Efficient peptide coupling involving sterically hindered amino acids

Article abstract of DOI:10.1021/jo0704255

(Chemical Equation Presented) Hindered amino acids have been introduced into peptide chains by coupling N-(Cbz- and Fmoc-α-aminoacyl) benzotriazoles with amino acids, wherein at least one of the components was sterically hindered, to provide compounds 3a-e, (3c +3 c′), 5a-d, (5a + 5a′), 6a-c, (6b + 6b′), 8a-c, 9a-e, 10a-d, and (10a + 10a′) in isolated yields of 41-95% with complete retention of chirality as evidenced by NMR and HPLC analysis. The benzotriazole activation methodology is a new route for the synthesis of sterically hindered peptides. (Note: compound numbers written within brackets represent diastereomeric mixtures or racemates; compound numbers without brackets represent enantiomers.)

Full text of DOI:10.1021/jo0704255

Efficient Peptide Coupling Involving Sterically Hindered Amino
Acids
Alan R. Katritzky,* Ekaterina Todadze, Parul Angrish, and Bogdan Draghici
Center for Heterocyclic Compounds, Department of Chemistry, UniVersity of Florida,
GainesVille, Florida 32611-7200
katritzky@chem.ufl.edu
ReceiVed March 1, 2007
Hindered amino acids have been introduced into peptide chains by coupling N-(Cbz- and Fmoc-R-
aminoacyl)benzotriazoles with amino acids, wherein at least one of the components was sterically hindered,
to provide compounds 3a-e, (3c +3 c′), 5a-d, (5a + 5a′), 6a-c, (6b + 6b′), 8a-c, 9a-e, 10a-d, and
(10a + 10a′) in isolated yields of 41-95% with complete retention of chirality as evidenced by NMR
and HPLC analysis. The benzotriazole activation methodology is a new route for the synthesis of sterically
hindered peptides. (Note: compound numbers written within brackets represent diastereomeric mixtures
or racemates; compound numbers without brackets represent enantiomers.)
Introduction
Sterically hindered amino acids, such as Aib (2-methylala-
nine), Iva (2-ethylalanine), and Deg (2,2,-diethylglycine) (Figure
1) are constituents of naturally occurring peptaibols,1-4 a large
group of peptide antibiotics isolated from soil fungi.
The presence of an extra alkyl group at CR significantly
restricts the accessible conformational space in R,R-disubstituted
amino acids; this forces the chain to bend in peptides containing
these sterically hindered amino acids.5-7 Thus, peptides incor-
porating R,R-disubstituted amino acids, such as Aib or Iva, have
rigid backbones through the formation of helices and â-turns.8
These conformational properties of R,R-disubstituted amino
acids confer interesting biological activity to peptaibols, which
can form voltage dependent ion channels in bilayer cellular
FIGURE 1. Sterically hindered amino acids.
membranes and can cause, at high concentration, cell lysis.2
These properties make R,R-disubstituted amino acids important
for peptide and medicinal chemistry. Thus, Aib, Iva, and Deg
are often used to study the relationships existing between
structure, stability, and function in bioactive peptides.9 However,
the reactivities of both (i) amino groups and (ii) activated
carboxyl groups of sterically hindered R,R-disubstituted amino
acids are significantly lower than those of typical R-amino acids;
hence their incorporation even into short peptides has been
challenging.10
(1) Wilkening, R. R.; Stevens, E. S.; Bonora, G. M.; Toniolo, C. J. Am.
Chem. Soc. 1983, 105, 2560-2561.
(2) Wenschuh, H.; Beyermann, M.; Haber, H.; Seydel, J. K.; Krause,
E.; Bienert, M.; Carpino, L. A.; Elfaham, A.; Albericio, F. J. Org. Chem.
1995, 60, 405-410.
N-Substitution at the peptide bond also induces steric
modification and mimics various biological environments.
N-Substituted peptides are important constituents of a number
of naturally occurring peptides like cyclosporines,11 didemnins12
(3) Bodo, B.; Rebuffat, S.; Elhajji, M.; Davoust, D. J. Am. Chem. Soc.
1985, 107, 6011-6017.
(4) Kenner, G. W.; Sheppard, R. C. Nature 1958, 181, 48-48.
(5) Toniolo, C.; Benedetti, E. Macromolecules 1991, 24, 4004-4009.
(6) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747-6756.
(7) Paterson, Y.; Rumsey, S. M.; Benedetti, E.; Nemethy, G.; Scheraga,
H. A. J. Am. Chem. Soc. 1981, 103, 2947-2955.
(9) Samanen, J.; Cash, T.; Narindray, D.; Brandeis, E.; Adams, W.;
Weideman, H.; Yellin, T.; Regoli, D. J. Med. Chem. 1991, 34, 3036-3043.
(10) Humphrey, J. M.; Chamberlin, A. R. Chem. ReV. 1997, 97, 2243-
2266.
(8) Slomczynska, U.; Beusen, D. D.; Zabrocki, J.; Kociolek, K.;
Redlinski, A.; Reusser, F.; Hutton, W. C.; Leplawy, M. T.; Marshall, G. R.
J. Am. Chem. Soc. 1992, 114, 4095-4106.
(11) Wenger, R. M. Angew. Chem., Int. Ed. 1985, 24, 77-85.
