Antibiotic optimization and chemical structure stabilization of thiomuracin a

Article abstract of DOI:10.1021/jm300783c

Synthetic studies of the antimicrobial secondary metabolite thiomuracin A (1) were initiated to improve chemical stability and physicochemical properties. Functional group modifications of 1 included removing the C2-C7 side chain, derivatizing the C84 epoxide region, and altering the C44 hydroxyphenylalanine motif. The resulting derivatives simplified and stabilized the chemical structure and were evaluated for antibacterial activity relative to 1. The simplified structure and improved organic solubility of the derivatives facilitated isolation yields from fermentation broths and simplified the procedures involved for the process. These advancements increased material supply for continued medicinal chemistry optimization and culminated in the identification of 2, a structurally simplified and chemically stable analogue of 1 which retained potent antibiotic activity.

Full text of DOI:10.1021/jm300783c

Article
pubs.acs.org/jmc
Antibiotic Optimization and Chemical Structure Stabilization of
Thiomuracin A
Matthew J. LaMarche,*,† Jennifer A. Leeds,‡ Joanne Dzink-Fox,‡ Eric Gangl,† Philipp Krastel,⊥
Georg Neckermann,‡ Deborah Palestrant,§ Michael A. Patane,† Elin M. Rann,† Stacey Tiamfook,‡
and Donghui Yu‡
‡
§
†Global Discovery Chemistry, Infectious Disease Area, and Protein Structure Unit, Novartis Institutes for Biomedical Research,
Cambridge, Massachusetts 02139, United States
⊥Natural Products Unit, Novartis Institutes for Biomedical Research, Basel, Switzerland
S
* Supporting Information
ABSTRACT: Synthetic studies of the antimicrobial secondary
metabolite thiomuracin A (1) were initiated to improve
chemical stability and physicochemical properties. Functional
group modifications of 1 included removing the C2−C7 side
chain, derivatizing the C84 epoxide region, and altering the
C44 hydroxyphenylalanine motif. The resulting derivatives
simplified and stabilized the chemical structure and were
evaluated for antibacterial activity relative to 1. The simplified
structure and improved organic solubility of the derivatives
facilitated isolation yields from fermentation broths and
simplified the procedures involved for the process. These
advancements increased material supply for continued medicinal chemistry optimization and culminated in the identification of
2, a structurally simplified and chemically stable analogue of 1 which retained potent antibiotic activity.
properties of the thiomuracins (e.g., low solubility of the major
fermentation metabolite, 1) would require extensive optimiza-
tion. The low solubility in organic solvents limited isolation
yields from silica gel based chromatography. In addition, during
fermentation and associated isolation processes, the C84
epoxide region was prone to undesired chemical trans-
formations. Collectively, poor organic solubility and chemical
decomposition complicated isolation and supply efforts.1
Due to our interest in the area of natural product based
antimicrobial drug discovery3 and the potent in vitro biological
profile of the thiomuracins, we embarked on a medicinal
chemistry optimization program. Our ability to build an
antibiotic drug discovery program based on the thiomuracin
scaffold would depend on two key factors: (1) stabilizing the
C81−84 region of the structure to avoid undesired epoxide
transformations and (2) increasing the organic solubility, which
would facilitate purification, thereby increasing material supply
for continued medicinal chemistry investigation. Herein, we
describe the results of our effort directed toward improving
these factors while retaining biological activity.
INTRODUCTION
■
In 2009 we reported the fermentation, isolation, structural
elucidation, antimicrobial activity, and biosynthetic gene cluster
of a new class of thiazoyl actinomycetales metabolites, the
thiomuracins (Figure 1).1 These architecturally complex
secondary metabolites, fermented from the rare soil actino-
mycetes species Nonomurae, are characterized by their highly
modified, sulfur-containing macrocyclic peptide structures. The
thiomuracins are structurally related to another class of
actinomycetes metabolites, GE2270 A (3, Figure 1),2 upon
which we have based extensive drug discovery efforts.3 Both the
thiomuracins and 3 derive their antibiotic activity via binding to
elongation factor Tu and disrupting prokaryotic protein
synthesis. Currently, LFF571 (4), a derivative of 3, is an
investigational new drug in clinical trials for the treatment of
Clostridium difficile infection.4
Clinical resistance to marketed drugs is becoming increas-
ingly common.5 Therefore, the discovery of antibiotics that act
via novel mechanisms of action remains a high priority in
infectious disease drug development. The thiomuracins possess
potent antibiotic activity against methicillin-resistant staph-
ylococci (MRSA), vancomycin-resistant enterococci (VRE),
group A streptococci, and the anaerobic gut pathogen
Clostridium difficile, with minimum inhibitory concentrations
(MIC) < 1 μg/mL. This profile compared favorably to 3 and
associated derivatives, specifically with respect to group A
streptococci inhibition. Much like 3, the poor physicochemical
RESULTS
■
1 was available in high purity but in limited amounts (<100
mg) due to its limited chemical stability and challenges related
Received: June 5, 2012
Published: July 19, 2012
© 2012 American Chemical Society
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Figure 1. Structures of 1, 3, and 4.
