An efficient preparation of N-Methyl-α-amino acids from N-Nosyl-α-amino acid phenacyl esters

Article abstract of DOI:10.1021/jo901643f

Chemical Equation Presented In this paper we describe a simple and efficient solution-phase synthesis of N-methyl-TV-nosyl-α-amino acids and N-Fmoc-N-methyl-α-amino acids. This represents a very important application in peptide synthesis to obtain N-methylated peptides in both solution and solid phase. The developed methodology involves the use of N-nosyl-α-amino acids with the carboxyl function protected as a phenacyl ester and the methylating reagent diazomethane. An important aspect of this synthetic strategy is the possibility to selectively deprotect the carboxyl function or alternatively both amino and carboxyl moieties by using the same reagent with a different molar excess and under mild conditions. Furthermore, the adopted procedure keeps unchanged the acid-sensitive side chain protecting groups used in Fmoc-based synthetic strategies.

Full text of DOI:10.1021/jo901643f

pubs.acs.org/joc
An Efficient Preparation of N-Methyl-r-amino Acids from
N-Nosyl-r-amino Acid Phenacyl Esters
Antonella Leggio, Emilia Lucia Belsito, Rosaria De Marco, Angelo Liguori,*
Francesca Perri, and Maria Caterina Viscomi
ꢀ
Dipartimento di Scienze Farmaceutiche, Universita della Calabria, Via Ponte P. Bucci Cubo 15/C,
I-87036 Arcavacata di Rende (CS), Italy
a.liguori@unical.it
Received July 29, 2009
In this paper we describe a simple and efficient solution-phase synthesis of N-methyl-N-nosyl-R-amino
acids and N-Fmoc-N-methyl-R-amino acids. This represents a very important application in peptide syn-
thesis to obtain N-methylated peptides in both solution and solid phase. The developed methodology
involves the use of N-nosyl-R-amino acids with the carboxyl function protected as a phenacyl ester and the
methylating reagent diazomethane. An important aspect of this synthetic strategy is the possibility to selec-
tively deprotect the carboxyl function or alternatively both amino and carboxyl moieties by using the same
reagent with a different molar excess and under mild conditions. Furthermore, the adopted procedure
keeps unchanged the acid-sensitive side chain protecting groups used in Fmoc-based synthetic strategies.
Introduction
protected on the amino function with the p-nitrobenzenesul-
fonyl (nosyl) group.1a
The interesting biological activity of N-methylated pep-
tides has encouraged the development of various synthetic
procedures for both the preparation of N-methyl-R-amino
acids1 and their subsequent insertion into natural peptide
chains to enhance and improve their activity. A successful
synthesis of N-methyl-R-amino acid methyl esters has been
recently achieved by a novel methodology based on the use of
diazomethane as methylating reagent and R-amino acids
The developed procedure proved very effective also in the
site-specific methylation of N-nosyl-peptides protected on
the carboxyl function as methyl esters.2
The obtainment of N-methyl-N-nosyl-R-amino acids not
protected on the carboxyl function represents an interesting
goal to achieve. In fact, N-methyl-N-nosyl-R-amino acids
and N-Fmoc-N-methyl-R-amino acids are useful building
blocks in solution and solid phase synthesis of N-methylated
peptides.3
(1) (a) Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Napoli, A.;
Siciliano, C.; Sindona, G. J. Org. Chem. 2003, 68, 7416. (b) Luke, R. W.;
Boyce, P. G.; Dorling, E. K. Tetrahedron Lett. 1996, 37 (2), 263. (c) Prashad,
M.; Har, D.; Hu, B.; Kim, H.; Repic, O.; Blacklock, T. J. Org. Lett. 2003, 5,
125. (d) Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B. Org. Lett. 2002, 4,
3767. (e) Aurelio, L.; Box, J. S.; Brownlee, R. T. C.; Hughes, A. B.; Sleebs,
M. M. J. Org. Chem. 2003, 68, 2652. (f)Aurelio, L.; Brownlee, R. T. C.; Hughes,
A. B.; Sleebs, B. E. Aust. J. Chem. 2000, 53, 425. (g) Vidyasagar Reddy, G.;
Iyengar, D. S. Chem. Lett. 1999, 299. (h) Dorow, R. L.; Gingrich, D. E. J. Org.
Chem. 1995, 60, 4986. (i) Grieco, P. A.; Perez-Medrano, A. Tetrahedron Lett.
1988, 29, 4225. (j) Vedejs, E.; Kongkittingam, C. J. Org. Chem. 2000, 65, 2309.
(k) Yang, L.; Chiu, K. Tetrahedron Lett. 1997, 38, 7307. (l) Aurelio, L.;
Brownlee, R. T. C.; Hughes, A. B. Chem. Rev 2004, 104, 5823 and references
cited therein. (m) Zhang, S.; Govender, T.; Norstrom, T.; Arvidsson, P. I. J. Org.
Chem. 2005, 70, 6918.
(2) Di Gioia, M. L.; Leggio, A.; Liguori, A. J. Org. Chem. 2005, 70, 3892.
For previously reported N-methylation methods see also the references cited
therein.