10.1021/jo0704255 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/20/2007
5794
J. Org. Chem. 2007, 72, 5794-5801

Efficient Preparation of Hindered Peptides
(and references therein), and dolastatins13 (and references
therein). N-Methyl peptides exhibit antibiotic,14 anticancer,15-17
and antiviral activity.18
SCHEME 1
The cyclic side chain of the naturally occurring N-alkylated
amino acid proline locks a peptide backbone at a dihedral angle
of approximately -75°. Thus proline has exceptional confor-
mational rigidity compared to other amino acids and conse-
quently is often found in very tight turns in protein structures.
Proline also introduces kinks into R helices, since it is unable
to adopt a normal helical conformation. These conformational
properties of proline and proline rich sequences exhibit interest-
ing biological and medicinal roles: (1) drug delivery activity,19
(2) important components of collagen,19 (3) intracellular cell
signaling, and (4) quenching reactive oxygen species.20
Previously, we reported the successful preparation of peptides
without detectable racemization (<5% by NMR analysis and
<1% by HPLC analysis) in average yields of 88%.21-25 The
present paper reports convenient procedures for the incorporation
of hindered amino acids into peptide chains.
TABLE 1. Preparation of N-Terminus Aib Dipeptides 3a-e and
(3c + 3c′) from N-(Cbz- and Fmoc-Aib-)Bt 1a,b and Free Amino
Acids 2a-c and (2c + 2c′)
retention
yield
(%)a
time
(min)
entry
amino acid
product
1
2
3
4
L-Phe-OH 2a
L-Trp-OH 2b
L-Met-OH 2c
Cbz-Aib-L-Phe-OH 3a
Cbz-Aib-L-Trp-OH 3b
Cbz-Aib-L-Met-OH 3c
84
85
91
92
6.19
5.79
6.43
6.40, 7.34
DL-Met (2c + 2c′) Cbz-Aib-DL-Met-OH
(3c + 3c′)
Fmoc-Aib-L-Phe-OH 3d
Fmoc-Aib-L-Met-OH 3e
5
6
L-Phe-OH 2a
L-Met-OH 2c
67
70
5.39
5.82
a Isolated yield.
Results and Discussions
Recent literature approaches to realize coupling at the
carbonyl group of Aib to give hindered peptides (Scheme 2)
include (i) the use of DCC or DCC/HOBT26 (and references
within), (ii) 2-mercaptopyridone-1-oxide based uronium salts
(HOTT and TOTT),27 (iii) the active ester coupling method,28
(iv) CIP (2-chloro-1,3-dimethylimidazolidium hexaflurophos-
phate) in the presence of additives such as HOAt or HODhbt,29,30
(v) enzymatic coupling,31 (vi) PyBOP and PyBroP,26 and (vii)
polymer-supported BOP coupling reagents.32 The methods of
Scheme 2 give Aib-containing dipeptides in variable yields
(23-96%) depending upon the protecting group (Pg) and -R
group incorporated. Most need labor intensive procedures (ref
26 and references therein), utilize expensive or unstable
reagents,31 and/or need an additional deprotection28 step, because
amino acid esters are used.26-29,32 In comparison, our methodol-
ogy offers simple preparative and workup procedures, takes
less time to complete, uses inexpensive reagents, gives high
yields, and frequently allows free amino acids as coupling
components.
(b) By Coupling at the Amino Group of Aib. Our attempts
under varying conditions (temperature, solvent systems, time)
and reagents (base, additives) to couple N-(protected-R-
aminoacyl)benzotriazoles with the NH2 of free Aib (i.e.,
H2NMe2CCO2H), resulted in extensive hydrolysis of 1c-f and
(1c + 1c′). However, Aib methyl ester 4 was successfully
coupled in the presence of Et3N (in acetonitrile) at 20 °C in
24-36 h (Scheme 3 and Table 2). Under microwave irradiation
in the presence of 1 equiv of Et3N in THF as solvent at 55 and
60 °C (Scheme 3 and Table 2) the preparation of compounds
5a-d and (5a + 5a′) needed 1 h. Under conventional heating
I. Preparation of Aib-Containing Dipeptides. (a) Using
Carboxyl Activated N-Protected Aib. We now show that
amino acids 2a-c and (2c + 2c′) (compound numbers written
within brackets represent diastereomeric mixtures or race-
mates; compound numbers without brackets represent enantio-
mers) couple with N-(Cbz- and Fmoc-Aib-)Bt 1a,b (Bt )
benzotriazol-1-yl) in partially aqueous solution (CH3CN/H2O)
in the presence of Et3N in 1 h at 20 °C to give dipeptides 3a-e
and (3c + 3c′) (67-92%) isolated without chromatography
(Scheme 1 and Table 1). The enantiopurity of the dipeptides
3a-e was supported by HPLC analyses using a Chirobiotic T
column (detection at 254 nm, flow rate 0.5 mL/min, and MeOH
as solvent). As expected, HPLC analysis of the enantiopure
dipeptides 3a-e showed a single peak. In contrast, two peaks
of equal intensity were observed for the corresponding racemic
mixture (3c + 3c′) (Table 1).
(12) Jouin, P.; Poncet, J.; Dufour, M. N.; Pantaloni, A.; Castro, B. J.
Org. Chem. 1989, 54, 617-627.
