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`ILMN EXHIBIT 1033
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`Page 1 of 10
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`ILMN EXHIBIT 1033
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`

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`
`
`TheJournalllofic
`Chemistry
`
`EDITOR-IN-CHIEF
`
`CLAYTON I-I. I-IEATHCOCK
`Department of Chemistry
`University of California
`Berkeley, California 94720
`
`SENIOR ITORS
`Peter Beak
`University of Illinois. Urbano
`Robert M. Coates
`University of Illinois. Urbano
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`University of Utah
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`University of California, Berkeley
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`University of California. Berkeley
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`The Pennsylvania State University
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`Indiana University
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`

`
`
`
`J. Org. Chem. 1991, 56, 6659-6666
`
`6659
`
`was removed and rotary evaporated and the residue Kugalrohr
`distilled to give 120 mg (4%), bp 115-120 °C. ‘H NMR: 5 1.31
`(d. 3 H). 2.53 (In. 1 H). 2.6-1(m, 1 H). 3.17 (m. 2 H).
`I-Methyl-.1-methylenecyclobutane (6). Dimethyl sulfoxide
`(distilled from CaH,) was stirred under Ar for 30 min. Me-
`thyltriphenylphoephoniurn iodide (1.45 g, 3.6 mmol) was added
`and the reaction stirred for 2 h. 3-Methylcyclobutanone (120 mg,
`3.6 mmol) was dissolved in 2 mL of DMSO and added to the
`reaction via syringe. A cannula was connected from the reaction
`flask to a cold trap cooled in dry ice-acetone. A small amount
`of liquid was collected, and NMR and GC—MS were taken. 1H
`NMR.:'3 6 1.13 (d. 3 H). 2.24 (d. 2 H), 2.36 (In, 1 H), 2.80 (In, 2
`H), 4.71 (d. 2 H). GC-MS showed M“' at m/e 82.
`Metl|ylhieyclo[l.l.l]pentano (B).‘’' To crude 7 (5.1 mmol.
`1.1 g) in a round-bottomed flask that had been flushed with N,.
`cooled to -15 °C. and protected from light with Al foil was added
`thiophenol (10 mL. 0.1 mol) via syringe. The foil was removed
`and the stirred solution irradiated with a 300-77 tungsten Flament
`lamp for 30 min. A cannula was connected from the reaction flask
`to a cold trap cooled to -78 °C. and the reaction mixture was
`warmed to 70 °C. A small amount of clear distillate was collected
`
`(13) Della. E. W.; Pigou. P. E. J. Am. Chem. Soc. 1984, 106, 1035.
`
`‘H NMR:‘ 2: 1.1 (s. 3 H),1.66(s,6 H). 2.48 (1
`(200 mg, 50%).
`H. s). GC-MS showed M* peak at rn/e 82.
`
`Acknowledgment. The authors wish to thank Prof.
`Josef Mich] and Mr. Piotr Kszynski for the generous gift
`of a sample of bicyclo[1.l.1]pentylacetic acid as well as
`many helpful discussions. This work was supported by
`NSF Grant DMB 8717730. The GE QE-300 NMR used
`in this work was purchased with funds provided by NIH
`RR 02336 and NSF CHE 8411177. The VG ZAB-2F!-IF
`MS and VG 12/ 253 Quad MS instruments used in this
`work were purchased with funds provided by NIH RR
`03001, NSF DMB 8414627, and the M. J. Murdock
`Charitable Trust.
`Registry No. 3, 136379-2l~0; B, 15189-13-1: 7, 136379-22-1'.
`3, 10555-48-3'. 1-iJicyclo[1.l.llpentylacetic acid, 131515-31-B;
`.N-hydroxy-2-(1-H)-Pyridinethione. 1121-30-3; 2-(2-rnetl1yl-3-
`chloropropyl)-1.3-dithiane. 53198-70-2; dithisne, 51330-42-B; 1-
`bromo-3-chloro-2-methylpropane, 6974-77-2; 3-rnethylcyclo-
`butanone trimethylene thioketal, 136379-23-2; 3-methylcyclo-
`butanone, 1192-08-1; methyltriphenylphosphonium iodide.
`2065-66-9.
`
`Direct Cleavage of Peptides from a Solid Support into Aqueous Buffer.
