Preparation, isolation, and characterization of Nα-Fmoc- peptide isocyanates: Solution synthesis of oligo-α-peptidyl ureas

Article abstract of DOI:10.1021/jo0611723

(Chemical Equation Presented) The N^α-Fmoc-peptide isocyanates 3a-q, 4a-c, and 5a-c were prepared by the Curtius rearrangement of N^α-Fmoc-peptide acid azides in toluene under thermal, microwave, and ultrasonic conditions. All the N

Full text of DOI:10.1021/jo0611723

Preparation, Isolation, and Characterization of Nr-Fmoc-peptide
Isocyanates: Solution Synthesis of Oligo-r-peptidyl Ureas
Vommina V. Sureshbabu,* Basanagoud S. Patil, and Rao Venkataramanarao
Department of Studies in Chemistry, Central College Campus, Bangalore UniVersity, Dr. B. R. Ambedkar
Veedhi, Bangalore 560 001, India
hariccb@rediffmail.com
ReceiVed June 8, 2006
The NR-Fmoc-peptide isocyanates 3a-q, 4a-c, and 5a-c were prepared by the Curtius rearrangement
of NR-Fmoc-peptide acid azides in toluene under thermal, microwave, and ultrasonic conditions. All the
NR-Fmoc-oligo-peptide isocyanates made were isolated as stable crystalline solids with 71 to 94% yield
and were fully characterized by 1H NMR, 13C NMR, and mass spectroscopy. Their utility for the synthesis
of oligo-R-peptidyl ureas 7a-f and 8a-c by the divergent coupling approach was demonstrated. The
coupling of NR-Fmoc-dipeptide isocyanates with amino acid ester or with N,O-bis(trimethylsilyl)amino
acids resulted in NR-Fmoc-tripeptidyl urea ester and acids containing one each of peptide bond and urea
bond. The divergent approach is extended to the synthesis of tetrapeptidyl ureas by the 2 + 2 strategy
using bis-TMS-peptide acid as an amino component. To incorporate urea bonds in adjacent positions,
NR-Fmoc-peptidyl urea isocyanates 9a-d were prepared and employed in the synthesis of three
tetrapeptidyl ureas 10a-b and 11 containing one peptide bond and two urea bonds in series from the
N-terminal end. The protocol was then employed for the synthesis of five urea analogues 13-15, 18,
and 21 of [Leu5]enkephalin containing urea bonds at the 2, 3, 4 positions as well as at the 2, 4 and 2, 3,
4 positions. The analogue 2l was made by the convergent synthesis by the N f C terminal chain extension.
Finally, two urea analogues 22 and 23 of repeat units of bioelasto polymers, namely Val-Pro-Gly-Val-
Gly-OH and Pro-Gly-Val-Gly-Val-OH, were synthesized incorporating the urea bond by the concomitant
isocyanate generation and urea bond formation under thermal conditions.
Introduction
which are mimics of proteins.4 The molecular scaffold holds a
number of peptide strands in proximity. The peptide ester
isocyanates were employed successfully as building blocks for
the introduction of peptide strands during the construction of
artificial â-sheets.5-8 Guichard, et al., have reported (p)2.5
Peptides, sequence specific oligomers, are ubiquitous in living
cells and have diverse roles. Each one of their roles is due to a
unique three-dimensional structure. Nature, in this way, has
refined the overall structure of bioactive peptides. However, the
development of peptides as drug candidates needs to overcome
several of their disadvantageous characteristics such as low oral
bioavailability, poor metabolic stability in vivo, etc. Peptido-
mimetics, on the other hand, are often protease-resistant, and
may reduce immunogenecity when compared to the naturally
occurring R-peptide analogues and thus generate viable phar-
maceutical therapies.1,2
(1) (a) Houben-Weyl: Synthesis of Peptides & Peptidomimetics; Good-
man, M., Felix, A., Moroder, L., Toniolo, C., Eds.; Georg Thieme Verlag:
Stuttgart, New York, 2003; Vol. E22c and references cited therein. (b)
Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D.
A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.;
Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.;
Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367. (c) Patch, J.
A.; Barron, A. E. Curr. Opin. Chem. Biol. 2002, 6, 872. (d) Emmons, T.
K.; Murali, R.; Greene, M. I. Curr. Opin. Biotech. 1997, 8, 435.
(2) (a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173. (b) Kirshenbaum,
K.; Zuckermann, R. N.; Dill, K. A. Curr. Opin. Struct. Biol. 1999, 9, 530.
(c) Iverson, B. L. Nature 1997, 385, 113. (d) Borman, S. Chem. Eng. News.
1997, 75, 32. (e) Soth, M. J.; Nowick, J. S. Curr. Opin. Chem. Biol. 1997,
1, 120.
Among several classes of pseudopeptides,3 peptidyl urea
peptidomimetics are one of the important classes of molecules
with increasing applications both in de novo design of proteins
as well as in developing lead molecules in drug therapy. Nowick,
et al., developed methods for the preparation of artificial â-sheets
10.1021/jo0611723 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/29/2006
J. Org. Chem. 2006, 71, 7697-7705
7697

Sureshbabu et al.
helical structure in solution for a heptapeptidyl urea bearing
amino acids Tyr, Ala, and Val.9 A high degree of self-
complementarity that allows a self-assembly process resulting
in C4-symmetry (all S) structure by a cyclictetraalanylurea [-â-
HAlau-â-HAlau-â-HAlau-â-HAlau-] which possesses nano-
tubular structure was also demonstrated by X-ray analyis.10
Some of the illustrative examples befitting the incorporation of
urea moiety into native peptides to obtain peptidyl ureas as
potential inhibitors are TAR-binding portion of the Tat protein,11
γ-secretase,12 aspartic acid protease,13 HIV-1 proteases,14,15
microbial alkaline proteinase inhibitors (MAPI),16 and aspartic
FIGURE 1. Peptide ester isocyanate and NR-Fmoc-peptide isocyanate.
peptidases.17 As anticipated by Nowick, et al.,4 peptide isocy-
anates, being reactive with nucleophiles, are attractive building
blocks for the development of new biomolecules through
combinatorial chemistry and drug discovery efforts.
Peptide ester isocyanates (OCN-CHR1-CONH-CHR2-
COOMe, Figure 1), prepared by the treatment of peptide ester
hydrochloride salts with phosgene in toluene, in the presence
of saturated aqueous NaHCO3 under modified Schotten-
Baumann conditions or with triphosgene [bis(trichloromethyl)-
carbamate] in the presence of diisopropylethylamine (DIEA),
were coupled with amino acid esters to obtain peptidyl ureas
possessing one urea bond (MeO2C-CHR3-NHCONH-CHR2-
CONH-CHR1-CO2Me).18 Peptide ester isocyanate generation
is usually followed by trapping with an amino component.
Unlike amino acid ester isocyanates, peptide ester isocyanates
cannot be purified by Kugelrohr distillation. Instead, crude
isocyanates are trapped, and the final products are purified.