to purification.1 However, the crude fermentation extract was
available in multigram quantities and contained approximately
50% 1 by LCMS analysis. Initially, synthetic transformations
designed to increase the organic solubility of 1 were conducted
on the purified natural product. Ultimately, any prospective
improvements would require demonstration on the crude
extract itself. Simplifying the purification process and/or
increasing the isolation yields of 1 (or an alternative, potent
analogue) would constitute success.
Accordingly, the solubility of 1 was evaluated in a variety of
solvents. DMSO and DMF emerged as the only solvents that
would dissolve the material efficiently and reproducibly. The
C2 carboxylic acid was selectively esterified under basic
conditions (Scheme 1: Cs2CO3, MeI, DMF) in the presence
of the two hydroxyls and the epoxide, which furnished 5.
Dissolution of methyl ester 5 easily proceeded in acetone, and
the phenolic group was effectively alkylated (Cs2CO3, MeI,
acetone), which afforded 6. Thus, selective derivatization of the
carboxylate and the phenol functionalities was achievable.
Additionally, all hydroxyl groups in 5 were acetylated under
standard conditions, which furnished analogue 7. Unfortu-
nately, when applying these protecting group protocols to the
crude fermentation extract, the use of DMF as a solvent was
ineffective, as product recoveries were low. We suspected that
the low yields may be related to undesired side reactions of the
C84 epoxide (vide infra). Additionally, troubleshooting
reactions on the crude extract proved difficult because of the
low purity of the starting material.
The sensitivity of 1 toward acid and base-mediated reactions
was characterized as a means to understand possible
decomposition pathways and overall chemical stability. Under
acidic conditions (HCl, MeOH, H2O) at room temperature, 1
proved quite stable (Scheme 2). When the temperature was
elevated however (50 °C), the C2−C10 dehydroalanine-
containing side chain was hydrolyzed and the epoxide ring
was opened, affording the primary C10-thiazolyl amide
chlorohydrin 6 in good yield. The chlorohydrin 6 was smoothly
transformed into epoxide 7 under basic conditions (NaOH in
MeOH/DCM, H2O) and was further derivatized with base
(NH4OH) and under aqueous acidic conditions (TFA, H2O)
providing amine 8 and diol 9 analogues, respectively.
The chlorohydrin 8 and related derivatives were also treated
with various organic bases (e.g., DBU, Scheme 3). These
reactions cyclized N70 to C84 of the chlorohydrin to form a
pyrrolidine. Protecting the alcohols first as acetates avoided
competing epoxide formation and increased the yield of
pyrrolidine cyclization. The acetates were then removed
under standard conditions that afforded pyrrolidine 2.
Importantly, pyrrolidine 2 was chemically stable, soluble in
mixtures of MeOH/DCM, and purified by flash chromatog-
raphy with high recovery. Under forcing basic conditions
(NH4OH, 65 °C) however, the hydroxyl phenylalanine in the
southern region was removed in a retroaldol-like fashion (inset)
affording 12, likely facilitated via thiazolyl stabilization at the
pseudobenzylic C44 position. This undesired retroaldol
reaction could be avoided by using mild temperatures. So a
three-step synthetic protocol (acetylation, cyclization, and
deprotection) could be efficiently executed on chlorohydrin 8
(itself resulting from crude extract acidic hydrolysis) as a one-
pot procedure. Thus, the synthesis of a chemically stable,
organic solvent soluble, and structurally simplified thiomuracin
analogue 2 was accessible directly from the crude fermentation
extract (ca. 50% 1) in good overall yield (70%).
