Convenient synthesis of N-methylamino acids compatible with Fmoc solid-phase peptide synthesis

Article abstract of DOI:10.1021/jo050477z

N^α-Methylamino acid containing peptides exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and unavaibility of many N^α

Full text of DOI:10.1021/jo050477z

Convenient Synthesis of N-Methylamino Acids Compatible with
Fmoc Solid-Phase Peptide Synthesis
Eric Biron and Horst Kessler*
Department Chemie, Lehrstuhl II fu¨r Organische Chemie, Technische Universita¨t Mu¨nchen,
Lichtenbergstrasse 4, Garching D-85747, Germany
horst.kessler@ch.tum.de
Received March 10, 2005
NR-Methylamino acid containing peptides exhibit interesting therapeutic profiles and are increas-
ingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by
the high price and unavaibility of many NR-methylamino acids. An efficient and practical preparation
of NR-methyl-NR-(o-nitrobenzenesulfonyl)-R-amino acids without extensive purification is described.
The procedure is based on the well-known N-alkylation of NR-arylsulfonylamino esters which was
improved by using dimethyl sulfate and DBU as base. Ester cleavage is efficiently achieved by
using an SN2-type saponification with lithium iodide, avoiding racemization observed with lithium
hydroxide hydrolysis. Compatibility of the synthesized NR-methylamino acids with Fmoc solid-
phase peptide synthesis is demonstrated by using normal coupling conditions to efficiently prepare
N-methyl dipeptides. The described procedure allows the preparation of NR-methylamino acids in
a very short period of time and a rapid synthesis of N-methyl peptides using Fmoc solid-phase
peptide synthesis.
Introduction
sion of an agonist into an antagonist.8 NR-Methylamino
acid containing peptides are also potentially useful
therapeutics since N-methylation has been shown to
improve important pharmacological parameters such as
lipophilicity,9,10 bioavailability,10b proteolytic stability,9,11
conformational rigidity,5,9 and duration of action.8
Commercially available NR-methylamino acids are still
very expensive even if various methods have been
developed for the synthesis of optically active NR-meth-
ylamino acids.12 The most widely used methods are direct
NR-Methylamino acids are found in a wide range of
natural peptides and depsipeptides exhibiting a great
range of biological effects, including antibiotic,1 antican-
cer,2 antiviral,3 and immunosuppressive activity.4 Incor-
poration of NR-methylamino acids into biologically active
peptides changes the chemical and physical properties
and thus may provide important information about
backbone conformation.5 It may also result in enhanced
potency,6 new receptor subtype selectivity,7 and conver-
* Corresponding author. Tel: +49-089-289 13300. Fax: +49-089-
289 13210.
(6) (a) Tonelli, A. E. Biopolymers 1976, 15, 1615-1622. (b) Manava-
lan, P.; Momany, F. A. Biopolymers 1980, 19, 1943-1973. (c) Ron, R.;
Gilon, C.; Hanani, M.; Vromen, A.; Selinger, Z.; Chorev, M. J. Med.
Chem. 1992, 35, 2806-2811. (d) Dechantsreiter, M. A.; Planker, E.;
Matha¨, B.; Lohof, E.; Ho¨lzemann, G.; Jonczyk, A.; Goodman, S. L.;
Kessler, H. J. Med. Chem. 1999, 42, 3033-3040.
(7) (a) Laufer, R.; Wormser, U.; Friedman, Z. Y.; Gilon, C.; Chorev,
M.; Selinger, Z. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7444-7448. (b)
Laufer, R.; Gilon, C.; Chorev, M.; Selinger, Z. J. Biol. Chem. 1986, 261,
257-263. (c) Rajeswaran, W. J.; Hocart, S. J.; Murphy, W. A. J. Med.
Chem. 2001, 44, 1305-1311 and 1416-1421. (d) Lewis, I.; Bauer, W.;
Albert, R.; Chandramouli, N.; Pless, J.; Weckbecker, G.; Bruns, C. In
Peptide Revolution: Genomics, Proteomics & Therapeutics; Chorev, M.,
Sawyer, T. K., Eds.; American Peptide Society: San Diego, 2003; pp
721-722.
(1) Ebata, M.; Takahashi, Y.; Otsuka, H. Bull. Chem. Soc. Jpn. 1996,
39, 2535-2538 and references therein.
(2) (a) Jouin, P.; Poncet, J.; Difour, M.-L.; Pantaloni, A.; Castro, B.
J. Org. Chem. 1989, 54, 617-627. (b) Li, W.-R.; Jouillie´, M. M. In
Studies in natural Products Chemistry. Vol. 10. Stereoselective Syn-
thesis (Part F); Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1992; pp
241-302. (c) Ramanjulu, I. M.; Ding, X.; Joullie´, M. M. J. Org. Chem.
1997, 62, 4961-4969.
(3) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Fujii, Y.; Kizu, H.; Boyd,
M. R.; Boettner, F. E.; Doubek, D. L.; Schmidt, J. M.; Chapuis, J. C.;
Michel, C. Tetrahedron 1993, 49, 9151-9170.
(4) Wenger, R. M. Helv. Chim. Acta 1984, 67, 502-525.
(5) (a) Vitoux, B.; Aubry, A.; Cung, M. T.; Marraud, M. Int. J. Peptide
Protein Res. 1986, 27, 617-632. (b) Goodfellow, V. S.; Marathe, M. V.;
Kuhlman, K. G.; Fitzpatrick, T. D.; Cuarado, D.; Hanson, W.; Zuzack,
J. S.; Ross, S. E.; Wieczorek, M.; Burkard, M.; Whalley, E. T. J. Med.
Chem. 1996, 39, 1472-1484. (c) Takeuchi, Y.; Marshall, G. R. J. Am.
Chem. Soc. 1998, 120, 5363-5372.
(8) Mazur, R. H.; James, P. A.; Tyner, D. A.; Hallinan, E. A.; Sanner,
J. H.; Schulze, R. J. Med. Chem. 1980, 23, 758-763 and references
therein.
(9) Fairlie, D. P.; Abbenante, G.; March, D. R. Curr. Med. Chem.
1995, 2, 654-686.
10.1021/jo050477z CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/20/2005
J. Org. Chem. 2005, 70, 5183-5189
5183

Biron and Kessler
or no application to NR-methylamino acids with function-
alized side-chains and are incompatible with Fmoc solid-
phase peptide synthesis (Fmoc-SPPS) side-chain protect-
ing groups (Boc, Trt, t-Bu). Therefore, we were interested
in developing a fast, easy, and highly efficient procedure
to prepare side-chain protected NR-methylamino acids in
large quantities with the possibility of using them directly
in Fmoc-SPPS.
