Transcript Document

Development of a Combichem Initiative at a Primarily Undergraduate Institution
Philip L. Kumler; SUNY College at Fredonia
BACKGROUND
Over the last two years we have been fortunate to receive a number of equipment
donations which have allowed us to initiate a combinatorial chemistry initiative within our
department. This equipment has been donated by Boehringer-Ingelheim Inc. of Ridgefield,
CT due primarily to the efforts of an alumnus of our department (Dr. Gregory Roth; BS 1979;
MS 1984). The instrumentation acquired consists of:
OBJECTIVES OF THE INITIATIVE
The following objectives have been incorporated into the initiative.
(A) Incorporate combinatorial experiments into the undergraduate organic program.
(B) Develop a lecture/laboratory course in Combinatorial Chemistry.
 Rainin modular HPLC system for analytical and preparative HPLC (Figure 1)
(C) Incorporate combinatorial methodology in other laboratory courses.
 Waters Alliance 2690 HPLC with autosampler (Figure 2)
(D) Incorporate combichem into my personal research program.
 Two Advanced Chem Tech Model 496 Automated Synthesizers (Figure 3)
(E) Facilitate use of combichem techniques and instrumentation into the research
programs of other departmental faculty.
 An Argonaut Quest 210 Synthesizer with Automated Solvent Wash module and
Gaseous Reaction and Concentration Module (Figure 4)
 Perkin Elmer Sciex API-III+ LC/MS with electrospray interface (Figure 5)
COMBICHEM IN UNDERGRADUATE ORGANIC LABORATORY
Figure 1
During the FA2002 semester the class of 40 students was to synthesize a small
library of 40 oligopeptides (36 tetrapeptides and 4 pentapeptides) based on four different
amino acids (Ala, Gly, Leu, and Phe). The synthesis was carried out using the ACT-496
synthesizer using standard solid phase methods and Fmoc chemistry. Incorporation of the
Phe would allow analysis of the product by either TLC or HPLC using UV detection.
Figure 2
Figure 3
Based upon their assigned peptide each student received a Wang resin with the Cterminal amino acid already attached and was assigned one well in the reaction block.
After examining the software commands and dispense matrix to verify that it would
generate their peptide, the synthesis was completed under software control. The peptides
were cleaved from the resin using standard TFA cleavage protocols.
For as yet undetermined reasons only about one-half of the syntheses resulted in
any significant amount of solid oligopeptide. There were several instrumental problems
during the synthesis which may have been the problem. The peptides that gave solid
residues were analyzed by TLC and found to contain one major component, but no further
analysis was attempted.
Figure 4
Figure 5
Figure 6
Now that we have considerably more experience with the ACT-496 (see below) we
will repeat this experiment (with modification) in the Fall 2003 semester.
COMBICHEM LECTURE COURSE
COMBICHEM LABORATORY COURSE
In the Spring 2003 semester, I taught a course entitled Combinatorial Chemistry. The
lecture portion was two credit hours and met for the first 10 weeks of the semester for a total
time of 27 hrs. The laboratory portion was one credit hour and met the last 5 weeks of the
semester; about 30 hrs of lab work was expected. The text was Czarnik & DeWitt’s book “A
Practical Guide to Combinatorial Chemistry” (see Figure 6). The were 8 students (6 undergrad,
2 grad) in the lecture and 7 students (5 undergrad and 2 grad) in the laboratory. The lecture
portion was taught in a combination lecture/discussion format. There were written assignments
for most class meetings and a single final exam, Each class was typically composed of a
presentation by the instructor (40 minutes) followed by a discussion section (40 minutes) with
an assigned student moderator. The objective of the lecture portion of the course was to
provide the students with a broad overview of a large number of topics rather than an in-depth
treatment of fewer topics. The topics covered are summarized below.
The laboratory portion of the course took up the last 5 weeks; each student had to carry
out a combichem project of their own design. It was suggested that the projects be based
upon published work to maximize their chance of success. A large collection of published
papers was available from the instructor or the students were free to select a project of their
own choosing based upon their library research. The proposed project had to use either the
Quest 210 or the ACT-496.