`
`Application in Simultaneous Multiple Peptide Synthesi‘
`
`Andrew M. Bray. N. Joe Maeji,* Robert M. Valerio, Rhonda A. Campbell, and H. Mario Geysen
`
`Chiron Mimotopes Pty. Ltd., P.O. Ba: 40, Clayton, Victoria. 3168. Australia
`
`Received May 20, 1991 [Revised Manuscript Received July 29, 1991')
`
`A method of simultaneous multiple peptide synthesis which integraus synthesis, side-chain deprotection, cleavage.
`and pu.ri.fication so as to afford peptide solutions suitable for immediate biological testing is described. The approach
`utilizes a novel diltetopiperazine-forming cleavable linker 1. Upon side-chain cleprotection. 1 gives 2, which is
`stable to a protocol designed to remove contexninants from the support-bound peptide prior to cleavage. Peptide
`cleavage is then effected by treating 2 with a neutral or near neutral buffer to give peptide 4, which carries a
`C-terminal diketopiperazine moiety, in good yield. In this study the glycolarnido and 4-loxyniethyllbenzsmido
`esters of 1 have been appraised. The approach is demonstrated in model studies on 7 and 8 and in the preparation
`and characterization of peptides 17-21. The general approach allows 10-100-nmol quantitim of many hundreds
`of peptides to be concurrently prepared in a relatively short period of time when used in conjunction with the
`multipin method of multiple peptide synthesis.
`
`Introduction
`
`Growth in the demand for synthetic peptides has been
`partly addressed by a range of techniques facilitating rapid
`peptide synthesis through parallel
`Simultaneous
`multiple peptide synthesis has been performed on resin,“
`cellulose” and grafted polyethyleneH° or polypropylene”
`
`(1) Preliminary communication: Bray. A. Mr. Maeji. N. J.; Geysen. H.
`M. Tetrahedron Lett. 1990. 3.‘. 5811-5314.
`(2) Hougliten. R. A. PPIIC. NEH. cloud. Sci. U.-3.5.. 1985. 32, 5131-5135.
`(3) Wolfe, H. R. Manual to RIMPS multiple peptide aynfliesis system,
`DuPont/NEN Co.. Wilmington. DE. 1987.
`(4) Tjoeng. F. S.; Towery, D. 5.: Bulock, J. W.: Whipple. D. E.-, Fok.
`K. F.; Williams, M. H.;Z1.|pec. M. E...‘ Adams, S. P. Int. J. Pepi. Protein
`Res. 1990. 35. 141-146.
`(5) Hudson. D. J. Org. Chem. 1988, 53. 817-624.
`(5) Frank. 11.; During, 11. Tetrahedron 1988. 44, 6031-6040.
`(7) Krchnak. V.; Vsgner, J.; Novac. J.'. Suchsukova. A.: Rouba]. J.
`Anal. Biochem. 1990. 1'59. B0"B3.
`(B) Geysen. H. M.; Meloen, R. H.: Bsrteling. S. J. Proc. Natl. Acad.
`Sci. U.S.A. 198-1.. 81'. 3998-4002.
`
`supports. Despite the speed at which peptides can be
`assembled by a parallel synthesis strategy, the need for
`individual handling at the side-chain deprotection, cleav-
`age, and purification steps limits the number of peptides
`that can be conveniently prepared. Several methods of
`overcoming the postsynthesis bottleneck have been pro-
`posed. For example, closely related peptides can he syn-
`thesized on the same support and subsequently separated
`by HPLC.‘ Purification and characterization must be
`straightforward. however. if this method is to succeed. The
`use of specialized apparatus designed for multiple peptide
`cleavage simplifies cleavage and side-chain deprotection”
`
`(9) Geysen. H. M.; Rodda, 3. J.; Mason, '1'. J.; Tribbick, G.; S-ciroofa.
`P. G. J. hnmunol. Methods 1987. 102. 259-274.
`(10) Berg. R. I-1.; Alrndal, K.; Pederaen, W. B.; Holm, A4 Tarn. J. P.;
`Merrifield. R. B. J. Am. Chem. Soc. 1989. In‘, 3024-8026.
`(11) Daniels. S. B.: Bernatowice. M. S.; Coull. J. M.; Koster. H. Tet-
`mhedran Lett. 11189. 30. 4345-4348.
