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Accurate analysis of boronic pinacol esters using low residual

silanol silica based reversed phase HPLC

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DOI: 10.1080/10826076.2013.825856

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ACCURATE ANALYSIS OF BORONIC

PINACOL ESTERS USING LOW RESIDUAL
SILANOL SILICA BASED REVERSED PHASE
HPLC
a a a
Archana Kumar , Tania Ng , Sushant Malhotra , Jason
a a
Gruenhagen & Larry Wigman
a
Small Molecule Pharmaceutical Sciences, Genentech Inc. , South
San Francisco , California , USA
Accepted author version posted online: 12 Sep 2013.Published
online: 03 Apr 2014.

To cite this article: Archana Kumar , Tania Ng , Sushant Malhotra , Jason Gruenhagen & Larry Wigman
(2014) ACCURATE ANALYSIS OF BORONIC PINACOL ESTERS USING LOW RESIDUAL SILANOL SILICA BASED
REVERSED PHASE HPLC, Journal of Liquid Chromatography & Related Technologies, 37:14, 1985-1998,
DOI: 10.1080/10826076.2013.825856

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 Journal of Liquid Chromatography & Related Technologies, 37:1985–1998, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2013.825856

ACCURATE ANALYSIS OF BORONIC PINACOL ESTERS USING

LOW RESIDUAL SILANOL SILICA BASED REVERSED PHASE HPLC

Archana Kumar, Tania Ng, Sushant Malhotra, Jason Gruenhagen, and

Larry Wigman
Small Molecule Pharmaceutical Sciences, Genentech Inc., South San Francisco,
California, USA
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& Pinacol esters of boronic acids are widely used as starting materials in organic synthesis for
Suzuki-Miyaura chemistry. The accurate analysis of these esters is complicated by competing hydroly-
sis to the corresponding boronic acids under typical RP-HPLC testing conditions. To address this
challenge, we evaluated conditions to minimize on-column hydrolysis during RP-HPLC analysis.
The effects of several HPLC parameters on hydrolysis were explored through the design of experiment
(DoE) studies. Three columns bearing different silanol activities were selected to analyze boronate
esters: Agilent Zorbax Eclipse XDB C18, Phenomenex Onyx Monolithic, and Waters XTerra
MS C18. It was determined that the stationary phase has a significant influence on the rate of
on-column hydrolysis, as did formic acid concentration in mobile phase. Column temperature
had a minor effect. The optimized method, using an XTerra MS C18 column and mobile phase with
no pH modifier, was applied to various boronate esters and minimal to no on-column hydrolysis was
observed.

Keywords boronic acid, boronic acid pinacol ester, on-column hydrolysis, RP-HPLC,
stationary phase, Suzuki coupling

INTRODUCTION
Boronic acids and their pinacol esters are widely used as starting mate-
rials in the formation of C–H, C–O, C–N, C–Br, C–I, and C–C bonds.[1–5]
In the context of the C–C bond forming process, the palladium-catalyzed
Suzuki cross-coupling reaction[6–10] has been widely used in the pharma-
ceutical industry in order to synthesize various APIs (Figure 1). Although
traditionally boronic acids were employed in this reaction, recent work
has led to the finding that pinacol esters of boronic acids are often

Address correspondence to Archana Kumar, Small Molecule Pharmaceutical Sciences, Genentech

Inc., South San Francisco, CA 94080, USA. E-mail: kumar.a@gene.com
Color versions of one or more of the figures in the article can be found online at www.tandfonline.
com/ljlc.
 1986 A. Kumar et al.
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FIGURE 1 (a) Representative Suzuki coupling reaction and hydrolysis products for ethyl 2-methyl-2-
(4-boronic acid pinacol ester)-1H-pyrazol-1-yl)propanoate; and (b) Representative Suzuki coupling
reaction and hydrolysis products for 1-ethyl-phenylurea pinacol boronate ester.

