Pyroglutamic acid as a pseudoproline moiety: A facile method for its introduction into polypeptide chains

Article abstract of DOI:10.1016/S0040-4039(01)00981-9

The acylation of benzyl (S)-pyroglutamate is reported by reaction with the pentafluorophenyl ester of protected alanine and threonine. The ¹H NMR analysis of the acylated products shows that the α hydrogens of the protected amino acids are very deshielded. This effect is due to the presence of the carbonyl of the lactam ring and shows that the peptide bond is in the trans conformation. The deprotection of the amino acids is also reported.

Full text of DOI:10.1016/S0040-4039(01)00981-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5211–5214
Pyroglutamic acid as a pseudoproline moiety: a facile method
for its introduction into polypeptide chains
Claudia Tomasini* and Marzia Villa
Dipartimento di Chimica ‘G.Ciamician’, Universita` degli Studi di Bologna, Via F. Selmi 2, 40126 Bologna, Italy
Received 6 June 2001; accepted 7 June 2001
Abstract—The acylation of benzyl (S)-pyroglutamate is reported by reaction with the pentafluorophenyl ester of protected alanine
1
and threonine. The H NMR analysis of the acylated products shows that the a hydrogens of the protected amino acids are very
deshielded. This effect is due to the presence of the carbonyl of the lactam ring and shows that the peptide bond is in the trans
conformation. The deprotection of the amino acids is also reported. © 2001 Elsevier Science Ltd. All rights reserved.
The amino acyl-proline cis/trans isomerisation is a rate-
limiting step of protein folding1 and modulates the
biological activity of peptides.2 Therefore the possibility
of controlling this isomerisation is very important in the
design of peptidomimetics. For example, it has been
demonstrated by 1H NMR analysis that proline
oligomers (n=2–4) contain nearly random distribution
of cis and trans peptide bonds in CDCl3,3 while in D2O
the dimer (n=2) exists in a 35:65 cis/trans mixture and
in the higher oligomers (n=3–5) the percentage of
peptide bonds in the trans conformation remains
approximately constant (90%).4
we have suggested that the oligomerisation affords
efficiently very rigid structures, which exist in a single
stable conformer, where the Xaai-1ꢀXaai bond are
exclusively in the trans conformation. The 1H NMR
spectra of the trimer and of the tetramer suggest that
these molecules fold in ordered structures, where the
C-4 hydrogen of a ring is always close to the carbonyl
of the next ring. This phenomenon has been confirmed
by semi-empirical calculations and can be ascribed to
the presence of a carboxy group in the pseudoproline
ring near the peptide bond.
In order to use cheap natural compounds which are
able to form imidic bonds, we have checked the possi-
bility of utilising pyroglutamic acid as a pseudoproline
moiety. Although pyroglutamic acid is available in both
configurations at low price, it is usually present in
polypeptide chains only as N-terminal amino acid,
owing to the low reactivity of the nitrogen.8 Very few
examples of acylation of the nitrogen are described,9
and some oligopeptides containing N-acylated pyroglu-
tamic acid have been isolated from the glandular secre-
tion of Litoria rubella.10
As a consequence, many proline surrogates have been
designed for the study and control of conformational
transitions in peptides and proteins.5 For instance,
many pseudoprolines (C-Pro) have been obtained by
reaction of amino acids with aldehydes and preferen-
tially assume the cis conformation.6
In a previous paper we have described the synthesis of
poly 4-carboxy 5-methyl oxazolidin-2-ones,7 whose
monomer belongs to the family of pseudoprolines, and
2,5: R =
PhtN
3,6: R =
O
O
O
NHBoc
OBn
O
OBn
:B
+
R
OC6F5
2-4
N
NH
5-7
4,7: R =
1
R
O
O
N
O
Boc
O
Scheme 1. General procedure for the acylation of benzyl (S)-pyroglutamate 1.
* Corresponding author. E-mail: tomasini@ciam.unibo.it
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00981-9

5212
C. Tomasini, M. Villa / Tetrahedron Letters 42 (2001) 5211–5214
We wish to describe here the results we have obtained
in the acylation of benzyl (S)-pyroglutamate 1 with
protected alanine and threonine, whose acidic moiety
was activated as pentafluorophenyl ester.11
results were obtained with NaH and with LiHMDS,
which turned out to be the reagent of choice.