Similarly, several reagents such as N,N1-carbonyldiimidazole
(CDI),19 1,11-carbonylbisbenzotriazole,20 and p-nitrophenyl car-
bamate,21 have also been used for the synthesis of peptidyl ureas.
The coupling of isocyanates derived from peptide ester with
amino acid ester or peptide ester results in peptidyl ureas
comprising carboxyl group at both the terminal ends.
Alternatively, organic synthesis also permits insertion of an
amino group in place of R-carboxyl group that can then be
converted to its isocyanate. This functional group transformation
was accomplished by converting NR-Boc-R-amino acid amide
to nitrile followed by its reduction to amine.22a Alternatively,
NR-Fmoc-amino acid was converted to its â-amino alcohol
which was transformed into an amine via azide.22b A similar
protocol was employed by Burgess et al.23 Consequently, the
backbone in each repeating unit of oligourea peptidomimetic
is generally extended by one carbon atom. Further, the mono-
protected diamines were either converted to the corresponding
isocyanates (using phosgene) or were treated with chlorofor-
(3) Other classes include peptoids, oligocarbamates, hydrazinopeptides,
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Simon, R.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D.
A.; Banville, S.; Ng, S.; Wang, L.; Resenberg, S.; Marlowe, C. K.;
Spellmeyer, D. C.; Tans, R.; Frankelo, A. D.; Santi, D. V.; Cohen, F. E.;
Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367. (c) Zuckermann,
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Holmes, D. L.; Noronha, G.; Smith, E. M.; Nguyen, T. M.; Huang, S.-L. J.
Org. Chem. 1996, 61, 3929. For the synthesis of amino acid ester isocyanates
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7698 J. Org. Chem., Vol. 71, No. 20, 2006

Na-Fmoc-peptide Isocyanates
SCHEME 1. Synthesis of Nr-Fmoc-peptide Acid Azides and Their Isocyanates
mates (p-nitrophenyl chloroformate and 2,4,5-trichlorophenyl
chloroformate) to obtain carbamates which serve as monomeric
building blocks for the synthesis of peptidyl ureas.21,24 Almost
all of these routes give oligo-â-peptidyl ureas. Boeijen and
Liskamp have described that this was for reasons of synthetic
accessibility as well as product stability.25
acids are hitherto unreported compounds which are demon-
strated, in this paper, to be useful as key intermediates in the
synthesis of oligo-R-peptidyl ureas both by the divergent as well
as convergent N f C terminal extension strategies. The utility
of the protocol is demonstrated by the synthesis of five urea
analogues of [Leu5]enkephalin and two analogues of bioelastic
polymer repeat units Val-Pro-Gly-Val-Gly-OH and Pro-Gly-
Val-Gly-Val-OH.
The oxidative Hoffmann type rearrangement of Boc-/Z-R-
amino acid/peptide acid amides using bis(trifluoroacetoxy)-
iodobenzene (IBTFA) mediated by the isocyanate intermediate
is another known approach for the synthesis of peptidyl ureas.26
In addition to longer duration of reaction period, less yields,27
and carboxamide formation in the case of Gln, Asn, etc., this
protocol was found to be ineffective in Fmoc chemistry.27
Sureshbabu, et al.,28 isolated the isocyanates derived from NR-
Fmoc-amino acids including the ones with side chain protection
possessing tert-butyl, benzyl, and Boc groups, as crystalline
solids and were fully characterized. The removal of Boc or Z
groups from protected gem-diamine systems29,30 under normal
acidic or basic conditions hampers the synthesis of oligo-R-
peptidyl ureas by the C f N terminal stepwise chain extension.31
However, NR-Fmoc-peptide isocyanates comprising coded amino
Results and Discussion
NR-Fmoc-peptide Isocyanates. Fmoc-peptide acids 1a-q32,33
were converted to the corresponding acid azides by employing
the standard procedures. As demonstrated by us in the case of
NR-Fmoc-amino acid azides,34 all the NR-Fmoc-peptide acid
azides 2a-q prepared were isolated as shelf-stable crystalline
solids and fully characterized before use. They were then
subjected to Curtius rearrangement (Scheme 1) by refluxing the
toluene solution at 65 °C. The conversion, as monitored by TLC
as well as by IR spectra, was found to be complete in about 45
min. Evaporation of toluene solution under reduced pressure
resulted in the isolation of the isocyanates 3a-q as solids. The
crude NR-Fmoc-peptide isocyanates were recrystallized using
ethyl acetate (EtOAc) and n-hexane to obtain the pure crystalline
compounds in 71 to 85% yields (Table 1). The tri- and
tetrapeptide isocyanates Fmoc-Val-Ala-Leu-NCO 4a, Fmoc-Tyr-
(But)-Gly-Gly-NCO 4b, Fmoc-Val-Pro-Gly-NCO 4c, Fmoc-
Gly-Phe-Leu-Val-NCO 5a, Fmoc-Tyr(But)-Gly-Gly-Phe-NCO
5b, and Fmoc-Pro-Gly-Val-Gly-NCO 5c were also prepared
starting from the corresponding peptide acid azides and isolated
as analytically pure solids in 89 to 94% yields. The NR-Fmoc-
oligo-R-peptide isocyanates 3-5 were characterized by IR, 1H,
13C NMR, ES-MS, and MALDI-mass spectral analysis.
When stored for longer periods at 4 °C, the NR-Fmoc-peptide
isocyanates 3-5 did not undergo noticeable degradation or show
any change in their 1H and 13C NMR spectral properties recorded
after several weeks of their preparation. The NR-Fmoc-peptide
isocyanates containing the amino acids Ser, Thr, Tyr, Asp, Glu,
Gln, His, Arg, bearing side chains protected by But, Boc, Trt,
Pmc, etc., were made and isolated as pure compounds after a
single recrystallization step. NR-Fmoc-peptide acid azides as well
as their isocyanates were found to be stable while carrying out
HPLC analysis by an isocratic method using the mobile phase
acetonitrile and water (60:40) during which the chromatographic
run was initiated and completed in 20 min. Thus, purity as well
as the progress of the reaction involving isocyanates synthesis
can be qualitatively monitored using HPLC analysis.
(24) (a) Hutchins, S. M.; Chapman, K. T. Tetrahedron Lett. 1995, 36,
2583. (b) Lipwowski, A. W.; Tam, S. W.; Portoghese, P. S. J. Med. Chem.
1986, 29, 1222. (c) Adamiak, R. W.; Stawinski, J. Tetrahedron Lett. 1977,
1935.
(25) Boeijen, A.; Liskamp, R. M. J. Eur. J. Org. Chem. 1999, 2127.
(26) (a) Boutin, R. H.; Loudon, G. M. J. Org. Chem. 1984, 49, 4277.
