, H-3),
2.93–2.89 (m, 1H, H-5), 2.39 (dt, J 13.6, 9.2, 1H, H-4), 2.19 (s, 3H,
NCH3), 2.02–1.97 (m, 1H, H-4), 1.83 (s, 3H, CH3). 13C NMR
TM
using a semi-preparative C18 Jupiter column (21.2 ꢀ 250 mm,
10 lm, Phenomenex, Inc.) to P98% purity. MALDI-TOF mass spec-
trometry was used to verify the identity of the purified product
(Abz-LPETG-Dap(DNP)-NH2: m/z = 885.3). The purified, lyophilized
peptide were stored at ꢁ20 °C.
9. Inhibition assays
(100 MHz; DMSO-d6):
d
164.81, 152.18, 143.05, 138.52,
9.1. Steady-state activity assays
128.72(2C), 128.44, 128.23(2C), 120.93, 108.57, 71.45, 63.53,
48.70, 38.75, 35.20, 31.49, 12.40. Anal. Calcd for C18H20N4O2: C,
66.65; H, 6.21; N, 17.27. Found: C, 66.79; H, 6.06; N, 17.15.
Purified recombinant SrtA
at 1 lM was incubated with
D
N24
2 mM Abz-LPETG-Dap(DNP)-NH2, 2 mM NH2-Gly5-OH, and
increasing amounts of inhibitor at 37 °C in 2% DMSO, 0.1% CHAPS,
150 mM NaCl, 5 mM mM CaCl2 and 300 mM Tris (pH 7.5), in a total
6.12. Crystal Structure Determination of Compound (9)
volume of 100 lL. To control for the possibility of time-dependent
The single crystal of
9
of approximate dimensions
inactivation, the enzyme and inhibitor were pre-incubated in buf-
fer at 37 °C for 30 min. Reactions were initiated by addition of sub-
strate mix (Abz-LPETG-Dap(DNP)-NH2 and NH2-Gly5-OH), and
allowed to incubate at 37 °C. After 30 min, the reactions were
0.30 ꢀ 0.20 ꢀ 0.20 mm was mounted in inert oil on the top of glass
fiber and transferred on the Bruker SMART APEX diffractometer.
Crystal data: C18H20N4O2, M = 324.38, monoclinic, a = 12.097(3),
b = 7.6630(18), c = 18.508(4) Å, b = 105.559(3)°, V = 1652.7(7) Å3,
quenched by removal of 80
ume) of 1.2 M HCl. Next, 70
injected onto a reverse phase octadecylsilica analytical fast-flow
m, Vydac, Inc.). Products were sep-
lL of reaction mix into 40
lL (1/2 vol-
space group P21/c, Z = 4, Dc = 1.304 g/cm3, F(000) = 688,
l
(Mo
Total of 12,508 reflections (3244 unique,
Rint = 0.0617) were measured using graphite monochromatized
Mo K radiation (k = 0.71073 Å) at 293(2) K. Data were collected
lL of the quenched reaction mix was
Ka .
) = 0.088 mmꢁ1
HPLC column (4.6 ꢀ 50 mm, 3
l
a
arated using a linear gradient from 0% to 45% CH3CN/0.1%TFA over
5 min. The elution of the dinitrophenol-containing substrate (Abz-
LPETG-Dap(DNP)-NH2) and product (NH2-G-Dap(DNP)-NH2) were
monitored at 355 nm, and integration of the areas under the sub-
strate and product peaks was used to determine the percentage
of substrate converted to product.
in the range 2.89 < h < 26.00 (ꢁ14 ꢃ h ꢃ 14, ꢁ9 ꢃ k ꢃ 9,
ꢁ22 ꢃ l ꢃ 22). Omega scan mode with the step of 0.3° (30 s per
step) was used. The structure was solved by direct methods28
and refined by full matrix least-squares on F2.29 All H atoms (ex-
cept 1H) were placed in calculated positions and refined using a
riding model. The final residuals were: R1 = 0.0577, wR2 = 0.1403
for 1638 reflections with I > 2
and 223 parameters. Goof = 1.047, maximum
r
(I) and 0.1228, 0.1686 for all data
= 0.215 e/Å3.
