261Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
DOI: 10.17344/acsi.2021.6673
Scientific paper
The Synthesis of (2R)-Aziridine-2-carboxylic  
Acid Containing Dipeptides
Samo Kuzmič, Martina Hrast and Rok Frlan*
University of Ljubljana, Faculty of Pharmacy, The Chair of Pharmaceutical Chemistry,  
Aškerčeva 7, 1000 Ljubljana, Slovenia
* Corresponding author: E-mail: rok.frlan@ffa.uni-lj.si;  
Tel.: +386 1 4769 674; Fax: +386-1-4258031
Received: 03-05-2021
Abstract
Optimized conditions for the synthesis of fully deprotected (2R)-aziridine containing dipeptides are described. Prepa-
ration of fully protected N- and C- terminal aziridine containing dipeptides was found to be straightforward and high 
yielding for the majority of compounds, whereas their full deprotection was possible only for C-terminal analogs. De-
protection of N-terminal derivatives using standard procedures of peptide chemistry was found difficult providing only 
mixtures of unidentifiable products. The described molecules have potential as building blocks in synthetic chemistry, in 
the chemical biology arena, as covalent modifiers, and as biomarkers.
Keywords: Dipeptides; aziridines; biomarkers; warheads; antibacterial agents
1. Introduction
Small molecules that mimic the structures of bioac-
tive peptides are an important synthetic tool in organic 
chemistry. A special interest has been given to the synthe-
sis of conformationally restricted β-turn dipeptide mimet-
ics, peptide bond isosteres and nonproteinogenic deriva-
tives to obtain drug-like target molecules.1–3 A common 
strategy, that has been also widely applied, especially in the 
design of several protease inhibitors is to incorporate an 
electrophilic warhead that forms a covalent bond between 
amino acids in the active site and the inhibitor.4,5
The importance of aziridine-2-carboxylic acid, as 
well as aziridine-containing peptides as useful interme-
diates in the synthesis of various amino acid and peptide 
derivatives, has been extensively recognized, both from a 
medicinal and a synthetic point of view.6–10 Further-
more, despite their reactivity aziridine-containing pep-
tides and related compounds may be found in numerous 
bioactive compounds of natural origin, such as madura-
statin A111 and miraziridine A12 (Figure 1). In addition, 
this heterocyclic fragment has also been incorporated in 
numerous synthetic peptides as reactive electrophilic 
warheads to obtain promising irreversible inhibitors of 
different proteases, such as cathepsins,13,14 papain,15 as-
partate proteases,16 HIV proteases17 and SARS-CoV 
main protease.18
Their unusual reactivity due to ring strain renders 
aziridine-2-carboxylic acid derivatives important for the 
preparation of amino acids,19–21 amino alcohols,22,23 pep-
tides and other peptide-like compounds.17,24–33 In addi-
tion, they have also been used for labelling biomolecules 
with fluorine-18.34,35
Consequently, the preparation of aziridine-containing 
dipeptides, which is a subject of this communication, is of 
great importance for medicinal as well as organic chemistry. 
There have been many reports in the literature describing 
their synthesis.36,37 Nonetheless, up to now, literature re-
ports on the preparation of partially or fully deprotected di-
peptide derivatives have been rare. To the best of our knowl-
edge, there has been only one report by Korn36 describing 
the synthesis of fully deprotected H-Azy-Leu-OH and only 
a couple of papers describing the synthesis of N-deprotect-
ed21,27,38,39 and C-deprotected dipeptides.34,36 Most of them 
suffer from low isolated yields following work-up and chro-
matography which reflects the inherent instability of these 
compounds. At the same time, it is known that aziri-
dine-containing peptides possess the potential antifun-
gal,16,40 antiviral18 antiprotozoal13,14 and cytostatic activi-
ty.13–15,41 Clearly, a more versatile approach to deprotected 
aziridine-containing dipeptides is desired, given their po-
tential to serve as electrophiles in reactions with appropriate 
nucleophiles, as biomarkers and bioactive compounds.
262 Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
During our ongoing study concerning the dipeptide 
D-Ala-D-Ala, we were interested in preparing its electro-
philic aziridine-containing analogs with potential biologi-
cal activity. D-Ala-D-Ala sequence is found in the stem 
termini of peptidoglycan side-chain pentapeptide and is 
recognized by multiple essential bacterial enzymes. Any 
molecule that could mimic its structure thus offers an at-
tractive potential toward the development of new inhibi-
tors or as a false substrate for any of those enzymes.42 In-
deed, it was reported that dipeptides with D-Ala as a first 
amino acid inhibit bacterial enzyme D-Alanine:D-Alanine 
ligase (Ddl) in micromolar range (1, Figure 1).43 (R)-aziri-
dine-2-carboxylic acid was therefore chosen as a rigid sur-
rogate of D-Ala because of its electrophilic character and 
close resemblance to D-Ala.
Herein, we report details of our efforts to develop a 
new approach to the preparation of a series of fully depro-
tected N-terminal aziridine containing dipeptides with 
general structure 2. In addition, an attempt on the synthe-
sis of C- terminal aziridine containing dipeptides with 
general structure 3 is also reported (Figure 2).
The designed molecules are very attractive not only 
because of their biological potential but also because aziri-
dines have become important building blocks in synthetic 
chemistry. Such small dipeptides have a potential as covalent 
modifiers and are therefore useful in the chemical biology 
arena with different applications including bioconjugation, 
activity-based protein profiling and target identification.6,8
2. Experimental
2. 1. General Methods
The reactions were monitored by TLC carried out on 
Merck silica gel (60 F254) by using UV light as a visualizing 
agent, KMnO4 in water, phosphomolybdic acid in ethanol 
and ninhydrin in ethanol and heat as developing agents. 
Column chromatography was performed using Merck Sili-
ca Gel 60. Proton nuclear magnetic resonance spectra (1H 
NMR) were obtained at 400 MHz on Bruker Avance III 400 
spectrometers. Spectra were recorded in CDCl3, MeOD and 
DMSO-d6 solutions. Chemical shifts are reported in ppm, 
referenced to tetramethylsilane (TMS) as the external refer-
ence. Carbon-13 nuclear magnetic resonance spectra (13C 
NMR) were obtained at 100 MHz on Bruker 400 spectrom-
eter. Chemical shifts are reported in ppm, referenced to the 
Figure 1. Natural products with aziridine ring and D-Ala:D-Ala ligase (Ddl) inhibitor 1.
Figure 2. N- and C-terminal aziridine containing dipeptides.
263Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
solvent peak of CDCl3. Low-resolution mass spectra were 
obtained with a Shimadzu GC-MS-QP2010 mass spec-
trometer. High resolution mass spectra (HRMS) were re-
corded on Q Executive Plus LC-MS/MS system (Thermo 
Scientific). Melting points were found on the Cambridge 
instruments melting point apparatus and are corrected. Op-
tical rotation was found with Perkin Elmer 1241MC po-
larimeter at wavelength 589,3 nm (l = 10 cm). Ethyl acetate 
and methanol were used as solvents. Infrared spectra were 
recorded on a Perkin-Elmer FTIR 1600 spectrometer. Melt-
ing points were determined using a Reichert hot-stage mi-
croscope and are corrected. HPLC analyses were performed 
on an HPLC Dionex UltiMate 3000 instrument with a UV-
VIS detector. Three different analytical methods were used. 
Method 1: Column: Phenomenex Luna® 5 µm C18 100 Å; 
Injection volume: 10 μL; Flow rate: 1,5 mL/min; Detection 
wavelengths: λ = 210 nm, 220 nm, 254 nm and 280 nm; 
Column temperature: 25 °C; Mobile phase: 40% ACN in 
water to 100% ACN in 15 min. Method 2: Column: Supelco 
SUPELCOSIL™ LC-1 HPLC; Injection volume: 1 μL; Flow 
rate: 1 mL/min; Detection Wavelengths: λ = 195 nm, 210 
nm, 220 nm and 254 nm; Column temperature: 25 °C; Mo-
bile phase: 5% ACN in 20 mM phosphate buffer (pH = 2.10) 
to 60% ACN in 20 min. Method 3: Column: Agilent ZOR-
BAX Extend-C18; Injection volume: 5 μL; Flow rate: 1 mL/
min; Detection wavelengths λ = 210 nm, 220 nm, 254 nm 
and 280 nm; Column temperature25 °C; Mobile phase: 40% 
ACN in water to 100% ACN in 20 min.
2. 2. Synthesis and Characterization
(R)-3-hydroxy-1-methoxy-1-oxopropan-2-aminium 
chloride (4).
To a solution of D-serine (20.00 g, 190.3 mmol, 1.00 
equiv.) in 500 mL MeOH, SOCl2 (24.90 mL, 342.6 mmol, 
1.80 equiv.) was added at 0 °C. The reaction mixture was 
stirred at 80 °C for 2 h and stirred at room temperature for 
an additional 20 h. The solution was concentrated under 
reduced pressure and diethyl ether (250 mL) was added to 
remove the excess of HCl. The solvent was evaporated un-
der reduced pressure yielding 28.95 g (97.8%) of com-
pound 2 as white crystals. mp 178.1–179.0 °C (lit. 175-
176 °C44). Rf 0.11 (CH2Cl2:MeOH = 7:1 + 3% Et3N).
Methyl trityl-D-serinate (5)
To a cooled solution of 4 (29.27 g, 188.1 mmol, 1.00 
equiv.) and Et3N (55.10 mL, 396.1 mmol, 2.11 equiv.) in 
CH2Cl2 (500 mL), trityl chloride (52.45 g, 188.1 mmol, 
1.00 equiv.) was added and the mixture was stirred for 2 
hours at 0 °C. The reaction mixture was washed with 10% 
citric acid solution (3 × 150 mL) and brine (150 mL). The 
organic phase was dried over anhydrous Na2SO4, the dry-
ing agent was filtered off and the solvent was evaporated 
under reduced pressure. The product was purified via flash 
column chromatography (hexane:EtOAc = 2:1) to obtain 
55.70 g (81.9%) of 5 as white solid. mp 158.8-161.5 °C (lit. 
152–154 °C45). Rf: 0.27 (hexane:EtOAc = 2:1). 1H NMR 
(400 MHz, CDCl3): δ 2.29 (dd, J 6.2 Hz, 6.0 Hz, 1H, OH), 
2.97 (bs, 1H, NH), 3.30 (s, 3H, CH3), 3.50‒3.61 (m, 2H, 
CH2 and CH), 3.65‒3.75 (m, 1H, CH2), 7.16‒7.32 (m, 9H, 
CPh3), 7.43‒7.51 (m, 6H, CPh3) ppm.
Methyl (R)-1-tritylaziridine-2-carboxylate (6)
To a cooled solution of 5 (5.01 g, 13.9 mmol, 1.00 
equiv.) and Et3N (4.25 mL, 30.5 mmol, 2.20 equiv.) in anhy-
drous THF (40 mL), methanesulfonyl chloride (1.08 mL, 
14.0 mmol, 1.01 equiv.) was added dropwise. Reaction mix-
ture was stirred for 30 minutes at room temperature. The 
temperature was then raised to 65 °C (reflux temperature) 
and reaction mixture was refluxed for 48 hours. The solvent 
was evaporated under reduced pressure, the solid residue 
was dissolved in EtOAc (40 mL) and washed with 10% cit-
ric acid solution (3 × 10 mL), saturated NaHCO3 solution 
(3 × 10 mL) and brine (1 × 10 mL). The organic phase was 
dried over anhydrous Na2SO4, the drying agent was filtered 
off and the solvent was evaporated under reduced pressure. 
The product was purified via flash column chromatography 
(hexane:EtOAc = 4:1) to yield 3.42 g (71.9%) 6 as white 
crystals. mp 122.0–123.0 °C (lit. 122-123 °C46). Rf: 0.36 
(Hexane:EtOAc = 4:1). 1H NMR (400 MHz, CDCl3): δ1.41 
(dd, J 6.3 Hz, 1.7 Hz, 1H, CH2), 1.89 (dd, J 6.3 Hz, 2.7 Hz, 
1H, CH), 2.26 (dd, J 2.7 Hz, 1.7 Hz, 1H, CH2), 3.76 (s, 3H, 
CH3), 7.19‒7.31 (m, 9H, CPh3), 7.47‒7.52 (m, 6H, CPh3) 
ppm. IR (ATR): ῡ 3705, 3467, 2973, 1742, 1596, 1489, 1445, 
1394, 1328, 1234, 1181, 1011, 893, 843, 756, 697 cm–1.