(b) Yamamoto, Y.; Yatagai, H.; Maruyama, K. J. Org. Chem. 1979, 44,
1746. (c) Chorev, M. Biopolymers 2005, 80, 67. (d) Myers, A. C.; Kowalski,
J. A.; Lipton, M. A. Bioorg. Med. Chem. Lett. 2004, 14, 5219.
(27) Hoffman rearrangement of Fmoc-protected asparagine using the
reagent bis(trifluoroacetoxy)iodobenzene (IBTFA) has failed to give 2,3-
diaminopropionic acid. Englund, E. A.; Gopi, H. N.; Apella, D. H. Org.
Lett. 2004, 6, 213.
(28) (a) Patil, B. S.; Vasanthakumar, G. R.; Suresh Babu, V. V. J. Org.
Chem. 2003, 68, 7274. (b) Patil, B. S.; Suresh Babu, V. V. Lett. Pept. Sci.
2003, 10, 93. (c) Suresh Babu, V. V.; Kantharaju; Tantry, S. J. Int. J. Pept.
Res. Ther. 2005, 11, 131.
(29) For a report on the generation of Z-Tyr-NCO, Z-Gly-NCO and
Z-Phe-NCO from the corresponding azides see: Kawaskai, K.; Maeda, M.;
Watanabe, J.; Kaneto, H. Chem Pharm Bull. 1988, 36 (5), 1766.
(30) For the synthesis of ureido analogues of angiotensins using Z
chemistry and catalytic hydrogenation for deporotection see: (a) Antsan,
Y. E.; Chipens, G. I. SoV. J. Bioorg. Chem. (Engl. Transl.) 1975, 1, 998.
(b) Antsan, Y. E.; Makarova, N. A.; Chipens, G. I. SoV. J. Bioorg. Chem.
(Engl. Transl.) 1982, 3, 185.
(31) For the decomposition of Z-peptidyl urea ethyl ester during the
deprotection of the ester group under both alkaline and acidic conditions
see: (a) Vegner, R. E.; Chipens, G. I. J. Gen. Chem. USSR (Engl. Transl.)
1975, 45, 431. (b) For the formation of hydantoins during the cleavage
from Tentagel resin bound oligourea peptidomimetics by the transesterifi-
catin in basic methol due to the abstraction of a proton by base from the
urea N-H, see ref 25. (c) Semetey, V.; Hemmerlin, C.; Didierjean, C.;
Schaffner, A.-P.; Giner, A. G.; Aurby, A.; Briand, J.-P.; Marraud, M.;
Guichard, G. Org. Lett. 2001, 3, 3843. For the studies concerning the
decomposition of gem-diaminoalkyl compounds under acidic and basic
conditions see: (e) Moad, G.; Benkovic, S. J. J. Am. Chem. Soc. 1978,
100, 5495. (f) Fife, T. H.; Hutchins, J. E. C.; Pellino, A. M. J. Am. Chem.
Soc. 1978, 100, 6455. (g) Loudon, G. M.; Almond, M. R.; Jacob, J. N. J.
Am. Chem. Soc. 1981, 103, 4508. (h) Parham, M. E.; Louden, G. M.
Biochem. Biophys. Res. Commun. 1978, 80, 1. (i) For a detail study
concerning side reactions in the synthesis of N,N′-diacyl-1,1-diamino-2-
phenylethane derivatives see: Chorev, M.; MacDonald, S. A.; Goodman,
M. J. Org. Chem. 1984, 49, 821.
(32) For the synthesis of peptide acids see Katritzky, A. R.; Angrish. P.;
Suzuki, K. Synthesis 2006, 411.
(33) (a) For the synthesis of bis-TMS-amino acids see: Bolin, D. R.;
Sytwu, I.-I.; Humiec, F.; Meienhofer, J. Int. J. Pept. Protein Res. 1989, 33,
353. (b) For the synthesis of Fmoc-peptide acids employing bis-TMS-amino
acids by the mixed anhydride method using EtOCOCl and NMM see Tantry,
S. J.; Suresh Babu, V. V. Indian J. Chem. 2004, 43B, 1282-1287.
(34) Suresh Babu, V. V.; Ananda, K.; Vasanthakumar, G. R. J. Chem.
Soc., Perkin Trans. 1 2000, 4328.
J. Org. Chem, Vol. 71, No. 20, 2006 7699

Sureshbabu et al.
TABLE 1. Nr-Fmoc-peptide Isocyanates Fmoc-NH-CHR1-CONH-CHR2-NCO
product
R1
R2
methoda
time
yieldb (%)
mp (°C)
3a
H
CH2C6H5
CH3
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
30 min
40 s
20 min
30 min
40 s
25 min
30 min
40 s
30 min
30 min
40 s
30 min
30 min
35 s
30 min
30 min
45 s
30 min
30 min
45 s
30 min
40 min
45 s
83
87
86
84
92
91
85
91
88
84
88
91
81
87
82
79
82
80
79
82
80
85
91
86
84
88
84
72
74
74
71
72
71
208-209
3b
3c
3d
3e
3f
CH(CH3)2
178-180
193-194
178-180
198-199
172-174
168-169
168-170
148-149
164-165
167-169
CH2CH(CH3)2
CH3
CH(CH3)2
CH2CH(CH3)2
CH2C6H5
CH(CH3)2
CH(CH3)2
C6H5
3g
3h
3i
CH(CH3)2
N-(CH2)3-C
CH2OCH2C6H5
CH2CH(CH3)2
CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH2COOC(CH3)3
CH2CH2CONH(Trt)
25 min
35 min
35 s
30 min
30 min
35 s
30 min
40 min
45 s
3j
3k
25 min
3l
3m
3n
3o
3p
3q
(CH2)3NCN(NHPmc)
CH(SCH2C6H5)CH3
CH2OCH2C6H5
CH2CH(CH3)2
CH(CH3)2
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
A
C
D
35 min
45 s
30 min
40 min
45 s
25 min
35 min
45 s
30 min
40 min
30 s
25 min
45 min
60 s
20 min
30 min
30 s
82
86
82
79
82
80
82
88
83
81
86
80
76
78
76
88
89
85
182-183
154-155
188-189
168-169
139-140
123-125
CH2C6H5
CH2OC(CH3)3
CH2C6H5
CH(CH3)OC(CH3)3
CH2C6H4OC(CH3)3
CH2CH2COOCH2C6H5
H
20 min
a Method A, reflux in toluene; method B, microwave irradiation in toluene; method C, microwave irradiation using solid powder; method D, conversion
by ultrasonication. Method B results are not shown in the table. b Isolated yield after crystallization.
