`
`Page 1 of 10Page 1 of 10
`
`
`
`ILMN EXHIBIT 1032ILMN EXHIBIT 1032
`
`
`
`Page 1 of 10
`
`ILMN EXHIBIT 1032
`
`

`

`1A
`
`JOURNAL
`
`OF THE AMERICAN CHEMICAL SOCIETY
`
`
`EDITOR
`
`MARSIIALL GATES
`
`ASSISTANT EDITORS
`
`R. L. AUTREY
`
`FRED BASOL0
`
`A. B. F. DUNCAN
`
`E. H. Srorz
`
`SENIOR PRODUCTION EDITOR
`
`CHARLES R. Bmrrscrr
`
`EDITORIAL BOARD
`
`ROBERT A. ALBERTY
`V. BOEKELHEIDE
`Jomv M. BUCHANAN
`CLAYTON F. CALLIS
`BRYCE CRAWFORD, J1:.
`PAUL DELAI-KAY
`H. S. Gurowsmr
`
`WILLIAM H. HAMILL
`WILLIAM S. JOHNSON
`HAROLD S. Jonusron
`HANS B. JONASSEN
`H. Gosnm KHDRANA
`Nnrson J. LEONARD
`
`WILLIAM N. LIPSCCIM13
`L. G. Lorqcswonrn
`LE0 MANDELKERN
`F. A. MATSEN
`A. L. OPPEGARD
`C. G. OVERBERGER.
`
`JOHN D. Roman-rs
`CHARLES TANFORD
`Canvas WALLING
`F. H. WESTHEIMER
`S. WINSTEIN
`Osman WINTERSTEINER
`
`
`
`© Copyright, 1963, by the American Chemical Society.
`Published semi-monthly by the American Chemical
`Society at’ 20th and Northampton Sts., Esston, Pa. Sec-
`ond-class postage paid at Easton, Pa.
`review
`for
`Manuscripts
`for publication and hooks
`should be sent
`to the Editor, Marshall Gates, Depart-
`ment of Chemistry, University of Rochester, Rochester 20,
`N. Y.
`Correspondence regarding accepted papers, proofs and
`reprints should be directed to Senior Production Editor,
`Charles R. Bertsch, ACS Ofiice, Mack Printing Company,
`20th and Northampton Sts., Easton, Pa.
`'
`Director of Planning, Fundamental Journals: Richard H.
`Belknap, 1155 Sixteenth St., N. W., Washington 6, D. C.
`Advertising Ofliee: Reinhold Publishing Corporation,
`430 Park Avenue, New York 22, N. Y.
`The American Chemical Society and the Editors of the
`Journal of the American Chemical Society assume no re-
`sponsibility for the statements and opinions advanced by
`contributors.
`
`Chemical Society must be. received on or before the 18th of
`the preceding month.
`if
`Claims for missing numbers will not be allowed (1)
`received more than sixty days from date of issue plus time
`normally required for postal delivery of journal and claim,
`(2) if loss was due to failure of notice of change in address
`to be received before the date specified in the preceding
`paragraph, or (3)
`if the reason for the claim is “missing
`from files."
`to members, $13.00 per year,
`Subscription rates for 1963:
`to non-members, $30.00 per year. Postage to countries in
`the Pan-American Union, $2.50; Canadian, $1.10-, all other
`countries, $3.60. Back volumes: Vol. 36-84, $36.00 per
`volume;
`foreign postage, per volume, $3.60; Canadian,
`$1.10; Pan-American Union, $2.50.
`for current year, $1.50;
`Single copies: back issues, $2.00;
`postage, single copies:
`foreign, $0.20; Canada and Pam
`American Union, $0.15.
`Publications of the American Chemical Society include:
`Analytical Chemistry, Biochemistry, Chemical Abstracts,
`CA Section Groupings, Chemical and Engineering News,
`Business and Subscription Information
`Chemical Reviews, Chemical Titles, Chemistry,
`Industrial
`Remittances and orders for subscriptions and for single
`and Engineering Chemistry, Inorganic Chemistry, Journal of
`copies, notices of changes of address and new profemional
`Agricultural and Food Chemistry, Journal of the American
`connections, and claims for missing numbers should be sent
`Chemical Society,
`Journal of Chemical Documentation,
`to the Subscription Service Department, American Chemi-
`Journal of Chemical and Engineering Data, Journal of
`cal Society, 1155 Sixteenth Street, N.W., Washington 6,
`Medicinal Chemistry, The Journal of Organic Chemistry,
`D. 0‘. Changes of address for the Journal of the American
`and The Journal of Physical Chemistry. Rates on request.
`Notice to Authors last printed in the issue of May 5, 1963.
`
`Page 2 of10
`
`
`
`Page 2 of 10
`
`

`

`JOURNAL
`
`OF THE AMERICAN CHEMICAL SOCIETY
`
`VOLUME 85, NUMBER 14
`]UI.‘£' 20. 1963
`
`
`3A
`
`I. PHYSICAL
`Edward A. Boudreaux, Hans B. Jonassen and Leroy J.
`Theriot: Physical and Theoretical Studies on Some
`Solid Complex Diazonium Salts of 1)-Dimetliylarninm
`benzene Diazonium Chloride.
`I. Magiictic Investi-
`.
`gations .
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.-
`. . .
`.
`.
`.
`. . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`H. Komazawa, A. P. Stefani and M. Sz-ware: Studies of
`Irreversibility of CF; Addition. Reactions with Some
`cis and trims Isomers and the Problem of ::is—tr£ms
`Isornerization .
`.
`. . .
`.
`.
`.
`.
`.
`. . .
`.
`.
`. . .
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`Ernest Grunwald and S. Meiboom: Kinetics of Proton
`Transfer in Methanol for a Series of Carboxylic Acids
`of Varying Strength .
`.
`.
`.-
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`Ernest Grunwald and Charles F. Jumper: Kinetics and
`Meclianisni of the Proton Transfer between Methyl-
`oxoniurn Ion and Benzoic Acid in Metliariol Solution.
`13. C. Roquitte and M. H. J. Wijnen : The Photolysis of
`Carbon Tetracliloride in the Presence of Ethane and
`Ethy1ene...........'. .
`.
`.
`.
`. . .
`.
`.
`. . . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. ..
`M. M. Labes and O. N. Rudyj: Effect of Gases on the
`Conductivity 0'5 Organic Solids.
`I. The Anthraeene-
`.
`Iodine Interaction .
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`P. J. Reucroft, 0. N. Rudyj and M. M. Labes: Effect of
`Gases on the Conductivity of Organic Solids.
`II.
`The 15-Chlorani1—Amine Interaction . . . .
`. . . .
`.
`.
`.
`.
`.
`.
`II. ORGANIC
`I-Iydroboration.
`Herbert G. Brown and A. W. Moerikofer:
`XV. The Influence of Structure on the Relative
`Rates of Hydroboration of Representative Unsatu-
`rated Hydrocarbons with Diborane and with I}i5~[3-
`methyl-2-butyl)—borane .
`. .
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`. . . .
`.
`.
`.
`.
`.
`.
`.
`.
`George Zweifel and Herbert C. Brown: Hydroboration.
`XVI. The Hydroboration of Olefins, Acetylenes and
`Dienes with Thexylborane . .
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`-.
`.
`.
`.
`George Zweifel, Nagaraj R. Ayyangar and Herbert C.
`Brown: Hydroboration. XVII. An Examination of
`Several Representative Dialkylboranes as Selective
`I-Iydroborating Agents .
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`M. P. Cara, D. R. Napier and R. J. Pohl: Condensed
`Cyclobutane Aromatic Compounds. XXVI. Benze-
`cyclobutadienoqulnone: Synthesis and Simple Trans-
`formations .
`.
`.
`.
`.
`.
`.
`. .
`. .
`.
`.
`.
`. . . .
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. .
`M. P. Cava, R. J. Pohl and M. J. Mitchell: Condensed
`Cyclobutane Aroinatie Compounds. XXVII.
`1.2-
`Dimethylenebetlzocyclobutenc
`and Related Sub-
`stances .
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. . . .
`.
`. ..
`.
`.
`Cheves Walling and Andreas A. Zavitsas: The Copper-
`Catalyzed. Reaction of Percstcrs with Hydrocarbons.
`
`,
`
`.
`
`D. H. Williams, J. M. Wilson, H. Bucizikiewicz and Carl
`Djerassi: Mass Spectrometry in Structural and
`Stereoehemical Problems. XXIV. A Study of the
`Hydrogen Transfer Reactions Accompanying Frag-
`mentation Processes of 11-Keto Steroids. Synthesis
`of Deuterated Androstan-11-ones . . .
`. . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`. .
`Edwin F. Ullman and Erwin Buncel: An Approach to a
`Bcnzocyelopropene .
`. . .
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`. _ .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`A. Schriesheim, C. A. Rowe, Jr., and L. Naslund: Anionic
`Activation of C-H Bonds in Olefins. V.
`Influence
`.
`.
`of Structure on Reaction Rate .
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`S. Bank, C. A. Rowe, Jr.. and A. Schriesheim: Anionic
`Activation of C~H Bonds in Olefins. VI.
`Intra-
`molecular Nature and Kinetic Isotope Effect of Base-
`Catalyzed Olefin Isomerization . .
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`D. J. Pasto and J. L. Miesel: Transfer Reactions Involv-
`ing Boron.
`III. Hydrolioration Studies with Ene-
`thiol Ethers .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`E. S. Pysh and N. C. Yang:
`Polarographic Oxidation
`Potentials of Aromatic Compounds .
`. .
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`William G. Dauben and David A. Cox_:_ Photochemical
`Traiisformations. XIV.
`Isocolclncinc .
`.
`.
`.
`.
`. . . .
`.
`._
`.
`Charles J. Si]: and K. C. Wang: Meclianisins of_ Steroid
`Oxidation by Mierotirganis1ns.
`II.
`Isolation and
`Characterization of 360:-I“I-4a-[3l'P1'O9i0“l-C 3-C‘-di-
`7a,B-inetllyllaexahydro-l.5-indanedione . . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`William. R. Nes and Dwain L. Ford: The Anthrasteroid
`Rearrangement. XI.
`The Conversion of
`a‘»'-9-
`Anthrapregnatrien-20—one
`to
`4:’,10-D11_t1ethy1-1,2-
`henzanthraeenc by a Model of a. Biochemical}-1.oute.
`Alan R. Friedman and Edward Leete: Biosyntliesis of the
`Nicotiana. Alltaloids.
`IX. The Non-random Incor-
`poration of Aeetate—2—C“ into the Pyridine Ring of
`Anahasine .
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.1 . .
`.
`.
