`
`Page 1 of6Page 1 of6
`
`
`
`ILMN EXHIBIT 1010ILMN EXHIBIT 1010
`
`
`
`Page 1 of 6
`
`ILMN EXHIBIT 1010
`
`

`

`TETRAHEDRON LETTERS
`
`The International Journal for the Rapid Publication of Preliminary Communications
`in Organic Chemistry
`
`EXECUTIVE BOARD OF EDITORS FOR TETRAHEDRON PUBLICATIONS
`
`Chairman: Professor Sir Derek Barton, FRS, Department of Chemistry. Texas A&M University. College Station.
`TX 77843-3255, USA (FAX:
`I 409 845 4719)
`
`Profemr J. E. Baldwin, FRS, D son Perrins Laboratory.
`Oxford, England (FAX: 44 865 75632)
`Professor D. L. Boger. Department of Chemistry. Purdue
`University, West Lafayette, Indiana 47907, USA (FAX: I 317
`494 9695)
`Dr S. G. Davies, Dyson Perrins Laboratory. Oxford, England
`(FAX: 44 865 275633)
`Professor L. Ghosez, Laboratoire tie Chimie Organique dc
`Synthése. Université Catholi ue de Louvain, Place L. Pasteur
`1. H4348 Lzouvain-la-Neuve,qBelgium (FAX: 32 10 474 I68)
`Professor S. Ito, Faculty of Phannaceutical Sciences,
`Tokushima Bunri University, Yamashiro-cho. Tokushima
`770, Japan (FAX: 81 886 55 8774)
`Professor A. R. Katritztty, FRS, Department of Chemistry.
`Universit of Florida, Gainesville, FL 32611, USA
`(FAX: 1
`392 9199)
`Professor N. K. Koehetltov, N. D. Zelinsky Institute of
`Organic Chemistry. Academy of Sciences USSR. Leninslty
`Prospeltt 47, Moscow B-334. USSR (FAX: 7 095 135 5328]
`Professor A. M-cKilIop, University of East Anglia. School of
`Chemical Sciences. University Plain. Norwich NR4 TFJ.
`England (FAX: 44 603 507773)
`
`Professor A. Nicknn, Department of Chemistry. The Johns
`Hopkins University. Baltimore. MD 2l2l8. USA
`(FAX: 1301338 8420)
`Professor W. D. Ollis, FRS, University of Sheffield. Sheffield
`S3 7HF. England (FAX: 44 742 668318)
`Professor G. Ourisson, Centre National dc la Recherche
`Scientifique. Centre dc Neurochimic. 5 rue Blaise Pascal,
`67084 Strasbourg. Cedex, France (FAX: 33 88 60762tl)
`Professor T. Shioiri. Faculty of Pharmaceutical Sciences.
`Na oya City University, Tanahe-dori, Nll2’.Ul'l0-l£LI. Nagoya
`46 , Japan (FAX: 81 52 334 4172)
`Professor W. Steglich, lnstitut ltir Or anische Che-mic und
`Bioehemie dcr Universitiit Bonn, I)-_ 300 Bonn l, Gerhard-
`Domagk-Strasse 1. Germany (FAX: 49 228 258216)
`Professor H. H. Wasserman, Department of Chemistry. Yale
`University. PO Box 6666. New Haven. CT 0651 l . USA
`(FAX: 1 203 432 9990)
`Professor W. T. Wipke, Thimann Laboratories, University of
`California, Santa Cruz, CA 95tlh::t. USA
`(FAX: 1408 459 4716)
`
`F. Areamoue. Florence
`D. Arigoni, Zurich
`P. D. Bartlett. Fort Worth. TX
`A. R. Battersby. Cambridge
`A. I... J. Beet-nvlth. Canberra
`A. J. Birch, Canberra
`R. Brestow. New York. NY
`H. (2. Bmvrrt, Lafayette. IN
`E. .l. Corey, Cambridge. MA
`J.
`W. Cornforth. Brighton
`D.
`,1. Cram, Los Angeles. CA
`P. Deslongchalntls. Quebec
`M. .l. S. Dewar. Gainesvillc. FL
`C. Djerassi. Stanford. CA
`W. van E. Doering. Cambridge.
