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Combinatorial Chemistry with Laser Optical Encoding.

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all-E content characteristic of the double elimination process
holds for the present case as well: all-E:9Z:[(lIZ) + (13Z)l =
91 :4:5.
We compared this new process with those used commerciall ~ . [ The
~ ] Hoffmann-La-Roche process involves a C,, + C ,
route, and BASF makes use of the Wittig reaction between C,,
and C, building blocks. Rh6ne-Poulenc also employs the
C,, + C, combination based on the Julia sulfone coupling. Our
strategy is novel in that two C , , components are coupled. The
C,, skeletons are one of the most common units in terpenoid
derivatives and, accordingly, readily available. As a whole, our
process has the twofold benefit of ready availability of the starting materials and simple operation.[61
We have shown that the following reactions can be integrated:
a) addition of carbanion to carbonyl, b) 0-alkylation of the
addition product, and c) successive elimination of the alkoxy
and sulfinyl groups. The only modification of the stepwise process is made for the 0-alkylation. This results in not only compaction of the process but increase in the total yield. Although
we took up a rather simple case in this study, it has proved that
even a slight modification can bring about critical improvements of the chemical process. This suggests the importance and
potential of the integrated chemical process for designing a synthesis.
Experimental Section
NaI (225 mg. 1.5 mmol), cyclogeranyl sulfone 1 [3c] (362 mg, 1.3 mmol), and T H F
(2 mL, dried over sodium benzophenoneketyl) were placed in a flame-dried, twonecked flask. A solution of BuLi in hexane (1.6 M, 0.75 mL, 1.2 mmol) was added,
and the mixture stirred at -78 "C (dry ice/MeOH) under argon for 1 h. After thc
addition of aldehyde 2 [3c] (211 mg, 1.0 mmol) in T H F (1 mL) followed by MOMCI
(0.09 mL, 1.2 mmol), the solution was stirred for 4 h at -78°C and 1 h at room
temperature. Cyclohexane (3 mL) and KOMe (701 mg, 10 mmol) were added, and
the mixture stirred for 1 h at room temperature and 1 h at 40°C. The reaction was
quenched with aqueous NaHCO, solution (10 mL) and ethyl acetate (15 mL). After
separation of the organic layer and extraction of the aqueous phase with ethyl
acetate ( 2 x 15 mL), the combined organic fractions were washed with brine
(20 mL), dried over MgSO,, and concentrated under reduced pressure (crude,
452 mg). Hexane ( 5 mL), pyridine (0.5 mL), acetic anhydride (2 mL), and DMAP
(48 mg, 0.4 mmol) were added to the crude product under argon, and the mixture
stirred for 1 h at room temperature. Aqueous NaHCO, solution (20 mL) and ethyl
acetate (15 mL) were added at 0"C, and the organic layer separated. The aqueous
phase was extracted with ethyl acetate (2 x 15 mL), and the combined organic fractions washed with brine (20 mL), dried over MgSO, and concentrated under reduced pressure (crude, 504 mg). The crude oil was analyzed by HPLC; 76% yield,
248 mg, all-E:9Z:[(llZ) (13Z)l = 91:4:5 (column: ZORBAX SIL 4 . 6 m m x
25 cm; eluent: terr-butylmethyl ether/hexane 51100; internal standard: 2,6-xylenol).
Combinatorial Chemistry with
Laser Optical Encoding
Xiao-yi Xiao," Chanfeng Zhao, Hanan Potash, and
Michael P. Nova
In the rapid development of synthetic and combinatorial
chemistry,"] solid-phase combinatorial synthesisr2]continues to
be one of the most effective techniques for building diverse
libraries. Advantages of solid-phase synthesis over solution
chemistry include ease of intermediate isolation, use of excess
reagents to drive reactions to completion, and potential use of
the highly efficient pool and split r31 synthesis technique. Two
elements are essential for successful solid-phase combinatorial
library generation and evaluation: an efficient solid-support
chemical reaction sequence and a reliable means for structural
elucidation and confirmation[41of library members. Although
considerable efforts have been devoted to the development of
solid-support chemistry,L2I the choice of methods for structural
elucidation (spatial a d d r e s ~ i n g ,mixture
~~]
deconvolution,[61direct microanalysis,[71or chemical tagging[']) is rather limited.
Tagging-especially nonchemical, noninvasive tagging-is potentially the most efficient and reliable encoding method. We
have recently developed a new, nonchemical, noninvasive radiofrequency encoding technology.[g1Here we report another
strategy for noninvasive synthetic chemistry encoding, namely,
laser optical tagging, with LOSCs (Laser Optical Synthesis
Chips). This technology is applicable to library synthesis of all
types of compounds including small molecules, peptides, and
oligonucleotides in multimilligram quantities.
