Assessment and use of two silicon carbide multi-well plates for library synthesis and proteolytic digests using microwave heating

Article abstract of DOI:10.1039/b902112d

The use of two silicon carbide plates is reported for the preparation of three libraries of organic molecules using microwave heating. In addition, a preliminary study has been carried out, showing that one of the plates can also be used in a proteomics setting. Both the 24-position and 48-position plates heated evenly when irradiated with microwave energy. The 48-position plate was used to prepare a library of N-aryl functionalized β-amino esters via an aza-Michael reaction between anilines and Michael acceptors. The 24-position plate was used to prepare a library of biaryls via a Suzuki coupling methodology and a library of 1,4-dihydropyridines via a Hantzsch synthesis. The 48-position plate was also used to perform the proteolytic digestion of insulin chain B by trypsin. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b902112d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Assessment and use of two silicon carbide multi-well plates for library
synthesis and proteolytic digests using microwave heating
Lauren M. Stencel, Chad M. Kormos, Keri B. Avery and Nicholas E. Leadbeater*
Received 2nd February 2009, Accepted 24th March 2009
First published as an Advance Article on the web 21st April 2009
DOI: 10.1039/b902112d
The use of two silicon carbide plates is reported for the preparation of three libraries of organic
molecules using microwave heating. In addition, a preliminary study has been carried out, showing that
one of the plates can also be used in a proteomics setting. Both the 24-position and 48-position plates
heated evenly when irradiated with microwave energy. The 48-position plate was used to prepare a
library of N-aryl functionalized b-amino esters via an aza-Michael reaction between anilines and
Michael acceptors. The 24-position plate was used to prepare a library of biaryls via a Suzuki coupling
methodology and a library of 1,4-dihydropyridines via a Hantzsch synthesis. The 48-position plate was
also used to perform the proteolytic digestion of insulin chain B by trypsin.
uniform heating has been shown by IR thermography. The plate
has been used for the preparation of a library of oxindoles and
Introduction
Microwave heating offers an alternative to conventional heating
substituted benzimidazoles.
methods and proves to be very useful in preparative organic
In our laboratory, we have performed a study on the heating
chemistry.1,2 Although microwave heating of reactions may not
characteristics of both silicon carbide plates (Fig. 1) using organic
always offer a direct rate enhancement over conventional heating
reactions as probes in addition to using the plates for the
under strictly comparable conditions, it is a very versatile tool be-
preparation of three libraries of compounds. In addition, we have
cause it offers reproducible non-contact heating as well as precise
used the 48-position plate to perform the digestion of insulin chain
temperature monitoring and data recording. Taking advantage
B using trypsin. We report our results here.
of the benefits of microwave heating and parallel processing,
chemists have become interested in performing reactions in well
plates.3,4 Although an attractive proposition, the use of well plates
in a microwave unit has some significant problems. The most
important issue to overcome is uneven heating across the plate.
Many of the early reports using polypropylene, Teflon or high-
temperature polyethylene plates showed that wells located on the
periphery were at a significantly lower temperature than those on
the inside due to radiative heat loss as well as lower microwave
coupling. In an attempt to overcome this problem, plates doped
with strongly microwave absorbing materials such as graphite
have been used. More recently a 48-position plate made of silicon
carbide has been used for parallel synthesis in a microwave unit
with success.5 Silicon carbide is an inert, highly microwave
absorbing material and has been previously used as a heating
insert for reaction mixtures containing non-absorbing reagents or
solvents.6 The use of a silicon carbide plate allows for equal heating
of the wells. This was shown using IR thermal imaging. The plate
was used for preparing a library of 2-aminopyrimidines.5 While it
proves useful for rapid library preparation, a drawback of using
the plate is that each well has a working volume of only 0.1–0.3 mL.
In a recent development, a silicon carbide plate capable of holding
up to 24 standard glass vials has become available.7 Each vial
is capable of holding from 0.3–3 mL of reaction mixture. Again
Department of Chemistry, University of Connecticut, 55 North Eagleville
Rd, Storrs, CT 06269-3060, USA. E-mail: nicholas.leadbeater@uconn.edu;
Fax: +1 860 486 2981; Tel: +1 860 486 5076
Fig. 1 (a) A 48-position silicon carbide plate; (b) A 24-position silicon
carbide plate capable of holding standard glass vials.
