Diversity-oriented synthesis of biaryl-containing medium rings using a one bead/one stock solution platform

Article abstract of DOI:10.1021/ja017248o

Diversity-oriented synthesis of structurally complex and diverse small molecules can be used as the first step in a process to explore cellular and organismal pathways. The success of this process is likely going to be dependent on advances in the synthesis of small molecules having natural product-like structures in an efficient and stereoselective manner. The development, scope, and mechanism of the oxidation of organocuprates was investigated and exploited in the atropdiastereoselective synthesis of biaryl-containing medium rings (9-, 10-, and 11-membered rings). The methodology was performed on high-capacity, large polystyrene beads by metalating aryl bromides with-iPrBu₂MgLi, followed by transmetalating with CuCN·2LiBr and then oxidizing with 1,3-dinitrobenzene, and was used in a diversity-oriented synthesis of biaryl-containing medium rings (library total theoretical maximum 1412 members). The high capacity beads were arrayed into 384-well plates and, using a process optimized during the development of a one bead/one stock solution technology platform, converted into arrays of stock solutions, with each stock solution containing largely one compound. These stock solutions were used in numerous phenotypic and proteinbinding assays. The process described outlines a pathway that we feel will contribute to a comprehensive and systematic chemical approach to exploring biology (chemical genetics).

Full text of DOI:10.1021/ja017248o

Published on Web 01/22/2002
Diversity-Oriented Synthesis of Biaryl-Containing Medium
Rings Using a One Bead/One Stock Solution Platform
David R. Spring,† Shyam Krishnan, Helen E. Blackwell, and Stuart L. Schreiber*
Contribution from the Department of Chemistry and Chemical Biology, Howard Hughes Medical
Institute (HHMI), Institute of Chemistry and Cell Biology (ICCB), HarVard UniVersity,
Cambridge, Massachusetts 02138
Received October 5, 2001. Revised Manuscript Received November 21, 2001
Abstract: Diversity-oriented synthesis of structurally complex and diverse small molecules can be used
as the first step in a process to explore cellular and organismal pathways. The success of this process is
likely going to be dependent on advances in the synthesis of small molecules having natural product-like
structures in an efficient and stereoselective manner. The development, scope, and mechanism of the
oxidation of organocuprates was investigated and exploited in the atropdiastereoselective synthesis of biaryl-
containing medium rings (9-, 10-, and 11-membered rings). The methodology was performed on high-
capacity, large polystyrene beads by metalating aryl bromides with i-PrBu2MgLi, followed by transmetalating
with CuCN‚2LiBr and then oxidizing with 1,3-dinitrobenzene, and was used in a diversity-oriented synthesis
of biaryl-containing medium rings (library total theoretical maximum 1412 members). The high capacity
beads were arrayed into 384-well plates and, using a process optimized during the development of a one
bead/one stock solution technology platform, converted into arrays of stock solutions, with each stock solution
containing largely one compound. These stock solutions were used in numerous phenotypic and protein-
binding assays. The process described outlines a pathway that we feel will contribute to a comprehensive
and systematic chemical approach to exploring biology (chemical genetics).
Introduction
of diversity-oriented synthesis to screening in a way that led to
the discovery of several new probes of biological processes.
Small molecules can be used to understand biological
pathways in a process analogous to genetics (chemical genet-
ics).1 To enhance the generality of this approach, a method is
required to discover small molecule partners (activators/inhibi-
tors) for any protein target. A promising method begins by
synthesizing structurally complex and diverse small molecules
efficiently using diversity-oriented synthesis.2 Next, these small
molecules are screened individually for their ability to either
induce a desired cellular or organismal change (phenotypic
screening) or bind to a protein (proteomic screening; the term
proteomic is used to emphasize protein-binding screens that use
many proteins in parallel).3 This paper illustrates the coupling
Nature provides guidelines for the characteristics of small
molecules that are desirable for effective interactions with
proteins, such as rigidity and stereochemical complexity. Natural
products such as vancomycin and pterocaryanin C, which have
an axially disymmetric biaryl moiety implanted within a ring,
are particularly engaging due to their atropisomerism (Figure
1).4 Indeed, pterocaryanin C was especially stimulating on
account of its biosynthesis via a stereoselective, C-C bond
formation to provide the biaryl-containing medium ring.4b
Medium rings are not only considered generally to be the most
difficult ring sizes to synthesize,5 but they also require special
attention since their formation en masse using split-pool
synthesis presents special challenges.6 Thus, a general synthesis
of medium rings would be useful, especially one that is operative
on a polymer support and is high yielding and selective.
Any approach that aims to construct large collections of small
molecules for individual use in biological assays needs to take
† Current address: Department of Chemistry, Cambridge University,
Lensfield Road, Cambridge, CB2 1EW, U.K.
(1) This small molecule approach has the following features: (i) Effects of
small molecules on cells and organisms are conditional; they are induced
only following the addition of the small molecule. This allows the immediate
effects of modulating protein function, rather than the steady-state effects
resulting from mutations, to be observed. (ii) A dose-response can be used
to gain more confidence of cause and effect and to determine the effects
of only partial loss or gain of function. (iii) Protein function can be
investigated in cases where a cell having a gene encoding a protein of
interest deleted is not viable. For more information see: (a) Schreiber, S.
L. Bioorg. Med. Chem. 1998, 6, 1127-1152. (b) Mitchison, T. J. Chem.
Biol. 1994, 1, 3-6. (c) http://www-schreiber.chem.harvard.edu. (d) http://
sbweb.med.harvard.edu/∼iccb/.
(3) Following the analogy with genetics, these two complementary screening
approaches are known as forward and reverse chemical genetics, respec-
tively.
(4) (a) Nicolaou, K. C.; Boddy, C. N. C.; Brase, S.; Winssinger, N. Angew.
Chem., Int. Ed. 1999, 38, 2097-2152. (b) Quideau, S.; Feldman, K. S.
Chem. ReV. 1996, 96, 475-503.
(5) This difficulty is due to their associated torsional, transannular, and large-
angle strain, which makes these rings relatively rigid conformationally.
Illuminati, G.; Mandolini, L. Acc. Chem. Res. 1981, 14, 95-102. For a
recent review of medium ring synthesis: Yet, L. Chem. ReV. 2000, 100,
2963-3007.
(6) Lee, D.; Sello, J. K.; Schreiber S. L. J. Am. Chem. Soc. 1999, 121, 10648-
10649.
(2) (a) Schreiber, S. L. Science 2000, 287, 1964-1969. (b) Lee, D.; Sello, J.
K.; Schreiber, S. L. Org. Lett. 2000, 2, 709-712. For leading references
on split-pool synthesis see: (a) Furka, A.; Sebestye´n, F.; Asgedom, M.;
Dibo´, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (b) Lam, K. S.;
Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R.
J. Nature 1991, 354, 82-84. (c) Houghten, R. A.; Pinilla, C.; Blondelle,
S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84-86.