1 and novel analogues 2, 5−12 were evaluated in MIC
(minimum inhibitory concentration) assays for G+ bacteria
growth inhibition. Five organisms comprised our antibacterial
screen and included Enterococcus faecalis, Enterococcus faecium,
Staphylococcus aureus, Streptococcus pyogenes, and the anaerobic
intestinal pathogen, Clostridium difficile. Streptococcus pneumo-
nia was also evaluated (not shown) but did not differ
significantly from S. pyogenes. An in vitro transcription/
translation cell extract assay was also employed to confirm
that the analogues inhibited protein synthesis and to determine
whether cellular penetration was affecting antibacterial activity.6
1 was potent in all five organisms tested with MICs ranging
from 0.03 to 0.5 μg/mL. The protected analogues of 1 (i.e., 5,
6, and 7) lost significant antibacterial activity (MICs: 1 to >32
μg/mL), which suggests that the carboxylic acid functionality
and two hydroxyl groups located on the macrocycle may play
important roles related to antibacterial activity. These analogues
were inactive in the in vitro transcription/translation assay as
well, which may be related to their low solubility (undetermin-
able). Surprisingly, the chlorohydrin and epoxy analogues with
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Scheme 1. Esterification and Hydroxyl Protection of 1
the C2−C10 side chain removed retained potent antibacterial
activity in four out of five target organisms (i.e., 8, 9: MICs =
0.03−0.5 μg/mL). Group A streptococcus remained the least
susceptible (8, 9: MICs = 2 μg/mL). Furthermore, both amino
and hydroxy derivatives of the epoxide (10, 11, respectively)
reduced the antibacterial activity as compared to the
chlorohydrin 8, while the retroaldol byproduct analogue 12
retained moderate activity in only three organisms (MIC = 2−4
μg/mL). The pyrrolidine 2 retained potent activity in all five
target organisms with MICs ranging from <0.008 to 1 μg/mL.
Importantly, analogues 8−12 and compound 2 retained activity
in a transcription/translation assay measuring protein synthesis
inhibition in cell extracts (50−75% inhibition @ 2uM). This
data suggests that the observed SAR were likely due to
differences in cellular penetration.
within 5 Å of the binding site). The macrocyclic conformation
of 2 was quite similar as compared to 3 (PDB code 2C77), 4
(PDB code 3U2Q), and other derivatives of 3, with
intermolecular H-bond interactions noted between the primary
amide (N35, thiomuracin numbering) and two residues
(Asn273 and Phe296) and also the backbone nitrogen of
Phe261 and O90 of thiomuracin. Further and unique
interactions for 2 and the protein include intramolecular
hydrogen bond stabilization between the tyrosine O68 and
carbonyl O91 (3.1 Å) and also amide N35 and carbonyl O69
(3.0 Å). Phe218 also formed a T-stack interaction with the
pyridine ring of 2. In addition, the pyrrolidine methyl of 2
(C85) favorably fills the pocket formed by the side chain of
Glu215. The pyrrolidine OH is also within reasonable
hydrogen bonding distance (3.4 Å) with the Glu215 carboxylic
acid. Interestingly, several functional groups appear to access
solvent-exposed areas; these structural motifs may represent
areas amenable to further derivatization for improved
Thiomuracin derivative 2 was cocrystallized with E. coli EF-
Tu at 2.3 Å (Figure 2, PDB code 4G5G). The homology
between E. coli EF-Tu and C. difficile EF-Tu is high (86%
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Scheme 2. Acid- and Base-Mediated Transformations of 1
physicochemical properties. In particular, the hydroxyphenyla-
lanine (O52), the pyrrolidine OH, and the amide (C10) all are
oriented toward solvent.
Amide 2 was investigated in rat pharmacokinetic (Figure 3)
and mouse efficacy experiments. Accordingly, 2 was dosed
intravenously to male Sprague−Dawley rats (1 mg/kg).7 Good
systemic exposure (AUC = 1354 nM·h), low clearance (10.1
mL/min/kg), low volume of distribution (Vss = 0.134 L/kg),
and a moderate half-life (T1/2 = 3.4 h) was observed.
