Biocatalytic resolution of bis-tetrahydrofuran alcohol

Article abstract of DOI:10.1021/op100254z

A simple and efficient process has been developed to effect the kinetic resolution of the racemic alcohol 2 using immobilized lipase to afford the desired optically pure (R)-bis-tetrahydrofuran (bis-THF) alcohol 3, to facilitate the rapid progression of a clinical candidate. Rapid optimization and development of reproducible and scalable processes are essential to meet aggressive timeframes for preclinical, safety, and early clinical drug development. Process parameters were initially scoped and optimized using a combination of a rational bioprocess screening design and parallel microscale empirical studies, specifically accounting for scale-up and downstream processing considerations. The choices of reaction solvent, acyl donor, and immobilized biocatalyst proved to be critical factors in the design of a conveniently scalable and enantioselective enzymatic resolution process. The improved process was initially validated on 3-g and then 90-g scale in simple impeller-stirred reactors, exhibiting excellent reproducibility. This methodology was successfully implemented on a multikilogram scale to give the target alcohol 3 with >99% ee.

Full text of DOI:10.1021/op100254z

Organic Process Research & Development 2011, 15, 279–283
Biocatalytic Resolution of Bis-tetrahydrofuran Alcohol
Yuri L. Khmelnitsky,*,† Peter C. Michels,† Ian C. Cotterill,† Michael Eissenstat,‡ Venkataiah Sunku,§
Venugopal R. Veeramaneni,§ Hariprasad Cittineni,§ Gopal R. Kotha,§ Shyamsunder R. Talasani,§ Krishna K. Ramanathan,⊥
Vakula K. Chitineni,⊥ and Bhaskar R. Venepalli⊥
AMRI Inc., Department of Metabolism and Biotransformations, 21 Corporate Circle, Albany, New York 12203, United States,
Sequoia Pharmaceuticals Inc., 401 Professional DriVe, Suite 200, Gaithersburg, Maryland 20879, United States, Indus
Biosciences PriVate Limited, Plot No. 72/A, Part 2, Phase-1, IDA Jeedimetla, Hyderabad 500 055, AP, India, and
CiVentiChem, 1001 Sheldon DriVe, Cary, North Carolina 27513, United States
Scheme 1
Abstract:
A simple and efficient process has been developed to effect the
kinetic resolution of the racemic alcohol 2 using immobilized lipase
to afford the desired optically pure (R)-bis-tetrahydrofuran (bis-
THF) alcohol 3, to facilitate the rapid progression of a clinical
candidate. Rapid optimization and development of reproducible
and scalable processes are essential to meet aggressive timeframes
for preclinical, safety, and early clinical drug development. Process
parameters were initially scoped and optimized using a combina-
tion of a rational bioprocess screening design and parallel micros-
cale empirical studies, specifically accounting for scale-up and
downstream processing considerations. The choices of reaction
solvent, acyl donor, and immobilized biocatalyst proved to be
critical factors in the design of a conveniently scalable and
enantioselective enzymatic resolution process. The improved
process was initially validated on 3-g and then 90-g scale in simple
impeller-stirred reactors, exhibiting excellent reproducibility. This
methodology was successfully implemented on a multikilogram
scale to give the target alcohol 3 with >99% ee.
alcohol (Scheme 1). In general, achieving both the desired
diastereoselectivity and enantioselectivity has remained a chal-
lenge, ultimately requiring additional steps and further enhance-
ment of chiral purity. We present here some of our investiga-
tions aimed at improving the resolution step for the scaled-up
synthesis of chiral alcohol 3, to facilitate the rapid progression
of SPI-256 as a clinical candidate.
