Synthesis of Prolylproline

Article abstract of DOI:10.1134/S1070428019070169

Prolylproline has been synthesized by both classical peptide synthesis method utilizing tert-butoxycarbonyl or trifluoroacetyl protection of the NH group and carbodiimide-promoted peptide bond formation and by opening of the dioxopiperazine ring in octahydrodipyrrolo[1,2-a:1′,2′-d]pyrazine-5,10-dione obtained by thermolysis of proline methyl ester.

Full text of DOI:10.1134/S1070428019070169

ISSN 1070-4280, Russian Journal of Organic Chemistry, 2019, Vol. 55, No. 7, pp. 1005–1008. © Pleiades Publishing, Ltd., 2019.
Russian Text © The Author(s), 2019, published in Zhurnal Organicheskoi Khimii, 2019, Vol. 55, No. 7, pp. 1110–1114.
Synthesis of Prolylproline
V. A. Gaidukevicha, L. A. Popovaa, Z. P. Zubreichuka, and V. A. Knizhnikova*
a Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus
*e-mail: knizh@ifoch.bas-net.by
Received January 31, 2019; revised March 6, 2019; accepted March 15, 2019
Abstract—Prolylproline has been synthesized by both classical peptide synthesis method utilizing tert-butoxy-
carbonyl or trifluoroacetyl protection of the NH group and carbodiimide-promoted peptide bond formation and
by opening of the dioxopiperazine ring in octahydrodipyrrolo[1,2-a:1′,2′-d]pyrazine-5,10-dione obtained by
thermolysis of proline methyl ester.
Keywords: prolylproline, octahydrodipyrrolo[1,2-a:1′,2′-d]pyrazine-5,10-dione, tert-butoxycarbonyl and tri-
fluoroacetyl N-protecting groups, carbodiimide method.
DOI: 10.1134/S1070428019070169
A possible synthetic route to peptides with frag-
ments consisting of sequences of the same amino acid
residues involves the use of such fragments as inter-
mediate products. The goal of the present work was to
find an optimal method for the synthesis of L-prolyl-
L-proline (1) which can be used as starting material for
the preparation of a number of physiologically active
peptides [1–3].
L-proline with L-proline methyl ester in the presence
of N,N′-dicyclohexylcarbodiimide (DCC) gave N-Boc-
L-prolyl-L-proline methyl ester 2. The latter was also
prepared by a modified procedure [1]. Mixed anhy-
dride derived from N-Boc-L-proline and isobutyl
chloroformate (IBCF) was brought (without isolation)
into reaction with L-proline methyl ester prepared from
the corresponding hydrochloride by the action of tri-
ethylamine. Hydrolysis of the methyl ester moiety of 2
with sodium hydroxide and subsequent acidification of
the reaction mixture with citric acid afforded N-Boc-
Dipeptide 1 was obtained by the classical peptide
synthesis utilizing tert-butoxycarbonyl and trifluoro-
acetyl protecting groups. The condensation of N-Boc-
Scheme 1.
O
Me
O
N
Me
O
IBCF
Et3N
–15°C
O
HProOMe·HCl
Et3N
Boc
O
O
(1) NaOH
(2) Citric acid
HProOMe, DCC
N
N
COOH
COOMe
EtONa
COOH
N
N
N
Boc
Boc
Boc
2
3
O
O
HCl, dioxane
N
N
H
H
COOH
COOH
N
N
· HCl
4
1
1005

GAIDUKEVICH et al.
1006
Scheme 2.
(1) NaOH
(2) H+
N
HProOMe, DCC
COOH
N
N
1
COOMe
O
F3C
O
CF3
O
5
Scheme 3.