The LOSC technology combines the most advanced developments in laser bar code etching and identification as well as
organic synthesis on novel solid supports. The initial LOSC is
shown in Figure 1. It is fabricated by combining a two-dimen-
+
Received: November 12, 1996 [Z9760IE]
German version: A n g e w Chem. 1997, 109, 804-805
Key words: eliminations
one-pot reactions
*
vitamins
Figure 1. Illustration of the laser optical synthesis chip (LOSC), viewed from the
top (top) and in cross-section (bottom). The size of the LOSC is 10 x 10 x 2 mm.
G. H. Posner, Chem. Rev. 1986,86, 831 ; L. E Tieze, U. Beifuss, 4ngrw. Chrm.
1993, i05,137; Angew. Chem. f n t . Ed. Eizgl. 1993,32,131; a special issue on this
subject (Ed.: P.A. Wender): Chem. Rev. 1996, 96, no. 1.
Hudlicky pointed out that "a multistep synthesis must therefore be considered
as a dynamic continuum in which every event depends on all others": T.
Hudlicky, Chem. Rev. 1996, 96, 3.
a) T. Mandai, T. Yanagai, K. Araki, Y Morisaki, M. Kawada, J. Otera, J. Am.
Client SOC.1984, 106, 3670; b) J. Otera, H. Misawa, T. Mandai, T. Onishi, S.
Suzuki. Y Fujita, Chem. Lett. 1985, 1883; c) J. Otera, H. Misawa, T. Onishi, S.
S,izuki, Y Fujita, J. Org. Chrm. 1986,5/, 3834; d) J. Otera in Curotehoids, Vol. 2
(Eds.: G. Britton, S . Liaaen-Jensen, H . Pfander), Birkhauser, Basel, 1996, pp.
103, 31 1 .
Note that the yield of step 3 (78%) should come from the successive high-yielding elimination reactions (2 x 88 Yo).
J. Paust, Pure Appl. Chem. 1991,63,45; J. Paust in Curotenoids, b l . 2 (Eds.: G.
Britton, S. Liaden-Jensen. H. &'pander),
Birkhiuser, B a d , 1996, p. 259.
[6] Of course, accessibility of the key building blocks is an important factor for
assessing the overall brevity of the process. A more detailed discussion will be
given in a full paper.
780
m
C VCH VerlagsgesellschaftmhH, 0-69451
Weinhrim, 1997
sional 16-digit bar code for encoding and a polymeric support
for chemical synthesis. The 2-D bar codes are laser-etched with
a CO, laser on 6 x 6 segments of a chemically inert alumina
ceramic plate (the actual size of each bar code is 3 x 3 mm). The
surrounding synthesis support is a stable polypropylene or
fluoropolymer square (10 x 10 x 2 mm), which is radiolytically
[*I Dr. X.-Y Xiao, Dr. C. Zhao, Dr. H. Potash, Dr. M P. Nova
IRORI Quantum Microchemistry
11 025 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: Int. code +(619)546-3083
e-mail: xyxiao@jirori.com
[**I We thank Prof. K. C. Nicolaou, who is an advisor to IRORI Quantum Microchemistry, and Dr. A. W. Czarnik for valuable discussions, and Mr. Bill Ewing
for software development.
0570-0833~97/3607-07rYO$ 17.50+ ,5010
An,qen'. Chen?. Inr Ed. En,TI. 1997. 36, No. 7
grafted with low cross-linking polystyrene.['o1 Very small
LOSCs can be manufactured, since the laser-etching optical resolution of an entire bar code can be extended well below a total
diameter of 0.5 mm. Two-dimensional bar coding allows more
data compression in a much smaller surface area than regular
linear bar coding.
The LOSCs are then subjected to a modified arninomethylation[''] (or other procedures for introducing groups other than
aminomethyl['21) to functionalize the polystyrene surface graft.
A loading of 5-8 pmol per chip is typically obtained, as measured by Fmoc (Fmoc = 9-fluorenylmethoxycarbonyl) analysis. The chips are now ready for use in chemical synthesis.
A "directed sorting" strategy (instead of statistical pool and
split) is used with the LOSC for constructing combinatorial
libraries with zero redundancy (that is, the number of LOSCs is
equal to the number of library members). A small camera
(Quickcam) that is linked to a proprietary pattern recognition
and combinatorial synthesis manager software is used to scan
the bar codes and register the chips with the library members.
