Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A TEST TUBE REACTION FOR THE DETERMINATION OF THE ABSOLUTE CONFIGURATIONOF s-AMINO ACIDS V. Toome and G. Reymond Hoffmann-La Roche Inc., Chemical Research Department Nutley, New Jersey 07110 Received June 30,1975
SUMMARY 2-Methoxy-2,4-diphenyl-3(2H)-furanone
(MDPF) reacts readily with
s-amino acids to form pyrrolinone-type chromophores with long wavelength absorption maxima at 370-390 nm.
Measurement of the chiroptical
properties
of such reaction mixtures, without isolation of products, enables one to determine the absolute configuration of s-amino acids in s i t u . INTRODUCTION We have recently described the application of 2-methoxy-2,4-diphenyl3(2H)-furanone (MDPF) I as a new chromophoric reagent for the determination of the absolute configuration of e-amino acids ( I ) .
A number of chromo-
phores I I were synthesized with various s-amino acids and t h e i r ORD and CD spectra were recorded in R COOH "-CH/ I
OOH
R-CH-NH2 >
II
I
ethanol.
Without exception, the L-amino acid derivatives showed f i r s t
positive Cotton effects centered at ca 380 nm and second negative Cotton effects centered at ca 325 nm.
Within the experimental error, the ORD
and CD curves of the D-amino acid condensation products were mirror images of those of L-configuration. Copyright © 1975 by Academic Press, Inc, All rights o f reproduction in any form reserved
The chromophoric reaction between MDPF and 7S
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
an m-amino acid can be carried out in a test tube and CD spectra can be obtained from the resulting reaction mixtures without the isolation of the new derivatives I I .
This procedure provides a rapid and simple method
for the determination of the absolute configuration of s-amino acids in solution (2). A number of methods for the preparation of chromophoric derivatives of m-amino acids have been described in the l i t e r a t u r e of t h e i r disadvantages have been noted (5,6).
(3,4,5), and some
In some instances the sign
of the Cotton effect of the derivative is not only the function of the configuration, but is also dependent upon the nature of the substituents of the m-carbon.
Furthermore, derivatization is often accompanied by
partial racemization or the reagent (chromophoric or chelating) may also react with the hydroxy or sulfhydryl groups (6,7). EXPERIMENTAL A.
Reagents MDPF was obtained from Hoffmann-La Roche Inc., Nutley, New Jersey.
Methanol (AR-grade) and dioxane (histological grade) were purchased from Fischer S c i e n t i f i c Co., Fair Lawn, New Jersey, and the amino acids from Fox Chemical Co., Los Angeles, California.
Triethylamine was obtained
from Eastman Kodak Co., Rochester, New York. B.
Method General procedure:
0.5 ml of a 0.02 M (concentration may range between
0.I and 0.002 M) of an m-amino acid in 50% methanol/water (or in 50% dioxane/water) is neutralized with 1 equivalent of triethylamine (0.025 ml of a 0.4 molar solution in methanol or dioxane).
Then 0.475 ml of a
0.0421 molar (2 equivalents) solution of MDPF in methanol (or in dioxane) is added under s t i r r i n g .
The reaction mixture is heated at 50°C for
5 minutes, cooled, and transferred into a 0.01 cm cell
(total volume of
0.5 ml required) and the CD spectra are recorded on a Durrum-JASCO Spectropolarimeter, Model ORD/CD/UV-5 between 450 and 270 nm. are d i f f i c u l t
The spectra
to obtain below 270 nm because of the high absorption of the
reagent. On a microscale, useful CD spectra (signal to noise r a t i o higher than I0) were obtained with 0.01 mg of amino acids.
In order to avoid working
with d i l u t e solutions, where the reaction is slow or incomplete, 0.01 mg of an amino acid was dissolved in 0.01 ml 50% methanol/water and reacted in a test tube with 2 equivalents of MDPF in 0.01 ml methanol containing 1 equivalent of triethylamine.
After heating at 50°C for 5 minutes, 5 ml
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Vol. 66, No. 1, 1 9 7 5
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of methanol were added and the CD spectra recorded in 5 cm c e l l s ( t o t a l volume of 2.5 ml needed). RESULTS AND DISCUSSION As previously described, ~-amino acids react with MDPF I to form N-substituted-3,5-diphenyl-5-hydroxy-2-pyrrolin-4-ones I I .