`MA
`
`BOARD OF CONSULTING EDITORS
`J. E. Duhois, Paris
`J. Michalsltl. Lodz
`J. Elgtlero. Madrid
`M. L. Mthallovie. Belgrade
`A. Eschenmnser. Zurich
`K. Mori. Tokyo
`D. A. Evans, Cambridge. MA
`T. Multaiyama. Tokyo
`(3. Fudllr, Morgantown, WV
`K. Naltanishi. New York. NY
`A. Gonzalez Gonzalez. Tenerife
`T. Ntlrill, Stockitolln
`R. No}-orl, Nagoya
`Y. Hirata. Nagoya
`S. Nozoe. Sendai
`R. Huisgen. Munich
`W. S. Johnson. Stanford. CA
`W. Oppolzer. Geneva
`M. Julia, Paris
`H. Pauls-en. Hamburg
`A. Kjaer. Lynghy
`P. Potter, Gif~sur~Yvette
`V. A. Koptyug. Novosibirsk
`S. K. Praclhan, Bombay
`R. U. Lernieux. Alberta
`V. Prelog, Zurich
`(3. Qulnkert, Frankfurt am Main
`X. T. Liang, Beijing
`R. A. Raphael, Cambridge
`P. de Mayo, Ontario
`(T. W. Rees, London
`A. 1. Meyers, Fort Collins. ('0
`
`.l.
`D. Roberts. Pasadena. (‘A
`A. L Scott, College Station. TX
`K. B. Sharpless. Cambridge. MA
`G. Snatzlte. Bochum
`H. A. Staab. Heidelberg
`G. Stork. New York, NY
`Sulth Dev. New Delhi
`W. Tochtermann. Kiel
`A. Todd. Cambridge
`H. G. Viehe. Louvain-la-Ncuvc
`E. Vogel. Cologne
`Y. ‘Wang. Shanghai
`K. B. Wiberg, New Haven. CT
`E. Winterfeldt, Hannover
`N. S. Zefirov, Moscow
`
`Publishing. Subscription and Advertising Offices
`Pergamon Press plc, Headington Hill Hall, Oxford OX3 OBW
`(phone Oxford 794141: FAX: 60285) and Pcrgamon Press
`lnC., Maxwell House, Fairview Park, Elmsford. NY 10523.
`USA {phone 914 S92 7700:
`telex 137328).
`
`Fifty-two issues per year
`
`Annual Subscription Rates (1991)
`Annual institutional subscription rate (1991) DM 6160.00.
`Two—year institutional rate (1991192) DM 11704.00.
`Personal subscription rate for those whose library subscribes
`at
`the regular rate (1991).
`is available from your nearest
`Pergamon office.
`Rates include postage and insurance. Subscription rates for
`Japan include despatch by air and prices are available on
`application.
`to change without notice.
`Prices are subject
`Microfonn Subscriptions and Back Issue
`Back issues of all previously published volumes are available direct from Pcrgamon
`Press otfices,
`
`E‘) M The paper used in this publication meets the minimum requirements of
`American National Standard for Information Sciences — Permanence of Paper [or
`Printed Library Materials. ANS] 23943-1984.
`
`Copyright © 1991 Pcrgarnon Press pic.
`It is a condition of puhtication that manuscripts submitted to this journal have not
`been published and will not be simultaneously submitted or published elsewhere.
`By submitting a manuscript. the authors agree ll1itt the copyright for their article is
`transferred to the publisher it and when the article is accepted tor publication.
`However. assignment of copyriyrt
`is not required from authors who work for
`organizations which do not permit such assignment. The copyright covers the
`exclusive
`rights
`to reproduce and distribute the article including reprints.
`pliotographic reproductions. rrticrotnrm. or any other reproduction of similar
`nature. and translations. No part of this publication may be reproduced. stored l.I'1. a
`retrieval
`system or
`transmitted in any form or by any means. electronic.
`electrostatic, magnetic tape. mechanical. photocopying. recording. or otherwise.
`without permission in writing from the copyright holder.
