IV. Combinatorial Chemistry 1. Library synthesis a) in solution, parallel synthesis b) on solid support c) split and combine, one bead one compound 2. Deconvolution and Tagging 3. Dynamic Combinatoric Chemistry Parallel Library Synthesis • 12 reactions provide 9 compounds • The library members are spatially separated, so this technique can be used for solution as well as solid phase synthesis diversification divide diversification reaction reaction Split-pool Synthesis • 6 reactions lead to 9 compounds • Each library member must be compartmentalized (each compound on its own bead) to allow pooling of the library splitdiversification pool and split diversification reaction reaction An Example of Split-Pool Synthesis O NHBoc NH 1) Pd2dba3, Cl Me 2 O O SnMe O 3X 3 2) TFA 3X Me NH2 O O O 3X Me NH2 Cl NHBoc NH2 NHBoc 1) Pd2dba3, O O O 3X O O SnMe O SnMe 3X 9X 3 3X 3 2) TFA NH2 O OCl O 3X Cl NH2 Cl NHBoc 1) Pd dba , 2 3 O O 3X 3X O SnMe3 2) TFA Cl splitdiversification pool reaction Ellman, J. et al. J. Am. Chem. Soc., 1995, 117, 3306. O NH2 NH2 NH O O O O O NH 1) O NH 2 F O 2 NH NH Me NH 2 NHFmoc O O NH2 O O O O O Me O 2) piperidine Cl Cl HO2C NH2 O CO H NH2 O O 2 HO C O NH HO2C 2 1) F O O O O NH2 NH2 NHFmoc NH2 Me NH NH O NH2 O O O 2) piperidine O O Me O O Cl Cl NH2 S O S O O O 1) NH F 2 S NH2 S NHFmoc NH O NH2 O O O O NH O NH 2 Me O 2 NH O 2) piperidine Me NH Cl O O O O split diversification Cl reaction Overview of the Entire Split-Pool Library R2 O 2 O R NHFmoc NH F NHBoc Pd dba , 2 2 3 TFA NHFmoc NH O O O O SnMe O 3 1 35 Amino Acids O R 1 Cl R1 R 20 Acid 20 cpds 20 x 35 = 700 cpds Chlorides R3 H O O N N 1) piperidine Base, R3X TFA R2 R2 2) AcOH N N O 16 Alkylating O R1 Agents R1 700 cpds 20 x 35 x 16 = 11,200 cpds Split-Pool step: Ellman, J. et al. J. Am. Chem. Soc., 1995, 117, 3306. Example of the Efficiency of the Split-pool Strategy • Optimization of 154 reactions affords 105 amplification in the number of compounds R1 R1 I O H R2 N 1 O H 2 NH R R NH2 O O N H O N N O O H 62 amines H 30 alkynes N HO O H O H N O O O O 18 iodides O R1 R2 O NH R3 OH O N 3 O R O H N 30 + 62 + 62 = 154 reactions 62 acids 18 X 30 X 62 X62 = 2.1 million compounds O O O Schreiber SL et al. JACS, 1998, 120, 8565. Structural Characterization: Direct Methods Off-bead Analysis • Cleavage, then use of analytical techniques used in TOS (e.g. LC, MS, NMR) • Requires high sensitivity and high throughput format •Example: LC-UV/ MS OH OH S O Ph HO N Ph Structural Characterization: Direct Methods On-bead Analysis I • Can be used to monitor the progress of a reaction • MAS-NMR ( Magic angle spinning NMR ) is necessary due to polymer Magic angle rotor (left), rotor spinning at the magic angle (right) MAS- NMR spectrum (600 MHz) Si O OMe O O O O O O O Structural Characterization: Direct Methods On-bead Analysis II • Example: Single-bead FT-IR microspectrometry • Can be used to monitor the progress of a reaction OO OO H O HO O O O DIC, DMAP, DMF Beads in IR cell Wavelength (cm-1) Structural Characterization: Indirect Methods Deconvolution • Screen as a mixture of compounds then re- synthesize and re-assay possible candidates in active pools Drawbacks: • Interference by unwanted properties of other compounds (e.g. cytotoxicity) • Possible synergistic interaction of multiple compounds • Sub-library synthesis is cumbersome Encoding • Encoding should provide a fast and simple way to identify the structure of all library members • Classification – Spatial encoding: position of the compound provides the information about its structure (possible only in parallel synthesis) – Graphical encoding: bar codes or other graphical tags are displayed on the solid support used in the library synthesis – Chemical encoding: every reaction used in the library synthesis is recorded on the solid support by the chemical attachment of a tag • binary coding (presence or absence of a tag) or polymer based (polypeptide, DNA) – Spectrometric encoding: using a spectrometric technique (NMR, MS, Fluorescence microscopy, NMR etc.) to read tags directly from the solid support – Electronic encoding: radio frequency memory chip attached to the solid support records and emits coded information A. C. Czarnik Current. Opp. Chem. Biol. 1997, 1, 60 Encoding in Split-pool Synthesis • Optimization of 6 reactions leads to 9 compounds • Each library member