N-methylation of peptides on selected positions during the elongation of the peptide chain in solution phase

Article abstract of DOI:10.1021/jo0478959

An efficient and general solution-phase method for the site-specific N-methylation of peptides has been developed. This novel procedure involves synthesis of N-nosyl protected peptides and their subsequent N-methylation with diazomethane. Its efficiency w

Full text of DOI:10.1021/jo0478959

N-Methylation of Peptides on Selected Positions during the
Elongation of the Peptide Chain in Solution Phase
M. Luisa Di Gioia, Antonella Leggio,* and Angelo Liguori
Dipartimento di Scienze Farmaceutiche, Universita` della Calabria, Via Ponte P. Bucci cubo 15/C,
I-87036 Arcavacata di Rende (CS), Italy
A.Leggio@unical.it
Received November 26, 2004
An efficient and general solution-phase method for the site-specific N-methylation of peptides has
been developed. This novel procedure involves synthesis of N-nosyl protected peptides and their
subsequent N-methylation with diazomethane. Its efficiency was proved by the successful synthesis
of various hindered oligopeptides containing N-methyl amino acid residues with excellent yield
and purity. The method is particularly attractive in that the adopted conditions do not cause any
detectable racemization of the peptide stereocenters and the process does not require chromato-
graphic purification of the methylated products. A further advantage is the compatibility of this
methodology with Fmoc solution-phase peptide synthesis.
Introduction
therapeutic potential of the peptide.2 N-Methyl peptides
also represent important systems for structure-activity
studies.
Several naturally occurring peptides that exhibit in-
teresting biological activities such as cyclosporines,3
dolastatins,4 and didemnins5 are characterized by the
presence in their chain of N-methylated peptide bonds.
Peptide analogues that possess an increased biological
activity and bioavailability with respect to the corre-
sponding naturally occurring peptides can be obtained
by modifying appropriately the peptide backbone.1
N-Methylation of a peptide bond provides a bulky steric
modification that can change the conformation of the
peptide. It can also block potential intramolecular hy-
drogen bonding sites and proteolytic enzyme cleavage
sites. These properties, together with the enhanced
hydrophobicity, can increase the bioavailability and the
(2) (a) Ali, F. E.; Bennett, D. B.; Calvo, R. R.; Elliott, J. D.; Hwang,
S.-M.; 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.-K.; Samanen, J.
M. J. Med. Chem. 1994, 37, 769-780. (b) Manavalan, P.; Momany, F.
A. Biopolymers 1980, 19, 1943-1973. (c) Rajeswaran, W. G.; Hocart,
S. J.; Murphy, W. A.; Taylor, J. E.; Coy, D. H. J. Med. Chem. 2001, 44,
1416-1421. (d) 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. (e) Stimson, E. R.; Meinwald, Y. E.;
Mintelion, G. T.; Scheraga, H. A. Int. J. Pept. Protein Res. 1986, 27,
569-573. (f) Mammi, S.; Goodman, M. Int. J. Pept. Protein Res. 1986,
27, 29-34. (g) Schimidt, R.; Kalman, A.; Chung, N. N.; Lemieux, C.;
Horvath, C.; Schiller, P. W. Int. J. Pept. Protein Res. 1995, 46, 47-55.
(h) Takenchi, Y.; Marshall, G. R. J. Am. Chem. Soc. 1998, 120, 5363-
5372. (i) Mazur, R. H.; James, P. A.; Tyner, D. A.; Hallina, E. A.;
Sanner, J. H.; Schulze, R. J. Med. Chem. 1980, 23, 758-763.
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Kantoci, D.; Burkett, D. D.; Barko`czy, J.; Hogan, F.; Wardlaw, T. R.
J. Org. Chem. 1994, 59, 6287-6295 and references therein.
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J. M.; Moos, W. H Bioorg. Med. Chem. Lett. 1994, 4, 2657-2662. (b)
Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Siciliano, C. Eur.
J. Org. Chem. 2004, 463-467. (c) Di Gioia, M. L.; Leggio, A.; Liguori,
A.; Napoli, A.; Siciliano, C.; Sindona, G. J. Org. Chem. 2001, 66, 7002-
7007. (d) Leggio, A.; Liguori, A.; Procopio, A.; Sindona, G. J. Chem.
Soc., Perkin Trans. 1 1997, 1969-1971. (e) Steer, D. L.; Lew, R. A.;
Perlmutter, P.; Smith, A. I.; Aguilar, M.-I. Lett. in Peptide Sci. 2001,
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Int. Ed. Engl. 1993, 32, 1244-1267. (b) Gante, J. Angew. Chem., Int.
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C.; Lansky, A.; Zechel, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 2289-
2337.
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Org. Chem. 1989, 54, 617-627 and references therein.