The conversion of NR-Fmoc-peptide acid azides to their
isocyanates was accomplished under microwave irradiation in
an open vessel using both toluene solution as well as naked
solid powder. The rearrangement was completed in about 30-
60 s. In the later protocol, after the reaction, a single recrys-
tallization has directly resulted in the isolation of the pure
isocyanates. This simplified procedure avoids tedious workup
such as evaporation of toluene. Both the yields and the purity
of all the isocyanates made by this route were satisfactory. In
addition, under the influence of the ultrasound, NR-Fmoc-peptide
acid azides in toluene solution could also be converted to their
corresponding isocyanates at ambient temperature in about 20-
30 min. It is noteworthy that the mild conditions employed are
compatible with the use of a number of protecting groups in
the side chains of bifunctional amino acids.
NR-Fmoc-oligo-R-peptidyl Ureas. NR-Fmoc-peptide isocy-
anates 3 were coupled with bis-TMS/tris-TMS amino acids to
obtain NR-Fmoc-peptidyl urea acids (Scheme 2). The reaction
was found to be complete in about 20-30 min at room
temperature, and the routine workup of the reaction mixture
gave the crude peptidyl urea acids which were purified by
crystallization or using column chromatography whenever
necessary. The 2 + 2 fragment coupling strategy has been
adopted to couple 3e, 3f, and 3b with appropriate bis-TMS-
dipeptides to obtain the tetra-R-peptidyl ureas Fmoc-Val-Phe-
ψ(NH-CO-NH)-Ala-Leu 8a, Fmoc-Val-Phg-ψ(NH-CO-
NH)-Val-Gly 8b, and Fmoc-Val-Ala-ψ(NH-CO-NH)-Val-Gly
8c as analytically pure compounds in 88-89% yield. Similarly,
the coupling of the isocyanates 3 with amino acid esters as well
as peptide esters led to peptidyl urea esters.
7700 J. Org. Chem., Vol. 71, No. 20, 2006

Na-Fmoc-peptide Isocyanates
SCHEME 2. Synthesis of Nr-Fmoc-tripeptidyl Ureasa
a (i) (6) X ) Y ) TMS, (7a-c) Y ) H; (ii) (7d-f) X ) HCl or p-TsOH salt, Y ) CH3, CH2C6H5 group. [ 7a: Fmoc-Val-Phe-ψ(ΝΗ-CO-ΝΗ)-Ala-
OH. 7b: Fmoc-Val-Pro-ψ(ΝΗ-CO-ΝΗ)-Gly-ÃΗ. 7c: Fmoc-Ser(Bzl)-Phe-ψ(NH-CO-NH)-Ile-OH. 7d: Fmoc-Gly-Phe-ψ(NH-CO-NH)-Gly-OCH3.
7e: Fmoc-Ser(But)-Phe-ψ(NH-CO-NH)-Leu-OCH3. 7f: Fmoc-Thr(But)-Gly-ψ((NH-CO-NH)-Leu-OBzl].
Urea Analogues of [Leu5]enkephalin. Employing NR-Fmoc-
peptide isocyanates, the synthesis of urea analogues of [Leu5]-
enkephalin containing urea bonds at the 2nd and 3rd as well as
at 4th position was carried out by the fragment condensation
strategy. The analogues synthesized were H-Tyr(But)-Gly-ψ-
(NH-CO-NH)-Gly-Phe-Leu-OH 13, H-Tyr(But)-Gly-Gly-ψ-
FIGURE 2. NR-Fmoc-R-peptidyl urea isocyanates: 9a, Fmoc-Ala-
Leu-ψ(NH-CO-NH)-Val-NCO; 9b, Fmoc-Pro-Val-ψ(NH-CO-NH)-
Gly-NCO; 9c, Fmoc-Leu-Phe-ψ(NH-CO-NH)-Ala-NCO; 9d, Fmoc-
Tyr(But)-Gly-ψ(NH-CO-NH)-Gly-NCO.
(NH-CO-NH)-Phe-Leu-OH 14 and H-Tyr(But)-Gly-Gly-Phe-
ψ(NH-CO-NH)-Leu-OH 15. After the deprotection of the
NR-Fmoc group, the final compounds were isolated as ana-
lytically pure compounds in good yield. The divergent strategy
was then extended for the synthesis of 18 possessing urea
bonds at alternative positions. The analogue 21 possessing
urea bonds in place of peptide bonds at positions 2, 3, and 4
were synthesized by the convergent strategy. The steps in-
volved and the key intermediates as well as the protected and
free final compounds isolated and characterized during the
synthesis of 18 and 21 are given in the Schemes 4 and 5,
respectively. The urea analogues were tested for opioid activity
in the isolated GPI and MVD preparations. The insertion of
the urea bond resulted in decrease or loss of the opioid activity
in vitro when compared with the natural peptide in both the
assays.
FIGURE 3. NR-Fmoc-R-tetrapeptidyl ureas containing urea bonds at
2nd and 3rd positions.
NR-Fmoc-R-peptidyl Urea Isocyanates. For the synthesis
of oligo-R-peptidyl ureas containing urea bonds in both adjacent
positions as well as continuously, NR-Fmoc-R-peptidyl urea
isocyanates are the key intermediates (Figure 2). For this part
of study, NR-Fmoc-peptidyl urea acids 7 were converted to the
corresponding isocyanates 9 via their azides. Thus, 9a-d have
been obtained in 89-95% yield and were found to be soluble
as well as stable compounds. The isocyanate 9c was then
coupled with tris-TMS-Ser and Gly-OMe to obtain the tetrapep-
tidyl ureas 10a,b containing two consecutive urea bonds in 90-
94% yield (Figure 3). Then, 9c, using 3 + 2 strategy, was
coupled with tris-TMS-Val-Tyr to obtain the pentapeptidyl urea
11 in 88% yield.
Urea Analogues of Repeat Units of Bioelasic Polymers:
Concomitant Isocyanate Generation and Urea Bond Forma-
tion. Bioelastic materials are elastomeric polypeptides composed
of repeating sequences having their origins found in the
mammalian elastic protein, elastin. The polymers of the repeat-
ing sequences Val-Pro-Gly-Val-Gly-OH, Pro-Gly-Val-Gly-Val-
OH, Gly-Val-Gly-Val-Pro-OH, Val-Gly-Val-Pro-Gly-OH, and
Gly-Val-Pro-Gly-Val-OH carried out by polymerizing the
pentamer permutations were cross linked into sheets, rods, and
tubes.35 Some of the polypentapeptides were found to be
elastomeric capable of an elastic modulus similar to that of the
natural elastic fibers.36 As a part of our interest in such
molecules, the urea analogues Val-Pro-Gly-ψ(NH-CO-NH)-
Val-Gly-OH 22 and Pro-Gly-Val-Gly-ψ(NH-CO-NH)-Val-
OH 23 (Figures 4 and 5) were prepared in the present study. In
general, incorporation of urea moiety into the peptidyl urea
peptidomimetics was accomplished initially by generating
isocyanates from azides and then coupling them with an amino
component. Since the isocyanates are reactive, we envisaged
that both the generation and coupling steps could be carried
NR-Amino Free Oligo-R-peptidyl Ureas. The deprotection
of Fmoc group from the NR-Fmoc-peptidyl ureas was carried
out employing the normal deprotection conditions. Thus, NR-
Fmoc-peptidyl urea on treatment with diethylamine (DEA,
Scheme 3) at room temperature has resulted in complete
deprotection of the Fmoc group in 20-25 min. And, all the
resulting amino free peptidyl urea acids 12a-d made have been
isolated as crystalline solids and fully characterized.