9
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Synthesis of Biaryl-Containing Medium Rings
A R T I C L E S
Scheme 1
century, for example, in the Ullmann reaction.13 Whitesides,14
Kauffmann,15 van Koten,16 Ziegler,17 Bertz,18 and others19 have
used oxidants on aryl cuprates to give biaryls. More recently,
Lipshutz and co-workers have expanded this work considerably
in two ways: first, by using “kinetic” cuprates to cross-couple
aryl units intermolecularly, generating unsymmetrical biaryls;20
and second, by using a chiral tether to synthesize biaryls
intramolecularly and atropdiastereoselectively.21 The Lipshutz
method was successfully extended to 3a, a substrate bearing a
new and readily accessible chiral tether (Scheme 1).22 Acyclic
substrates, such as 3a, were synthesized readily in three steps:
(a) amino alcohols were treated with an ortho-halobenzaldehyde,
and the resultant imine was reduced with NaBH4 to give 1a,23
(b) Eschweiler-Clarke N-methylation24 or reductive alkylation
using a borane-pyridine complex25 yielded 2a, and finally (c)
3a was generated by O-alkylation with an ortho-halobenzyl
halide via the sodium alkoxide.26 Using the Lipshutz method,
treatment of the cyclization precursor 3a with tert-butyllithium,
then with CuCN, and last with 3O2, gave a 58% yield of biaryl
Figure 1. Both vancomycin and pterocaryanin C have axially disymmetric
biaryl units contained within 12- and 10-membered rings, respectively
(highlighted in red).
account of the format that will be required for the screen. When
a diverse range of screens is desired, then a flexible and
preferably automated approach will be essential. We have
developed a procedure for synthesizing and arraying small
molecules individually as stock solutions in quantities sufficient
to permit hundreds of phenotypic and proteomic assays to be
performed per compound - the one bead/one stock solution
technology platform.7 The research described herein makes use
of that platform.
We report the development, scope, and mechanism of general,
efficient, and atropdiastereoselective reactions leading to com-
pounds having biaryl-containing medium rings.8 The reactions
were studied in solution and on a high capacity polymeric
support, and the structural and conformational properties of the
medium ring products were determined. Details of the strategies
adopted in the library’s design and specification, and its
subsequent apportioning, which led to our being able to perform
a range of biological assays on each of the resulting compounds,
are also included.
(13) (a) Ullmann, F.; Bielecki, J. Chem. Ber. 1901, 34, 2174-2185. (b) Fanta,
P. E. Synthesis 1974, 9-21.
(14) (a) Whitesides, G. M.; San Filippo, J.; Casey, C. P.; Panek, E. J. J. Am.
Chem. Soc. 1967, 89, 5302-5303. (b) Whitesides, G. M.; Fischer, W. F.;
San Filippo, J.; Bashe, R. W.; House, H. O. J. Am. Chem. Soc. 1969, 91,
4871-4882. (c) Whitesides, G. M.; Stedronsky, E. R.; Casey, C. P.; San
Filippo, J. J. Am. Chem. Soc. 1970, 92, 1426-1427.
(15) Kauffmann, T. Angew. Chem., Int. Ed. Engl. 1974, 13, 291-305.
(16) (a) van Koten, G.; Jastrzebski, J. T. B. H.; Noltes, J. G. Chem. Commun.
1977, 203-204. (b) van Koten, G.; Jastrzebski, J. T. B. H.; Noltes, J. G.
J. Org. Chem. 1977, 42, 2047-2053. (c) Janssen, M. D.; Corsten, M. A.;
Spek, A. L.; Grove, D. M.; van Koten, G. Organometallics 1996, 15, 2810-
2820.
(17) Ziegler, F. E.; Chliwner, I.; Fowler, K. W.; Kanfer, S. J.; Kuo, S. J.; Sinha,
N. D. J. Am. Chem. Soc. 1980, 102, 790-798.
(18) Bertz, S. H.; Gibson, C. P. J. Am. Chem. Soc. 1986, 108, 8286-8288.
(19) Konduirov, N. V.; Fomin, D. A. J. Russ. Phys. Chem. Soc. 1915, 47, 190-
198 (CA 1915, 9, 1473).
(20) (a) Lipshutz, B. H.; Siegmann, K.; Garcia, E. J. Am. Chem. Soc. 1991,
113, 8161-8162. (b) Lipshutz, B. H.; Siegmann, K.; Garcia, E. Tetrahedron
1992, 48, 2579-2588. (c) Lipshutz, B. H.; Siegmann, K.; Garcia, E.;
Kayser, F. J. Am. Chem. Soc. 1993, 115, 9276-9282. (d) Lipshutz, B. H.;
Kayser, F.; Siegmann, K. Tetrahedron Lett. 1993, 34, 6693-6696.
(21) (a) Lipshutz, B. H.; Kayser, F.; Liu, Z.-P. Angew. Chem., Int. Ed. Engl.
1994, 33, 1842-1844. (b) Lipshutz, B. H.; Kayser, F.; Maullin, N.
Tetrahedron Lett. 1994, 35, 815-818. (c) Lipshutz, B. H.; Liu, Z.-P.;
Kayser, F. Tetrahedron Lett. 1994, 35, 5567-5570.
Results and Discussion
A library of small molecules related to pterocaryanin C was
envisaged to be synthesized via a differentially acylated or
alkylated 1,2-amino alcohol followed by intramolecular biaryl
formation, where the atropdiastereoselectivity of the biaryl
would be directed by the chiral amino alcohol. Unfortunately,
all attempts at the medium ring-forming reactions, using Stille,9
Suzuki and Miyaura,10 and other methodologies,11 were not
sufficiently promising for a split-pool synthesis, which requires
reactions to proceed in excellent yield and purity.12
Reaction Development. Biaryl synthesis by way of oxidation
of organocopper complexes has been recognized for about a
(7) (a) Blackwell, H. E.; Pe´rez, L.; Stavenger, R. A.; Tallarico, J. A.; Eatough,
E. C.; Foley, M. A.; Schreiber, S. L. Chem. Biol. 2001, 8, 1167-1182. (b)
Clemons, P. A.; Koehler, A. N.; Wagner, B. K.; Sprigings, T. G.; Spring,
D. R.; King, R. W.; Schreiber, S. L.; Foley, M. A. Chem. Biol. 2001, 8,
1182-1195.
(22) Use of the Lipshutz method: (a) Coleman, R. S.; Grant, E. B. Tetrahedron
Lett. 1993, 34, 2225-2228. (b) Coleman, R. S.; Grant, E. B. J. Am. Chem.
Soc. 1994, 116, 8795-8796. (c) Coleman, R. S.; Grant, E. B. J. Am. Chem.
Soc. 1995, 117, 10889-10904. (d) Sugimura, T.; Yamada, H.; Inoue, S.;
Tai, A. Tetrahedron: Asymmetry 1997, 8, 649-655. (e) Lin, G.-Q.; Zhong,
M. Tetrahedron: Asymmetry 1997, 8, 1369-1372. (f) Andrus, M. B.;
Asgari, D.; Sclafani, J. A. J. Org. Chem. 1997, 62, 9365-9368. (g)
Kyasnoor, R. V.; Sargent, M. V. Chem. Commun. 1998, 2713-2714. (h)
Carbonnelle, A.-C.; Zamora, E. G.; Beugelmans, R.; Roussi, G. Tetrahedron
Lett. 1998, 39, 4471-4472. (i) Kabir, S. M. H.; Iyoda, M. Chem. Commun.
