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Gas Chromatographic Identification of the Thiohydantoins of Degradation Products of Peptides and Proteins.

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yellow needles, hmax = 314 n m (log E = 4.43) in n-hexane;
N M R spectrum (in CDCI3): multiplets centered a t T = 3.09
(H-5), 3.79 (H-4), 4.09 (H-6), broad singlet a t T = 6.75
(N(CH&), multiplet centered at T = 6.97 (Hz-1, Hz-~)].
Intramolecular Michael addition is followed by basecatalyzed isomerization of the intermediate ( 3 ) t o the
thermodynamically more stable 6-arninofulvene derivatives
(1,2-dihydropentalenes) ( 4 ) .
Like 6-dimethylaminofulvene [61, the bicyclic fulvenes ( 4 )
also add lithium tetrahydridoaluminate and alkyl- or aryllithiums in ether at 20 OC t o give the organometallic compounds ( 5 ) and ( 6 ) respectively. On hydrolysis a t 0°C
followed by amine elimination a t 25OC, compound ( 5 )
affords 1,Zdihydropentalene (7) [yellow, air-sensitive and
thermolabile oil, b.p. 15 oC/10-2 torr; Amax = 378 (2.68),
272 (3.70), 263 (4.04), 258 (4.08), 255 (4.09), 250 (4.04) n m
(log E) in n-hexane; N M R spectrum (in CC14): multiplets
centered at 7 = 3.29 (H-3, H-5), 3.96 (H-4), 4.18 (H-6),
6.93 ( H z - ~ )7.39
,
(Hz-l)] and ( 6 ) gives the corresponding
1,Zdihydropentalene (8).
1. H20
____)
2. -RzNH
Gas Chromatographic Identification of the
Thiohydantoins of Degradation Products of
Peptides and Proteins [**I
By Harald Tschesche, Rainer Obermeier, and
Sigrid Kupfer [*I
Although the Edman degradation has been developed for
the stepwise sequential determination of peptides and proteins and has been extensively schematized and even automated [I], a simple method (cf. [21) for the identification of
the cleaved amino acids- mainly in the form of phenylthiohydantoins ( I ) - has not yet been reported. T h e compounds ( I ) are not well suited for identification by gas
chromatography 131 because of their decomposability and
low volatility, which can only be overcome in part by silylation [41.
We have found, however, that the methylthiohydantoins
(2) are suitable for gas chromatographic identification.
With the exception of arginine and histidine all the naturally
occurring amino acids, including Phe, Asn, Gln, Tyr, and
Trp, can be chromatographed in the form of their methylthiohydantoins; Asp, Glu, and His can be chromatographed after methylation o r silylation; and. the separation
and identification of leucine and isoleucine d o not present
any difficulties. The dehydrated derivatives of Ser a n d T h r
were prepared and identified (see Table). Preliminary experiments indicate the possibility of coupling with automatic
sequenators 161.
Mass spectra were used for the characterization of methylthiohydantoins (2) (see Table).
Table. Detection of naturally occurring amino acids in the form of
their methylthiohydantoins ( 2 ) . Varian gas chromatograph 1520 with
linear temperature programming of 4 "C/min, starting temperature
130"C, 2 m column 1 / 4 ' ' , 5 % OV-1 o n Chromosorb W-AW-DMCS,
50 ml Helmin.
The composition a n d structure of the isolated compounds
( 4 ) , (7), and (8) were confirmed by elemental analysis and
by UV, N M R , and mass spectrometry. Experiments on the
conversion of (3), (7), and (8) into the corresponding
pentalenes are in progress.
Received: August 7, 1970
[Z 252 IEI
German version: Angew. Chem. 82, 877 (1970)
[*I Dip1.-Chem. R. Kaiser and Prof. Dr. K. Hafner
Institut fur Organische Chemie der Technischen Hochschule
61 Darmstadt, Schlossgartenstrasse 2 (Germany)
111 K . Hafner, Liebigs Ann. Chem. 606, 79 (1957).
12.1 K . H . Vopel, Diplomarbeit, Universitat Marburg, 1958;
K . Hafnei-, W . Bauer, and W. aus der Fiinten, unpublished experiments.
[3] E . Cioranescu, A . Bucur, G . Mihai, G . Mateescu, and D .
Nenitzescu, Chem. Ber. 95, 2325 (1962); E . Le Goff, J. Amer.
chem. SOC.84, 3975 (1962); L. Hurner, H . G . Schmelzer, H . U.
v. d . Eltz, and K . Habig, Liebigs Ann. Chem. 661, 44 (1963);
T . J . Katr, M . Rosenberger, and R . K . O'Hara ( J . Amer. chem.