The racemic form of the alcohol (2) was derived by reduction
of ketone 1.7 Lipase-catalyzed kinetic resolution promised to
be the most effective and practical route for achieving high
chiral purity of 3. A published biocatalytic procedure6 for
enantioselective acylation of 2 with acetic anhydride catalyzed
by Pseudomonas lipase in 1,2-dimethoxy ethane (DME) was
found to result in relatively low ee values for 3, modest
volumetric productivity, and modest yield due to overacylation
of the (R)-alcohol and challenging product isolation and
purification in our hands. Scale-up processes of this general
approach using lipases in DME have been reported but still
suffer from challenges in control of water activity for hygro-
scopic DME, modest ee, product isolation issues, and practical
process scale-up issues that ultimately necessitated the design
of a continuous reactor.4
Introduction
The synthetic intermediate (3R,3aS,6aR)-hexahydrofuro[2,
3-b]furan-3-ol, 3, is a key component for the synthesis of various
clinical HIV protease inhibitors (PIs) including darunavir,
brecanavir, GS-9005, and SPI-256.1-5 The bis-THF moiety has
imbued these PIs with high potency and excellent resistance
profiles. Not surprisingly, it has thus been the subject of
numerous synthetic investigations.1-4,6 The synthesis of the PIs
requires the enantiomerically pure (3R)-form 3 of bis-THF-
The goal of this work was to develop a more enantioselec-
tive, robust, and scalable biocatalytic process for kinetic
resolution of 2. An optimized process was developed to
successfully resolve and conveniently isolate both enantiomers
3 and 4 on a multikilogram scale.
* Corresponding author. E-mail yuri.khmelnitsky@amriglobal.com. Telephone
(518)-512-2890. Fax (518)-512-2078.
† AMRI Inc.
‡ Sequoia Pharmaceuticals Inc.
§ Indus Biosciences Private Limited.
Results and Discussion
⊥ CiVentiChem.
Optimization of Reaction Conditions. Catalyst EValuation.
Initially, several commercial immobilized Pseudomonas lipases
from Amano were tested, including PS-C I, PS-C II, PS-D I,
and AK-C I. The enzymes were immobilized on ceramic
particles (code “C” in the enzyme designation) or diatomaceous
earth (code “D”). The reactions were carried out in both DME
(1) Quaedflieg, P. J. L. M.; Kesteleyn, B. R. R.; Wigerinck, P. B. T. P.;
Goyvaerts, N. M. F.; Vijn, R. J.; Liebregts, C. S. M.; Kooistra,
J. H. M. H.; Cusan, C. Org. Lett. 2005, 7, 5917–5920.
(2) Canoy, W. L.; Cooley, B. E.; Corona, J. A.; Lovelace, T. C.; Millar,
A.; Weber, A. M.; Xie, S.; Zhang, Y. Org. Lett. 2008, 10, 1103–1106.
(3) Black, D. M.; Davis, R.; Doan, B. D.; Lovelace, T. C.; Millar, A.;
Toczko, J. F.; Xie, S. Tetrahedron: Asymmetry 2008, 19, 2015–2019.
(4) Yu, R. H.; Polniaszek, R. P.; Becker, M. W.; Cook, C. M.; Yu, L. H. L.
Org. Process Res. DeV. 2007, 11, 972–980.
(5) Erickson, J. W., Eissenstat, M., Silva, A. M., Afonina, E. I., Gulnik,
S. V. Poster H-1266; 48th Annual Interscience Conference on
Antimicrobial Agents and Chemotherapy (ICAAC), Washington, DC,
October 25-28, 2008.
(7) Ghosh, A. K.; Kincaid, J. F.; Walters, D. E.; Chen, Y.; Chaudhuri,
N. C.; Thompson, W. J.; Culberson, C.; Fitzgerald, P. M. D.; Lee, H. Y.;
McKee, S. P.; Munson, P. M.; Duong, T. T.; Darke, P. L.; Zugay, J. A.;
Schleif, W. A.; Axel, M. G.; Lin, J.; Huff, J. R. J. Med. Chem. 1996,
39, 3278–3290.
(6) Ghosh, A. K.; Chen, Y. Tetrahedron Lett. 1995, 36, 505–508.
10.1021/op100254z  2011 American Chemical Society
Published on Web 12/07/2010
Vol. 15, No. 1, 2011 / Organic Process Research & Development
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Figure 1. GC analysis of enzymatic acylation of bis-THF alcohol in DME with vinyl butyrate using lipase PS-C I. (A) Starting
racemic bis-THF alcohol; (B) reaction after 3 h. Retention times: (S)-bis-THF alcohol, 17.9 min; (R)-bis-THF alcohol, 19.3 min;
(S)-bis-THF alcohol butyrate, 27.3 min.
and methyl tert-butyl ether (MTBE) at 25 °C using vinyl
butyrate as the acyl donor. Each reaction contained 26 g/L
enzyme, 20 g/L racemic bis-THF alcohol, and 2-fold molar
excess of acyl donor, in a total volume of 1 mL. The reactions
were done in 2-mL screw-capped glass vials with agitation in
an orbital shaker at 300 rpm.