`.
`.
`. . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`. -
`Louis A, Carpinot Synthesis and Oxidation of 2-Amino-
`2,3~dihydro-1H-benzldc]isoquinoline
`_and
`12,331-
`Tetrahydronaphtho[1,8—de] [1,2]diazepine
`and Re-
`lated Cyclic 1.2-Dihcnzylhydrazines . .
`.
`.
`.
`.
`.
`.
`.
`.
`. .
`. . .
`Robert Warner Chambers and Viktor Kurltov : The Struc-
`turc and Properties of Some Sulfur Analogs of Ace-
`toneuridine .
`.
`. . .
`.
`.
`.
`. .
`.
`.
`.
`. . . . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`-
`-
`-
`
`2091
`
`2100
`
`2111
`
`2115
`
`2118
`
`2124
`
`2130
`
`2135
`
`213?
`
`2141
`
`-
`
`2 144
`
`2160
`
`III. BIOLOGICAL
`
`I. The
`R. B. Merrifield: Solid Pliase Peptide Synthesis.
`SynthesisofaTetrapept1de......................
`Sidney Belman: Beryllium Binding by Bovine Serum
`Albumin at Acid pH . . . .
`.
`. . .
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`
`2149
`
`2154
`
`2039
`
`2043
`
`2047
`
`2051
`
`2053
`
`2055
`
`2059
`
`2063
`
`2066
`
`2072
`
`2076
`
`2080
`
`2084
`
`COMMUNICATIONS TO THE EDITOR
`
`John J. Eiseh and William C. Kaska: A Novel Carbon-
`Carbon Bond Scission and the Isomerization Mecha-
`nism of Vinylaluminum Compounds . . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`J. R. Thomas:
`Influence of Cumene Hydroperoxide upon
`the Inhibited Oxidation of Cumcne .
`. .
`.
`.
`.
`. . . .
`.
`.
`.
`.
`.
`.
`George M. Whitesides, Fred Kaplan and John D. Rob-
`erts: The Configurational Stability of Primary Gri-
`gnard Reagents .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`_
`.
`_
`_
`_
`_
`.
`.
`.
`_
`_
`.
`_
`.
`.
`.
`.
`_
`Emil H. White and Joan E. Stttber: The Dca.:ninati.(m of
`Tertiary Carbinailiiries . . . .
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`Walter L. Meyer and Robert B. Meyer: Rates of Rota-
`tion of Asymmetric Diphenyls by Nuclear Magnetic
`Resonance Spectroscopy .
`.
`.
`.
`.
`. .
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`. 2
`.
`Jerome F. Eastham and Gerald W. Gibson: Solvent
`Effects in Organometallic Reactions.
`II .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`Thomas J. Katz and Ralph Dessau: Hydrogen Isotope
`Effects and the Mecliaiiislli of Cycloaddition .
`.
`.
`.
`.
`.
`.
`Norbert Muller and Philip I. Rose: Unusual Nuclear
`Magnetic Resonance Dilution Shift for Acetic Acid in
`Acetic Anhydride .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`. . . .
`.
`.
`.
`D. P. Tate and M. Augl: A Novel Acetylenic Complex
`of Tungstcn(0) Carbonyl . . .
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`, _ .
`.
`.
`,
`.
`
`2185
`
`2166
`
`2167
`
`2168
`
`2170
`
`2171
`
`2172
`
`2 173
`
`2174
`
`The Cyclohept(a)ace:1aphtl1ylene
`Merle A. Battiste:
`Cation; Synthesis of a. Pcntaphenyl Derivative. .
`.
`.
`C. H. DePuy and F. W. Breitbeil: The Stcrcospecific
`Acid- and Base—Catalyzed Ring Opening of a Substi-
`tuted Cyclonropanol .
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`,
`.
`. . . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`Charles A. Buehler and Harold Hart: Peroxytri[luoro-
`‘acetic Acid—Boron Fluoride as :1. Source of Positive
`Hydroxyl .
`.
`.
`.
`.
`. . .
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`,_.
`.
`.
`.
`.
`.
`. . . .
`Gilbert Star}: and Susan R. Dowd: A New Method for
`the Allcylation of Ketones and Aldeliydest
`the C-
`Alkylation of the Maguesiuin Salts of N -Substituted
`Imines .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`,
`.
`_ _ _
`_
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`. .
`.
`.
`Allan J. Lundeen and Robert Van Hoozer:
`Selective
`Catalytic Deliydration of 2-Alcohols;
`a New Syn-
`thesis of l-Olefms .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. .
`.
`.
`. . .
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`Henry Weiner and Richard A. Sneen: Acetone as :3.
`Nuclcopllile .
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`Alfred I-Iassner and John Larkin: A Novel Rearrange-
`ment of Cyclic o:—Nitr0 Ketones.
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`. . .
`Don C. Ifliand and Thomas M. Davies: Conversion of a
`Scrnicarbazonc to 3. Carbamate. A Novel Rearrange-
`ment .
`. .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`,
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. ..
`
`2175
`
`2176
`
`2177
`
`2178
`
`2180
`
`2181.
`
`2181
`
`Page 3 of10
`
`
`
`Page 3 of 10
`
`

`

`4A
`
`Irving J. Borowitz and Rein Virkhaus: On the Mecha-
`nism of the Reactioris of oz-Brumulcetones with Tri-
`pllenylphosplline .
`.
`.
`. . .
`.
`.