`Photocopying information for users in the USA. The item-[cc Code for this
`publication indicates that authorization to photocopy Items for internal or petsoltal
`use is granted by the copyright holder for libraries and other users registered with
`the Copyright Clearance Center [(.‘(.'(') Transactional Reporting Service provided
`the stated fee for copying beyond that permitted by Section to? or llllt or the
`United States Copyright Law is paid. The appropriate remittance of $3.tltI per copy
`per article is paid directly to the Copyright Clearance Center Inc . 37 Congress
`Street. Salem. MA tli‘.l7ll.
`Permission for other use. The copyright owner's consent does not extend to copying
`for general distribution. for pmnlolinn. for creating new works. or for resale.
`Specific written permission must be obtained from the publisher for such copying.
`
`The Ilemfee (‘ode for this publirariorr is".
`
`IIlI<m—.1tI3'J.l9l S3.IltI + .lttt
`
`Page 2 of6
`
`
`
`Page 2 of 6
`
`

`

`Tetrahedron Letters. Vol32, No.7. pp 319-832. 1991
`Printed in Great Britain
`
`oo4u.4039;91 53_oo + .00
`Pcrgamon Press plc
`
`FACILE PREPARATION OF TOLIGONUCLEOTIDE-PEPTIDE CONJUGATES
`
`Carl D. Juby, Christopher D. Richardson, and Roland Brousseau“
`
`Genetic Engineering Section. Biotechnology Research Institute, National Research Council Canada, Montreal,
`Quebec, Canada l-l4P 2R2
`
`5_tymn_a_1: The solid phase synthesis of 3' deoxyoligonucleotide (I7 mer)-peptide conjugates is described. The
`oligonucleotide is complementary to the template strand of the measles virus in the region of the nucleocapsid protein
`on the 60 nucleotide leader RNA. The conjugates were synthesized on teflon support using a combination of solid phase
`FMOC peptide chemistry and phosphoramidite chemistry linked by a branched modifier.
`
`The concept of specific control of gene expression through the use of oligonucleotides promises rational
`
`therapeutic approaches to viral‘ diseases as well as to certain cancers‘. Several distinct modes of action for inhibition
`
`of gene expression’ have been demonstrated; they all have in common a requirement that oligonucleotides be readily
`
`incorporated into living cells. There is currently intense research activity focussed on the synthesis of oligonucleotide
`
`conjugates capable of enhanced cellular uptake.
`
`In attempts to increase the level of cellular incorporation, oligonucleotides have been covalently linked to a variety
`
`of hydrophobic groups such as cholesterol‘, or lipids’ or to positive! y charged groups such as poly-L-lysine‘.
`
`In this
`
`context. the synthesis of peptide-oligonucleotide conjugates offers great potential in terms of increased uptake and also
`
`through the possibility of targeting the antisense construct to certain types of cells using specific peptides. A generally
`
`applicable approach has been recently published’; this approach however requires several preliminary steps to modify
`
`the commercially available solid support before synthesis of the conjugate proper can begin.
`
`In this paper, we describe a synthetic method which is rapid and simple to carry out and which allows preparation
`
`of peptidcoligonucleotide conjugates starting directly from commercially available starting materials.
`
`The peptide (Z-D-Phe-I.-Phe-Gly, Z=benzy1o:tycarbonyI) was found to be a specific inhibitor of cell fusion and
`
`hemolysis‘. The oligonucleotide part of the conjugate is a 17-mer with the sequence 5'CAAA.G'I'I‘GGG'I‘AAGGAT.
`
`it was designed as a positive (rnRNA) sense oligomer hybridizing with the genomic. negative strand of this
`
`paramyoxvirus in the leader RNA region. starting 50 bases upstream of the mRNA cap site. Complexes which bind the
`
`viral genome in this region have shown ability to inhibit transcription. The hydrophobic conjugate (Trp), and the
`
`positively charged conjugate (Lys), are expected to provide enhanced uptake of the oligonucleotide moiety.
`
`The strategy is outlined in Figure I; it is based on a commercially available teflon resin which contains a
`
`our. Dimathuxyifityl;
`CNEl= 2—cyanoeihy|
`
`ootrr _.
`
`TCAJCI-l2C12
`1)
`2t Branched modifier!
`tetrazole
`
`Figure
`
`1
`
`Fmuc = Fluorenylmethoxycarbonyl; TCA= Trichloroacetic acid
`
`(“J
`.