All of the tested lipases showed high enantioselectivity,
selectively acylating (S)-bis-THF alcohol to form ester 4, and
leaving behind the desired (R)-enantiomer 3 of bis-THF alcohol.
However, the enzymes differed in their catalytic activity and
convenience for use and reuse. The reaction rate was the highest
with PS-C I, followed by PS-C II, PS-D I and AK-C I.
Specifically, after 1 h incubation in MTBE, the reaction afforded
alcohol 3 with >99% ee using PS-C I, 99% ee using PS-C II,
83% ee using PS-D I, and 75% ee using AK-C I. In all cases,
the reaction gave alcohol 3 with >99% ee after 3 h. As an
example, Figure 1 shows the reaction profile using lipase PS-C
I after 3 h. No reaction took place in the absence of enzyme.
Selection of Acyl Donor and Reaction SolVent. The acyl
donors vinyl acetate, vinyl butyrate, and isopropenyl acetate
were selected for evaluation. Vinyl and isopropenyl esters
typically offer better reaction control for enantioselective
acylation, and longer-chain (butyl, isopropenyl) esters typically
react more efficiently with better enantioselectivity using lipases.
Isopropenyl esters also typically afford few side reactions and
better biocatalyst recycle. Reactions were again done in two
different solvents, DME and MTBE containing 15% v/v
methylene chloride (MTBE/CH2Cl2). Lipase PS-C I was used
in this set of experiments at a lower concentration of 7 g/L, to
reduce the reaction rate and thus allow for a more precise
comparison of the different conditions. Test reactions were
carried out on a 1-mL scale at 25 °C and orbital shaking at 300
rpm, using 20 g/L racemic bis-THF alcohol and 2-fold molar
excess of acyl donor.
All different acyl donor/solvent combinations were tested,
and reactions were analyzed by chiral GC after 1 and 3 h.
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Table 1. Effect of solvent, acyl donor, and reaction time on
enantioselective resolution of bis-THF alcohol catalyzed by
lipase PS-C I
was found that changing the molar excess to 0.75-fold did not
noticeably affect reaction rates, yields, and observed ee values
but offered improved workup and lower reagent costs.
While the commercial lipase PS-C I was determined to be
an adequate catalyst for the process on the basis of results of
initial enzyme testing, a more cost-effective catalyst was desired
for large-scale production of (R-)bis-THF alcohol. Lipase PS-
30, a commercial enzyme from the same microbial source as
that for lipase PS-C I (Pseudomonas cepacia), was immobilized
on Kieselgur Celite-521 using an internally developed protocol
(see Experimental Section for details) and compared under
conditions selected for lipase PS-C I: 100 g/L bis-THF alcohol
in MTBE/CH2Cl2 using vinyl butyrate as acyl donor (0.75-fold
molar excess over racemic bis-THF alcohol), 25 g/L enzyme,
reaction temperature 25 °C. It was found that the immobilized
PS-30 lipase afforded a full conversion of (S)-bis-THF alcohol,
resulting in >99% ee of (R)-bis-THF alcohol after 3 h (Table
4). Thus, the immobilized PS-30 enzyme showed enantiose-
lectivity and overall product yield similar to those observed with
lipase PS-C I. On the basis of these parameters, immobilized
PS-30 was chosen in all subsequent scale-up optimization
reactions as a cost-effective alternative to the commercial
immobilized lipase. Similarly, on the basis of cost considerations
and easier downstream processing, vinyl acetate was chosen
instead of vinyl butyrate for the large-scale resolution of bis-
THF alcohol. Both acyl donors showed similar performance
during the scoping studies (Table 1).