(13) Pettit, G. R.; Singh, S. B.; Herald, D. L.; Lloydwilliams, P.; Kantoci,
D.; Burkett, D. D.; Barkoczy, J.; Hogan, F.; Wardlaw, T. R. J. Org. Chem.
1994, 59, 6287-6295.
(14) Abe, H.; Sato, K.; Kato, T.; Izumiya, N. Bull. Chem. Soc. Jpn. 1976,
49, 3113-3118.
(15) Aoki, S.; Cao, L. W.; Matsui, K.; Rachmat, R.; Akiyama, S.;
Kobayashi, M. Tetrahedron 2004, 60, 7053-7059.
(16) Haviv, F.; Henkin, J.; Bradley, M. F.; Kalvin, D. M. PCT Int. Appl.,
038397, 2001; Chem. Abstr. 2001, 135, 5822.
(17) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Fujii, Y.; Kizu, H.; Boyd,
M. R.; Boettner, F. E.; Doubek, D. L.; Schmidt, J. M.; Chapuis, J. C.;
Michel, C. Tetrahedron 1993, 49, 9151-9170.
(18) Printsevskaya, S. S.; Solovieva, S. E.; Olsufyeva, E. N.; Mirchink,
E. P.; Isakova, E. B.; De Clercq, E.; Balzarini, J.; Preobrazhenskaya, M.
N. J. Med. Chem. 2005, 48, 3885-3890.
(19) Sanclimens, G.; Shen, H.; Giralt, E.; Albericio, F.; Saltzman, M.
W.; Royo, M. Biopolymers 2005, 80, 800-814.
(26) Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M. N.; Jouin, P.
Tetrahedron 1991, 47, 259-270.
(20) Alia, J. M.; Backendorf, C. M. P. PCT Int. Appl., 075903, 2003;
Chem. Abstr. 2003, 139, 240387.
(27) Bailen, M. A.; Chinchilla, R.; Dodsworth, D. J.; Najera, C. J. Org.
Chem. 1999, 64, 8936-8939.
(21) Katritzky, A. R.; Suzuki, K.; Singh, S. K. Synthesis 2004, 2645-
2652.
(28) McGahren, W. J.; Goodman, M. Tetrahedron 1967, 23, 2017-2030.
(29) Akaji, K.; Kuriyama, N.; Kiso, Y. Tetrahedron Lett. 1994, 35, 3315-
3318.
(22) Katritzky, A. R.; Angrish, P.; Hur, D.; Suzuki, K. Synthesis 2005,
397-402.
(23) Katritzky, A. R.; Angrish, P.; Suzuki, K. Synthesis 2006, 411-
(30) Akaji, K.; Tamai, Y.; Kiso, Y. Tetrahedron Lett. 1995, 36, 9341-
9344.
424.
(24) Katritzky, A. R.; Todadze, E.; Cusido, J.; Angrish, P.; Sheshtopalov,
A. A. Chem. Biol. Drug Des. 2006, 68, 37-41.
(25) Katritzky, A. R.; Todadze, E.; Cusido, J.; Sheshtopalov, A. A.;
Angrish, P. Chem. Biol. Drug Des. 2006, 68, 42-47.
(31) Sekizaki, H.; Itoh, K.; Toyota, E.; Tanizawa, K. Tetrahedron Lett.
1997, 38, 1777-1780.
(32) Filip, S. V.; Lejeune, V.; Vors, J. P.; Martinez, J.; Cavelier, F. Eur.
J. Org. Chem. 2004, 1936-1939.
J. Org. Chem, Vol. 72, No. 15, 2007 5795

Katritzky et al.
SCHEME 2. Literature Peptide Coupling Procedures for Activating the Carboxyl Group of Aib
SCHEME 3
and (vi) 2-chloro-4,6-[(heptadecaflurocarbonyl)oxy]-1,3,5-tri-
azine (FCDMT).36 The literature methods of Scheme 4 led to
dipeptides in moderate to high yields (35-97%) depending upon
the Pg and -R group incorporated. Reaction times ranged from
4 to 20 h. Some involved low temperatures or two-step
procedures.35,36 Our methodology provides yields of 71-80%
in 1 h with convenient preparative procedures, simple workup
procedures (the byproduct, BtH, is easily dissolved using dilute
acids or bases), and insensitivity to water.
TABLE 2. Preparation of C-Terminus Aib Dipeptides 5a-d and
(5a + 5a′) from N-(Cbz-r-aminoacyl) Benzotriazoles 1c-f, (1c +
1c′)
II. Preparation of Dipeptides Containing an N-Methyl
Amino Acid. (a) From a Carboxyl Activated N-Protected
N-Methyl Amino Acid. Amino acids 2c-e and (2d + 2d′) were
coupled successfully with Cbz-L-N(Me)-Phe-Bt 1g in partially
aqueous solution (CH3CN/H2O) in the presence of Et3N during
1 h at room temperature (Scheme 5 and Table 3). After washing
with 4 N HCl, pure dipeptides 6a-c and (6b + 6b′) were
obtained in yields ranging of 72-93%.
