New chiral derivatizing agents: Convenient determination of absolute configurations of free amino acids By 1H NMR

Article abstract of DOI:10.1021/ol8028719

The chiral carbonate reagents 5 allow for the direct and unambiguous determination of the absolute configurations of a wide range of free amino acids using ¹H NMR. By using a ~3:1 mixture of (S)-5 and (R)-5, absolute configurations of the corresponding carbamates are determined by only analyzing the nitrogen protons.

Full text of DOI:10.1021/ol8028719

ORGANIC
LETTERS
2009
Vol. 11, No. 4
911-914
New Chiral Derivatizing Agents:
Convenient Determination of Absolute
Configurations of Free Amino Acids by
1H NMR
Michio Kurosu* and Kai Li
Department of Microbiology, Immunology, and Pathology, College of Veterinary
Medicine and Biomedical Sciences, Colorado State UniVersity,
1682 Campus DeliVery, Fort Collins, Colorado 80523-1682
michio.kurosu@colostate.edu
Received December 12, 2008
ABSTRACT
The chiral carbonate reagents 5 allow for the direct and unambiguous determination of the absolute configurations of a wide range of free
amino acids using 1H NMR. By using a ∼3:1 mixture of (S)-5 and (R)-5, absolute configurations of the corresponding carbamates are determined
by only analyzing the nitrogen protons.
Isosteric replacement of a scissile peptide bond represents a
viable and popular approach in the rational design of
peptidomimetics, which find applications as drugs.1 To date,
numerous synthetic methods for the preparation of optically
active unnatural amino acid building blocks have been
reported, and development of efficient synthetic methods for
unnatural amino acids is still a subject of interest in modern
organic synthesis.2 In synthetic studies on such molecules,
the unambiguous determination of absolute configurations
of target molecules often requires comparison of physical
properties of pure authentic samples or their derivatives. An
indirect method via chiral derivatizing agents (i.e., Marfey’s
reagent) and subsequent HPLC analysis is a standard for
determination of the absolute configurations or enantiomeric
purities of amino acids.3 On the other hand, the methods for
determination of absolute configurations of free amino acids
based on NMR analysis are limited, due to the lack of (1)
proper chiral derivatizing reagents which react quantitatively
with free amino acids in water containing solvents and (2)
understanding of conformational preference of amino acid
derivatives.4 In addition, it is not possible to unambiguously
determine the absolute configuration of chiral nonracemic
amines or alcohols in a single chemical derivatization step
with NMR spectroscopic analysis.5 We now report a con-
(1) (a) Vagner, J.; Qu, H.; Hruby, V. J. Curr. Opin. Chem. Biol. 2008,
12, 292. (b) Aguilar, M.-I.; Purcell, A. W.; Devi, R.; Lew, R.; Rossjohn,
J.; Smith, A. I.; Perlmutter, P. Org. Biomol. Chem. 2007, 5, 2884.
(2) (a) Perdih, A.; Dolenc, M. S. Curr. Org. Chem. 2007, 11, 801. (b)
Ager, D. J.; Fotheringham, I. G. Curr. Opin. Drug Disc. DeVel. 2001, 4,
800. (c) Gentilucci, L.; Tolomelli, A.; Squassabia, F. Curr. Med. Chem.
2006, 13, 2449. (d) Ager, D. J. Curr. Opin. Drug Disc. DeVel. 2002, 5,
892. (e) Ager, D. J.; Fotheringham, I. G. Curr. Opin. Drug DiscoV. DeVel.
2001, 4, 800.
(3) (a) Marfey, P. Calsberg Res. Commun. 1984, 49, 591. (b) Bhushan,
R.; Bru¨ckner, H. Amino Acids 2004, 27, 231.
(4) (a) Seco, J. M.; Quio`oa´, E.; Riguera, R. Chem. ReV. 2004, 104, 17.