An interesting outcome was obtained from the analysis
1
of the H NMR spectra: compounds 5, 6 and 7 show
signals between 5 and 6 ppm17 (Fig. 1), which corre-
spond to the a-hydrogens of protected alanine and
threonine and are very deshielded compared with Boc-
L
-Alanine was transformed into Pht-L-Ala-OH (Pht=
phthalimide) in quantitative yield by heating the amino
acid and phthalic anhydride in the absence of solvent,12
L
-Ala-L
-Pro-OBn (l=4.46 ppm).18
and Boc-
L-Ala-OH was purchased. On the other hand
L
-threonine was transformed into (4S,5R)-N-benzyl-
This outcome should be ascribed to the presence of the
carbonyl of the g-lactam which strongly deshields the
hydrogen. Therefore Ha must be nearby the carbonyl,
so that the peptide bond is in the trans conformation,
as we had previously shown for oligomers of 4-carboxy
oxazolidin-2-ones.7 The 1H NMR spectrum of com-
pound 5 shows the presence of two stable conformers,
both with a very deshielded quartet (5.66 and 5.85
oxycarbonyl-4-carboxy 5-methyl oxazolidin-2-one by
reaction with triphosgene and sodium hydroxide in
water/dioxane13 and subsequent protection of the nitro-
gen with (Boc)2O and DIEA/DMAP (DIEA=iPr2EtN)
in DMF. This compound can also be synthesized by
rearrangement of the corresponding N-Boc aziridine:
following this route a wide range of five-substituted
4-carboxy oxazolidin-2-ones can be obtained.14
1
ppm), which do not collapse even by recording the H
NMR spectrum at 120°C in DMSO-d6.
Then the acid moieties were transformed into the corre-
sponding pentafluorophenyl esters 2–4 by reaction with
pentafluorophenyl trifuoroacetate and pyridine in dry
DMF.15 The coupling with benzyl pyroglutamate was
performed by means of several bases and all the reac-
tions were carried out under an inert atmosphere
(Scheme 1 and Table 1).
Dipeptides 5, 6 and 7 were deprotected, respectively, by
reaction with hydrazine or with trifluoroacetic acid
As could be foreseen, the reactivity of the g-lactam ring
is very poor, compared with the oxazolidin-2-one ring,7
whose nitrogen is far more basic, owing to the back
donation of the oxygen adjacent to the carbonyl.
Thus in the reaction of benzyl pyroglutamate 1 with
Pht- -Ala-OC6F5 2, mild reaction conditions such as
L
the use of DIEA and DIEA/DMAP (entries 1 and 2) as
bases failed completely, and poor results were obtained
with DBU (entry 3). In addition, by using Cs2CO3
(entry 4) a complex mixture was recovered with no
required product. On the other hand, the formation of
an anion by reaction with NaH or LiHMDS (entries 5
and 6) afforded the desired dipeptide 5 in good to high
yield. The coupling with activated esters 3 and 4 proved
to be even harder, so that DBU afforded no results at
all (entries 7 and 10). Once again good to excellent
Figure 1. 1H NMR (the areas regarding the a hydrogens are
reported) and conformation of Boc-L-Ala-L-Pyr-OBn 6. This
conformation is in agreement both with 1H NMR analysis
and with AM1 calculations and accounts for the anomalous
downfield chemical shift of Ha hydrogen of
L
-Ala.
Table 1. Reaction conditions and chemical yields for the acylation of benzyl (S)-pyroglutamate 1
Entry
Acylating agent
Method16 Base (equiv.)
Solv.
React. time (h)
React. temp.
Yield (%)
1
2
3
4
5
6
7
8
2
2
2
2
2
2
3
3
3
4
4
4
A
A
A
A
C
B
A
C
B
A
C
B
DIEA (3)
DIEA (3), DMAP (0.25)
DBU (3)
Cs2CO3 (3)
NaH (1.2)
LiHMDS (1.2)
DBU (3)
NaH (1.2)
LiHMDS (1.2)
DBU (3)
NaH (1.2)
LiHMDS (1.2)
DMF
DMF
DMF
DMF
DMF
THF
DMF
DMF
THF
16
16
16
60
2.5
2.5
16
2.5
2.5
16
Rt
Rt
Rt
Rt
0°C–rt
0°C–rt
Rt
0°C–rt
0°C–rt
Rt
–
–
44
–
77
95
–
50
95
–
71
77
9
10
11
12
DMF
DMF
THF
2.5
2.5
0°C–rt
0°C–rt

C. Tomasini, M. Villa / Tetrahedron Letters 42 (2001) 5211–5214
5213
O
Zhang, L. S.; Tam, J. P.; Shegara, H. A. J. Am. Chem.
Soc. 1999, 121, 11558–11566; (d) Be´lec, L.; Slaninova, J.;
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3. Dever, C. M.; Bovey, F. A.; Carver, J. P.; Blout, E. R. J.
Am. Chem. Soc. 1970, 92, 6191–6198.