yield (%)a
entry
reactant
product
20 °Cb MWc
1
2
Cbz-L-Phe-Bt 1c
Cbz-DL-Phe-Bt
(1c + 1c′)
Cbz-L-Phe-Aib-OMe 5ad
Cbz-DL-Phe-Aib-OMe
(5a + 5a′)e
41
41
72
71
3
4
5
Cbz-L-Met-Bt 1d Cbz-L-Met-Aib-OMe 5bf
78
41
64
79
80
79
Cbz-L-Trp-Bt 1e
Cbz-Gly-Bt 1f
Cbz-L-Trp-Aib-OMe 5c
Cbz-Gly-Aib-OMe 5d
1
Variable temperature H NMR confirmed that the complex
peaks obtained for 6a-c at 20 °C were due to restricted rotation
at the amide bonds (Figure 2). For example, the expected doublet
arising from the methyl protons of the L-Ala fragment in 6b
collapsed to an apparent triplet at 25 °C which was subsequently
resolved at 100 °C into a clean doublet, with improved
resolution, demonstrating the absence of racemization (Figure
2). Similar behavior was shown by 6b,c and (6b + 6b′). HPLC
analysis (Table 3) of the enantiopure LL-dipeptides 6a-c showed
for each a single peak. In contrast, two peaks of equal intensity
were observed for the corresponding diastereomeric mixture (6b
+ 6b′).
a Isolated yield. b Reaction time ) 24-36 h. c Power 200 W for 30 min
at 55 °C and 300 W for 30 min at 60 °C. d HPLC for 5a: 6.35 min. e HPLC
for (5a + 5a′): 6.43 and 7.17 min. f HPLC for 5b: 7.17 min.
conditions at 60 °C lower yields and longer reactions times in
comparison to those obtained with microwave technique were
obtained.
Analysis by HPLC (Table 2) of the compounds 5a,b and (5a
+ 5a′), prepared by the MW technique, confirmed the absence
of racemization. Single peaks were obtained for the enantiomers
5a and 5b at 6.35 and 7.17 min, respectively, whereas the
racemate (5a + 5a′) gave two peaks at 6.43 and 7.17 min.
Previous couplings at the NH group of Aib (Scheme 4)
utilized (i) the active ester method,33 (ii) polymer-supported
coupling,34 (iii) coupling reagent HOTT,27 (iv) coupling reagent
TOTT,27 (v) 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT),35
Literature methods for the synthesis of peptides incorporating
N-alkylated amino acids (Scheme 6) use the usual carbodiimide
based coupling reagents including (i) DCCI-HOBt,37 (ii) DCCI-
HONSu,37 and (iii) EDC-HOBt.38 Additional methods utilize
(35) Kaminski, Z. J. Synthesis 1987, 917-920.
(36) Markowicz, M. W.; Dembinski, R. Synthesis 2004, 80-86.
(37) Davies, J. S.; Mohammed, A. K. J. Chem. Soc., Perkin Trans. 1
1981, 2982-2990.
(33) Jelokhaniniaraki, M.; Yoshioka, K.; Takahashi, H.; Kato, F.; Kondo,
M. J. Chem. Soc., Perkin Trans. 2 1992, 1187-1193.
(34) Barrett, A. G. M.; Bibal, B.; Hopkins, B. T.; Kobberling, J.; Love,
A. C.; Tedeschi, L. Tetrahedron 2005, 61, 12033-12041.
(38) Hitotsuyanagi, Y.; Hasuda, T.; Aihara, T.; Ishikawa, H.; Yamaguchi,
K.; Itokawa, H.; Takeya, K. J. Org. Chem. 2004, 69, 1481-1486.
5796 J. Org. Chem., Vol. 72, No. 15, 2007

Efficient Preparation of Hindered Peptides
SCHEME 4. Literature Peptide Coupling Procedures with Amino Group of Aib
SCHEME 5
1c-d,f with the NH group of N-methyl amino acid ester 7a.
Dipeptides 8a-c were thus prepared (Scheme 7 and Table 4)
in 72-80% yield during 1 h using microwave irradiation (for
conditions see Table 4). Without microwave irradiation the
reactions needed 12-18 h.
1
The room-temperature H NMR showed the existence of
rotamers for 8a-c. The results obtained from high-temperature
NMR were difficult to interpret precisely; all peaks appeared
as broadened singlets because of the low solubility of com-
pounds 8a-c in DMSO. However, the elemental analyses of
8a-c confirmed their formation, and HPLC analyses of 8a,b
revealed single retention times for each, supporting their
enantiopurity (Table 4).
Additionally, the structure of compound 8b was confirmed
by the 2D homonuclear correlations COSY and NOESY. The
COSY spectrum presented (see Figure 3 in Supporting Informa-
tion) confirmed the sequence of the amino acids in peptide 8b
with the vicinal and geminal proton peaks. For the major
rotamer, coupling was observed between the proton (CH) at
4.92 ppm, the NH at 5.65 ppm, and the prochiral protons at
2.06 and 1.87 ppm. For the minor rotamer, the corresponding
proton at 4.72 ppm coupled with the NH at 5.55 ppm and with
the prochiral protons at 1.87 and 1.84 ppm.
The NOESY experiment (see Figure 4 in Supporting Infor-
mation) confirmed the existence of two rotameric forms by
showing exchange peaks between protons of both populations
and transfer peaks between spacially near protons in the same
species. For example, the N-methyl group from major species
at 3.19 ppm gave NOE transfer peaks with a proton at 4.92
ppm and S-methyl at 2.11 ppm and gave transfer peaks with
proton at 4.73 ppm and S-methyl at 2.07 ppm. The two different
rotameric species are designated in Figure 5.