2000, 2329-2330.
(8) Spring, D. R.; Krishnan, S.; Schreiber, S. L. J. Am. Chem. Soc. 2000, 122,
5656-5657.
(9) Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508-524.
(10) (a) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95, 2457-2483. (b)
Carbonnelle, A.-C.; Zhu, J. Org. Lett. 2000, 2, 3477-3480.
(11) Ni0 has been shown to be effective at medium ring formation; however,
cyclization of 3a (X ) I) under literature conditions furnished only 18%
of 4a [2:3 (P:M)] and 18% of dehalogenated 3a (i.e., X ) H). Similarly,
3m (X ) I) yielded only 26% of 4m [1:1 (P:M)] and 22% of dehalogenated
3m.
(12) For recent reviews of biaryl synthesis: (a) Bringmann, G.; Menche, D.
Acc. Chem. Res. 2001, 34, 615-624. (b) Lloyd-Williams, P.; Giralt, E.
Chem. Soc. ReV. 2001, 30, 145-157. (c) Bringmann, G.; Breuning, M.;
Tasler, S. Synthesis 1999, 525-558. (d) Stanforth, S. P. Tetrahedron 1998,
54, 263-303.
(23) Saavedra, J. E. J. Org. Chem. 1985, 50, 2271-2273.
(24) Wikening, R. R.; Ratcliffe, R. W.; Doss, G. A.; Mosley, R. T.; Ball, R. G.
Tetrahedron 1997, 53, 16923-16944.
(25) (a) Pelter, A.; Rosser, R. M.; Mills, S. J. Chem. Soc., Perkin Trans. 1 1984,
717-720. (b) Moormann, A. E. Synth. Commun. 1993, 23, 789-795. (c)
Bomann, M. D.; Guch, I. C.; DiMare, M. J. Org. Chem. 1995, 60, 5995-
5996.
(26) (a) Stoochnoff, B. A.; Benoiton, N. L. Tetrahedron Lett. 1973, 14, 21-24.
(b) Brown, C. A.; Barton, D.; Sivaram, S. Synthesis 1974, 434-436.
9
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A R T I C L E S
Spring et al.
Table 1. Kinetic and Thermodynamic Ratios
Figure 2. Optimized conditions for the atropdiastereoselective synthesis
of the biaryl-containing medium ring 4a. The LCMS total ion count (TIC)
trace for the crude reaction mixture depicts the efficiency and selectivity
of the cyclization. The signal-to-noise ratio decreased along the solvent
gradient from H2O (0.1% HCO2H) to MeCN (0.1% HCO2H) on the reverse-
phase column and accounts for the baseline angle.
4a with a diastereomeric ratio (dr) of 1:6 (P:M) in favor of the
thermodynamically more stable atropisomer. In attempts to
improve the reaction’s suitability for split-pool synthesis, the
yield and diastereoselectivity were found to depend on the choice
of reaction solvent,27 temperature of oxidation,28 and the choice
of oxidant.29 It is intriguing that the oxidant plays a crucial role
in determining the degree and direction of atropdiastereoselec-
tivity.
Optimized conditions entailed the use of 2-methyltetrahy-
drofuran (2-MeTHF) as the reaction solvent and 1,3-dinitroben-
zene (1,3-DNB) as the oxidant at -40 °C. This improved
procedure resulted reproducibly in the near-quantitative forma-
tion of the biaryl (88%, combined yield of biaryls after
chromatography) with excellent atropdiastereoselectivity [20:1
(P:M)]. Dehalogenated starting materials are the only other
byproducts (Figure 2) and originate from the presence of
moisture.30 The reaction is all the more remarkable in that high
dilution conditions were not necessary to give the biaryl. In
fact, the acyclic substrate concentration has been as high as 0.15
M (1.4 g of 3r in 15 mL of 2-MeTHF), and oligomers were
not detected. This eventuality permits the reaction to be
performed on polymer supports since, as the reaction favors
intrinsically an intramolecular pathway, it avoids the problem
of site-site (intermolecular) interactions.31
The scope of the reaction was investigated by using the
optimized conditions on a wide range of substrates (Table 1).
The reaction proceeded efficiently with different aromatic rings
(e.g., phenyls; electron-poor aryl fluorides, aryl chlorides,
naphthylenes, and pyridines; electron-rich aryl ethers, thiophenes,
(27) Reaction solvent: 2-MeTHF [dr ) 9:1 (P:M)]; Et2O [dr ) 6:1]; PhMe [dr
) 6:1]; THF [dr ) 5:1]; DME [dr ) 1:1]. Conditions: 3a to 4a; oxidant,
1,3-DNB; temperature of oxidation, 0 °C.
(28) Temperature of oxidation: -78 °C [dr ) 2:1 (P:M)] unclean reaction; -40
°C [dr ) 5:1]; -20 °C [dr ) 5:1]; 0 °C [dr ) 5:1]; 25 °C [dr ) 4:1].
Conditions: 3a to 4a; oxidant, 1,3-DNB; reaction solvent, THF. Temper-
ature of oxidation: -100 °C [dr ) 2:1 (P:M)]; -78 °C [dr ) 1:6]; 0 °C
[dr ) 1:1] unclean reaction. Conditions: 3a to 4a; oxidant, 3O2; reaction
solvent, THF.
(29) Oxidant: PhNO2 [dr ) 5:1 (P:M)]; 1,2-DNB [6:1]; 1,3-DNB [20:1]; 1,4-
DNB [14:1]; 2,4-dinitrotoluene (2,4-DNT) [dr ) 3:1]; 3,4-DNT [dr ) 1:1].
Conditions: 3a to 4a; temperature of oxidation, -40 °C; reaction solvent,
2-MeTHF.
(30) Other products, such as phenols (presumably from ArLi + oxidant) and
tert-butyl adducts (from the use of more than 4.0 equiv of t-BuLi), can be
avoided if exact reagent quantities are used. The crude reaction mixture
also contains the excess 1,3-DNB and 3,3′-dinitroazoxybenzene (from
oxidized 1,3-DNB).
a Kinetic atropisomer drawn; 3b-z were dibromides. b Combined yield.
c P ≡ S and M ≡ R for a stereogenic axis. d Unable to determine
thermodynamic dr. e No atropisomerism observed at room temperature.
f Ratio refers to the configuration of the enantiomer drawn. g Atropisomeric
stereochemistry not assigned. h Debromo-4x was also isolated (12%).
and benzothiophenes), different ring sizes (9-, 10-, and 11-
membered rings), substrates with different substituents (alkyl,
(31) (a) Blackwell, H. E.; Clemons, P. A.; Schreiber, S. L. Org. Lett. 2001, 3,
1185-1188. (b) Hodge, P. Chem. Soc. ReV. 1997, 26, 417-424.