O
OBn
O
OBn
O
CF3COOH
CH2Cl2
N
N
H
0 °C, 24 h
6
H
8
O
O
NHBoc
NH2
4. Zhang, R.; Madalengoitia, J. S. Tetrahedron Lett. 1996,
37, 6235–6238.
O
5. (a) Andres, C. J.; Macdonald, T. L.; Ocain, T. D.;
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(i) Lenman, M. M.; Ingham, S. L.; Gani, D. J. Chem.
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G.; Madison, V. Int. J. Pept. Protein Res. 1982, 19,
543–548; (k) Magaard, V. W.; Sanchez, R. M.; Bean, J.
W.; Moore, M. L. Tetrahedron Lett. 1993, 34, 381–384;
(l) Beausoleil, E.; L’Archeveque, B.; Be´lec, L.; Atfani, M.;
Lubell, W. D. J. Org. Chem. 1996, 61, 9447–9454; (m)
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OBn
O
Boc-Ala-OC6F5
DIEA, CH2Cl2
N
O
9
70%
H
O
NH
NHBoc
O
O
CF3COOH
CH2Cl2
O
O
Ph
O
O
Ph
O
N
N
r.t., 4 h,
80%
NBoc
O
NH
O
7
10
O
O
O
Scheme 2. Deprotection and derivatization of the dipeptides 6
and 7.
(Scheme 2). The deprotection of Pht-L-Ala-L-Pyr-OBn
5 with hydrazine failed completely and (S)-benzyl
pyroglutamate 1 was obtained in quantitative yield. On
the other hand, the deprotection of 6 and 7 with
trifluoroacetic acid affords 8 and 10 in quantitative
yield, which can be further derivatized in order to
obtain polypeptide chains. So H2N-
was derivatized by reaction with Boc-
the presence of DIEA in methylene chloride and Boc-
Ala- -Ala- -Pyr-OBn 9 was obtained in good yield.
L
-Ala-
L
-Pyr-OBn 8
L
-Ala-OC6F5 2 in
L
-
L
L
6. (a) Wittelsberger, A.; Keller, M.; Scarpellino, L.; Patiny,
L.; Acha-Orbea, H.; Mutter, M. Angew. Chem., Int. Ed.
2000, 39, 1111–1115; (b) Mutter, M.; Haack, T. Tetra-
hedron Lett. 1992, 33, 1589–1592; (c) Tam, J. P.; Miao, Z.
J. Am. Chem. Soc. 1999, 121, 9013–9022.
In conclusion, we have shown that pyroglutamic acid
can be easily introduced in a polypeptide chain and
forces the newly formed peptide bond into the trans
conformation.
7. Lucarini, S.; Tomasini, C. J. Org. Chem. 2001, 66, 727–
732.
Acknowledgements
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This work was supported in part by MURST Cofin
2000 and 60% (Roma) and by the University of
Bologna (funds for Selected Research Topics).
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11. All new compounds have been fully characterized.
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1993, 23, 2839–2844.
14. Tomasini, C.; Vecchione, A. Org. Lett. 1999, 1, 2153–
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15. Green, M.; Berman, J. Tetrahedron Lett. 1990, 31, 5851–
5854.
Water (10 mL) was added and the mixture was concen-
trated (rotary evaporator) to remove THF, the aqueous
layer was extracted with ethyl acetate (3×10 mL). The
combined organic layers were dried over sodium sulfate
and the solvent was removed under reduced pressure. The
residue was purified by silica gel chromatography (cyclo-
hexane/ethyl acetate, 8:2 as eluant).
16. Typical experimental procedure: Method A: The penta-
fluorophenyl ester 2, 3 or 4 (0.6 mmol) was added to a
stirred solution of benzyl (S)-pyroglutamate 1 (0.5 mmol,
110 mg) and the base reported in Table 1 in dry DMF
(1.5 mL) under a nitrogen atmosphere at 0°C. The mix-
ture was stirred at room temperature for a variable
period of time (see Table 1), then ethyl acetate (10 mL)
was added, the mixture was washed with 1 M aqueous
HCl (2×7 mL) and with an aqueous saturated solution of
NaHCO3 (1×10 mL). The combined organic layers were
dried over sodium sulfate and the solvent was removed
under reduced pressure. The residue was purified by silica
gel chromatography (cyclohexane/ethyl acetate, 8:2 as
eluant). Method B: LiHMDS (2.4 mmol, 1 M sol. in
THF, 2.4 mL) was added to a stirred solution of benzyl
(S)-pyroglutamate 1 (2.0 mmol, 438 mg) in dry THF (7
mL) under a nitrogen atmosphere at 0°C. The mixture
was stirred for 20 min at 0°C and 40 min at room
temperature. A solution of pentafluorophenyl ester 2, 3
or 4 (2.4 mmol) in dry THF (4 mL) was added dropwise
at 0°C. The mixture was stirred for 20 min at 0°C and 3
h at room temperature. Water (10 mL) was added and
the mixture was concentrated (rotary evaporator) to
remove THF, the aqueous layer was extracted with ethyl
acetate (3×10 mL), the combined organic layers were
dried over sodium sulfate and the solvent was removed
under reduced pressure. The residue was purified by silica
gel chromatography (cyclohexane/ethyl acetate, 8:2 as
eluant). Method C: NaH (0.6 mmol, 14 mg) was added to
a stirred solution of benzyl (S)-pyroglutamate 1 (0.5
mmol, 110 mg) in dry THF (7 mL) under a nitrogen
atmosphere at 0°C. The mixture was stirred for 20 min at
0°C and 20 min at room temperature. A solution of
pentafluorophenyl ester 2, 3 or 4 (0.6 mmol) in dry THF
(2 mL) was added dropwise at 0°C. The mixture was
stirred for 20 min at 0°C and 3 h at room temperature.