Once the project was approved each student had to submit to the instructor a detailed list
of all chemicals required, including source, size, and cost. The list had to include all resins,
reagents, solvents, and catalysts.
In many cases we chose to acquire the materials from sources other than those submitted
by the student, but the pedagogical value of having the students generate this list was clear.
The students were especially surprised at the cost of many of the materials, especially
catalysts and solid phase resins. The students could not schedule time on the appropriate
instrument until all chemicals and supplies had been received.
LECTURE TOPICS
1) Merrifield’s classic paper (1963) on solid phase peptide synthesis.
2) Overview of Combinatorial Chemistry (C & D; Chapter 1)
STUDENT COMBICHEM PROJECTS
3) Solid-Phase Resins
4) Solid-Phase Synthesis (C & D; Chapter 3)
SOLID-PHASE SYNTHESIS OF BIARYLS ON THE QUEST 210 MANUAL SYNTHESIZER
5) Automated Synthesizers (Advanced Chem TECH 496; Argonaut Quest 210)
Four different biaryls were synthesized via the Suzuki coupling reaction using a palladium (0)
catalyst and four different boronic acids. The products were analyzed by reversed-phase
HPLC. [Source: Deegan, T., MacDonald, A; Munyak, E., Wright, P., Porco, J. ; Argonaut
Technologies Application Note APN #011].
6) Combinatorial Chemistry & New Drugs
7) Linear Free Energy Relationships and the Hammett Equation
8) QSAR and Lipinksi’s “Rule of Five”
9) On-Resin Analysis in Combichem (C & D; Chapter 5)
PRODUCTION OF A TETRADECAPEPTIDE ON THE ACT-496
10) Solution-Phase Synthesis (C & D Chapter 7)
A 14-residue tetrapeptide was synthesized using standard Fmoc chemistry on the ACT-496.
The peptide was analyzed by RP-HPLC. [Source: “Synthesis Guidelines for the Acyl Carrier
Protein (65-74)”; Advanced Chem Tech Training Manual.]
11) Combichem Resources & Catalogs (ACT, NovaBiochem, Argonaut)
12) Ion Exchange Resins
13) Solid Phase Extraction
SOLID PHASE SYNTHESIS OF ARYL AMINES
14) Polymeric Reagents
A small library of 5 aryl amines was prepared by the palladium-mediated aryl amination of
4-bromobenzamide on the Quest 210. The products were analyzed by HPLC and IR. [Source:
Deegan, T ; Porco, J. ; Argonaut Technologies Application Note APN #017].
15) Fluorous Phase Chemistry
16) Microwave Enhanced Chemistry
17) STUDENT PRESENTATIONS
COMBICHEM IN MY RESEARCH PROGRAM
STUDENT COMBICHEM PROJECTS (CONTINUED)
SOLUTION PHASE SYNTHESIS OF ARYL SULFONAMIDES
A small library of 10 sulfonamides was synthesized on the Quest 210; this project
demonstrated the feasibility of using the Quest for product isolation by liquid-liquid extraction.
The products were analyzed by capillary GC. [Source: Deegan, T ; Munyak, E., Wright, P.,
Porco, J. ; Argonaut Technologies Application Note APN #010].
We have recently initiated a collaboration with the research group of Professor Jeffery Kelly
at The Scripps Research Institute. The Kelly group is actively studying the self-assembly of hairpin peptides at the air-water interface. As part of this collaboration we will synthesize and
purify a number of peptides to incorporate in their program. One tetradecapeptide has already
been prepared and several more will be prepared during the Fall 2003 semester. [Sources:
(a) Powers, E.T.; Kelly, J.W. J. Am. Chem. Soc. 2001, 123, 775-776. (b) Powers, E.T.;
Yang, S.I.; Lieber, C.M.; Kelly, J.W. Angew. Chem. Int. Ed. 2002, 41, 127-130. (c) Colfer,
S.; Kelly, J.W.; Powers, E.T. Langmuir 2003, 19, 1312-1318]
SOLID PHASE BIGINELLI DIHYDROPYRIMIDINE SYNTHESIS
A small library of 6 dihydropyrimidines was prepared by the Biginelli reaction. The products
were analyzed by TLC and H-NMR. [Source: Wipf, P.; Cunningham, A.; Tetrahedron Letters,
36, 7819 (1995).]
