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Oligonucleotide Synthesis with a Soluble Polymer as Carrier.

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This method can therefore be used to prepare trifluoroalanyl peptides directly, for the isonitriles are readily obtainable from N-formylamino esters [61. For example, the
methyl ester of N-benzyloxycarbonyl-3,3,3-trifluoroalanyl-~leucine, m.p. 165 "C, was obtained in 64 % yield in this way.
It was shown by conversion of the product into the N-trifluoroacetyl dipeptide methyl ester and separation of the
diastereoisomers by gas chromatography "1 that a n asymmetric induction takes place during the synthesis; the ratio of
the diastereomers was 72: 28.
The scope of these reactions is extended by using N - ( N
benzyloxycarbonylaminoacy1)-1 -chIoro-2,2,2- trifluoroethylamines ( I ) , X = CI, Ac= C6Hs~CH20-CO-NH-CHR-CO,
which are obtained by reaction of N-benzyloxycarbonylamino acid arnides with trifluoroacetaldehyde and subsequent
treatment with phosphorus pentachloride. The reaction
sequence described above then leads to N-benzyloxycarbonyl
tripeptide esters with trifluoroalanine in the center; for
example, the methyl ester of N-benzyloxycarbonylglycyl3,3,3-trifluoroalanyl-~-leucine
was obtained in the form of
an oil, and the amino-acid sequence was confirmed by mass
spectrometry [81 after conversion of the product into the
N-trifluoroacetyl compound.
(3),
(4j,
x : -011
x : -(.l
The polymer (4) was treated with 3'-0-acetylthymidine in
pyridine, wherupon 40 % of the functional groups reacted.
The acetyl groups were then removed with 0.01 M sodium
methoxide or ammonia in dioxane, and the polymeric
residue was brought into reaction with 3'-0-acetyl-5'thymidylic acid. Repetition of the procedure gave a polymer
(5) containing a trinucleotide. After each reaction step the
polymer can be precipitated by a n nonpolar solvent or by
water and freed from reagents and unchanged starting
material by washing or reprecipitation.
Received: April 25th, 1966
[Z 228 1El
German version: Angew. Chem. 78, 640 (1966)
[ I ] N-Acyliminotrihalogenoethanes as Reactive Intermediates in
Elimination-Addition Reactions, Part IV. - Part 111: F. Weygand, W . Steglicli, I . Lengyel, F. Fraunberger, A. Maierhofer, and
W . Oetlrneier, Chem. Bcr., in the press. - We wish to thank the
Deutsche Forschungsgemeinschaft for material support of this
work, Dr. 0. Scherer, Farbwerke Hoechst AG., Frankfurt (Germany) for supplying trifluoroacetaldehyde, and Dr. A . pro.^ for
measuring thc mass spectra.
[2] F. Weygond and W. Strglich, Chcm. Ber. 98, 487 (1965).
13) E . J . Cohr! and J. T. Edsnll: Proteins, Amino Acids and Peptides. Reinhold, Ncw York 1943, p. 83, give pK,* = 2.34 and
pKz'%= 9.69 for alanine at 25 "C.
[4] R . Buyle and H . G. Viehe, Angew. Chcm. 76, 572 (1964); A n gcw. Chem. internat. Edit. 3, 582 (1964).
151 The formation of oxazoline derivatives from 2-(N-benzoylimino)propane and isonitriles is described by N . P . Ganzb~rynn
CI ~ 7 / . , Habilitation Thcsis, Moscow 1965. - Hydrolysis: cf. E . M .
Roclrliii, 3rd Internat. Fluorine Symposium, Munich 1965.
[6] I. Ugi, W. Betz, U . Fetzer, and K . Offerinann, Chem. Bcr. 94,
2814 (1961); F. Sukiyrinta and B. Witkop, J . org. Chemistry 30,
1905 (1965).
[7] F. Wrygatid, A . Prox, L. Schniidhaminer, and W. Konig, Angew.Chcm.73,282(1963);Angew.Chem.internat.Edit.2,!83(1963).
(81 F. Weygnnd, A. Prox, H . H . Frssel, and K . K . Sun, Z . Naturforsch. 20h, 1169 (1965).
Oligonucleotide Synthesis with a Soluble Polymer
as Carrier
By Prof. Dr. F. Cramer, Dip1.-Chem. R. Helbig,
Dr. H. Hettler, Dr. K. H. Scheit, and Dipl.-Chem. H. Seliger
Max-Planck-Institut fur Experimentelle Medizin,
Chemische Abteilung, Gottingen (Germany)
Stepwise synthesis of oligonucleotides requires column
separations in aqueous systems after each step with consequent loss of time and material. By analogy with Merrifield's
method "1, we have tried to build oligonucleotides on a polymeric carrier [21, where use of a polystyrene derivative soluble
in pyridiner31 proved expedient. In order to be able to link
all four nucleotides to the polymers, we used a polystyrene
containing p-methoxyphenylphenylmethyl chloride functional groups. Polystyrene ( I ) (mol. wt. 170000) was converted by benzoyl chloride/AlCI3 into the polymeric ketone
( 2 ) , which was treated with p-methoxyphenylmagnesium
bromide io give the tertiary alcohol (3). This was converted
into the chloride ( 4 ) with aceryl chloride. About 20 7; of the
phenyl groups of the polystyrene were thus transformed into
functional groups.
A n g e w . Chem. internat. E d i t .
Vol. 5 (1966)
No . 6
OAc
After the last reaction the nucleotide mixture was split o f f
by 1 ml of 5 0 % trifluoroacetic acid in 100 ml of dioxane
(1 hr at room temperature), and the products were separated
chromatographically and analysed. In this way thymidine
and the nucleotides pT, TpT, and TpTpT were obtained in
molar proportions of 28 :7 :25 :40. The yield of TpTpT was
11 calculated on thymidine.
Linear aminopolystyrenes can be loaded with nucleotides by
formation of the (acid-labile) phosphoric amide linkage. The
polymer used is one containing o n an average one aromatic
amino group o n every fourth styrene unit. 9 % of the amino
groups react with 5'-thyniidylic acid (condensing agent: dicyclohexylcarbodiimide). Fission from the polymeric carrier
is effected with 80 % acetic acid at 80 "C (24 hr).
Received: May 9th, 1966
[Z 233 IE]
German version: Angew. Chem. 78, 640 (1966)
~~~
~~~~~
~
[ I ] R . B. Merrifield, J. Amer. chem. SOC.85, 2149 (1963); 86,
304 ( I 964).
[2] Cf. R. L. Letsinger and V . Mnhadevan, J. Amer. chem. Soc.
87, 3526 (1965).
[3] M . M . Sheinynkiii, Yu. A . Ovchinnikov, A . A . Kiryitshkin,
and I . V. Kozhevnikova, Tetrahedron Letters 1965, 2323.
A Model for the Action of Chymotrypsin
By Prof. Dr. F. Cramer and Dipl.-Chem. G . Mackensen
Max-Planck-Institut fur Experimentelle Medizin,
Chemische Abteilung, Gottingen (Germany)
Chymotrypsin catalysis consists in an acid-base catalysis by
imidazole, i n which a histidine imidazole group of the enzyme
increases the nucleophilicity of a serine hydroxyl group of
the protein to such an extent that at pH 7 to 9 ester transfer
with subsequent splitting of the enzyme-product complex
occurs[11. I n the first step of this reaction the substrate must
be bound to the enzyme by a very specific, non-covalent bond
601
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