`(12) Hougliton. R. A.; Bray, M. K.; Degraw, S. T.; Kirby, C. J. Int. J.
`Pept. Protein Res. 1985, 27, 673-678.
`
`0022-3263 f9l/ 1956-6659$02.50/ 0 © 1991 American Chemical Society
`
`Page 3 of10
`
`
`
`Page 3 of 10
`
`

`
`6660 J. Org. Chem, Vol. 56, No. 23, 1991
`
`Scheme 1. Peptide Cleavage via Diketopiperasine
`Formation
`
`F'EDtide- NH
`
`FaDtida- NH
`
`Boo-NH
`
`N
`
`HM
`
`N
`
`°"""
`
`HO-Pin
`
`II DH I IIJFIII ifldl
`
`Peptida- NH
`
`Peptide-NH
`
`1ll'IArIVIIlUIDTl fin-1| uflhtnulflf
`T
`4-
`DUI‘!wuss El
`
`|-|3N
`
`---—-I>
`
`H2N
`
`N
`
`0 - Pin
`
`0
`
`0-Pin
`
`but does not obviate the need for individual peptide pu-
`rification. The multipin approach of Geysen” bypassed
`the need for individual handling by synthizing peptides
`on plastic pins arranged to complement the 96-well mi-
`crotitre plate used in enzyme-linked immunosorbent assay
`(ELISA). By using the same support for both synthesis
`and testing, the screening of large numbers of peptides by
`ELISA was greatly simplified. Peptides bound to a solid
`support, however, have limited application. Until re-
`cently)” no method of simultaneous synthesis integrated
`peptide assembly, side-chain deprotection, cleavage, and
`purification so as to allow immediate biological testing.
`An integrated approach requires that the following
`strategy he adopted. Side-chain deprotection and peptide
`cleavage must be distinct processes, separated by a rigorous
`washing protocol designed to remove all organic contam-
`inants, which may prove toxic in subsequent assays.
`Peptide purity is then dependent solely on the efficiency
`of peptide synthesis and side-chain deprotection. The
`second requirement is that side-chain-deprotected peptides
`can be cleaved directly into a medium compatible with
`their final use. Generally, this would be aqueous. Po-
`stcleavage handling is then minimal and a fully integrated
`method for the simultaneous handling of large numbers
`of discrete peptides becomes viable.
`A linker which meets the dual requirement of stability
`under the conditions of peptide synthesis and side-chain
`deprotection and lability under biologically compatible
`conditions has been designed. As shown in Scheme I, a
`diketopiperazine-forming linker (I) is interposed between
`the solid support and the target peptide. Side-chain de-
`protection primes the linker for cleavage with the forma-
`tion of ammonium salt 2. Provided the assembly remains
`in the protonated form, it is stable to washing steps in
`organic solvents, aqueous,’ organic solvents. and low pH
`buffers. With the equilibrium generation of 3 in neutral
`aqueous buffer, cyclization, and hence cleavage to give 4,
`proceeds with good efficiency. The resulting peptides carry
`a diketopiperazine moiety at the C-terminal.
`In this study, the strategy outlined above has been ex-
`plored in conjunction with the multipin method of peptide
`
`(13) Maeji. N. J.: Bray. A. M.; Geyaen, H. M. J. Immunol. Methods
`mo, :34, 23-33.
`
`Page 4 of10
`
`Bray at a].
`
`synthesis. Studies on model system 1, where “Peptide”
`= (2,4-dinitrophenyl)-5-alanine (Dnp-flAla), show that l
`is stable to the conditions of peptide synthesis. 2 is stable
`to the precleavage washing protocol, and cleavage proceeds
`rapidly and efficiently in pH 7 phosphate buffer. The
`utility of the approach has also been demonstrated with
`the synthesis and characterization of a selection of test
`peptides. This approach has: allowed us to concurrently
`prepare thousands of discrete solution-phase peptides in
`a matter of weelra.‘~‘3 As the peptides are produced in the
`10-100-nmol range, they may be characterized by con-
`ventional techniques. These quantities are adequate for
`many biological, immunological, and pharmacological ap-
`plications. The peptides are suitable for applications where
`native C-termini are not required.