Potassium (R)-1-tritylaziridine-2-carboxylate (7, C22H18 
KNO2)
To a cooled solution of 6 (5.78 g, 16.8 mmol, 1.00 
equiv.) in THF (17 mL), 1M solution of KOH was added 
(16.84 mL, 16.84 mmol, 1.00 equiv.). Reaction mixture was 
stirred at room temperature for 24 hours. The solvent was 
removed under reduces pressure to obtain 6.15 g (99.4%) 
of 7 as lightly yellow solid. Reaction product was stable in 
salt form, but unstable in acid form. Yield: 99.4. mp Ther-
mal Tdec > 250.0 °C. Rf: 0.30 (hexane:EtOAc = 1:1 + 0.3% 
CH3COOH). 1H NMR (400 MHz, CDCl3): δ 1.80‒1.88 (m, 
2H, CH2), 3.71‒3.78 (m, 1H, CH), 7.18‒7.33 (m, 15H, 
CPh3) ppm. 13C NMR (100 MHz, CDCl3): δ 28.82, 34.95, 
74.28, 126.67‒128.36 (signals overlap), 129.51, 144.32, 
178.15 ppm. MS m/z (relative intensity): 327.7 ([M-H]–, 
100%). HRMS-ESI: [M-H]– calcd for C22H18NO2, 
328.1343. found, 328.1341. IR (ATR): ῡ 3366, 3060, 2972, 
2167, 1960, 1580, 1488, 1423, 1325, 1217, 1155, 1057, 1013, 
900, 847, 750, 699 cm–1. [α]D20 +184.0 (c 0.31, EtOAc).
2. 3.  General Procedure for Coupling reactions 
(Procedure A, Compounds 8a-8e)
To a cooled solution of compound 7 (1.00 equiv.) 
and protected D-amino acid (1.00–1.10 equiv.) in 50 mL 
dichloromethane, HOBt (1.10 equiv.), NMM (3.00 equiv.) 
264 Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
and EDC (1.10 equiv.) were added at 0 °C. The reaction 
mixture was stirred at room temperature for 24 h. The 
mixture was washed with 10% citric acid solution (3 × 50 
mL), saturated NaHCO3 solution (3 × 50 mL) and brine 
(1 × 50 mL). The organic phase was dried over anhydrous 
Na2SO4, the drying agent was filtered off and the solvent 
was evaporated under reduced pressure.
Methyl ((R)-1-tritylaziridine-2-carbonyl)-D-alaninate (8a)
The compound was synthesized according to the 
general procedure A. The product was purified via flash 
column chromatography. Initial mobile phase was CH2 
Cl2:hexane = 2:1, which was gradually replaced by hex-
ane:EtOAc = 1:1. Yield: 1.198 g (53.1%) of 8a as white crys-
tals. Mp 161.0–165.2 °C. Rf: 0.48 (hexane:EtOAc = 1:1). 1H 
NMR (400 MHz, CDCl3): δ 1.44‒1.51 (m, 4H, CHCH3 and 
aziridine CH2), 1.98‒2.02 (m, 2H, aziridine CH and aziri-
dine CH2), 3.81 (s, 3H, OCH3), 4.69 (qd, J 7.5 Hz, 7.2 Hz, 
1H, NHCHCH3), 7.20‒7.34 (m, 10H, CPh3 and CONH), 
7.41‒7.48 (m, 6H, CPh3) ppm. 13C NMR (100 MHz, 
CDCl3): δ 18.79, 30.12, 34.17, 47.61, 52.79, 74.82, 127.34, 
128.00, 129.60, 143.45, 170.74, 173.46 ppm. MS (ESI+): 
m/z = 436.85 ([M+Na]+, 100%). HRMS-ESI (m/z): 
[M+H]+ calcd for C26H27N2O3, 415,2016. found, 415.2017. 
IR (ATR): ῡ 3705, 3293, 2972, 2869, 1746, 1645, 1532, 
1491, 1448, 1359, 1276, 1206, 1165, 1056, 1009, 956, 905, 
857, 771, 747, 703 cm–1. HPLC: Method 1: tr: 6.25 min 
(95.1% at 220 nm). [α]D20 +132.1 (c 0.33, EtOAc).
Methyl (R)-3-(1-tritylaziridine-2-carboxamido)propa-
noate (8b, C26H26N2O3)
The compound was synthesized according to the 
general procedure A. The product was purified via flash 
column chromatography (hexane:EtOAc = 2:1), which was 
gradually replaced by hexane:EtOAc = 1:1. Yield: 1.24 g 
(62.9%) of 8b as white crystals. mp 65.0-67.0 °C. Rf: 0.32 
(hexane:EtOAc = 1:1). 1H NMR (400 MHz, CDCl3): δ 1.45 
(dd, J 6.6 Hz, 0.7 Hz, 1H, aziridine CH2), 1.96 (dd, J 2.7 Hz, 
0.7 Hz, 1H, aziridine CH2), 1.99 (dd, J 6.6 Hz, 2.7 Hz, 1H, 
aziridine CH), 2.57‒2.71 (m, 2H, CH2COO), 3.44‒3.55 
(m, 1H, NHCH2), 3.65‒3.79 (m, 4H, NHCH2 and OCH3), 
7.20‒7.30 (m, 9H, CPh3), 7.33 (dd, J 6.2 Hz, 6.2 Hz, 1H, 
CONH), 7.38‒7.44 (m, 6H, CPh3) ppm. 13C NMR (100 
MHz, CDCl3): δ 29.94, 34.15, 34.29, 34.61, 52.10, 74.73, 
127.30, 127.91, 129.48, 143.47, 171.12, 172.99 ppm. MS 
m/z (relative intensity): 436.7 ([M+Na]+, 100%). m/z = 
412.5 ([M-H]–, 100%). HRMS-ESI (m/z): [M+H]+ calcd 
for C26H27N2O3, 415.2016. found, 415.2014. IR (ATR): ῡ 
3706, 3333, 2949, 1733, 1663, 1522, 1442, 1365, 1256, 1180, 
1059, 1009, 901, 858, 749, 702 cm–1. HPLC: Method 1: tr: 
5.69 min (99.4% at 220 nm). [α]D20 +109.4 (c 0.33, EtOAc).
Methyl ((R)-1-tritylaziridine-2-carbonyl)-D-phenylala-
ninate (8c)
The compound was synthesized according to the 
general procedure A. The product was purified via flash 
column chromatography (hexane:EtOAc = 2:1), yielding 
1.480 g (62.8%) of 8c as white crystals. mp 120.5–122.0 °C. 
Rf: 0.23 (hexane:EtOAc = 2:1). 1H NMR (400 MHz, 
CDCl3): δ 1.37 (dd, J 6.7 Hz, 0.7 Hz, 1H, aziridine CH2), 
1.69 (dd, J 2.7 Hz, 0.7 Hz, 1H, aziridine CH2), 1.95 (dd, J 
6.7 Hz, 2.7 Hz, 1H, aziridine CH), 3.18 (dd, J 13.9 Hz, 6.2 
Hz, 1H, CH2Ph), 3.21 (dd, J 13.9 Hz, 6.0 Hz, 1H, CH2Ph), 
3.80 (s, 3H, OCH3), 4.93 (ddd, J 8.7 Hz, 6.2 Hz, 6.0 Hz, 1H, 
CH2Ph), 7.15‒7.39 (m, 21H, CPh3, Ph and CONH) ppm. 