O
(1) NaOH
(2) HCl
∆
N
1
COOMe
N
N
H
O
6
were recorded on a Bruker Avance-500 spectrometer
relative to the residual proton and carbon signals of
the solvent. The optical rotations were measured on
an ATAGO AP-300 polarimeter. All operations were
carried out using anhydrous solvents. L-Proline methyl
ester was prepared by treatment with triethylamine of
the corresponding hydrochloride which was synthe-
sized as described in [4]. N-Trifluoroacetyl- and
N-Boc-prolines were synthesized according to [5, 6].
Intermediate products isolated from the reaction mix-
tures were used without further purification.
L-prolyl-L-proline 3 which was converted to L-prolyl-
L-proline hydrochloride 4 by treatment with a solution
of hydrogen chloride in dioxane. Finally, the reaction
of 4 with an equimolar amount of sodium hydroxide
yielded targeted dipeptide 1 (Scheme 1).
The number of synthetic steps can be reduced by
using N-trifluoroacetyl-L-proline methyl ester as start-
ing compound. Its condensation with L-proline methyl
ester in the presence of DCC gave intermediate N-tri-
fluoroacetyl-L-prolyl-L-proline methyl ester 5, and the
latter was deprotected in one preparative step by treat-
ment with sodium hydroxide and subsequent acidifica-
tion (Scheme 2).
L-Prolyl-L-proline (1). a. A solution of 0.69 g
(30 mmol) of sodium in 30 mL of ethanol was added to
a solution of 7.46 g (30 mmol) of L-prolyl-L-proline 4
in 30 mL of ethanol. The mixture was stirred for 2 h,
10 mL of chloroform was added, the precipitate was
filtered off, and the filtrate was evaporated under
reduced pressure. The residue was reprecipitated from
ethanol with diethyl ether. Yield 4.52 g (71%),
mp 144–145°C, [α]D20 = –138.3° (c = 1, H2O). IR spec-
The above described procedures make it possible to
obtain fairly large amounts of dipeptide 1, but they
involve consumption of large amounts of various
reagents. We have found that it is convenient to
synthesize dipeptide 1 from octahydrodipyrrolo-
[1,2-a:1′,2′-d]pyrazine-5,10-dione 6 which can be
obtained by thermolysis of L-proline methyl ester in
boiling dioxane or butan-1-ol or without a solvent at
105–110°C. Opening of the piperazine ring in 6 by the
action of aqueous sodium hydroxide, followed by
acidification, gives dipeptide 1 (Scheme 3).
1
trum, ν, cm–1: 1654, 1612 (C=O). H NMR spectrum
(D2O), δ, ppm: 1.80–2.15 m, 2.20–2.35 m, and 2.46–
2.56 m (8H, CH2); 3.32–3.50 m and 3.53–3.67 m
(4H, CH2), 4.18–4.32 m and 4.54–4.59 m (2H, CH).
13C NMR spectrum (D2O), δC, ppm: 22.15, 23.93,
24.00, 24.55, 28.12, 28.56, 29.31, 31.48, 46.51, 46.66,
47.39, 47.58, 59.03, 59.13, 61.88, 62.30, 167.24,
168.00, 178.35, 178.87. Found, %: C 56.33; H 7.44;
N 13.01. C10H16N2O3. Calculated, %: C 56.59; H 7.60;
N 13.20.
b. A solution of 4.4 g (110 mmol) of sodium
hydroxide in 50 mL of water was added to a solution
of 16.11 g (50 mmol) of N-trifluoroacetyl-L-prolyl-
L-proline 5 in 100 mL of methanol. The mixture was
stirred for 20 h, a solution of 1.14 g (10 mmol) of
trifluoroacetic acid in 10 mL of water was added
The structure of the isolated compounds was
confirmed by IR and NMR spectra and elemental
analyses. In the NMR spectra of 1–3, some signals
were doubled. Similar signal doubling was noted pre-
viously for compound 2 [1] and was rationalized by
the presence of stereoisomers.