Methods for selective N-methylation of peptides on
solid support have been reported.20,21 The most efficient
procedure is a three-step sequence involving amine
activation by an o-nitrobenzenesulfonyl group (o-NBS),
followed by alkylation20a or Mitsonobu reaction20b,c on the
activated nitrogen and then removal of the sulfonamide
group. Three steps are added to the normal coupling/
deprotection sequence with this method and can be time-
consuming and confusing when N-methylated peptide
libraries are synthesized. Another problem is that only
very small quantities can be prepared on solid support.
It has been shown that o-NBS-amino acids are compat-
ible with side-chain protections used in Fmoc-SPPS.22 In
this paper, we report the successful synthesis of o-NBS-
protected NR-methylamino acids and their direct utiliza-
tion in Fmoc-SPPS.
FIGURE 1. Amino acid N-methylation by two different
procedures.13,14
N-methylation of NR-protected R-amino acids or R-amino
esters13 and 5-oxazolidinone formation and reduction
(Figure 1).14 Methods involving reductive amination,15
Mitsunobu reaction,16 Diels-Alder/retro-Diels-Alder se-
quences,17 and alternative procedures have also been
developed.18 Unfortunately, most of these methods are
limited to aliphatic amino acids or are characterized by
harsh reaction conditions or long synthetic sequences,
and some cause partial racemization of the sub-
strate.13f,15b,19 In general, the methods above show limited
(10) (a) Ali, F. E.; Bennet, D. B.; Calvo, R. R.; Elliot, J. D.; Hwang,
S.; Ku, T. W.; Lago, M. A.; Nichols, A. J.; Romoff, T. T.; Shah, D. H.;
Vasko, J. A.; Wong, A. S.; Yellin, T. O.; Yuan, C.; Samanen, J. M.; J.
Med. Chem. 1994, 37, 769-780. (b) Cody, W. L.; He, J. X.; Reily, M.
D.; Haleen, S. J.; Walker, D. M.; Reyner, E. L.; Stewart, B. H.; Doherty,
A. M. J. Med. Chem. 1997, 40, 2228-2240.
(11) Haviv. F.; Fitzpatrick, T. D.; Swenson, R. E.; Nichols, C. J.;
Mort, N. A.; Bush, E. U.; Diaz, G.; Bammert, G.; Nguyen, A.; Nellans,
H. N.; Hoffman, D. J.; Johnson, E. S.; Greer, J. J. Med. Chem. 1993,
36, 363-369.
Results and Discussion
The o-nitrobenzenesulfonyl group (o-NBS), first de-
scribed by Fukuyama et al.,23 has been commonly used
for selective N-alkylation.13l,20,22-24 Our strategy was to
use the o-NBS group to protect and activate the amino
function of the amino acid derivatives allowing the use
of weaker base and softer conditions for the N-methyla-
tion step. By doing every step in solution, NR-methyl-NR-
o-NBS-R-amino acids could be prepared and directly used
in Fmoc-SPPS. Therefore, three supplementary steps
on solid support could be eliminated. Our initial at-
tempts involved the direct N-methylation of NR-o-NBS-
R-amino acids (Scheme 1). Phenylalanine was protected
with o-NBS-Cl using the Schotten-Baumann proce-
dure.25 N-Methylation of NR-o-NBS-phenylalanine (1) was
achieved by the Vigneaud and Behrens procedure13b using
iodomethane in NaOH 0.5 N at 65 °C. Unfortunately, the
reaction gave a mixture of NR-o-NBS-phenylalanine
methyl ester (2a), NR-methyl-NR-o-NBS-phenylalanine
(12) Recently reviewed. Gilon, C.; Dechantsreiter, M. A.; Burkhart,
F.; Friedler, A.; Kessler, H. In Houben-Weyl Methods of Organic
Chemistry. Vol E22c. Synthesis of Peptides and Peptidomimetics;
Goodman, M., Felix, A., Moroder, L., Tonolio, C., Eds.; Georg Thieme
Verlag: Stuttgart, Germany, 2002; pp 215-291 and references therein.
(13) (a) Fischer, E.; Lipschitz, W. Ber. Dtsch. Chem. Ges. 1915, 48,
360-378. (b) Du Vigneaud, V.; Behrens, O. K. J. Biol. Chem. 1937,
117, 27-36. (c) Olsen, R. K. J. Org. Chem. 1970, 35, 1912-1915. (d)
McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51, 1915-1919.
(e) Hlava´cek, J. Poduska, K.; Sorm, F. Slama, K. Collect. Czech. Chem.
Commun. 1976, 41, 2079-2087. (f) Cheung, S. T.; Benoiton, N. L. Can.
J. Chem. 1977, 55, 906-909. (g) Hlava´cek, J.; Fric, I.; Budesinsky,
M.; Blaha, K. Collect. Czech. Chem. Commun. 1988, 53, 2473-2494.
(h) Easton, C. J.; Kociuba, K.; Peters, S. C. J. Chem. Soc. Chem.
Commun. 1991, 1475-1476. (i) Xue, C.-B.; Degrado, W. F. Tetrahedron
Lett. 1995, 36, 55-58. (j) Vedejs, E.; Kongkittingam, C. J. Org. Chem.
2000, 65, 2309-2318. (k) Di Gioia, M. L.; Leggio, A.; Le Pera, A.;
Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G. J. Org. Chem. 2003,
68, 7416-7421. (l) Prashad, M.; Har, D.; Hu, B.; Kim, H.-Y.; Repic,
O.; Blacklock, T. J. Org. Lett. 2003, 5, 125-128.
(14) (a) Ben-Ishai, D. J. Am. Chem. Soc. 1957, 79, 5736-5738. (b)
Itoh, M. Chem. Pharm. Bull. 1969, 17, 1679-1680. (c) Freidinger, R.
M.; Hinkle, J. S.; Perlow, D. S.; Arison, B. H. J. Org. Chem. 1983, 48,
77-81. (d) Williams, R. M.; Yuan, C. J. Org. Chem. 1994, 59, 6190-
6193. (e) Reddy, G. V.; Rao, G. V.; Iyengar, D. S. Tetrahedron Lett.
1998, 39, 1985-1986. (f) Aurelio, L.; Brownlee, R. T. C.; Hughes, A.
B.; Sleebs, M. M. Aust. J. Chem. 2000, 53, 425-433. (g) Luo, Y.;
Evindar, G.; Fishlock, D.; Lajoie, G. A. Tetrahedron Lett. 2001, 42,
3807-3809. (h) Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B. Org.
Lett. 2002, 4, 3767-3769. (i) Aurelio, L.; Box, J. S.; Brownlee, R. T.
C.; Hughes, A. B.; Sleebs, M. M. J. Org. Chem. 2003, 68, 2652-2667.