10.1021/jo0478959 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/07/2005
3892
J. Org. Chem. 2005, 70, 3892-3897

N-Methylation of Peptides
N-Methylated peptides are generally prepared by
incorporation of previously synthesized protected N-
methylated amino acids6 into the peptide chain in solu-
tion or solid phase.7 The difficulty of these syntheses is
often due to the sterically hindered N-methyl amino acids
which resist coupling.8 Therefore, preparation of N-methyl-
ated peptides can be challenging because racemization9
and diketopiperazine formation10 are common side reac-
tions.
Recently we introduced a new and efficient solution
method for preparing N-methylated amino acid methyl
esters by methylation of N-p-nitrobenzensulfonyl-R-
amino acid methyl esters with diazomethane.6a
atom by the nosyl group as reagents for peptide coupling.
Nosyl protected amino acids chlorides should allow
practical synthesis of hindered peptides containing N-
methyl amino acid residues using solution-phase meth-
odology. In addition, amino acid chlorides protected on
the amino function with arenesulfonyl group can avoid
the risk of oxazolone formation.11
Only a few examples of direct N-methylation of pep-
tides are reported, the majority of which on the solid
phase.12 Some of these procedures are characterized by
nonselective permethylation of peptides,12f or modest
yields12d or incompatibility with standard Fmoc peptide
synthesis procedures.12e
Direct methylation of the terminal amino function of
N-nosyl peptides and the subsequent elongation of the
methylated peptide chains could represent an interesting
goal in the peptide synthesis. The successful application
of the direct N-methylation to peptides N-nosyl protected
could be used afterward to carry out N-methylation of
peptide bond in key positions of peptide chain and to
study the effects of N-methylation at every residue of a
biologically active peptide.
N-Methylation of N-nosyl-peptides could circumvent
the problem of racemization since it avoids the use of
protected N-methyl amino acids as activated components.
In fact, protected N-methyl-amino acids activated on the
carboxyl function are more subjected to racemization,7a
proceeding through R-deprotonation or enolization of the
oxazolonium ion, than the corresponding amino acids not
methylated on the protected amino function.
In this work we describe a new, straightforward and
general method for site-specific N-methylation of peptides
during the elongation of peptide chain in solution phase.
It involves the synthesis of N-nosyl-protected peptides
and their subsequent N-methylation.6a The latter process
requires simply an ethereal solution of diazomethane,6a
that acting as a base, generates in situ the methyl
diazonium ion13 responsible for the N-methylation. The
use of bases in the methylation reaction can be crucial,
since weak bases can provide poor yield of methylated
peptide while strong bases can cause methylation of the
amide backbone. The choice of diazomethane as methy-
lating reagent could be significant to achieve specific
N-methylation of the amino terminal function.
The proposed methodology is developed for solution
peptide synthesis and consequently it is applied to short
peptide sequences considering the poor solubility of larger
peptides in most of organic solvents.
Furthermore, we are interested in the use of highly
reactive aminoacyl chlorides protected at the nitrogen
Results and Discussion
(6) (a) Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Napoli,
A.; Siciliano, C.; Sindona, G. J. Org. Chem. 2003, 68, 7416-7421. (b)
Luke, R. W. A.; Boyce, P. G. T.; Dorling, E. K. Tetrahedron Lett. 1996,
37, 263-266. (c) Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B. Org.
Lett. 2002, 4, 3767-3769. (d) Prashad, M.; Har, D.; Hu, B.; Kim, H.-
Y.; Repic, O.; Blacklock, T. J. Org. Lett. 2003, 5, 125-128. (e) Aurelio,
L.; Box, J. S.; Brownlee, R. T. C.; Hughes, A. B.; Sleebs, M. M. J. Org.
Chem. 2003, 68, 2652-2667. (f) Yang, L.; Chiu, K. Tetrahedron Lett.
1997, 38, 7307-7310. (g) Papaioannou, D.; Athanassopoulos, C.;
Magafa; V.; Karamanos, N.; Stavropoulos, G.; Napoli, A.; Sindona, G.;
Aksnes, D. W.; Francis, G. W. Acta Chem. Scand. 1994, 48, 324-333.
(h) Dorow, R. L.; Gingrich, D. E. J. Org. Chem. 1995, 60, 4986-4987.
(i) Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B. Chem. Rev. 2004,
104, 5823-5846.
(7) (a) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97,
2243-2266. (b) Akaji, K.; Hayashi, Y.; Kiso, Y.; Kuriyama, N. J. Org.
Chem. 1999, 64, 405-411. (c) Li, P.; Xu, J. C. Tetrahedron 2000, 56,
9949-9955. (d) Jao, E.; Cooper, A. B.; Rane, D. F.; Saksena, A. K.;
Desai, J.; Wang, J.; Girijavallabhan, V. M.; Ganguly, A. K: Tetrahedron
Lett. 1996, 37, 5661-5664. (e) Angell, Y. M.; Thomas, T. L.; Flentke,
G. R.; Rich, D. H. J. Am. Chem. Soc. 1995, 117, 7279-7280. (f) Raman,
P.; Stokes, S. S.; Angell, Y. M.; Flentke, G. R.; Rich, D. H. J. Org. Chem.