EXPERIMENTAL
The IR spectra were recorded on a Nicolet Protégé-
460 spectrometer with Fourier transform from samples
1
prepared as KBr discs. The H and 13C NMR spectra
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 55 No. 7 2019

SYNTHESIS OF PROLYLPROLINE
1007
dropwise, and the solvent was removed under reduced
pressure. The residue was ground with 50 mL of
acetone, and the precipitate was filtered off, washed on
a filter with acetone (3×50 mL), dried under reduced
pressure, and dissolved in 75 mL of ethanol. To the
resulting solution we added 25 mL of a 2 N solution of
hydrogen chloride in ethanol, the mixture was stirred
for 1 h and filtered, and the filtrate was evaporated
under reduced pressure. The residue was reprecipitated
from ethanol with diethyl ether. Yield 7.22 g (68%).
The IR and NMR spectra of the product were identical
to those given above.
152.94, 153.68, 170.37, 170.87, 171.70, 171.94.
Found, %: C 58.97; H 8.21; N 8.32. C16H26N2O5.
Calculated, %: C 58.88; H 8.03; N 8.58.
b. A solution of 21.53 g (100 mmol) of N-Boc-
L-proline and 10.12 g (100 mmol) of triethylamine in
350 mL of methylene chloride was cooled to –16°C,
and 13.66 g (100 mmol) of isobutyl chloroformate was
added. The mixture was stirred for 15 min at that tem-
perature, 17.39 g (105 mmol) of L-proline methyl ester
hydrochloride was added, and 10.63 g (105 mmol) of
triethylamine was then added dropwise. The mixture
was stirred for 2 h at –12±2°C, allowed to slowly
warm up to room temperature, and stirred for 5 h more.
The mixture was filtered, and the filtrate was washed
in succession with 0.7 N aqueous HCl (3×50 mL),
water (2 ×50 mL), a saturated solution of sodium
hydrogen carbonate (3 ×50 mL), and water again
(3×50 mL) and dried over anhydrous sodium sulfate.
The solution was filtered, the solvent was removed
from the filtrate under reduced pressure, and the
residue was washed with cold hexane and dried under
reduced pressure until constant weight. Yield 22.85 g
(70%). The IR and NMR spectra of the product were
identical to those given above.
c. Octahydrodipyrrolo[1,2-a:1′,2′-d]pyrazine-5,10-
dione (6), 9.71 g (50 mmol), was added to a solution of
2.2 g (55 mmol) of sodium hydroxide in 20 mL of
water. The mixture was stirred for 24 h, 27.5 mL of
2 N aqueous HCl was added dropwise with vigorous
stirring, and the solvent was distilled off under reduced
pressure. The residue was extracted with ethanol, the
extract was filtered and concentrated to a volume of
20 mL. Diethyl ether, 150 mL, was added, and the
precipitate was filtered off, washed with diethyl ether,
and dried under reduced pressure until constant weight.
Yield 7.45 g (73%). The IR and NMR spectra of the
product were identical to those given above.
N-Boc-L-prolyl-L-proline (3). A solution of 2.8 g
(70 mmol) of sodium hydroxide in 50 mL of water was
added to a solution of 21.22 g (65 mmol) of N-Boc-
L-prolyl-L-proline methyl ester 2 in 200 mL of
methanol. The mixture was stirred for 20 h and diluted
with 250 mL of water, methanol was distilled off under
reduced pressure, the mixture was filtered, and the
filtrate was acidified with citric acid. The product was
extracted into methylene chloride, and the extract was
dried over sodium sulfate, filtered, and concentrated
under reduced pressure to a volume of 50 mL. Hexane,
250 mL, was added to the residue, and the precipitate
was filtered off, washed with hexane, and dried under
reduced pressure. Yield 15.43 g (76%), mp 181–
N-Boc-L-prolyl-L-proline methyl ester (2).