13C NMR (100 MHz, CDCl3): δ 30.04, 33.99, 38.06, 52.08, 
52.66, 74.64, 127.25, 127.50, 127.90 (signals overlap), 
128.91, 129.46, 135.98, 143.35, 170.54, 171.99 ppm. MS 
m/z (relative intensity): 488.6 ([M-H]–, 100%). HRMS-ESI 
(m/z): [M+H]+ calcd for C32H31N2O3, 491.2329. found, 
491.2326. IR (ATR): ῡ 3709, 3360, 3281, 2974, 2037, 1743, 
1679, 1596, 1501, 1446, 1356, 1277, 1197, 1164, 1129, 1057, 
1009, 903, 858, 808, 748, 703 cm–1. HPLC: Method 1: tr: 
7.69 min (99.5% at 220 nm). [α]D20 +74.9 (c 0.31, EtOAc).
Methyl ((R)-1-tritylaziridine-2-carbonyl)-D-valinate (8d)
The compound was synthesized according to the 
general procedure B. The product was purified via flash 
column chromatography (hexane:EtOAc = 4:1), yielding 
1.17 g (64.7%) of 8f as white crystals. mp 111.0–113.0 °C. 
Rf: 0.34 (hexane:EtOAc = 3:1). 1H NMR (400 MHz, 
CDCl3): δ0.97 (d, J 6.8 Hz, 3H, CH(CH3)2), 0.98 (d, J 6.8 
Hz, 3H, CH(CH3)2), 1.51 (dd, J 6.2 Hz, 1.0 Hz, 1H, aziri-
dine CH2), 1.99‒2.03 (m, 2H, aziridine CH2 and aziridine 
CH), 2.25 (qqd, J 6.8 Hz, 6.8 Hz, 5.0 Hz, 1H, CH(CH3)2), 
3.81 (s, 3H, COOCH3), 4.59 (dd, J 9.3 Hz, 5.0 Hz, 1H, CH-
COO), 7.21‒7.32 (m, 9H, CPh3), 7.34 (d, J 9.3 Hz, 1H, 
CONH), 7.42‒7.47 (m, 6H, CPh3) ppm. 13C NMR (100 
MHz, CDCl3): δ 17.95, 19.39, 30.33, 31.50, 34.28, 52.45, 
56.53, 74.77, 127.32, 127.99, 129.54, 143.44, 170.93, 172.39 
ppm. MS m/z (relative intensity):440.6 ([M-H]–, 100%). 
HRMS-ESI (m/z): [M+H]+ calcd for C29H33N2O3, 
457.2486. found, 457.2486. IR (ATR): ῡ 3273, 2965, 1742, 
1649, 1554, 1490, 1444, 1314, 1264, 1210, 1150, 1011, 902, 
749, 701, 634 cm–1. HPLC: Method 1: tr 7.32 min (100.0% 
at 220 nm). [α]D20 +145.0 (c 0.32, EtOAc).
Dimethyl ((R)-1-tritylaziridine-2-carbonyl)-D-glutamate 
(8e)
The compound was synthesized according to the 
general procedure A. The product was purified via flash 
column chromatography (hexane:EtOAc = 2:1), yielding 
0.555 g (23.9%) of 8e as white crystals. mp 151.5–152.5 °C. 
Rf: 0.23 (hexane:EtOAc = 2:1). 1H NMR (400 MHz, 
CDCl3): δ 1.49 (dd, J 6.0 Hz, 1.5 Hz, 1H, aziridine CH2), 
1.96‒2.00 (m, 2H, aziridine CH2 and aziridine CH), 
2.05‒2.16 (m, 1H, CH2COO), 2.23‒2.34 (m, 1H, CH-
2COO), 2.36‒2.54 (m, 2H, CH2), 3.67 (s, 3H, OCH3), 3.82 
(s, 3H, OCH3), 4.68 (ddd, J 8.3 Hz, 8.3 Hz, 5.1 Hz, 1H, 
CHCOO), 7.21‒7.32 (m, 9H, CPh3), 7.44‒7.50 (m, 7H, 
CPh3 and CONH) ppm. 13C NMR (100 MHz, CDCl3): δ 
27.30, 30.05, 30.37, 34.13, 51.44, 52.13, 52.82, 74.84, 
265Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
127.29, 127.95, 129.59, 143.44, 171.33, 172.23, 173.59 ppm. 
MS m/z (relative intensity):508.7 ([M+Na]+, 80%). m/z = 
484.5 ([M-H]–, 70%). HRMS-ESI (m/z): [M+H]+ calcd for 
C29H31N2O5, 487.2227, found, 487.2228. IR (ATR): ῡ 3696, 
3362, 3059, 2956, 2068, 1977, 1738, 1670, 1596, 1509, 1441, 
1373, 1344, 1312, 1205, 1181, 1129, 1098, 1065, 1013, 904, 
869, 820, 768, 747, 705 cm–1. HPLC: Method 1: tr: 6.33 min 
(99.9% at 220 nm). [α]D20 +119.4 (c 0.32, EtOAc).
2. 4.  General Procedure for Removal of 
Trityl Protection Group (Procedure B, 
Compounds 9a-9e)
To a cooled solution of the starting compound (8a-
8e, 1.00 equiv.) and triethylsilane (1.75 equiv.) in 30 mL 
dichloromethane, trifluoroacetic acid (3.50 equiv.) was 
added drop-wise at 0 °C and stirred for another 30 min on 
ice bath. After the completion of the reaction (monitored 
by TLC), the solvent was evaporated under reduced pres-
sure and the solid residue was washed with 25 mL diethyl 
ether and water (25 mL). NaHCO3 was added to the aque-
ous phase to give a solution with a pH = 10. To further re-
duce solubility of the product, NaCl was added and the 
aqueous phase was washed with ethyl acetate (6 × 25 mL). 
The collected organic phases were washed with brine 
(1×50 mL) and dried over anhydrous Na2SO4. The drying 
agent was filtered off and the solvent was evaporated under 
reduced pressure.
