Tritium-labelled amino acids find wide application in both chemistry and biochemistry(2) and are therefore in much demand. They are usually prepared by ...
Journal of Labelled Compounds and Radiopharmaceuticals
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VoL. XII, No. 2
265
TRITIUM NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY. PART IV[zef. (111. DISTRIBUTION OF TRITIUM IN [G-3H] PHENYLALANINE AND OTHER AMINO ACIDS Jasim M.A. Al-Rawi, John A. Elvidge, and John R. Jones, Chemistry Department, University of Surrey, Guildford, GU2 5XH. Victor MIA. Chambers and 8 . Anthony Evans, The Radiochemical Centre Ltd., Amersham, Buckingham HP7 9LL Received on ITovember 12, 1975
SUMMARY
The usefulness of 3H n.m.r. spectroscopy as an analytical tool for the determination of tritium distribution in both generally and specifically labelled compounds is illustrated by reference to the results for phenylalanine and several other amino acids. Key words:- Tritium n.m.r., phenylalanine, tyrosine, tryptophan, proline INTRODUCTION Tritium-labelled amino acids find wide application in both chemistry and biochemistry(2)
and are therefore in much demand.
They are usually prepared by catalytic reduction of unsaturated precursors(3), catalytic halogen-tritium replacement(4), or metal-catalysed heterogeneous exchange(5).
The first two methods
usually lead to specific labelling and the third to a generally labelled product.
For many purposes it is necessary to know the
distribution of the label in the compound before embarking on e.g. biochemical experiments, and hitherto this has required stepwise degradation and counting of the isolated products.
Not only is
the process tedious and time consuming but it is only as reliable as the integrity of the reactions employed.
H ' N.m.r. ( 6 ) however
provides a rapid, accurate and non-destructive method of determining both positions and extent of tritium labelling. This is because 0 1976 by John WiZey & Sons, Ltd.
J. At-Raw;,
266
J . EZuidge, J . Jones, V. Chambers and E. Evans
both chemical shifts and intensities of signals have the same significance as in H '
n.m.r.
spectroscopy(7) and the wealth of
information available from the latter field is immediately applicable to the interpretation of 3H n.m.r. spectra. Here we report our detailed findings on [ G - 3H] phenylalanine and other amino acids. RESULTS AND DISCUSSION Phenylalanine [G- 3H] Phenylalanine, prepared by platinum catalysed exchange
of phenylalanine with tritiated water at 135OC for 18h, has long been available.
The pattern of labelling has been established by
degradation and counting, which incidentally took
z. 1 tnan-month(8).
Recently(9) it was reported that the tritium in this product was confined to the aromatic ring, with 4 5 % in the para-position and the rest in the ortho-positions. This conclusion is now shown to be erroneous (8). The 3H n.m.r. spectrum of the [ G - 3 Hlphenylalanine (Figure l), recorded with 1H decoupling, shows a tritium line from each position in the molecule (excluding the carboxyl and amino groups) and hence that the label is indeed generally distributed. assignments follow from the established H ' and the results are summarised in Table 1.
The
spectrum of phenylalanine(l0) It is apparent that on
average 26% of the label is in the side chain, mostly at the 8positions, and 7 4 % in the ring, fairly equally distributed.
The
total time taken for a determination (as given in Table 1) was little more than the spectral acquisition time of 8.5h for a 5 4 mCi sample. It is noteworthy that the chemical shifts of the two anisochronous B-methylene hydrogens are available by inspection as also are those of the ring hydrogens. before, even from the H'
These latter shifts have not been derived spectrum of phenylalanine(l1) measured at
220 MHz, although this did indicate that the 2-hydrogens gave rise
to the highest field of the ring signals.
3
T r i t i u m in 1 0 HI Phenglulanfne and other Anrhw Acids
I
I 5
I
7
6
267
I 3
I h
6
ppm
Figure 1
Table 1.
