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.
Chem., 2005, 7, 353; (c) M.-D. H. De Bas and D. F. O’Shea, J. Comb.
Chem., 2005, 7, 947; (d) G. A. Strohmeier and C. O. Kappe, J. Comb.
Chem., 2002, 4, 154; (e) M. C. Coleman, J. M. D. MacElroy, J. F.
Gallagher and D. F. O’Shea, J. Comb. Chem., 2002, 4, 87.
5 J. M. Kremsner, A. Stadler and C. O. Kappe, J. Comb. Chem., 2007, 9,
285.
6 J. M. Kremsner and C. O. Kappe, J. Org. Chem., 2006, 71, 4651.
7 M. Treu, T. Karner, R. Kousek, H. Berger, M. Mayer, D. B. McConnell
and A. Stadler, J. Comb. Chem., 2008, 10, 863.
8 K. M. Amore, N. E. Leadbeater, T. A. Miller and J. R. Schmink,
Tetrahedron Lett., 2006, 47, 8583.
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,
Org. Proc. Res. Dev., 2006, 10, 833; (b) R. K. Arvela, N. E. Leadbeater
and M. J. Collins, Tetrahedron, 2005, 61, 9349; (c) R. K. Arvela and
N. E. Leadbeater, Org. Lett., 2005, 7, 2101; (d) R. K. Arvela, N. E.
Leadbeater, M. S. Sangi, V. A. Williams, P. Granados and R. D. Singer,
J. Org. Chem., 2005, 70, 1786.
13 See for example: (a) A. E. Thompson and J. E. Pope, Rheumatology,
2005, 44, 145; (b) P. A. Poole-Wilson, B. A. Kirwan, Z. Voko´, S. de
Brouwer, F. J. van Dalen and J. Lubsen, Cardiovas. Drugs Ther., 2006,
20, 45; (c) A. Di Stilo, S. Visentin, C. Clara, A. M. Gasco, G. Ermondi
and A. Gasco, J. Med. Chem., 1998, 41, 5393; (d) R. A. Coburn, M.
Wierzba, M. J. Suto, A. J. Solo, A. M. Triggle and D. J. Triggle, J. Med.
Chem., 1988, 31, 2103.
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.
15 (a) S. Torchy, G. Cordonnier, D. Barbry and J. J. Vanden Eynde,
Molecules, 2002, 7, 528; (b) L. Ohberg and J. Westman, Synlett, 2001,
1296; (c) R. Alajarin, P. Jordan, J. J. Vaquero and J. Alvarez-Builla,
Synthesis, 1995, 389; (d) Y.-W. Zhang, Z.-X. Shan, B. Pan, X.-H. Lu
and M.-H. Chen, Synth. Commun., 1995, 25, 857; (e) B. M. Khadilkar,
V. G. Gaikar and A. A. Chitnavis, Tetrahedron Lett., 1995, 36, 8083;
(f) M. Suarez, A. Loupy, E. Perez, L. Moran, G. Gerona, A. Morales
and M. Autie´, Heterocycl. Commun., 1996, 2, 275; (g) V. Sivamurugan,
A. Vinu, M. Palanichamy and V. Murugesan, Heteroatom Chem., 2006,
17, 267; (h) R. Alajarin, J. J. Vaquero, J. L. Garcia Navio and J. Alvarez-
Builla, Synlett, 1992, 297.
16 M. D. Bowman, J. L. Holcomb, C. M. Kormos, N. E. Leadbeater and
V. A. Williams, Org. Proc. Res. Dev., 2008, 12, 41.
17 For a review see: R. Pontremoli, G. Leoncini and A. Parodi, Expert
Rev. Cardiovasc. Ther., 2005, 3, 43.
18 W. N. Sandoval, V. Pham, E. S. Ingle, P. S. Liu and J. R. Lill, Comb.
Chem. High Throughput Screening, 2007, 10, 751.
19 J. R. Lill, E. S. Ingle, P. S. Liu, V. Pham and W. N. Sandoval, Mass
Spectrom. Rev., 2007, 26, 657.
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.
20 J. M. Collins and N. E. Leadbeater, Org. Biomol. Chem., 2007, 5, 1141.
21 X. Liu, K. Chan, I. K. Chu and J. J. Li, Carbohydrate Res., 2008, 343,
2870.
22 S. Lin, G. P. Yao, D. W. Qi, Y. Li, C. H. Deng, P. Y. Yang and X. M.
Zhang, Anal. Chem., 2008, 80, 3655.
23 B. N. Pramanik, U. A. Mirza, Y. H. Ing, Y.-H. Liu, P. L. Bartner, P. C.
Weber and A. K. Bose, Protein Sci., 2002, 11, 2676.
24 W. Sun, S. Gao, L. Wang, Y. Chen, S. Wu, X. Wang, D. Zheng and Y.
Gao, Mol. Cell Proteomics, 2006, 5, 769.
25 H. W. Vesper, L. Mi, A. Enada and G. L. Myers, Rapid Commun. Mass
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Notes and references
1 For books on the use of microwave heating in organic synthesis,
see: (a) A. Loupy, Ed., Microwaves in Organic Synthesis, Wiley-
VCH, Weinheim, 2006; (b) C. O. Kappe and A. Stadler, Microwaves
in Organic and Medicinal Chemistry, Wiley-VCH, Weinhiem, 2005;
(c) P. Lidstro¨m and J. P. Tierney, Eds., Microwave-Assisted Organic
Synthesis, Blackwell, Oxford, 2005.
2 For recent reviews see: (a) C. O. Kappe, Chem. Soc. Rev., 2008, 37,
1127; (b) V. Polshettiwar and R. S. Varma, Chem. Soc. Rev., 2008, 37,
1546; (c) C. O. Kappe, Angew. Chem., Int. Ed., 2004, 43, 6250.
26 H.-F. Juan, S.-C. Chang, H.-C. Huang and S.-T. Chen, Proteomics,
2005, 5, 840.
27 CEM Corp. http://www.cem.com/biosciences/.
This journal is
The Royal Society of Chemistry 2009
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