(3) (a) 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., Toniolo,
C., Eds.; Georg Thieme Verlag: Stuttgart, Germany, 2002; pp 215-291 and
references cited therein. (b) Biron, E.; Kessler, H. J. Org. Chem. 2005, 70, 5183.
(c) Biron, E.; Chatterjee, J.; Kessler, H. J. Pept. Sci. 2006, 12, 213. (d) Miller,
S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301. (e) Miller, S. C.; Scanlan,
T. S. J. Am. Chem. Soc. 1998, 120, 2690. (f) Di Gioia, M. L.; Leggio, A.; Liguori,
A.; Perri, F. J. Org. Chem. 2007, 72, 3723. (g) Leggio, A.; Liguori, A.; Perri, F.;
Siciliano, C.; Viscomi, M. C. Chem. Biol. Drug Des. 2009, 73, 287.
1386 J. Org. Chem. 2010, 75, 1386–1392
Published on Web 02/01/2010
DOI: 10.1021/jo901643f
r
2010 American Chemical Society

Leggio et al.
JOCArticle
SCHEME 1
TABLE 1. Results of the Synthesis of N-Nosyl-R-amino Acid Phenacyl
Esters 3a-i
In solution phase synthesis of N-methyl-R-amino acids
and N-methyl-peptides the protection of the carboxyl func-
tion as a methyl ester is advantageous since it is stable during
the entire synthetic process.1a,2 However, the regeneration of
the free carboxyl function presents some difficulties. In
peptide synthesis, the most used method for methyl ester
cleavage is base hydrolysis.4 This procedure, even when it is
carried out under strictly controlled conditions, could cause
racemization and other side reactions.5
entry
R
yield (%)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH(CH3)CH2CH3
CH(CH3)2
CH2CH(CH3)2
CH3
CH2(C6H5)
95
98
95
99
92
89
86
98
79
CH2S-(Bzl)
(CH2)4NH-(Boc)
CH2CONH-(Trt)
CH2C6H4O-(t-Bu)
Results and Discussion
aIsolated yield.
In the light of the above-discussed limits, it is essential to
use a carboxyl protecting group that remains stable during
the N-methylation reaction and is easy to remove after the
methylation of the sulfonamide nitrogen atom. Further-
more, the unblocking conditions of the carboxyl function
should be compatible with the presence of protecting groups
of side chain functionalized R-amino acids.
SCHEME 2
We developed a practical and general method for the
preparation of N-methylated amino acids N-nosyl and N-
Fmoc protected by using diazomethane as methylating
reagent and protecting the carboxy function with an appro-
priate easily removable protecting group.
Subsequently, the N-methylation of 3a-i performed with
use of a dichloromethane solution of diazomethane9 pro-
vided the corresponding N-methyl-N-nosyl-R-amino acid
phenacyl esters 4a-i in quantitative yields (Scheme 2).
Sodium benzenethiolate (PhSNa) was used as sulfur nu-
cleophile to deprotect the carboxyl function of phenacyl
esters 4a-i.10
The deprotection reaction of the carboxyl function was
investigated by using lipophilic N-methyl-N-nosyl-R-amino
acid phenacyl esters 4a-e as model systems.
In a preliminary experiment the deprotection reaction
was performed with N-methyl-N-nosyl-L-isoleucine phenacyl
ester (4a) as starting material. A DMF solution of 4a was
treated with PhSNa in the molar ratio 1:5 (Scheme 3, Table 2).
After 30 min the TLC analysis (diethyl ether/petroleum ether
70:30 v/v) of the reaction mixture showed the disappearance of
4a. The GC/MS analysis of the organic extracts obtained from
the extraction of the basified reaction mixture revealed the
formation of the deprotection adduct 1-phenyl-2-phenylsulfa-
nylethanone (6). It was produced by the interaction of benze-
nethiolate with the carbinol carbon atom of the phenacyl ester
function. The aqueous phase was then acidified and extracted to
give the N-methyl-N-nosyl-L-isoleucine (5a) in 70% yield.
The synthetic strategy reported in this work is based on the
use of N-nosyl-R-amino acids protected on the carboxyl
function as phenacyl esters. The choice of the phenacyl group
is due to the need to obtain not only the formation of N-
methyl-N-nosyl-R-amino acids but also N-methyl-R-amino
acids exploiting the same reagent to deprotect both the
carboxyl and amino function. The phenacyl group is easily
introduced on the carboxyl function and requires for its
removal the treatment of the protected product with a sulfur
nucleophile. This could also be used to remove the nosyl
group from the amino functions.6 The cleavage of the
phenacyl ester is performed through the nucleophilic attack
at the carbinol carbon atom under relatively mild conditions.
N-Nosyl-R-amino acid phenacyl esters 3a-i were prepared
inhigh yields bytreatment of cesiumsalts2a-i, obtained from
the reaction of the corresponding N-nosyl-R-amino acids2,7
1a-i with cesium carbonate, with phenacyl bromide8 in N,N-
dimethylformamide (DMF) (Scheme 1, Table 1).
(4) (a) Answer, K. L.; Audhya, T. K.; Goldstein, G. Tetrahedron Lett.