`.
`. . . .
`.
`.
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`Edward E. Smissman and Michael A. Oxrnan: A Stereo-
`specific Synthesis of di-Quinic Acid .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`Alex Nickon, Margaret A. Castle, Rolmro Harada, Charles
`E. Berkofi and Roger 0. Williams: Chemical Shifts
`
`of Axial and Equatorial a-Protons in the N,n1.r. of
`Stcroidal mlialoketones . . . .
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`. . . .
`M. V. Sargent and C. J. Timmons: The Pliotocheinical
`Conversions of Stilbenes
`to 9.1C|~Di.1’1yIZ1I‘0p11t':1'l€ltl-
`threnes .
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`. .
`Robert G. Line}: and Henry Taube: The Quinone Oxida-
`tion of Ethanol Catalyzed bjr Clirornic Ion . .
`.
`.
`.
`.
`.
`.
`.
`
`2185
`
`2186
`
`2187
`
`B 0 OK REVIEWS
`
`Les Cyclitols. Chimie.l3i0c11imie,'Biologie. By Theodore
`Posternak. Reviewed by C. E. McCasland .
`.
`.
`.
`.
`.
`.
`.
`Developments in Inorganic Polymer Chemistry. Edited
`by M. F. Lappert and G. J. Leigh. Reviewed by
`john C. Bailar, Jr. .
`. . . .
`.
`.
`.
`.
`.
`.
`.
`.
`. . . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`. .
`.
`Mass Spectrometry. Organic Chemical Applications.
`. . .
`.
`Klaus Biernann. Reviewed by Carl Djerassi .
`.
`.
`Theories of Electrons in Molecules. By William T. Simp-
`son. Reviewed by Charles S. Johnson, Jr. .
`.
`.
`.
`.
`.
`.
`.
`.
`Inorganic Polymers. Edited by F. G. A. Stone and
`u - . .
`.
`.
`.
`-
`. . . .
`.
`.
`.
`. .
`3?. A. G. Graham. Reviewed by G. Bart'h-Wel1ren-
`p .
`.
`.
`.
`. .
`.
`.
`. . .
`.
`.
`.
`.
`.
`. . .
`.
`.
`.
`.
`.
`.
`.
`.
`
`2189
`
`2100
`
`2100
`
`2192
`
`2192
`
`The Chernistry of Rhenium. By K. B. Lebedev. Re-
`viewed by Charles L. Rulfs . . .
`.
`.
`.
`. . .
`.
`.
`.
`.
`. . .
`. .
`.
`.
`.
`.
`.
`Ions in Hydrocarbons. By Andrew Gernzmt. Reviewed
`by M. ]. Allen .
`. . . .
`.
`.
`.
`.
`.
`.'
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`. .
`.
`.
`.
`.
`.
`Elements of Chenlical Thermodynamics. By Leonard K.
`Nnsli. Reviewed by Frederick D. Rossini . . .
`. .
`.
`.
`.
`.
`Eiectroni-2:: for Scientists. Principles and Experiments for
`Those W110 Use Instrurnents. By I-I.- V. Malmstadt
`and C.
`Enke. R.evicwed- by Daniel W. Healy, Jr.
`Comprehensive Biochemistry. Volume 3. Methods for
`the Study of Molecules. Edited by Marcel Florkin
`and Elmer II. Stotz. Reviewed by Idhn T. Edsall.
`
`2193
`
`2193
`
`2193
`
`2193
`
`219-!
`
`AUTHOR INDEX
`
`.A11en._M. _T., 2193.
`Aug], J. M., 2174.
`Ayyangar, N. R.. 2072.
`Bailar, J". C., IL, 2190.
`B:1n1:.S..211.'3.
`Barth-Welirenalp, C.,,
`2192.
`Baitiste, M. A, 2175.
`Belrnan. S.. 2154;.
`Berkofl, C. 13.. 2135.
`Borowitz, 1. _T., 2183.
`Boudreaux. E. A... 2039.
`Breitbeil, F. W., 2176.
`Brown, H. C., 2063, 2066,
`2072.
`Budzikiewicz, 1-1., 2091.
`Buehler, C. A.. 2177.
`Buncel,
`2105.
`Carpiuo. L. A., 2144.
`Castle, N1. 11., 2185.
`Czwa. M. P., 2076. 2080.
`Chambers, R. W., 2160.
`Cox, D. A.. 2130.
`Daubcrl. W. G., 2130.
`
`Davies. T. M.. 2182.
`DePuy. C. H.. 2170.
`Dessau, R., 2172.
`Djerassi, C., 2091, 2190.
`Dowd, S. P... 2178.
`
`Kaplan. F.. 2167.
`Kaslca. W. C., 2165.
`Katz, '1‘. _T.. 2172.
`Kornazawa, IL, 2043.
`Kurlcov, V., 2160.
`
`Oxrnan. M. A., 2184.
`
`Prism. D. _I'., 2118.
`Fold, R. J.. 2076, 2030.
`Pysh, E. S., 2124.
`
`E€I.$tl1£l.!'1‘1, J. F.. 2171.
`Edsall. J. T., 2194.
`Eisch. J. J.. 2165.
`Ford, D. L.. 2137.
`Friedman, A. 1?... 2141.
`
`Gibson. G. W., 2171.
`Grunwald. [C., 2047, 2051.
`
`Haracla. R.. 2185.
`Hart, 1-1., 2177.