(15) (a) O’Donnell, M. J.; Polt, R. L. J. Org. Chem. 1982, 47, 2663-
2666. (b) O’Donnell, M. J.; Bruder, W. A.; Daugherty, B. W.; Liu, D.;
Wojciechowski, K. Tetrahedron Lett. 1984, 25, 3651-3654. (c) Ohfune,
Y.; Kurokawa, N.; Higuchi, N.; Saito, M.; Hashimoto, M.; Tanaka, T.
Chem. Lett. 1984, 441-444. (d) Ramanjulu, J. M.; Jouillie´, M. M. Synth.
Commun. 1996, 26, 1379-1384. (e) Chruma, J. J.; Sames, D. Polt, R.
Tetrahedron Lett. 1997, 38, 5085-5086.
(18) (a) Auerbach, J.; Zamore, M.; Weinreb, S. M. J. Org. Chem.
1976, 41, 725-726. (b) Coulton, S.; Moore, G. A.; Ramage, R.
Tetrahedron Lett. 1976, 4005-4008. (c) Effenberger, F.; Burkard, U.;
Willfahrt, J. Angew. Chem., Int. Ed. Engl. 1983, 22, 65-66. (d) Dorow,
R. L.; Gingrich, D. E. J. Org. Chem. 1995, 60, 4986-4987. (e) Luke,
R. W. A.; Boyce, P. G. T.; Dorling, E. K. Tetrahedron Lett. 1996, 37,
263-266. (f) Spengler, J.; Burger, K. Synthesis 1998, 67-70.
(19) (a) McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51,
2555-2561. (b) Cheung, S. T.; Benoiton, N. L. Can. J. Chem. 1977,
55, 916-921.
(20) (a) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119,
2301-2302. (b) Reichwein, J. F.; Liskamp, M. J. Tetrahedron Lett.
1998, 39, 1243-1246. (c) Yang, L.; Chiu, K. Tetrahedron Lett. 1997,
38, 7307-7310.
(21) Laplante, C.; Hall, D. G. Org. Lett. 2001, 3, 1487-1490.
(22) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1998, 120, 2690-
2691.
(23) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995,
36, 6373-6374.
(16) (a) Papaioannou, D.; Athanassopoulos, C.; Magafa, V.; Kara-
manos, N.; Stavropoulos, G.; Napoli, A.; Sindona, G.; Aksnes, D. W.;
Francis, D. W. Acta Chem. Scand. 1994, 48, 324-333. (b) Wisˇniewski,
K.; Kolodziejczyk, A. S. Tetrahedron Lett. 1997, 38, 483-486.
(17) Grieco, P. A.; Bashsas, A. J. Org. Chem. 1987, 52, 5746-5749.
(24) Bowman, W. R.; Coghlan, D. R. Tetrahedron 1997, 53, 15787-
15798. (b) Albanese, D.; Landini, D.; Lupi, V.; Penso, M. Eur. J. Org.
Chem. 2000, 8, 1443-1449.
(25) Milne, H. B.; Peng, C.-H. J. Am. Chem. Soc. 1957, 79, 639-
644.
5184 J. Org. Chem., Vol. 70, No. 13, 2005

Preparation of NR-Methylamino Acids
products were observed after N-methylation of NR-o-NBS-
methionine methyl ester (2p). Both products were iso-
lated, and NMR studies revealed that one is the expected
NR-methyl-NR-o-NBS-methionine methyl ester (3p) and
that the other one has been also methylated on the side-
chain to give NR-methyl-NR-o-NBS-S-methyl-methionine
methyl ester (3p′). This side-chain methylation can be
observed in 1H NMR by the presence of a new singlet at
3.01 ppm integrating for six protons due to two methyl
groups on the δ-thio function plus the normal new singlet
at 2.93 ppm corresponding to N-methylation and by the
absence of the methyl thioether signal at 2.00 ppm and
of the N-H resonance at 5.60. This overmethylated
product is water soluble and explains the lower yields
obtained for N-methylation of 2p-r. HPLC analysis of
NR-methyl-NR-o-NBS-isoleucine methyl ester (3b) and NR-
methyl-NR-o-NBS-threonine-O-tert-butyl ether methyl
ester (3h) showed the presence of only one diastereoiso-
mer, and 1H NMR showed the presence of signals
corresponding to only one diastereoisomer.
SCHEME 1. Direct Methylation of o-NBS-Phe-OH
(1)
methyl ester (3a), and NR-methyl-NR-o-NBS-phenylala-
nine (4a). The desired NR-methyl-NR-o-NBS-phenylala-
nine (3a) was only obtained in 35% yield after purifica-
tion. These low yields become expensive when using side-
chain protected amino acids and led us to explore the
possibility of using R-amino acid methyl esters.
The NR-o-NBS amino acid methyl esters 2a-r were
prepared in 92-99% yields of isolated products (Table
1) by treatment of the corresponding methyl esters with
o-nitrobenzenesulfonyl chloride in the presence of tri-
ethylamine in dry methylene chloride (Scheme 2).20b,24
Protection of the amino acid methyl esters did not involve
racemization as shown by 1H NMR and HPLC of the
crude product of NR-o-NBS-isoleucine methyl ester (2b)
and NR-o-NBS-threonine-O-tert-butyl ether methyl ester
(2h).
It is known that NR-protected-NR-methylamino acid
methyl esters racemize more readily during saponifica-
tion than the corresponding amino acid esters.15c,19 Ra-
cemization was also observed in our case during saponi-
fication of 3b and 3h using 2 M LiOH in THF or
1
methanol. Racemization could be observed by H NMR
with the appearance of a new doublet next to the normal
RH doublet. 8% racemization has been observed for NR-
methyl-NR-o-NBS-isoleucine (4b) and 10% racemization
for NR-methyl-NR-o-NBS-threonine O-tert-butyl ether (4h).
Racemization could also be observed by HPLC with the
appearance of a new peak next to the main one. Higher
racemization of NR-methyl-NR-o-NBS R-amino acid meth-
yl esters during saponification could be explained by the
fact that the N-H will generally be more acidic than the
R-C-H. Thus, in basic conditions, this group will be
deprotonated first and so protect the neighboring C-H
bond from deprotonation. With NR-substituted NR-meth-
yl-R-amino acid esters, no such effect is obtained, and
the R-C-H will ionize more readily. Similarly, ionization
of the free carboxyl group of NR-substituted NR-methyl-
R-amino acids will prevent further ionization at the
R-carbon atom.