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Synthesis of Biaryl-Containing Medium Rings
A R T I C L E S
aryl), and substrates with different substituent stereochemistry;32
however, the reaction is constrained by the aryllithium inter-
mediate on account of its basicity and nucleophilicity. Although
the yield of the reaction remained high for the broad range of
substrates, the atropdiastereoselectivity had a lower tolerance.
As can be seen from the series 4a-e, the diastereomeric ratio
was influenced dramatically by aryl substituents and ring size.
Similarly, looking down the series 4j-l and 4o-t, the nitrogen
substituent and the amino alcohol substituent(s) affected acutely
the atropdiastereoselectivity of the biaryl formation; sterically
large substituents tended to reduce both the kinetic and the
thermodynamic selectivity. At room temperature the heteroaro-
matic biaryls 4g-i did not display atropisomerism,33 presumably
due to an insufficient restriction of rotation about the biaryl
bond.34
The atropisomer that was formed kinetically was not favored
thermodynamically, allowing a thermal isomerization (10- and
11-membered rings only) to reverse the stereochemistry of the
biaryl. This discovery permits ready access to both atropisomers
in the case of 10- and 11-membered rings. The atropisomers
were often separable by chromatography, so to determine the
thermodynamic ratio for 10- and 11-membered ring products,
each atropisomer was heated neat under argon at 120-150 °C
for between 24 and 48 h.35 In the cases where the atropisomers
were inseparable, the mixtures were heated until no change in
the diastereomeric ratio was observed.
In the case of nine-membered ring products (4c, 4u, and 4w-
z), the barrier to rotation about the biaryl bond was too high
energetically such that even heating with a flame failed to
interconvert atropisomers. In light of this finding, efforts were
made to investigate whether the use of different oxidants in the
ring-closing reaction would favor the opposite atropisomer
kinetically. Although the investigation was unsuccessful, an
intriguing result was obtained. Using a suspension of CuCl2 (10
equiv) in THF at -50 °C as the oxidant in the cyclization of 3z
gave a 90% yield of 4z [1:14 (P:M)];36 however, when a solution
of CuCl2‚2LiCl (10 equiv; THF; -50 °C) was used as the
oxidant, biaryl 4z was not formed. Instead, only high molecular
weight products were detected, indicative of extensive inter-
molecular coupling.37
One of the many concerns involved in preparing a collection
of small molecules for use in many biological screens over an
extended period of time is the thermal stability of the com-
pounds. All biaryl products (4a-z) were stable configurationally
at room temperature indefinitely. Indeed, heating (P)-4a at 70
°C for 24 h resulted in little conversion to its atropisomer.
Structural Analysis. The biaryl configurations of the prod-
ucts were determined by X-ray crystallographic analysis in 18
Figure 3. X-ray crystal structures of selected biaryl-containing medium
ring compounds. Below each structure is listed the compound identifier,
ring size, angle Φ, and whether the atropdiastereomer is the thermodynami-
cally more stable atropisomer. Multiple torsion angles (Φ) observed in
different crystallographic polymorphs are depicted within parentheses.
Key: M ) minus; P ) positive; Y ) yes; N ) no; NA ) not applicable;
U ) unknown; Φ ) biaryl torsion angle within the medium ring (see inset).
cases, including 9-, 10-, and 11-membered ring sizes. In all but
one of the remaining cases the stereochemistry of the biaryl
atropisomers was identified reliably by the presence of nuclear
Overhauser effect interactions between specific hydrogen atoms
around the ring. With X-ray crystal structures of a variety of
biaryl-containing medium rings in hand, structural analysis of
these products was revealing (Figure 3).
Both 4g and 4h do not display atropisomerism at room
temperature; however, the solid-state conformations of the biaryl
axes are the same as the configuration of the thermodynamically
more stable atropisomer (M) of biphenyl analogues, for example,
(M)-4a. In the case of 10-membered ring products, the solid-
state structure of the atropisomer favored kinetically had inter-
ring torsion angles (Φ) between 76.4° and 99.5° (median Φ )
82.0°; standard deviation ) 10.5°), whereas the atropisomer
(32) All the cyclization precursors shown contain bisortho-iodo/bromoaromatic
rings. Attempts to cyclize different substrates to give the biaryl implanted
within a medium ring in an ortho,meta-relationship (e.g., within the 12-
membered ring of vancomycin) failed.
(33) Atropisomerism has been defined arbitrarily as existing where the isomers
can be isolated and have a half-life, t1/2, of at least 1 000 s. Oki, M. Top.
Stereochem. 1983, 14, 1-81.
(34) Note that when the ortho-N is replaced by C-H (compare 4i with 4a) the
increase to the energy barrier of rotation about the biaryl bond is enough
to observe atropisomerism at room temperature.
(35) Higher temperatures were required to determine the thermodynamic
diastereomeric ratio of 4f.
(36) A suspension of FeCl3 (10 equiv) in THF improved the atropdiastereose-
lectivity [1:>50 (P:M)].
(37) This result was confirmed with other nine-membered ring substratres. Also,
a suspension of CrCl3 (10 equiv) in THF yielded high molecular weight
products only.
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A R T I C L E S
Spring et al.
Figure 4. The molecule shown on the left [(M)-4p] was found not only to
cause a pericardial edema and a tube heart at a 5 µM concentration, but
also to increase the ratio of atrium to ventricle contractions to 2:1, instead
of the usual ratio of 1:1. The atrium is partially obscured by surface
pigmentation. The P atropisomer, shown on the right [(P)-4p], had no
unusual observable effects on zebrafish development.
Figure 5. Synthesis and X-ray crystal structure of the organomercurial 8,
presumably analogous structurally to the reaction intermediate 6.
formed in 6 around the CuI atom, which is bonded linearly; (b)
a fast, energetically favorable single electron transfer occurs
from 6 to form a 1,3-dinitrophenyl radical that is stable
kinetically;40 and (c) diorganocopper(II) complexes, such as 7,
are unstable with respect to reductive elimination.
Scheme 2. Postulated Mechanism for the Oxidative Coupling of
Organocuprates
To gain an insight into the foundation of the atropdiastereo-
selectivity, stable analogues of the intermediates 6 and 7 were
sought.41 The linear bonding mode of copper(I), illustrated in
6, is adopted by mercury(II) complexes. The substitution of
CuCN with HgBr2 in the cyclization reaction furnishes the
crystalline 8 in 65% yield,42 after recrystallization from EtOH
(Figure 5).43 Confidence in the resemblance of the mercury
analogue 8 to the speculated copper intermediate 6 is enhanced,
because the M-C(Ar) bond length of HgII (HgII-CAr ) 2.07 Å)
is similar to that of CuI (CuI-CAr ) 1.92 Å).44 The mercury
complex 8 does not display atropisomerism in solution at room
temperature; however, it is interesting to note that the solid-
state conformation that is adopted by the two aromatic rings
along the biaryl axis is the same as the configuration of the
atropisomer favored kinetically (P). This observation is depicted
in the illustrations of 6 and 7 in Scheme 2 and assumes that
this is the ground-state conformation, that is, P rather than M.
