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Novel Polymeric Materials from Monodisperse Copolypeptides by Biotechnological Methods.

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Novel Polymeric Materials from Monodisperse Copolypeptides
by Biotechnological Methods **
By Helmut Ritter*
The multiplicity of chemical methods that has been described and employed for the synthesis of peptides impressively demonstrates that an ideal synthetic method is apparently still lacking. Emil Fischer already recognized this need
at an earlier date, and neatly summed up the situation at that
time with the poignant remark “der Ochs kann’s besser”
(“the ox can do it better”). Meanwhile, however, there has
been a change of approach with increasing application of
biotechnological methods, and success has not only been
reported in the pharmaceutical and agricultural sectors, but
quite topically a first breakthrough is also recognizable in
the directed preparation of novel and interesting protein materials with predetermined structures.“]
The effort to realize the kinetically and thermodynamically controlled synthesis of peptides, not only by more improved chemical methods but also biocatalytically using free
or supported enzymes has long surpassed the stage of an
experimental exercise, and, e.g. in the case of racemizationfree preparation of the sweetener aspartame (AspPhe) with
thermolysin, has found industrial use.[*]As an extension to
the widely developed synthesis of peptides on polymer supports‘’] we are currently also concerned with the employment of pure enzymes, e.g. a-chymotrypsin, for the coupling
of peptides to side chains in comblike structured
The use of the enzyme apparatus of whole cells has recently led to the synthesis of chiral polyesters with the side chains
containing phenyl groups and fluorine atoms. The molecular
weights of such polyesters generated by Pseudomonas oleovoruns reached M, values of up to 350000.[51
The procedure recently elaborated by TireN et al. for the
directed synthesis of proteins with predetermined molecular
weights and repetitive sequences by gene manipulation provides a completely new entry to materials similar to naturally
occurring proteins (e.g. silk or elastin) and having interesting
properties such as elasticity in fibers or the adhesiveness of
excretory products of certain types of mussels. Whereas biotechnologically produced enzymes or hormones exhaust the
full spectrum of the twenty common amino acids, the new
biomaterials consist of only few repeating amino acids. This
means, however, that the respective DNAs and mRNAs
must to a large extent also be built up in a repeating fashion.
Since this simple regular structural assemblage presents
problems, e.g. stiffening of the mRNA by secondary hydrogen bonds, whereby translation is made difficult, the genetic
code was alternatingly varied. For example, the coding of
glycine was alternatingly carried out by GCT and GGC, that
of alanine by GCC and GCT. In order to keep the errors in
the gene synthesis as small as possible DNA sequences were
first prepared by conventional chemical methods with two
repeat units, e.g. of a nonapeptide, and these subsequently
cloned by insertion in a plasmid which was introduced into
[*] Prof. Dr. H. Ritter
Bergische Universitat-GH Wuppertal
Fachbereich 9
Gausstrasse 20, W-5600 Wuppertal (FRG)
[**I I thank Professor Dr. W Reineke, Wuppertal, for valuable information.
Angew. Chem. h i . Ed. Engl. 30 (1991) No. 6
0 VCH
Escherichia coli as host cell and reproduced. The DNA segments amplified in this way were coupled to a series of multimers by self ligation and recloned with E. coli in nutrient
containing antibiotics. Individual colonies were separately
further reproduced and the respective molecular weight of
the protein produced therefrom ascertained by gel electrophoresis. Staggered monodisperse proteins with predetermined molecular weights can be produced in large amounts
(ca. 40 mgL- ’) from the separate cultures. A copolypeptide
with exactly 14 repeat units of the undecapeptide sequence 1
was very effectively prepared by the above-mentioned simple, general method.[’]
-(AlaGly),ProGluGly-
1
The novel “pol ycondensate” has a glass temperature of
182”C and decomposes above ca. 250 “C. Homogeneous
films can be readily cast from formic acid solution. The
respective oligonucleotide 2 containing 82 base pairs was
synthesized on a support using the phosphoramidite method.
For comparison the chemical synthesis of a protein with
repeating nonapeptide units was also carried out in a rather
tedious way according to Scheme 1.I6] The total yield after
Boc-Ala-OH
I
DCC, HOSu
Boc-GIu(OBZ)-OH
I
Boc-Ala-OSu
DCC, HOSu
BOC-GIU(OBZ)-OSU H-Gly-OH
1,
H-Gly-OBz
L
i
Boc-Ala-Gly-OH
B~~-GI~(oB~)-GI~-oH
I
H-Gly-oH
Boc-Ala-Gly-OBz
I
Boc-Ala-Gly-OSu
HCI , H-Ala-Gly-OBz
I
I
I
DCC, HOSu
CF3C0,H3
HCI
HCI ’ H-Glu(OBz)Gly-OH
CF,CO,H. HCI
BOC-Pro-OSu
Boc-Pro-Glu(0Bz)-Gly-OH
I
Boc-Ala-GIy-Ala-Gly-OBz
i
DCC, HONB
Boc-Pro-Glu(0Bz)-Gly-ONB
1 ) H,, Pd
2 ) CF,CO,H, HCI
HCI H-Ala-Gly-Ala-Gly-OH
i
&
Boc-Pro-Glu(0Bz)-Gly-(Ala-Gly),-OH
i
1) CF,CO,H, HCI
2) DMFIH,O, Et,N
BOC-AG-OSu
Boc-Ala-Gly-Pro-Glu(0Bz)-Gly-(Ala-GI
y),-OH
I
CF,CO,H
CF,COO . H,N-Ala-Gly-Pro-Glu(0Bz)-Gly-(Ala-Gly),-OH
i
1 fAla-Gly-Pro-Glu(0Bz)-Gly-(Ala-Gly),k1
Scheme 1. Conventional peptidechemical method for thesynthesis o f a protein
with repeating nonapeptide sequences. DCC = dicyclohexylcarhodiimide,
HOSu = N-hydroxysuccinimide, HONB = N-hydroxy-5-norbornene-2,3-dicarhoximide.