To overcome this problem, methyl ester cleavage by
SN2 dealkylation was tested on the NR-methyl-NR-o-NBS-
R-amino acid esters 3a-r (Scheme 4). This mild and
neutral method occurs with displacement of the carboxy-
late by SN2 dealkylation and is usually used for selective
methyl ester cleavage.26 Ester cleavage works best when
a powerful nucleophile such as iodide or cyanide is used
in a dipolar aprotic solvent. SN2 methyl ester cleavage
using lithium iodide in pyridine has been used lately to
minimize racemization during synthesis.27 A first inves-
tigation of the reaction was achieved by treating 3a with
LiI in refluxing pyridine for 30 min.27 Unfortunately,
three products were observed after 30 min. It appears
that the methyl on the nitrogen is also electrophile due
to the neighboring o-NBS group and can also undergo
substitution. To overcome this nonselective substitution,
N-Alkylation of NR-o-NBS amino acid methyl esters has
already been reported and can be achieved with diazo-
methane in 24 h,13l with iodomethane and K2CO3 or
Cs2CO3 in acetonitrile or dimethylformamide in 3 h,20b,24
or under Mitsonobu conditions.20b,c Miller and Scanlan
reported a site-selective N-methylation of o-NBS peptides
on solid support using methyl p-nitrobenzenesulfonate
in combination with the hindered, nonionic guanidium
base MTBD (7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene)
in DMF and completed in 30 min.20a,22 This procedure
was chosen and transferred into solution. A first inves-
tigation of the reaction with NR-o-NBS-phenylalanine
methyl ester (2a) revealed that a minimum of 2 equiv of
MTBD is needed for full conversion to NR-methyl-NR-o-
NBS-phenylalanine methyl ester (3a) and is achieved in
5 min in dimethylformamide. N-Methylation in tetrahy-
drofuran or dichloromethane was not achieved after 2 h.
Unfortunately MTBD is too expensive to be used for the
synthesis of NR-methyl-R-amino acids at the gram scale.
Therefore, TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene) and
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene, two structurally
similar hindered bases, were tested.
Investigation of the reaction revealed that both bases
achieve complete conversion of 2a under same conditions
as MTBD. The less expensive and readily available DBU
was chosen and tested on the other NR-o-NBS-amino acid
methyl esters 2b-r (Scheme 3). N-Methylation was then
performed using DBU and dimethyl sulfate in DMF at 0
°C for 15 min and gave the corresponding NR-methyl-NR-
o-NBS amino acid methyl esters 3b-o in 92-99% yields
of isolated product without any purification (Table 1).
Lower yields were obtained for NR-methyl-NR-o-NBS-
methionine methyl ester (3p) (79%), NR-methyl-NR-o-
NBS-S-tritylcysteine methyl ester (3q) (65%), and NR-
methyl-NR-o-NBS-Nimid-tritylhistidine methyl ester (3r)
(73%). These lower yields could be possibly explained by
the presence of side-chain methylation. For example, two
(26) Mu¨ller, P.; Siegfried, B. Helv. Chim. Acta 1974, 57, 987-994.
(b) McMurry, J. Org. React. 1976, 24, 187-224.
(27) (a) Borch, R. F.; Grudzinskas, C. V.; Peterson, D. A.; Weber, L.
D. J. Org. Chem. 1972, 37, 1141-1145. (b) Magnus, P.; Gallagher T.
J. Chem. Soc., Chem. Commun. 1984, 389-390.
J. Org. Chem, Vol. 70, No. 13, 2005 5185

Biron and Kessler
TABLE 1. Three-Step Synthesis of Nr-Methyl-Nr-o-NBS Amino Acids 4a-r
o-NBS protection
N-methylation
saponification
R1
amino acid
entry (yield (%))
entry (yield (%))
entry (yield (%))
CH2Ph
Phe
2a (97)
2b (96)
2c (90)
2d (95)
2e (93)
2f (96)
2g (98)
2h (95)
2i (86)
2j (95)
2k (98)
2l (97)
2m (97)
2n (97)
2o (98)
2p (90)
2q (95)
2r (99)
3a (98)
3b (96)
3c (99)
3d (98)
3e (98)
3f (99)
3g (92)
3h (97)
3i (96)
3j (93)
3k (98)
3l (99)
3m (98)
3n (96)
3o (95)
3p (79)
3q (65)
3r (73)
4a (96)
4b (85)
4c (90)
4d (94)
4e (97)
4f (93)
4g (83)
4h (88)
4i (88)
4j (91)
4k (95)
4l (93)
4m (89)
4n (99)
4o (88)
4p (85)
4q (35)
4r (33)
CH(CH3)CH2CH3
CH3
Ile
Ala
PHg
Ph
(CH2)4NHZ
Lys(Z)
Lys(Boc)
Ser(tBu)
Thr(tBu)
Asp(tBu)
Glu(tBu)
Arg(Pbf)
Asn(Trt)
Gln(Trt)
Trp
Tyr(tBu)
Met
Cys(Trt)
His(Trt)
(CH2)4NHBoc
CH2OtBu
CH(CH3)OtBu
CH2COOtBu
CH2CH2COOtBu
(CH2)3NHC(NH)NHPbf
CH2CONHTrt
CH2CH2CONHTrt
CH2indol-3-yl
CH2PhOtBu
CH2CH2SCH3
CH2STrt
CH2-Nim-Trt-imidazol-4-yl
SCHEME 2. Synthesis of 2a-r
acids 4a-r to resin-bound leucine using normal Fmoc-
SPPS coupling conditions.29 Racemization of NR-protected
NR-methylamino acids during peptide bond formation
with mixed anhydride coupling has been reported.30
Racemization was expected to be less important when
using the benzotriazole-based coupling reagent TBTU in
combination with HOBt in the presence of a very weak
base. The NR-methyl-NR-o-NBS-amino acids 4a-r were
coupled to the amino free resin-bound leucine using 3
equiv of amino acid, TBTU, HOBt, and collidine in NMP
for 30 min. Resulting dipeptides 5a-r were cleaved from
the resin, and only one diastereoisomer was observed by
HPLC analysis (96-99% purity). In an additional experi-
ment, NR-methyl-NR-o-NBS-D-phenylalanine (4a′) and NR-
methyl-NR-o-NBS-D-tryptophan (4n′) were also coupled
to resin-bound leucine. HPLC analysis of 5a′ and 5n′
showed a different retention time compared to their
corresponding diastereoisomeric dipeptides 5a and 5n
excluding any detectable racemization throughout the
entire synthetic procedure. To continue the peptide
synthesis, the o-NBS protecting group can be easily and
selectively removed with mercaptoethanol and DBU in
30 min.20a,22 Couplings on resin-bound N-methyl peptide
can be achieved with HATU/HOAt or with triphosgene
methods.20a,31
By comparing the herein described procedure with
other known procedures to prepare N-methylamino acids,
it can be easily observed that no procedure is the best
one to prepare every amino acid with respect to yield,
number of steps, and economy (Table 2). Therefore, it is
crucial to choose the right procedure to prepare the
desired amino acid. For example, the 5-oxazolidinone
procedure is without a doubt the best method to prepare
Fmoc-NR-methyl aliphatic amino acids (Table 2). Only
two steps and 2 days are needed to prepare the NR-
methylamino acids. On the other hand, the o-NBS
SCHEME 3 N-Methylation of 2a-r
SCHEME 4. Methyl Ester Cleavage of 3a-r
pyridine was replaced by ethyl acetate which has a lower
boiling point and is neutral.28 NR-Methyl-NR-o-NBS-
phenylalanine (4a) was isolated in 96% yield after
treatment of 3a with LiI in refluxing ethyl acetate for
16 h. Methyl ester cleavage with lithium iodide was
tested on the other NR-methyl-NR-o-NBS amino acid
methyl esters 3b-r, and the corresponding NR-methyl-
NR-o-NBS amino acids 4b-p were obtained in 83-99%
yield without purification (Table 1). Both compounds 3p
and 3p′ gave the desired NR-methyl-NR-o-NBS-methion-
ine (4p) in 85% yield after methyl ester cleavage. Lower
yields were obtained again for 4q (35%) and 4r (33%).