9.2. IC50 determination
D
q
For IC50 determination, 1
l
M SrtA
was pre-incubated with
N24
D
7. Expression and purification of 6-His tagged SrtA
D
N24
the inhibitor for 30 min at 37 °C, to allow for any time-dependent
inactivation to occur. Assays were then initiated by addition of a
mixture of Abz-LPETG-Dap(DNP)-NH2 and NH2-Gly5-OH. Assays
N-Terminally His6-tagged SrtA lacking the amino-terminal 24
amino acids was expressed in BL21(DE3) cells containing the plas-
were performed in 100
then were quenched by addition of 50
HCl. All measurements were performed in triplicate. 70
l
L final volume, were run for 30 min, and
L (1/2 volume) of 1.2 M
L of each
mid pET15bSrtA N24. Cells were grown in Luria Broth containing
D
l
100
Protein expression was induced by the addition of 1 mM isopropyl
-D-thiogalactopyranoside (IPTG), and the cells were grown for an
lg/ml of ampicillin at 37 °C until the OD600 reached 0.5–0.6.
l
quenched reaction mixture was analyzed by HPLC using the meth-
od described above. Fractional activity remaining relative to unin-
hibited controls (vi/vo) was calculated by comparing the difference
in percent product formation (as measured at 355 nm). GRAFIT v.4.0
(Erithacus Software) was used to generate plots of fractional activ-
ity remaining versus inhibitor concentration, which were fit using
the equation:
b
additional 3 h at 37 °C then harvested by centrifugation at 3000 g
for 10 min. Cells were resuspended in 150 mM NaCl, 50 mM Tris-
Cl, 5 mM imidazole, 10% glycerol, pH 7.5, and lysed with an Emul-
siFlex-C5 high-pressure homogenizer (Avestin, Inc.). Lysate was
clarified by centrifugation and applied to a chelating sepharose fast
flow column. The column was washed with 50 mM imidazole,
SrtA N24 was eluted using a linear gradient of 50–500 mM imidaz-
D
vi
vo
1
¼
ole and fractions were collected. SrtA N24-containing fractions
ꢀ
ꢁ
D
h
½Iꢄ
IC50
1 þ
were pooled, concentrated, and dialyzed overnight into 150 mM
NaCl, 50 mM Tris-Cl, 5 mM CaCl2, 0.1% b-mercaptoethanol, 10%
glycerol, pH 7.5. Purified SrtA
was concentrated using
where vi is the initial velocity in the presence of the inhibitor at con-
D
N24
10,000-MWCO Centriplus centrifugal filters (Amicon, Inc.).
SrtA concentration was determined using a calculated extinc-
centration [I], vo is the initial velocity in the absence of inhibitor,
IC50 is the concentration of inhibitor at which one-half of the origi-
nal activity remains, and h is the Hill coefficient.
D
N24
tion coefficient (e
280 = 17,420 Mꢁ1 cmꢁ1).

K. V. Kudryavtsev et al. / Bioorg. Med. Chem. 17 (2009) 2886–2893
2893
9.3. Measurement of second-order rate constants for SrtA
References and notes
inactivation
1. Talbot, G. H.; Bradley, J.; Edwards, J. E., Jr.; Gilbert, D.; Scheld, M.; Bartlett, J. G.
Clin. Infect. Dis. 2006, 42, 657.
2. Marraffini, L. A.; DeDent, A. C.; Schneewind, O. Microbiol. Mol. Biol. Rev. 2006,
70, 192.
Assays were performed in 100 lL volume, containing 1 lM
SrtA N24, inhibitor, 2 mM Abz-LPETG-Dap(DNP)-NH2, 2 mM NH2-
D
3. Suree, N.; Jung, M. E.; Clubb, R. T. Mini-Rev. Med. Chem. 2007, 7, 991.
4. Frankel, B. A.; Bentley, M.; Kruger, R. G.; McCafferty, D. G. J. Am. Chem. Soc.
2004, 126, 3404.
5. Oh, K. B.; Kim, S. H.; Lee, J.; Cho, W. J.; Lee, T.; Kim, S. J. Med. Chem. 2004, 47,
2418.
6. Oh, K. B.; Oh, M. N.; Kim, J. G.; Shin, D. S.; Shin, J. Appl. Microbiol. Biotechnol.
2006, 70, 102.
7. Maresso, A. W.; Wu, R.; Kern, J. W.; Zhang, R.; Janik, D.; Missiakas, D. M.; Duban,
M. E.; Joachimiak, A.; Schneewind, O. J. Biol. Chem. 2007, 282, 23129.
8. Chenna, B. C.; Shinkre, B. A.; King, J. R.; Lucius, A. L.; Narayanab, S. V. L.; Velua, S.
E. Bioorg. Med. Chem. Lett. 2008, 18, 380.
9. Kang, S. S.; Kim, J. G.; Lee, T. H.; Oh, K. B. Biol. Pharm. Bull. 2006, 29, 1751.
10. Oh, K. B.; Mar, W.; Kim, S.; Kim, J. Y.; Oh, M. N.; Kim, J. G.; Shin, D.; Sim, C. J.;
Shinc, J. Bioorg. Med. Chem. Lett. 2005, 15, 4927.
11. Park, B. S.; Kim, J. G.; Kim, M. R.; Lee, S. E.; Takeoka, G. R.; Oh, K. B.; Kim, J. H. J.
Agric. Food Chem. 2005, 53, 9005.