Verlagsgeselischaft mbH, W-6940 Weinheim. 1991
OS70-0833/91/0606-0677 8 3.50
+ ,2510
677
18 steps was only 11 % in the conventional method. The
presence of a polydisperse mixture having only a relatively
small molecular weight of ca. 10000 was established by gel
chromatography.
According to recent findings,“] the entry to proteins with
repeating amino acid sequences by biotechnological methods seems to be very promising. The great efforts made to
prepare materials with such relatively simple structures is
justified in particular by the fact that applications in medicine are now already recognizable. Thus, e.g., block cocondensates containing repeating amino acid sequences which
are analogues of silk, and sequences which are capable of
binding strongly to cell surfaces, may be useful for the “adhesion” of individual cells to give large aggregates such as is
required, e.g., in cases of severe burning. The coupling of
hard and soft segments of appropriate repeating structural
amino acid sequences is considered as the reason for the high
elasticity of spider’s silk.
The materials now accessible biotechnologically should
show similar properties in blocklike coupling and be of particular interest in the form of films for covering wounds or
coatings for implants.[’] The route outlined by Tirrell et al.
for the synthesis of proteins which contain few repetitive
amino acid sequences and have a predetermined molecular
weight has proven better than classical methods as regards
outlay and results. Hence, polymer chemists and protein
chemists now have new materials on hand that should be
further convertible by chemical and enzymatic processes, e.g.
into block and graft cocondensates. Finally, there remains
the question as to what extent the structural variants of the
monodisperse proteins can be further enlarged, e.g. by fluorinated amino acids in the nutrient.
German version; Angew. Chem. 103 (1991) 694
~~~~
(11 H. S. Creel, M. J. Fournier, T. L. Mason, D. A. Tirrell, Macromolecules 24
(1991) 1213.
[2] H. D. Jakubke, P. Kuhl, A. Konnecke, Angew. Chem. 97(1985) 79; Angew
Chem. Inr. Ed. Engt. 24 (198.5) 85.
[3] E. Bayer, Angew. Chem. 103 (1991) 117; Angew. Chem. Inf. Ed. Engl. 30
(1991) 113.
[4] H. Rehse, H. Ritter, Makrornol. Chem. 189 (1988) 529; M. Gormanns, H.
Ritter, ibid. f92 (1991) p. 74.5.
[Sl K. Fritzsche, R. W. Lenz, R. C. Fuller, Makromol. Chem. 191 (1990) 1957;
C . Abe, Y Tairna, Y. Nakamura, Y. Doi, Polym. Commun. 31 (1990) 404.
[6] K. P. McGrath, D. A. Tirrell, M. Kawai, T. L. Mason, M. J. Fournier.
Biolechnol. Pmg. 6 (1 990) 188.
171 D. A. Tirrell, Amherst, MA, USA, private communications.
C6,,:From Soot to Superconductors
By Frangois Diederich* and Robert L. Whetten
In 1990, a team of astronomers led by Kratschmer and
H u f l r n ~ n [ reported
‘~
that the new allotrope of carbon C,,
(buckminsterfullerene, so named after Buckminster Fuller,
an architect renowned for his geodesic domes)[2]can be produced in macroscopic quantities through resistive heating of
graphite under inert atmosphere. In an Angewandte Chernie
Highlight article in January 1991, Fraser Stoddart summarized the early results in the vigorous worldwide research
efforts that were triggered by this surprising development.r31
Only half a year later, a series of exciting new findings require that yet another Highlight article be written (closing
date May 20, 1991).
X-ray Crystal Structure Analysis of Osmylated C,,
Although the icosahedral-cage structure of C,, was
strongly suggested by I3C NMRI3I, IR13-,’, RamanC6],and
photoelectron spectro~copy,[’~
as well as by scanning tunneling microscopy (STM) imaging,[*the ultimate confirmation and detailed parameters of the soccerball-shaped
framework had to await X-ray crystallography. However,
rapid isotropic rotational motion of the orderly packed
spherical molecules in the crystal to temperatures down to
[*I Prof. Dr. F. Diederich, Prof. R. L. Whetten
Department of Chemistry and Biochemistry
University of California, Los Angeles, CA 90024-1569 (USA)
678
0 VCH Verlagsgeseltsehaft nzbH, W-6940 Weinheim, 1991
100 K[”* ”1 have so far prevented a successful high resolution X-ray crystal structure analysis. To break the symmetry
of C,, and prevent its rapid spinning in the crystal, Huwkins
et al. prepared by osmylation [Eq. (a)] a remarkable 1:I
C,,-osmium tetraoxide a d d ~ c t . ~l4]‘ ~The
. crystal structure
of this adduct was subsequently solved by the Berkeley
group and clearly confirms the soccerball structure of C,, ,
composed of 20 six-membered rings fused to 12 five-membered rings (Fig. l).1141All tricoordinate carbon atoms of
C,, are pyramidalized and lie at an average distance of
3.512(3) 8, from the center of the sphere. The unfunctionalized five- and six-membered rings are all planar within
& 0.05 A. The two bond lengths in these rings are 1.388(9)8,
for the fusion between two six-membered rings and
1.432(5) 8, for the fusion between a six- and a five-membered
ring. Values of 1.40 ( f 0.015) 8, and 1.45 (_+0.015) 8, were
determined for the two bond lengths in C,, by solid-state
13C NMR.[”]
OS70-0833pl10606-0678 $3.50 t .2Sj0
Angew. Chem. Int. Ed. Engl. 30 (1991) No. 6
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