1991, 32, 327. (b) Salomon, C. J.; Mata, E. G.; Mascaretti, O. A. Tetrahedron
1993, 49, 3691.
(5) (a) McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51, 2555.
(b) Benoiton, N. L.; Cheung, S. T. Can. J. Chem. 1977, 55, 916. (c) Schroder,
E.; Lubke, K. In The Peptides. Methods of Peptide Synthesis; Academic Press:
New York, 1965; Vol. 1.
(6) Fukuyama, T.; Jow, C. K.; Cheung, M. Tetrahedron Lett. 1995, 36,
6373.
(7) Leggio, A.; Di Gioia, M. L.; Perri, F.; Liguori, A. Tetrahedron 2007,
63, 8164.
(8) Clark, J. H.; Miller, J. M. Tetrahedron Lett. 1997, 38, 599.
(9) Arndt, F. Org. Synth. 1943, 2, 165. Diazomethane is dangerous
because, in the presence of relatively acid protons, it forms the methyl
diazonium ion that could methylate the alcoholic hydroxyl, amino, thiol,
and carboxyl functions of biological structures. Diazomethane is explosive
when it is prepared by distillation and used in hydrocarbon solvents which
cannot solvate it. Commercially available trimethylsilyldiazomethane was
also used as a methylating agent as a safe substitute for diazomethane (ref 3g).
(10) Seehan, J. C.; Daves, G. D. J. Org. Chem. 1964, 29, 2006.
J. Org. Chem. Vol. 75, No. 5, 2010 1387

Leggio et al.
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SCHEME 3
TABLE 2. Results of the Synthesis of N-Methyl-N-nosyl-R-amino
Acids 5a-e
was then basified and treated with an excess of acetic
anhydride to convert the completely deprotected N-methyl-
L-valine (8b) into the corresponding N-acetyl derivative 9b
(Scheme 4). The latter was characterized by GC/MS analysis
as methyl ester derivative (10b).
The conditions adopted to obtain N-methyl-L-valine start-
ing from the N-methyl-N-nosyl-L-valine phenacyl ester (4b)
led to a partial deprotection of the amino function. In fact
two products corresponding to the N-methyl-N-nosyl-L-
valine (5b) (40%) and the N-acetyl-N-methyl-L-valine (9b)
(25%) were recovered.
On the basis of these results, the deprotection reaction was
performed with use of a large excess of sodium benzenethio-
late. N-Methyl-N-nosyl-L-valine phenacyl ester (4b) (1
mmol) was then treated in DMF with sodium benzenethio-
late in the molar ratio 1:10, respectively (Scheme 5, Table 3).
The deprotection of the amino function went to comple-
tion in 3 h. GC/MS analysis of the organic extract of the
acidified reaction mixture did not reveal the presence of the
N-methyl-N-nosyl-L-valine (5b). This confirmed that the
amino function was completely deprotected. The aqueous
phase, containing the completely deprotected N-methyl-L-
valine (11b), was then basified and treated with 9-fluorenyl-
methyloxycarbonyl chloride (Fmoc-Cl) in order to directly
obtain the N-Fmoc-N-methyl-L-valine (12b). This was finally
recovered in 72% yield (Scheme 5, Table 3).
entry
R
yield (%)a
5a
5b
5c
5d
5e
CH(CH3)CH2CH3
CH(CH3)2
CH2CH(CH3)2
CH3
CH2(C6H5)
70
70
87
76
70
aIsolated yield.
The detailed examination of the spectroscopic data of the
crude product N-methyl-N-nosyl-L-isoleucine (5a) allowed
investigation of stereochemical aspects of the reaction. The
1H NMR spectrum of 5a showed resonances for a single
reaction product as a single diastereoisomer.
N-Methyl-N-nosyl-L-isoleucine (5a) was converted into
the corresponding methyl ester 7a by treatment with diazo-
methane (Scheme 3). The GC/MS analysis of 7a, showed also
the presence of a single chromatographic peak and then of a
single diastereoisomer, the mass spectrum of which corresponds
to the N-methyl-N-nosyl-L-isoleucine methyl ester (7a).1a
Hence no measurable epimerization was observed in the
deprotection reaction of the N-methyl-N-nosyl-L-isoleucine
phenacyl ester 4a with sodium benzenethiolate.
N-Methyl-N-nosyl-R-amino acids 5b-e were also ob-
tained in good yields and high purity (Scheme 3, Table 2)
as described for 5a.
Sodium benzenethiolate used in 5-fold molar excess al-
lowed selective deprotection of the phenacyl esters 4a-e
providing the corresponding N-methyl-N-nosyl-R-amino
acids 5a-e.
Subsequently we also explored the possibility of obtaining
from 4a-i the corresponding N-methyl-R-amino acids de-
protected on both the amino and the carboxyl function using
the same deprotecting reagent. In this additional experiment
the N-methyl-N-nosyl-L-valine phenacyl ester (4b) was trea-
ted in DMF with PhSNa in the molar ratio 1:5 respectively
for longer times (Scheme 4).