1998, 63, 5734-5735. (g) Ko, S. Y.; Wenger, R. M.; Helv. Chim. Acta
1997, 80, 695-705.
To explore the direct methylation of oligopeptides with
diazomethane, the synthesis of N-nosyl-protected dipep-
tides was undertaken. The preparation of dipeptides 5-9
required the synthesis of N-nosyl-R-amino acids and their
subsequent activation.
The starting N-nosyl-R-amino acids 2a-g were pre-
pared from p-nitrobenzensulfonyl chloride (Ns-Cl) and
the corresponding R-amino acids 1a-g dissolved in an
aqueous solution of NaOH 1N11a,14 (Scheme 1, Table 1).
The reaction of 2a-g with thionyl chloride gave the
corresponding N-nosyl amino acids chlorides 3a-g in
quantitative yields (Scheme 2).
The synthesis of N-nosyl-dipeptides 5-9 was then
accomplished by coupling of N-nosyl amino acids chlo-
rides 3a-g with R-amino acid methyl esters 4a,b (Scheme
2). The proceeding of the coupling reaction was initially
verified by treating N-nosyl-l-phenylalanine chloride (3c),
(8) (a) Angell, Y. M.; Garcia-Echeverria, C.; Rich, D. H. Tetrahedron
Lett. 1994, 35, 5981-5984. (b) Coste, J.; Frerot, E.; Jouin, P.; Castro,
B. Tetrahedron Lett. 1991, 32, 1967. (c) Wenger, R. M. Helv. Chim.
Acta 1983, 66, 2672. (d) Tung, R. D.; Dhaon, M. K.; Rich, D. H. J. Org.
Chem. 1986, 51, 3350
(11) (a) Vedejs, E.; Lin, S.; Klapars, A.; Wang, J. J. Am. Chem. Soc.
1996, 118, 9796-9797 and references therein. (b) Carpino, L. A.;
Ionescu, D.; El-Faham, A. Henklein, P.; Wenschuh, H.; Bienert, M.;
Beyermann, M. Tetrahedron Lett. 1998, 39, 241-244.
(12) (a) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119,
2301-2302. (b) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1998,
120, 2690-2691. (c) Vedejs, E.; Kongkittingam, C. J. Org. Chem. 2000,
65, 2309-2318. (d) Reichwein, J. F.; Liskamp, R. M. J. Tetrahedron
Lett. 1998, 39, 1243-1246. (e) Kaljuste, K.; Unden, A. Int. J. Pept.
Protein Res. 1993, 42, 118-124. (f) Ostresh, J. M.; Husat, G. M.;
Blondelle, S. E.; Dorner, B.; Weber, P. A.; Hounghten, R. A. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 11138-11142.
(9) (a) Fre`rot, E.; Coste, J.; Poncet, J.; Jouin, P.; Castro, B.
Tetrahedron Lett. 1992, 33, 2815-2816. (b) McDermott, J. R.; Benoiton,
N. L. Can. J. Chem. 1973, 51, 2555-2561. (c) McDermott, J. R.;
Benoiton, N. L. Can. J. Chem. 1973, 51, 2562-2570. (d) Calmes, M.;
Cavelier-Frontin, F.; Jacquier, R.; Mercadier, J.-L.; Sabil, S.; Verducci,
J.; Quiot, J.-M.; Vey, A. Int. J. Pept. Protein Res. 1993, 41, 528-535.
(e) Wenger, R. M. Helv. Chim. Acta 1983, 66, 2672-2681.
(10) (a) Khosla, M. C.; Smeby, R. R.; Bumpus, F. M. J. Am. Chem.
Soc. 1972, 94, 4721-4724. (b) Rich, D. H.; Bhatnagar, P.; Mathia-
paranam, P.; Grant, J. A.; Tam, J. P. J. Org. Chem. 1978, 43, 296-
302. (c) Gisin, B. F.; Merrifield, R. B. J. Am. Chem. Soc. 1972, 94,
3102-3106. (d) Thern, B.; Rudolph, J.; Jung, G. Tetrahedron Lett. 2002,
43, 5013-5016.
(13) Leggio, A.; Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G. J.
Org. Chem. 2001, 66, 2246-2250.
(14) Milne, H. B.; Peng, C.-H J. Am. Chem. Soc. 1957, 79, 639-
644.
J. Org. Chem, Vol. 70, No. 10, 2005 3893

Di Gioia et al.
SCHEME 1
N-Nosyldipeptide methyl esters 7-9 were also ob-
tained as previously described in high yields and purity
(Scheme 2, Table 2).
Methylation reaction was performed, at room temper-
ature, by treating N-nosyldipeptides 5-9 with an ethe-
real solution of diazomethane (0.66 N) in the molar ratio
1:8 (Scheme 3). The reaction was completed within 30
min and the corresponding N-nosyl-N-methyl-dipeptides
5a-9a were recovered with quantitative yields and high
purity after evaporation of the solvent under reduced
pressure (Scheme 3).