SCHEME 3. Deprotection of Fmoc-Group from Nr-Fmoc-peptidyl Ureasa
a 12a: Val-Pro-ψ(ΝΗ-CO-ΝΗ)-Gly-OH. 12b: Ser(Bzl)-Phe-ψ(ΝΗ-CO-ΝΗ)-Leu-ÃΗ. 12c: Tyr(But)-Gly-ψ(NH-CO-NH)-Gly-OH. 12d: Val-
Phe-ψ(ΝΗ-CO-ΝΗ)-Ala-Leu-OH.
J. Org. Chem, Vol. 71, No. 20, 2006 7701

Sureshbabu et al.
SCHEME 4. Synthesis of [Leu5]enkephalin Containing Urea Bonds at 2nd and 4th Positions
SCHEME 5. Synthesis of [Leu5]enkephalin Possessing Three Urea Bonds
out in a single step. Thus the synthesis of 22 was carried out
by the concomitant isocyanate generation starting from NR-
Fmoc-Val-Pro-Gly-N3 in toluene and peptidyl urea formation
under thermal conditions by coupling with bis-TMS-Val-Gly.
The concomitant reaction resulted in pure pentapeptidyl urea
after a routine workup and a single recrystallization. A similar
approach was used for the synthesis of 23 also. The final free
peptidyl ureas 22 and 23 were obtained, after the deprotection
of Fmoc group, in 76-79% yield.
diastereomeric of peptidyl urea adducts 25 made by the coupling
of Fmoc-Leu-Val-NCO with R-(+)-, S-(-)-, and a racemic
mixture of 1-phenylethylamine in the presence of DIEA
(Scheme 6). The urea adducts 25a-c were isolated as solids.
Their 1H NMR analysis was carried out. The methyl protons of
1-phenylethylamine urea adducts 25a,b were observed as
doublets with the separation of 0.02 ppm, while the methyl
protons of the urea adduct 25c was observed as a triplet. On
the basis of these observations, it was demonstrated that the
present protocol does not lead to racemization of Fmoc-Leu-
Test for Racemization. The conversion of NR-Fmoc-peptide
acid azides to its isocyanates was found to retain their enan-
tiomeric purity. This was confirmed by 1H NMR of the
1
Val-NCO at levels detectable by H NMR.
FIGURE 4. Bioelastic polymer fragment Val-Pro-Gly-ψ(NH-CO-
NH)-Val-Gly-OH.
FIGURE 5. Bioelastic polymer fragment Pro-Gly-Val-Gly-ψ(NH-
CO-NH)-Val-OH.
7702 J. Org. Chem., Vol. 71, No. 20, 2006

Na-Fmoc-peptide Isocyanates
SCHEME 6. Preparation of Urea Adducts for Racemization
Studya
Method B: Employing Acid Chloride Method. To a cold
solution of NR-Fmoc-peptide acid chloride (1 mmol) in acetone (5
mL) was added aqueous NaN3 (0.1 g, 1.5 mmol in 1 mL of water).
The resulting reaction mixture was stirred for 15 min. at -10 °C.
The separated solid was filtered and washed with cold water. The
resulting solid was dissolved in CH2Cl2 (5 mL), dried over
anhydrous Na2SO4, and crystallized using n-hexane.
Fmoc-Gly-Phe-N3 2a. A 0.44 g (1 mmol) of Fmoc-Gly-Phe-
OH was converted to its azide following the general procedure for
NR-Fmoc-peptide acid azides 2 (method A). Yield 0.43 g (92%);
mp 205-206 °C. IR: 2142 cm-1. H NMR (δ, CDCl3): 2.9 (2H,
d), 3.25 (2H, d), 4.2-4.45 (3H, m), 5.2 (1H, d), 6.3 (1H, s), 7.2-
7.8 (13H, m).
1
a 25a: Fmoc-Leu Val-ψ(ΝΗ-CO-ΝΗ)-(R)-phenylethylamine (R at /).
25b: Fmoc-Leu-Val-ψ(ΝΗ-CO-ΝΗ)-(S)-phenylethylamine. 25c: Fmoc-
Leu-Val-ψ(ΝΗ-CO-ΝΗ)-1-phenylethylamine (R and S at /).
NR-Fmoc-peptide Isocyanates 3: General Procedure. Method
A: Employing Conventional Method. A solution of NR-Fmoc-
peptide acid azide (1 mmol) in toluene (5 mL) was heated at 65
°C under nitrogen. After the completion of the rearrangement, the
solvent was removed under reduced pressure to obtain the crude
isocyanate that was recrystallized using DCM and n-hexane.
Method B: Employing MW Irradiation. NR-Fmoc-peptide acid
azide (1 mmol) was dissolved in toluene (5 mL) and exposed to
microwave irradiation until the rearrangement was complete. The
solvent was removed under reduced pressure to obtain the isocy-
anate that was recrystallized as described in the method A.
Method C: Employing MW Irradiation under Solvent Free
Condition. Powdered NR-Fmoc-peptide acid azide (1 mmol) placed
in a glass beaker was exposed to microwave irradiation until the
rearrangement was complete. The resulting mass was recrystallized.
Method D: Employing Ultrasonic Waves. A solution of NR-
Fmoc-peptide acid azide (1 mmol) in toluene (5 mL) was sonicated
for 30 min, until the completion of the reaction. The solvent was
removed under reduced pressure to obtain the crude isocyanate that
was recrystallized using DCM and n-hexane.
Fmoc-Gln(Trt)-Ile-NCO 3j. An amount of 0.764 g (1 mmol)
of Fmoc-Gln(Trt)-Ile-CON3 2j was converted to its isocyanate
following the general procedure for NR-Fmoc-peptide isocyanates
3 (method A). IR: 2252 cm-1. 1H NMR (δ, CDCl3): 0.93 (6H, m),
1.21 (1H, m), 1.71 (2H, m), 2.12 (2H, t), 2.56 (2H, m), 3.71-4.48
(5H, m), 5.6 (1H, d), 6.2-6.45 (3H, m), 7.2-7.9 (23H, m). 13C
NMR (δ, CDCl3): 11.3, 15.5, 23.0, 31.4, 34.8, 47.0, 54.7, 57.3,
66.7, 69.6, 119.9, 124.9, 125.8, 126.9, 127.6, 127.9, 128.1, 128.9,
141.1, 143.7, 145.3, 156.1, 158.2, 161.3. ES MS m/z observed,
722.2.