3H N.m.r.
results for [G-3Hlphenylalanine in d6-DMSO
at 96 MHz with ’H decoupling
Chemical shifts 6 PPm
3.06 3.22
A s sigm e n t
1
I
Relative Intensity % (different samples)
14.4, 12:7, B-CH2
3.90 7.34
26.6,
27.4,
7.39 7.43
29.3, 28.5,
11.3, 13.0
J . EZvage, J . Jones, V . Chambers and E. Evans
J. AZ-Raw&
268
1 4-3HI Phenylalanine This specifically labelled compound was prepared by catalytic tritiodehalogenation of p-chlorophenylalanine and examined as before, but using a deuterium oxide solution. The 3H spectrum showed a strong line at 67.39 from the E - ~ H ,together with minor lines at 67.25 and 6.86 from unknown impurities. concentration in deuterium oxide ,
At a similar the 3H spectrum of [ G-3HI phenyl-
alanine provided lines at 67.36, 7.40, and 7.44 from the 0-, p-, and E - ~ Hrespectively, confirming the deductions. p-Fluoro[G-3Hlphenylalanine The 3H spectrum (with H' decoupling) (Figure 2) resembled that of [G- 3Hlphenyfalanine in the aliphatic region but showed four lines
I 9
I 8
I 7
I
6
I 5
I 4
I 3
I 2
I 1
1
0
6 p.p.m.
Figure 2
of roughly equal intensity in the aromatic region. Since on average J(o-lHF) is 9. 8.5 Hz and J(;- 1HF) is z. 7.0 Hz (12), 1 and knowing that J(3HX) = 1.06664 J ( HX) (l), it was possible to assign the higher field pair of lines (centred at 67.17) with
Tritium in 1 0 3 H I PhenyklaZmbine and other Amino Acids
269
-J
9.7 Hz to the (ortho) 3,5-3H, and the other doublet with 2 = 5.3 Hz to the 2,6-3H. No other pairing gave reasonable values for the coupling constants.
The results are summarised in Table 2.
Again
it is noteworthy that the chemical shifts could not be extracted from the H'
Table 2 .
spectrum measured at the same field strength (90 MHz).
3H N.m.r.
results for 2-fluoro [G-3Hlphenylalanine
' decoupling in D20 at 96 MHz with H
Chemical shifts 6 PPm
3.22
Relative intensity %
I
3.09
B-CH2
11.3 (equally distributed]
-CH
9.4
7.17 (doublet, 2 9.7 Hz)
3,5-H
37.8
7.33 (doublet, 2 5.3 Hz)
2,6-H
41.5
3.93
[
Assignment
Q
2,6-3H1Tyrosine With 1H decoupling, the 3H spectrum showed a single line at
67.20, in accord with the labelling expected from tritiodeiodination of 2,6-di-icdo-tyrosine. [3,5-3HlTyrosine Measured as in the previous case, the 3H spectrum showed a singlet at 66.90 from the more shielded tritons ortho to the hydroxyl group. method
-
Again, the complete integrity of the labelling
tritiodeiodination of 3,5-di-iodotyrosine
-
was
demonstrated unequivocally. t5-
3HI Tryptophan As
expected for specific labelling at the 5-positionD
270
J. A l - R d ,
J. E l v i d g e , J . Jones, V. Chambers and E. Evans
achieved by tritiodebromination of 5-bromotryptophan, the
3 H
spectrum showed a singlet at 67.01 when measured with H' decoupling.
Without H' decoupling, the signal appeared as a
(distorted)triplet, as a result of 2-coupling of the triton to a proton on each side at the 4- and 6-positiont with 2
z. 7 Hz.
There were signs of further doublet splitting from E-coupling to the 7-proton, with 2
z. 2 Hz.
Thus the assignment of the
triton signal was fully confirmed. [ G-3H1 Tryptophan
The 1H decoupled 3H n.m.r. spectrum of this sample in d6-DMSO showed signals in the region 62.29 to 3.48 from the labelled C-H s of the side chain amounting to 8.25% of total tritium content. Unfortunately the spectral acquisition time had to be curtailed and so the intensity was too low for the lines to be properly resolved
from base-line noise.
At lower field, there was a line from label
at the 2-position of the ring, with 66.65 (6.6%), and then four lines in the aromatic region, as expected, with 67.07 (18.1%), 7.16 (19.8%), 7.30 (17.0%), and 7.44 (22.0%).
Finally to lower
field at G . 68.3 was a broadened signal, presumably from labelled NH (8.25%). 3HI Proline
[ 3,4-
This compound was prepared by catalytic reduction of A3*4
-
dehydroproline with tritium and hydrogen gas, a procedure which might be expected to yield a *-3,4-ditritiated
proline.