TABLE 1. Results of the Syntheses of N-Nosyl Amino
Acids 2a-g
The methylation reaction is chemospecific, as deter-
mined by analyzing the products 5a-9a by NMR and GC/
MS methods; in fact, only products monomethylated on
the sulfonamide nitrogen atom were identified, no traces
of other methylated products were observed. Mass spec-
tra of the obtained N-methylated dipeptides allowed the
unequivocal assignment of the methylation site in each
dipeptide. The N-nosyl-N-methylated residue was easily
identified by a considerable N-nosyl-N-methylimmonium
ion peak. Analysis of the other mass fragments and of
the 1H NMR spectra confirmed the location of the
N-methyl group in the peptide chain.
compound
R1
R2
yield (%)a
2a
2b
2c
2d
2e
2f
CH3CH2(CH3)CH
H
H
H
84
83
95
93
84
82
84
(CH3)2CH
C6H5CH2
H
C6H5CH2
CH3
H
H
CH3
H
2g
(CH3)2CHCH2
a Isolated yield.
TABLE 2. Results of the Syntheses of N-Nosyl
Dipeptides Methyl Esters 5-9
The application of the adopted N-methylation condi-
tions to two diastereomeric dipeptides 5 and 6 allowed
us to exclude the formation of epimeric methylated
products. The diastereomeric N-methyl-dipeptides 5a and
6a were readily resolved by GC/MS. Furthermore careful
compound
R1
R2
R3
yield (%)a
5
6
7
8
9
C6H5CH2
H
CH3
94
85
94
74
76
H
C6H5CH2 CH3
CH3CH2(CH3)CH
H
(CH3)2CHCH2
H
CH3
(CH3)2CH
CH3
CH3
H
1
examination of H NMR spectra of both N-methylated
a Isolated yield.
products 5a and 6a showed the presence of signals
corresponding to only one diastereomer. In particular,
comparison of the peaks of N-methyl and N-H amide
groups of both products 5a and 6a was employed to
analyze the presence in each spectrum of the other
diastereomer.
dissolved in ethanol free chloroform, with an aqueous
basic solution of l-alanine methyl ester hydrochloride 4a
at room temperature (Scheme 2). The reaction was
complete within 1h and afforded the dipeptide N-nosyl-
l-phenylalanyl-l-alanine (5) in 94% yield (Scheme 2, Table
2).
To study the stereochemical aspects of coupling reac-
tion, the dipeptide N-nosyl-d-phenylalanyl-l-alanine (6)
diastereomer of 5 was synthesized for comparison under
the same conditions using N-nosyl-d-phenylalanine chlo-
ride (3d). GC/MS analysis of the single crude products 5
and 6 showed the presence of only one diastereomer in
both samples.
GC/MS analysis performed on an appropriately pre-
pared mixture of the two diastereomers 5 and 6 showed
instead the presence of two peaks corresponding to the
two diastereomers. Hence N-nosyldipeptide methyl ester
5 was obtained without measurable epimerization. Also,
the comparison of H NMR spectrum of the mixture to
those obtained from the single crude products 5 and 6
excluded the formation of epimerized products.
The comparison signals were found with chemical
shifts that differs in the two diastereomers as showed
1
by H NMR spectrum of a mixture of 5a and 6a (Figure
1).
In light of the excellent results obtained with N-
nosyldipeptides, the methylation reaction with diazo-
methane was then investigated also with tripeptide
systems.
The tripeptide N-nosyl-l-Ile-d-Ala-l-Val-OMe (11), pre-
pared by coupling of N-nosyl-l-isoleucine chloride (3a)
with the dipeptide d-alanyl-l-valine-OMe (10), was readily
N-methylated according to the described procedure
(Scheme 4). The resulting N-nosyl-protected tripeptide
11a methylated on the nitrogen atom of the N-terminal
residue was isolated in quantitative yield as the sole
reaction product. 1H NMR and mass spectra of tripeptide
11a confirmed the specific N-methylation of the sulfona-
mide group.
1
SCHEME 2
3894 J. Org. Chem., Vol. 70, No. 10, 2005

N-Methylation of Peptides
FIGURE 1. 1H NMR spectrum of a mixture of N-nosyl-(Me)-l-Phe-l-Ala-OMe (5a) and N-nosyl-(Me)-d-Phe-l-Ala-OMe (6a).
SCHEME 3
SCHEME 5
SCHEME 4
TABLE 3. Results of the Syntheses of Tripeptides
Methyl Esters 12-14
compound
R1
R2
R3
yield (%)a
12
13
14
C6H5CH2
H
CH3
86
88
72
To demonstrate that our procedure could be used to
methylate each amino acid in a peptide chain, polym-
ethylated peptides were prepared by sequential N-
methylation of the N-terminal amino acid residue during
the elongation of the peptide.