These lower yields could be possibly explained by the
sensibility of the trityl protecting group. Methyl ester
1
cleavage did not involve racemization as shown by H
NMR and HPLC of the crude product of NR-methyl-NR-
o-NBS-isoleucine (4b) and NR-methyl-NR-o-NBS-threo-
nine O-tert-butyl ether (4h).
(29) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35,
161-214.
(30) McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51,
2562-2570.
Compatibility with Fmoc-SPPS was investigated by
coupling the synthesized NR-methyl-NR-o-NBS amino
(28) (a) Pinho e Melo, T. M. V. D.; Soares, M. I. L.; Rocha Gonzales,
A. M. d’A.; Paixa˜o, J. A.; Beja, A. M.; Silva, M. R.; Alte da Veiga, L.;
Pessoa, J. C. J. Org. Chem. 2002, 67, 4045-4054. (b) Pinho e Melo, T.
M. V. D.; Santos, C. I. A.; Rocha Gonzales, A. M. d’A.; Paixa˜o, J. A.;
Beja, A. M.; Silva, M. R. Tetrahedron Lett. 2003, 44, 8285-8287.
(31) (a) Falb, E., Yechezkel, J., Salitra, Y., Gilon, C. J. Peptide Res.
1999, 53, 507-517. (b) Thern, B.; Rudolf, J.; Jung, G. Tetrahedron Lett.
2002, 43, 5013-5016. (c) Thern, B.; Rudolf, J.; Jung, G. Angew. Chem.,
Int. Ed. 2002, 41, 2307-2309. (d) Sewald, N. Angew. Chem., Int. Ed.
2002, 41, 4661-4663.
5186 J. Org. Chem., Vol. 70, No. 13, 2005

Preparation of NR-Methylamino Acids
TABLE 2. Preparation of Nr-Methylamino Acids by Different Proceduresa
oNBS procedure
5-oxazolidinone
direct N-methylation
amino acid
Y
yield (%)
X
Y
% (no. of steps)
X
Y
% (no. of steps)
aliphatic
Asn
78-91
89
91
73
22
85
80
24
88
86
75
75
81
Fmoc
Cbz
51-96 (2)14
62 (2)14f
39 (7)14h
38 (2)14f
62 (2)14i
42 (2)14i
54 (2)14f
30 (5)14h
21 (6)14i
51 (2)14c
64 (4)14i
94 (2)14c
64 (5)14i
Boc
83-90 (1)13
Trt
Pbf
tBu
Trt
Trt
tBu
Trt
Arg
Cbz
Boc2
Bzl
Boc
Cbz
Cbz2
62 (4)13i
Asp
Cbz
tBu
86 (1)13f
Cys
Cbz
Bzl
Gln
Cbz
Glu
Cbz
Cbz
tBu
70 (1)13f
His
Cbz
Cbz
DNP
Bzl
Pht
Lys
Boc
Cbz
Fmoc
Cbz
Fmoc
Cbz
Met
Ser
Thr
Trp
Tyr
Cbz
Boc
Boc
71 (1)13f
80 (1)13d
75 (1)13d
tBu
tBu
Bzl
Bzl
Bzl
92
82
tBu
Fmoc
Bzl
60 (2)14i
Boc
Bzl
70 (1)13d
a Suggested procedures to prepare the NR-methylamino acid are shown in boldface type.
procedure is the method of choice to prepare directly
Fmoc-SPPS compatible tryptophan, lysine, asparagine,
glutamine, and arginine. It is important to note that some
NR-methylamino acids reported in Table 2 still need some
transformation to be compatible with Fmoc-SPPS. One
advantage of the o-NBS procedure is that o-NBS-Cl is
actually less expensive than Fmoc-Cl. Another advantage
is that the starting material (side-chain protected amino
acid methyl esters) is also quite inexpensive or can be
easily prepared from the less expensive amino acid
methyl esters or side-chain protected amino acids. Un-
fortunately, cysteine and histidine are still problematic
and are obtained in low yields or with Fmoc-SPPS
incompatible protecting groups in every reported proce-
dure.
can be directly used in Fmoc-SPPS with normal coupling
conditions to synthesize N-methyl peptide analogues. The
entire synthetic sequence is compatible with Fmoc-SPPS
protecting groups and can be done in 1 day, allowing a
rapid and efficient synthesis of N-methyl peptide ana-
logues.
Experimental Section
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-L-phenylala-
nine Methyl Ester (3a). To an ice-cold solution of NR-(o-
nitrobenzenesulfonyl)phenylalanine methyl ester 2a (1 mmol)
in dimethylformamide (30 mL) were slowly added first di-
methyl sulfate (3 mmol) and then 1,8-diazabicyclo[5,4,0]undec-
7-ene (2 mmol), and the mixture was stirred at 0 °C for 15
min. After the addition of acetic acid (1.1 mmol), the solvent
was evaporated under reduced pressure and the residue taken
up in ethyl acetate and washed with a 10% aqueous solution
of NaHCO3 (3 × 25 mL) and water, dried (Na2SO4), and
evaporated to dryness. Diethyl ether was added and evapo-
rated two times, and the residue was precipitated with CHCl3
and n-hexanes to afford a pale yellow solid (98%): 1H NMR
(250 MHz, CDCl3) δ 7.81-7.75 (m, 1H), 7.69-7.51 (m, 3H),
7.31-7.19 (m, 5H), 5.01-4.92 (m, 1H), 3.65 (s, 3H), 3.42-3.31
(dd, J ) 6.2 Hz, 14.2 Hz, 1H), 3.08-2.91 (m, 4H); MS (ESI)
m/z 379.1 (M + H)+, 401.1 (M + Na)+; RP-HPLC tR ) 23.9
(10-100%).