preferred due to the improved chemical stability.[11,12] These boronate

esters can be hydrolyzed in situ to generate the boronic acid necessary
for the cross-coupling reaction.[13–17] Furthermore, boronic acids may exist
as mixtures of oligomeric anhydrides, especially the cyclic six-membered
boroxines which further complicates their analysis.[18]
In manufacturing processes involving the Suzuki coupling, the monitor-
ing of starting materials and intermediates is essential as imprecise analyses
can lead not only to lowered yields but also the generation of undesired
downstream impurities. Whereas conventional RP-HPLC is suitable for
the purity analysis of most starting materials, it may lead to artifacts in the
analysis of molecules susceptible to hydrolysis[19,20] under the sample
preparation and testing conditions used in traditional RP-HPLC (Figure 1).
To minimize such analytical artifacts, a fast LC method[21] has been
reported to mitigate the on-column hydrolysis of some pinacol boronate
esters. Recently a method using a polymer column and high pH mobile
phase has been reported for the analysis of highly reactive pinacol boronate
esters with electron deficient aryl ring.[22] However, limited pressure
 Accurate Analysis of Boronic Pinacol Esters 1987

resistance, hindered mass transfer, and swelling=shrinking properties lead-

ing to a reduction in plate number make these columns less desirable than
silica-based columns.[23] Zirconia columns with lack of silanol groups are
an alternative.[24] However, these columns were not thoroughly investi-
gated due to peak broadening issues. Thus, an accurate low residual silanol
silica based reversed phase method was developed for the analysis of less
reactive pinacol boronate esters with electron-rich aryl rings containing
electron-donating functionalities.
Purity analysis using quantitative 1H-NMR (qNMR) is another alternative
although low sensitivity (LOQ
0.5%) makes its use less desirable. For rou-
tine quality control purposes, HPLC therefore remains desirable to quantify
both the major component and various by-products present in a sample mix-
ture.[25] However, the role of stationary phase for the on-column hydrolysis
of pinacol boronate esters has not been investigated and understood.
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Given the utility of pinacol boronate ester starting materials in drug

discovery and development, this study was aimed at understanding HPLC
method parameters using silica based columns that impact the in situ
hydrolysis of boronate esters and finding RP-HPLC method conditions,
which minimize the analytical artifacts associated with their analyses.

EXPERIMENTAL
Instrumentation
All HPLC experiments were performed using an Agilent 1200 series
HPLC equipped with a quaternary pump, a diode array detector, a standard
column oven, and an autosampler equipped with a thermostat. The UV
detector wavelength was set at 220 nm. Columns investigated include Agilent
Zorbax Eclipse XDB C18 4.6  50 mm 3.5 mm, Phenomenex Onyx Mono-
lithic C18 2.0  50 mm, and Waters XTerra MS C18 4.6  50 mm 2.5 mm.

Chemicals
HPLC grade acetonitrile (ACN) and methanol were obtained from EMD.
Tetrahydrofuran (THF) was purchased from Sigma-Aldrich. The 1-methyl
pyrazole-4-boronic acid pinacol ester and 1H-Benzimidazole-5-boronic acid
pinacol ester were purchased from Sigma-Aldrich. Ethyl 2-methyl-2-
(4-boronic acid pinacol ester)-1H-pyrazol-1-yl)propanoate (1) and 1-ethyl-
phenylurea pinacol boronate ester (3) were synthesized in-house. All other
boronic acid pinacol esters were purchased from Boron Molecular. All
boronic acids were obtained by treating the respective pinacol boronate
esters with 1:1 water=ACN.
 1988 A. Kumar et al.

Diluent Selection
Initial results indicated that composition of diluent in sample prep-
aration was essential for reliable HPLC analysis. Sample preparation using
1:1 ACN:water resulted in hydrolysis of boronate ester (1) to the corre-
sponding boronic acid prior to injection (Figure 1). To select a more appro-
priate diluent, samples prepared in 100% ACN, THF, and methanol (aprotic
and protic diluents) were evaluated. These samples were analyzed by
RP-HPLC periodically over a 24-hr period.