`
`Results
`
`Design and Preparation of Diketopipera2ine-
`Forming Handle 1. A cliketopiperazine-forming cleavable
`linker such as 1 has two requirements: a C-terminal N-
`alkylamino acid and an amino acid with a side-chain
`functional group upon which peptide synthesis can be
`performed. Rather than proline, another N-alkylamino
`acid such as sarcosine could have been used. The rate of
`diketopiperazine formation is, however. dependent on the
`N-alkylamino acid.“ Lysine was chosen as the second
`residue so that peptide synthesis could be continued on
`the side chain. The protecting groups in l were arranged
`for use with N"-Fmoc-protected amino acids with acid-
`labile side-chain protection. The stability/lability prop-
`erties of the linker can also be expected to be sensitive to
`the type of ester linkage to the solid support.“-1*‘
`In early
`work,” 1 was assembled on the side chain of serine. In this
`study linkers 5 and 6 based respectively on the glycol-
`amido" and 4-(oxymethyl)benzamido“ esters of lysyl-
`proline have been investigated.
`X
`
`Boo-Lira-Pro-O-Y-CO-Pin
`
`~NH
`
`Dnp-flAIa
`
`Rather than asseble the linker in piecemeal fashion
`a was done in earlier work,“ the ester bond was formed
`prior to coupling to the pin surface, hence bypassing the
`need to perform a moisture-sensitive dicyclohexylcarbc-—
`diimide [DOC]/4-ldimethylaminolpsridme (DMAP) me-
`diated coupling in an open container. Linker 5 was as-
`sembled onto the pins in two coupling step using pre-
`formed ester H and Boc—Lys(Fmoc)-OH. The dicyclo—
`hexylamine (DCHA) salt of compound 11 was prepared
`in 91% overall yield as shown in Scheme 11. The linker
`assembly was simplified further by incorporating 6 onto
`the pin in one step using dipeptide ester 16. An overall
`yield of 64% was achieved in the i'1ve—step synthesis of 16,
`presented in Scheme III.
`Stability and Cleavage Studies. As thousands of
`peptides are handled simultaneously, the method of syn-
`
`H4) Rothe. M.; Mazanslr, J. Liebigs Am. Chem. I974. 439-469.
`(15) Giralt. E.: Eritja, 11.; Pedroeo, E. Tetrahedron Letl. I981. 22.
`3779-3732.
`(16) Pedroso. E.; Grandma, A.; do In Hens, X4 Eritja. IL: Girllli. R.
`Tetrahedron Lett. 1985, 27, 743-746.
`22-2 .
`(131 Baleux, F.: Calaa. B.:M¢1'3F. J. Int. J. Pept. Protein Res. 1936. 3.
`
`
`
`
`
`Page 4 of 10
`
`

`
`Direct Cleavage of Peptides from a Solid Support
`
`J. Org. Chem, Vol. 56, No. 23, 1991 666!
`
`Scheme II. Synthesis of Compound 11 (Pac = phenacyl {CH,COC.H.)l
`Frnao-Pro-DH!
`DGCJDMAPICHZCIZ
`
`1lHI O
`2] DCHN'El20lplIrol
`
`HOCI-i2C02Pac en-
`
`Fmoc-Pro-OCH2C02Pac —--—- Fmoc-Pro-OCH2C02H.DCHA
`
`'10
`
`11
`
`AmountofPepnde
`
`CleavedperPin(nmol)
`
`
`
`Percentcleaved
`
`owcowcnwcnwenntnweowcn
`1
`'2
`I
`I
`5
`!
`
`60
`
`90
`
`P20
`
`190
`
`-hC
`U0'
`
`S I
`
`-.l C3
`
`U!
`
`AmountofPeptide
`
`FlemainingonPin(nmol)
`
`Time (min)
`Figure 2. Cleavage of H-Lys(Dnp-6Ala)—Pro-OCl-l3C0-pin with
`0.1 M pH 7 phosphate buffer. Each data point is an average of
`30 pins.
`
`50
`
`o?