H
C6H5CH2 CH3
CH3CH2(CH3)CH
H
CH3
a Isolated yield.
N-Methylated dipeptides 5a and 7a were easily depro-
tected on the terminal amino function by the reagent
system mercaptoacetic acid/sodium methoxide6a and then
coupled with N-nosyl-l-valine chloride (3b) (Scheme 5,
Table 3). The resulting monomethylated tripeptides 12
and 14 were recovered in high yields.
sponding dimethylated tripeptides N-nosyl-(Me)-l-Val-
(Me)-l-Phe-l-Ala-OMe (12a), N-nosyl-(Me)-l-Val-(Me)-d-
Phe-l-Ala-OMe (13a), and N-nosyl-(Me)-l-Val-(Me)-l-Ile-
l-Ala-OMe (14a) (Scheme 5).
Finally, to complete our study, it was also investigated
the compatibility of standard Fmoc solution peptide
synthesis techniques (Fmoc-SPS) with the use of p-
nitrobenzensulfonyl protecting group required when N-
methylation is desired.
Therefore in an additional experiment N-nosyl-N-
methyldipeptides 6a, 8a, and 9a, after removal of the
sulfonamide group, were coupled with N-Fmoc-amino
acid chlorides 15a,b (Scheme 6, Table 4) to give the
The tripeptide 13 epimer of 12 was also prepared
starting from the methylated dipeptide 6a diastereomer
of 5a to test the epimerization. There was no cross-
1
contamination of epimers according to H NMR spectra
of both tripeptides.
The methylated N-nosyltripeptides 12-14 were further
methylated with diazomethane to provide the corre-
J. Org. Chem, Vol. 70, No. 10, 2005 3895

Di Gioia et al.
SCHEME 6
N-methyl substituent of the deprotected methylated
dipeptides in the coupling step. Therefore products
derived from incomplete peptide coupling or competitive
cyclization reactions were not detected.
Conclusions
In conclusion, a novel and efficient solution-phase
method for the site-specific N-methylation of peptides
was developed.
The method described allows the specific methylation
of the sulfonamide nitrogen atom of N-nosyl peptides and,
after removal of the nosyl group, to elongate easily the
N-methylated peptide chain in order to obtain stereo-
chemically pure peptides N-methylated on specific amide
bonds.
The highly efficient coupling, the chemospecific and
quantitative N-methylation reaction and the rapid and
complete deprotection step allowed us to obtain N-
methylated peptides with high yields and excellent purity
without time-consuming chromatographic workup.
In addition, the elongation of peptide chain carried out
successfully using standard Fmoc SPS procedures to the
position where N-methylation was desired, demonstrated
that this methodology combines very well with solution
peptide synthesis based on Fmoc-chemistry.
TABLE 4. Results of the Syntheses of N-Fmoc
N-Methylated Tripeptides Methyl Esters 16-18
yield
(%)a
compound
R1
R2
R3
R4
CH3
16
17
18
H
H
C6H5CH2 CH3
CH3
H
92
(CH3)2CH (CH3)2CH 94
CH3
(CH3)2-
CHCH2
(CH3)2CH
93
a Isolated yield.
SCHEME 7
The adopted methodology represents a useful tool to
label with a methyl group the nitrogen atom of every
amino acid residue of biologically active peptide se-
quences for structure-activity studies.
The application on the solid phase of this new proce-
dure for the N-methylation of amino acids and peptides
is under investigation.
Experimental Section
Synthesis of N-Methyl-N-nosyldipeptides 5a-9a and
N-Methyl-N-nosyltripeptide 11a. General Procedure A.
A 0.66 M solution of diazomethane in diethyl ether (8 mmol)
was added cautiously dropwise to a magnetically stirred
solution of the N-nosyldipeptides methyl esters 5-9 (1 mmol)
or N-nosyltripeptide methyl ester 11 in dry dichloromethane
(10 mL). The resulting mixture was maintained at room
temperature and under N2 for 30 min. TLC analysis (chloroform/
methanol, 98:2 v/v) showed complete conversion of the precur-
sor. Evaporation of the solvent under reduced pressure af-
forded the N-methyl-N-nosyldipeptides methyl esters 5a-9a
or N-methyl-N-nosyltripeptide methyl ester 11a in quantita-
tive yields.
corresponding N-Fmoc-tripeptides 16-18 methylated on
the nitrogen atom of the second amino acid unit. Removal
of the Fmoc protecting group has been successfully tested
for these systems.16
Also, the N-nosyl-N-methyltripeptide 11a was sub-
jected to the above-described two-step sequence composed
of terminal amine deprotection, followed by coupling with
N-Fmoc-valine chloride (15b) to afford the corresponding
methylated tetrapeptide N-Fmoc protected 19 (Scheme
7).