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across the plate is uniform and the calibration factor accurately
gauges the reaction temperature.
Results and discussion
Organic chemistry
To probe the heating characteristics of the 24-position plate
we turned our attention to the Suzuki coupling reaction between
4-bromoanisole and phenylboronic acid. We chose to use a 1:1
water/ethanol (v:v) mix as solvent, sodium hydroxide as base and
ligandless palladium (0.002 mol%) as catalyst.11,12 We loaded eight
vials with reaction mixture. We also introduced an additional vari-
able, namely stirring. Given the relative size of the glass vials used
in the 24-position plate, adequate mixing becomes necessary in
order to ensure reaction-to-reaction reproducibility. The Synthos
3000 microwave unit is equipped with a magnetic stirrer under the
rotor. To probe the efficiency of stirring in the plate, we decided to
place small stir-bars in half the vials used. The loaded plates were
heated over a 5 min period to an external temperature of 140 ◦C
(internal temperature of ~156 ◦C) and held at this temperature for
20 min. Product conversions are shown schematically in Fig. 3.
We know these Suzuki conditions to be fairly robust, so we were
not surprised to see near complete conversion in six of the eight
vessels. Interestingly, the two reactions that did not reach complete
conversion were also reactions not containing stir-bars. Based on
the higher overall yields of the stirred reactions as compared to
the unstirred reactions, we concluded that the stirring mechanism
is efficacious in increasing reaction-to-reaction reproducibility.
Our first objective was to determine how even the heating was
across the plates as well as, in the case of the 24-position plate, the
applicability of standard glass vials as reaction vessels. Starting
with the 48-position plate, the previous literature report showed
uniformity in heating for an esterification reaction. We decided
to use the aza-Michael reaction between aniline and methyl
acrylate catalyzed by acetic acid to yield an N-aryl functionalized
b-amino ester as a probe for the heating characteristics of the
block. This reaction was chosen because we have studied it
extensively before.8,9 It is performed neat at 200 ◦C for 20 min.
The pressure of the reaction mixture during the run peaks
at around 200 psi. The chemistry is temperature dependent.
Insufficient heating leads to poor yields; too much heating leads
to side-product formation and decomposition. We loaded twelve
representative wells in the plate with reaction mixture, placed a
PFA foil across all the wells and then sealed the plate by attaching
the Viton cushion and alumina top plate. The microwave unit is
capable of holding four of the silicon carbide plates in a rotor.
Although we performed all of our tests in one plate, for even rotor
distribution we placed an empty plate in the vacant diagonally-
opposite position. The plates were heated over a 5 min period to
an external temperature of 180 ◦C as monitored using an IR sensor
located on the bottom of the microwave unit. This corresponds to
a temperature inside the wells of 200 ◦C since a calibration factor
of 1.11 has to be used to equate the temperature read remotely by
the infrared sensor to the actual temperature of the silicon carbide
block.10 The plates were held at this temperature for 20 min before
allowing them to cool to 50 ◦C, this taking approximately 15 min.
We then determined the product conversion of the contents of each
loaded well. The results are shown schematically in Fig. 2. With
one exception (well A7) all the conversions laid within the range of
71–75%, and no by-product formation was observed in the NMR
spectra of the product mixtures. This suggests that the heating
Fig. 3 Testing the temperature uniformity and stirring efficiency of the
24-position plate. Values shown represent conversions (%).
We next turned our attention back to the 48-position plate with
the objective of preparing a library of twelve N-aryl functionalized
b-amino esters on the 1 mmol scale. We chose four anilines and
three Michael acceptors. We knew from previous attempts to
prepare a library of N-aryl functionalized b-amino esters using
another set-up that homogeneity of temperature across each
reaction vessel was essential.9 In our previous work we had initially
attempted to prepare the library using a carousel of twelve sealed
glass tubes, measuring the internal temperature of one using a
fiber-optic probe and the external temperature of all twelve using
an external IR sensor. We found significant temperature variation
across the vessels. As a result the outcome of the reaction was
dependent on which vessel was used as the temperature control
during the course of the run. The effects could be mollified
Fig. 2 Testing the temperature uniformity of the 48-position plate. Values
shown represent product conversions (%).
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by adding a small quantity of a highly microwave absorbing
quaternary ammonium salt to each reaction vessel. Using the
48-position plate we believed that the highly microwave absorbing
silicon carbide should ensure homogeneity of temperature across
all the reaction mixtures. The results are shown pictorially in Fig. 4.