S O C . 86,249 (1964)) synthesized the 1,5-dihydropentalene which
gave dilithium pentalenediide on reaction with butyllithium.
141 K . Hafner, K . H . Hafner, C . Konig, M . Kreuder, G . Ploss,
G . Schulr, E . Sturm, and K . H . Yopel, Angew. Chem. 75, 35
(1963); Angew. Chem. internat. Edit. 2, 123 (1963).
[5] K . Hafner, K . F. Bangert, and V . Orfanos, Angew. Chem.
79, 414 (1967); Angew. Chem. internat. Edit. 6, 451 (1967).
161 E . Sturm and K . Hafner, Angew. Chem. 76, 862 (1964);
Angew. Chem. internat. Edit. 3, 749 (1964).
Angew. Chem. infernat. Edit.
1 Yol.9 (1970) No. I 1
Amino
acid
Effusion
temp. ("C)
Rel. retention
time (min)
Ala
Gly
Ser [a]
Val
Thr Ial
Ile
Leu
Pro
Met
Phe
Asn
Gln
Tyr
Trp
152
I57
157
164
178
190
190
206
208
208
23 1
24 1
252
277
6.3
7.6
7.65
10.0
12.7
15.4
16.0
19.6
19.9
20.1
24.5
26.8
28.5
34.8
I
nile [51
144
130
142
172
156
186
186
170
204
220
187
20 1
236
259
[a] Dehydrated compound.
Experimental
T h e peptide (1-5 pmole), dissolved in pyridine (0.5 ml,
spectral grade) o r pyridine/water (1 :l), was coupled with
methyl isothiocyanate (10 mg) under nitrogen a t 0 o r 40 O C
for 6+90 min in a microflask in a rotary evaporator; solvent and unreacted reagent were removed under vacuum.
After repeated extraction with toluene the residue was rubbed with ethyl acetate for separation of the dimethylthiourea a n d the carbamoyl peptide was centrifuged off. This
procedure was repeated once. Trifluoroacetic acid or
heptafluorobutyric acid were used for the cleavage a n d
cyclization (50 'C, 45 min). Although the methylthiohydan-
893
toins of Ser a n d T h r are partially dehydrated with trifluoroacetic acid, with heptafluorobutyric acid we have obtained only the dehydrated derivative. After removal of acid
and repeated extraction with toluene, the cleaved derivatives (2) were dried in vacuum and extracted with ethyl
acetate. The residual peptide can be used for the next
degradation.
meter a t 25 O C . 4000 scans each of 0.4 s were accumulated
using a pulse width of 40 ps, resulting in a n overall measuring time of less than 30 min. The two examples (Fig. 1)
illustrate how easily the 13C spectra of polypeptides can be
assigned from the 13C data of amino acids.
5 2
Received: August 17, 1970
[Z 268 IE]
German version: Angew. Chem. 82. 880 (1970)
[*I Priv.-Doz. Dr. H. Tschesche, Dr. R. Obermeier, and
S. Kupfer
Laboratorium fur Organische Chemie und Biochemie
der Technischen Universitat
8 Munchen 2, Arcisstrasse 16 (Germany)
[**I Note added in proof: A paper on the same subject has
recently come to our knowledge: M . Waterfield and E. Haber
Biochemistry 9, 832 (1970).
[l] P . Edman, Europ. J. Biochem. I , 80 (1967); cf. M . von
Wilm, Angew. Chem. 82, 304 (1970); Angew. Chem. internat.
Edit. 9, 267 (1970).
[2] For older methods see W . A . Schroeder in C . H . W . Hirs:
Methods in Enzymology. Academic Press, New York 1967,
Vol. 11, p. 445; F. Weygand, Lecture at the Anniversary Meeting of the Chemical Society, Exeter 1967; F. Weygand, Z. Anal.
Chem. 243, 2 (1968); H . Tschesche, E. Wachter, S. Kupfer, and
K . Niedermeier, Hoppe-Seylers Z. Physiol. Chem. 350, 1247
(1969); H . Tschesche and E . Wachter, Europ. J. Biochem. 16,
187 (1970); F. Weygand and R. Obermeier. unpublished.
131 J . J . Pisano, W . J . A . Vanden Heuvel, and E . C. Horning,
Biochem. Biophys. Res. Commun. 7, 82 (1962).
[4] R . A. Harman, J . L. Patterson, and W . J . A. Vanden Heuvel,
Anal. Biochem. 25, 452 (1968); J . J . Pisano and T . J . Bronzert,
J. Biol. Chem. 244, 5597 (1969).
IS] F. F. Richards, W. T . Barnes, R . E. Lovins, R . Salomone,
and M . D . Waterfield, Nature 221, 1241 (1969).