`Hassncr, A.. 2181.
`Healy, D. W., Jr., 2193.
`
`Ifiland, D. C., 2182.
`
`Johnson. C. S., Jr.. 2192.
`Jonassen, H. 13., 2039.
`Jumper, C. F., 2051.
`
`Labes, M. M.. 2055. 2059.
`Larkin, I., 2181.
`Leete, 13.. 2141.
`Linclc, R. G., 2187.
`Luncleen. A. _l'., 2180.
`
`McCasland, C. E.. 2189.
`Meiboom, 3., 2047.
`Merrifield, R. B., 2149.
`Meyer, R. 13., 2170.
`Meyer, W. L., 21.70.
`Miesel, J. L., 2118.
`Mitchell. M. _I., 2080.
`Moeriicofer, A. W., 2003.
`Muller, N., 2173.
`
`Napier. D. R., 2076.
`Naslund, L., 2111.
`Nes, W. R., 2137.
`Nickon, A... 2185.
`
`Rcucroft.-P. _T., 2059'.
`Roberts, J’. 1).. 2107.
`Ruquitte, B. C., 2053.
`Rose, P. 1., 2173.
`Rossini, F. D.. 2193.
`Rowe, C. A.. Jr.. 2111,
`2115.
`Rudyj, 0. N., 2055, 2059.
`Rulls, C. L.. 2193.
`
`Sargent. M. V.. 2186.
`Schrieshcim, JL. 2111,
`2115.
`S111, C. _I., 2135.
`Smissman, E. 13., 2184.
`Sneen. R. 11., 2181.
`Stefani, A. P.. 201:3.
`Stork. G., 2178.
`Stuber, J. E., 2168.
`Szwarc. M., 2043.
`
`Tate. D. P_, 2174.
`Taube. H., 2187.
`Tlieriot. L. 1.. 2039.
`Thomas, J. R... 2166.
`Timmons, C. J‘.. 2186.
`
`Uilman, E. F.. 2106.
`
`Van Hoozer, R., 2180.
`Virkhaus, 11.. 2183.
`
`Walling. C., 2084.
`Wang, K. C., 2135.
`Weiner, 1-1., 2181.
`White. E. H.. 2168.
`Wliitesides. G. M., 2167.
`Wijncn, M. H. _T.. 2053.
`Williams, D. H., 2091.
`Williams, R. 0., 2185.
`Wilson, I. M., 2091.
`
`Yan , N. C., 2124.
`g
`. 21.11.. 2084.
`'t
`Z
`an Sag
`Zweifel. G.. 2066. 2072.
`
`
`
`Page 4 of 10
`
`

`

`July 20, 1963
`
`SYNTIIESIS or A TETRAPEPTIDE
`
`2149
`
`spectrum was being taken in denteriocliloroforin the sample tube
`popped its cork and vigorous gas evolution was noted. The
`spectrum was run several times during the first 15 min. after
`which it became constant and appeared to be that of 9,10-
`diliydrophenantliteiie contaminated by an unidentified impurity
`which exhibited a strong peak at approximately 7.3 5. Positive
`identification was made by comparison of the infrared spectrum
`with that of an authentic sample.
`Thermal Decomposition of 1,4-Dihyclronephtholl,3-d,e][1,2]-
`diazepine (XX).-—In a semimicro distillation apparatus 0.4 1;.
`of XX. was heated gently with a free flame. The solid melted
`to an orange liquid and on continued heating sudden and vigorous
`decomposition occurred with the release of a cloud of smoke.
`The black liquid remaining in the pot was distilled into the re-
`ceiver by means of the free flame. The distillate solidified as a
`black solid (0.2 g., 59.2%). Recrystallization from cth:.inol~
`water (dccolorizing carbon) gave nearly white crystals, m.p.
`93.5-94.5“. The melting point was not depressed on admixture
`with an authentic sample of aceuaphthene.
`When the azo compound was heated in diglyn-1:: {b.p. 162”)
`or triglyme (b.p. 210°],
`isomerization to XXI was noted as
`proved by mixture melting point determination.
`Isomerization of
`1 .4-Dihydronaphtho [1,B-d,e] [1 ,2] diaze-pine
`(XX).—~A solution of 0.5 ml. of concentrated hydrochloric acid
`and 3 ml. of water was warmed with 0.2 g. of XX for 2-3 min.
`
`until solution occurred. Excess solid potassiiiin hydroxiilc was
`added_and the cream—colored powder filtcrcd and dried in air.
`The yield was 0.17 g‘. (85%). m.;:. 129-130°, identified as XXI
`by comparison with a sample prepared by selenium dioxide
`oxidation of X.
`1—Benzai-2-benzylhydrazine.—A mixture of 16.4 g. of benzal—
`azine, 4.66 g. of dry lithium chloride. 5.9-1 g. of potassium boro-
`liydride a_ntl 81'] ml. of tetrallyiirofuran was refluxed for 24 hours
`Wltll stirring.“ The mixture was decomposed by the addition of
`water and the tetraliydrofuran layer. separated and allowed to
`evaporate spontaneously.
`Snow—wl1ite _1'.1ak_cs
`(9.5 g.. 91%)
`remained, m.p. 64-T2
`ilec. Recrystallization from methanol
`gave ’I".6 g. (72%) of.the pure hydrazone, m.p.
`'.7lJ—73.5° dec.