(b) Lpez, B.; Quio, E.; Riguera, R. J. Am. Chem. Soc. 1999, 121, 9724. (c)
Hulst, R.; de Vries, K.; Feringa, B. L. Angew. Chem., Int. Ed. Engl. 1992,
31, 1092. (d) Parker, D. Chem. ReV. 1991, 91, 1441.
10.1021/ol8028719 CCC: $40.75
Published on Web 01/26/2009
2009 American Chemical Society

venient and reliable method for the determination of the
absolute configurations of free amino acids via a single
chemical operation followed by H NMR analysis of the
rt), Brønsted bases (i.e., NH4OH (in aq THF), LiOH (in aq
THF-MeOH), and DBU (in aq THF) at rt), and a wide
variety of nucleophiles. Analysis of a lowest energy con-
former of the acetate of 1 via a single-crystal X-ray analysis
revealed that the -COO- linkage and the ether methine
proton formed by two planar chloro-substituted benzenes
showed a deviation of ∼20° out of a preferential common
plane (Figure 1). Extensive 2D-NOESY experiments of the
esters of 1 supported that the conformation in solution
(CDCl3) agreed with that observed in the solid state.7 There
1
nitrogen protons (-OCONH-) of the resulting carba-
mate derivatives. To our knowledge, this is the first observa-
tion that the nitrogen proton can reliably be utilized for
determination of the absolute configurations of nonracemic
1
chiral primary amines by H NMR.
We have developed an unsymmetrical (2,6-dichloro-4-
methoxyphenyl)(2,4-dichlorophenyl)methanol (1, Scheme 1)
Scheme 1. Resolution of rac-1 and Syntheses of the Chiral Carbonate (S)-5 and (R)-5
which is stable against Brønsted and Lewis acids (i.e., 15%
TFA, 30% HF, 2 N HCl, HBr/AcOH, TiCl4, ZnCl2, AlCl3,
B(C6F5)3, BCl3, BBr3, BF3·OEt2, TMSOTf, and La(OTf)3 at
must be a significant electronic repulsion between the
o-chloro atoms in the two phenyl rings. The o-chloro atom
in the dichlorobenzene ring (the phenyl ring A in Figure 1)
is directed toward the carbonyl ester plane. Thus, the chloro
atoms at the ortho positions in both phenyl groups hinder
nucleophilic attack at the ester carbonyl from either the re-
or si-face. In addition, the 3,5-dichloro atoms in the anisole
moiety (the phenyl ring B) attenuate the electron-donating
character of the methoxy group. Therefore, it was concluded
that the esters derived from 1 would exhibit unusual stability
against bases, nucleophiles, and acids.8 Based on these
conformational analyses of the esters of 1, we speculated
that the optically pure (S)- and (R)-(2,6-dichloro-4-methox-
yphenyl)(2,4-dichlorophenyl)methanol, (S)-1 and (R)-1, would
be very useful chiral derivatizing agents for determination
of absolute configurations of chiral nonracemic carboxylic
acids and amino acids.
All attempts to selectively reduce (2,6-dichloro-4-meth-
oxyphenyl)(2,4-dichlorophenyl)methanone via chiral reduc-
ing agents9 or to resolve diastereomers of rac-1 using known
chiral derivatizing agents did not provide satisfactory selec-
tivity or separation.10 Gratifyingly, (S)-1 and (R)-1 could
readily be obtained through the resolution of rac-1 using the
glucose derivative 211 as illustrated in Scheme 1.12 Absolute
Figure 1.