The synthesis of methylated N-nosyl- or N-Fmoc-
protected structures occurs in a simple way and in high
yields. Furthermore, the high reactivity of the amino acid
chloride, formed cleanly using SOCl2 procedure, is more
than sufficient to overcome the steric hindrance by the
N-Methyl-N-nosyl-L-phenylalanyl-L-alanine Methyl Es-
ter (5a). The product was prepared by general procedure A
using 0.059 g (0.135 mmol) of 5 in dry dichloromethane and
1.64 mL (1.08 mmol) of 0.66 M diazomethane in diethyl ether.
The reaction was stirred for 30 min. Evaporation of the solvent
afforded the corresponding N-methyl dipeptide 5a in quantita-
tive yield: 1H NMR (300 MHz, CDCl3) δ 8.05 (d, 2 H, J ) 8.9
Hz), 7.48 (d, 2 H, J ) 8.9 Hz), 6.98-7.20 (m, 5 H), 6.83 (d, 1
H, J ) 7.0 Hz), 4.75 (dd, 1 H, J ) 10.3, 5.3 Hz), 4.52 (m, 1 H),
3.75 (s, 3 H), 3.21 (dd, 1 H, J ) 14.5, 5.2 Hz), 2.95 (s, 3 H),
2.82 (dd, 1 H, J ) 14.6, 10.3 Hz), 1.42 (d, 3 H, J ) 7.2 Hz). 13
C
NMR (75 MHz, CDCl3): δ 172.8, 168.5, 149.7, 144.5, 136.7,
129.0, 128.8, 128.00, 127.00, 124.2, 61.6, 52.7, 48.4, 34.0, 30.0,
18.1. MS (EI) m/z (rel intensity %) 390 (M+ - COOCH3•, 1),
358 (1), 319 (100), 303 (2), 263 (21), 233 (21), 186 (4), 132 (45),
122 (18), 91 (36), 76 (9), 59 (5). Anal. Calcd. For C20H23N3O7S:
C, 53.44; H, 5.16; N, 9.35; S, 7.13. Found: C, 53.46; H, 5.15;
N, 9.34; S, 7.11.
(15) Arndt, F. Org. Synth. 1943, 2, 165-168.
(16) (a) Leggio, A.; Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G.
Eur. J. Org. Chem. 2000, 573-575. (b) Di Gioia, M. L.; Leggio, A.; Le
Pera, A.; Siciliano, C.; Liguori, A. J. Peptide Res. 2004, 63, 383-387.
(b) Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Perri, F.;
Siciliano, C. Eur. J. Org. Chem. 2004, 4437-4441.
3896 J. Org. Chem., Vol. 70, No. 10, 2005

N-Methylation of Peptides
N-Methyl-N-nosyl-L-isoleucyl-D-alanyl-L-valine Methyl
Ester (11a). The product was prepared by general procedure
A using 0.50 g (1.01 mmol) of 11 in dry dichloromethane and
12 mL (8.01 mmol) of 0.66 M diazomethane in diethyl ether.
The reaction was stirred for 30 min. Evaporation of the solvent
afforded the corresponding N-methyl tripeptide 11a in quan-
titative yield: 1H NMR (300 MHz, CDCl3) δ 8.28 (d, 2 H, J )
8.9 Hz), 7.95 (d, 2 H, J ) 8.9 Hz), 6.82 (d, 1 H, J ) 6.5 Hz),
6.68 (d, 1 H, J ) 8.1 Hz), 4.55 (dd, 1 H, J ) 8.6, 4.9 Hz), 4.35
(m, 1 H), 4.06 (d, 1 H, J ) 6.7 Hz), 3.75 (s, 3 H), 2.93 (s, 3 H),
2.17 (m, 1 H), 1.95 (m, 1 H), 1.47-1.21 (m, 2 H), 1.34 (d, 3 H,
J ) 6.9 Hz), 0.96-0.82 (m, 12 H). 13C NMR (75 MHz, CDCl3):
δ 172.1, 171.5, 168.4, 149.9, 144.6, 128.5, 124.3, 64.0, 57.7, 52.4,
48.4, 33.0, 31.3, 30.2, 24.8, 19.3, 19.1, 18.9, 17.7, 10.5. MS (EI)
m/z (rel intensity %) 514 (M+• , 1), 384 (1), 356 (2), 328 (33),
285 (100), 270 (7), 255 (12), 229 (40), 186 (9), 122 (6). Anal.
Calcd. For C23H36N4O8S: C, 52.26; H, 6.86; N, 10.60; S, 6.07.
Found: C, 52.24; H, 6.88; N, 10.63; S, 6.04.
Synthesis of N-Nosyltripeptides 12-14, N-Fmoc-tri-
peptides 16-18, and N-Fmoc-tetrapeptide 19. General
Procedure B. Mercaptoacetic acid (3 mmol) was added to a
solution of 5a-9a or 11a (1 mmol) in dry acetonitrile (10 mL)
under N2 at 50 °C. Solid sodium methoxide (8 mmol) was then
added to the solution with a variable amount of methanol to
facilitate the sodium methoxide solubilization. The resulting
mixture was stirred for ∼1 h monitoring the conversion of 5a-
9a or 11a by TLC (diethyl ether/petroleum ether, 60:40 v/v).