`is
`as
`§s
`5;
`
`Cycle
`Figure I. Stability of Boo-Lys(Dnp-,8Ala)-Pro-0CHgCO-pin (7)
`during peptide synthesis. After each discrete step six pins were
`removed and cleaved with 0.25 M NaOH. and the amount of
`material cleaved from the pin was determined. D, deprotection
`with 20% piperidine in DMF‘; W, wash with MeOH; C, dummy
`coupling with Fmoc-Gly-OH/DCC HOBE/DMF; A. acetylation;
`SCD. side-chain deprotection wit TFA/Ph0HjHS(CH,),SH
`(95:2.5:2.5, v wfv); SON; sonication in 0.1% l-{Cl in MeOH/H20
`(lzl, v,/V); p 3, precleavage soak in pH 3 buffer; pH'i', cleavage
`with pH 7 buffer.
`
`thesis used in multipin synthesis deviates. by necessity.
`from conventional solid-phase peptide synthesis Coupling
`reactions are performed for 16 h under nonanhydrous
`conditions. MeOH is used in postcoupling and postde—
`protection washing steps and washes are performed in open
`containers. Although these conditions appear not to
`compromise peptide quality, the stability of the ester link
`to these conditions was unknown. Hence the stability of
`the glyoolamido and -1-(oxymethynhenzarnido ester groups
`were reassessed using the model systems 7 and 8 to dem-
`onstrate compatibility with the Frnoc synthesis strategy
`as applied to pins. A stability study performed on 7 is
`summarized in Figure 1. One hundred and fifty-six de-
`rivatized pins were subjected to a mock heptapeptide
`synthesis in which six pins were removed for analysis
`followed each discrete step. Analysis comprised tri-
`fluoroacetic acid (TFAI treatment of the pins followed by
`cleavage into the wells of a microtitre plate with 0.25 M
`NaOH (3 h) and subsequent spectrophotometric deter-
`mination of the cleavage solutions. Less than 5% of the
`model system was lost from the pin surface over seven
`deprotection/wash/coupling cycles. In practice, occasions
`have been found where inadvertant cleavage of the gly-
`colamido linker 5 has occurred during peptide synthesis
`(unpublished results). In contrast, the benzamido linker
`6 has superior stability. Sporadic loss of this system from
`the pin through synthesis has not been encountered; con-
`sequently 6 has been adopted for most of our routine work.
`A two-step post-side-chain deprotection protocol was
`developed earlier for use with s diketopiperazine-forming
`cleavable linker assembled on the side chain of serine.”
`The protocol was designed to afford nontoxic peptide so-
`
`IDO
`90
`80
`"JD
`60
`50
`40
`
`
`
`Percentcleaved
`
`30
`20
`10
`0
`210
`
`I20
`
`150
`
`160
`
`Tirna (min)
`Figure 3. Cleavage of H-Lys[Dnp—.6A|a)-Pro-0CHgC¢,H.C0—pin
`with 0.1 M phosphate buffer (pH 7). Each data point is an average
`of 28 pins.
`
`lutions free of organic contaminants upon cleavage. So-
`nication of the pins in 0.1% HCl in MeOH-water effec-
`tively removes scavengers and byproducts arising from
`side-chain deprotection. Counterion exchange of tri-
`fluoroacetate for chloride also occurs in this step. MeOH
`and any remaining organic contaminants are then removed
`in a subsequent low pH aqueous buffer soak. Previously,‘3
`a 16-h soak in 0.1 M pH 5 phosphate buffer was advocated.
`Alternatives to this procedure have been examined in order
`to reduce the risk of inadvertent. peptide cleavage. A 1-h
`pH 5 soak has been employed but is not favored in the
`preparation of peptides for use with sensitive-cell mediated
`assays. The preferred method and the primary one used
`in this study is a 5-h soak in pH 3 citrate—phosphate buffer.
`The rate and efficiency of the buffer-mediated cyclica-
`tion—cleavsge step was also investigated. Figure 2 shows
`the results of a time trial cleavage study performed on 30
`pins derivatized with model system 7. Following treatment
`with TFA, sonication, and a 1-h precleavage soak in pH
`
`
`
`Page 5 of 10
`
`

`
`6662 J. Org. Chem, Vol. 56', No. 23. 1991
`
`Bray et al.
`
`Schema III. Synthesis of Compound [6
`
`Boo-Pro-OH!