Fmoc-His(Trt)-Ile-NCO 3k. An amount of 0.757 g (1 mmol)
of Fmoc-His(Trt)-Ile-CON3 2k was converted to its isocyanate
following the general procedure for NR-Fmoc-peptide isocyanates
3 (method C). IR: 2256 cm-1. 1H NMR (δ, CDCl3): 0.92 (6H, m),
1.19 (1H, m), 1.71 (2H, m), 3.68-4.45 (10H, m), 5.6 (1H, d), 6.45
(1H, m), 7.2-8.4 (27H, m). 13C NMR (δ, CDCl3): 11.3, 15.5, 25.4,
35.8, 47.0, 57.3, 66.7, 119.9, 121.3, 124.9, 125.2, 126.3, 126.9,
127.6, 127.9, 128.1, 128.9, 132.4, 141.1, 143.7, 145.3, 156.1, 158.2,
161.3. ES MS m/z observed, 730.7.
Fmoc-Arg(Pmc)-Leu-NCO 3l. An amount of 0.80 g (1 mmol)
of Fmoc-Arg(Pmc)-Leu-CON3 2l was converted to its isocyanate
following the general procedure for NR-Fmoc-peptide isocyanates
3 (method D). IR: 2247 cm-1. 1H NMR (δ, CDCl3): 0.93 (6H, d),
1.33 (2H, m), 1.63 (1H, m), 3.77 (1H, m), 4.2 (1H, t), 4.4 (2H, m),
5.07 (1H, d), 6.58 (1H, m), 7.25-7.75 (8H, m). 13C NMR (δ,
CDCl3): 22.0, 23.0, 24.6, 40.3, 47.2, 51.2, 66.6, 119.8, 124.8, 126.8,
126.9, 127.6, 141.2, 143.8, 156.7, 169.1. ES MS m/z observed,
773.8.
Conclusions
A simple and efficient method for the rapid synthesis of stable
and crystalline NR-Fmoc-peptide isocyanates, including the one
at ambient temperature, employing ultrasound was developed.
The scale-up of the rearrangement up to 75 mmol was
accomplished smoothly. The protocol is particularly noteworthy
with regard to “chemistry of peptidomimetics: a green ap-
proach”. The use of NR-Fmoc-peptidyl urea isocyanates allowed
the incorporation of urea bonds in the adjacent positions as well
as continuously. The NR-Fmoc- group from NR-Fmoc-peptidyl
ureas was deprotected under normal deprotection conditions.
All the NR-Fmoc-protected as well as free oligo-R-peptidyl ureas
prepared resulted in good yield as well as purity.
The removal of Fmoc group under normal basic conditions
from NR-Fmoc-oligo-R-peptidyl ureas possessing a urea bond
in the place of a N-terminal peptide bond was not feasible. In
such cases, catalytic hydrogenation or catalytic transfer hydro-
genation techniques could be employed for deportection of the
Fmoc group. The use of NR-Fmoc-peptide isocyanates as useful
fragments in solid-phase fragment condensation (SPFC)37 for
the synthesis of bioelastic polymer fragment Val-Pro-Gly-ψ-
(NH-CO-NH)-Val-Gly-OH will be published soon.
Experimental Section
Some representative experimental procedures for key intermedi-
ates and final compounds are furnished here. Complete experimental
procedures and characterization data are available in the Supporting
Information.
NR-Fmoc-peptide Acid Azides 2: General Procedure. Method
A: Employing Mixed Anhydride Method. NR-Fmoc-peptide acid
(1 mmol) was dissolved in dry THF (5 mL) and cooled in an ice-
salt bath. EtOCOCl (0.1 mL, 1 mmol) and NMM (0.11 mL, 1
mmol) were added to the above cooled solution and stirred for 10
min. while maintaining the temperature at -10 °C. The resulting
reaction mixture was treated with aqueous NaN3 (0.1 g, 1.5 mmol
in 1 mL of water) at the same temperature. The reaction, as
monitored by TLC as well as IR analysis, was complete in about
15 min. The THF solution was evaporated under reduced pressure
at rt and the residue was taken in CH2Cl2 (15 mL). The CH2Cl2
layer was washed with cold water (2 × 10 mL), dried over
anhydrous Na2SO4, and concentrated to half of the volume.
n-Hexane (10 mL) was added to obtain pure NR-Fmoc-peptide acid
azide as a crystalline solid.
NR-Fmoc-peptidyl Urea Acids 7a-c: General Procedure. To
a solution of NR-Fmoc-peptide isocyanate 3 (1 mmol) in DCM (5
mL) at 0 °C was added freshly prepared bis-TMS- or tris-TMS-
amino acid or peptide (from 1.2 mmol of amino acid or peptide
refluxed with 2.5 mmol each of TEA and TMS-Cl in 10 mL of
DCM or 4 mmol each of TEA and TMS-Cl in the case of tris-
TMS derivatives) and stirring was continued for 15-30 min. After
completion of the reaction, the solvent was evaporated under
(35) (a) Sandberg, L. B.; Leslie, J. G.; Leach, C. T.; Torres, V. L.; Smith,
A. R.; Smith, D. W. Pathol. Biol. 1985, 33, 266.
(36) (a) Urry, D. W. Prog. Biophys. Mol. Biol. 1992, 57, 23. (b) Urry,
D. W. Angew. Chem., Int. Ed. Engl. 1993, 32, 819. (c) Urry, D. W.; Gowda,
D. C.; Harris, C.; Harris, R. D.; Cox, B. A. J. Am. Chem. Soc. 1992, 33,
84.
(37) For solid-phase fragment condensation see Benz, H. Synthesis 1994,
337.
J. Org. Chem, Vol. 71, No. 20, 2006 7703

Sureshbabu et al.
reduced pressure below 40 °C. The residue was dissolved in 10%
Na2CO3 (20 mL) and washed with diethyl ether (20 mL × 2). The
aqueous layer was acidified with dilute HCl until pH 2. The
separated product was extracted with EtOAc (20 mL × 3). The
water wash (20 mL × 3) was given to the combined organic layer
and dried over anhydrous Na2SO4. The organic layer was concen-
trated to obtain the title compound. The peptidyl ureas were purified
either by crystallization using DMF and water or by column
chromatography using the solvent system MeOH and CHCl3 (5%
to 10% of MeOH in CHCl3).
Fmoc-Ser(Bzl)-Phe-ψ(NH-CO-NH)-Ile-OH 7c. An amount
of 0.561 g (1 mmol) of 3n was coupled with freshly prepared bis-
TMS-Ile (from 0.421 g, 1.2 mmol of Ile-OH in 10 mL of DCM) to
obtain 7c as a white solid. Yield 0.567 g (88%); mp 192-193 °C.