Literature methods for coupling N-alkylated amino acids
(Scheme 8) have used (i) the usual carbodiimide based coupling
reagents, BOP,41 (ii) CF3NO2-PyBrOP,42 (iii) BEP and FEP,43
(iv) PyBroP,42 (v) active ester,44 and (vi) acid chloride method
followed by N-methylation with diazomethane.45 Yields of 62-
TABLE 3. Preparation of Dipeptides 6a-c and (6b + 6b′) from
Cbz-L-N(Me)-Phe-Bt 1g and Free Amino Acids 2c-e and (2d + 2d′)
retention
yield
(%)a
time
(min)
entry
reactant
product
1
2
3
L-Met-OH 2c Cbz-L-N(Me)-Phe-L-Met-OH 6a 93 7.17
L-Ala-OH 2d Cbz-L-N(Me)-Phe-L-Ala-OH 6b 72 6.56
DL-Ala-OH
(2d + 2d′)
Gly-OH 2e
Cbz-L-N(Me)-Phe-DL-Ala-OH
(6b + 6b′)
Cbz-L-N(Me)-Phe-Gly-OH 6c
72 6.60, 7.16
4
82 7.34
a Isolated yield.
(iv) a three-step procedure via oxazolidinones,39 and (v)
N-methylation with diazomethane after coupling of amino acid
chlorides with amino acid methyl esters.40 Reported yields range
from 40 to 96% depending upon the Pg and -R group
incorporated. The two-step procedure involving toxic diazo-
methane has a reaction time of 1.5 h.40 Other reaction times in
Scheme 6 vary from 12 to 48 h.37-39 Davis et al. observed
racemization (10-35%) during the coupling of benzoyl-N-
alkylated amino acids with amino acid methyl esters.37 Our one-
step methodology provides optically pure dipeptides in yields
of 72-93% using simple preparative and workup procedures
in reaction times of 1 h.
(b) By Coupling at the NH Group of Free N-methyl Amino
Acids. We adopted procedures similar to those of section I(b)
in the Results and Discussion to couple Cbz-(aminoacyl)Bt
(39) Dorow, R. L.; Gingrich, D. E. Tetrahedron Lett. 1999, 40, 467-
(41) Gerber, C.; Seebach, D. HelV. Chim. Acta 1991, 74, 1373-1385.
(42) Wijkmans, J.; Blok, F. A. A.; Vandermarel, G. A.; Vanboom, J.
H.; Bloemhoff, W. Tetrahedron Lett. 1995, 36, 4643-4646.
(43) Li, P.; Xu, J. C. Tetrahedron 2000, 56, 8119-8131.
470.
(40) Di Gioia, M. L.; Leggio, A.; Liguori, A. J. Org. Chem. 2005, 70,
3892-3897.
J. Org. Chem, Vol. 72, No. 15, 2007 5797

Katritzky et al.
1
FIGURE 2. Variable temperature H NMR of compound 6b.
SCHEME 6. Literature Peptide Coupling Procedures
Involving Activated Carboxyl Groups of N-methyl Amino
Acids
III. Preparation of Proline-Containing Dipeptides. (a)
Using N-Cbz-proline Acyl Bt. L-Amino acids 2a-d,f were
coupled with Cbz-Pro-Bt 1h22 to give products 9a-e in yields
of 70-95% (Scheme 9 and Table 5); the procedure was similar
to that utilized above for the preparation of 6a-c (Scheme 5).
At 20 °C, the 1H and 13C NMR spectra of 9c-e were
1
complex. However, the H NMR spectra at 100 °C for 9c-e
were simplified, demonstrating the existence of rotamers, and
confirming the absence of racemization (for the example of 9c,
see Figure 6). HPLC analysis revealed single retention times
for 9a-e, supporting their enantiopurity (Table 5).
Previously, proline peptides were prepared (Scheme 10) in
reaction times of 24-48 h via (i) carbodiimide,46 (ii) DCCI,47
(iii) EDCI, HOBt,48 or (iv) active ester methods using p-
nitrophenyl, alkyl chloroformate, or N-hydroxysuccinimide.47,49
in yields up to 85% for those reported depending upon the Pg
and -R group incorporated. Our methodology led to optically
pure dipeptides in yields of 70-95% using simple preparative
and workup procedures and reaction times of 1 h.
SCHEME 7
(b) By Coupling at the NH Group of Free Proline. N-(Cbz-
R-aminoacyl)benzotriazoles 1c,e,f,i and (1c + 1c′) were coupled
with L-Pro 2g in partially aqueous solution (CH3CN/H2O) in
the presence of Et3N for 1 h at room temperature to give
dipeptides 10a-d and (10a + 10a′) in 63-89% yields in high
purity (Scheme 11 and Table 6). NMR analysis of the enan-
tiopure LL-dipeptides 10a-d showed no detectable racemization
(<5%). The enantiopurity of dipeptides 10a-d was confirmed
by HPLC analyses using Chirobiotic T column (detection at
254 nm, flow rate 0.5 mL/min, and MeOH as solvent):
dipeptides 10a,d each gave a single retention time, whereas the
TABLE 4. Preparation of Dipeptides 8a-c from
N-(Cbz-r-aminoacyl)benzotriazoles 1c-d,f and N(Me)-Gly-OMe 7a
yield (%)a
entry
reactant
product
20 °Cb MWc
1
2
3
Cbz-L-Phe-Bt 1c Cbz-L-Phe-N(Me)-Gly-OMe 8ac
57
51
66
75
72
80
Cbz-L-Met-Bt 1d Cbz-L-Met-N(Me)-Gly-OMe 8bd
(44) Kawasaki, K.; Tsuji, T.; Hirase, K.; Miyano, M.; Imoto, Y.;
Iwamoto, M. Chem. Pharm. Bull. 1991, 39, 584-589.