`
`DCCIDMAPICP-I2C|2
`
`1) TFA
`2] HCUIHOH
`
`H0m2 Pac —. Boc-Pro-OCH2-@-CO2Pac ——- H-5-ro-ocH2<:>-co-2Pac.HcI
`13
`14
`
`12
`
`Boo-Lys1Fmoc|-OH!
` F
`
`hfMO O
`
`Boc—Lys(Fmoc]-Pro-0CH2-©-C02H -— Boo-Lys(Frnoc)-Pro-OCH2-©-CO2PaO
`15
`16
`
`S
`
`100
`3
`
`Table I. Peptides Used in Method Appraisal
`amount
`cleaved‘
`lnmol)
`31
`
`positive ion FAB MS
`data’ (m/z)
`1309, [M — H + 2Na]*;
`1287, [M + NaI*
`1336. {M + Na]*
`
`sequence
`17 Ac-ASQGGLEDPN
`6Ala-eycIo{KP)
`18 Ac-PGPSDTPILF
`Bhla-cyclo(K.P)
`19 Ac-VQAAIDYING-
`,8Ala-cyclo(KP)
`20 Ac-WEIPEPYVWD-
`,8Ala-cyclo(KP)
`2| Ac-SYSMEH FRWG-
`.6Ala~cyclo(KP)
`“Analysis performed on the cleavage product from a single pin.
`‘Not obtained in 20 and 21.
`
`-~ 40
`3
`0 5 J5
`o '3 30
`
`fig
`'6 lg 35
`E3
`20
`g; ..
`
`10
`5
`o
`n
`
`so
`
`10 E
`an
`in
`
`.. 2
`
`so
`10
`o
`210
`
`so
`
`so
`
`so
`
`no
`
`150
`
`use
`
`Time (min)
`Figure 4. Cleavage of H-Lyannmsala)-Pro-ocH,c,H.co-pin
`with 0.1 M Nal-ICO3 (pH 8.3). Each data point is an average of
`28 pins.
`
`5 buffer, each pin was immersed in 150 nL of a 0.1 M pH
`7 phosphate buffer solution within a 96-well microtitre
`plate. Absorbances of the cleavage solutions were read
`every 10 min over a 3-h period. Within 3 h, 70% cleavage
`was achieved, yielding 150 ,uL of a 0.17 mM solution of
`cleaved peptide per pin. Two similar studies. summarized
`in Figures 3 and 4, were performed on model system 8. In
`these studies the alternative 5-h, pH 3 precleavage soak
`was used. Twenty-eight pins were treated with 0.1 M pH
`7 phosphate buffer for 3 h, after which time 64% of the
`available peptide was cleaved. Twenty-eight pins were
`cleaved with 0.1 M NaHCO,, to demonstrate the use of
`other near neutral cleavage media.
`In this case 79%
`cleavage was effected after 3 h. Cleavage with 0.25 M
`NaOH for 3 h removed all of the color from the pin surface
`and was taken to be 100% cleavage. This was confirmed
`by amino acid analysis on the cleaved pins. When water
`was substituted for the cleavage buffer, less than 2%
`cleavage was observed at 3 h.
`Application in Peptide Synthesis. Five decapeptide
`sequences, shown in Table I, were selected for critical
`appraisal of the cleavage method used in conjunction with
`the multipin method of peptide synthesis. Peptides were
`prepared on pins derivatized with the linker system 5. A
`.6—alanine (,6Ala) spacer was included between the linker
`and target peptide. The peptide set included two common
`test sequences, acyl carrier protein (ACP) 65-74“ 19 and
`
`Page 6 of10
`
`66
`
`-10
`
`40
`
`18
`
`1428. [M - H + 2Na|*;
`1406. [M + Na!‘
`
`sdrenocorticotrophic hormone (ACTH) 1-10“ 21, which
`are widely used in the appraisal of automated peptide
`synthesizers. The peptides were synthesized using an
`Fmoc synthesis protocol, where couplings were effected
`using DCC/1-hydroxybenzotriazole (I-IOBt) in dimethyl-
`formamide (DMF). Following N-terminal capping by
`acetylation, the peptides were sidechain deprotected with
`TFA/phenol/ethanedithiol (EDT). Following sonication
`and a 5-h precleavage soak in pH 3 buffer, the peptides
`were cleaved from the pins into 150 aL of 0.1 M pH '7
`phosphate buffer in the wells of a microtitre plate.