17. (5): low melting solid—[h]D −80 (DCM, c 1.00).—IR
(Nujol): w=1752, 1713 cm−1.—1H NMR (CDCl3) (mix-
ture of conformers): l=1.73 (d, 3H, J=7.4 Hz, CH3) and
1.86 (d, 3H, J=7.2 Hz, CH3), 2.00–2.19 (m, 1H, CH2),
2.22–2.82 (m, 3H, CH2), 4.79 (dd, 1H, J=1.8, 9.0 Hz,
CHN) and 4.83 (dd, 1H, J=3.0, 9.2 Hz, CHN), 5.15
(AB, 2H, J=12.0 Hz, CH2Ph) and 5.22 (AB, 2H, J=12.2
Hz, CH2Ph), 5.76 (q, 1H, J=7.2 Hz, CHN) and 5.95 (q,
1H, J=7.4 Hz, CHN), 7.30–7.38 (m, 5H, Ph), 7.65–7.91
(m, 4H, Pht).—13C NMR (CDCl3) (mixture of conform-
ers): l=14.7 and 15.1, 21.6 and 21.7, 31.5, 50.2 and 50.4,
58.1 and 58.4, 67.2 and 67.4, 123.2, 128.0, 128.1, 128.2,
128.4, 128.5, 131.6, 131.7, 133.8 and 133.9, 134.8, 167.4
and 167.5, 170.0, 170.3 and 170.4, 173.6 and 173.7. (6):
low melting solid—[h]D −54 (DCM, c 1.05).—IR (Nujol):
w=3397, 1739, 1700 cm−1.—1H NMR (CDCl3): l=1.30
(d, 3H, J=6.9 Hz, CH3), 1.41 (s, 9H, t-Bu), 2.01–2.18 (m,
1H, CH2), 2.30–2.45 (m, 1H, CH2), 2.52–2.75 (m, 2H,
CH2), 4.85 (dd, 1H, J=3.3, 9.6 Hz, CHN), 5.07 (d, 1H,
J=6.9 Hz, NH), 5.16 (AB, 2H, J=12.3 Hz, CH2Ph), 5.35
(dq, 1H, J=6.9 Hz, CHN), 7.32–7.40 (m, 5H, Ph).—13C
NMR (CDCl3): l=17.6, 21.3, 28.2, 31.7, 49.7, 57.7, 67.4,
79.8, 128.2, 128.5, 134.8, 155.1, 170.4, 173.7, 174.5. (7):
low melting solid—[h]D −116 (DCM,
c 1.00).—IR
(Nujol): w=1825, 1752, 1726, 1700 cm−1.—1H NMR
(CDCl3): l=1.52 (s, 9H, t-Bu), 1.57 (d, 3H, J=6.6 Hz,
CH3), 2.08–2.22 (m, 1H, CH2), 2.32–2.78 (m, 3H, CH2),
4.43 (dq, 1H, J=1.8, 6.6 Hz, CHO), 4.90 (dd, 1H, J=3.0,
9.6 Hz, CHN), 5.19 (AB, 2H, J=12.1 Hz, CH2Ph), 5.46
(d, 1H, J=1.8 Hz, CHN), 7.30–7.42 (m, 5H, Ph).—13C
NMR (CDCl3): l=21.0, 22.0, 28.1, 31.5, 57.8, 62.7, 68.1,
72.7, 84.5, 128.5, 128.9, 134.7, 151.0, 168.6, 170.2, 175.2.
18. Paulsen, H.; Adermann, K. Liebigs Ann. Chem. 1989,
751–770.
.