Before each reaction with n splits, the chips are scanned and
sorted (split) into n groups as directed by the computer. After
completion, the structure of the compound synthesized on each
LOSC (if every reaction in the entire synthesis is successful) or
the reaction pathway to which each LOSC has been subjected
can be decoded simply by optically reading the 2-D image on the
chip (Figure 2).
was used for the coupling reactions, and ammonia in water/l,4dioxane (l/l v/v) for the cleavage. The cleaved, crude oligonucleotides were 67 Yo to 97 % pure, as determined by HPLC.
Between 2 and 5 mg of pure oligonucleotide were isolated for
each library member. The structures of the oligonucleotides
were confirmed by MS, 'H N M R spectroscopy,[141and sequence analysis.[' '1
The LOSC shown in Figure 1 can be widely varied in shape,
configuration, and polymeric support. The ceramic 2-D bar
codes carry at least 8 bytes of information. When etched on
ceramics they are inert to the majority of organic synthesis conditions, easy and reliable to use, inexpensive, and very amenable
to mass production. The low cross-linking polystyrene surface
graft (and other polymer grafts) on the stable and inert base
polymer provides an ideal solid support for chemical synthesis
with excellent functionalizability and chemokinetics. LOSC
technology can also be applied to the synthesis of other types of
compounds, especially small organic molecules.['61 Potential
advantages of LOSC technology over existing chemical tagging
combinatorial techniques include 1) low manufacturing cost, 2)
noninvasive encoding, 3) high encoding reliability and capacity,
4) total chemistry flexibility, 5 ) excellent chemokinetics, 6) easy
and clean washing between reactions, 7) utilization of the highly
efficient directed sorting strategy, 8) delivery of pure, discrete
compounds in multimilligrams quantities, and 9) amenability to
full automation. Integration of LOSC technology with automation will further enhance its applicability in high through-put
chemical synthesis and biological screening and is now in progress.
Received: September 16, 1996 [Z9561 IE]
German version: Angew. Chem. 1997,109, 799-801
Keywords: combinatorial chemistry
Figure 2. Schematic representation of a 3 x 3 pool and split combinatonal synthesis
with the LOSCs and directed sorting. A, B, and Care building blocks. The numbers
above each LOSC represent their 2-D bar codes (single-digit codes are used for
brevity).
To demonstrate the utility of the LOSCs in chemical synthesis, a library of 27 oligonucleotides of general structure X4-X,X,-T was synthesized on 27 LOSCs with the directed sorting
strategy. Standard oligonucleotide chemistry['31 was applied
with the following modifications : CH,CN/CH,CI, (2/3 v/v)
Angew. Chem. Int. Ed. Engl. 1997, 36, No. 7
C;
*
laser coding
*
peptides
[I] a) A. W Czarnik, Chemtracrs; Org. Chem. 1995, 8 , 1 3 ; b) D. J. Ecker, S. T.
Crooke, Biotechnologj 1995, 13, 351; c) M. A. Gallop. R. W. Barret, W. 1.
Dower, S. P. A. Fodor, E. M. Gordon, J Med. C'hem. 1994,37,1233;d) E. M.
Gordon, R. W. Barret. W. J. Dower, S. P. A . Fodor, M . A. Gallop, ibicl. 1994.
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Schroeder, D. M. Reynolds Cody, M. R. Pavia, Proc. Nafl. Acud. Sci. US.4
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Am. Chem. Soc. 1963, 85, 2149; g) S . J. Danishefsky,
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Knapp, Nufure 1991,354,82;b) A. Furka, F. Sevestye, M. Asgedom, G. Dibo,
Int. 1 Peptide Res. 1991, 37, 487.
[4] X.-Y Xiao, M. P. Nova in ComhiuatoFialChemistr)~:S~nrhesisundApplication
(Eds.: S. R. Wilson, A. W. Czarnik). Wiley, New York, 1996, chap. 7.
[5] a) H. V. Meyers, G. J. Dilley, T. L. Durgin, T. S. Powers, N . A. Winssinger, H.
Zhou, M. R. Pavia, Mol. Diversifj 1995, 1, 13; b) S. H. DeWitt, A. W.
Crarnik, Arc. Chem. Res. 1996, 29. 114.
[6] a) H. M. Geysen, S. J. Rodda, T. J. Mason, G. Tribbick. P. G . Schoofs, J
Immunol. Methods 1987. 102, 259; b) R. A. Houghten, C. Pinilla. S. E.
Blondelle, J. R. Appel, C. T. Dooley, J. H. Cuervo. Narure 1991, 354. 84;
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1994, 219, 261; b) B. B. Brown, D. S. Wagner, H. M. Geysen, Mol. D i w r s i f j
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Clwn. Soc. 1995, 11 7, 3900; d) C. L. Brummel, I. N. W Lee, Y Zhou. S. J.