The product yields obtained in the reactions of methyl acrylate
with the four aniline substrates are comparable to those from
our previous report using a monomode microwave apparatus in
which we ran the reactions one at a time. This shows that the
silicon carbide plate absorbs the majority of the microwave energy
and thus facilitates uniform heating across the wells. The trend in
reactivity across the aniline substrates is reflected in the product
yields: 2,6-dimethylaniline is least reactive and 3-methoxyaniline
(m-anisidine) most reactive.
Fig. 5 Preparing a library of twenty-four biaryls in the 24-position plate.
Values shown represent product conversions (%).
hydroxide (35%) was used both as the ammonia source and the
solvent for the reaction. Vials were loaded, sealed and placed into
the plate which was then heated to a target external temperature
◦
◦
of 120 C (internal temperature of ~140 C). The plate was then
held at this temperature for 10 min before allowing it to cool.
The product conversions are shown in Fig. 6. The results varied
considerably for this library; the best conversions being obtained
with ethyl acetoacetate as the dicarbonyl component. Of the
aldehydes screened, pyridine-2-carboxaldehyde and 3,4-methylene
dioxybenzaldehyde gave the best results. Our motivation for
screening 2-nitrobenzaldehyde as a substrate comes from the fact
that it is used in the synthesis of nifedipine (brand names Adalat,
Nifedical, and Procardia), a 1,4-dihydropyridine that serves as a
calcium channel inhibitor.17 Our library allowed us to prepare
three analogs of this compound.
Fig. 4 Preparing a library of twelve N-aryl functionalized b-amino esters
in the 48-position plate. Values shown represent product conversions (%).
Moving to the 24-position plate we prepared a library of twenty-
four biaryls employing the Suzuki coupling protocol previously
used. We screened four aryl bromides and six arylboronic acids,
performing each reaction on the 0.25 mmol scale in glass vials. The
results are shown in Fig. 5. Good yields were obtained with the
exception of the couplings with 2,5-difluorophenylboronic acid
and 2,6-dichlorophenylboronic acid, presumably because these
two substrates are electron-poor and sterically congested thus
impeding the transmetallation. Yields with 2-bromoanisole were
generally lower than with the 4-bromo analog, an expected result
based on steric arguments.
Our final organic chemistry library as part of this study
was a range of twenty-four 1,4-dihydropyridines, prepared in a
multicomponent reaction between an aldehyde, a b-dicarbonyl
compound and ammonia (Hantzsch synthesis). Our motivation
behind choosing this reaction is that the dihydropyridine products
have attracted considerable attention due to their biological
activity.13 In addition, microwave heating has been used as a tool
for preparing dihydropyridines.14–16 We screened six aldehyde sub-
strates and four b-dicarbonyl compounds using a stoichiometric
ratio of 1 mmol:5 mmol. A volume of 0.33 mL aqueous ammonium
Proteolytic digestion
Scientists working in the biosciences have found increasingly
that microwave heating can be an enabling technology in their
research fields. One area that has seen considerable activity is
the application of controlled microwave heating in proteolytic
digestion. Reports have suggested that microwave heating can
increase the efficiency of tryptic digestions compared to conven-
tional heating.18–26 Since only very small volumes of material are
generally used, it is difficult to know with certainty how much of
the microwave energy actually couples with the sample and thus
how evenly they are heated. A commercially available accessory is
available for use with a monomode microwave unit. It comprises
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Fig. 7 Testing the uniformity of the 48-position plate for the digestion of
insulin chain B using trypsin. Values shown represent amount of substrate
digested (%).
We next wanted to use the plate to probe the effects of varying
the protease-to-protein ratio on the extent of digestion. We filled
the wells in the plate with solutions of protease-to-protein ratio
1:5, 1:10 and 1:25. We loaded four wells at each different ratio, thus
filling a total of twelve wells. We sealed the plate and then heated it
using an identical protocol as in our first trial (50 ◦C internal temp
for 5 min ramp and 30 min hold) and upon completion analyzed
the contents of each well. The results are shown schematically
in Fig. 8. Not unexpectedly, the level of digestion decreases with
increasing protease-to-protein ratio. Our main objective was to see
whether, again, there was uniformity in results across the plate. We
found this was indeed the case, the values for the four wells at each
protease-to-protein ratio being comparable to each other.