After about 1 h the TLC analysis (diethyl ether/petroleum
ether 70:30 v/v) of the reaction mixture showed the forma-
tion of a second product. It presented the typical coloration
at the ninhydrin test, thus proving the deprotection of the
amino function.
After 12 h the reaction mixture was acidified with a
solution of 1 N HCl and extracted with chloroform. GC/
MS analysis of the organic extract revealed the presence of
the N-methyl-N-nosyl-L-valine (5b). The aqueous solution
N-Methyl-N-nosyl-R-amino acid phenacyl esters (4a,
4c-i) treated under the same conditions of 4b produced
the corresponding N-Fmoc-N-methyl-R-amino acids (12a,
12c-i) in 70-78% yield of isolated product (Scheme 5,
Table 3).
Such results show that using a large excess of sodium
benzenethiolate both the amino and carboxyl functions of
the N-methyl-N-nosyl-R-amino acid phenacyl esters are
effectively deprotected.
Furthermore, the treatment of the deprotected products
11a-i with Fmoc-Cl afforded the corresponding N-Fmoc-
N-methyl-R-amino acids (12a-i) with good yields and high
purities.
The adopted procedure for obtaining N-Fmoc-N-methyl-
R-amino acids was successfully applied also to N-methyl-N-
nosyl-R-amino acid phenacyl esters 4f-i bearing functional-
ized side chains protected by acid labile protecting groups.
The recovery of N-Fmoc-N-methyl-R-amino acids 12f-i
required special attention due to the presence of the acid-
sensitive side chain protecting groups. The workup was
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SCHEME 4
SCHEME 5
TABLE 3. Results of the Synthesis of N-Fmoc-N-methyl-R-amino
Acids 12a-i
presence of chiral lanthanide NMR shift reagent europium-
(III) tris[3-(heptafluoropropylhydroxymethylene)-d-campho-
rate] (Eu[hfc]3).12 In a typical experiment to 10 mg of N-
Fmoc-N-methyl-L-valine (12b) dissolved in 0.5 mL of deut-
erated methanol was added the shift reagent with different
entry
R
yield (%)a
12a
12b
12c
12d
12e
12f
12g
12h
12i
CH(CH3)CH2CH3
CH(CH3)2
CH2CH(CH3)2
CH3
CH2(C6H5)
70
72
75
70
70
78
70
70
78
1
molar ratio and the corresponding H NMR spectra were
obtained. The spectra obtained from each interaction com-
plex that 12b has formed with different amounts of the shift
reagent are shown in Figure 1b-e. The effect of molar ratio
of the shift reagent to the substrate on the change of the
chemical shift of N-CH3 protons (δ 2.76 - 2.82) and R-CH
protons (δ 3.92 - 3.99) was investigated (Figure 1). Increas-
ing the concentration of the shift reagent only a greater
separation of the characteristic signals of the two rotational
isomers is observed but no signals for N-CH3 and R-CH
protons corresponding to the enantiomer N-Fmoc-N-methyl-
D-valine were detected. Decoupling experiments were used
for the attribution of the signals of the two rotational isomers
and thus to determine more accurately their ratio.
Additional experiments were performed to demonstrate
that the two sets of signals, one for each rotational isomer,
coalesce at higher temperature. The coalescence of the
signals of the two rotational isomers of the N-Fmoc-N-
methyl-L-valine (12b) was observed at 50 °C.3f
Equally in the spectra obtained, under the same conditions of
12b, with the products 12a, 12c-i there was no evidence of the
presence of enantiomeric species. For the products 12a, 12c,
12e, and 12i we observed a better separation of the signals
corresponding to the rotational isomers. The adopted experi-
ments exclude the presence in the products 12a-i of the corres-
ponding enantiomers in concentrations higher than that defined
by the instrument sensitivity under the used conditions (3%).
CH2S-(Bzl)
(CH2)4NH-(Boc)
CH2CONH-(Trt)
CH2C6H4O-(t-Bu)
aIsolated yield.
performed with a 5% aqueous solution of KHSO4 in order to
prevent the undesired deprotection of side chain functional-
ities. N-Fmoc-N-methyl-R-amino acids 12f-i were recov-
ered in good yields keeping unchanged the protecting group
on the side chains.
We measured the optical rotations of the N-Fmoc-N-methyl-
R-amino acids 12b ([R]25D -32.0), 12d ([R]25D -21.2), and 12e
([R]25 -54.5) and compared them with the corresponding
D
values reported in literature.11,1k The comparison demonstrated
that their optical purity is greater than 99%. Similarly, the opti-
cal rotations of the other products of the series 12 were mea-
sured (12a: [R]25D -51.4; 12c: [R]25D -20.2; 12f: [R]25D -67.5;
12g: [R]25 -23.8; 12h: [R]25 -6.5; 12i: [R]25 -48.1) and
D
D
D
compared with the values obtained, under the same conditions,
from the corresponding authentic samples with defined stereo-
chemistry and purity (12a: [R]25D -52.1; 12c: [R]25D -20.5; 12i:
[R]25 -48.3). The results show that these products have an
D
optical purity greater than 98%.