(45) Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Napoli, A.;
Siciliano, C.; Sindona, G. J. Org. Chem. 2003, 68, 7416-7421.
(46) Ranganathan, D.; Singh, G. P.; Ranganathan, S. J. Am. Chem. Soc.
1989, 111, 1144-1145.
Cbz-Gly-Bt 1f
Cbz-Gly-N(Me)-Gly-OMe 8c
a Isolated yield. b Power 200 W for 30 min at 55 °C and 300 W for 30
min at 60 °C. c HPLC for 8a: 6.17 min. d HPLC for 8b: 6.23 min.
99% and reaction times of 1-16 h were reported depending
upon the Pg and -R group incorporated. Our methodology led
to optically pure dipeptides (75-80%), is insensitive to water,
and uses simple preparative and workup procedures and short
reaction times (1 h).
(47) Nicolaid, E.; Dewald, H.; Westland, R.; Lipnik, M.; Posler, J. J.
Med. Chem. 1968, 11, 74-79.
(48) Tang, Z.; Yang, Z. H.; Cun, L. F.; Gong, L. Z.; Mi, A. Q.; Jiang,
Y. Z. Org. Lett. 2004, 6, 2285-2287.
(49) Skripko, T. U.S. Patent, 079667, 2006; Chem. Abstr. 2006, 144,
331702.
5798 J. Org. Chem., Vol. 72, No. 15, 2007

Efficient Preparation of Hindered Peptides
FIGURE 5. Two rotameric forms of compound 8b.
SCHEME 8. Literature Peptide Coupling Procedures with Amino Group of N-Methyl Amino Acids
SCHEME 9
method synthesizes proline peptides in short reaction times
utilizing mild reaction conditions.
Conclusions
The methodology described in the present paper provides for
the convenient and efficient preparation of hindered peptides
in short reaction times utilizing simple workup procedures. It
is noteworthy that the reactivity of a sterically hindered amino
acid differs depending on whether the N- or the C-terminus is
undergoing coupling. Coupling using C-activated hindered
amino acids generally proceeds smoothly in high yields under
mild aqueous reaction conditions. In contrast, coupling at a
hindered NH group under similar conditions results in hydrolysis
of the (aminoacyl)Bt component. This behavior is probably due
to competitive hydrolysis; that is, hydrolysis is more important
when the steric strain is close to the NH site and hinders the
coupling.
TABLE 5. Preparation of Dipeptides 9a-e from N-Cbz-L-Pro-Bt
1h and Free Amino Acids 2a-d,f
yield
(%)a
retention
time (min)
entry
reactant
product
1
2
3
4
5
L-Phe-OH 2a
L-Trp-OH 2b
L-Met-OH 2c Cbz-L-Pro-L-Met-OH 9c
L-Ala-OH 2d
L-Val-OH 2f
Cbz-L-Pro-L-Phe-OH 9a
Cbz-L-Pro-L-Trp-OH 9b
91
78
82
70
95
7.13
7.14
7.48
7.16
7.14
Cbz-L-Pro-L-Ala-OH 9d
Cbz-L-Pro-L-Val-OH 9e
a
Isolated yield.
diastereomeric mixture (10a + 10a′) showed two retention times
as expected (Table 6).
Experimental Section
Previous preparations of proline peptides by coupling at
the proline NH were achieved via carbodiimide methods47,50,51
(i-iii) and active ester methods47,52,53 (iv-vi) (Scheme 12). Our
General Procedure for the Preparation of Dipeptides 3a-e,
(3c + 3c′), 6a-c, (6b + 6b′), 9a-e, 10a-d, (10a + 10a′).
N-(Protected-R-aminoacyl)benzotriazoles (1 mmol) were added at
20 °C to a solution of R-amino acid (1 mmol) in CH3CN (7 mL)/
H2O (3 mL) in the presence of Et3N (2 mmol). The reaction mixture
was stirred at 20 °C until the starting material was completely
consumed as observed by TLC using hexanes/ethyl acetate (2:1)
as the solvent. After 1 mL of 4 N HCl was added, the solution was
concentrated under reduced pressure to remove acetonitrile. The
solution was extracted with EtOAc (20 mL), and the organic extract
was washed with 4 N HCl (3 × 5 mL) and saturated NaCl (10
mL) and then dried (anhydrous MgSO4). Evaporation of the solvent
(50) Trotter, N. S.; Brimble, M. A.; Harris, P. W. R.; Callis, D. J.; Sieg,
F. Bioorg. Med. Chem. 2005, 13, 501-517.
(51) Moss, R. A.; Chiang, Y. C. P.; Hui, Y. Z. J. Am. Chem. Soc. 1984,
106, 7506-7513.