Methyl ((R)-aziridine-2-carbonyl)-D-alaninate (9a, 
C7H12 N2O3)
The compound was synthesized according to the 
general procedure B. Yield: 0.140 mg (52.7%) as white 
crystals. mp 99.5-102.3 °C. Rf: 0.45 (CH2Cl2 +:MeOH = 9:1 
+ 3% Et3N). 1H NMR (400 MHz, MeOD): δ 1.39 (d, I = 7.3 
Hz, 3H, CHCH3), 1.79‒1.86 (m, 2H, aziridine CH2), 2.55 
(dd, J 5.7 Hz, 3.2 Hz, 1H, aziridine CH), 3.70 (s, 3H, 
OCH3), 4.44 (q, J 7.3 Hz, 1H, CHCH3) ppm. 13C NMR 
(100 MHz, MeOD): δ 17.65, 26.28, 30.42, 52.95, 172.73, 
174.55 ppm, one signal covered by solvent. MS m/z (rela-
tive intensity):171,3 ([M-H]–, 50%). HRMS-ESI (m/z): 
[M+H]+ calcd for C7H12N2O3, 172.0848. found, 173.0920. 
IR (ATR): ῡ 3701, 3286, 3196, 2973, 1732, 1665, 1560, 
1451, 1405, 1374, 1344, 1211, 1137, 1058, 1011, 932, 826, 
713 cm–1. HPLC: Method 2: tr: 4.29 min (94.6% at 195 
nm). [α]D20 +32.9 (c 0.33, MeOH).
Methyl (R)-3-(aziridine-2-carboxamido)propanoate (9b)
The compound was synthesized according to the 
general procedure B. The product was purified via flash 
column chromatography using Al2O3 (CH2Cl2:MeOH = 
20:1), yielding 0.173 g (43.6%) of 9b as white crystals. mp 
93.8-95.8 °C. Rf: 0.26 (CH2Cl2:MeOH = 9:1 + 3% Et3N). 
1H NMR (400 MHz, CDCl3): δ 1.70‒2.00 (m, 2H, aziridine 
CHAHB), 2.46 (bs, 1H, aziridine CH), 2.56 (t, J 6.1 Hz, 2H, 
CH2COO), 3.49‒3.60 (m, 2H, NHCH2CH2), 3.71 (s, 3H, 
OCH3), 7.09 (bs, 1H, CONH) ppm. 13C NMR (100 MHz, 
CDCl3): δ 26.33, 30.34, 33.80, 34.98, 51.87, 171.11, 
172.77  ppm. HRMS-ESI (m/z): [M+H]+ calcd for C7H-
12N2O3, 172.0848. found, 173.0924. IR (ATR): ῡ 3704, 
3272, 3212, 2958, 2841, 1723, 1657, 1577, 1440, 1407, 1371, 
1295, 1266, 1227, 1195, 1172, 1112, 1056, 1017, 978, 909, 
884, 841, 740 cm–1. HPLC: Method 2. tr: 4.23 min (97.6% 
at 210 nm). [α]D20 +32,9 (c 0,33, MeOH).
Methyl ((R)-aziridine-2-carbonyl)-D-phenylalaninate 
(9c, C13H16N2O3)
The compound was synthesized according to the 
general procedure B. The product was purified via flash 
column chromatography (CH2Cl2:MeOH = 20:1), yielding 
0.450 g (67.9%) of 9c as white crystals. mp 95.7–98.2 °C. 
Rf: 0.24 (CH2Cl2:MeOH = 20:1). 1H  NMR (400  MHz, 
MeOD): δ 1.82 (bs, 2H, airidine CH2), 2.53 (dd, J 5.5 Hz, 
3.2 Hz, 1H, aziridine CH), 3.01 (dd, J 13.8 Hz, 8.8 Hz, 1H, 
CH2Ph), 3.20 (dd, J 13.8 Hz, 5.6 Hz, 1H, CH2Ph), 3.71 (s, 
3H, OCH3), 4.73 (dd, J 8.8 Hz, 5.6 Hz, 1H, CHCH2Ph), 
7.19‒7.34 (m, 5H, Ph) ppm. 13C NMR (100 MHz, MeOD): 
δ 26.18, 30.24, 38.45, 52.77, 55.31, 127.98, 129.54, 130.23, 
137.98, 172.72, 173.17  ppm. MS m/z (relative intensity): 
271,24 ([M+Na]+, 100%). HRMS-ESI (m/z): [M+H]+ cal-
cd for C13H16N2O3, 248.1161. found, 249.1230. IR (ATR): 
ῡ = 3703, 3208, 2945, 1736, 1661, 1543, 1446, 1401, 1362, 
1219, 1164, 1110, 1055, 1012, 954, 914, 827, 699  cm–1. 
HPLC: Method 2: tr: 14.84 min (99.3% at 210 nm). [α]D20 
+18.7 (c 0.31, MeOH).
Methyl ((R)-aziridine-2-carbonyl)-D-valinate (9d)
The compound was synthesized according to the 
general procedure C, yielding 0.365 mg (75.5%) of 9d. mp 
93.5-94.8 °C. Rf: 0.42 (CH2Cl2:MeOH = 9:1 + 3% Et3N). 
1H  NMR (400  MHz, MeOD): δ 0.96 (d, J 6.9  Hz, 3H, 
CH(CH3)2), 0.96 (d, J 6.9  Hz, 3H, CH(CH3)2), 1.85 (bs, 
2H, aziridine CH2), 2.16 (qqd, J 6.9 Hz, 6.9 Hz, 5.9 Hz, 1H, 
CH(CH3)2, 2.66 (dd, J 5.4 Hz, 3.1 Hz, 1H, aziridine CH), 
3.72 (s, 3H, COOCH3), 4.37 (d, J 5.9  Hz, 1H, CH-
COOCH3)  ppm. 13C  NMR (100  MHz, MeOD): δ 18.53, 
19.57, 26.37, 30.33, 32.00, 52.67, 59.53, 173.17, 173.44 ppm. 
HRMS-ESI (m/z): [M-H]– calcd for C9H16N2O3, 200.1161. 
found, 199.1081. IR (ATR): ῡ 3704, 3278, 3202, 2967, 1731, 
1666, 1558, 1463, 1439, 1391, 1346, 1318, 1285, 1235, 1203, 
1160, 1069, 1005, 940, 891, 840, 822, 722  cm–1. HPLC: 
Method 2. tr: 6.98 min (95.0% at 195 nm). [α]D20 +114.7 (c 
= 0.34, MeOH).
Dimethyl ((R)-aziridine-2-carbonyl)-D-glutamate (9e, 
C10H16N2O5)
The compound was synthesized according to the 
general procedure B. The product was purified via flash 
column chromatography using Al2O3 (CH2Cl2:MeOH = 
50:1), which was gradually replaced by CH2Cl2:MeOH = 
20:1, yielding 0.124 g (48.9%) of 9d as colourless viscous 
oil. Rf: 0.38 (CH2Cl2:MeOH = 9:1). 1H NMR (400 MHz, 
266 Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
CDCl3): δ 1.84 (bs, 1H, aziridine CH2), 1.88‒2.07 (m, 2H, 
aziridine CH2 and CH2COO), 2.16‒2.28 (m, 1H, CH2 
COO), 2.31‒2.48 (m, 2H, CH2), 2.54 (bs, 1H, aziridine 
CH), 3.69 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 4.58‒4.68 
(m, 1H, CHCO), 7.16 (d, J 8.0  Hz, 1H, CONH)  ppm. 