Furthermore, in a mouse systemic infection model,8 amide 2
displayed dose-dependent protection (via iv dosing) from lethal
infection with ED50 values of 8.0 and 1.5 mg/kg against S.
aureus and E. faecalis, respectively. Daptomycin was utilized as a
positive control in these experiments; amide 2 proved to be less
potent or commensurate depending on the organism
(daptomycin: S. aureus ED50 = 0.3 mg/kg; E. faecalis ED50
=
3.3 mg/kg). Thus, amide 2 was more than 10-fold less active
against S. aureus in the mouse systemic model as compared to
daptomycin but 2-fold more potent against E. faecalis. These
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Scheme 3. Pyrrolidine Cyclization
Table 1. Biological Activity of 1 and Analogues (MICs in μg/mL)
1
2
5
6
7
8
9
10
11
12
E. faecalis
E. faecium
S. aureus
0.5
0.5
0.5
0.5
0.03
0.25
0.5
8
8
8
8
32
32
0.25
0.5
0.5
2
0.25
0.125
0.5
1
>32
>32
>32
>32
2
4
4
1
2
0.25
1
>32
>32
1
>32
>32
1
>32
>32
>4
2
S. pyogenes
C. difficile
2
>32
1
>32
n/a
<0.008
0.25
0.003
results also parallel results obtained with other derivatives of
fermentation extract, which facilitated overall material isolation
and resupply. Amide 2 was also examined in pharmacokinetic
studies and proved efficacious in the mouse systemic model of
infection. Taken together, these studies culminated in the
discovery of a structurally simplified, chemically stable analogue
2 of 1, which retained antibacterial activity in vitro and in vivo,
and allowed for continued medicinal chemistry exploration and
optimization. The results of further SAR studies will be
reported in due course.
3.3,4
CONCLUSION
■
In summary, synthetic studies of the antimicrobial metabolite 1
were initiated to improve the chemical stability and
physicochemical properties to facilitate isolation and material
supply for further medicinal chemistry discovery. Novel
semisynthetic analogues (2, 5−12) of 1 included protecting
group derivatives of hydroxyl and acid functionalities as well as
derivatives stemming from acid- or base-mediated reactions.
The latter resulted in the removal of the C2−C7 side chain,
derivatization of the C84 epoxide region, and N70−C84
pyrrolidine cyclization. Most notably, removal of the C2−C10
side chain and cyclization of the C84 epoxide to a pyrrolidine
(i.e., 2) retained potent antibiotic activity in all five target Gram
+ organisms. The synthesis of pyrrolidine 2 was optimized (two
pots, four steps) and demonstrated directly on the crude
EXPERIMENTAL SECTION
■
Compound Synthesis and Characterization. Synthetic proce-
dures and compound characterization data are found in the Supporting
Information. Compound purity was assessed by two distinct 20 min
HPLC runs to confirm >95% purity.
MIC Assays. MIC assays were conducted according to broth
microdilution methods described by the Clinical and Laboratory
Standards Institute, Wayne, PA (CLSI M07-A8, 2009, Vol. 29, No. 2;
CLSI M11-A7, Vol. 27). Bacterial strains included E. faecalis (ATCC
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Figure 2. (a) Amide 2 cocrystyallized with E. coli EF-Tu at 2.3 Å. (b) Numbering of 1.
columns (2.1 mm × 30−50 mm × 3.5−5 μm particle size). The
mobile phase A was 0.1% formic acid in water and mobile phase B was
0.1% formic acid in acetonitrile. Gradients were run from 5% B to 95%
B in ∼3.5 min. Plasma samples were protein-precipitated with
acetonitrile containing glyburide as the internal standard (Sigma-
Aldrich, St. Louis, MO).
Figure 3. Efficacy profile of 2 in the mouse systemic infection model
(EC50 in mg/kg).
Mouse Systemic Infection Model. The studies were approved
by the Institutional Animal Care and Use Committee of the Novartis
Institutes for BioMedical Research Inc., Cambridge MA. Animals were
maintained under controlled conditions with free access to food and
water. Female CD1 mice (21−25 g, Charles River Laboratories,
Wilmington, MA) were used for infections with S. aureus (ATCC
49951) and E. faecalis (NB04025, a clinical isolate from the Novartis
bacterial collection that is resistant to erythromycin, tetracycline, and
gentamicin, courtesy of Dr. B. Willinger, Vienna General Hospital,
Austria). MICs of amide 2 were 1 μg/mL and 0.125 μg/mL for animal
strains S. aureus (ATCC 49951) and E. faecalis (NB04025),
respectively. Lethal infections were induced by intraperitoneal
injection of a freshly prepared bacterial suspension of 1 × 108
CFU/mouse in either 50% sterile rat fecal extract (E. faecalis) or
0.9% NaCl (S. aureus). The injected bacterial dose corresponded to
10−100 times the minimal lethal dose as determined from previous
lethal dose titration studies. Against E. faecalis infections the mice were
treated once immediately following the bacterial inoculation, while
against S. aureus infections mice were treated 1 and 5 h after
inoculation. Compound 2 was formulated in 5% DMA, 30% PEG400,
10% cremophor EL, 5% 0.1 N NaOH, 50% pH 7.4 buffer. Daptomycin
was formulated in saline. Compounds were administered iv via tail vein
bolus injection at several dose levels to groups of six mice each.