must be isolated on its own bead to allow pooling of the library How do you know what compound is on a given bead after a pool step? splitdiversification pool and split diversification reaction reaction Chemical Encoding in Split-Pool Synthesis • Every diversification reaction is followed by a tagging reaction in which a tag(s) that codes for a particular transformation is covalently attached to the solid support Decoding • Every bead has tags that provide information, once cleaved, about the chemical history of that bead • Conditions for cleavage of compound and tags have to be orthogonal compound cleavage tag cleavage small molecule Binary Chemical Coding 11 10 011101 110 111 1 Building blocks Binary 00 01 001 010 100 0 (base 2) codon Tags 2 digit codon 3 digit 1 digit 22 = 4 max 11 110 1 n binary tags code for 2n building blocks Another Example 10 011 0 11 10 011101 110 111 1 Building Blocks 00 01 001 010 100 0 Tags Halogenated Aromatics As Tags • Small amount of tag can be reliable detected (0.5-1 pmol/bead) using easily automated electron capture GC in the mixture of tags based on different retention times • Inert under most reaction conditions OMe Cl OO X n X=Cl or H N HC 2 Cl Cl O X linker variable variable region electrophore W. C. Still et al. J. Org. Chem. 1994, 59, 4723 Attachment and Cleavage of Tags • Tags are attached using rhodium carbene-insertion chemistry and can be cleaved using (NH4)Ce(NO3)6 (CAN) Cl HF O (CH2)n O X cleavable N2HC OMe Cl Cl Me MeMe X O TAG Me Si X = H or Cl * O small molecule MeMe Me Me n = 1-14 Si O * O small molecule X Rh2(O2CC(Ph)3)4 OMe Cl Cl CH2Cl2, 25 °C O (CH2)n O X Cycloheptatriene Cl TAG CAN cleavable Binary Chemical Encoding of a Peptide Library • A library of decapeptides was synthesized and screened for binding to 9E10 mAb. • 7 Amino acids were used at each position (S, I, K, L, Q, E, D) • Every amino acid was assigned a 3 digit binary codon (001=S, 010=I, 011=K, 100=L, 101=Q, 110=E, 111=D) where 1=presence of a tag and 0=absence • For each step in the library synthesis there are 3 tags designated nX (total of 18 tags for a library of 117,649 members, maximum encodable is 218 = 262,144) EQKLISEEDL known to bind 9E10 H2N-X-X-X-X-X-X-E-E-D-L-G-G-G-G- OO O HO2C n Arx O NO2 linker electrophoric tag nX general formula of tags used 110 101 011 100 010 001 identified as the best binder W.C. Still et al. PNAS 1993, 90, 10923 Dynamic Combinatoric Chemistry Virtual Dynamic Combinatoric Library Virtual Dynamic Combinatoric Library - macrocycles Virtual Dynamic Combinatoric Library – carboanhydrase inhibitors Lehn et al. 2106 Virtual Dynamic Combinatoric Library Lehn et al. 2106 Virtual Dynamic Combinatoric Library – metal grids Lehn et al, PNAS, 2003, 100, 11970 Virtual Dynamic Combinatoric Library – metal grids Lehn et al, PNAS, 2003, 100, 11970 Virtual Dynamic Combinatoric Library – neuraminidase inhibitors Eliseev et al, PNAS, 2002, 99, 3382 Virtual Dynamic Combinatoric Library – neuraminidase inhibitors Eliseev et al, PNAS, 2002, 99, 3382 Virtual Dynamic Combinatoric Library – acetylcholine esterase inhibitors Sharpless et al, PNAS, 2004, 101, 1449 Bourne, Yves et al. (2004) Proc. Natl. Acad. Sci. USA 101, 1449-1454 Pseudodynamic Dipeptide Library Protease from Streptomyces griseus Angew. Chem. Int. Ed. 2004, 43, 2432. Pseudodynamic Dipeptide Library Angew. Chem. Int. Ed. 2004, 43, 2432. Pseudodynamic Dipeptide Library CA = carbonic anhydrase Angew. Chem. Int. Ed. 2004, 43, 2432. V. Diversity Oriented Synthesis Convergent Synthesis – Target oriented synthesis (TOS) • complexity-generating Synthesis planning: rxns. retrosynthetic • fragment coupling rxns. one analysis molecule Divergent Synthesis – Diversity oriented synthesis (DOS) • complexity-generating rxns. many Synthesis planning: • multicomponent coupling different forward synthetic • diversity-generating rxns. molecules analysis Molecular Complexity TOS: Retrosynthetic Analysis of Saframycin A OMe OMe OMe HO Me O Me HO Me OMe H O H OMe H H H H OMe Me OMe Me O Me N Me N Me N Me NH N N MeO MeO H MeO H H N OH NC O CN OH CN NH FmocHN NHFmoc O O O Intramolecular 2 pictet-spengler reactions Strecker Reaction Me saframycin A OMe OMe H HO Me Me CHO OH OMe OMe H NH NH Me OMe MeO 2 N OH OHC Me H N H MeO 2 H OMe HCN OH NC N HN CHO O O FmocHN Imine formation and Strecker reaction A.