1H NMR (δ, DMSO): 0.91 (6H, m), 1.13 (1H, m), 1.53 (2H, m),
2.84-2.92 (4H, m), 3.5 (2H, m), 3.82-4.46 (6H, m), 5.65 (1H,
d), 6.5-6.8 (2H, m), 7.08-7.85 (18H, m), 8.35 (1H, m). 13C NMR
(δ, DMSO): 11.3, 15.5, 25.4, 35.8, 37.3, 37.4, 46.7, 51.8, 54.1,
57.3, 62.1, 66.5, 119.7, 124.8, 126.7, 126.7, 126.9, 127.3, 128.6,
128.6, 129.2, 129.3, 137.4, 137.5, 140.9, 143.6, 156.1, 157.2, 169.8,
175.2. ES MS m/z observed, 693.7.
NR-Fmoc-peptidyl Urea Esters 7d-f: General Procedure. To
an ice-cold mixture of amino acid or peptide ester hydrochloride
or p-toluenesulfonate salt (1.1 mmol) in THF (5 mL) and NMM
(1.21 mL, 1.1 mmol) was added a solution of NR-Fmoc-peptide
isocyanate (1 mmol) in DCM (5 mL), and the solutions was stirred
for about 15-20 min. After the completion of the reaction, the
solvent was removed under reduced pressure, and the residue was
dissolved in EtOAc. The organic layer was washed with 10% Na2-
CO3 (10 mL) and water (10 mL × 3) and finally dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure to obtain the NR-Fmoc-peptidyl urea esters. The peptidyl
ureas were purified either by the crystallization using DMF-water
or by column chromatography using the solvent system MeOH and
CHCl3 (2% to 10% of MeOH in CHCl3).
Fmoc-Ser(But)-Phe-ψ(NH-CO-NH)-Leu-OMe 7e. An amount
of 0.527 g (1 mmol) of 3o was coupled with Leu-OMe.HCl (0.199
g, 1.1 mmol) to obtain 7e. Yield 0.575 g (96%); mp 180-181 °C.
1H NMR (δ, DMSO): 0.8-0.86 (6H, d), 1.1 (9H, s), 1.39 (2H, m),
1.41 (1H, m), 2.92 (2H, d), 3.3 (2H, m), 3.59 (3H, s), 3.99-4.41
(6H, m), 5.29 (1H, d), 6.4-6.56 (2H, m), 7.17-7.9 (13H, m), 8.35
(1H, m). 13C NMR (δ, DMSO): 22.0, 23.1, 24.6, 27.6, 39.3, 40.9,
41.2, 41.4, 47.1, 51.1, 52.0, 56.0, 62.4, 66.2, 73.3, 120.5, 125.7,
126.6, 127.5, 128.1, 128.5, 129.7, 138.0, 141.1, 144.2, 144.3, 156.2,
156.6, 169.7, 174.4. ES MS m/z observed, 673.4.
129.7, 132.4, 137.6, 141.3, 143.9, 154.2, 154.2, 155.2, 156.4, 168.7,
169.6, 174.3. ES MS m/z observed, 865.1.
Deprotection of Fmoc Group from the NR-Fmoc-peptidyl
Urea Acids 12: General Procedure. A mixture of NR-Fmoc-
peptidyl urea (1 mmol) in DCM (5 mL) and DEA (5 mL) was
stirred until the deprotection was complete. After the completion
of the reaction, the solvent was removed under reduced pres-
sure below 40 °C, and the residue was recrystallized using EtOAc
and n-hexane to get the solid crystalline compounds in most of the
cases. Free peptidyl ureas were purified by column chromatography
using the solvent system CHCl3/MeOH (5% to 15% of MeOH in
CHCl3).
Val-Pro-ψ(NH-CO-NH)-Gly-OH 12a. A suspension of 0.493
g (1 mmol) of 9c in 5 mL of DCM was treated with 5 mL of DEA
to obtain 12a. Yield 0.235 g (87%); mp 146-148 °C. 1H NMR (δ,
DMSO): 0.95 (6H, d), 1.2-2.1 (5H, m), 3.2-3.85 (6H, m), 4.0
(1H, m), 6.25-6.75 (2H, m), 7.8 (1H, d), 8.34 (1H, m) 8.6 (1H, br
s). 13C NMR (δ, DMSO): 18.6, 19.5, 24.1, 28.6, 29.1, 47.3, 58.5,
60.8, 154.9, 158.3, 169.8. ES MS m/z observed, 286.4.
Tyr(But)-Gly-ψ(NH-CO-NH)-Gly-OH 12c. A suspension of
0.573 g (1 mmol) of Fmoc-Tyr(But)-Gly-ψ(NH-CO-NH)-Gly-
OH in 5 mL of DCM was treated with 5 mL of DEA, and the
reaction mixture was stirred until the deprotection was complete.
After the completion of the reaction, the solvent was removed under
reduced pressure below 40 °C, and the residue was recrystallized
using EtOAc and n-hexane to get the solid crystalline compound.
Free 12c compounds were purified by column chromatography
using the solvent system CHCl3/MeOH (5% to 15% of MeOH in
CHCl3). Yield 0.315 g (90%); mp 134-135 °C. 1H NMR (δ,
DMSO): 1.22 (9H, s), 2.87 (2H, d), 3.6-3.75 (4H, m), 3.87 (1H,
m), 6.46-6.9 (2H, m), 7.9-8.6 (3H, m). 13C NMR (δ, DMSO):
28.7, 37.4, 52.1, 78.5, 124.3, 129.4, 132.4, 154.2, 155.4, 159.1,
170.3. ES MS m/z observed, 366.3.
Tyr-Gly-ψ(NH-CO-NH)-Gly-ψ(NH-CO-NH)-Phe-ψ(NH-
CO-NH)-Leu-OH 21. A 0.556 g (0.95 mmol) of Fmoc-Tyr-Gly-
ψ(NH-CO-NH)-Gly-NCO 9d was coupled with bis-TMS-Phe
(1.1 mmol) to obtain the Fmoc-Tyr(But)-Gly-ψ(NH-CO-NH)-
Gly-ψ(NH-CO-NH)-Phe-OH 19. Yield 0.634 g (89%); mp 184-
187 °C. MS (MALDI-TOF) m/z observed: 772.8 [M + Na]+, 789.0
[M + K]+. An amount of 0.634 g (0.84 mmol) of 19 was converted
to the corresponding isocyanate. Yield 0.574 g (91%); mp 184-
187 °C. IR: 2252 cm-1. 1H NMR (δ, CDCl3): 2.86-2.95 (4H, m),
3.84-4.52 (8H, m), 5.36 (1H, d), 6.22-6.98 (5H, m), 7.12-7.95
(17H, m). 13C NMR (δ, CDCl3): 36.7, 37.4, 43.3, 43.4, 47.3, 54.2,
54.4, 66.7, 119.9, 124.5, 125.1, 126.7, 126.8, 127.3, 128.7, 127.9,
129.3, 129.8, 132.3, 137.6, 141.4, 144.3, 154.5, 156.7. ES MS m/z
observed, 748.7. A solution of 0.574 g (0.768 mmol) of Fmoc-
Tyr(But)-Gly-ψ(NH-CO-NH)-Gly-ψ(NH-CO-NH)-Phe-NCO in
5 mL of DCM was coupled with bis-TMS-Leu (0.92 mmol in 10
mL of DCM) and after the routine workup, 20 was obtained. The
recrystallization in methanol gave almost pure product, and it was
used directly for the deprotection. Yield 0.533 g (79%); mp 186-
187 °C. MS (MALDI-TOF) m/z observed: 901.0 [M + Na]+, 917.2
[M + K]+. After deprotection of Fmoc and But groups as described
above for 13, the free peptidyl urea 21 was obtained. It was purified
by flash column chromatography using the solvent system CHCl3/
MeOH (5% to 15% of MeOH in CHCl3) to obtain an analytically
pure compound. Yield 0.298 g (82%); mp 113-114 °C. 1H NMR
(δ, DMSO): 1.02 (6H, m), 1.42 (2H, m), 1.56 (1H, m), 2.78-4.63
(11H, m), 6.46-8.75 (18H, m). 13C NMR (δ, DMSO): 10.9, 13.8,
21.5, 22.9, 24.3, 36.1, 39.2, 41.3, 50.4, 53.8, 115.5, 115.9, 116.3,
118.9, 124.8, 126.5, 128.0, 129.3, 130.5, 131.4, 155.3, 156.8, 158.2,
158.6, 169.9. MS (MALDI-TOF) m/z observed: 623.3 [M + Na]+,
639.3 [M + K]+.