Furthermore in order to evaluate the enantiomeric purity
of the compounds of series 12 the proton NMR spectra of
these compounds were studied in deuterated methanol in the
(12) (a) Bucci, P.; Chidichimo, G.; Liguori, A.; Menniti, G.; Uccella, N.
Org. Magn. Reson. 1984, 22, 399. (b) Sweeting, L. M. J. Org. Chem. 1987, 52,
2273.
(11) Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S.; Arison, B. H. J. Org.
Chem. 1983, 48, 77.
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FIGURE 1. (a) The 300 MHz 1H NMR spectrum of N-Fmoc-N-methyl-L-valine (12b) in CD3OD. (b-e) Spectra of various mixtures of 12b
with the chiral shift reagent Eu(hfc)3.
These results are consistent with what is observed in
the NMR analysis of the N-Fmoc-N-methyl-L-Ile-OH
(12a) that showed the absence of epimerization products.3f
deprotects rapidly and selectively the carboxylic function
of the phenacyl esters 4a-e. The observed selectivity can be
explained by assuming that nucleophilic substitution by
sodium benzenethiolate at the carbinol carbon atom of the
phenacyl ester function is faster with respect to nucleo-
philic aromatic substitution involved in the removal of the
nosyl group from the amino function. The deprotection of
the amino function is subsequent to the removal of the
phenacyl group and occurs completely only when sodium
1
In the H NMR and 13C NMR spectra of 12a only signals
corresponding to one diastereoisomer were observed. The
protection of the carboxylic function of N-nosyl-R-amino
acids as phenacyl esters proved to be a successful choice to
obtain the corresponding N-methyl-N-nosyl-R-amino acids.
Sodium benzenethiolate used with a 5-fold molar excess
1390 J. Org. Chem. Vol. 75, No. 5, 2010

Leggio et al.
JOCArticle
benzenethiolate is used with a 10-fold molar excess and for
longer times.
(1 mmol) in N,N-dimethylformamide (DMF) was added slowly
a solution of phenacyl bromide (1 mmol) in DMF. During the
reaction a white solid of cesium bromide was formed. The
reaction mixture was stirred for about 1 h, monitoring the
conversion of 2a-i by TLC (diethyl ether/petroleum ether
70:30 v/v). The white solid was then separated by filtration
and the solvent was evaporated under reduced pressure. The
residue was treated with a 9% aqueous solution of sodium
carbonate and extracted with chloroform (3 ꢀ 10 mL). The com-
bined organic extracts were washed with water and a saturated
aqueous solution of NaCl, dried (Na2SO4), and evaporated to
dryness to afford the corresponding N-nosyl-R-amino acid
phenacyl esters 3a-i as pale yellow solids in 79-99% overall
yields.
N-Nosyl-L-isoleucine phenacyl ester (3a): yellow solid (95%);
mp 191-192 °C; [R]25D þ 12.5 (c 0.50, CHCl3); 1H NMR (300
MHz, DMSO-d6) δ 8.75 (d, 1H, J = 9.3 Hz), 8.39 (d, 2H, J = 9.0
Hz), 8.03 (d, 2H, J = 9.0 Hz), 7.91-7.84 (m, 2H), 7.67 (m, 1H),
7.56-7.49 (m, 2H), 5.31 (s, 2H), 3.92 (dd, 1H, J = 6.0 Hz, J =
9.3 Hz), 1.85 (m, 1H), 1.48 (m, 1H), 1.17 (m, 1H), 0.92 (d, 3H,
J = 6.9 Hz), 0.81 (t, 3H, J = 7.5 Hz); 13C NMR (75 MHz,
DMSO-d6) δ 192.5, 170.4, 149.9, 146.9, 134.5, 134.1, 129.3,
128.5, 128.2, 124.8, 67.2, 60.7, 37.5, 24.6, 15.6, 11.4; MS (EI) m/z
(rel intensity, %) 377 (0.4), 315 (0.8), 271 (26), 248 (2), 215 (17),
192 (21), 186 (9), 122 (15), 120 (55), 105 (100), 77 (24), 65 (3).
Anal. Calcd for C20H22N2O7S: C, 55.29; H, 5.10; N, 6.45; S,
7.38. Found: C, 55.11; H, 5.12; N, 6.48; S, 7.34.
Conclusions
The present paper shows how we developed an efficient
method to prepare N-methylated R-amino acids N-nosyl and
N-Fmoc protected, which are, in turn, useful to obtain N-
methylated peptides.
The synthetic strategy is based on the use of the phenacyl
group to temporarily protect the carboxyl function of N-
nosyl-R-amino acids. The phenacyl group has proved bene-
ficial in that it is easily introduced, it is stable to the
methylation reaction with diazomethane and it is easily
removed.
An important and attractive aspect of this protecting
system is represented by the possibility to employ for its
removal a reagent also useful to deprotect the amino func-
tion of N-methyl-N-nosyl-R-amino acid phenacyl esters.
In fact, the simple use of sodium benzenethiolate allows
the selective and rapid deprotection of the carboxyl function
of N-methyl-N-nosyl-R-amino acid phenacyl esters, thus
providing the corresponding N-methyl-N-nosyl-R-amino
acids.