Design of Experiment
On-column hydrolysis was studied through a design of experiment
(DoE) study by varying the column stationary phase, level of formic acid
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in mobile phase, and column temperature. Table 1 lists the experimental

factors, their variable types, and levels tested. The experiment was a
12-run full factorial design with three experimental factors evaluating:
three different stationary phases, two levels of formic acid, and two column
temperatures (3  2  2 ¼ 12).
The design was replicated for the two compounds (1, 3) giving a total of
24 runs (Table 2). The sequence was designed to avoid any carryover effect
of formic acid; such that the first 12 runs were conducted in the absence of
formic acid and the last 12 runs with 0.05% formic acid present in mobile
phase. Columns of similar dimensions were used. The gradient and flow rate
were adjusted to enable the resolution of boronate ester peak from respect-
ive boronic acids and process impurities; further, the method parameters
were altered to yield similar retention times for the boronic acid pinacol
ester peak on the three columns. Column packing particle size was not
assessed as part of the DoE study.

Stationary Phase (Column) Selection

The column is a key component of any HPLC method, enabling
separation of compounds based upon selectivity.[26–28] Given the range of

TABLE 1 Experimental Factors, Their Variable Types and Levels

Factor Type Low () High (þ) # of Levels

1 Column Temperature ( C) Numeric 15 C 35 C 2

2 Formic Acid Numeric 0% 0.05% 2
3 Solid Phase Categorical Onyx Monolithic C18 (A) 3
XTerra MS C18 (B)
Zorbax Eclipse XDB C18 (C)
Total # of runs per compound 12
 Accurate Analysis of Boronic Pinacol Esters 1989

TABLE 2 24-Run Full Factorial Experimental Design Using Compounds 1 and 3

Run Order Compound Stationary Phase Column Temp Formic Acid Pattern

1 1 Onyx Monolithic C18 15 0 A

2 3 Onyx Monolithic C18 15 0 A
3 1 Onyx Monolithic C18 35 0 Aþ
4 3 Onyx Monolithic C18 35 0 Aþ
5 1 Zorbax Eclipse XDB C18 15 0 C
6 3 Zorbax Eclipse XDB C18 15 0 C
7 1 Zorbax Eclipse XDB C18 35 0 Cþ
8 3 Zorbax Eclipse XDB C18 35 0 Cþ
9 1 XTerra MS C18 15 0 B
10 3 XTerra MS C18 15 0 B
11 1 XTerra MS C18 35 0 Bþ
12 3 XTerra MS C18 35 0 Bþ
13 1 Onyx Monolithic C18 15 0.05 Aþ
14 3 Onyx Monolithic C18 15 0.05 Aþ
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15 1 Onyx Monolithic C18 35 0.05 Aþþ

16 3 Onyx Monolithic C18 35 0.05 Aþþ
17 1 Zorbax Eclipse XDB C18 15 0.05 Cþ
18 3 Zorbax Eclipse XDB C18 15 0.05 Cþ
19 1 Zorbax Eclipse XDB C18 35 0.05 Cþþ
20 3 Zorbax Eclipse XDB C18 35 0.05 Cþþ
21 1 XTerra MS C18 15 0.05 Bþ
22 3 XTerra MS C18 15 0.05 Bþ
23 1 XTerra MS C18 35 0.05 Bþþ
24 3 XTerra MS C18 35 0.05 Bþþ

available reversed-phase stationary phases, we selected columns with differ-

ent degrees of residual silanol activity to assess its impact on hydrolysis of
pinacol boronate esters. Agilent Zorbax Eclipse XDB C18,[29] Phenomenex
Onyx Monolithic,[30,31] and Waters XTerra MS C18[32] were selected for the
study (Table 3).

TABLE 3 Types of Columns Studied and Their Properties

Carbon Surface
Silanol Load Area End
Column Type of Stationary Phase Activity[32–34] (%) (m2=g) Capping

Zorbax Eclipse A packed column with bonded 3.05 10 180 Yes

XDB C18 stationary phase on silica particles
Onyx Monolithic A column of bonded-phase on highly – 18 300 Yes
C18 porous rods of silica with a bimodal
pore structure
XTerra MS C18 A column based upon Hybrid Particle 1.12 15.5 179 Yes
Technology, combining the
properties of silica and polymer
with 33% fewer residual silanols
 1990 A. Kumar et al.