Fmoc-Tyr(But)-Gly-ψ(NH-CO-NH)-Gly-NCO 9d. An amount
of 0.588 g (1 mmol) of Fmoc-Tyr(But)-Gly-ψ(NH-CO-NH)-Gly-
OH was converted to its isocyanate following the general procedure
1
for 3. Yield 0.556 g (95%); mp 148-149 °C. IR: 2256 cm-1. H
NMR (δ, CDCl3): 2.88 (2H, d), 3.84-4.46 (8H, m), 5.31 (1H, d),
6.36-6.85 (3H, m), 7.2-7.78 (12H, m). 13C NMR (δ, CDCl3):
36.7, 43.4, 43.5, 47.3, 54.4, 66.6, 119.8, 124.5, 125.2, 126.6, 127.3,
127.8, 129.6, 132.3, 141.3, 144.2, 154.5, 156.8. ES MS m/z
observed, 586.7.
Fmoc-Leu-Phe-ψ(NH-CO-NH)-Ala-ψ(NH-CO-NH)-Val-
Tyr-OH 11. An amount of 0.583 g (1 mmol) of Fmoc-Leu-Phe-
ψ(NH-CO-NH)-Ala-NCO was coupled with tris-TMS-Val-Tyr
(from 0.336 g, 1.2 mmol of Val-Tyr-OH in 10 mL of DCM) to
obtain the title compound. It was further purified by column
chromatography using the solvent system CHCl3 /MeOH/AcOH
(5% to 20% of MeOH in CHCl3 containing 1% AcOH) to get the
analytically pure compound. Yield 0.76 g (88%); mp 218-219 °C.
1H NMR (δ, DMSO): 0.86-0.98 (12H, m), 1.16 (3H, d), 1.33 (2H,
m), 1.63 (1H, m), 1.85 (1H, m), 2.85-2.9 (4H, m), 3.68-4.48 (8H,
m), 5.26 (1H, s), 6.35-6.8 (4H, m), 7.08-7.85 (17H, m), 8.26
(1H, m), 8.45 (1H, m). 13C NMR (δ, DMSO): 17.2, 18.6, 19.5,
22.0, 23.0, 24.6, 29.1, 36.5, 37.3, 40.3, 47.2, 48.9, 51.2, 54.1, 54.2,
58.5, 66.6, 120.0, 124.3, 125.0, 126.7, 127.0, 127.6, 128.4, 129.3,
Test for Racemization. To a solution of NR-Fmoc-peptide
isocyanate 3c (1 mmol) was added 1-phenylethylamine (0.121 g,
1 mmol) in the presence of DIEA (0.172 mL, 1 mmol). After the
completion of the coupling, the separated solid was filtered and
7704 J. Org. Chem., Vol. 71, No. 20, 2006

Na-Fmoc-peptide Isocyanates
recrystallized using DMF and water (3:1), to obtain the urea adduct
25 as a solid crystalline powder.
(1H, m), 6.5 (1H, m), 7.2-8.0 (14H, m). ES MS m/z observed,
573.4.
Fmoc-Leu-Val-ψ(NH-CO-NH)-R-(+)-1-phenylethylamine
Acknowledgment. We thank Professor Fred Naider, CUNY,
New York, for useful suggestions and discussion throughout
the course of this work. We are grateful to the Department of
Science and Technology, Govt. of India, New Delhi, for
financial assistance to VSSB. B.S.P. and R.V.R thank the CSIR,
Govt. of India, for senior research fellowship.
1
25a. Yield 92%; mp 206-207 °C. H NMR (δ, DMSO): 0.79-
0.90 (12H, m), 1.28-1.30 (3H, d), 1.36-1.92 (4H, m), 4.0 (1H,
m), 4.2-4.3 (3H, m), 4.7 (1H, d), 4.9 (1H, d), 6.1 (1H, m), 6.6
(1H, m), 7.2-8.0 (14H, m). ES MS m/z observed, 573.4.
Fmoc-Leu-Val-ψ(NH-CO-NH)-S-(-)-1-phenylethyl-
1
amine 25b. Yield 94%; mp 205-207 °C. H NMR (δ, DMSO):
Supporting Information Available: IR spectrum for 2o, 2g,
and 3g; 1H NMR spectra for the compounds 3b, 7a,e, 22, and 25a-
c; 13C NMR spectra for the compounds 3b, 7a,e, and 14; mass
spectra for 3b,d, 4c, 7a,e, 13, 21, 22a, 23a, and 25b and the
analytical HPLC data of 2e and 3e. This material is available free
of charge via the Internet at http://pubs.acs.org.
0.8-0.86 (12H, m), 1.27-1.29 (3H, d), 1.31-1.88 (4H, m), 3.98
(1H, m), 4.2-4.29 (3H, m), 4.7 (1H, d), 4.9 (1H, d), 6.1 (1H, m),
6.5 (1H, m), 7.2-8.0 (14H, m). ES MS m/z observed, 573.4.
Fmoc-Leu-Val-ψ(NH-CO-NH)-(R&S)-1-phenylethyl-
1
amine 25c. Yield 89%; mp 208-209 °C. H NMR (δ, DMSO):
0.81-0.90 (12H, m), 1.27-1.30 (3H, t), 1.34-1.88 (4H, m),
4.0 (1H, m), 4.21-4.29 (3H, m), 4.7 (1H, m), 4.9 (1H, m), 6.1
JO0611723
J. Org. Chem, Vol. 71, No. 20, 2006 7705