The same reagent, used with a larger molar excess, depro-
tects both the amino and the carboxy function of N-methyl-
N-nosyl-R-amino acid phenacyl esters. The completely de-
protected N-methyl-R-amino acids were converted easily
into the corresponding N-Fmoc-N-methyl-R-amino acids.
Our approach was successfully applied also to a set of side
chain functionalized R-amino acids bearing acid-sensitive
protecting groups.
General Synthetic Procedure for N-Methyl-N-nosyl-r-amino
Acid Phenacyl Esters 4a-i. A 0.66 M solution of diazomethane9
in dichloromethane (6 mmol) was added cautiously dropwise to
a stirred solution of N-nosyl-R-amino acid phenacyl esters 3a-i
(1 mmol) in dry dichloromethane (10 mL). The resulting mixture
was maintained under an inert atmosphere (N2) and stirred at
room temperature for 1 h monitoring the conversion of the
precursor by TLC analysis (diethyl ether/petroleum ether 70:30
v/v). Evaporation of the solvent under reduced pressure af-
forded the N-methyl-N-nosyl-R-amino acid phenacyl esters
4a-i as oils in quantitative yields.
Experimental Section
N-Methyl-N-nosyl-L-isoleucine phenacyl ester (4a): yellow oil;
General Experimental Methods. Solvents were purified and
dried by standard procedures and distilled prior to use. Melting
points were recorded on a Kofler hot-stage apparatus and are
uncorrected. The 1H NMR and 13C NMR spectra were recorded
at 300 and 75 MHz, respectively, using CD3OD, CDCl3 or
DMSO-d6 as solvents. Chemical shifts are reported in units
of parts per million and all coupling constants are reported
in hertz. GC/MS analyses were performed with an HP-5MS
(30 m ꢀ 0.25 mm, PhMesiloxane 5%) capillary column. The
mass detector was operated in the electron impact ionization
mode (EI-MS) with an electron energy of 70 eV. All reactions
were monitored by thin-layer chromatography, using silica gel
60-F254 precoated glass plates. When required, the reactions
were carried out under an inert atmosphere (N2). The dichloro-
methane solution of diazomethane was prepared from N-methyl-
N-nitrosourea with a classical procedure.9 The concentration of
the diazomethane solution (0.66 M) was obtained by a back-
titration performed with a standard benzoic acid solution.
Caution:9 Diazomethane is highly toxic. Hence, this reagent
must be handled carefully. Dichloromethane solutions of diazo-
methane are stable for long periods if stored on KOH pellets at
-20 °C.
1
[R]25 -33.0 (c 0.50, CHCl3); H NMR (300 MHz, CDCl3) δ
D
8.30 (d, 2H, J = 9.0 Hz), 8.07 (d, 2H, J = 9.0 Hz), 7.78-7.72 (m,
2H), 7.61 (m, 1H), 7.51-7.43 (m, 2H), 5.19 (d, 1H, J = 16.2 Hz),
5.11 (d, 1H, J = 16.2 Hz), 4.50 (d, 1H, J = 10.5 Hz), 2.98 (s, 3H),
2.02 (m, 1H), 1.68 (m, 1H), 1.23 (m, 1H), 1.04 (d, 3H, J = 6.3
Hz), 0.98 (t, 3H, J = 7.5 Hz); 13C NMR (75 MHz, CDCl3) δ
190.5, 168.4, 149.9, 144.6, 134.2, 133.6, 129.1, 128.9, 127.5,
123.9, 65.9, 63.9, 33.6, 30.5, 24.9, 15.5, 10.7; MS (EI) m/z (rel
intensity, %) 405 (0.1), 391 (3), 329 (0.9), 285 (100), 262 (13), 229
(26), 188 (13), 186 (12), 122 (20), 120 (23), 105 (44), 77 (20), 65
(3). Anal. Calcd for C21H24N2O7S: C, 56.24; H, 5.39; N, 6.25; S,
7.15. Found: C, 56.41; H, 5.41; N, 6.27; S, 7.18.
General Synthetic Procedure for N-Methyl-N-nosyl-r-amino
Acid 5a-e. Sodium benzenethiolate (5 mmol) was added cau-
tiously to a stirred solution of N-methyl-N-nosyl-R-amino acid
phenacyl esters 4a-e (1 mmol) in DMF. The resulting mixture
was maintained under an inert atmosphere (N2) and stirred at
room temperature for 30 min monitoring the conversion of the
precursor by TLC analysis (diethyl ether/petroleum ether 70:30
v/v). After evaporation of the solvent under reduced pressure
the obtained residue was treated with an aqueous solution of 1 N
NaOH and extracted with chloroform (3 ꢀ 10 mL). The result-
ing aqueous basic solution was then acidified with a solution
of 1 N HCl and then extracted with chloroform (3 ꢀ 10 mL).