If this assumption is correct, then the atropdiastereoselectivity
could be explained by the one-electron oxidation and reductive
elimination following the principle of least nuclear motion.45
Along a similar vein, tetrahedral analogues of 7 were made
by substituting the CuCN with either Me2SiCl2 or Ph2SiCl2 to
favored thermodynamically had torsion angles between 65.4°
and 75.0° (median Φ ) 68.6°; standard deviation ) 2.8°). When
both atropisomers were characterized crystallographically [(M)-
and (P)-4e; (M)- and (P)-4k], insights into why one atropisomer
is more stable thermodynamically could then be gleaned by
visual inspection and molecular modeling. Rather than one
overriding factor, a cumulative combination of large-angle,
torsional, and transannular strain appears to be responsible.
Considering that the atropisomeric small molecules have acutely
different three-dimensional structures, it is hardly surprising to
discover different biological activities for each. As an example,
using a zebrafish development assay,38 we discovered in the
current study that (M)-4p affects the cardiovascular system
during zebrafish development, whereas (P)-4p had no effect
(Figure 4).
Reaction Mechanism. The mechanism of this remarkably
efficient ring-closing reaction is believed to involve the fol-
lowing: (a) lithium-halogen exchange to give the dilithium
intermediate 5 (Scheme 2), (b) transmetalation resulting in the
formation of the 11-membered copper(I) intermediate 6, where
the CuI atom is bonded linearly, and (c) this intermediate (6) is
oxidized upon addition of 1,3-dinitrobenzene to furnish the
square planar or tetrahedral copper(II) intermediate 7, which
itself eliminates reductively to form the biaryl C-C bond and
Cu0.39 The success of this approach to medium ring synthesis
is thought to be due to three factors: (a) a favorable chelate is
(40) The dinitrobenzene radical species persist until the reaction is quenched
with acid, at which point they disproportionate to form 3,3′-dinitroazoxy-
benzene and 1,3-dinitrobenzene.
(41) Also, attempts were made to crystallize the dilithium intermediate 5 via
standard Schlenk techniques in the hope of obtaining evidence for the
proposed structure illustrated in Scheme 2 (cf. Beno, M. A.; Hope, H.;
Olmstead, M. M.; Power, P. P. Organometallics 1985, 4, 2117-2121);
however, these efforts only resulted in the crystallization of moisture- and
temperature-sensitive [LiBr‚Et2O]4 (see Supporting Information), which has
been characterized previously: Neumann, F.; Hampel, F.; Schleyer, P. V.
R. Inorg. Chem. 1995, 34, 6553-6555.
(42) Ba¨hr, G.; Ku¨pper, F. W. Chem. Ber. 1967, 100, 3992-3995.
(43) The mercury complex 8 is stable to air, moisture, and room temperature.
A 10-membered ring analogue of 8 was made with 3y in 81% yield after
chromatography. Although the product was not crystalline, it was character-
ized spectroscopically; this showed an increased conformational rigidity
as compared with 8, but atropisomerism was not observed.
(38) Peterson, R. T.; Link, B. A.; Dowling, J. E.; Schreiber, S. L. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 12965-12969.
(39) It has been speculated that the organocuprate may be oxidized to a copper-
(III) intermediate. While this cannot be ruled out conclusively, it is
considered unlikely, especially since a slight excess of CuCl2 can be used
alternatively as the oxidant to give the biaryl in good yield.
(44) Leoni, P.; Pasquali, M.; Ghilardi, C. A. J. Chem. Soc., Chem. Commun.
1983, 240-241.
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1358 J. AM. CHEM. SOC. VOL. 124, NO. 7, 2002

Synthesis of Biaryl-Containing Medium Rings
A R T I C L E S
Scheme 3
Scheme 4
give 9 and 10, respectively (Scheme 3); however, these
silacycles failed to crystallize, and atropisomerism was not
observed at room temperature. Nevertheless, we find these
structures fascinating from the perspective of their unknown
potential as modulators of biological pathways. The reactions
that lead to metal insertion and ring formation are also of interest
from the perspective of planning diversity-oriented reaction
pathways. Together with the biaryl-forming reactions, they
constitute a branch point in the pathway, where the addition of
distinct reagents transforms the acyclic bis(bromoarenes) into
products having distinct scaffolds. Such reactions are of prime
significance in efforts to synthesize complex and diverse
compounds efficiently, a goal of diversity-oriented synthesis.
Polymer-Supported Synthesis. The small polystyrene beads
(e.g., diameter of 50 µm; capacity of 0.1 nmol per bead) that
have been used in traditional applications of solid-phase
synthesis, as a purification technique, provide a quantity of
compound insufficient for multiple assays. In the current study,
solid-phase supports are used to take advantage of the power
of split-pool synthesis, which results in a predominantly single
compound on each individual bead.
We have shown that by using larger “macrobeads” (e.g.,
diameter of 500 µm; capacity of 100 nmol per bead), we are
able to obtain enough compound to perform many assays.7 We
have also been able to adapt an encoding method to these
macrobeads to determine a macrobead’s chemical history; one
key feature of this method is that decoding is accomplished by
using a tiny fraction of the final stock solution46 (e.g., using
GC tags).47 In addition, by analyzing the compound after it has
been released from the bead (e.g., using liquid chromatograph/
mass spectrometry [LCMS]), we have been able to determine
directly the structure of a compound associated with a given
macrobead.48
The macrobeads are functionalized with a diisopropylalkyl-
silyl group,49 ideal for alcohol attachment.50 The most attractive
way to attach the amino alcohol building blocks to a polymer
support was via reductive amination onto a polymer-supported
aldehyde. Unfortunately, because of the poor atropdiastereose-
lectivity observed in the cyclization of N-benzyl substrates 4j
and 4k (Table 1), the wide range of commercially available
hydroxybenzaldehydes has not yet been exploitable. Instead,
the alkyl aldehyde 11 was used, generated by silyl ether
formation (via the silyl triflate) with Z-octen-5-ol, followed by
treatment with O3 and then Me2S (Scheme 4).51
The reductive amination was conducted by soaking the
macrobeads in a solution containing an excess of amino alcohol
to form the oxazolidine, which was reduced by the addition of
NaBH4 to furnish 12 (>90% pure by HPLC and NMR).52 The
reductive alkylation of the polymer-supported secondary amine
was achieved (>95% pure by HPLC and NMR) in high
conversion to give 13 by the use of 2-bromobenzaldehyde and
borane-pyridine complex as a reductant.53 O-Alkylation of 13
proved to be a difficult reaction to accomplish successfully on
the macrobeads.54 After screening many permutations of bases
(e.g., NaH/15-crown-5, KH/18-crown-6, BEMP,55 CsOH,56
t-BuOK, Ag2O) and solvents, the most successful combination
proved to be formation of the polymer-supported potassium
alkoxide with potassium bis(trimethylsilyl)amide (KHMDS) in
THF,57 followed by treatment with 2-bromobenzyl bromide. This
(48) Sternson, S. M.; Louca, J. B.; Wong, J. C.; Schreiber, S. L. J. Am. Chem.
Soc. 2001, 123, 1740-1747.