Conclusion
NR-Methyl-NR-o-NBS-amino acids are prepared from
amino acid methyl esters in three steps and excellent
overall yields without any purification. Side-chain pro-
tected amino acid methyl esters are commercially avail-
able or can be easily obtained from inexpensive amino
acid methyl esters or side-chain protected amino acids.
The o-NBS procedure is also an inexpensive way to
prepare NR-methyl-D- and unnatural amino acids. After
protection and activation of the amino function of the
amino acid methyl esters with o-NBS-Cl, the N-methy-
lation reaction is performed by treatment of o-NBS
derivatives with DBU and dimethyl sulfate. Methyl ester
hydrolysis of the NR-methyl-NR-o-NBS-amino acid methyl
esters with lithium hydroxide causes partial racemiza-
tion. To overcome this problem, a clean and racemization-
free SN2 methyl ester cleavage of NR-methyl-NR-o-NBS-
amino acid methyl esters is achieved with lithium iodide
Synthesis of N-Methyl-N-(o-nitrobenzenesulfonyl)-
amino Acids 4a-r. General Procedure. The N-methyl-N-
(o-nitrobenzenesulfonyl)-R-amino acid methyl ester 3a-r (1
mmol) and LiI (5 mmol) were dissolved in ethyl acetate (5 mL).
The reaction mixture was protected from light and heated at
reflux for 16 h or until full conversion was observed by TLC.
Water was added (5 mL), and the solution was acidified with
0.1 M HCl and quickly extracted with ethyl acetate. The
organic phase was washed with water and a saturated aqueous
solution of NaCl and extracted with a solution 0.1 M NaOH
(3 × 15 mL). The aqueous phase was washed with DCM (3 ×
15 mL), acidified with 1 M HCl, and quickly extracted with
ethyl acetate (3 × 15 mL). The organic phase was dried and
evaporated to dryness to afford a pale yellow solid or a pale
yellow oil. To obtain the amino acids as powder, they were
precipitated with CHCl3 and n-hexanes (Phe, Phg, Thr, Arg,
Asn, Gln, Trp, Cys, His) or lyophilized with a mixture of
dioxane/water (Ala, Lys, Ser, Asp, Glu, Tyr, Met). They can
also be recrystallized with ethyl acetate-benzene or ethyl
1
in boiling ethyl acetate as confirmed by HPLC and H
NMR spectroscopy. It has been shown, by comparing
literature data, that no single procedure is best for the
synthesis of all derivatives, suggesting that the right
procedure should be chosen to prepare the desired
N-methyl amino acid. NR-Methyl-NR-o-NBS-amino acids
J. Org. Chem, Vol. 70, No. 13, 2005 5187

Biron and Kessler
acetate-n-hexanes (Phe, Phg, Thr, Arg, Asn, Gln, Trp, Cys,
His) and EtOH-water (Ala, Lys, Asp, Glu, Tyr). Isoleucine,
serine, and methionine derivatives could not be recrystallized.
N-Methyl-N-(o-nitrobenzenesulfonyl)-L-phenylala-
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-L-aspartic acid
â-tert-butyl ester (4i): pale yellow solid (88%); mp 75-77 °C;
[R]25D +22.9 (c 0.1, MeOH); 1H NMR (250 MHz, CDCl3) δ 10.01
(br s, 1H), 8.10-8.03 (m, 1H), 7.78-7.64 (m, 3H), 5.13 (t, J )
7.1 Hz, 1H), 3.02-2.87 (m, 4H), 2.71 (dd, J ) 7.6 Hz, 16 Hz,
1H), 1.45 (s, 9H); 13C δ 174.6, 168.8, 147.9, 133.8, 132.2, 131.7,
131.0, 124.4, 82.4, 56.4, 36.4, 31.3, 27.9; MS (ESI) m/z 389.3
(M + H)+, 411.3 (M + Na)+, 777.2 (2M + H)+, 799.2 (2M +
Na)+; RP-HPLC tR ) 20.9 (10-100%).
nine (4a): pale yellow solid (96%); mp 56-59 °C; [R]25 +64
D
(c 0.1, MeOH); 1H NMR (250 MHz, CDCl3) δ 10.55 (br s, 1H),
7.78-7.52 (m, 4H), 7.31-7.19 (m, 5H), 5.08-4.97 (m, 1H),
3.46-3.35 (dd, J ) 5.4 Hz, 14.5 Hz, 1H), 3.12-2.95 (m, 4H);
13C δ 175.79, 147.81, 135.73, 133.57, 132.35, 131.71, 130.62,
128.96, 128.66, 127.19, 124.35, 60.72, 35.37, 30.86; MS (ESI)
m/z 365.1 (M + H)+, 387.1 (M + Na)+; RP-HPLC tR ) 20.8
(10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-L-glutamic acid
γ-tert-butyl ester (4j): pale yellow solid (91%); mp 58-62 °C;
[R]25D +66.9 (c 0.1, MeOH); 1H NMR (250 MHz, CDCl3) δ 8.87
(br s, 1H), 8.06-8.00 (m, 1H), 7.77-7.64 (m, 3H), 4.80-4.72
(m, 1H), 2.96 (s, 3H), 2.54-2.26 (m, 3H), 2.04-1.91 (m, 1H),
1.47 (s, 9H); 13C δ 175.4, 171.8, 147.8, 133.8, 131.9, 131.6,
130.7, 124.3, 81.2, 58.7, 31.6, 30.4, 28.0, 24.0; MS (ESI) m/z
403.1 (M + H)+, 425.1 (M + Na)+; RP-HPLC tR ) 21.8 (10-
100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-L-isoleucine
(4b): pale yellow oil (85%); 1H NMR (250 MHz, CDCl3) δ 10.55
(br s, 1H), 8.05-7.98 (m, 1H), 7.74-7.60 (m, 3H), 4.24 (d, J )
9.3 Hz, 1H), 3.04 (s, 3H), 2.04-1.83 (m, 1H), 1.59-1.44 (m,
1H), 1.28-1.04 (m, 1H), 1.02-0.82 (m, 6H); 13C δ 176.0, 147.9,
133.9, 132.2, 131.7, 130.9, 124.2, 63.7, 34.5, 31.1, 25.6, 15.4,
10.9; MS (ESI) m/z 331.1 (M + H)+, 353.1 (M + Na)+; RP-
HPLC tR ) 20.9 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-NG-(2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine
(4k): white solid (95%); mp 93-101 °C dec; [R]25D +49.3 (c 0.1,
MeOH); 1H NMR (250 MHz, CDCl3) δ 8.12-8.04 (m, 1H),
7.75-7.56 (m, 3H), 6.23 (s, 2H), 6.05 (s, 1H), 4.68-4.59 (m,
1H), 3.37-3.23 (m, 2H), 2.99 (s, 2H), 2.92 (s, 3H), 2.52-2.47
(d, 6H), 1.89-1.54 (m, 4H), 1.48 (s, 6H); 13C δ 174.1, 159.6,
156.5, 148.2, 139.0, 134.2, 132.5, 131.2, 125.5, 124.6, 118.2,
87.1, 59.5, 43.5, 41.0, 29.0, 26.4, 19.7, 18.2, 12.9; MS (ESI) m/z
626.5 (M + H)+, 648.5 (M + Na)+; RP-HPLC tR ) 21.9 (10-
100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-L-alanine (4c):
pale yellow solid (90%); mp 48-50 °C; 1H NMR (250 MHz,
CDCl3) δ 8.12-8.06 (m, 1H), 7.76-7.64 (m, 3H), 7.00 (br s,
1H), 4.78-4.69 (m, 1H), 2.91 (s, 3H), 1.45 (d, J ) 7.5 Hz, 3H).