Following bacterial inoculation, the mice were observed for 5 days. In
addition, body temperature was monitored by electronic micro-
transponders (Bio Medic Data Systems, Inc., Seaford, DE) that were
implanted in mice subcutaneously prior to infection. Mice that became
moribund as indicated by a combined score based on clinical
observations and drop in body temperature were preemptively
euthanized. The 50% effective dose (ED50), the dose providing
protection to 50% of mice, and the 95% confidence limits (95% CI)
were calculated from the survival data at day 5 by probit analysis using
the program Systat (SPSS Inc.). All animals in the vehicle-treated
control groups developed lethal infections.
29212), E. faecium (Prof. Chopra, Univ. Leeds UK, strain 7130724), S.
aureus (MRSA from Prof. Willinger, isolated from a pharyngeal smear,
AKH Vienna A4.018), S. pyogenes (ATCC BAA-595), and C. difficile
(ATCC 43255).
In Vitro Transcription/Translation Assays. The E. coli S30
extract system for circular DNA (Promega cat. no. L1020) was used
per recommendation of the manufacturer with slight modifications.
Briefly, 3.5 μL of 286 ng/μL template DNA (pBESTluc) was mixed
with 1.0 μL each of 1 mM “methionine minus” and “cysteine minus”
amino acid mixes, 8 μL of S30 premix, 6 μL of S30 extract, and 0.5 μL
of the test agents at 40× final concentration, in a total volume of 20
μL. The reaction mixtures were incubated for 2 h at 37 °C in 384-well
flat bottom white plate (Corning, cat. no. 3704). The formation of
luciferase was measured by adding an equal volume (20 μL) of Steady-
Glo luciferase reagent (Promega cat. no. 27104), and emitted light was
detected with a luminometer (Molecular Devices, LMaxll plate
reader). Kirromycin and puromycin, which are known protein
synthesis inhibitors, were used as positive controls. Ampicillin was
used as a negative control. The Rabbit Reticulocyte TnT Quick
Coupled Transcription/Translation system (Promega cat. no. L1170)
was used as recommended by the manufacturer except that the assay
volumes were scaled down from 50 to 20 μL. Briefly, the assay
components consisted of 16 μL of TnT Quick master mix, 3 μL of
(pT7luc) at 167 ng/μL, 0.5 μL of methionine, and 0.5 μL of the test
compound at 40× final concentration in a final volume of 20 μL. The
reaction mixtures were incubated at 37 °C for 75 min. Luminescence
was detected as described above.
Rat Pharmacokinetic Studies. Intravenous PK studies (N = 2)
were performed with male Sprague−Dawley rats, weighing 220−270 g
and approximately 6−10 weeks old, which were obtained from Harlan
Research Laboratories (South Easton, MA), each bearing dual
implanted jugular vein cannula. The rats were fasted overnight before
use and for 8 h after dosing. Blood samples were placed into K2-
EDTA-coated tubes and then centrifuged to yield plasma samples for
analysis by LC-MSMS. Bioanalysis of rat plasma from in-life
experiments were performed by LC-MSMS using a system with the
following configuration: Agilent liquid chromatograph (Santa Clara,
CA), LEAP Technologies CTC-PAL autosampler (Carrboro, NC),
and Applied Biosystems API 4000 mass spectrometer (Framingham,
MA). LC was performed in gradient mode with reversed phase C18
Crystallization and X-ray of Compound 2·EF-Tu Complex.
To form the EF-Tu·NVP-compound 2 complex, 10 mg/mL (227 μM)
E. coli EF-Tu protein in a buffer containing 50 mM Tris pH 8 and 50
mM NaCl was incubated with 1 mM compound 2 for 1 h at 4 °C. The
sample was centrifuged at 20 000g to remove any resulting precipitate.
Crystallization was carried out using 300 nL of the protein sample plus
300 nL of crystallization solution containing 0.1 mM Tris pH 8.23,
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24% PEG3350, 0.2 M MgSO4, using a sitting drop format and
equilibrated against a reservoir of the crystallization solution. The
crystal was flash frozen with liquid nitrogen after being stabilized in a
cryobuffer containing 0.1 mM Tris pH 8.23, 24% PEG3350, 0.2 M
MgSO4, 20% ethylene glycol.
