Application of the dimenthyl chlorophosphite for the chiral analysis of amines, amino acids and peptides

Article abstract of DOI:10.1016/S0957-4166(99)00139-1

The title reagent for the determination of the enantiomeric excess of chiral amino acids and peptides was prepared from (-)-(1R,2S,5R)-menthol and PCl₃. Its use as a chiral derivatizing agent for the determination of the enantiomeric content of

Full text of DOI:10.1016/S0957-4166(99)00139-1

Pergamon
Tetrahedron: Asymmetry 10 (1999) 1729–99
Application of the dimenthyl chlorophosphite for the chiral
analysis of amines, amino acids and peptides
∗
Oleg I. Kolodiazhnyi, Oleg M. Demchuk and Alexander A. Gerschkovich
Institute of Bioorganic Chemistry, National Academy of Sciences of Ukraine, Murmanskaya Street, 1, Kiev 253094, Ukraine
Received 29 March 1999; accepted 12 April 1999
Abstract
The title reagent for the determination of the enantiomeric excess of chiral amino acids and peptides was
prepared from (−)-(1R,2S,5R)-menthol and PCl3. Its use as a chiral derivatizing agent for the determination of
the enantiomeric content of amino acids and peptides by 31P NMR is described. © 1999 Elsevier Science Ltd. All
rights reserved.
1. Introduction
The determination of the enantiomeric purity of chiral amino acids and control of their racemization
during peptide synthesis is an important process in the theoretical and practical study of asymmetric
1,2
synthesis. Different methods (polarimetry, chiral GC and HPLC, NMR in chiral medium, etc.) for ee
3,4
determination of these compounds have been described. Among these, the formation of diastereomers
through the use of chiral derivatizing agents and determination of their ratio by NMR is a very useful and
practical technique. Mosher’s reagent, α-methoxy-α-(trifluoromethyl) phenylacetic acid, is currently the
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most widely employed in this case.
There is a continuing need for the development of new chiral derivatizing agents for the determination
of enantiomeric purities of amino acids and peptides potentially superior to Mosher’s reagent. In this
work we describe dimenthyl chlorophosphite as a convenient chiral derivatizing reagent. This reagent is
convenient in that the phosphorus atom bearing two chiral menthyl groups is symmetric, therefore either
retention or inversion at phosphorus during derivatization yields the same diastereomer (Scheme 1).
It is necessary to note that, although several chiral P(III) and P(V) chlorides have been examined
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previously as chiral derivatizing agents for alcohols, thiols, amines, and diols, no method has been
developed for the ee determination of amino acids and peptides.
The dimenthyl chlorophosphite can be prepared by reaction of PCl with (−)-(1R,2S,5R)-menthol in
3
ether at −20 -→ +20°C. This reagent is stable for weeks under an inert atmosphere and can be purified by
∗
Corresponding author. E-mail: oik@biophacs.kiev.ua
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00139-1

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distillation under vacuum. The preparation of dimenthyl chlorophosphite was accomplished according to
the synthetic route outlined in Scheme 1.
Scheme 1.
The dimenthyl chlorophosphite reacts easily with RNH compounds. Very large differences in the
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31
P NMR chemical shift (∆δ) for the diastereomeric phosphonamides were observed allowing accurate
integration and quantitative determination of the diastereomeric ratios. Analysis of the diastereomeric de-
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rivatives by P NMR spectroscopy and the results obtained with different racemic and enantiomerically
enriched amino acids or peptides are summarized in Table 1. An excellent agreement in all cases, with the
ee measured using other analytical methods, has been found. The 31P NMR spectroscopic observations
reveal the effectiveness of the dimenthyl chlorophosphite for the determination of enantiomeric ratios in
31
mixtures of racemic or enantiomerically enriched amino acids. The P NMR spectra show well separated
singlets corresponding to the two diastereomers (Fig. 1a). The enantiomeric purity can be accurately
measured, and no kinetic resolution has been observed. Indeed, the diastereomeric pairs of derivatives
were always observed in the case of racemic amino acids, and integration of the signals corresponded
to the expected 50:50ꢀ2% ratios. A range of enantiomerically enriched mixtures of the (D)- and (L)-
leucine enantiomers [(D):(L)=5:95, 20:80, 30:70, 40:60, 50:50] has been prepared. Integration of the
NMR signals of these mixtures corresponded to the expected proportions with average deviation of
ꢀ
2–5% (Fig. 1b).
One of the most useful characteristics of dimenthyl chlorophosphite is the substantial difference in P
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NMR chemical shifts (∆δ) exhibited by the resulting diastereomeric amidophosphites. The advantage
of this reagent is the possibility to discriminate diastereomers even with a significant distance of the
stereogenic center from the phosphorus atom (up to 10–11 atoms) (Table 1, compound 7a). Mosher’s
acid in analogous conditions can discriminate an asymmetric center at a distance of 5–6 atoms from the
CF group.
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In view of the easy oxidation of trivalent phosphorus compounds in air, it is desirable to perform
the spectroscopic studies of compounds directly after reaction completion. The oxidation of derivatized
products by oxygen or elemental sulfur gives stable P(V) derivatives, which can also be used for the ee
determination (Fig. 1c and Table 1, compounds 1b, 7b).
In conclusion, the new chiral derivatizing agent, dimenthyl chlorophosphite, is accessible, versatile
and an effective derivatizing agent for the determination of the enantomeric excess of amines, amino
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acids and peptides bearing a free amino group. It is easily prepared from cheap starting compounds and
reacts readily with a wide range of amines, amino acids and simple peptides bearing free amino groups.
2. Experimental
Commercially available chiral (−)-(1R,2S,5R)-menthol of 98% enantiomeric purity was used in this
work. Toluene was dried with phosphorus pentoxide and kept over sodium. Amino acid esters and
peptides were prepared according to standard procedures3b or were obtained from commercial sources.