Preferred conformations of the esters of 1.6
912
Org. Lett., Vol. 11, No. 4, 2009

configurations of (S)-1 and (R)-1 were established through
the advanced Mosher method and NOESY correlations of
the L-alanyl carbamate derivative.13
reactions of the primary amines with (S)-5 and (R)-5
furnished the corresponding carbamates in quantitative yield
1
(determined by H NMR) regardless of the structure of the
1H NMR analyses of the (S)-3-methylpentanoic acid esters
of (S)-1 and (R)-1 revealed that the syn-substituent with
respect to the dichloromethoxybenzene moiety (the phenyl
ring B in Figure 1) in the ester derived from (S)-1 was
downfield-shifted due to the anisotropy effects of the phenyl
ring; a large enough chemical shift nonequivalence of the
ꢀ-methyl group between the esters of (S)- and (R)-1 was
observed (i in Figure 1).14 Encouraged by these observations
of the esters of 1, we synthesized a series of carbamates by
the treatment of free L- and D-amino acids with the carbonate
(S)-5, which was synthesized with N,N′-disuccinimidyl
carbonate (DSC) in the presence of iPr2NEt in acetone-water
(3/1) (Scheme 1). Conformational analyses of the carbamates
of amino acids via 2D-NOESY experiments revealed that
the R-residues of L-amino acid derivatives exhibited a
correlation with the diphenyl methine proton (examples of
L- and D-Ala are shown in Figure 2). In addition, the
R-residue or the protection or nonprotection of carboxylic
acid group. The indole, imidazole, alcohol, and phenol groups
did not react with 5 under the conditions used for the
carbamation reactions. Thus, the carbonates 5 serve as
selective chiral carbamate reagents.15
Selected examples of the 1H NMR analyses of the nitrogen
protons of carbamate synthesized from L- (or (S)-)configu-
ration substrates are summarized in Table 1. The nitrogen
Table 1. Chemical Shift Difference of the -O(CO)NH-
protona
δ (S),
δ (R)
∆δ
(S-R)
entry
substrate
products
1
2
3
4
5
6
7
8
6a: R1 ) CH3, R2 ) CH3
9a, 10a
9b, 10b
5.52, 5.41 +0.11
5.45, 5.38 +0.07
5.56, 5.54 +0.02
5.63, 5.55 +0.08
6.10, 6.04 +0.06
5.57, 5.47 +0.10
5.85, 5.83 +0.02
5.60, 5.50 +0.10
5.71, 5.53 +0.18
5.51, 5.42 +0.09
5.48, 5.41 +0.07
5.50, 5.45 +0.05
t
6b: R1 ) Bu, R2 ) CH2Ph
6c: R1 ) CH3, R2 ) (CH2)2CO2CH3 9c, 10c
6d: L-Ala
6e: L-Hisb
6f: L-Phe
6g: L-Thrb
6h: L-Trpb
6i: L-Tyrb
6j: L-Lysc
6k: L-Val
9d, 10d
9e, 10e
9f, 10f
9g, 10g
9h, 10h
9i, 10i
9j, 10j
9k, 10k
9l, 10l
9m, 10m 5.53, 5.49 +0.04
9n, 10n
9o, 10o
9p, 10p
9q, 10q
9r, 10r
Figure 2. Plausible preferred conformers of the carbamates of
(S)-1.
9
10
11
12
13
14
15
16
17
18
a
t
6l: R1 ) H, R2 ) Bu
6m: R1 ) H, R2 ) CH2CN
6n: R1 ) H, R2 ) CH2CH3
6o: R1 ) H, R2 ) CH2-2-furyl
6p: R1 ) H, R2 ) CH2-cyclohexyl
7a
carbamate nitrogen proton exhibited strong NOESY correla-
tions with the R-proton and the diphenyl methine proton.
Therefore, the nitrogen proton of the L-amino acid derivatives
should be located toward the dichloromethoxybenzene
moiety (the phenyl ring B in ii in Figure 2); thus, the nitrogen
proton is shifted downfield due to the anisotropy effect in
5.53, 5.42 +0.11
5.66, 5.61 +0.05
5.37, 5.28 +0.09
5.99, 5.53 +0.46
5.79, 5.68 +0.11
8a: R3 ) Ph
1H NMR spectra were recorded on a 300 or 400 MHz NMR
spectrometer using CDCl3 as solvent. b The hydroxy or amino group in the
residue is intact. c The Nꢀ group of lysine formed the carbamate: its ∆δ (δS
- δR) value ) (0.