TABLE 4 Chromatographic Conditions for the Analysis of Boronic Pinacol Esters

Parameter Condition

Column Waters XTerra MS C18; 4.6  50 mm

Column temperature 35 C
Flow rate 1.2 mL=min
Injection volume 2 mL
Auto-sampler temperature 4 C
Detection wavelength 220 nm (DAD Reference: off; Bandwidth: 4 nm)
Mobile phase A: Water
B: Acetonitrile

Time (min) A (%) B (%)

Gradient 0.0 80 20
0.4 80 20
5.4 20 80
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7.0 20 80
7.2 80 20
10.0 80 20

Mobile Phase Composition Selection

Mobile phase with no pH modifier and with 0.05% formic acid were
selected for the study (Table 1).

Column Temperature Selection

Column temperature effect was evaluated at 15 C and 35 C (Table 1).

Optimized Method and Boronate Esters Screening

The optimized method employing the XTerra MS C18 column was used
to analyze other aromatic boronate esters. The chromatographic conditions

FIGURE 2 Representative Chromatogram of spiked Pyrazole boronic pinacol ester (1): showing
separation of Pyrazole boronic pinacol ester (1) from process impurities (5, 6) and hydrolyzed ester (2).
 Accurate Analysis of Boronic Pinacol Esters 1991

are listed in Table 4. The specificity of this method is demonstrated in

Figure 2, which shows the acceptable resolution for all potential process
impurities including the structural isomer.[5] Good linear response is
shown for the analyte concentration from 1.2 mg=mL to 1.4 mg=mL.

RESULTS
Effect of Sample Diluent
Samples prepared in 1:1 ACN:water and 100% methanol resulted in the
formation of significant amounts of boronic acid 2 (Figure 3). Whereas,
samples prepared in ACN and THF exhibited no increase in hydrolysis,
measured as area%, over time for boronate ester 1. However, peak splitting
was observed with acetonitrile and THF, both being strong solvents, despite
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using 5 mL of injection volume, which was mitigated by changing the injec-

tion volume to 2 mL. Being Lewis acidic in nature, boron is known to form
adducts readily with water and hydroxyl compounds.[36] Acetonitrile was
ultimately selected as the sample diluent over THF due to its enhanced
compatibility with HPLC systems.

Influence of Stationary Phase (Column)

Figure 4 shows the main effects of the three stationary phases for com-
pounds 1 and 3. These results demonstrate the influence of the column on
the level of hydrolysis observed. On-column hydrolysis was lowest for the
XTerra MS C18 and intermediate for Onyx Monolithic C18; it was most sig-
nificant with the Zorbax Eclipse XDB C18 column. Hydrolysis was 0.31%
(Table 5) higher for samples analyzed on Zorbax column than hydrolysis
of samples analyzed on Onyx column, which is significantly above the
impurity qualification threshold per ICH Q3B (R2) guidance. However
difference between Onyx and XTerra columns clearly depends upon the

FIGURE 3 Influence of Diluent on the Stability of ethyl 2-methyl-2-(4-boronic acid pinacol ester)-1H-
pyrazol-1-yl)propanoate (1).
 1992 A. Kumar et al.

FIGURE 4 Influence of Stationary Phase (column) and Mobile Phase composition [Horizontal position
indicates run #. Each group of four consecutive runs included effects of changing column temperature
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and compound – first 2 at 15 C and last 2 at 35 C; Runs 1, 3, 5, 7 . . . .are for compound 1 and runs 2, 4,
6, 8 . . . .are for compound 3].

presence or absence of formic acid: when present, the difference in

hydrolysis between the columns is greater than 1 percentage point, whereas
without formic acid, the differences are much smaller.