`”cH2_0,DMT
`9
`—- °'F"°“3
`O-P—O—CH
`'
`.
`~~-..
`_
`_
`0’
`OCNB
`CH2 NH Fmoc
`ll Peptide synth-
`21 Oligo synth.
`3} Deprotection
`
`Intermediate
`
`I
`
`|-(CH2-0-OllQ0nUCl90tld0
`“- CH2-NH-Gly-Phe-Pho-Z
`
`Product
`
`879
`
`Page 3 of6
`
`
`
`Page 3 of 6
`
`

`

`B80
`
`25 atom spacer arm terminating in a Dimethoxytrityl (DMT) protected hydroxyl. To this hydmxyl group is added the
`
`bifunctional branched modifier to create Intermediate I. This intermediate bears both a DMT-protected hydroxyl group
`
`and an Fmoc-protected amino group and therefore can be used directly both for solid phase Frnoc peptide synthesis and
`
`for subsequent oligonucleotirle synthesis by the solid phase phosphoramidite method.
`
`The stepwise synthesis of Z-D-Phe-L-Phe-Gly on Teflon was performed as follows:
`
`the commercially available
`
`Teflon support (Molecular Biosystetru, San Diego, CA, 1.0 ptnole) was placed in a DNA synthesis column. The DMT
`
`group was removed on the machine using 39!: trichloroacetic acid ('1'CA). A non-standard symhesis cycle was run
`
`introducing the branched modifier 1 (Glen Research, I-lemdon, VA., 0.1M) phosphoramidite with the amiditc reaction
`
`time extended to 110 seconds. The DMT group on the branched modifier was left on. The teflon packing was removed
`
`from the column and treated with 20% piperidinelDMF for [5 minutes to effect removal of the FMDC group. The teflon
`
`was washed with DMF and dry pyridine. Fmoc-Gly—OPFp (23.2 mg, 50.0 ptmole, Milligen, Cambridge, UK.) wa added
`
`to the teilon and dissolved in 700 pl of dry pyridine. The reaction mixture was stirred overnight at room temperature.
`
`Afier washing with DMF, 20% piperidine:‘DMF was added and stirred at room temperature for 15 minutes to effect
`
`removal of the Fmoc group. The solution was removed and the support was washed with dry pyridine. The previous step
`
`was repeated using Fmoc-L-Phe-0PFp (27.65 mg, 50.0 pmole) in 700 pl dry pyridine. Finally, Z-D-Phe-OH (29.9 mg,
`
`100.0 pmole) was reacted with the growing peptide chain using HOBT (15.3 mg, 100.0 umolc) and DCC (22.7 mg,
`
`100.0 pmole) in 700 pl of dry pyridine. Within 5 minutes a precipitate was formed and the mixture was left stirring at
`
`room temperature overnight.
`
`After the peptide coupling, the tellon was placed on the Applied Biosystems Model 380A and DNA synthesis
`
`was carried out using the cyanoethyl phosphoramidite chemistry with extended reaction and detritylation times (110 see.
`
`each) as specified by the manufacturer of the teflon resin. The trityl cation release was assayed and gave an average
`
`stepwise yield of 98.9%. The deprotection of the oligonucleotide was not carried out using Ni-l',0l-l because of the lability
`
`of the peptide bonds to strong base. For this reason,
`
`the alternative procedure originally reported by Miller and
`
`coworkers” for the deprotection of methyl phosphonate oligonucleotides was employed. The method involves treating the
`
`derivatized teflon with ethylenediarnine (EDA) in absolute ethanol (l:l "’.f.,.) for 1 hour at 55 “C. The conjugate was
`
`removed from the support” by treating with a 1 ml solution of 100 mM sodium periodate, 100 mM sodium phosphate
`
`buffer, pH 7.2, in acetonitrile/water (l:4 WV). The mixture was agitated at room temperature with exclusion of light.
`
`After 4 hours the solution was removed and the support was washed with excess water and methanol. The solution was
`
`removed and the tetlon was treated with n-propylamine (50 ul), acetonitrilc (I00 ul), and water (400 Lil) for 3 hours at
`
`55 "C. The solution was evaporated to dryness i. The product was purified by reverse phase HPLC to yield pure
`
`conjugate (14.8 0.D., 10.4% for 1 step synthesis or 15.1 0.1)., 10.7% for stepwise synthesis).