Fig. 6 Preparing a library of twenty-four 1,4-dihydropyridines in the
24-position plate. Values shown represent product conversions (%). [n.d. =
not determinable due to complex mixture of product, starting material and
by-products.]
a holder capable of containing up to 14 Eppendorf tubes.27 It
holds the tubes in a bath of moderating fluid (usually water) and
IR thermography shows that, when irradiated with microwave
energy, the tubes can be heated uniformly. For higher throughput,
it would be useful to be able to use standard microtiter plates
in conjunction with microwave heating. This way they could be
interfaced with peripheral robotic instrumentation for loading,
analyzing and cataloging. It occurred to us that the 48-position
silicon carbide plate could be ideal for this and so decided to
perform a preliminary study as part of our investigations.
Our first objective was to determine how uniform the results
from a proteolytic digest were across the plate. We chose to perform
the digestion of insulin chain B using trypsin as a test. Using
an insulin chain B concentration of 1 mg/mL and a protease-to-
protein ratio of 1:5 (w/w), we loaded twelve wells in the 48-position
plate with 100 mL aliquots of protein and trypsin solution. The
plate was heated over a 5 min period to an external temperature
of 45 ◦C (internal temperature of ~50 ◦C) where it was then held
for a further 30 min. We then removed the plate and quenched the
reactions by injecting a small volume (20 ml) of 2 M hydrochloric
acid into the filled wells. We then analyzed the contents of each well
using HPLC to determine the level of digestion. We performed the
heating study in triplicate and our results are shown schematically
in Fig. 7. We find that the level of digestion is not highly dependent
on the location on the plate, suggesting the heating is even across
the wells.
Fig. 8 Using the 48-position plate to probe the effects of varying the
protease-to-protein ratio on the extent of digestion of insulin chain B
using trypsin. Values shown represent amount of substrate digested (%)
using 1:5, 1:10 and 1:25 ratios of protease-to-protein.
Conclusion
In summary, we have used two silicon carbide plates for the
successful preparation of three libraries of organic molecules
using microwave heating. In addition, we have performed a
preliminary study that shows the 48-position plate can also be
used in a proteomics setting. We first confirmed that both the
24- and 48-position plates heated evenly upon irradiation with
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microwave energy. We then used the 48-position plate to prepare
a library of N-aryl functionalized b-amino esters using an aza-
Michael reaction between anilines and Michael acceptors. We
used the 24-position plate to prepare libraries of biaryls and 1,4-
dihydropyridines using Suzuki coupling and Hantzsch synthesis
methodologies respectively. We used the 48-position plate to
perform the digestion of insulin chain B using trypsin.
onto the rotor together with an empty plate on the diagonally
opposite position, the protective top cover locked in place and
then the entire rotor assembly placed into the microwave◦unit.
The plates were heated to an external temperature of 180 C as
◦
measured by the IR sensor (~200 C internal temperature) over
a 5 min period ramping up to a microwave power of 1200 W.
The plate was then held at this temperature for an additional
20 min. Upon cooling, an aliquot from each occupied well was
removed, analyzed using 1H-NMR spectroscopy and the product
conversion determined. Anilines used: aniline (90 mL), N-ethyl
aniline (126 mL), 2,6-dimethylaniline (123 mL), 3-methoxyaniline
(112 mL). Michael acceptors used: methyl acrylate (90 mL), n-butyl
acrylate (143 mL), acrylonitrile (66 mL).
Experimental
General experimental
For the organic chemistry, all materials were obtained from
1
commercial suppliers and used without further purification. H-
and 13C-NMR spectra were recorded at 293 K on a 400 MHz
spectrometer. For the proteolytic digestions, Trizma hydrochloride
and insulin chain B oxidized from bovine pancreas were purchased
from Sigma and TPCK trypsin was purchased and used as
obtained from Pierce. HPLC analysis was performed using a
Hewlett Packard Series 1100 HPLC.