(52) Kawasaki, K.; Kawasaki, C.; Tsuda, Y.; Yagyu, M.; Okada, Y.;
Yamaji, K.; Takagi, T.; Tanizawa, O. Chem. Pharm. Bull. 1980, 28, 2699-
2706.
(53) Kouge, K.; Koizumi, T.; Okai, H.; Kato, T. Bull. Chem. Soc. Jpn.
1987, 60, 2409-2418.
J. Org. Chem, Vol. 72, No. 15, 2007 5799

Katritzky et al.
1
FIGURE 6. Variable temperature H NMR of compound 9c.
SCHEME 10. Literature Peptide Coupling Procedures with
Activated Carboxyl Group of Proline
7.05 (br t, J ) 7.2 Hz, 1H), 7.10 (d, J ) 1.8 Hz, 1H), 7.26-7.38
(m, 7H), 7.45 (d, J ) 7.8 Hz, 1H), 7.51 (d, J ) 7.8 Hz, 1H), 10.87
(s, 1H), 12.67 (br s, 1H). 13C NMR (DMSO-d6): δ 24.8, 25.2, 26.9,
52.8, 55.9, 65.1, 109.5, 111.2, 118.1, 118.2, 120.8, 123.5, 127.2,
127.6, 128.2, 136.0, 136.8, 154.6, 173.1, 173.9. Anal.Calcd for
C23H25N3O5: C, 65.24; H, 5.95; N, 9.92. Found: C, 64.88; H, 6.12;
N, 9.67.
(2S)-2-((2S)-2-[[(Benzyloxy)carbonyl](methyl)amino]-3-phen-
ylpropanoylamino)propanoic Acid (Cbz-L-N(Me)-Phe-L-Ala-
OH, 6b). Recrystallized from diethyl ether/hexanes to give colorless
microcrystals (72%). mp 89-91 °C. [R]D23 ) -4.6 (c 1.90, DMF).
1H NMR (DMSO-d6, 100 °C): δ 1.31 (d, J ) 7.3 Hz, 3H), 2.82
(s, 3H), 2.95 (dd, J ) 14.7, 10.6 Hz, 1H), 3.24 (dd, J ) 14.6, 5.1
Hz, 1H), 4.30 (q, J ) 7.3 Hz, 1H), 4.91 (dd, J ) 10.6, 5.1 Hz,
1H), 4.97 (d, J ) 12.5 Hz, 1H), 5.03 (d, J ) 12.5 Hz, 1H), 7.23-
7.35 (m, 10H), 7.84 (d, J ) 6.7 Hz, 1H). 13C NMR (DMSO-d6,
100 °C): δ 16.6, 30.2, 34.0, 47.2, 59.2, 65.8, 125.6, 126.6, 127.0,
127.5, 127.7, 128.2, 136.4, 137.5, 155.3, 169.2, 172.9. Anal. Calcd
for C21H24N2O5: C, 65.61; H, 6.29; N, 7.29. Found: C, 65.89; H,
6.33; N, 7.01.
SCHEME 11
General Procedure for the Preparation of Dipeptides 5a-d
and (5a + 5a′). Method A. N-(Protected-R-aminoacyl)benzotria-
zoles (1 mmol) were added at 20 °C to a solution of R-amino acid
esters (1 mmol) in CH3CN (7 mL) in the presence of Et3N (2 mmol).
The reaction mixture was then stirred at 20 °C until the starting
material was completely consumed as observed by TLC using
hexanes/ethyl acetate (2:1) as the solvent. The solution was
concentrated under reduced pressure to remove acetonitrile. The
solution was extracted with EtOAc (20 mL), and the organic extract
was washed with saturated Na2CO3 (3 × 5 mL) and saturated
NaCl (10 mL) and then dried (anhydrous MgSO4). Evaporation
of the solvent gave the desired product in pure form, which was
further recrystallized from CH2Cl2/hexanes unless otherwise speci-
fied.
TABLE 6. Preparation of Dipeptides 10a-d and Diastereomeric
Mixture (10a + 10a′) from N-(Cbz-r-aminoacyl)benzotriazoles
1c,e,f,i, (1c + 1c′) and L-Pro 2g
yield
entry
reactant
product
(%)a
1
2
Cbz-L-Phe-Bt 1c
Cbz-DL-Phe-Bt
(1c + 1c)
Cbz-L-Phe-L-Pro-OH 10ab
Cbz-DL-Phe-L-Pro-OH
(10a + 10a′)c
81
90
3
4
5
Cbz-L-Trp-Bt 1e
Cbz-Gly-Bt 1f
Cbz-L-Ala-Bt 1i
Cbz-L-Trp-L-Pro-OH 10b
Cbz-Gly-L-Pro-OH 10c
Cbz-L-Ala-L-Pro-OH 10dd
89
74
63
a Isolated yield. b HPLC result for 10a: 6.20 min. c HPLC for (10a +
10a′): 6.10 and 7.09 min. d HPLC for 10d: 7.16 min.
Method B. A mixture of N-(protected-R-aminoacyl)benzotri-
azoles (1 mmol), R-amino acid esters (1 mmol), and Et3N (2 mmol)
in THF (7 mL) was stirred in a sealed tube (10 mL) under
microwave irradiation (200 W) at 55 °C for 30 min and an
additional 30 min at 60 °C (300 W). The solution was concentrated
under reduced pressure to remove THF, and the concentrate was
extracted with EtOAc (20 mL). The organic extract was washed
with saturated Na2CO3 (3 × 5 mL) and saturated NaCl (10 mL)
and then dried (anhydrous MgSO4). Evaporation of the solvent gave
gave the desired product in pure form, which was further recrystal-
lized from CH2Cl2/hexanes unless otherwise specified.