SOLID PHASE SUZUKI REACTION ON THE ACT-496
Four different biaryls were synthesized (using the ACT-496) via the Suzuki coupling reaction
using a palladium (0) catalyst and four different boronic acids. The products were analyzed by
reversed-phase HPLC. [Source: Christensen, J; Bennett, W.D.; Advanced ChemTech
Application Note #007-0522-BB].
SYNTHESIS OF AN ARGININE-RICH PEPTIDE
A 23 residue arginine-rich peptide was synthesized using standard Fmoc chemistry on the
ACT-496. This peptide will be used in the research program of another faculty in our department
to use in probing specific RNA hairpin loops. Analysis was by RP-HPLC. [Source: Austin, R.J.;
Xia, T.; Ren, J.; Takahashi, T.; Roberts, R. J. Am. Chem. Soc. 2002, 124, 10966-10967.]
Unfortunately, time limitations precluded complete purification and
products. Part of the problem was due to insufficient consideration,
logistics involved and part was due to normal student procrastination.
course the planning, ordering, etc. will begin during the second half of
course (approximately at week 6).
analysis of most of the
by the instructor, of the
In future offerings of this
the lecture portion of the
FIELD TRIP
Thanks to the hospitality of Professor Peter Wipf, the class was able make a field trip to the
Combinatorial Chemistry Center at the University
`` of Pittsburgh. In addition to lunch and a tour of
the facility the students were able to meet with current graduate students and staff of the center.
The field trip was a highlight of the course.
COMBICHEM RESEARCH PROGRAMS OF OTHER FACULTY`
Two other faculty members in our department have expressed an interest in
incorporating combinatorial instrumentation and methodology into their ongoing research
programs. It is expected that these collaborations will be instituted over the next year. The
specific proposed collaborations are as follows:
Dr. Matthew Fountain is currently focusing on the synthesis of modified nucleotides to
probe specific interactions in RNA hairpin loops. The plan is to modify various parts of the
structure (sugar, base, phosphate backbone) to be able to determine their contribution to
the structure and stability of the hairpin loop in RNA. Combinatorial methodology would
facilitate the incorporation of molecular diversity into the various components. [Source:
Fountain, M.A.; Serra, M.J.; Krugh, T.R.; Turner, D.H. Biochemistry 1996, 35, 65396548.]
Dr. Mark Janik is currently collaborating with Dr. Hiroaki Suga at the University of
Buffalo on a project involving the synthesis of new antibiotics to treat diseases caused by
the bacteria Pseudomonas aeruginosa. This organism typically infects people with
impaired host defense systems, such as in AIDS, cancer, diabetes, and cystic fibrosis. This
organism has already developed resistance to the traditional anti-pseudomonal antibiotics,
highlighting the need for new antibiotics. Dr. Janik and Dr. Suga are collaborating on the
synthesis of a combinatorial library of potential antagonistic compounds. Now having the
ability to do automated synthesis at Fredonia will facilitate this Buffalo-Fredonia
collaboration. [Sources: (a) Kovacs, K.; Paterson, D.L.; Yu, V.L. Infect. Med. 1998, 15,
464-472. (b) Oliver, A.; Canton, R.; Campo, P.; Baquero, F.; Blazquez, J. Science
2000, 288, 1251-1253.]
ACKNOWLEDGMENTS
Boehringer-Ingelheim Inc. (Dr. Greg Roth): Equipment Donations
Dr. Peter Wipf : Tour of CCC at Univ. of Pittsburgh
Mr. Lee Servatius: Technical help at all stages.