`
`HPLC conditions were as follows: reverse phase C-18 column 5 pm particle size, LKB type Pep-S analytical,
`
`using a linear gradient between Solvent A, 0.1 M triethylamine acetate (TEAA) in water and Solvent B, 75% acetonitrile
`
`in 0.1 M TEAA. The gradient was run from 0-l00% in 100 minutes. The conjugate elutes between the oligonucleotide
`
`(23.5 min.) and the peptide (56.8 min.) giving a sharp single peak at 42.4 minutes.
`
`Page 4 of6
`
`
`
`Page 4 of 6
`
`

`

`88]
`
`The ‘H NMR spectrum of the conjugate shows a phenyl absorption at 7.1-7.8 ppm, while the peptide (Z-D-Phe-
`
`L-Phe-Gly) shows a phenyl absorption at 7.1-7.3 ppm. This peak broadening of the phenyl groups is consistent with
`
`previous results".
`
`U.V. data (B20) is as follows: (hm 262.6 nm., e = 150,000 (estimate), hm 260.6 nm., hm 256.6 nm., it,-_
`
`253.0 nm., hm 250.4 nm., Aug 247.4 um.) and peptide, Z-D—Phe~L—Phe~Gl3f UN... 263.9 nm., 1,-_ 261.8 nm., )t_,
`
`258.] nm., e = 840, Lb 255.4 nm., }t,_, 252.3 nm.). These three maxima are indicative of the peptide.
`
`Stepwise §ynth§i§ of ([45), gm fljgpj, on tgflpn "l"he procedure described above was applied to the synthesis of the
`
`(Lys),- and (Tq)),- oligonucieotide conjugates. Fmoc-Lys-Boc—OPFp (31.8 mg, 50.0 pmole) or Fmoc-Trp-0PFp (29.6
`
`mg, 50.0 pmole) was added to the teflon and dissolved in 700 pl of dry pyridine. The reaction mixture was stirred for
`
`at least 4 hours at room temperature. The solution was pipetted off and the support was washed with DMF. Following,
`
`20% piperidine!DMF was added and stirred at room temperature for 15 minutes to effect removal of the Fmoc group.
`
`The solution was removed and the support was washed with dry pyridine. The previous addition step was repeated four
`
`times. The final Fmoc group was left on.
`
`The peptide synthesis mixture was pipetted off and the support was washed with dry acetonitrile. The teflon was
`
`placed in a DNA synthesis column. The oligonucleotide was synthesised as described above. The terminal Fmoc group
`
`was removed by treating with 20% piperidinefDMF for 15 minutes. The oligonucleotide was deprotected by treating with
`
`ethylenediaminelabsethanol, Ill at 55°C for 1 hour. The Boc groups on the lysine conjugate were removed by treating
`
`with 90% TFA)'ethanedithiol for 5 minutes". The teflon was washed with acetonitrile and 20% Et,NIDCM then again
`
`with acetonitrile. The conjugates were removed from the support by treating with sodium periodate as described above.
`
`The DNA-(Trp), product was purified by reverse phase HPLC to yield pure conjugate (1.8 0.D., 1.3% overall
`
`yield). HPLC conditions: Conjugate retention time of 47.2 min., DNA retention time of 24.6 min., using the same HPLC
`
`column and conditions as above. U.V. data (H20) was obtained for the Trp conjugate. 7\,,_,_ 256.0 nm. with a shoulder
`
`at 280 nm. ).,,,i,, 235 nm.
`
`The DNA—(Lys), product was purified by DEAE ion exchange HPLC to yield pure conjugate( 12 0.1)., 8.595
`
`overall yield ). HPLC conditions: Conjugate retention time of 38.4 min., DNA retention time of 42.5 min., column,
`
`Protein-Pak, DEAE SPW, Waters. analytical: gradient 0-100% of B in 100 minutes at a flow rate of 1.0 tnllmin. Solvent
`
`A = 25 mM Tris pH 7.5, Solvent B = 20% acetonitrile in 25 mM Tris, 1 M NaCl pH 7.5. The conjugate was desalted
`
`on a sephadex G-25 Fine column. U.V. data (H20) was obtained for the Lys conjugate. Am 256 nm., R,-_ 237 nrrl.