(49) Tallarico, J. A.; Depew, K. M.; Pelish, H. E.; Westwood, N. J.; Lindsley,
C. W.; Shair, M. D.; Schreiber, S. L.; Foley, M. A. J. Comb. Chem. 2001,
3, 312-318.
(50) The compounds are attached to the bead by way of a silyl ether. Release
of the compound from the bead is achieved by treatment with HF‚pyridine;
then excess reagent is removed by the addition of TMSOMe followed by
concentration under reduced pressure.
(51) Yields on the polymer support were calculated by performing the reactions
with 100 mg of macrobeads. The products were released from the polymer
support, and the purified mass was compared with the mass of starting
material released from 100 mg of unreacted beads.
(52) This reaction illustrates two concerns when transforming a solution-phase
reaction on to large polystyrene beads. First, the reaction rate decreases,
requiring increased reaction times. Second, the choice of solvent is critical
to the success of the reaction.
(53) Khan, N. M.; Arumugam, V.; Balasubramanian, S. Tetrahedron Lett. 1996,
37, 4819-4822.
(54) This may be due to unfavorable charge-charge interactions required when
the reactants are held in close proximity to each other on the polymer
support.
(45) It is likely that, even at the low temperatures used in the reaction, the cuprate
6 will entertain rotation about the biaryl axis; however, the ground-state
conformation will be most populated. If the hypothesis that the later
mechanistic steps follow the principle of least nuclear motion is correct,
then the atropisomer stereochemistry and selectivity are ascertained by the
relative conformational population (P vs M) of the cuprate 6. Following
this conjecture through, however, does not immediately provide an
explanation for the atropdiastereoselectivity of other oxidants. For reviews
of the principle of least nuclear motion, see: (a) Hine, J. AdV. Phys. Org.
Chem. 1977, 15, 1-61. (b) Sinnott, M. L. AdV. Phys. Org. Chem. 1988,
24, 113-204.
(46) (a) Stavenger, R. A.; Schreiber, S. L. Angew. Chem., Int. Ed. 2001, 40,
3417-3421. (b) Blackwell, H. E.; Pe´rez, L.; Schreiber, S. L. Angew. Chem.,
Int. Ed. 2001, 40, 3421-3425.
(55) O’Donnell, M. J.; Zhou, C.; Scott, W. L. J. Am. Chem. Soc. 1996, 118,
6070-6071.
(56) Dueno, E. E.; Chu, F.; Kim, S. I.; Jung, K. W. Tetrahedron Lett. 1999, 40,
1843-1846.
(47) Barnes, C.; Balasubramanian, S. Curr. Opin. Chem. Biol. 2000, 4, 346-
350.
9
J. AM. CHEM. SOC. VOL. 124, NO. 7, 2002 1359

A R T I C L E S
Spring et al.
Scheme 5
alkylation procedure resulted in an 80% conversion of the
starting material to O-benzylated product 14. Nevertheless, since
the reaction was apparently without side-products, it could be
repeated to drive the reaction to completion.
With the polymer-supported cyclization precursor 14 in hand,
the copper-mediated cyclization was investigated. It was surpris-
ing initially to discover that the lithium-halogen exchange,
which had precedent on polymer supports,58 would not proceed
on the macrobeads.59 Eventually, the exchange was found to
occur cleanly at room temperature over 24 h in benzene using
an excess of t-BuLi. Using this dilithium intermediate, trans-
metalation was achieved in a cooled solution of CuCN‚2LiBr
in 2-MeTHF, and oxidation of the cuprate was accomplished
by the addition of 1,3-DNB to give 15. The biaryl-containing
medium ring compound 15 could be released from the macro-
bead at this stage to give 16 in 55% yield [dr ) 6:1 (P:M);
>80% pure by HPLC and NMR]. Alternatively, the macrobeads
(15) could be heated prior to compound release to reverse the
atropdiastereomeric ratio [dr ) 1:7 (P:M); yield 65%]. Although
it was gratifying to have accomplished the medium ring
synthesis on the macrobeads, it was evident that to use this
reaction in a split-pool synthesis, a less aggressive procedure
for the metal-halogen exchange was required.
Not only would the present procedure not be compatible with
many of the building blocks that might be considered, but also
it would rule out the use of the Still method60 of encoding that
had previously been adapted to the macrobeads.7a Alternative
metalation procedures were attempted, specifically application
of zinc,61 copper,62 or magnesium63 ate complexes (e.g., Me4-
ZnLi2, Me3Zn(CN)Li2, t-Bu3ZnLi,64 Me2Cu(CN)Li2, Me3Cu-
(CN)Li3, i-PrBu2MgLi, t-Bu3MgLi)65 and preferably with the
more available aryl bromides rather than iodides. It was
rewarding to discover that Me4ZnLi2, i-PrBu2MgLi, and t-Bu3-
MgLi all work very well with arylbromides in solution;66
however, on the polymer-supported cyclization precursor 14,
i-PrBu2MgLi gave consistently the best results (Scheme 5).67
At this stage it was interesting to look at the yield of compound
(16) available per macrobead. HPLC was used to analyze the
amount of compound released from 20 beads. The mean average
was found to be a yield of 76 nmol/macrobead, which ranged
between 53 and 106 nmol/macrobead with a standard deviation
of 16 nmol/macrobead.
With a series of suitable reactions available, consideration
was given to their use in a split-pool synthesis. Unfortunately,
this modified procedure was still not orthogonal to polychlo-
rinated aromatics used for GC encoding of macrobeads;68 in
fact it was observed that a pentachloro-aromatic ring is metalated
before a monobromo-aromatic ring. This nonorthogonality
dictated that each library member, released from an individual
bead in the one bead/one stock solution format,7 should be
identified by direct analysis of the material, for example, by
LCMS. Fortunately, electrospray mass spectrometry was ex-
tremely sensitive in detecting these tertiary amine products.
However, consideration of the building blocks must be made
to avoid making library members with the same empirical
formula, which would have indistinguishable molecular ions.
Fragmentation of the molecular ion,48 next to the heteroatoms,
could be used to differentiate the two halves of the molecule;
nevertheless, building block constitutional isomers, such as
leucinol and isoleucinol, still should be avoided. In the case of
atropisomers, these will, by definition, have the same empirical
formula; hence, macrobeads that have undergone a thermal
equilibration would not be mixed with beads that have not been
heat-treated. In this way, by not pooling beads at the last step,
the library would be encoded positionally. Similarly, enanti-
omers cannot be differentiated by mass spectrometry; thus, the
library was carried out in parallel with (S)- and (R)-amino
alcohols.
(57) The macrobeads were washed after treatment with base, since KHMDS
reacts with benzyl bromides to form stilbenes. For example, the addition
of 2-bromobenzyl bromide (1 equiv) to a THF solution of KHMDS at room
temperature affords an instantaneous and quantitative formation of (E)-1,
1′-(1,2-ethenediyl)bis(2-bromobenzene).
(58) (a) Farrall, M. J.; Fre´chet, J. M. J. J. Org. Chem. 1976, 41, 3877-3882.
(b) Darling, G. D.; Fre´chet, J. M. J. J. Org. Chem. 1986, 51, 2270-2276.