12. Jang, K. H.; Chung, S. C.; Shin, J.; Lee, S. H.; Kim, T. I.; Lee, H. S.; Oh, K. B. Bioorg.
Med. Chem. Lett. 2007, 17, 5366.
13. Zong, Y.; Bice, T. W.; Ton-That, H.; Schneewind, O.; Narayana, S. V. J. Biol. Chem.
2004, 279, 31383.
Gly5-OH, 2% DMSO, 0.1% CHAPS, 150 mM NaCl, 5 mM CaCl2, and
300 mM Tris (pH 7.5). No pre-incubation of enzyme with inhibitor
was performed. Assays were initiated by addition of enzyme and
allowed to run at 37 °C. At 10, 20, 30, 40, 50 and 60 min, assays
were quenched by removing 90
lL of reaction mix into 45
lL
(1/2 volume) of 1.2 M HCl. Quenched samples (70
l
L) were in-
jected onto an octadecylsilica column and run using the standard
HPLC method described above. Progress curves were generated,
and fit using SIGMAPLOT v.8.0 to the following equation:
À
Á
obst
P ¼ A 1 ꢁ eꢁk
where P is product concentration, A is the amplitude (
vi/kobs), kobs is
the observed rate constant for inactivation, and t is time. For several
cases outlying values of kobs were observed due to a lack of curva-
ture in the progress lines, and these were removed prior to subse-
quent analysis. Plots of kobs versus inhibitor concentration were
generated, and SIGMAPLOT v.8.0 was used to fit these plots to either
a linear fit or a saturation fit, using the equations below:
14. Meadows, D. C.; Gervay-Hague, J. Med. Res. Rev. 2006, 26, 793.
15. Santos, M. M. M.; Moreira, R. Mini-Rev. Med. Chem. 2007, 7, 1040.
16. Grigg, R. Chem. Soc. Rev. 1987, 16, 89.
17. Tsuge, O.; Kanemasa, S.. In Advances in Heterocyclic Chemistry; Katritzky, A. R.,
Ed.; Academic Press: SanDiego, 1989; Vol. 45, pp 231–349.
18. Kudryavtsev, K. V.; Nukolova, N. V.; Kokoreva, O. V.; Smolin, E. S. Russ. J. Org.
Chem. 2006, 42, 412.
ꢂ
ꢃ
kinact
KI
kobs
¼
½Iꢄ
ðLinearÞ
19. Kudryavtsev, K. V.; Tsentalovich, M. Yu.; Yegorov, A. S.; Kolychev, E. L. J.
Heterocycl. Chem. 2006, 43, 1461.
kinact ꢅ ½Iꢄ
KI þ ½Iꢄ
20. Kudryavtsev, K. V.; Tsentalovich, M. Yu. Moscow Univ. Chem. Bull. 2007, 62, 252.
21. Barr, D. A.; Donegan, G.; Grigg, R. J. Chem. Soc., Perkin Trans. 1 1989, 1550.
22. Tsuge, O.; Kanemasa, S.; Yoshioka, M. J. Org. Chem. 1988, 53, 1384.
23. Crystallographic data (excluding structure factors) for the compound 9 have
been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication no. CCDC-701397. Copies of the data can be
obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK [Fax: int.code + 44(1223)336-033; E-mail: deposit@ccdc.cam.ac.uk].
24. Frankel, B. A.; Kruger, R. G.; Robinson, D. E.; Kelleher, N. L.; McCafferty, D. G.
Biochemistry 2005, 44, 11188.
kobs
¼
ðSaturationÞ
Subsequent analysis determined the best fit, and provided the
apparent second-order rate constant for inactivation, kainpapct=KIapp
.
This value was subsequently corrected for the fraction of active en-
zyme concentration of 0.06% previously determined for SrtA24 to
yield the second-order rate constant of inactivation kinact=KI.
25. Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.;
Berthelette, C. Bioorg. Med. Chem. Lett. 2007, 17, 998.
Acknowledgments
26. Montfort, W. R.; Perry, K. M.; Fauman, E. B.; Finer-Moore, J. S.; Maley, G. F.;
Hardy, L.; Maley, F.; Stroud, R. M. Biochemistry 1990, 29, 6964.
27. Zhou, J.; Shevlin, P. B. Synth. Commun. 1997, 27, 3591.
28. Sheldrick, G. M. Acta Crystallogr., Sect. A 1990, 46, 467.
29. Sheldrick, G. M. SHELXL-97, Program for the Refinement of Crystal Structures;
University of Gottingen, 1997.
This research was kindly supported by a National Institutes of
Health Allergy and Infectious Disease research Grant AI46611 to
D.G.M. K.V.K. acknowledges a partial support from the Russian
Foundation for Basic Research (Grant 08-04-01800a).