Influence of Mobile Phase Composition

Figure 4 demonstrates the main effects of the presence of formic acid in
mobile phase on boronic ester hydrolysis for compounds 1 and 3. There was
no hydrolysis observed using an XTerra MS C18 column with the addition of
formic acid in mobile phase. However, for Onyx and Zorbax stationary
phases, one percentage point increase in hydrolysis was observed with the
addition of formic acid. The available formate anion (conjugate base of
formic acid) is an electron donor and presumably coordinating with the
Lewis acidic boron atom making it more reactive.
The regression model was fitted to the observations using Onyx and
Zorbax stationary phases to determine the interaction between formic acid
and stationary phase. Data from XTerra Stationary phase was not included

TABLE 5 Statistically Significant Main Effects from the Regression Model

for Compounds 1 and 3

Factor Effect Size

Formic Acid 0.87% (0.72%a, 1.02%b)

Solid Phase (Change from Onyx to Zorbax) 0.31% (0.16%a, 0.45%b)
a
Lower 95% confidence limit.
b
Upper 95% confidence limit.
 Accurate Analysis of Boronic Pinacol Esters 1993

FIGURE 5 Influence of Column Temperature [Horizontal position indicates run #. Each group of four
consecutive runs included effects of changing column temperature and compound – first 2 at 15 C and
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last 2 at 35 C; Runs 1, 3, 5, 7 . . . .are for compound 1 and runs 2, 4, 6, 8 . . . .are for compound 3].

in the regression model as no formic acid effect was observed (Figure 4). A
synergistic interaction between the stationary phase and formic acid was
observed for Onyx Monolithic C18 and Zorbax Eclipse XDB C18 columns
since adding 0.05% formic acid to the mobile phase led to increased degra-
dation by 0.87 percentage points (Table 5).

Influence of Column Temperature

Figure 5 shows the main effects of column temperature on boronic
ester hydrolysis for compounds 1 and 3. Contrary to expectation, increasing

FIGURE 6 Effect of all 3 factors on both compounds 1 and 3; For each Compound and Stationary
Phase, solid black lines connect the mean Degradation for runs with Formic Acid ¼ 0% to the mean
Degradation for runs with Formic Acid ¼ 0.05%.
 1994 A. Kumar et al.

TABLE 6 Analysis of Various Aryl Boronic Esters

Sample Sample Preparation

Preparation in 50:50 ACN=Water
in CAN
2 hr; RT 6 d; 4 C
Hydrolyzed Hydrolyzed Hydrolyzed
Compound Compound Name Structure Product (%) Product (%) Product (%)

1-Methylpyrazole-4-
7 boronic acid 0 10.9 96.1
Pinacol ester
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1H-Benzimidazole-
8 5-boronic acid 0 11.4 89.8
pinacol ester

4-Hydroxyphenyl
9 Boronic acid 0 6.0 72.9
pinacol ester

3-Hydroxyphenyl
10 Boronic acid 0 25.7 96.7
pinacol ester

4-Aminophenyl
11 Boronic acid 0 1.0 25.8
pinacol ester

3-Aminophenyl
12 Boronic acid 0 11.5 92.0
pinacol ester

Phenyl Boronic acid

13 0.19 15.0 94.5
pinacol ester
 Accurate Analysis of Boronic Pinacol Esters 1995

the column temperature from 15 C to 35 C resulted in little to no variation

in degradation, presumably due to silanol catalysis lowering activation
energy for the hydrolysis.

Optimized Method and Boronate Esters Screening

Figure 6 summarizes the data for compounds 1 and 3 grouped by
stationary phase, mobile phase composition, and column temperature.
The extent of hydrolysis that occurred on the HPLC column was of practi-
cal interest. However, total degradation after HPLC analysis was used as the
response in the statistical analysis. Compound 3 had 2% initial hydrolyzed
product; analyzed by qNMR. As shown in Figure 6, the XTerra MS C18
column was most suitable for the analysis of pinacol boronic esters via
RP-HPLC as no significant hydrolysis was observed with changes in column
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temperature and formic acid concentration.

From an overall method capability, a mobile phase with formic acid may
be acceptable using an inert column. Column temperature was established
at 35 C. This optimized method was able to resolve and quantify the
amount of the major component 1 and the various by products (2, 5, 6)
present in a sample mixture with no on column hydrolysis (Figure 2).
As shown in Table 6, this method is also applicable for the analysis of
various boronate esters with electron rich aromatic ring.