Scale-Up of Optimized Enzymatic Process. In the initial
scale-up experiments two identical reactions were set up,
differing only in impeller stirring speed (165 and 350 rpm, with
the catalyst fully suspended at both stirring speeds), in order to
verify the reaction performance upon scale-up and to explore
the effect of stirring speed in larger reactors. The reaction was
carried out on a 3-g scale of bis-THF alcohol in 30 mL of
MTBE/CH2Cl2, 0.75-fold molar excess vinyl acetate, and 20
g/L immobilized PS-30 enzyme in a jacketed vessel at 25 °C
(total reaction volume 30 mL). Both reactions were complete
in 4 h with no overacylation, giving the desired (R)-alcohol
with >99% ee by chiral GC analysis. Thus, the reaction was
not diffusion-controlled, and the reaction rate and yield were
not affected by the stirring speed, as long as the catalyst
remained fully suspended in solution.
enantiomeric excess of (R)-bis-THF
alcohol, %
DME
MTBE
acyl donor
1 h
3 h
1 h
3 h
vinyl acetate
vinyl butyrate
isopropenyl acetate
59
44
26
>99
98
89
85
91
48
>99
>99
>99
Table 2. Chromatographic properties of bis-THF alcohol
and its esters
compound
GC retention time, min TLC Rfa
(R)-bis-THF alcohol
19.3
17.9
11.5
27.3
0.12
0.12
0.29
0.38
(S)-bis-THF alcohol
(S)-bis-THF alcohol acetate
(S)-bis-THF alcohol butyrate
a Silica gel, MTBE/n-hexane ) 4:1 v/v.
Selected results of this study are presented in Table 1; Table 2
shows the comparison of chromatographic properties of the
corresponding reactants and products that are important for
purification assessment. In all cases the enzyme showed higher
activity in MTBE/CH2Cl2 compared to DME (Table 1).
Furthermore, the boiling point of MTBE (55 °C) is substantially
lower than that of DME (85 °C), and therefore the postreaction
removal of MTBE/CH2Cl2 is easier and more cost-effective and
provides a higher recovered yield of the volatile alcohol
product.8 Using the reaction rate and the achieved alcohol ee
as selection criteria, the best performing system was lipase PS-C
I using vinyl butyrate or acetate as acyl donor. The use of vinyl
butyrate offers easier extractive or chromatographic separation
of the target (R)-bis-THF alcohol from (S)-bis-THF alcohol
ester, as predicted on the basis of the chromatographic and
physical characteristics of these compounds (Table 2), while
vinyl acetate is a less costly reagent at scale.
Process Scoping. An optimization study for enzymatic
resolution of bis-THF alcohol was initially carried out using
immobilized lipase PS-C I and vinyl butyrate in MTBE/CH2Cl2.
A broad range of reaction conditions was tested, as summarized
in Table 3. The optimization study was done in 1 mL of reaction
volume using shaking at 300 rpm. The reaction temperature
was maintained at 25 °C. Reaction rates under essentially all
conditions were typically very high (even at high alcohol
concentrations full conversions in many cases were achieved
after approximately 3 h), and therefore heating the reaction was
not necessary.
After completion of the reaction, the enzyme was separated
by filtration, and the solvent and excess acyl donor were
removed by evaporation under reduced pressure. This resulted
in a clean mixture of the desired (R)-enantiomer of bis-THF-
alcohol and the acetic acid ester of the (S)-enantiomer, ready
for chromatographic separation. No decomposition or reduction
in ee of the alcohol was observed after the workup of the
reaction mixture.
Having verified the reaction conditions on a 3-g scale, the
reaction was further scaled up to a 90 g scale, using the
following protocol. To 90 g (0.692 mols) of bis-THF alcohol
in a 1-L medium bottle were added 47.9 mL (44.74 g, 0.520
mols) of vinyl acetate (0.75-fold molar excess with respect to
racemic alcohol) and methylene chloride (112.5 mL). The
mixture was swirled until a homogeneous solution was obtained.
MTBE (637.5 mL) was added with mixing to obtain a
Increasing enzyme concentrations resulted in higher reaction
rates but also caused the undesirable overacylation of the target
(R-)bis-THF alcohol (Table 3), especially at longer reaction
times and higher starting concentrations of bis-THF alcohol.
This could make the control of the reaction outcome at scale
more difficult, and therefore moderate enzyme concentrations
were preferable. The data in Table 3 were obtained using a
2-fold molar excess of vinyl butyrate over bis-THF alcohol. It
(8) A significant problem encountered when acetic anhydride was used in
preliminary studies was the difficulty in its removal due to the relatively
high boiling point.