Aqueous HCl 1 N was then added and the acidified solution
(pH 2) was extracted with ethyl acetate (3 × 10 mL). The
aqueous phase was basified with saturated aqueous NaHCO3
(pH ) 8). The basic liquors, containing the N-deprotected
products, were then treated with a solution of N-nosyl-L-valine
chloride 3b (1 mmol) or N-Fmoc amino acid chlorides 15a-b
in dry methylene chloride (10 mL). The reaction mixture was
stirred at room temperature for ∼1 h, and the organic layer
was separated. The aqueous phase was extracted with three
additional portions of methylene chloride (3 × 10 mL). The
combined organic extracts were dried with Na2SO4 and the
solvents evaporated under vacuum to afford the corresponding
N-Nosyl-tripeptides 12-14 in 72-88% yields, the N-Fmoc-
tripeptides 16-18 in 92-94%, and the N-Fmoc-tetrapeptide
19 in 83% yield.
toacetic acid, and 0.19 g (3.56 mmol) of sodium methoxide in
methanol (5 mL). The reaction was stirred at 50 °C for 40 min.
The afforded unmasked dipeptide in an aqueous 9% solution
of NaHCO3 was treated with 0.14 g (0.45 mmol) of 15a in dry
methylene chloride. The reaction was stirred at RT for 45 min.
The subsequent work up afforded 0.22 g of the title compound
16 (0.39 mmol, 92%): 1H NMR (300 MHz, CDCl3) δ 7.81-7.15
(m, 13 H), 6.80 (d, 1 H, J ) 7.4 Hz), 5.66 (dd, 1 H, J ) 11.2,
5.6 Hz), 5.60 (d, 1 H, J ) 6.8 Hz), 4.69-4.43 (m, 2 H), 4.36-
4.17 (m, 3 H), 3.76 (s, 3 H), 3.46 (dd, 1 H, J ) 14.9, 5.6 Hz),
2.99 (s, 3 H), 2.95 (dd, 1 H, J ) 14.9, 11.2 Hz), 1.42 (d, 3 H, J
) 7.2 Hz), 1.35 (d, 3 H, J ) 7.3 Hz). 13C NMR (75 MHz,
CDCl3): δ 173.1, 172.5, 171.1, 156.4, 143.0, 141.4, 129.2, 128.9,
128.2, 127.7, 127.4, 127.1, 125.1, 120.0, 58.3, 56.5, 51.7, 51.0,
49.9, 38.9, 35.1, 31.7, 18.9, 18.2. FAB+ MS m/z 558 (M+H)+.
Anal. Calcd. For C32H35N3O6: C, 68.92; H, 6.33; N, 7.54.
Found: C, 68.94; H, 6.31; N, 7.52.
Synthesis of N-Fmoc-L-valyl-N-methyl-l-isoleucyl-D-
alanyl-L-valine Methyl Ester (19). The product was pre-
pared by general procedure B using 0.069 g (0.13 mmol) of
11a in dry acetonitrile (10 mL), 0.028 mL (0.40 mmol) of
mercaptoacetic acid, and 0.058 g (1.07 mmol) of sodium
methoxide in methanol (2 mL). The reaction was stirred at 50
°C for 1 h. The afforded unmasked dipeptide in an aqueous
9% solution of NaHCO3 was treated with 0.048 g (0.13 mmol)
of 15b in dry methylene chloride. The reaction was stirred at
RT for 1h. The subsequent work up afforded 0.073 g of the
title compound 19 (0.11 mmol, 83%): 1H NMR (300 MHz,
CDCl3) δ 7.85-7.26 (m, 8 Η), 6.94 (d, 1 H, J ) 8.5 Hz), 6.80
(d, 1 H, J ) 8.2 Hz), 5.78 (d, 1 H, J ) 8.7 Hz), 4.55-4.18 (m,
6 H), 3.71 (s, 3 H), 3.11 (s, 3 H), 1.98-2.28 (m, 3 H), 1.42-
1.31 (m, 2 H), 1.36 (d, 3 H, J ) 7.1 Hz), 1.04 (d, 3 H, J ) 6.9
Hz), 0.96-0.82 (m, 15 H). 13C NMR (75 MHz, CDCl3): δ 173.4,
172.1, 171.4, 170.1, 156.5, 143.8, 141.3, 128.1, 127.8, 127.3,
125.2, 67.0, 57.0, 56.0, 52.0, 48.6, 47.2, 46.4, 31.4, 31.3, 31.0,
30.8, 24.4, 19.5, 18.9, 17.7, 17.4, 17.3, 15.7, 10.5. MS m/z (%)
673.0778 [(M + Na)+, 100] 689.0432 [(M+K)+, 54]. Anal. Calcd.
For C36H50N4O7: C, 66.44; H, 7.74; N, 8.61. Found: C, 66.44;
H, 7.74; N, 8.61.