Benkovic, N. Winograd, Science 1994, 264, 399
VCH Vrrlngsge.crNvchaftmhIl, D-69451 Weinheim, 1997
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78 1
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[81 a) S. Brenner, R. A. Lerner, Proc. Nail. Acod. Sci. USA 1992, 89, 5381;
b) M. C. Needles, D. G. Jones, E. H. Tate, G. L. Heinkel, L. M. Kochersperger, W J. Dower, R. W. Barret, M. A. Gallop, ibid. 1993, YO, 10700;
C) M. H. J. Ohlmeyer, R. N. Swanson, L. W Dillard, J. C. Reader, G. Asouline, R. Kobayashi, M. Wigler, W. C. Still, ibid. 1993, YO, 10922; d) P. Eckes,
Angew. Cbem. 1994, 106. 1649; Angew. Chem. Int. Ed. Engl. 1994,33, 1573;
e) 2. J. Ni, D. Maclean, C. P. Holmes, M. Murphy, B. Ruhland, J. W. Jacobs,
E. M. Gordon, M. A. Gallop, J. Med. Chem. 1996, 39. 1601.
[9] K.C. Nicolaou, XY
:
Xiao, 2. Parandoosh, A. Senyei, M. P. Nova, Angew.
Chem. 1995, 107, 2476; Angew. Chem. h t . Ed. Engl. 1995.34, 2289.
[I01 H. A. J. Battaerd, G. W. Tregear, Gru/r Copolymer.$,Wiley, Interscience, New
York, 1967.
[Ill a) A. R. Mitchell, S. B. H. Kent, M. Engelhard, R. B. Merrifield, J Org.
Chem. 1978, 43, 2845; b) A. R. Mitchell, B. W. Erickson, M. N. Ryabstev,
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[I 31 M. J. Gait, Oligonucleotide Synzhesis, A Prartical Approach, IRL. Oxford,
1990.
[14] Selected 'HNMR datl (500 MHz, D,O) ofcrude oligonucleotides: ACCT. 6
= 8.47 (s, 1 H, CH-adenine), 8.37 (s, 1 H, CH-adenine), 8.09 id, J =7.8 Hz,
1 H, CH-cytosine), 8.06 (d, J =7.8 Hz, 1 H, CH-cytosine), 7.68 (s, 1H, CHthymine), 6.15-6.32 (3m, 6H, CH-cytosine and 0-C(N)H/, 3.81-5.03 (61x1,
16H, 0-CH, and 0 - C H I , 2.28-2.92 (4m, XH. CH,), 1.87 (s. 3H, CH,).
ACAT: 6 = 8.47 (s, 1H, CH-adenine), 8.44 (s, 1 H, CH-adenine). 8 35 (s, 1H,
CH-adenine), 8.34 (s, I H, CH-adenine),8.05 (d, J = 8.0 Hz. 1 H, CH-cytosine), 7.59 (s, l H , CH-thymine), 6.15-6.43 (3m, 5H, CH-cytosine and
0-C(N)H/, 3.78-5.01 (7m, 16H, 0-CH,andO-CHI, 2.17-2.87(5m, XH,
CH,). 1.79 (s, 3H, CH,).
182
f i , V T H Vprlnvspesellschaft mbH. D-6Y451 Weinheim. 1997
115) Two library members with the same mass (ACGT and AGCT) were sequenced
with electrospray tandem mass spectrometry. For details of this method, see
a) G. Siuzdak. Mass Spectrometry f o r Biotechnology, Academic Press, San
Diego, 1996, p. 41; b) J. W Metzger, C. Kempter, K.-H. Wiesmiiller, G. Jung,
Anal. Biocbem. 1994,219,261, cj J. Ni, S. C. Pomerantz, J. Rozenski, Y Zhang,
1. McCloskey, Anal. Chem. 1996,68, 1989.
[t 61 A manuscript on application of LOSC in small molecule library synthesis is in
preparation.
Corrigendum
In the communication "Isolation of a Nonicosahedral Intermediate in the Isomerization of an Icosahedral Metallacarbarone" by S. Dunn, G. M. Rosair, Rh. L1. Thomas, A. S.
Weller, and A. J. Welch (Angew. Chem. Innt. Ed. Engl. 1997, 36,
645-647) incorrect "B{'H)NMR values were given in the
Experimental Section for BTMA+ 1 --.The correct values are as
follows: "B{'H)NMR (128.4 MHz, (D,CN): 6 = -0.9 (1 B),
-4.7 (2B), -8.1 (1 B), -9.8 (2B), -10.9 (2B), -16.5 (1 B).
OS7O-O833~Y7~3607-07112$175O+ SO10
Angew. Chem. Int. Ed. Engl. 1997. 36. N O . 7
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