ACKNOWLEDGMENTS
■
The authors thank all of the contributors to the EFT project
team.
X-ray Data Collection. Initial data from a single crystal of the EF-
Tu·compound 2 complex were collected on a ADSC Quantum 210
CCD detector using synchrotron radiation (λ = 1 Å) at the IMCA-
CAT beamline 17-ID of the Argonne Photon Source. Data were
collected using φ rotations of 0.5° and 180° of total data collected.
X-ray Data Processing, Structure Determination, and
Refinement. Data from the EF-Tu·compound 2 complex were
processed using the HKL2000 Suite, version 0.95.9 Data processing
statistics are shown in Table 2. Crystals were diffracted to 2.3 Å
ABBREVIATIONS USED
■
MIC, minimum inhibitory concentration; EC50, 50% effective
dose; G+, Gram positive; MRSA, methicillin-resistant Staph-
ylococcus aureus; VRE, vancomycin-resistant enterococci; S.
aureus, Staphylococcus aureus; E. faecalis, Enterococcus faecalis; E.
faecium, Enterococcus faecium; S. pyogenes, Streptococcus pyogenes;
EF-Tu, elongation factor-Tu
REFERENCES
Table 2
■
(1) Morris, R. P.; Leeds, J. A.; Naegeli, H. U.; Oberer, L.; Memmert,
K.; Weber, E.; LaMarche, M. J.; Parker, C. N.; Burrer, N.; Esterow, S.;
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(2) Selva, E.; Beretta, G.; Montanini, N.; Saddler, G. S.; Gastaldo, L.;
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701. The structure was later corrected and verified: Tavecchia, P.;
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Restelli, E.; Ciabatti, R. Tetrahedron 1995, 51 (16), 4867−4890 . For
acidic decomposition, see above.
(3) (a) LaMarche, M. J.; Leeds, J. A.; Dzink-Fox, J.; Gunderson, K.;
Krastel, P.; Memmert, K.; Patane, M. A.; Rann, E. M.; Schmitt, E.;
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compound 2
data collection parameters (highest resolution shell)
resolution range (Å)
total observations
unique reflections
completeness (%)
I/σ
50−2.3 (2.38−2.3)
148 584
21 195
99.7 (100)
44.8 (8.0)
0.073
Rsym
refinement parameters
work/Rfree
R
0.212/0.258
3047
protein atoms
heterogen atoms (Mg/GDP/compd)
solvent atoms
87
117
average B-factor (Å2)
rms Deviations from ideal value
bond lengths (Å)
43.6
0.009
1.6
bond angles (deg)
resolution in the space group P2(1)2(1)2 with a unit cell of a = 80.305
Å, b = 123.577 Å, c = 45.092 Å, α = β = γ = 90°. Data collection
statistics are shown in Table 2. The structure of the EF-
Tu·GDP·compound 2 complex was determined by the molecular
replacement as implemented in PHASER,10 using E. coli EF-Tu
protein as a search model (1D8T). The resulting molecular
replacement solution contained one EF-Tu·GDP·compound 2 protein
complex in the asymmetric unit. Refinement was carried out with
CNX11 using a single round of rigid-body refinement, following several
cycles of simulated annealing refinement, B-factor refinement, and
model building with the COOT software package.12 Water molecules,
GDP, and the Mg2+ ion were added prior to addition of the ligand 2.
Refinement produced a final model with excellent geometry (rmsd
bond lengths 0.009 Å, rmsd bond angles 1.6°) and R-factors of Rwork
and Rfree of 21% and 26%, respectively.
(6) (a) Zubay, G. In Vitro Synthesis of Protein in Microbial Systems.
Annu. Rev. Genet. 1973, 7, 267−287. (b) Zubay, G. Isolation and
Properties of CAP, the Catabolite Gene Activator. Methods Enzymol.
1980, 65, 856−877.
(7) Because of the lack of aqueous solubility, organic excipients were
necessary to enable formulation of 2 (5% DMA, 30% PEG400, 10%
cremophor EL, 5% 0.1 N NaOH, 50% pH 7.4 buffer).
ASSOCIATED CONTENT
■
S
* Supporting Information
(8) Frimodt-Moller, N.; Knudesen, J. D.; Espersen, F. Handbook of
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: (617) 871-7729. Fax: 617-871-4081. E-mail: matthew.
lamarche@novartis.com.
Notes
The authors declare no competing financial interest.
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