a. A solution of 21.53 g (100 mmol) of N-Boc-
L-proline in 100 mL of tetrahydrofuran was cooled to
0°C, a solution of 21.01 g (102 mmol) of DCC in
100 mL of THF and 17.06 g (103 mmol) of L-proline
methyl ester hydrochloride were added in succession
with stirring, and a solution of 10.43 g (103 mmol) of
triethylamine in 50 mL of THF was added dropwise
with stirring. The mixture was stirred for 24 h, the
precipitate was filtered off, the solvent was distilled off
from the filtrate under reduced pressure, and the
residue was washed with cold hexane and extracted
with diethyl ether. The extract was filtered, the solvent
was removed under reduced pressure, and the oily
residue was dried under reduced pressure until con-
stant weight. Yield 25.13 g (77 %), [α]D20 = –102.1°
(c = 2, MeOH). IR spectrum, ν, cm–1: 1747, 1699,
1662 (C=O). 1H NMR spectrum (CDCl3), δ, ppm: 1.0 s
and 1.05 s (9H, t-Bu), 1.40–1.53 m (2H, CH2), 1.53–
1.75 m (4H, CH2), 1.75–1.87 m (2H, CH2); 2.98–
3.08 m, 3.10–3.20 m, 3.20–3.29 m, and 3.36–3.43 m
(4H, CH2); 3.30 s and 3.32 s (3H, OCH3), 4.01–4.07 m
and 4.08–4.17 m (2H, CH). 13C NMR spectrum
(CDCl3), δC, ppm: 22.81, 22.33, 24.30, 24.34, 27.57,
27.73, 28.02, 28.13, 28.30, 29.20, 45.80, 45.87, 45.92,
46.15, 51.23, 51.31, 57.03, 57.06, 58.03, 78.48,
183°C; published data [7]: mp 185–187°C [7]; [α]D20
=
–131° (c = 2, H2O). IR spectrum, ν, cm–1: 1753, 1688,
1
1609 (C=O). H NMR spectrum (CDCl3), δ, ppm:
1.26 s and 1.32 s (9H, t-Bu), 1.68–1.78 m and 1.85–
2.14 m (8H, CH2); 3.25–3.38 m, 3.40–3.55 m, and
3.62–3.70 m (4H, CH2); 4.24–4.30 m, 4.38–4.43 m,
and 4.45–4.53 m (2H, CH). 13C NMR spectrum
(CDCl3), δC, ppm: 23.49, 23.99, 24.77, 28.15, 28.19,
28.35, 28.97, 29.80, 46.57, 46.71, 46.84, 57.60,
57.69, 58.97, 59.07, 79.64, 79.69, 153.68, 154.59,
172.11, 172.50, 173.67, 174.08. Found, %: C 58.00;
H 7.51; N 8.78. C15H24N2O5. Calculated, %: C 57.68;
H 7.74; N 8.97.
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 55 No. 7 2019

GAIDUKEVICH et al.
1008
L-Prolyl-L-proline hydrochloride (4). To a solu-
Octahydrodipyrrolo[1,2-a:1′,2′-d]pyrazine-5,10-
dione (6). Triethylamine, 10.12 g (100 mmol), was
added to a solution of 16.56 g (100 mmol) of L-proline
methyl ester hydrochloride in 100 mL of methanol.
The mixture was stirred for 1 h, the solvent was
removed under reduced pressure, and the residue was
extracted with diethyl ether. The extract was filtered
and evaporated, the residue was dissolved in 50 mL of
dioxane on heating, and the solution was refluxed for
20 h with vigorous stirring. After cooling, the precip-
itate was filtered off, washed twice with diethyl ether,
and dried under reduced pressure. The product was
reprecipitated from chloroform with diethyl ether.
Yield 13.21 g (68%), mp 137–140°C, [α]D20 = –102.1°
(c = 0.5, H2O). IR spectrum: ν 1656 cm–1 (C=O).