Library of twenty-four biaryls. Aqueous sodium hydroxide
(1.00 mL, 0.5 M), a solution of aryl bromide (0.500 mL, 0.50 M
in ethanol), a solution of aryl boronic acid (0.500 mL, 0.65 M in
ethanol), and palladium (50 mL, 10 mg/mL in 0.05 wt.% aq. HCl,
prepared by dilution of an ICP standard solution) were combined
R
in a Wheatonꢀ 15 ¥ 46 mm screw cap vial equipped with a small
Teflon-coated magnetic stir-bar. The vial was sealed, capped and
placed into a well of the 24-position silicon carbide plate. Once
all the vials had been loaded, the filled plate was placed onto the
rotor together with an empty plate on the diagonally opposite
position, the protective top cover locked in place and then the
entire rotor assembly placed into the microwave unit. The plates
were heated to an external temperature of 140 ◦C as measured
by the IR sensor (~156 ◦C internal temperature) over a 5 min
period ramping up to a microwave power of 1200 W. The plate
was then held at this temperature for an additional 20 min. Upon
cooling, the contents of each vial were each rapidly acidified with
2 M HCl to stop any further reaction. Each product mixture was
then individually worked up. This involved emptying the contents
of the vial into a separatory funnel, extracting with diethyl ether
twice, combining the organics, drying with magnesium sulfate and
finally evaporating the solvent under reduced pressure. Product
Description and use of the microwave apparatus
A commercially available multimode microwave unit (Anton
Paar Synthos 3000) was used for the reactions. The instrument
is equipped with two magnetrons, with combined continuous
microwave output power from 0 to 1400 W. Reactions were
performed using either a ROTOR 4 ¥ 48 MC Wellplate or a
ROTOR 4 ¥ 24 MG5. When using the 48-position plate, the
individual wells of the plate are filled using a micropipettor. After
filling, a PFA foil is used to cover the entire plate, the Viton cushion
put in place followed by the alumina top plate and this held in
place by six hexagonal bolts. The whole assembly is then placed
on a dedicated plate rotor and a protective top cover locked in
place. When using the 24-position plate, sealed glass vials are put
into the wells, the plate placed on a dedicated plate rotor and
a protective top cover locked in place. When using either plate,
for weight-balance purposes either two or four plates need to be
placed onto the rotor. The temperature of the plates is monitored
using an IR sensor located on the bottom of the microwave unit.
To ensure the proper internal reaction temperature, a calibration
factor of 1.11 is applied (the temperature of the inside of the wells
being 1.11 times that measured on the outside of the plate). After
cooling, the vials in the 24 position rotor can be removed and
the contents easily accessed by puncturing the PTFE seal with a
syringe or else removing the vial top entirely. In the case of the
48-position rotor, the contents of each well can be removed using a
syringe. The wells can be rinsed with solvent to obtain the product
quantitatively, again this operation can be performed on each
individual well with a syringe. When needed, reaction mixtures
were stirred by means of a rotating magnetic plate located below
the floor of the microwave cavity and a Teflon-coated magnetic
stir bar in the wells.
1
conversions were determined by using H-NMR spectroscopy in
the presence of an internal standard.
Library of twenty-four 1,4-dihydropyridines. Aldehyde
(1 mmol), b-ketoester (5 mmol), and ammonium hydroxide
R
(0.33 mL) were combined in a Wheatonꢀ 15 ¥ 46 mm screw
cap vial equipped with a small Teflon-coated magnetic stir-bar.
The vial was sealed, capped and placed into a well of the
24-position silicon carbide plate. Once all the vials had been
loaded, the filled plate was placed onto the rotor together with an
empty plate on the diagonally opposite position, the protective
top cover locked in place and then the entire rotor assembly
placed into the microwave unit. The plates were heated to an
external temperature of 125 ◦C as measured by the IR sensor
(~140 ◦C internal temperature) over a 5 min period ramping
up to a microwave power of 1000 W. The plate was then held
at this temperature for an additional 10 min. Upon cooling,
methanol was added to the contents of each vial and crude NMR
conversions obtained.