`
`Amino acid analysis of this conjugate gave lysine and glycine peaks. The glycine comes from hydrolysis of the
`
`uligonucleotide.
`
`The DNA peptide conjugates were run beside the oligonucleotide itself on a 15% pnlyacrylarnide gel containing
`
`7 M urea (Figure 2). ‘The Z-D-Phe-L—Phe—Gly conjugate and (Trp), conjugate run slower than the oligonucleotide. The
`
`(Lys), conjugate does not migrate on the gel due to the positively charged lysine residues. The DNA was visualized on
`
`the gel by U.V. shadowing.
`
`The conjugate was shown to be resistant to snake venom phosphodiesterase under conditions (0.8 0.D. of
`
`oligonucleolide, 0.1 unit enzyme, 0.1M Tris—HCI pH 8.9, 0.1 M l'~laCl, 0.014 M MgCl,, 3? “C,
`degraded the tmconjugated oligonucleotide (data not shown).
`
`1 hour) which severely
`
`Page 5 of6
`
`
`
`Page 5 of 6
`
`

`

`882
`
`In conclusion the results describe a facile synthesis starting directly with commercially available reagents of
`
`oligonucleotide-peptide conjugates with potential biological activity against
`measles virus.
`
`We would like to thank Sylvie Bilodeau for obtaining the NMR
`
`spectra and Bernard Gibbs for the amino acid analysis.
`
`Refergngfi
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`to.
`
`11.
`
`12.
`
`M. Rothenberg, G. Johnson, C. Laughlin, 1. Green, I. Cradock, N.
`
`Sarver and J.3. Cohen, J. Nat. Cancer Inst., 31, 1539-1544 (1989).
`
`C.A. Stein and LS. Cohen.
`
`, pp. 79-97 i_n Important Advances in
`
`Oncology 1989, V.T. devita 11-., S. Hellman and S.A. Rosenberg
`
`eds., Lippincott
`
`(1989).
`
`C. Helene and J .1. Toulmé, Biochim. Biophys. Acta, 1&2, 99-125
`
`(1990).
`
`A.S. Boutorin, L.V. Gus‘kova, E.M. Ivanova. N.D.Kobetz, V.F.
`
`Zarytova, A.S. Ryte, L.V. Yurchenko and V.V. Vlassov, FEES
`
`I..ett.. 251. 129-132 (1959).
`
`R.G. Shea, .l'.C. Marsters and N. Bischotberger, Nucleic Acids Ru,
`
`l_E, 3777-3783 (1990).
`
`Lemaitre, M., Bayard, 8., Lebleu, B. (I987) Proc. Natl. Acad. Sci.
`
`Figure 2.
`Lane 1 + 7, Bromphenol Blue and
`USA E’ 648652‘
`I. Haralambidis, L. Duncan, K. Angus and G.W. Tregear, Nucleic Xylene Cyano1_
`Lane 2 + 5
`Acids Res" ‘I-8-’ 493499 (1990)
`oligonucleotide 17-mer. 1.3.11.6 3 2-D-
`_
`_
`_
`Phe-L-Phe-Gly conjugate.
`Lane 4
`C. 1). Richardson, A. Scheid, A.and P.W. Choppm, Virology 1_0j_,
`(vrrph mnjugam [me 5 (Lysh
`205-222 (1980).
`conjugate. 0.8 0.D. of sample loaded
`1=.s. Mi1|er,M.P. Reddy, A. Murakami, K.R. Blake, S.B. Lin, and W "”°"'
`C.H. Agris. Biochemistry 25, 5092-5097 (1986).
`
`R. Lohrmann and J. Ruth, DNA 3, 122 (1934).
`
`EJ. Gal:-bay. K. Sanford, and C.S. Baxter, C.S., Biochemistry _l_1, 3429-3435 (1972).
`
`J. Haralambidis, M. Chai and G.W. Tregear, Nucleic Acids Res. LE, 4857-4876 (1937)
`
`(Received in USA 24 September 1990)
`
`Page 6 of6
`
`
`
`Page 6 of 6
`
`