(c) Itsuno, S.; Darling, G. D.; Lu, P. Z.; Fre´chet, J. M. J. Polym. Mater.
Sci. Eng. 1987, 57, 570-574. (d) Tempest, P. A.; Armstrong, R. W. J.
Am. Chem. Soc. 1997, 119, 7607-7608.
(59) It is believed that this problem is due to the difficulty in the t-BuLi
penetrating through the bead, since the reaction proceeded normally if small
(diameter 75 µm) polystyrene beads were used or if the large beads were
crushed before the reaction.
(60) (a) Ohlmeyer, M. J. H.; Swanson, R. N.; Dillard, L. W.; Reader, J. C.;
Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 10922-10926. (b) Nestler, H. P.; Bartlett, P. A.; Still,
W. C. J. Org. Chem. 1994, 59, 4723-4724.
(61) (a) Kondo, Y.; Takazawa, N.; Yamazaki, C.; Sakamoto, T. J. Org. Chem.
1994, 59, 4717-4718. (b) Uchiyama, M.; Kameda, M.; Mishima, O.;
Yokoyama, N.; Koike, M.; Kondo, Y.; Sakamoto, T. J. Am. Chem. Soc.
1998, 120, 4934-4946. (c) Kondo, Y.; Komine, T.; Fujinami, M.;
Uchiyama, M.; Sakamoto, T. J. Comb. Chem. 1999, 1, 123-126.
(62) Kondo, Y.; Matsudaira, T.; Sato, J.; Murata, N.; Sakamoto, T. Angew.
Chem., Int. Ed. Engl. 1996, 35, 736-738.
Building blocks for the library synthesis were selected
individually on the basis of sufficient success (>80% purity by
HPLC and NMR after release from the polymer support) with
a chosen substrate in the reaction where they were introduced.69
(63) (a) Kitagawa, K.; Inoue, A.; Shinokubo, H.; Oshima, K. Angew. Chem.,
Int. Ed. 2000, 39, 2481-2483. (b) Inoue, A.; Kitagawa, K.; Shinokubo,
H.; Oshima, K. J. Org. Chem. 2001, 66, 4333-4339.
(66) Me4ZnLi3 (2.2 equiv); 3m to 4m; yield: 84%; dr 13:1 (P:M). t-Bu3MgLi
(2.2 equiv); 3m to 4m; yield: 90%; dr 12:1 (P:M).
(67) It is notable that metalation with i-PrBu2MgLi is more functional-group
tolerant than that with t-BuLi. This advance has allowed the reaction to be
performed successfully in solution in the presence of esters, and no doubt
allows the cyclization reaction to be compatible with a much wider range
of functionalities.
(68) Blackwell, H. E.; Pe´rez, L.; Schreiber, S. L. Angew. Chem., Int. Ed. 2001,
40, 3421-3425.
(69) All the reactions were performed on a relatively large scale, 100 mg of
macrobeads, followed by release from the polymer support to ascertain
purity and yield.
(64) This reagent succeeds in exchange with aryl iodides; however, treatment
with CuCN and then 1,3-DNB results in a 21% isolated yield of the biaryl
[10:1 (P:M)] and a 76% yield of the dehalogenated starting material.
(65) Some of the other reagents attempted included Li/naphthalene, Rieke Cu
(Rieke, R. D. Aldrichimica Acta 2000, 33, 52-60), Rieke Cu- (Rieke, R.
D.; Dawson, B. T.; Stack, D. E.; Stinn, D. E. Synth. Commun. 1990, 20,
2711-2721), and cyclopentylMgBr (Boudier, A.; Bromm, L. O.; Lotz, M.;
Knochel, P. Angew. Chem., Int. Ed. 2000, 39, 4414-4435). Rieke Cu (4.0
equiv) was successful in solution; for example, 3v (X ) Br) gave 50% 4v
[9.5:1 (P:M)] using 1,3-DNB as the oxidant. However, the procedure was
not transferable to polymer-supported substrates.
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Synthesis of Biaryl-Containing Medium Rings
A R T I C L E S
Table 2. Kinetic Diastereomeric Ratios and Yields with a Soluble
Substrate or with a Substrate on an Insoluble Support
number of compounds from the split-pool library was 1412.71
In total, over 10 000 beads were used in the split-pool library
synthesis. This not only ensured that there would be a high
statistical coverage of the library members, but also, more than
adequately, it allowed for any bead loss and breakage.
Figure 6. Building blocks for the biaryl-containing medium ring library.
The library was synthesized over 2 weeks and comprised 42
individual steps from the polymer-supported aldehyde 11
(Scheme 4). To ensure the purity of the library, five or more
macrobeads from each step were analyzed by LCMS before the
macrobeads were pooled. The finished library consisted of three
pools of macrobeads (kinetic and thermodynamic 15 and 17)72
for both the (S) and (R) series. Ten macrobeads chosen randomly
from each pool (total of 60 beads) were analyzed by HPLC
and LCMS; 37 samples (62%) were over 70% pure, whereas
11 samples (18%) were less than 50% pure, and 1 sample
showed no compound was present.73 Although not perfect, the
library has enormous potential for use in biological screens. The
macrobeads from each pool that looked in best shape physically
were arrayed into 384-well plates, one macrobead per well.74
Each plate was treated with HF‚pyridine and then TMSOMe
following an optimized, automated operation of compound
release (Figure 7).7b This was followed by another automated
procedure to divide each compound into “daughter” 384-well
plates. Most of the compound was divided into plates of varying
concentration in DMSO to be used in phenotypic assays; 20%
was apportioned for small molecule printing, which is used to
make the small molecule microarrays used in protein-binding
assays, and 10% was sidelined for LCMS analysis.
Figure 6 illustrates all of the building blocks that were selected
for use in the split-pool synthesis.70
To gauge the similarity of atropdiastereoselectivity when the
cyclization is performed in solution versus on a polymer support,
several cyclization precursors were prepared where the C-5
alcohol was either protected by a triisopropylsilyl (TIPS) group
or attached to large polystyrene beads. Analysis of the cycliza-
tion reaction products is presented in Table 2. Clearly, a similar
solution- and gel-phase result was observed each time. This
result indicates that the previous solution-phase results, displayed
in Table 1, should be relevant to library members prepared on
a polymer support.
During the biaryl-forming reaction, the only side-product that
was always observed, albeit in less than 5% yield, was
dehalogenated starting material 17, that is, where the bromines
in 14 were substituted by hydrogens. The side-product’s
appearance was due, presumably, to the presence of moisture
during the reaction. Because it proved difficult practically to
eliminate these products completely, and to enhance the diversity
of library members, the dehalogenated products (17) were
synthesized deliberately by quenching the reaction with MeOH
after metalation with i-PrBu2MgLi. Again, for the sake of library
diversity, the cyclization precursors were included for biological
screening (12-14). Computing all the combinations of building
blocks and products revealed that the theoretical maximum
(71) The totals of (S) ) 854 and (R) ) 558 include both atropisomers with
benzyl bromide building blocks a-d.
(72) The macrobeads of the cyclization precursors were distributed equally
between the three other pools; since they all contain bromine they will be
distinguishable instantly, once released from the macrobead, by virtue of
the isotope pattern observed in the mass spectrum.