13C δ 177.0, 147.9, 133.7, 132.5, 132.0, 131.1, 124.4, 55.5, 30.2,
15.2; MS (ESI) m/z 311.0 (M + Na)+, 599.1 (2M + Na)+; RP-
HPLC tR ) 16.9 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-L-phenylgly-
cine (4d): pale yellow solid (94%); mp 55-59 °C; [R]25D +12.5
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-Nâ-trityl-L-as-
paragine (4l): pale yellow solid (93%); mp 105-112 °C dec;
[R]25D +11.5 (c 0.1, MeOH); 1H NMR (250 MHz, CDCl3) δ 10.25
(br s, 1H), 8.16-8.05 (m, 1H), 7.64-7.58 (m, 3H), 7.31-7.17
(m, 15H), 6.83 (s, 1H), 4.99-4.95 (m, 1H), 3.07-2.81 (m, 5H);
13C δ 170.8, 170.4, 147.9, 143.7, 133.7, 132.2, 132.0, 130.9,
128.7, 128.0, 127.2, 124.3, 71.3, 56.3, 38.1, 31.8; MS (ESI) m/z
574.5 (M + H)+, 596.7 (M + Na)+; RP-HPLC tR ) 25.1 (10-
100%).
1
(c 0.1, MeOH); H NMR (250 MHz, CDCl3) δ 9.23 (br s, 1H),
8.08-8.02 (m, 1H), 7.75-7.67 (m, 3H), 7.45-7.31 (m, 5H), 6.03
(s, 1H), 2.82 (s, 3H); 13C δ 175.2, 147.7, 133.9, 132.8, 132.2,
131.8, 130.7, 129.1, 129.0, 129.0, 124.5, 62.9, 31.3; MS (ESI)
m/z 351.1 (M + H)+, 373.1 (M + Na)+, 701.2 (2M + H)+, 723.2
(2M + Na)+; RP-HPLC tR ) 20.6 (10-100%).
ꢀ
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-N -benzyloxy-
carbonyl-L-lysine (4e): pale yellow solid (97%); mp 85-87
1
°C; [R]25 +46.5 (c 0.1, MeOH); H NMR (250 MHz, CDCl3) δ
D
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-Nγ-trityl-L-
glutamine (4m): pale yellow solid (89%); mp 105-112 °C dec;
1H NMR (250 MHz, CDCl3) δ 8.33 (br s, 1H), 8.00-7.94 (m,
1H), 7.68-7.57 (m, 3H), 7.35-7.20 (m, 15H), 6.90 (s, 1H),
4.68-4.61 (m, 1H), 2.93 (s, 3H), 2.56-2.41 (m, 2H), 2.39-2.21
(m, 1H), 2.02-1.83 (m, 1H); 13C δ 171.9, 171.7, 148.0, 145.0,
133.5, 132.4, 132.0, 130.2, 128.5, 127.4, 126.2, 123.8, 69.1, 61.7,
33.9, 30.2, 26.0; MS (ESI) m/z 588.4 (M + H)+, 610.4 (M +
Na)+; RP-HPLC tR ) 25.3 (10-100%).
8.37 (br s, 1H), 8.09-8.01 (m, 1H), 7.73-7.60 (m, 3H), 7.43-
7.32 (m, 5H), 6.11 (m, 1H), 5.10 (s, 2H), 4.71-4.61 (m, 1H),
3.25-3.09 (m, 2H), 2.95 (s, 3H), 2.07-1.88 (m, 1H), 1.84-1.32
(m, 5H); 13C δ 174.7, 147.9, 133.6, 132.4, 131.6, 130.8, 128.5,
128.1, 124.3, 66.7, 59.1, 40.6, 30.3, 29.0, 28.5, 23.0; MS (ESI)
m/z 480.1 (M + H)+, 502.1 (M + Na)+, 959.2 (2M + H)+, 981.2
(2M + Na)+; RP-HPLC tR ) 21.6 (10-100%).