DISCUSSION
Boron, an sp2 hybridized atom, possessing six valence electrons and a
vacant p-orbital, behaves as a mild Lewis acid. This Lewis acidic nature of
boron facilitates the coordination between electron donors and boronate
esters. The hydrolysis of a boronate ester, presumably involves the coordi-
nation of a nucleophile such as water, formate anion, or a silanol to the
trivalent Lewis acidic boron.[36,37]
This hypothesis was tested with various aryl boronate esters containing
different electron releasing groups and their positions (Table 6). As illu-
strated in Table 6, the rates of hydrolysis vary based on the substituents on
the aromatic ring. A rationale for this differential hydrolysis is based on
the electronic properties of the boronic ester. Boronate esters possessing
electron rich groups on the aromatic ring can donate electrons via
p-conjugation and undergo slower hydrolysis compared to boronate esters
where the ring substituent cannot enter into resonance with the vacant
orbital on boron. In the case of boronate ester 9, the para-phenol donates
electrons to the vacant boron making it less Lewis acidic. However, in
meta-phenol boronate ester 10, this conjugation is absent and therefore this
 1996 A. Kumar et al.

stabilization does not exist; making the boron more electrophilic thereby
promoting faster hydrolysis.
Analogously, this effect is observed with para- and meta-aniline based
substrates (11 and 12, respectively). Analyzing from the perspective of
boron-carbon bond length, the C–B bond length in phenyl boronic acid
14 is 1.568 Å; for boronic acid 15 an electron withdrawing p-carboxylic acid
increases the bond length to 1.588 Å and an electron donating p-methoxy
group in 16 decreases the bond length to 1.556 Å.[38] Changing boron–
carbon bond lengths suggest a small degree of p-bonding depending on
the substituent on the aromatic ring in various aryl boronic acids and
support the hypothesis that decreased rates of hydrolysis stem from
p-conjugation decreasing the Lewis acidity of boron atom.
Whereas the structure of the boronic ester determines its susceptibility
to hydrolysis, hydrolysis does not occur in the absence of an appropriate
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nucleophile such as silanols and protic solvents. The free silanol groups
present on standard RP-HPLC columns such as Onyx and Zorbax Eclipse
can act as good nucleophiles for hydrolysis reaction even if the analysis time
is short. However, when an RP-HPLC column with reduced silanol activity
such as XTerra MS C18 is employed, the lack of a good nucleophile allows
for successful analysis of most boronic acid pinacol esters. This low nucleo-
phile environment such as XTerra MS column is readily applicable for the
analysis of a wide variety of boronate esters; however, in some cases, aryl
boronate esters with an electron deficient ring will exhibit more hydrolysis
and need more rigorous control of conditions, such as high pH mobile
phase reported earlier.[22]

CONCLUSION
A reversed-phase HPLC method was developed for the purity analysis of
boronic acid pinacol esters containing electron-rich aromatic rings. Aprotic
solvents (e.g., acetonitrile) were found to be most suitable for sample prep-
aration. The DoE studies reported herein, demonstrated that the stationary
phase has the most significant impact to on-column degradation=hydrolysis
and that of the various columns evaluated, a passive column such as XTerra
MS C18 is optimal for the analysis of boronic acid pinacol esters. The pres-
ence of formic acid in mobile phase is only important synergistically with
silanols present on the stationary phases such as Onyx and Zorbax.
Unexpectedly, the rate of hydrolysis was insensitive to changes in column
temperature from 15 C to 35 C. The optimized method using the XTerra
stationary phase with a neutral mobile phase at 35 C column temperature
was successfully used for the release testing of compound 1 for a GMP
manufacturing campaign. Finally, the general utility of this method was
 Accurate Analysis of Boronic Pinacol Esters 1997

demonstrated by applying it to various boronate esters with widely differing

aromatic structures and substitutions.

ACKNOWLEDGMENT
The authors thank Dr. Lisa Bernstein for designing DoE experiment,
Dr. Alan Deese for running the qNMR, Dr. Travis Remarchuk and Herbert
Yajima for providing compounds 1 and 3.
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FUNDING
The authors thank Genentech Inc. for financial support.

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