13C NMR (100 MHz, CDCl3): δ 26.73, 27.35, 30.13, 30.38, 
51.64, 52.00, 52.69, 171.27, 172.13, 173.25  ppm. HRMS-
ESI (m/z): [M+H]+ calcd for C10H16N2O5, 244.1059. 
found, 245.1131. IR (ATR): ῡ 3704, 3668, 2970, 2868, 1736, 
1657, 1535, 1437, 1341, 1215, 1170, 1056, 1011, 833 cm–1. 
HPLC: Method 2: tr: 5.66 min (96.6% at 210 nm). [α]D20 
+54.0 (c 0.25, MeOH).
2. 5.  General Procedure for Removal of 
Methyl protection group (procedure C, 
compounds 2a-2e)
To a cooled solution of 8a-8e (1.00 equiv.) in MeOH 
(20 mL), 0.1 M LiOH solution (5.00 equiv.) was added 
drop-wise at 0 °C. After the completion of the reaction 
(monitored by TLC), the pH of the reaction mixture was 
adjusted to pH=7 with a 0.1 M HCl solution and the sol-
vent was evaporated under reduced pressure.
((R)-aziridine-2-carbonyl)-D-alanine (2a)
The compound was synthesized according to the 
general procedure C. The product was purified via flash 
column chromatography (EtOAc:MeOH:H2O = 4:2:1), 
yielding 0.080 g (98%) of 2a as lightly yellow crystals. mp 
thermal Tdec > 250 °C. Rf: 0.26 (EtOAc:MeOH:H2O = 
4:2:1). 1H NMR (400 MHz, MeOD): δ 1.36 (d, J 7.1 Hz, 3H, 
CHCH3), 1.78‒1.87 (m, 2H, aziridine CH2), 2.58 (dd, J 
5.8 Hz, 3.2 Hz, 1H, aziridine CH), 4.24 (q, J 7.1 Hz, 1H, 
CHCH3)  ppm. 13C  NMR (100  MHz, MeOD): δ 19.48, 
26.17, 30.85, 52.26, 171.82, 179.66 ppm. HRMS-ESI (m/z): 
[M+H]+ calcd for C6H10N2O3:158.0691. found, 159.0764. 
IR (ATR): ῡ 3659, 3324, 3100, 2970, 2872, 1643, 1606, 
1562, 1457, 1405, 1365, 1319, 1269, 1236, 1167, 1104, 1054, 
1017, 981, 942, 919, 868, 837, 756, 685 cm–1. HPLC: Meth-
od 2. tr: 3.46 min (95.1% at 195 nm). [α]D20 +66.7 (c 0.33, 
MeOH).
(R)-3-(aziridine-2-carboxamido)propanoic acid (2b, 
C6H10N2O3)
The compound was synthesized according to the 
general procedure C. The product was purified via flash 
column chromatography (EtOAc:MeOH:H2O = 4:2:1), 
yielding 0.096 g (65%) 2b as white crystals. mp thermal 
Tdec > 250 °C. Rf: 0.24 (EtOAc:MeOH:H2O = 4:2:1). 
1H NMR (400 MHz, D2O): δ 1.83 (d, J 3.1 Hz, 1H, aziri-
dine CH2), 1.87 (d, J 6.0 Hz, 1H, aziridine CH2), 2.36 (t, J 
6.9  Hz, 2H, CH2COO), 2.56 (dd, J 6.0  Hz, 3.1  Hz, 1H, 
aziridine CH), 3.39 (t, J 6.9  Hz, 2H, NHCH2)  ppm. 
13C NMR (100 MHz, D2O): δ 24.82, 29.59, 33.32, 53.91, 
172.50, 180.15 ppm. HRMS-ESI (m/z): [M+H]+ calcd for 
C6H10N2O3, 158.0691. found, 159.0764. IR (ATR): ῡ 3702, 
3659, 3258, 3086, 2975, 2871, 1646, 1556, 1404, 1312, 1259, 
1162, 1126, 1061, 1016, 908, 832, 622 cm–1. [α]D20 +143.6 
(c 0.30, MeOH).
((R)-aziridine-2-carbonyl)-D-phenylalanine (2c)
The compound was synthesized according to the 
general procedure C. The product was purified via flash 
column chromatography (EtOAc:MeOH:H2O = 4:1:1), 
yielding 0.163 g (71.4%) of 2c as white crystals. mp ther-
mal Tdec > 180 °C. Rf: 0.37 (EtOAc:MeOH:H2O = 4:2:1). 
1H  NMR (400  MHz, D2O): δ 1.75 (bs, 2H, aziridine 
CHAHB), 2.52 (bs, 1H, aziridine CH), 2.91 (dd, J 14.0 Hz, 
8.8  Hz, 1H, CH2Ph), 3.18 (dd, J 14.0  Hz, 4.9  Hz, 1H, 
CH2Ph), 4.45 (dd, J 8.8 Hz, 4.9 Hz, 1H, CHCOO), 7.21‒7.36 
(m, 5H, Ph) ppm. 13C NMR (100 MHz, MeOH): δ 25.05, 
29.48, 38.63, 54.38, 126.89, 128.61, 129.30, 137.59, 172.08, 
178.00 ppm. HRMS-ESI: [M-H]- calc for C12H14N2O3, 
234.1004. found, 233.0928. IR (ATR): ῡ 3371, 2976, 2117, 
2005, 1595, 1418, 1273, 1162, 1106, 1056, 923, 696 cm–1. 
HPLC: Method 2. tr: 7.22 min (86.5% at 210 nm). [α]D20 
+25.7 (c 0.30, MeOH).