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Table 3. Optimization of resolution of bis-THF alcohol catalyzed by lipase PS-C I
reaction time 1 h reaction time 3 h
conv. of overacylation of conv. of overacylation of conv. of
alcohol]g/L [lipase]g/L (S)-isomer % ee % (R)-isomer % (S)-isomer % ee % (R)-isomer % (S)-isomer % ee % (R)-isomer %
reaction time 6 h
overacylation of
[bis-THF
20
50
50
100
100
100
7
25
100
25
100
200
89
100
100
100
100
100
81
100
100
100
100
100
0
0
0
0
1.7
<2
100
100
100
100
100
100
100
100
100
100
100
100
0
nda
100
100
100
100
100
nda
100
100
100
100
100
nda
<2
1.5
2.6
2.2
1.9
7.2
7.1
1.8
10.1
15.6
a Not determined.
Table 4. Comparison of lipase PS-C I and immobilized
In order to avoid the laborious and time-consuming chro-
matography step in the purification of 3, an alternative highly
efficient purification strategy was developed on the basis of
chemical protection/deprotection of (R)-bis-THF alcohol. In this
process, the crude mixture of reaction products 3 and 4 was
dissolved in acetonitrile and treated with disuccinimidyl carbon-
ate (DSC), resulting in the formation of a DSC derivative 5
(Scheme 2). The latter could be easily separated from the acetate
4 by sequential trituration with ethyl acetate and MTBE. It could
then be used directly for further chemistry or subjected to
alkaline hydrolysis to remove the DSC protection (see Experi-
mental Section for details). The purity of the final product 3,
as determined by chiral GC analysis, was 99.5%, with ee >
99%, comparable to the purity obtained after purification with
column chromatography.
lipase PS-30 in enantioselective resolution of bis-THF alcohol
after 1.5 h reaction
after 3 h reaction
enzyme
yield, %
ee, %a
yield, %
ee, %a
lipase PS-30
lipase PS-C I
93
100
>99
>99
100
100
>99
>99
a Enantiomeric excess of (R)-bis-THF alcohol.
homogeneous solution, and the reaction mixture was slowly
poured onto the immobilized PS-30 lipase enzyme (18 g, 20
g/L) in a 2-L jacketed glass vessel maintained at 25 °C by a
circulating water bath. The reaction was mixed with the use of
an overhead stirrer assembly for 4 h at 350 rpm, which was
sufficient to keep the enzyme fully suspended. The enzyme was
removed by filtration, and the reaction mixture was analyzed
by chiral GC, affording the desired (R)-alcohol with >99% ee
(Figure 2). The reaction was repeated twice under the same
conditions, giving identical results.
Experimental Section
Chemicals. Lipase PS-30 and immobilized lipases PS-C I
(1638 U/g), PS-C II (1090 U/g), PS-D I (899 U/g), and AK-C
(597 U/g) were received from Amano Enzyme U.S.A. (Elgine,
IL). Immobilized lipase PS-30 (1425 U/g) and racemic bis-
THF alcohol were prepared at Indus Biosciences (lot # IB/TSR/
049/071). All solvents and acyl donors were purchased from
Aldrich (Milwaukee, WI) and were of the highest grade
available.
Reaction conditions identified and verified in the preliminary
scale-up experiments were applied to perform the lipase-
catalyzed resolution of racemic bis-THF alcohol on a multiki-
logram scale. A total of 10 kg of 3 was produced in several
reaction batches, as described in the Experimental Section, after
purification by flash chromatography. The purity of the final
product, as determined by chiral GC analysis, was 99.7%, with
ee > 99%.
Figure 2. GC analysis of enzymatic acylation of bis-THF alcohol with vinyl acetate on a 90-g scale. Retention times: (R)-bis-THF
alcohol, 19.1 min; (S)-bis-THF alcohol acetate, 11.3 min.
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Scheme 2
Enzyme Immobilization. A suspension of Kieselgur Celite-
was separated and evaporated under reduced pressure. Obtained
solids (12.6 g) were triturated in MTBE (20 mL) at 0 °C for
30 min. The solvent was removed by filtration, and the solid
residue was washed with chilled hexane (5 mL) to produce 5.1 g
of DSC derivative 5 (melting point 118-120 °C). The DSC
derivative (0.5 g) was dissolved in THF (5 mL) and the solution
stirred at 30 °C for 10 min, followed by the addition of 3 mL
of 20% aqueous NaOH solution and stirring for 30 min at 30
°C. As a result of this treatment a biphasic mixture was formed,
which was separated into aqueous and organic layers. The
aqueous layer was extracted with ethyl acetate (5 mL). The
organic extract was dried over sodium sulfate and concentrated
under reduced pressure to give pure alcohol 3 (56% isolated
yield). The purity of the final product 3, as determined by chiral
GC analysis, was 99.5%, with ee > 99%.