161 Corresponding investigations carried o u t in collaboration
with Beckman Instruments GmbH will be reported separately.
Fourier Transform I3C-NMR Spectroscopy of
Biologically Active Cysteine Peptides [**I
By Gunther Jung, Eberhard Breitmaier, Wolfgang Voelter,
Toni Keller, and Christian Tanzer [*I
Nuclear magnetic resonance was hitherto unable to provide
direct information concerning the carbon skeleton of large
organic molecules owing to the low natural abundance of
the 13C isotope (1.1%).The Fourier transformation of
accumulated pulse interferograms solves this problem,
since it provides assignable 13C-NMR spectra within a
short time despite the small nuclear concentration.
The results of our Fourier transform 13C measurements o n
amino acid derivatives"] with natural abundance of 13C,
in conjunction with earlier measurements on 13C-enriched
amino acids 121 relative to tetramethylsilane as external
standard, permit the following conclusions:
1) The 13C-carbonyl signals of carboxyl, ester, amide,
hydrazide, and peptide groups appear between -185 and
-170 ppm. Acyl protecting groups such as benzyloxycarbonyl and tert-butyloxycarbonyl are characterized by
signals between -160 and -150 ppm.
2) The 13Ca signals of amino acids appear between -65
and -40 ppm. They depend characteristically o n the side
chain.
3) The 13Cfi signals of amino acids appear between -70
and -15 ppm and are strongly influenced by hetero substituents such as S H and OH.
4) The 13C signals of aromatic rings in the side chain are
observed between -140 and -120 ppm.
The 'H-noise-decoupled 22.63-MHz Fourier transform
13C-NMR spectra (Fig. 1) of glutathione and its oxidized
form in 0.2 M aqueous solution were measured relative t o
external T M S o n a Bruker HX-90 multinuclear spectro-
894
3 8.9
6 8.9
4
7
5
-176 4
-1763-1740
-175.3
-553-416 29J
-56 5 -44 6 -3&1
' 1
Fig. 1. '3C-NMR spectra of glutathione (above) and its oxidized form
(below).
I n this way the carboxyl signals of the two peptides can be
assigned by comparison with glutamic acid (-175.6 ppm)
and glycine (-173.5 ppm). Further, the carbonyl resonance
region contains the signals of the two peptide groups, which
are split for glutathione (-175.2 and -175.4 ppm) but are
superimposed for the oxidized form (-175.3ppm). By a
further comparison of the spectra of glutathione and its
oxidized form it is shown that the 13C signals of the methylene groups of glutamic acid (Cp -29.0, C, -34.1 ppm)
and glycine (-44.6 ppm) are almost identical for the two
peptides.
The most important results of our measurements are the
large differences between the 13CEand 13Cp shifts of cysteine
and cystine in glutathione and its oxidized form. The 13C
signal of Cp next t o sulfur is shifted by 13 ppm to lower
field on transition from glutathione (-CHzSH) t o the
oxidized form (-CH2SSCHz-),
and the 13C signal of C , is
shifted by 3 ppm t o higher field. Further increase in the
oxidation state causes even greater shifts: thus the 13C
signal of Cp of cysteic acid (-CHzS03H) at -52ppm is
shifted downfield by about 2 4 p p m in comparison with
that of cysteine; the 13C, signal of cysteic acid, however,
appears about 5 ppm upfield.
These differences open up new pathways to the elucidation
of the structure of cysteine peptides without loss of material.
For example, we have also been able t o recognize the 13C
signal of the -CHzSSCHzgroup a t -41.6ppm in the
Fourier transform 13C-NMR spectra of the peptide hormones oxytocin, vasopressin, and insulin.
Received: August 26, 1970
[Z 273 IE1
German version: Angew. Chem. 82, 882 (1970)
[*] Dr. G. Jung, Dr. E. Breitmaier, and Dr. W. Voelter
Chemisches Institut der Universitat
74 Tiibingen, Wilhelmstrasse 33 (Germany)
T. Keller and Dr. Ch. Tanzer
Bruker Physik AG
7501 Karlsruhe-Forchheim, Silberstreifen (Germany)
[**I Part 2 of Fourier Transform 13C-NMR Spectroscopy. Part 1: W . Voelter, E. Breitmaier, G . Jumg, T. Keller, and D . Hiss,
Angew. Chem. 82, 812 (1970); Angew. Chem. internat. Edit. 9,
803 (1970).
[ l ] E. Breitmaier, W. Voelter, and G . Jung, unpublished.
121 W. Horsley, H . Sternlicht, and I . S . Cohen, J. Amer. Chem.
SOC.92, 680 (1970).
Angew. Chem. internat. Edit.
/
Vol. 9 (1970) J No. 11
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