((R)-aziridine-2-carbonyl)-D-valine (2d, C9H16N2O3)
The compound was synthesized according to the gen-
eral procedure C. The product was purified via flash column 
chromatography (EtOAc:MeOH:H2O = 4:2:1), yielding 
0.109 g (63.7%) of 2e. mp thermal Tdec > 220 °C. Rf: 0.31 
(EtOAc:MeOH:H2O = 4:2:1). 1H NMR (400 MHz, D2O): δ 
0.87 (d, J 7.0  Hz, 3H, CH(CH3)2), 0.90 (d, J 7.0  Hz, 3H, 
CH(CH3)2), 1.84 (d, J 3.4 Hz, 1H, aziridine CH2), 1.90 (d, J 
5.9  Hz, 1H, aziridine CH2), 2.10 (qqd, J 7.0  Hz, 7.0  Hz, 
5.6 Hz, 1H, CH(CH3)2, 2.68 (dd, J 5.9 Hz, 3.4 Hz, 1H, aziri-
dine CH), 4.06 (d, J 5.6 Hz, 1H, CHCOO) ppm. 13C NMR 
(100 MHz, MeOD): δ18.36, 20.33, 26.30, 30.89, 32.67, 61.76, 
172.45, 178.40  ppm. HRMS-ESI: [M-H]– calc for 
C8H13N2O3, 185.0932. found, 185.0933. IR (ATR): ): ῡ 3707, 
3666, 3288, 2970, 2871, 1644, 1590, 1546, 1424, 1235, 1162, 
1057, 1011, 939, 915, 835, 752, 669 cm–1. HPLC: Method 2. 
tr: 4.11 min (85.4% at 210 nm). [α]D20 +52.6 (c 0.33, MeOH).
Benzyl aziridine-2-carboxylate (10)
To a cooled solution of 7 (15.01 g, 40.84 mmol, 1.00 
equiv.) in acetonitrile (400 mL) benzyl bromide (4.85 mL, 
40.8 mmol, 1equiv.) was added. After stirring at room tem-
perature for 3 h, solvent was evaporated under reduced 
pressure. The oily residue was dissolved in CH2Cl2 (200 
mL), and washed with water (200 mL), and brine (150 
mL). The organic phase was dried over anhydrous Na2SO4, 
the drying agent was filtered off and the solvent was evap-
orated under reduced pressure to obtain 14.34 g (83.4%) 
viscous oil, which was used for further reaction without 
additional purification. The trityl group was removed fol-
lowing the standard procedure B, yielding 0.697 g (83.9%) 
of 10 as colourless oil. Rf: 0.32 (hexane:EtOAc = 1:2 + 3% 
Et3N). 1H NMR (400 MHz, CDCl3): δ 1.89 (dd, J 5.5 Hz, 
1.4 Hz, 1H, CH2), 2.04 (dd, J 2.9 Hz, 1.4 Hz, 1H, CH2), 2.58 
267Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
(dd, J 5.5  Hz, 2.9  Hz, 1H, CH), 5.18 (d, J 12.3  Hz, 1H, 
CH2Ph), 5.22 (d, J 12.3 Hz, 1H, CH2Ph), 7.32‒7.42 (m, 5H, 
Ph) ppm. IR (ATR): ῡ 3285, 3229, 3065, 3037, 1725, 1564, 
1456, 1402, 1360, 1187, 1113, 1007, 970, 871, 825, 744, 
697 cm–1. 1H NMR spectra was found to be identical to the 
ones described in ref.19
Benzyl (R)-1-((tert-butoxycarbonyl)-D-alanyl)aziridine 
-2- carboxylate (11)
The compound was synthesized according to the 
general procedure A. The product was purified via flash 
column chromatography (hexane:EtOAc = 3:1), yielding 
0.471 g (25.7%) of 11 as viscous oil. Rf: 0.23 (MF: hex-
ane:EtOAc = 3:1). 1H NMR (400 MHz, CDCl3): δ 1.42 (s, 
9H, C(CH3)3), 1.45 (d, J 7.1 Hz, 3H, CHCH3), 2.63 (dd, J 
3.1  Hz, 1.9  Hz, 1H, aziridine CH2), 2.72 (dd, J 5.8  Hz, 
1.9 Hz, 1H, aziridine CH2), 3.28 (dd, J 5.8 Hz, 3.1 Hz, 1H, 
aziridine CH), 4.30 (qd, J 7.1  Hz, 7.1  Hz, 1H, CHCH3), 
5.01 (d, J 7.1  Hz, 1H, CONH), 5.20 (s, 2H, CH2Ph), 
7.32‒7.42 (m, 5H, Ph) ppm. 13C NMR (100 MHz, MeOD): 
δ18.98, 28.42, 30.96, 34.45, 51.30, 67.71, 80.02, 128.65, 
128.73, 128.78 (overlapping of signals), 135.06, 155.33, 
168.31, 184.23 ppm. HRMS-ESI (m/z): [M+H]+ calcd for 
C18H24N2O5, 348.1685. found, 349.1753. IR (ATR): ῡ 3354, 
3177, 3036, 1972, 2935, 2878, 1681, 1497, 1452, 1367, 1324, 
1273, 1248, 1168, 1063, 1018, 944, 913, 854, 750, 699 cm–1. 
HPLC: Method 3. tr: 6.64 min (95.1% at 220 nm). [α]D20 
+84,4 (c = 0,27, EtOAc).
Benzyl (R)-1-(((benzyloxy)carbonyl)-D-phenylalanyl)
aziridine-2-carboxylate (13)
The compound was synthesized according to the 
general procedure B. The product was purified via flash 
column chromatography (hexane:EtOAc = 3:1), yielding 
1.67 g (92.4%) 11 as viscous oil. Rf: 0.62 (hexane:EtOAc = 
1:1). 1H  NMR (400  MHz, CDCl3): δ 2.57‒2.63 (m, 2H, 
aziridine CH2), 3.05‒3.15 (m, 2H, CH2Ph and aziridine 
CH), 3.25 (dd, J 14.0 Hz, 5.8 Hz, 1H, CH2Ph), 4.55‒4.63 
(m, 1H, CH), 5.0 (d, J 12.3 Hz, 1H, OCH2Ph), 5.06 (d, J 
12.3 Hz, 1H, OCH2Ph), 5.17 (s, 2H, OCH2Ph), 5.23 (d, J 
8.1 Hz, 1H, OCONH), 7.12‒7.42 (m, 15H, 3 × Ph) ppm. 
13C NMR (100 MHz, MeOD): δ 31.68, 35.63, 38.98, 58.87, 
67.65, 68.65, 127.87, 128.75, 129.04, 129.57, 129.61, 129,76 
(overlapping of 3 signals), 130.52, 136.95, 138.32, 138.70, 
158.37, 169.74, 183.98  ppm. MS m/z (relative intensity): 
480.6 ([M+Na]+, 100%). HRMS-ESI: [M-H]+ calcd for 
C27H26N2O5, 458.1842. found, 457.1773. IR (ATR): ῡ 3328, 
3062, 3033, 2952, 1699, 1505, 1452, 1375, 1248, 1191, 1077, 
1047, 1027, 910, 747, 696  cm–1. HPLC: Method 3. tr: 
9.24 min (70.5% at 210 nm). [α]D20 +45,6 (c = 0.43, EtOAc).