The combined organic extracts were washed with a saturated
aqueous solution of NaCl, dried (Na2SO4), and evaporated
to dryness to afford the corresponding N-methyl-N-nosyl-R-
amino acids 5a-e as oils in 70-87% overall yields. N-Methyl-
N-nosyl-R-amino acids 5a-e (1 mmol) by treating with a
General Synthetic Procedure for N-Nosyl-r-amino Acid Phe-
nacyl Esters 3a-i. The N-nosyl-R-amino acids2,7 1a-l (1 mmol)
were dissolved in ethanol and cooled to 0 °C. An aqueous
solution of cesium carbonate (0.5 mmol) was added slowly
and the reaction mixture was stirred for 1 h at room tempera-
ture. Then the solvent was evaporated under reduced pressure to
afford the corresponding cesium salts of N-nosyl-R-amino acids
2a-i as yellow solids in quantitative yields. To a solution of 2a-i
J. Org. Chem. Vol. 75, No. 5, 2010 1391

Leggio et al.
JOCArticle
diazomethane solution (3 mmol) were converted into the corre-
sponding methyl esters 7a-e and analyzed by GC/MS.
N-Methyl-N-nosyl-L-isoleucine (5a): yellow oil (70%); 1H
NMR (300 MHz, CD3OD) δ 8.40 (d, 2H, J = 9.0 Hz), 8.05
(d, 2H, J = 9.0 Hz), 4.16 (d, 1H, J = 10.5 Hz), 2.90 (s, 3H), 1.90
(m, 1H), 1.60 (m, 1H), 1.17 (m, 1H), 0.98-0.91 (m, 6H); 13C
NMR (75 MHz, CD3OD) δ 171.2, 149.9, 143.9, 128.6, 123.8,
63.7, 33.2, 29.6, 24.8, 14.3, 9.3. Anal. Calcd for C13H18N2O6S: C,
47.26; H, 5.49; N, 8.48; S, 9.71. Found: C, 47.32; H, 5.47; N,
8.46; S, 9.68.
N-Fmoc-N-methyl-L-isoleucine (12a): white solid (70%); mp
182-183 °C; [R]25D -51.4 (c 1.00, CHCl3); 1H NMR (300 MHz,
DMSO-d6) [mixture of two rotational isomers A and B (80:20)] δ
7.94-7.24 (m, 8H), 4.48-3.92 (m, 4H), 2.71 (A) and 2.69 (B) (2s,
3H), 1.80 (m, 1H), 1.34-1.07 (m, 2H), 0.93-0.72 (m, 6H); 13
C
NMR (75 MHz, DMSO-d6) [two rotational isomers] δ 172.5,
156.4, 144.3, 144.2, 141.3, 128.1, 127.5, 125.4, 120.5, 67.2, 62.7,
59.9, 47.2, 33.1, 30.4, 25.1, 22.1, 16.2, 10.8. Anal. Calcd for
C22H25NO4: C, 71.91; H, 6.86; N, 3.81. Found: C, 71.69; H, 6.87;
N, 3.82.
General Synthetic Procedure for N- Fmoc-N-methyl-r-amino
Acid 12f-i. Sodium benzenethiolate (10 mmol) was added to a
stirred solution of N-methyl-N-nosyl-R-amino acid phenacyl
esters 4f-i (1 mmol) in dimethylformamide (DMF). The result-
ing mixture was maintained under an inert atmosphere (N2) and
stirred at room temperature for 3 h monitoring the progress of
the deprotection reaction by TLC analysis (diethyl ether/petro-
leum ether 70:30 v/v). After evaporation of the solvent under
reduced pressure the obtained residue was treated with a 5%
aqueous solution of KHSO4 (pH 3-4) and extracted with
ethyl acetate (3 ꢀ 10 mL). The aqueous phase was basified with
saturated aqueous Na2CO3 (pH 9). To the basic liquor kept at
0 °C by an ice bath, containing the corresponding completely
deprotected N-methyl-R-amino acids 11f-i, was added drop-
wise a solution of 9-fluorenylmethyloxycarbonyl chloride
(1 mmol) in dioxane. The reaction mixture was stirred at 0 °C
for 3 h monitoring the complete conversion of the precursor
11f-i by TLC analysis (chloroform/methanol 90:10 v/v). After
evaporation of the solvent under reduced pressure the basic
aqueous solution was extracted with diethyl ether. The aqueous
solution was then acidified with a 5% aqueous solution of
KHSO4 (pH 3-4) and extracted with ethyl acetate (4 ꢀ 10
mL). The combined organic extracts were dried (Na2SO4) and
evaporated to dryness to afford the N-Fmoc-N-methyl-R-amino
acids 12f-i (70-78% overall yields).
N-Methyl-N-nosyl-L-isoleucine methyl ester (7a): MS (EI) m/z
(rel intensity, %) 287 (32), 285 (100), 229 (31), 186 (23), 122 (20),
57 (3).