(2S)-2-[(2-[(Benzyloxy)carbonyl]amino-2-methylpropanoyl)-
amino]-3-(1H-indol-3-yl)propanoic Acid (Cbz-Aib-L-Trp-OH,
3b). Colorless microcrystals (85%). mp 75-77 °C. [R]D23 ) +13.2
(c 2.16, DMF). 1H NMR (DMSO-d6): δ 1.30 (s, 3H), 1.32 (s, 3H),
3.13 (J ) 6.6 Hz, 2H), 4.47 (q, J ) 6.9 Hz, 1H), 4.93 (d, J ) 12.6
Hz, 1H), 5.00 (d, J ) 12.6 Hz, 1H), 6.96 (br t, J ) 7.2 Hz, 1H),
5800 J. Org. Chem., Vol. 72, No. 15, 2007

Efficient Preparation of Hindered Peptides
SCHEME 12. Peptide Coupling with N-Terminus Proline
the desired product in pure form, which was further recrystallized
from CH2Cl2/hexanes unless otherwise specified
A): δ 1.84-1.95 (m, 1.5H), 1.99-2.06 (m, 0.5H), 2.08 (s, 0.5H),
2.11 (s, 2.5H), 2.48-2.63 (m, 2H), 2.95 (s, 0.5H), 3.19 (s, 2.5H),
3.74 (s, 2.5H), 3.77 (s, 0.5H), 3.84 (d, J ) 16.8 Hz, 0.2H), 4.11 (d,
J ) 18.0 Hz, 0.8H), 4.42 (d, J ) 16.0 Hz, 0.8H), 4.46 (d, J ) 18.1
Hz, 0.2H), 4.67-4.77 (m, 0.2H), 4.88-4.98 (m, 0.8H), 5.10 (br s,
2H), 5.56 (d, J ) 8.7 Hz, 0.2H), 5.66 (d, J ) 8.7 Hz, 0.8H), 7.30-
7.38 (m, 5H). 13C NMR (CDCl3, method A, two rotameric forms):
δ 15.4, 15.6, 29.8, 31.5, 32.2, 36.6, 49.8, 53.0, 67.1, 67.2, 128.0,
128.2, 128.3, 128.5, 128.6, 136.0, 136.2, 156.1, 156.2, 172.4, 173.0,
175.7. Anal. Calcd for C17H24N2O5S: C, 55.42; H, 6.57; N, 7.60.
Found: C, 55.11; H, 6.81; N, 7.51.
Methyl 2-[((2S)-2-[(Benzyloxy)carbonyl]amino-3-phenylpro-
panoyl)amino]-2-methylpropanoate (Cbz-L-Phe-Aib-OMe, 5a).
Recrystallized from EtOAc/hexanes to give colorless microcrystal
needles (method A 41%, method B 72%). mp 105-107 °C, lit.33
mp 88-90 °C. [R]D23 ) -10.2 (c 1.05, DMF). 1H NMR (CDCl3):
δ 1.40 (s, 3H), 1.43 (s, 3H), 2.98 (dd, J ) 13.7, 8.0 Hz, 1H), 3.14
(dd, J ) 13.7, 6.0 Hz, 1H), 3.70 (s, 3H), 4.30-4.42 (m, 1H), 5.10
(s, 2H), 5.36 (d, J ) 7.1 Hz, 1H), 6.11 (s, 1H), 7.19-7.38 (m,
10H). 13C NMR (CDCl3): δ 24.4, 24.5, 38.6, 52.7, 56.2, 56.5, 67.0,
127.1, 128.0, 128.2, 128.5, 128.7, 129.5, 136.3, 136.4, 155.9, 169.7,
174.5. Anal. Calcd for C22H26N2O5: C, 66.32; H, 6.58; N, 7.03.
Found: C, 66.36; H, 6.85; N, 7.24.
General Procedure for the Preparation of Dipeptides
(8a-c). Compounds 8a-c were prepared according to the general
procedure for preparation of compounds 5a-d (method A and
method B using 4 mmol of Et3N instead of 2 mmol.
Methyl 2-[[(2S)-2-[(Benzyloxy)carbonyl]amino-4-(methylsul-
fanyl)butanoyl](methyl)amino]acetate (Cbz-L-Met-N(Me)-Gly-
OMe, 8b). Oil (method A 51%, method B 72%). lit.14 mp not
reported. [R]D23 ) -10.9 (c 1.83, DMF). 1H NMR (CDCl3, method
Acknowledgment. We thank Dr. Alexander A. Shestopalov
for help with some of the initial experimental work and Dr.
Ion Ghiviriga for his interest and useful suggestions.
Supporting Information Available: Compound characterization
data for 1a,b,g; 3a,c-e, (3c + 3c′); (5a + 5a′), 5b-d; 6a, c, (6b
+ 6b′); 8a,c; 9a-e; and 10a-d, (10a + 10a′) is available free of
charge via the Internet at http://pubs.acs.org.
JO0704255
J. Org. Chem, Vol. 72, No. 15, 2007 5801