3. Results and Discussion
There are two common strategies concerning the 
synthesis of aziridine containing peptides. The first strate-
gy starts from a partially protected serine dipeptides fol-
lowed by cyclization. This approach is reportedly less effi-
cient because several various by-products are formed in 
the cyclization step.27,28,37,39,47 The second more efficient 
strategy, which starts from the aziridine-2-carboxylic acid, 
was therefore used.34,39,48
The synthesis of N-terminal aziridine containing di-
peptides 2a-e was straightforward and high yielding. It 
started with the introduction of a trityl protective group 
onto D-Ser-OMe (4)49 in 82% yield followed by cyclization 
using methanesulfonyl chloride50,51 to obtain the aziridine 
6 in 72% yield. Next, the screening of different bases 
(NaOH, LiOH, KOH) and solvents (THF, CH3CN and 
EtOH) for the subsequent saponification was performed. 
1M KOH in THF was found to be the most optimal in 
terms of yield (99%) and purity of the final carboxylate 7. 
Our attempts to neutralize 7 with diluted HCl or acetic 
acid were not successful because of its decomposition 
upon neutralization. Potassium salt was therefore used in 
the coupling reactions between (R)-aziridine-2-carboxyl-
ate 7 and different amino acids to obtain the aziridine con-
taining dipeptide products 8a-e in 24–65% yield.39 Acido-
lytic treatment of 8 following the literature reported 
procedures37,39 using CF3COOH in CH2Cl2/MeOH or 
CH2Cl2 generated a mixture of degradation products and 
was therefore unsatisfactory for the synthesis of larger 
amounts of final products. The analysis of reaction mix-
tures by NMR confirmed that the products were present in 
very small quantities, which we were unable to purify. Dif-
ficulties in removing trityl as well as Boc protective group 
from aziridine peptides using CF3COOH, formic acid or 
HCl have been previously reported.7,36 Instead, optimiza-
tion of reaction conditions following different reaction 
procedures demonstrated that the addition of Et3SiH to 
the reaction mixture was essential for the successful reac-
tion.52 Hence, reductive deprotection of N-tritylaziridines 
8 and basification with triethylamine prior to isolation was 
successfully applied to obtain deprotected aziridines 9a-e 
in 31–98% yield. Finally, basic hydrolysis with LiOH in a 
mixture of MeOH and H2O yielded compounds 2a–d in 
31–98% yield. A reaction with Glu derivative 9e yielded 2e 
as confirmed by MS and NMR analysis. However, a prod-
uct was unstable on silica or Al2O3 and purification was 
therefore not possible. The majority of compounds were 
stable for up to 3 weeks if stored under argon and in the 
fridge. However, after prolonged storage, significant de-
composition was observed, which is in agreement with 
literature data. It is not uncommon that many aziridine 
derivatives exhibit sequence-dependent instability in both 
reaction and purification steps, as well as on storage. This 
is caused by self-protonation to generate a reactive aziri-
dinium species that subsequently decompose (Figure 
3).31,53,54
The synthesis of compounds with general structure 3 
was started from benzyl protected (R)-aziridine-2-carbox-
ylic acid 10 which was then coupled with Boc-D-Ala and 
268 Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
Cbz-D-Phe to obtain tert-butoxycarbonyl and benzyloxy-
carbonyl protected dipeptides 11 and 13 in 26% and 32% 
yields, respectively. N-acylated aziridine derivatives have 
major issues with stability due to acylation and subsequent 
lower electronic density of the aziridine ring.25 It was 
therefore expected that the removal of a protective group 
could potentially yield unstable compounds if free NH2 
group was present. Boc and Cbz protective groups were 
therefore chosen because they are easy to remove to hypo-
thetical products 12 and 14 that have NH2 groups in the 
form of a HCl salt or as a zwitterion, respectively. However, 
the deprotection of Boc to obtain 12 using CF3COOH or 
HCl in anhydrous THF or diethyl ether did not provide us 
with the final product and only a mixture of unidentifiable 
compounds could be isolated. NMR analysis indicated 
that the opening of the aziridine ring could be the cause. 
Interestingly, NMR analysis also indicated that the open-
ing of the aziridine ring is favoured compared to the re-
moval of Boc protective group at concentrations of HCl 
below 0.1 M. Deprotection under neutral conditions using 
catalytic hydrogenation was next applied to remove both 
benzyl protective groups from 13 and to yield 14 in ˝zwit-
terionic˝ form. However, a mixture of unidentifiable prod-
ucts was obtained, again (Figure 4).
Figure 3. Synthesis of aziridine containing dipeptides.
Figure 4. Study on the preparation of 12 and 14.
269Acta Chim. Slov. 2022, 69, 261–270
Kuzmič et al.:   The Synthesis of (2R)-Aziridine-2-carboxylic   ...
4. Conclusions
In conclusion, fully deprotected aziridine-containing 
dipeptides can be easily synthesized from trityl protected 
aziridine-carboxylic acid. In most cases, the deprotection of 
the trityl group from the aziridine ring and final hydrolysis 
proceeds smoothly in moderate to high yields. However, 
significant decomposition of deprotected dipeptides was 
observed after 3 weeks even if stored under argon in the 
fridge. The synthesis of derivatives with aziridine-2-carbox-
ylic acid in place of a second amino acid in the dipeptide 
sequence was not possible due to the instability of the acy-
lated aziridine ring in the presence of a free amino group.
Acknowledgement 
This research was funded by Slovenian Research 
Agency (ARRS, research core funding no. P1-566 0208).
Author Contribution
All authors contributed to the study conception and 
design. Material preparation, data collection and analysis 
were performed by Samo Kuzmič and Rok Frlan. The first 
draft of the manuscript was written by Rok Frlan and all 
authors commented on previous versions of the manu-
script. All authors read and approved the final manuscript. 
The authors declare no competing financial interest.
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Povzetek
V prispevku so opisani optimizirani pogoji za sintezo dipeptidov, ki vsebujejo popolnoma odščiten (2R)-aziridin. Prip-
rava popolnoma zaščitenih N- in C- terminalnih dipeptidov, ki vsebujejo aziridin, je enostavna in poteka z visokim 
izkoristkom za večino spojin, medtem ko je njihova popolna odščita možna le za C-terminalne analoge. Odstranjevanje 
zaščite z N-terminalnih derivatov z uporabo standardnih postopkov peptidne kemije se je izkazalo kot težko, saj vodi do 
mešanice nedoločljivih produktov. Opisane molekule imajo velik potencial kot gradniki v sintezni kemiji, na področju 
kemijske biologije, kot kovalentni modifikatorji in kot biomarkerji.
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