1H NMR spectrum (CDCl3), δ, ppm: 1.78–1.85 m (2H,
CH2), 1.88–1.95 m (2H, CH2), 2.02–2.10 m (2H, CH2),
2.17–2.24 m (2H, CH2), 3.38–3.45 m (4H, CH2), 4.08 t
(2H, J = 8.0 Hz, CH). 13C NMR spectrum (CDCl3), δC,
ppm: 23.24, 27.60, 45.08, 60.41, 166.28. Found, %:
C 62.03; H 7.39; N 14.19. C10H14N2O2. Calculated, %:
C 61.84; H 7.27; N 14.42.
tion of 12.48 g (40 mmol) of N-Boc-L-prolyl-L-proline
3 in 50 mL of dioxane we added 40 mL of a 6.25 N
solution of hydrogen chloride in dioxane. The mixture
was stirred for 2 h, and the product was precipitated by
adding 250 mL of diethyl ether. The precipitate was
filtered off, washed twice with diethyl ether, and dried
under reduced pressure until constant weight. Yield
8.95 g (90%), mp 164–166°C, [α]D20 = –146° (c = 2,
H2O). IR spectrum, ν, cm–1: 1754, 1665 (C=O).
1H NMR spectrum (CD3OD), δ, ppm: 1.95–2.14 m
(6H, CH2), 2.29–2.40 m and 2.52–2.62 m (1H each,
CH2), 3.34–3.42 m and 3.42–3.50 m (1H each, CH2),
3.56–3.64 m and 3.73–3.80 m (1H each, CH2), 4.51 t
(1H, J = 6.9 Hz, CH), 4.66 t (1H, J = 7.0 Hz, CH).
13C NMR spectrum (CD3OD), δC, ppm: 25.06, 25.82,
29.45, 30.03, 47.67, 48.14, 60.30, 60.52, 168.33,
174.60. Found, %: C 48.04; H 7.12; Cl 14.51; N 11.08.
C10H17ClN2O3. Calculated, %: C 48.29; H 6.89;
Cl 14.25; N 11.26.
N-Trifluoroacetyl-L-prolyl-L-proline methyl
ester (5). A solution of 21.11 g (100 mmol) of
N-trifluoroacetyl-L-proline in 100 mL of THF was
cooled to 0°C, a solution of 21.63 g (105 mmol) of
DCC in 150 mL of THF and 17.39 g (105 mmol) of
L-proline methyl ester hydrochloride were added in
succession, and a solution of 10.63 g (105 mmol) of
triethylamine in 50 mL of THF was then added drop-
wise. The mixture was stirred for 20 h and filtered, and
the solvent was removed from the filtrate under
reduced pressure. The residue was extracted with
diethyl ether, the extract was filtered and concentrated
under reduced pressure until a solid began to precip-
itate, 150 mL of hexane was added, and the precipitate
was filtered off, washed with hexane, and dried under
reduced pressure. The product was reprecipitated from
diethyl ether with hexane. Yield 23.85 g (74%),
mp 99–102°C, [α]D20 = –92.7° (c = 2, THF). IR spec-
trum, ν, cm–1: 1748, 1697, 1660 (C=O). 1H NMR spec-
trum (CDCl3), δ, ppm: 1.87–2.05 m (5H) and 2.12–
2.23 m (3H) (CH2); 3.53–3.62 m (1H), 3.66–3.73 (1H),
and 3.73–3.84 m (2H) (CH2); 3.65 s (3H, OCH3),
4.49 q (1H, J = 4.5 Hz, CH), 4.65 q (1H, J = 4.0 Hz,
CH). 13C NMR spectrum (CDCl3), δC, ppm: 24.89,
24.94, 27.71, 28.81, 46.74, 47.47, 52.19, 58.80, 59.26,
116.12 q (J = 227 Hz), 155.62 q (J = 30 Hz), 168.95,
172.53. Found, %: C 48.33; H 5.12; N 8.48.
C13H17F3N2O4. Calculated, %: C 48.45; H 5.32; N 8.69.
CONFLICT OF INTERESTS
The authors declare the absence of conflict of
interests.
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