`Peptides were examined by reverse phase HPLC, amino
`acid analysis. and in selected cases positive ion FAB mass
`spectrometry. Figure 5 presents chromatograms of four
`of the test peptides recorded at 214 um. None of these
`peptides were subjected to prior purification. In each case
`a single major peak was observed. In all cases a minor peak
`due to phenol, one of the scavengers used in side-chain
`deprotection, was observed at £3 15.77 min. The methio-
`nine-containing peptide 21 was obtained in both the oxi-
`dized a.nd reduced forms. Aerial oxidation of the solution
`in the presence of base afforded a product of in 18.13 min.
`Amino acid ratios were determined for all five cleaved
`peptides and are presented in Table II. Analyses were
`performed on solutions of high salt content and low pep-
`tide content, nonideal conditions for peptide hydrolysis
`
`(13) Atberton, EL: Logan. C. J.: Sheppard, R. C. J. Chem. Soc., Perkin
`I Trans. 1981, 538-546.
`(19) Bergot. B. J .; Noble. R. L: Geiser. T. User Bulletin No. 16. Pep-
`tide Synthesizer, Appliecl Biosynterns, Inc., 1986.
`
`
`
`Page 6 of 10
`
`

`
`Direct Cleavage of Peptides from a Solid Support
`
`J. Org. Chem. Vol. 56, No. 23, 1991 6663
`
`Table II. Amino Acid Analysis of Peptides Cleaved into
`0.1 M Phosphate Buffer (pH 7.0)
`ratios‘
`19
`1.3 (2)
`1.6 (2)
`0.7 (1)
`
`20
`
`21
`
`0.8 (1)
`1.3 (2)
`
`119(1)
`1.0 (1)
`0.8 (1)
`0.9 (1)
`
`1.1 (1)
`
`1.1 (1)
`1.8 (1)
`1.0 (1)
`1.1 (2)
`
`1.0 (1)
`1.1 (1)
`1.4 (1)
`
`amino acid
`A
`D
`E
`F
`G
`H
`1
`K’
`L
`M.
`Pl’
`R
`S
`T
`V
`W
`Y
`Mile’
`
`17
`2.0 (2)
`0.7 (1)
`2.1 (2)
`
`18
`
`0.7 (1)
`
`1.4 (2)
`
`0.6 (1)
`
`0.9 (1)
`
`0.7 (1)
`1.2 (1)
`
`1.0 (1)
`0.7 (1)
`1.0 (1)
`
`1.9 (2)
`1.1 (1)
`
`1.0 (1)
`1.2 (I)
`
`3.0 (2)
`
`6.5 (5)
`
`1.6 (1)
`
`3.? 93)
`
`0.6 (1)
`
`0.6 (1)
`0.8 (1)
`
`1.3 (1)
`
`1.1 (1)
`
`0.9 (1)
`
`1.0 (1)
`1.5 (1)
`
`0.8 (1)
`1.8 (2)
`0.8(1l
`1.1 (1)
`
`‘Values in parentheses are expected values. ‘Residues in linker.
`
`and phenyl isothiocyanate (PITC) derivatization.3°'“
`Nevertheless, the analysis results give ratios consistent with
`the target sequences. The quantity of peptide cleaved from
`the pin support is listed in Table 1. Typically, cleavage
`solutions contain between 20 and 60 nmol of peptide.
`Variation in the individual loading of the pins reflects the
`discrete nature of this solid support. The identity of three
`of the peptides was further confirmed by positive ion FAB
`mass spectrometry; the data are included in Table 1. Due
`to the high sodium concentration of the cleavage solution,
`[M + Na]" rather than [M + H]* signals were observed.
`
`Discussion
`
`In general, peptides prepared _by conventional solid-
`phase synthesis strategies are side-chain deprotected and
`cleaved from the solid support in a single step. This ne-
`cessitates individual purification of crude peptide. usually
`by preparative HPLC. Once pure, the peptide is further
`handled in preparation for biological assay. This type of
`approach is not practical when handling small quantities
`of thousands or even hundreds of peptides. The challenge
`is to devise strategies where large numbers of peptides can
`be synthesized, side-chain deprotected, purified, cleaved,
`and presented for biological testing in a simultaneous
`fashion.