However,
experience with e.g. the hydrogenation with tritium of crotonic acid (13) or of 4-isopropylidene-2-phenyloxazolone (for labelled valine) (14) suggested that some non-specific labelling might be encountered. The 3H n.m.r. spectrum of the labelled proline in deuterium oxide, observed with H ' decoupling, showed strong doublets with
Tritium i n f O ' H 3 PheqjZaZanine and other Amino A c i d s
271
J _ 8 . 2 Hz at 61.96 and 2 . 3 2 , chemical shifts consistent with a
doubly-labelled
cis
[
3,4-3H21proline.
In addition, there were
single lines at each origin position, evidently from tritons present in singly-labelled $3- 3HI and 14-3HI proline species. There were also two weak lines at 63.24 and 3 . 3 2 from label in the 5-methylene group by (I) (see Ref. 15).
-
in the 61 and S 2 positions as indicated In the precursor dehydroproline the 5-
methylene group is an allylic methylene and so subject to exchange.
The 3H n.m.r. spectrum indicated that this exchange is non-stereospecific and only takes place to a small extent.
Interestingly there
is no labelling at the 2 - ( a ) position in the tritiated proline. Comparison with the data for the fully analysed 31'
spectrum of
proline at various pH in deuterium oxide, measured at 2 5 0 MHz (16), facilitated the assignments given in Table 3 .
The results demonstrate
that the hydrogenation of A 3 4-dehydroproline under specified conditions occurs on the face of the molecule opposite to the carboxyl group and that side reactions do not proceed extensively. Percentage labelling results (b) in Table 3 were obtained after the sample solution at high radioactive concentration has been kept at 2OC for 1 month.
On storage, self-decomposition may cause
J. Al-Rmi,
272
J. Elvidge, J. Jones, V . Chambers and E. Evans
t h e l a b e l t o be l o s t s l o w l y from t h e 3 , 4 - p o s i t i o n s e q u a l l y w i t h t h e tritium a p p e a r i n g a s an unknown i m p u r i t y (62.52).
Table 3 .
r e s u l t s f o r [3,4-3H]proline 1 96 MHz w i t h H d e c o u p l i n g
3H N . m . r .
Chemical s h i f t s 6 PPm
A s s i g nmen t
i n D20 a t
Relative i n t e n s i t y , % (a) (b)
i n (I)
1.96
48.6
43.9
2.32
45.7
41.3
1
3.24
3.32 unknown
2.52
4.6 4.3
4.1 1.4
6.1
EXPERIMENTAL The samples, m o s t l y 25-50 m C i of h i g h s p e c i f i c a c t i v i t y (from t h e Radiochemical C e n t r e ) , w e r e t r a n s f e r r e d t o t h e s t a t e d d e u t e r a t e d s o l v e n t ( t o p r o v i d e t h e l o c k ) c o n t a i n i n g TMS o r DSS a s a p p r o p r i a t e (7), and s e a l e d i n c y l i n d r i c a l microcells (100 pl; Wilmad) which were t h e n i n s e r t e d i n t o s t a n d a r d 5mm. n.m.r. c a p s w e r e added.
tubes:
The 3H spectra w e r e o b t a i n e d a t 25+1° w i t h a
Bruker WH90 p u l s e s p e c t r o m e t e r o p e r a t i n g a t 96 MHz.
The p u l s e
width was u s u a l l y 2.5 ps, and t h e r e p e t i t i o n i n t e r v a l 1.7s.
l o 4 - 4x104 T r a n s i e n t s w e r e a c q u i r e d i n t o 8K c h a n n e l s and F o u r i e r t r a n s f o r m e d t o p r o v i d e a s p e c t r a l d i s p l a y of w i d t h 1280 Hz. ACKNOWLEDGEMENTS
W e thank t h e S.R.C.
and The Radiochemical C e n t r e f o r s u p p o r t
and t h e D i r e c t o r of t h e l a t t e r , D r . W.P. Grove, f o r p e r m i s s i o n t o publish.
W e also thank t h e U n i v e r s i t y of Basrah, I r a q , f o r s t u d y
l e a v e t o D r . Al-Rawi.
3 Tritium in [G HI Pheriylatanine and other Amino Acids
273
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