`(softening at 69.5% lit.“ m.p. 69-70“). Since the hyclrazone
`decomposed readily on standing as noted by previous workers, the
`n.m.r. spectrum was run. at once.
`
`Acl-mow1edgment.——We are indebted to Prof. Thomas
`Stenglc for the n.m.r. spectra and discussions concerning
`their interpretation.
`ll.-I. Davis [.47. Chem. Sac.,
`(33) The procedure is based on the work of
`3981 (iQ5I‘:Jl , who showed that tertiary amides could be reduced under these
`conditions.
`(34) A. Wiihl and C. Oesterlin, Ban, 3!, 27.36 (1000).
`
`l(.‘.o1~:'rRInU1'ioN FROM THE ROCKEFELLER Institute, New YORK 21, N. Y.]
`
`1. The Synthesis of a Tetrapeptide‘
`Solid Phase Peptide Synthesis.
`Bv R. B. MERRIFIELD
`RECIIIVED JANUARY 31, 1963
`
`It involved the stepwise
`A new approach to the chemical syntliesis of polypeptides was investigated.
`addition of protected amino acids to a growing peptide chain which was ‘bound by ‘:3. covalent bo11d to a solid
`resin particle. This provided a. procediire whereby reagents and by-products were removed by filtration, and
`the recrystallization of intermediates was eliminated. The advantages of the new method were speed and sim-
`plicity of operation.
`The feasibility of the idea was demonstrated by the synthesis of the model tetfzlpepltlclt‘.
`1.-lcucyl-L-alauylglycyI—L—valinc.
`The peptide was identical with a. sample prepared by the standard p—nitro-
`plienyl ester procedure.
`
`The classical approach to peptide synthesis has
`yielded impressive successes in recent years in the prep-
`aration of several biologically active peptides.“ With
`the development of new reagents and techniques the
`synthesis of most small peptides has been placed within
`cosy reacl1.3 However,
`these procedures are not
`ideally suited to the synthesis of long chain polypep-
`tides because the technical dilliculties with solubility
`and purification become formidable as the number of
`amino acid residues increases. A new approach to
`peptide synthesis has been investigated in an effort to
`overcome some of these difficulties. The present re
`port deals with the basic idea behind the new method
`and with a demonstration of its feasibility through the
`synthesis of 21 simple model tetrapcptide.
`The general concept underlying the new method is
`outlined in Fig. 1.
`It depends on the attachment of
`the first amino acid of the chain to a solid polymer by a
`covalent bond,
`the addition of the succeeding amino
`acids one at a time in a stepwise manner until the de-
`sired sequence is assembled, and finally the removal of
`the peptide from the solid support. The reason for this
`approach is that when the growing peptide chain is
`(1)
`(:1) Supported in port by Grant A 1260 from the U. 5. Public Health
`Service.
`(b) An abstract of this work was presented at the 46th Annual
`Meeting or the Federation or American Societies for Experimental Biology,
`A.IIti|.1![.l[i2: R. B. Mcrrifield, Fad‘. Print... 21., -412 (1952).
`I’. G.
`ii’)
`(:1) V. du Vigneaud, C. Ilcssler, I. M. Swan,
`(2. W. Roberts,
`Kaisoyannis and S. Gordon. J. A m. Cimn. Son, 1'5, 4879 (1953);
`(b) R, B_
`Men-ilicid and I). W. Woollcy, 1'b:‘:i., 78, -10-16 (1956);
`(cl 1!. Schwarz, M.
`Burripus and I. H. Page,
`r'bI'd'., 19. 5697 {1l3.3?};
`{d} R, A. Boissonnau, S,
`Guttmann and 1’. A. Jnqnenoud, Hcfv. Chim. Asia, 43, 13-19 (1960):
`(e)
`K. ‘lloiirinnn, II. Vajima, N. Yariuihara, '1‘. Lin and S. Laurie. J’. Am. Clmaz.
`305-. 93. 487 (1951):
`ft} C. II. Li, J’. 1\-Ieienlrofer, E. Schnabel. I). Clluug,
`T. Lo and _'I'. Ramnchnndrau, Ebiiit. 53, 4-H0 (1961):
`lit} 1].’. Kaimcler and R.
`Schwyzcr, {Iain Cltim. Acid, {-1. I136 (1061).
`(3) See J’, P. Gi-er-nstein and M. W. Wluitz, "Chemistry of the Amino
`Al’-W5." VOL 2. Jfihtl Wiley and Sons. Inc., New York, N. Y.. 1981.
`
`firmly attached to a completely insoluble solid particle
`it is in a convenient form to be filtered and washed free
`of reagents and by—products. Thus the intermediate
`peptides are purified, not by the usual recrystallization
`procedures, but by dissolving away the impurities.
`This greatly simplifies the manipulations and ‘shortens
`the time required for the synthesis of the peptides.
`It
`is hoped that such a method will lend itself to automa-
`tion and provide a. route to the syn thesis of some
`of
`the higher molecular weight polypeptides which
`have not been accessible by conventional procedures.
`The Polymer.—The first requirement was for a, suit-
`able polymer.
`It had to be insoluble in all of the sol-
`vents which were used and have a stable physical form
`which permitted ready filtration.
`It also had to contain
`a functional group to which the first protected amino
`acid could be firmly linked by EL covalent bond. Many
`polymers and modes of attachment were investigated.