ꢀ
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-N -tert-butyl-
oxycarbonyl-L-lysine (4f): pale yellow solid (93%); mp 54-
55 °C; [R]25D +67.5 (c 0.1, MeOH); 1H NMR (250 MHz, CDCl3)
δ 8.85 (br s, 1H), 8.09-8.01 (m, 1H), 7.76-7.64 (m, 3H), 6.03
(br s, 1H), 4.68-4.62 (m, 1H), 3.17-3.01 (m, 2H), 2.96 (s, 3H),
2.08-1.93 (m, 1H), 1.84-1.65 (m, 1H), 1.58-1.31 (m, 13H);
13C δ 174.8, 156.0, 147.9, 133.6, 132.4, 131.6, 130.8, 124.3, 79.0,
59.2, 40.1, 30.3, 29.0, 28.4, 23.1; MS (ESI) m/z 446.1 (M + H)+,
468.1 (M + Na)+, 991.2 (2M + H)+, 1013.2 (2M + Na)+; RP-
HPLC tR ) 21.1 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-L-tryptophan
(4n): pale yellow solid (99%); mp 83-85 °C; [R]25 +40.1 (c
D
0.1, MeOH); 1H NMR (250 MHz, DMSO-d6) δ 13.10 (br s, 1H),
10.84 (s, 1H), 7.82-7.65 (m, 2H), 7.52 (d, J ) 7.7 Hz, 1H),
7.46-7.41 (m, 2H), 7.33 (d, J ) 7.7 Hz, 1H), 7.21-7.16 (m,
1H), 7.12-6.95 (m, 2H), 4.85-4.76 (m, 1H), 3.42-3.31 (m, 1H),
3.17 (dd, J ) 10.1 Hz, 15.2 Hz, 1H), 3.00 (s, 3H); 13C δ 171.5,
147.2, 136.1, 133.9, 131.6, 131.2, 129.4, 126.7, 123.9, 123.7,
121.0, 118.5, 117.8, 111.5, 108.9, 59.7, 30.1, 24.9; MS (ESI)
m/z 404.1 (M + H)+, 426.1 (M + Na)+, 807.2 (2M + H)+, 829.2
(2M + Na)+; RP-HPLC tR ) 20.1 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-O-tert-butyl-L-
serine (4g): pale yellow gummy solid (83%); [R]25 +47.1 (c
D
0.1, MeOH); 1H NMR (250 MHz, CDCl3) δ 9.08 (br s, 1H),
8.17-8.08 (m, 1H), 7.76-7.63 (m, 3H), 4.93-4.86 (m, 1H),
3.92-3.76 (m, 2H), 3.09 (s, 3H), 1.18 (s, 9H); 13C δ 173.2, 148.0,
133.5, 132.5, 131.6, 130.8, 127.3, 74.6, 60.9, 59.6, 32.1, 27.1;
MS (ESI) m/z 361.3 (M + H)+, 383.3 (M + Na)+, 721.6 (2M +
H)+, 743.6 (2M + Na)+; RP-HPLC tR ) 21.3 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-O-tert-butyl-L-
threonine (4h): pale yellow solid (88%); mp 142-145 °C;
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-O-tert-butyl-L-
tyrosine (4o): pale yellow solid (88%); mp 75-80 °C; [R]25
D
+50.9 (c 0.1, MeOH); 1H NMR (250 MHz, DMSO-d6) δ 8.97
(br s, 1H), 7.91-7.53 (m, 4H), 7.12 (d, J ) 8.5 Hz, 2H), 6.86
(d, J ) 8.5 Hz, 2H), 5.01-4.92 (m, 1H), 3.33 (dd, J ) 5.4 Hz,
14.5 Hz, 1H), 3.07-2.90 (m, 4H), 1.34 (s, 9H); 13C δ 175.4,
154.3, 147.7, 133.5, 132.3, 131.6, 130.6, 130.5, 129.4, 124.3,
124.2, 78.6, 60.7, 34.7, 30.8, 28.8; MS (ESI) m/z 437.2 (M +
H)+, 459.2 (M + Na)+, 873.4 (2M + H)+, 895.4 (2M + Na)+;
RP-HPLC tR ) 23.5 (10-100%).
[R]25 +156.3 (c 0.1, MeOH); 1H NMR (250 MHz, CDCl3) δ
D
10.44 (br s, 1H), 8.11-8.00 (m, 1H), 7.79-7.61 (m, 3H), 4.70
(d, J ) 3.6 Hz, 1H), 4.47-4.35 (m, 1H), 3.20 (s, 3H), 1.31 (d, J
) 6.3, 3H), 1.16 (s, 9H); 13C δ 175.1, 147.7, 133.5, 131.6, 131.5,
130.9, 124.5, 74.8, 69.2, 64.8, 33.8, 28.4, 20.3; MS (ESI) m/z
375.1 (M + H)+, 396.1 (M + Na)+, 749.2 (2M + H)+, 771.2 (2M
+ Na)+; RP-HPLC tR ) 24.1 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-S-methyl-L-me-
1
thionine (4p): pale yellow solid (85%); mp 109-111 °C; H
NMR (250 MHz, CDCl3) δ 9.66 (br s, 1H), 8.10-8.05 (m, 1H),
7.77-7.66 (m, 3H), 4.88-4.82 (m, 1H), 2.99 (s, 3H), 2.62-2.46
5188 J. Org. Chem., Vol. 70, No. 13, 2005

Preparation of NR-Methylamino Acids
(m, 2H), 2.33-2.18 (m, 1H), 2.14-1.95 (m, 4H); 13C δ 175.8,
147.8, 133.8, 132.3, 131.8, 131.1, 124.5, 58.3, 30.6, 30.3, 28.8,
15.4; MS (ESI) m/z 349.0 (M + H)+, 371.0 (M + Na)+, 697.0
(M + H)+, 719.0 (M + Na)+; RP-HPLC tR ) 19.6 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-S-trityl-L-cys-
teine (4q): pale yellow solid (35%); mp 62-66 °C dec; 1H NMR
(250 MHz, CDCl3) δ 8.09-7.96 (m, 1H), 7.67-7.50 (m, 3H),
7.44-7.13 (m, 15H), 4.57-4.44 (m, 1H), 2.85-2.68 (m, 4H),
2,55-2.42 (m, 1H); 13C δ 170.7, 147.9, 143.8, 134.0, 133.2,
132.2, 130.8, 128.6, 128.0, 126.9, 123.9, 69.3, 62.3, 31.2, 24.8;
MS (ESI) m/z 563.1 (M + H)+, 586.1 (M + Na)+, 1125.2 (2M +
H)+, 1147.2 (2M + Na)+; RP-HPLC tR ) 26.2 (10-100%).
NR-Methyl-NR-(o-nitrobenzenesulfonyl)-Nimid-trityl-L-
H)+, 619.3 (M + Na)+, 1193.6 (2M + H)+, 1215.6 (2M + Na)+;
RP-HPLC tR ) 21.5 (10-100%).
Acknowledgment. We gratefully acknowledge tech-
nical assistance from B. Cordes. E.B. thanks the Foun-
dation Alexander von Humboldt for Postdoctoral Fel-
lowships. Financial support was provided by the
Deutsche Forschungsgemeinschaft.
Supporting Information Available: General procedure
for the synthesis of NR-o-NBS amino acid methyl esters 2a-
r, synthesis of NR-methyl-NR-o-NBS amino acid methyl esters
3b-r, compound characterization data of 2a-r, 1H NMR
spectra of 2-4b and 2-4h, general procedure for the synthesis
of dipeptides 5a-r, and HPLC analyses of 5a-a′,b,f,n-n′.
This material is available free of charge via the Internet at
http://pubs.acs.org.
histidine (4r): white solid (33%); mp 81-85 °C dec; [R]25
D
+37.5 (c 0.1, MeOH); 1H NMR (250 MHz, CDCl3) δ 8.27 (s,
1H), 8.15-8.06 (m, 1H), 7.72-7.55 (m, 3H), 7.48-7.38 (m, 9H),
7.20-7.10 (m, 6H), 7.00 (s, 1H), 5.21-5.10 (m, 1H), 3.66-3.50
(m, 1H), 3.34-3.18 (m, 1H), 2.94 (s, 3H). 13C δ 170.0, 148.1,
139.4, 134.7, 133.7, 132.0, 131.7, 130.2, 129.2, 127.9, 124.0,
121.9, 79.0, 59.2, 30.9, 24.9, 15.4; MS (ESI) m/z 597.3 (M +
JO050477Z
J. Org. Chem, Vol. 70, No. 13, 2005 5189