Experimental procedures
Library of twelve N-aryl functionalized b-amino esters. The
substituted-aniline (1.0 mmol), Michael acceptor (1.0 mmol), and
acetic acid (0.35 mmol, 20 mL) were combined directly in a well of
the 48-position silicon carbide block. The block was sealed, placed
Digestion of insulin chain B to test the heating characteristics of
the SiC plate. Two stock solutions were prepared, one containing
insulin chain B (~1 mg) in 1 mL of 100 mM Trizma hydrochloride
solution, the other trypsin (~1 mg) in 5 mL of 100 mM Trizma
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hydrochloride solution. A 100 mL of the trypsin solution and
100 mL of the protein solution were combined directly in each
required well of the 48-position silicon carbide block, a total of
12 wells being filled. The block was sealed, placed onto the rotor
together with an empty plate on the diagonally opposite position,
the protective top cover locked in place and then the entire rotor
assembly placed into the microwave unit. The plates were heated
to an external temperature of 45 ◦C as measured by the IR sensor
(~50 ◦C internal temperature) over a 5 min period ramping up to a
microwave power of 600 W. The plate was then held at this temper-
ature for an additional 30 min. Upon cooling, the contents of each
occupied well was quenched by the addition 20 mL of 2M HCl.
This was followed by immediate HPLC analysis. A 15 mL aliquot
of the reaction was separated on a reverse phase C18 column with
a 25 minute linear gradient from 5 to 55% of 0.1% trifluoroacetic
acid in acetonitrile at a 1 min/mL flow rate with UV detection at
210 nm. On the chromatogram, the area under each peak was
integrated (one peak at 15.6 min for undigested insulin chain
B [FVNQHLCoxGSHLVEALYLVCoxGERGFFYTPKA, and two
peaks at 14.1 min [FVNQHLCoxGSHLVEALYLVCoxGER] and
11.6 min [GFFYTPK] corresponding to digested fragments). The
relative areas of undigested and digested protein were used to
determine the level of digestion.
3 For reviews see: (a) C. O. Kappe and M. Matloobi, Comb. Chem. High
Throughput Screening, 2007, 10, 735; (b) W. M. Dai and J. Y. Shi, Comb.
Chem. High Throughput Screening, 2007, 10, 837; (c) M. Nu¨chter and
B. Ondruschka, Mol. Divers., 2003, 7, 253.
4 For examples see: (a) L. Pisani, H. Prokopcova´, J. M. Kremsner and
C. O. Kappe, J. Comb. Chem., 2007, 9, 415; (b) J. Alca´zar, J. Comb.
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Chem., 2005, 7, 947; (d) G. A. Strohmeier and C. O. Kappe, J. Comb.
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6 J. M. Kremsner and C. O. Kappe, J. Org. Chem., 2006, 71, 4651.
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9 N. E. Leadbeater and J. R. Schmink, Tetrahedron, 2007, 63, 6764.
10 The difference between internal and external temperature arises due to
air flow through the rotor. This flow actively maintains an appropriate
temperature for rotor parts that may otherwise overheat during
extended runs.
11 For a review of the Suzuki reaction using water as a solvent in
conjunction with microwave heating see: N. E. Leadbeater, Chem.
Commun., 2005, 2881.
12 (a) N. E. Leadbeater, V. A. Williams, T. M. Barnard and M. J. Collins,
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Using the 48-position plate to probe the effects of varying the
protease-to-protein ratio on the extent of digestion of insulin chain B
using trypsin. Two stock solutions were prepared, one containing
insulin chain B (~1 mg) in 1 mL of 100 mM Trizma hydrochloride
solution, the other trypsin (~1 mg) in 1 mL of 100 mM Trizma
hydrochloride solution. From the trypsin stock solution, three
dilutions were prepared, one a 5-fold, one a 10-fold and one a
25-fold. A 100 mL of the requisite trypsin solution and 100 mL of
the protein solution were combined directly in each required well
of the 48-position silicon carbide block, a total of 12 wells being
filled (4 at a protease-to-protein ratio of 1:5, 4 at 1:10 and 4 at 1:25).
The block was heated and the products analysed in an identical
protocol to that used for assessing the heating characteristics of
the SiC plate.
14 For a review see: J. J. Vanden Eynde and A. Mayence, Molecules, 2003,
8, 381.
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Acknowledgements
Anton Paar USA are thanked for access to the silicon carbide
plates and assistance from Michael Canavan and Reinhardt
Klopper is acknowledged. Funding from the American Chemical
Society Petroleum Research Foundation (45433-AC1) and the
National Science Foundation REU program at the University of
Connecticut is acknowledged.
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Notes and references
1 For books on the use of microwave heating in organic synthesis,
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26 H.-F. Juan, S.-C. Chang, H.-C. Huang and S.-T. Chen, Proteomics,
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27 CEM Corp. http://www.cem.com/biosciences/.
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