`Among the polymers were cellulose, polyvinyl alcohol,
`polymethacrylatc and sulfonated polystyrene. The
`one which worked best was a chloromctliylated copoly-
`mer of styrene and divinylbenzene. The resin, in the
`form of 20(J—rl[)0 mesh beads, possessed a porous gel
`structure which allowed ready penetration of reagents,
`especially in the presence of swelling solvents. Al-
`though diffusion and steric hindrance were no doubt
`important factors, they were not serious enough to pre~
`vent the desired reactions from proceeding to comple-
`tion. The reaction rates were slower than correspond-
`ing ones in solution, but conditions were found which
`permitted all of the reactions to occur at useful rates in
`spite of the fact that the growing peptide chain was in
`the completely insoluble solid phase at all times.
`It
`was for this reason that the term solid phase peptide
`synthesis was introduced to describe the new method.
`
`
`
`Page 5 of 10
`
`

`

`2150
`
`R. B. MERRIFIELD
`
`vm;s5
`
`chlorornethylation step. Table I shows that while the
`carbobenzoxy group cleaved rapidly from the unsub~
`stitutcd carbobenzoxya.-valyl polymer even in 10%
`HBr-acetic acid there was also considerable loss of eS~
`ter. After nitration the rate of removal of carbo-
`benzoxy was decreased, but the loss of ester was re~
`duced to a very small level. With 30% HBr the car-
`bobenzoxy group was removed in 2 to 4 111:, while the
`ester cleavage remained at a low level for at least 6 hr.
`In fact, the peptides could not be removed completely
`from the nitratcd resin even with prolonged HBr treat
`ment. The carbobenzoxy-L~vaIyl brornopolymer had
`properties intermediate between the nitrated and un-
`substituted derivatives with respect to the stability of
`the earbobcnzoxy and ester bonds. The carbobenzoxy
`group could be removed from it with dilute (10%) H131:
`and the ester could be cleaved with concentrated (30%)
`HBr.
`
`TABLE I
`
`CLEAVAGE orr Cnnjaonnuzoxv AND Esrca Gnours nv
`HBr-Acerrc ACID
`
`,__——-s——-—--——-—-~l3xtcr1t of cleavage, %—-—-
`Cb:r.-vnl-
`a—bromo1:olyrner--
`Ch;-val~
`cbzwa:
`30%
`,_p,,]ym¢.-a .«--—-—--:nitropolymer'“
`,—1o% H13r— ,—1o% }IDr—~ .—30% Hnra r10% I113:- HBr
`Cb: Ester Ch: Ester Cb:
`‘Ester Ester
`Ester
`Chz
`'52
`6
`8
`0.5
`8
`73
`8
`1'?
`1.1
`13
`90
`12
`32
`2.2
`21
`IOU
`15
`28
`100
`18
`45
`52
`58
`
`1 .2
`
`3 . 8
`4.3
`
`77
`
`8?
`91
`
`6
`
`10
`14
`
`5. 4
`4.8
`
`100
`
`18
`
`59
`
`Time,
`min.
`5
`10
`20
`30
`60
`Q0
`120
`180
`240
`300
`
`6
`12
`19
`38
`57
`67
`83
`9-1
`99
`
`.
`
`1.2
`2.0
`2.5
`
`3 .2
`3.2
`
`14
`28
`43
`67
`90
`100
`99
`108
`98
`
`.
`
`The Peptide-Fonning Step.—After removal of the _N—
`terminal protecting group by HBr the hydrobrormde
`was neutralized with excess triethylarnine and the free
`base was coupled with the next protected amino acid.
`Although the p-nitrophenyl ester method’ at first ap-
`peared to be ideal for this step it finally proved to be un-
`satisfactory alter many experiments. The yields were
`not high enough even with elevated temperatures (80"l
`and there was evidence for partial racemization (Table
`II). The N,N’-dicyclohexylcarbodiimide method,‘ on
`the other hand, proved to be very satisfactory. The
`reaction went in virtually quantitative yield in about
`30 minutes at room temperature. The relatively In-
`soluble by-product dicyclohexylurea and the rearrange-
`ment product carbobenzoxyaminoacylclicyclohexylurea,
`which was formed in appreciable quantities, were both
`easily removed by thorough washing.
`In conventional
`syntheses this is not always the case, especially when
`the peptide derivatives have become large and relatively
`insoluble themselves. The choice of solvent for the
`condensation was very important. Of the several sol-
`vents which were examined dimethylformamide. W213
`the best, methylene chloride was satisfactory, but
`dioxanc, benzene, ethanol, pyridine and water gave very
`low yields and were not useful. The effectiveness of
`the solvent depended partly on its ability to swell the
`resin and partly on other factors. For example, the
`two effective solvents (dirnethylformamidc and methyl~
`cne chloride) had high dielectric constants and £1150
`swelled the resin whereas benzene, wh1ch_swelled the
`resin but had a low dielectric constant, was ineiiective.
`The coupling reaction was shown to occur between
`glycylvalyl polymer and the carbobenzoxy derivatives
`(7) M. Bodanszlcy, Nature, 115, 685 (1955).
`(3) J. c. Shcchan and G. P. I-1css,J. Am. Chem. 595.. 11', 1067 (19551.
`
` N02
`
`C|CHa—©—Polys1yrene
`
`
`~02
`M’
`Cbzo-NHCHCO-C H3-®-Polyslyrene
`HBr-HOAC
`
`
`
`
`
`Diimide
`
`
`we
`NHgCHC0—CH3-®-Polystyrene
`
`
`'?=‘.i M
`Cbzo-NHCHC-NHCHCO-CHg
`
`
`
`
`
`I
`I
`ll
`R,p
`R20
`NHgCHC-NHCHCOH
`
`NGOH
`
`Fig. 1.—The scheme for solid phase peptide synthesis.