O. I. Kolodiazhnyi et al. / Tetrahedron: Asymmetry 10 (1999) 1729–99
1731
Table 1
Chiral analysis of some amines, amino acids and peptides
Figure 1. The 31P NMR spectra of: (a) derivatized peptide 6a; (b) enantiomerically enriched mixtures of the derivatized leucine
ester 4a; (c) derivatized peptide 7b

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O. I. Kolodiazhnyi et al. / Tetrahedron: Asymmetry 10 (1999) 1729–99
The 31P NMR spectra were recorded on a JEOL-90Q (40.5 MHz) and a Varian VXR-300 MHz (121
MHz) spectrometer using 85% H PO as external standard.
3
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2.1. Preparation of dimenthyl chlorophosphite
A solution of phosphorus trichloride (1.35 g, 0.01 mol) in dry diethyl ether (25 ml) was added
dropwise with stirring under argon to a solution of (−)-(1R,2S,5R)-menthol (3.1 g, 0.2 mol) and 4 ml
of triethylamine in 25 ml of diethyl ether at −20°C. The mixture was stirred for 2 h at room temperature.
Then the triethylamine chlorohydrate was filtered off and the solvent was evaporated under reduced
pressure. The residue was purified by distillation under vacuum. Yield of 80%, bp 125°C (0.02 mmHg);
3
1
3
[
α]D −83.8 (3.6, toluene); P NMR (δ, ppm, CDCl ): 145 (t, J 10 Hz); found (%): Cl 7.81;
3
PH
C H ClO P calculated (%): Cl 7.62.
20
38
2
2
.2. Procedure for the determination of the enantiomeric purity of chiral RNH compounds with
2
dimenthyl chlorophosphite to give diastereomeric amidophosphites
A solution (5 ml) of dimenthylchlorophosphite (0.001 mol) in the appropriate solvent was introduced
into a round bottomed flask and a solution of Et N (0.002 mol) and amino acid ester or peptide (0.001
3
mol) was added slowly. After the addition the mixture was stirred at 0°C. When the formation of the
derivative was complete (∼5 min), the suspension was filtered, transferred into an NMR tube along with
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0
.1 ml of C D for locking. After that the P NMR spectrum was recorded.
6 6
References
1. (a) Kolodiazhnyi, O. I. Tetrahedron: Asymmetry 1998, 9, 1279; (b) Kolodiazhnyi, O. I. New Achievement in Asymmetric
Synthesis of Organophosphorus Compounds. In Advances in Asymmetric Synthesis; Hassner, A., Ed.; JAI: Greenwich, CT,
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998; Vol. 3, pp. 273–357.
. Valentine Jr., D. Asymmetric Synthesis; Morrison, J. D.; Scott, J. W., Eds.; Academic Press: New York, 1984; Vol. 4, pp.
67–312.
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. (a) Kamp, D. S. Racemization in Peptide Synthesis. In The Peptides. Analysis, Synthesis, Biology; Gross, E.; Meienhofer,
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. Takeuchi, Y.; Itoh, N.; Satoh, T.; Koizumi, T.; Yamaguchi, K. J. Org. Chem. 1993, 58, 1812–1820.
. Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543.
. (a) Brunel, J. M.; Faure, B. Tetrahedron: Asymmetry 1995, 6, 2353–2356; (b) Brunel, J. M.; Pardigon, O.; Maffel, M.;
Buono, G. Tetrahedron: Asymmetry 1992, 3, 1243–1246; (c) Alexakis, A.; Mutti, S.; Normand, J. F.; Mangeney, P.
Tetrahedron: Asymmetry 1990, 1, 437–440; (d) Alexakis, A.; Mutti, S.; Mangeney, P. J. Org. Chem. 1994, 59, 3326–3334.
. We also used dimenthyl chlorophosphite for the chiral analysis of alcohols and thiols. See: Kolodiazhnyi, O. I.; Demchuk,
O. Submitted for publication. The determinations of ee of some alcohols are shown in the table:
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