Synthesis of N-Acetyl-N-methyl-L-valine (9b). Sodium benzen-
ethiolate (2.37 mmol) was added cautiously to a stirred solu-
tion of N-methyl-N-nosyl-L-valine phenacyl esters (4b) (0.47
mmol) in DMF. The resulting mixture was maintained under an
inert atmosphere (N2) and stirred at room temperature for 12 h
monitoring the conversion of the precursor by TLC analysis
(diethyl ether/petroleum ether 70:30 v/v) and verifying the
deprotection of the amino function by ninhydrin test. After
evaporation of the solvent under reduced pressure the obtained
residue was treated with an aqueous solution of 1 N NaOH and
extracted with chloroform (3 ꢀ 10 mL). The resulting aqueous
basic solution was then acidified with a solution of 1 N HCl and
then extracted with chloroform (3 ꢀ 10 mL). The evaporation of
the organic solvent provided the N-methyl-N-nosyl-L-valine
(5b) (0.061 g) in 40% yields. The aqueous phase was made basic
with a saturated Na2CO3 solution (pH 9.0) and treated with
acetic anhydride (0.95 mmol) in chloroform at room tempera-
ture for 1 h. The mixture, after extraction with chloroform, was
acidified with 1 N HCl and again extracted with chloroform (3 ꢀ
8 mL). The combined organic extracts were dried and evapo-
rated to dryness to afford the N-acetyl-N-methyl-L-valine (9b)
(0.020 g) in 25% yield. The N-acetyl-N-methyl-L-valine (9b)
(0.020 g, 0.12 mmol) by treating with a dichloromethane solu-
tion of diazomethane (0.36 mmol) was converted into the
corresponding methyl esters 10b and analyzed by GC/MS.
N-Acetyl-N-methyl-L-valine methyl ester (10b): MS (EI) m/z
(rel intensity, %) 187 (2), 144 (5), 128 (54), 102 (64), 86 (100), 43
(22).
General Synthetic Procedure for N-Fmoc-N-methyl-r-amino
Acid 12a-e. Sodium benzenethiolate (10 mmol) was added to a
stirred solution of N-methyl-N-nosyl-R-amino acid phenacyl
esters 4a-e (1 mmol) in DMF. The resulting mixture was
maintained under an inert atmosphere (N2) and stirred at room
temperature for 3 h monitoring the progress of the deprotection
reaction by TLC analysis (diethyl ether/petroleum ether 70:30 v/
v). After evaporation of the solvent under reduced pressure the
obtained residue was treated with 1 N HCl (pH 2) and the
aqueous solution was extracted with ethyl acetate (3 ꢀ 10 mL).
The aqueous phase was basified with saturated aqueous
Na2CO3 (pH 9). To the basic liquors cooled at 0 °C, containing
the corresponding completely deprotected N-methyl-R-amino
acids 11a-e, was added dropwise a solution of 9-fluorenyl-
methyloxycarbonyl chloride (1 mmol) in dioxane. The reaction
mixture was stirred at 0 °C for 3 h with monitoring the complete
conversion of the precursor 11a-e by TLC analysis (chloro-
form/methanol 90:10 v/v). After evaporation of the solvent
under reduced pressure the basic aqueous solution was extracted
with diethyl ether. The aqueous solution was then acidified with
1 N HCl (pH 2) and extracted with ethyl acetate (4 ꢀ 10 mL).
The combined organic extracts were dried (Na2SO4) and eva-
porated to dryness to afford the N-Fmoc-N-methyl-R-amino
acids 12a-e (70-75% overall yields).
N-Fmoc-N-methyl-O-tert-butyl-L-tyrosine (12i):(78%);[R]25
-
D
48.1 (c 1.00, DMF); 1H NMR (300 MHz, DMSO-d6) [mixture of
two rotational isomers A and B (60:40)] δ 12.85 (br s, 1H),
7.88-7.25 (m, 8H), 7.08 (d, 2H, J = 8.4 Hz), 6.80 (d, 2H,
J = 8.4 Hz), 4.71 (A) and 4.63 (B) (2 dd, 1H, J = 4.8, 11.4 Hz),
4.31-4.10 (m, 3H), 3.19-2.89 (m, 2H), 2.68 (A) and 2.66 (B) (2s,
3H), 1.20 (B) and 1.15 (A) (2s, 9H); 13C NMR (75 MHz, DMSO-
d6) [two rotational isomers] δ 172.5, 172.3, 156.1, 153.8, 144.2,
144.1, 141.2, 133.0, 132.8, 129.7, 128.1, 127.5, 125.4, 123.9,
120.6, 78.0, 67.2, 60.9, 47.1, 46.9, 33.7, 32.2, 31.7, 28.9. Anal.
Calcd for C29H31NO5: C, 73.55; H, 6.60; N, 2.96. Found: C,
73.69; H, 6.62; N, 2.97.
Acknowledgment. We gratefully acknowledge Dr. Anna
Piperno and Dr. Caterina Carnovale of Farmaco-Chimico
Department University of Messina for optical rotation
measurements.
Note Added after ASAP Publication. The Supporting
Information file was incomplete in the version published ASAP
on February 1, 2010; the complete file was posted on February 5,
2010.
Supporting Information Available: General experimental
methods, experimental details for the synthesis of compounds
3b-i, 4b-i, 5b-e, and 12b-h, and copies of 1H NMR
and 13C NMR spectra of compounds 3a-i and 4a-i. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1392 J. Org. Chem. Vol. 75, No. 5, 2010