1
the H NMR spectra. On the contrary, the protons of the
R-residue and nitrogen proton of the carbamates derived from
D-amino acids did not show NOESY correlations with the
diphenyl methine proton, but the proton(s) of the R-residue
exhibited a NOESY correlation with the dichlorobenzene
proton (the phenyl ring A in iii). These observations in the
NOESY experiments led to the preferred conformation of
the D-amino acid derivatives as illustrated in iii (Figure 2);
thus, the nitrogen protons of D-Ala-(S)-5 carbamates may
not be suitably influenced by the anisotropic effect of the
dichloromethoxybenzene ring (the phenyl ring B).
In order to generalize the use of (S)-5 and (R)-5 for the
determination of absolute configurations of nonracemic chiral
primary amines by 1H NMR, over 50 carbamate derivatives
were synthesized from natural and unnatural free amino acids
and their esters and from amino alcohols. In all cases,
protons of carbamates derived with (S)-5 were shifted
downfield relative to those obtained with (R)-5. In all cases,
good signal separations of the carbamate nitrogen protons
were observed in the 1H NMR spectra. It is worth mentioning
that unlike other carbamate derivatives, concentration-
dependent changes in chemical shift of the nitrogen proton
of our carbamates have never been observed in CDCl3. This
could be attributed to the enhancement of the hydrophobic
physicochemical property of free amino acids via incorpora-
tion of 1. Due to the fact that (1) the absolute configuration
of amino acids can conveniently be determined solely on
1
the basis of H NMR analysis of the carbamate nitrogen
protons (Table 1), full assignments of all protons are not
Org. Lett., Vol. 11, No. 4, 2009
913

necessary,5,16 and (2) chemical shifts of the nitrogen protons
of carbamates are largely separated from the other protons,
we envisioned using a mixture of (S)-5 and (R)-5 (a ∼3:1
ratio) to determine the absolute configurations of nonracemic
chiral primary amines via a single chemical operation and
In conclusion, we have developed unprecedented chiral
derivatizing agents that allow for the determination of the
absolute configurations of a wide range of amino acids by
1
only analyzing the carbamate nitrogen protons in H NMR
spectra. The carbonate reagents (S)-5 and (R)-5 react
selectively with alkylamines in aqueous conditions and form
the corresponding carbamates in quantitative yield. Signal
separations of the carbamate nitrogen protons are large
enough, and thus, as summarized in Figure 3, the absolute
configurations of nonracemic amino acids can be determined
unambiguously by the 1H NMR analysis of the relative ratio
of the nitrogen protons of carbamates, which are synthesized
quantitatively by the carbamation reactions using a ∼3:1
mixture of (S)-5 and (R)-5. The method using these new
derivatizing agents appears to have a significant advantage
over currently available methods for determination of the
1
subsequent H NMR analysis. In this method, the relative
ratio of the signal area of L-amino acid derivatives should
afford L-(S)/L-(R) ratio of ∼3:1,17 whereas D-amino acid
derivatives are expected to give a D-(R)/D-(S) ratio of ∼1:3.
As summarized in Figure 3, the method for determination
1
absolute configuration of free amino acids via H NMR
spectroscopy.4 The further scope and limitations of this
method will be reported elsewhere.
Acknowledgment. We thank Colorado State University
for generous financial support. We also thank Dr. Crick
(Colorado State University) for useful discussions.
Supporting Information Available: Experimental pro-
cedures and copies of NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL8028719
(9) For example, asymmetric reduction of (2,6-dichloro-4-methoxyphe-
nyl)(2,4-dichlorophenyl)methanone via a chiral oxazaborolidine provided
a 2.5:1 mixture of alcohols.