Chiral GC Analysis. Prior to chiral GC analysis, reaction
samples were filtered to remove immobilized enzyme, and the
filtrate was directly injected into a gas chromatograph. GC
analysis was carried out on an Hewlett-Packard 5890 gas
chromatograph with a flame ionization detector using a Chiral-
dex ꢀ-cyclodextrin trifluoroacetyl (BTA) capillary column (20
m × 0.25 mm) supplied by Astec (Whippany, NJ). The
chromatography was conducted under isothermal conditions at
oven temperature 155 °C, inlet temperature 200 °C, and detector
temperature 250 °C. The flow rate of the carrier gas (helium)
was 2.0 mL/min.
521 (3 kg) was prepared in 30 L of 30 mM potassium phosphate
buffer pH 7.0. The slurry was filtered and Celite was washed
with 3 × 3 L of the buffer. After drying, the neutralized Celite
was mixed with a suspension of 750 g of lipase PS-30 in 10 L
of buffer. The resulting slurry was spread on plastic dishes and
allowed to air-dry for 4 days.
Large-Scale Resolution of Racemic Bis-THF Alcohol. A
solution of bis-THF alcohol (4.0 kg, 30.74 mols) in dichlo-
romethane (4 L) and MTBE (25 L) was placed in a 50-L reactor,
followed by vinyl acetate (1.984 kg, 23.05 mols). The mixture
was stirred for 15 min at ambient temperature. Immobilized
lipase PS-30 (0.70 kg, 20 g/L) was then added, and the resulting
suspension was stirred at 400 rpm at room temperature. Samples
of the reaction mixture were periodically withdrawn and
analyzed by chiral GC to monitor the reaction progress. Once
the ee of the target alcohol 3 exceeded 99% (∼5 h reaction
time), the enzyme was removed by filtration.
Purification of (R)-Bis-THF Alcohol Using Column
Chromatography. The filtrate obtained after the biocatalytic
reaction was concentrated under reduced pressure. The resulting
mixture was separated by column chromatography (100-200
mesh silica gel, 10 kg), eluting with a step gradient of ethyl
acetate in hexane (from 5 vol % to 30 vol % ethyl acetate in
5% steps) at 10 L/h flow rate. Fractions containing 3 were
pooled and evaporated under reduced pressure to remove the
solvent. The target chiral alcohol 3 was isolated in the amount
of 1.23 kg (30% isolated yield) with purity of 99.7% as
determined by chiral GC. 1HNMR (CDCl3, 200 MHz): δ 5.72
(d, 1H), 4.38-4.56 (q, 1H), 3.87-4.12 (m, 3H), 3.67-3.75 (m,
2H), 2.78-2.92 (m,1H), 2.28-2.34 (m,1H), 1.78-1.93 (m, 1H).
Purification of (R)-Bis-THF Alcohol Using Chemical
Protection/Deprotection. The biocatalytic resolution reaction
was carried out as described above on a 10-g scale. The filtrate
obtained after the biocatalytic reaction was concentrated under
reduced pressure. The crude product mixture (10.8 g) was
dissolved in acetonitrile (60 mL) and treated with disuccinimidyl
carbonate (1.5 equiv) in the presence of triethylamine (2 equiv)
at room temperature for 24 h. Acetonitrile was then removed
under reduced pressure, and the solids were dissolved in ethyl
acetate (100 mL). The organic solution was washed with water
(20 mL) and saturated NaCl solution (20 mL). The organic layer
Conclusion
Bis-THF alcohol 3 has proven to be a critical intermediate
in the synthesis of many of the most potent and effective HIV
PIs described in the past decade. In this work we show that by
making suitable adjustments to the originally described enzy-
matic resolution procedure this key intermediate can be prepared
in high yield and purity on large scale.
Acknowledgment
We acknowledge helpful discussions with Dr. M. C. Kang
in the preliminary stages of this work. The research was
sponsored by Sequoia Pharmaceuticals, Inc.
Received for review September 17, 2010.
OP100254Z
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