`The propensity of resin-bound dipeptide esters with a
`C-terminal N-alkylamino acid residue to undergo intra-
`rnolecular aminolysis was recognized in the early 19703.99“
`Diltetopiperazine formation is a side reaction where
`quantitative loss of peptide from resin can occur under
`very mild conditions. However, provided the deprotected
`dipeptide remains protonated as in 2, it is relatively stable.
`As the rate of cyclization is dependent on the N-allrylamino
`acid“ and on the type of peptide~ester linkage to the solid
`support,”«“’ there is scope to tune the reaction to a desired
`set of cleavage conditions. Though regarded as a problem
`reaction, it satisifies the criteria required for a buffer-labile
`cleavsge reaction.
`
`(20) Cohen, S. An, Stryclom, D. J. Anni. Biochem. 1938. I74, 1-6.
`(21) Inglis. A. 3.; Ba:-tone. N. A.; Finlayscm, J. R. J. Biochem. Binphys.
`Methods 1988. .15, 219-254.
`(22) Gisin, B. F.; Msrrifield, R. B. J. Am. Chem. Soc. 1912. 94.
`3102-3106.
`(23) Khosla, M. C.: Smeby. R. FL: Bumpus. F. M. J. Am. Chem. Soc.
`1912. .94, 4721-4724.
`(24) Rothe. M.; Msnnek, J. Angew. Chem, Int. Ed. Engi. 1972. H.
`293.
`
`Page 7 of10
`
`Figure 5. HPLC chromatograms of peptides cleaved into 0.1 M
`pH 7 phosphate buffer. Each trace was performed on 80 .uL of
`the cleaved peptide solution. Detection at 214 nm. Solvent A,
`H20 (0.1% TFA); solvent B. 60% CH3CN (0.1% TFA). Linear
`gradient A to B from 5 min to 20 min. Column: Merck LiCh-
`rosphere 100 RP-18 5 pm.
`
`Using the lysylproline assembly presented in Scheme I,
`cyclization, and hence cleavage, could be triggered by a pH
`7 buffer solution. In practice, the cleavage properties of
`5 and B were found to be similar. In preliminary work,
`where the cleavable linker was assembled on the side chain
`of serine. cleavage was shown to take place in phosphate
`buffer solutions of pH 6 to pH 8 over a range of ionic
`streng1‘..hs.13 Other near neutral buffer solutions have also
`proved useful. As expected,” cyclization was found not
`to occur to an appreciable extent during post-side-chain
`deprotection steps, where the assembly 2 was maintained
`in a protonated form. This is the first described integrated
`
`
`
`Page 7 of 10
`
`

`
`66641 J. Org. Chem, Vol. 56, No. 23. 1991
`
`Bray et al.
`
`hydrin in Me0H/AcOH (99:1.l or by viewing under 254-um UV
`light as appropriate. DMF was vacuum distilled from ninhydrin.
`Ether was distilled from sodium]benzophenone. EtOAc, TFA.
`and CH,C1, were distilled. Dioxan, Ac0H, MeOH, and petroleum
`spirits (40-60 °C fraction, hereon refered to as petrol) were of
`AR grade. Et,,N and N-methylmorpholine (NMM) were distilled
`from calcium hydride. DCC, HOBt, DCHA, phenacyl bromide
`(Pac-Br), phenol, and EDT were from Fluka. Switzerland. DMAP
`and 2,4-dinitrofluorobenzene (FDNB) were purchased from the
`Aldrich Chemical Company. Milwaukee. WI.
`4-(Hydroxy-
`methyllbenzoic acid (HMB). Boo-L-Lys(Fmoc)-O1-I, Boc-1.-Pro-
`OH. and Fmoc-protected amino acids were from Novabiochem,
`Switzerland. Glyeolic acid (Glyc) was from Sigma, St. Louis, MO.
`Solutions were dried using anhydrous Na2SO,.
`Phenseyl Glycolato (9). Et,N (14.0 ml’... 100 mmol) and
`Pat:-Br (20.0 g, 100 mmol) were added to a stirring suspension
`of glyeolic acid (7.6 g, 100 mmol) in Et0Ae (300 mL). After 20
`h, the solution was diluted with warm Et0Ac (300 mL) and
`extracted with war