Figure 3. Chemical shifts of the nitrogen protons of carbamates
derivatized using a ∼3:1 mixture of (S)-5 and (R)-5 (400 MHz
(10) Kasai, Y.; Taji, H.; Fujita, T.; Yamamoto, Y.; Akagi, M.; Sugio,
A.; Kuwahara, S.; Watanabe, M.; Harada, N.; Ichikawa, A.; Schring, V.
Chirality 2004, 16, 569.
spectra).
(11) Kurosu, M.; Li, K. Unpublished data.
(12) The esters 3 and 4 could be separated via flash column chroma-
tography (SiO2, hexanes/EtOAc; see the Supporting Information).
(13) For determination of the absolute stereochemistries of (S)-1, see
the Supporting Information.
(14) For assignment of the absolute configuration of R-chiral carboxylic
acids by 1H NMR, see: (a) Freire, F.; Quinoa, E.; Riguera, R. Chem.
Commun. 2008, 35, 4147. (b) Dickins, R. S.; Badari, A. Dalton Trans. 2006,
25, 3088. (c) Yabuuchi, T.; Kusumi, T. J. Org. Chem. 2002, 65, 397. (d)
Ferreiro, M. J.; Latypov, S. K.; Quin˜oa´, E.; Riguera, R. J. Org. Chem. 2000,
65, 2658. (e) Ferreiro, M. J.; Latypov, S. K.; Quinoa, E.; Riguera, R.
Tetrahedron: Asymmetry 1997, 8, 1015. (f) Tyrrell, E.; Tsanga, M. W. H.;
Skinnera, G. A.; Fawcettb, J. Tetrahedron 1996, 52, 9841.
(15) Neither kinetic resolution nor racemization has been observed during
the carbamation reactions.
(16) For a general method for determination of the absolute configura-
tions by 1H NMR, see: (a) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa,
H. J. Am. Chem. Soc. 1991, 113, 4092.
(17) “L-(R)” denotes the L- or (S)-amino acid-(R)-1 carbamate deriva-
tive.
(18) The R,R-disubstituted amino acids utilized in this study were
synthesized according to the reported procedures; see: (a) Davis, F. A.;
Lee, S.; Zhang, H.; Fanelli, D. L. J. Org. Chem. 2000, 65, 8704.
(19) In this empirical rule, the carboxylic acid attached group is
considered as the second highest priority of the substituent groups: -NH2
> the carboxylic acid attached group > the other residue. Thus, the relative
ratio of the nitrogen protons of 13b (R-configuration) agrees with that of
the L-amino acid derivatives.
of the absolute configurations via a (S)-5/(R)-5 ratio of ∼3:1
could apply to a wide range of chiral nonracemic primary
amines; the unnatural R-amino acids, a ꢀ-amino acid, and
an amino alcohol agreed with this empirical rule (11-14 in
Figure 3). Significantly, large enough chemical shift non-
equivalences of the carbamate nitrogen protons of the R,R-
disubstituted amino acids18 were observed, and thus, the
absolute configurations of nonracemic R,R-disubstituted
amino acids can be conveniently determined by this method
(15 and 16 in Figure 3).19
(5) Absolute configurations of esters have been determined by careful
analyses of ∆δ (δS - δR) values of esters derived from both (S)- and (R)-
chiral derivatizing agents (CDA) via high-field 1H NMR spectroscopy. Thus,
two separate transformations of alcohol with CDA are necessary.
(6) A plausible conformer of the (S)-3-methylpentanoic acid ester of 1
(Figure 1) was obtained by using the semiempirical AM1 method.
(7) For the NOESY correlations of the ester of 1, see the Supporting
Information.
(8) Kurosu, M.; Biswas, K.; Narayanasamy, P.; Crick, D. C. Synthesis
2007, 16, 2513.
914
Org. Lett., Vol. 11, No. 4, 2009