These
d e r i v a t i v e s e x h i b i t m u l t i p l e Cotton e f f e c t s between 400 and 200 nm. the case of L-amino acids, the f i r s t
In
c h a r a c t e r i s t i c Cotton e f f e c t at
ca 385 nm is p o s i t i v e and the second one at ca 325 nm is negative.
There
are three to four additional Cotton e f f e c t s between 300 and 200 nm, depending on the character of the amino acid. For many purposes, the reaction between I and ~-amino acids can be carried out in a fashion which obviates the need f o r the i s o l a t i o n of the
14
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Fig. 1
CD spectra of synthesized ( i s o l a t e d ) chromophores of D-phenylalanine (--) and L-leucine ( - - - ) and of the in s i t u reaction products of D-phenylalanine (0-0) and L-leucine (o--o) with MPDF.
77
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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VoI. 66, No. l, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chromophores I I ,
and allows one to obtain useful c h i r o p t i c a l
spectra with
the reaction mixture i t s e l f . CD data obtained with such reaction mixtures are summarized in Table I.
The s p e c i f i c spectra obtained with the reaction mixtures of
D-phenylalanine and L-leucine are depicted in Figure I. The f i r s t
two c h a r a c t e r i s t i c Cotton e f f e c t s of the chromophore II
produced in s i t u are easily accessible f o r configurational purposes.
or analytical
In the presence of excess reagent (UV max in methanol at 241 nm,
= 18800 and at 308 nm, ~ = 3500), the c u t o f f wavelength (under standard conditions) is approximately 260-270 nm and the t h i r d Cotton e f f e c t i s , in most cases, d i f f i c u l t
to measure.
Only in the case of phenylalanine could
the CD spectrum be recorded between 450 and 220 nm (Figure I ) . As seen from the Table, the i n t e n s i t i e s of the f i r s t
two a n a l y t i c a l l y
accessible Cotton e f f e c t s of the reaction mixtures are ca 30-70% of those of the isolated standards ( I ) .
Since the Cotton effects are strong, these
yields are s u f f i c i e n t for in s i t u determination of the absolute configuration of s-amino acids in solution.
On a microscale, the color
reaction was successfully carried out with as l i t t l e
as 0.01 mg of amino
acids (8). The signs of measured and predicted Cotton effects (based on those of the synthesized ones) are in agreement. alanine, where the sign of the f i r s t
The only exception observed is
Cotton e f f e c t in s i t u is opposite to
that of the standard, but the sign of the second one at 325 nm remains as expected. likely,
The reason for t h i s could be due to a solvent e f f e c t , or more
to solvent induced mutarotation r e s u l t i n g from epimerization of the
carbinolamine function.
Therefore, i t is advisable to measure the f i r s t
and
the second Cotton e f f e c t .
ACKNOWLEDGEMENTS We thank Dr. M. Weigele f o r helpful discussions and Mrs. B. Wegrzynski for s k i l l f u l
technical assistance.
REFERENCES AND FOOTNOTES I. 2.
3.
Toome, V., DeBernardo, S. and Weigele, M. (1975) Tetrahedron (in press). The c h i r o p t i c a l properties of reactions of s-amino a c i ~ w i t h the amine reagent fluorescamine [Weigele, M., DeBernardo, S. L., Tengi, J. P, and Leimgruber, W. (1972) J. Amer. Chem. Soc., 9_44, 5927-5928] are presently also being investigated. Toniolo, C. and Signoz, A. (1972) Experientia, 28, 753-759.
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Vol. 66, No. 1, 1975
4. 5. 6. 7. 8.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Suzuki, T., Igarashi, K., Hase, K. and Tuzimura, K. (1973) Agr. Biol. Chem., 3__77,411-416. Polo~ski, T., Chimiak, A. and Kochman, M. (1974) Tetrahedron, 30, 641643 and older references therein. Auterhoff, H. and Hansen, J.-G. (1970) Pharmazie, 2__5_5,336-340. Kerek, F. and Snatzke, G. (1975) Angew. Chem., 8__77,133-134. Only I-2 micrograms of the synthesized chromophores (isolated) are needed for useful CD spectra.
80