`
`Attachment of the First Amino Acid to the Po1yn1er.—-
`To provide a point of attachment for the peptide the
`polystyrene resin was partially chloromethylatedxt
`For reasons to be discussed later the product was then
`nitratcd or brorninated. The resulting substituted
`chlorornethyl polystyrene was treated with the tri-
`ethylatnmoniurn salt of the first protected amino acid
`in the proposed peptide chain to give a substituted
`benzyl ester linkage. This was the stable covalent
`bond which held the growing peptide chain in the solid
`phase on the supporting resin. The reaction was
`shown to go to completion with carbobenzoxyglycine at
`refluxing temperatures in dry solvents such as ethyl
`acetate, benzene or dioxane giving carbobenzoxyglycyl
`polymer.”
`It was also demonstrated to proceed with
`i_:arbobenzoxy—L-valine and carbobenzoxynitro—L-argin-
`me. Racemization of the C-terminal amino acid was
`not observed during the reaction. The quantity of
`amino acid attached to the resin was purposely limited
`to approximately 0.5 mmole per gram of substituted
`polymer. To avoid undesired alkylations in subse-
`quent steps the uureacted chloromethyl groups were
`then csterified with an excess of
`triethylammonium
`acetate. Complete reaction was indicated by the fact
`that the product was nearly free of halogen.
`Cleavage of
`the Amino-Protecting Group.--The
`proteetmg group which was used throughout the syn-
`theses to be reported was the carbobenzoxy group.
`It was selected because it could be removed readily and
`completely by hydrogficn bromide in glacial acetic acid.“
`Since the latter reagent also attacked the benzyl ester
`at a slow rate the loss of peptide from the resin was a
`serious problem. The diificulty was largely overcome
`by nitration or bromination of the polymer after the
`(vi-) K. W. Pepper. H. M. Paisley and M. A. Young, J. Chin. Sea, 4097
`(19.13).
`(5) The abbreviated nan-ic c.-irbobcnzoxyglycyl polymer was used to desig-
`nate the compound in which cntbobenxoxyglycine was bound in ester link-
`age to the aromatic rings of the styrene-divinylbenzene copolymer through
`hydrant)-methyl side chains. The number of substltuerits per molecule of
`polymer varied and the exact location on the ring or distribution among the
`rings was not known. The other polymer derivatives were named in an
`analogous manner.
`(6)
`I). Ben-Ishai and A. Berger, J. Org. Cl1em., 17, 156-1 (1952).
`
`Page 6 of10
`
`
`
`Page 6 of 10
`
`

`

`July 90. 1953
`
`SYNTHESIS or A Tnrimrnrrrrae
`
`2151
`
`P0lY11‘l'=|'
`subst.
`NC:
`
`Cl°3V=l3l>
`method
`NaOH
`
`Val
`0 . 58
`
`TABLE II
`Ammo Acm Aunmrsns on L-LIEUC'lr'L-L-ALANY1'.2C‘vLYCYL~L-VALINE
`enzym e llydrol ysnte
`Is‘. at in ‘ —- _
`acid hydrolysate
`Lcu
`Ala
`Gly
`0 . 83
`0 . 73
`0 . 01
`
`_
`C9“Pil"i5
`method
`Active
`ester
`.
`.
`0.97
`1.05
`1.00
`No.01-I
`Diimide
`N02
`1.01
`1.02
`1.00
`1.05
`l’IB1'
`Diirnide
`Br
`" The concentration of each amino acid in a leucine an1i11o-
`peptidase digest divided by the corresponding value in :1 6 N
`HCl liydroiysate.
`
`of leucinc, isoleucine, valine, alanine, glycine, phenyl-
`alanine, O-benzyltyrosine, proline, serine,
`threonine,
`methionine, S-benzylcysteine, -Em-benzylhistidine, nitro-
`arginine, 7-methyl glutamate and asparagine. Lysine
`and tryptophan derivatives were not studied.
`Since it was very important that no unreacted amine
`remain after the peptide-forming reaction,” several pre-
`cautions were taken. First, an excess of carbobenzoxy-
`amino acid and of diimide was used and each con-
`densation step was repeated with fresh reagents.
`Finally, after the coupling reactions were completed
`any trace of unreacted amine was acetylated with a
`large excess of acetic anhydride and triethylamine.
`’I‘his reduced the free amine to less than 0.1% of that
`originally present. The acetamido bond was com-'
`pletely resistant to 30% I-IBr~acetic acid for 18 hours
`at 25°. Consequently, the danger of re-exposure of
`these amino groups during subsequent steps was elimi-
`nated.
`The steps described to this point completed one cycle,
`i_e.,
`the peptide chain was lengthened by one amino
`acid residue. Further cycles were carried out in the
`same way by alternately deprotccting and coupling with
`the appropriate carbobenzoxyamino acid. The com-
`pleted protected peptides were finally decarbohenzox-
`ylated by HBr and the free peptides were liberated from
`the polymer by saponification, or,
`in the case of the
`brominatcd resin, by more vigorous HBr treatment.
`The liberated peptides were desalted and purified by
`ion-exchange chromatography, countercu