Synthesis of Dimethylated N-Nosyltripeptides 12a-
14a. General Procedure A.
N-Methyl-N-nosyl-L-valyl-N-methyl-L-phenylalanyl-L-
alanine Methyl Ester (12a). The product was prepared by
general procedure A using 0.069 g (0.13 mmol) of 12 in dry
dichloromethane and 1.57 mL (1.04 mmol) of 0.66 M diazo-
methane in diethyl ether. The reaction was stirred for 30 min.
Evaporation of the solvent afforded the corresponding dim-
ethylated tripeptide 12a in quantitative yield: 1H NMR (300
MHz, CDCl3) δ 8.40-8.32 (m, 2 H), 8.06-7.88 (m, 2 H), 7.40-
7.10 (m, 5 H), 6.47 (d, 1 H, J ) 8.1 Hz), 5.38 (m, 1 H), 4.52 (m,
1 H), 4.48 (m, 1 H), 3.68 (s, 3 H), 3.38 (dd, 1 H, J ) 14.3, 7.6
Hz), 3.19 (s, 3 H), 2.96 (dd, 1 H, J ) 14.1, 5.2 Hz), 2.87 (s, 3
H), 2.30 (m, 1 H), 1.34 (d, 3 H, J ) 7.2 Hz), 0.95 (d, 3 H, J )
6.7 Hz), 0.77? (d, 3 H, J ) 6.7 Hz). 13C NMR (75 MHz, CDCl3):
δ 172.6, 171.3, 169.0, 149.1, 145.3, 136.6, 128.9, 128.5, 127.7,
126.7, 123.5, 60.4, 57.2, 52.3, 48.0, 33.7, 31.6, 29.8, 28.3, 19.5,
18.7, 17.9. FAB+ MS m/z 563 (M + H)+. Anal. Calcd. For
C26H34N4O8S: C, 55.50; H, 6.09; N, 9.96; S, 5.70. Found: C,
55.52; H, 6.07; N, 9.96; S, 5.71.
N-Nosyl-L-valyl-N-methyl-L-phenylalanyl-L-alanine
Methyl Ester (12). The product was prepared by general
procedure B using 0.15 g (0.33 mmol) of 5a in dry acetonitrile
(10 mL), 0.069 mL (1.00 mmol) of mercaptoacetic acid, and
0.14 g (2.67 mmol) of sodium methoxide in methanol (2 mL).
The reaction was stirred at 50 °C for 50 min. The afforded
unmasked dipeptide in an aqueous 9% solution of NaHCO3
was treated with 0.11 g (0.33 mmol) of 3b in dry methylene
chloride. The reaction was stirred at RT for 1 h. The subse-
quent work up afforded 0.156 g of the title compound 12 (0.28
mmol, 86%): 1H NMR (300 MHz, CDCl3) δ 8.37-8.24 (m, 2
H), 8.07-7.95 (m, 2 H), 7.31-7.10 (m, 5 H), 7.10 (d, 1 H, J )
8.1 Hz), 6.48 (d, 1 H, J ) 7.8 Hz), 5.16 (dd, 1 H, J ) 8.7, 6.6
Hz), 4.41 (m, 1 H), 3.77 (m, 1 H), 3.66 (s, 3 H), 3.21 (dd, 1 H,
J ) 13.9, 8.7 Hz), 2.96 (s, 3 H), 2.50 ((dd, 1 H, J ) 13.9, 6.6
Hz), 2.03 (m, 1 H), 1.23 (d, 3 H, J ) 7.2 Hz), 1.03 (d, 3 H, J )
6.7 Hz), 0.87 (d, 3 H, J ) 6.7 Hz).13C NMR (75 MHz, CDCl3):
δ 172.8, 171.4, 168.5, 149.8, 146.1, 136.1, 129.7, 128.6, 128.7,
128.6, 124.1, 58.5, 57.8, 52.2, 47.8, 33.7, 31.1, 30.7, 19.5, 17.1,
16.6. FAB+ MS m/z 549 (M + H)+. Anal. Calcd. For C25H32-
N4O8S: C, 54.73; H, 5.88; N, 10.21; S, 5.84. Found: C, 54.70;
H, 5.89; N, 10.23; S, 5.85.
Supporting Information Available: Experimental de-
tails for the synthesis of compounds 2a-g, 5-9, 11, 6a-9a,
1
13-14, 17-18, and 13a-14a. GC/MS analyses and H NMR
Synthesis of N-Fmoc-L-alanyl-N-methyl-D-phenyl-
alanyl-L-alanine Methyl Ester (16). The product was pre-
pared by general procedure B using 0.20 g (0.45 mmol) of 6a
in dry acetonitrile (10 mL), 0.09 mL (1.34 mmol) of mercap-
spectra of dipeptides 5a and 6a. This material is available free
of charge via the Internet at http://pubs.acs.org.
JO0478959
J. Org. Chem, Vol. 70, No. 10, 2005 3897