(73) See Supporting Information.
(74) Columns 23 and 24 were left empty allowing for their use as internal
controls in assays.
(70) Note that “amino alcohol” (S)-17i was derivatized as an aryl ether; thus, it
did not react in the O-alkylation step and furnishes ultimately a nine-
membered ring product. Also, 1,3-amino alcohol (S)-17j yields ultimately
an 11-membered ring product.
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J. AM. CHEM. SOC. VOL. 124, NO. 7, 2002 1361

A R T I C L E S
Spring et al.
Figure 7. Postsynthesis formatting of the split-pool library.
Members of the library were next screened in many pheno-
typic assays, including zebrafish Danio rerio38 and plant
Arabidopsis75 developmental assays and cell-based assays, and
in protein-binding assays using small molecule microarrays. The
microarrays were constructed by covalently attaching each
alcohol-containing small molecule from the library onto a single
chlorinated glass slide, by spotting 1 nL of the DMF stock
solution using a quill pin robot.76 Proteins of interest, labeled
with fluorescent tags, were used to probe the glass slide to
identify small molecule partners.
As a graphic illustration of the potential of this chemical
genetic approach, the results from two developmental screens
are described below. Observational (phenotypic) screening after
random genetic mutagenesis has revealed much information
about the development of organisms. Small molecules can
modulate gene products to give observable phenotypes; however,
they can be used with temporal control and have other
advantages, as we have reported elsewhere.38
Figure 8. Plant (a and b) and zebrafish (c and d; 2 dpf) developmental
assays. (a) Seven day old Arabidopsis seedlings germinated on agar
containing 1% DMSO and (P)-4k (10 µM). (b) Seven day old control
seedlings germinated on agar containing 1% DMSO. (c) Synchronized
zebrafish embryos were treated with 13ab 100 nM. Zebrafish are delayed
developmentally; they exhibit lower than normal pigmentation, weak hearts,
abnormal brains, and misshapen jaws. (d) Synchronized embryos were
treated with 13ab 5 µM; zebrafish look indistinguishable to untreated
controls.
concentration of 100 nM. By the second day post-fertilization,
all fish were delayed developmentally; they exhibited lower than
normal pigmentation, weak hearts, abnormal brains, and mis-
shapen jaws. Remarkably, in side-by-side experiments per-
formed at a concentration of either 500 or 50 nM, zebrafish
developed normally.77 Compounds 13aa, 13ac, and 13ad do
not reproduce this phenotype, suggesting that 13ab may be
modulating a particular gene product specifically.78
Although the targets of (P)-4k and 13ab have not been
pursued yet, they may be discovered by a variety of techniques,
such as affinity chromatography followed by microsequencing
of the target protein.79 Identification of the targets of the
developmental modulators should provide insight into devel-
opmental processes at the molecular level and contribute to the
understanding of protein function.
Arabidopsis thaliana seeds (thale cress, ecotype Lansberg
erecta [Ler]) were germinated and grown on DMSO (1%) and
small molecule-containing (10 µM) agar in 96-well plates at
25 °C under continuous white light. The seedlings were
inspected visually under a dissecting light microscope from 1
to 7 days post-germination. Seedlings grown on DMSO-
containing agar alone developed normally, with a hypocotyl
(seedling stem), two cotyledons, and primary root apparent by
2 days of growth. Plants treated with (P)-4k were found to
exhibit stunted development that eventually led to noticeable
pigment loss by day 4 (potential inhibition of chlorophyll and/
or carotenoid biosynthesis) and death by day 7 (Figure 8). (M)-
4k was only very weakly active in this assay.
Vertebrate Danio rerio (zebrafish) embryos develop ex utero
and are largely transparent, allowing visual inspection of most
anatomical systems during development. Synchronized and
fertilized zebrafish embryos were collected and arrayed in 96-
well plates (three eggs per well) containing embryo buffer.
Library members were added to the embryo buffer from the
DMSO stock solutions, one compound per well. The embryos
were incubated at 28.5 °C and inspected visually using a
dissecting light microscope for developmental defects 1, 2, and
3 days post-fertilization (dpf). Compound 13ab was identified
to interfere reproducibly with zebrafish development at a
Conclusions
In conclusion, methodology involving the synthesis and
oxidation of bis-aryl organocuprates, producing biaryls contained
within a medium ring, was developed into an efficient, general,
and atropdiastereoselective ring-closing reaction. The reaction
pathway was modified for use on high-capacity macrobeads,
key elements of a one bead/one stock solution platform, and
used to synthesize a collection of small molecules (1412
theoretical total). Mass spectrometry was demonstrated as an
effective way to identify biologically active compounds. Phe-
notypic and protein-binding assays were performed on members
of the library, and results from zebrafish and plant develop-
(77) Zebrafish developed normally at 5 µM, 2.5 µM, 500 nM, and 50 nM
concentrations; however, developmental defects were observed reproducibly
at 10 µM and 100 nM concentrations (repeated five times). The develop-
mentally delayed zebrafish die 3 dpf if they continue to be exposed to
embryo buffer containing 13ab; however, they can be rescued if transferred
to 13ab-free embryo buffer and allowed to progress.
(75) Grozinger, C. M.; Chao, E. D.; Blackwell, H. E.; Moazed, D.; Schreiber,
S. L. J. Biol. Chem. 2001, 276, 38837-38843.
(76) Hergenrother, P. J.; Depew, K. M.; Schreiber, S. L. J. Am. Chem. Soc.
2000, 122, 7849-7850.
(78) These and other experiments indicate that the small molecules from the
library are cell-permeable and capable of interacting directly with intrac-
ellular protein targets.
(79) King, R. W. Chem. Biol. 1999, 6, R327-R333.
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Synthesis of Biaryl-Containing Medium Rings
A R T I C L E S
mental assays were described. The ability to use complexity-
generating reactions on high capacity beads suitable for a one
bead/one stock solution platform and the subsequent use of
library members in phenotypic and protein-binding screens
demonstrate key elements of the systematic (chemical genetic)
approach to exploring biology.
supervision of automated compound release and liquid handling,
Angela Koehler for printing small molecule microarrays of the
library, and Randall Peterson and Stanley Shaw for their
specialist assistance in the zebrafish assays. D.R.S. is supported
by a Wellcome Trust Postdoctoral Fellowship (No. 054741).
H.E.B. is supported by a postdoctoral fellowship from the Jane
Coffin Childs Fund for Medical Research. S.L.S. is an Inves-
tigator at the HHMI.
Acknowledgment. We thank the Donald W. Reynolds
Foundation, Cardiovascular Clinical Research Center, and
NIGMS for support of this research. The NCI, Merck KGaA,
and Merck & Co. are gratefully acknowledged for their support
of the Harvard ICCB. We also thank Paul Clemons for expert
Supporting Information Available: Complete experimental
procedure, characterization data, and library LCMS traces
(PDF). An X-ray crystallographic file (CIF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA017248O
9
J. AM. CHEM. SOC. VOL. 124, NO. 7, 2002 1363