Synthesis of dipeptides from N-hydroxy-3-azaspiro[5,5]undecane-2,4-dione activated α-amino acids

Article abstract of DOI:10.1016/j.tetasy.2010.12.021

A simple two step procedure for the synthesis of a dipeptide from N-hydroxy-3-azaspiro[5,5]undecane-2,4-dione (HO-ASUD) activated α-amino acids is described. In presence of DCC, N-hydroxy-3-azaspiro[5,5]undecane-2,4- dione readily esterifies the carboxylic acid group of all the N-protected amino acids to yield crystalline N-hydroxy-3-aza spiro[5,5]undecane-2,4-dione activated carboxy ester. The N-hydroxy-3-aza spiro[5,5]undecane-2,4-dione activated carboxy esters of N-protected amino acids readily condensed with other amino acids and gave a dipeptide. This new method is effective for the DCC coupling of a variety of chiral amino acids without loss of enantiomeric purity. Synthesis of fifteen dipeptides including the hitherto unreported Fmoc-l-Orn(Boc)-Val-OMe, Fmoc-l-Cys(trt)-Gly-OEt and Boc-l-Tyr-Gly-OEt is presented.

Full text of DOI:10.1016/j.tetasy.2010.12.021

Tetrahedron: Asymmetry 22 (2011) 22–25
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Synthesis of dipeptides from N-hydroxy-3-azaspiro[5,5]undecane-2,4-dione
activated a-amino acids
⇑
Shaik Nowshuddin, A. Ram Reddy
Department of Chemistry, University College of Science, Osmania University Campus, Osmania University, Hyderabad, Andhra Pradesh 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple two step procedure for the synthesis of a dipeptide from N-hydroxy-3-azaspiro[5,5]undecane-
Received 27 October 2010
Accepted 23 December 2010
Available online 2 February 2011
2,4-dione (HO-ASUD) activated
a-amino acids is described. In presence of DCC, N-hydroxy-3-azaspi-
ro[5,5]undecane-2,4-dione readily esterifies the carboxylic acid group of all the N-protected amino acids
to yield crystalline N-hydroxy-3-aza spiro[5,5]undecane-2,4-dione activated carboxy ester. The
N-hydroxy-3-aza spiro[5,5]undecane-2,4-dione activated carboxy esters of N-protected amino acids
readily condensed with other amino acids and gave a dipeptide. This new method is effective for the
DCC coupling of a variety of chiral amino acids without loss of enantiomeric purity. Synthesis of fifteen
dipeptides including the hitherto unreported Fmoc-
Boc- -Tyr-Gly-OEt is presented.
L
-Orn(Boc)-Val-OMe, Fmoc-
L-Cys(trt)-Gly-OEt and
L
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
1.1 equiv of DCC, its activated ester, Boc-alanine–ASUD, is
obtained. Similarly conversion of Fmoc, Boc and Z-protected amino
acids to their respective ASUD esters was carried out by stirring
equimolar amounts of the protected amino acids and HO-ASUD
overnight at room temperature in the presence of DCC in THF.
Similarly the hydroxyl group of HO-ASUD can also readily be
esterified with N- and side-chain-protected amino acids. Therefore,
formation of a protected amino acid-ASUD ester using DCC occurs
with all of the amino acids. During the course of the reaction, the
reaction mixture developed a white suspension due to the forma-
tion of insoluble dicyclohexylurea. The activated amino acid spiro
ester is stable and readily crystallizes, during which process the
unreacted HO-ASUD was removed. The formation of activated
amino acid-ASUD ester can be readily monitored by TLC as well
as by the appearance of a new band around 1745 cmꢀ1 in the infra-
red spectrum, due to the spiro ASUD ester.
Peptide synthesis is one of the most important and widely stud-
ied chemical transformations in organic synthesis. Suitability of
the protecting and activating groups1–5 is one of the major deter-
rent problems that necessitate modification of the prevailing pep-
tide methodologies.
There are numerous reports on the use of activated spiro esters
in peptide synthesis.6–11 The N-hydroxy compounds used to
activate the N-protected amino acids were NOSU (N-hydroxy-
succinimide)12–14 1-hydroxybenzotriazole15 (HOBT) and N-hydroxy-
phthalimide (HOPh).16 Recently, it was observed that HOBT is not
safe to use under ambient conditions. In the present investigation,
we report a facile two step synthesis of fifteen dipeptides including
the hitherto unreported Fmoc-
Cys(trt)-Gly-OEt and Boc- -Tyr-Gly-OEt involving N-hydroxy-3-
L-Orn(Boc)-Val-OMe, Fmoc-L-
L
azaspiro[5,5]undecane-2,4-dione (OH-ASUD) activated amino acid
esters as intermediates and DCC as a coupling reagent in THF.
It was found that esterification of N-protected L-amino acids
with HO-ASUD can be carried out not only in THF but also in other
organic solvents such as acetone, dichloromethane, chloroform,
toluene and acetonitrile, but not in n-hexane and water.
2. Results and discussion
When the activated amino acid-ASUD ester is charged and stir-
red overnight with equimolar amounts of another a-amino acid in
The HO-ASUD is a stable reagent. It was prepared starting from
cyclohexanone following Stevens et al.17 A typical procedure for
the synthesis of activated Boc-alanine–ASUD ester is shown in
Scheme 1. When equimolar amounts of Boc-alanine and HO-ASUD
are stirred overnight in THF at room temperature in the presence of
water–THF (1:1 v/v) and 2.5–3.0 equiv of Na2CO3 at room temper-
ature, an N-protected dipeptide was obtained. Similarly an N-pro-
tected dipeptide methyl ester was prepared using 2.5 equiv of
triethylamine in place of Na2CO3 in THF at room temperature along
the lines of Scheme 1. The overall yield of dipeptides obtained is
very high ranging between 69% and 86% as shown in Table 1. No
premature deblocking of the N-protecting and other side chain
groups occurred either during esterification or peptide bond
⇑
Corresponding author.
E-mail address: aramreddy2008@yahoo.com (A. Ram Reddy).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.12.021

S. Nowshuddin, A. Ram Reddy / Tetrahedron: Asymmetry 22 (2011) 22–25
23
O
CH3
HO
N
O
Boc
N
H
COOH
+
DCC (1.1 equiv.), THF
95%
Boc-L-Alanine
(1 equiv.)
HO-ASUD
(1.0 equiv.)
O
N
O
CH3
O
Alanine (1.0 equiv.)
Boc
N
H
Alanine methyl ester (1.0 equiv.)
THF, TEA (2.5 equiv.)
H2O-THF
O
Na2CO3 (3.0 equiv)
activated amino acid-ASUD ester
74%
80%
CH3
CH3
CH3
CON
CH3
OH
OMe
Boc
N
H
CON
H
Boc
N
H
H
O
O
Boc-L-Ala-Ala-OH
Boc-Ala-Ala-OMe
Scheme 1. Synthesis of Boc-Ala-Ala-OH and Boc-Ala-Ala-OMe involving N-hydroxy-3-azaspiro[5,5]undecane-2,4-dione activated amino acid ester intermediate.
Table 1
Dipeptides prepared involving the activated amino acid-ASUD ester intermediate
S. No.
Product
Obtained yield
Reported yield
Obtained ORDb
Reported ORD c 1 ½a D20
ꢁ
1
2
3
4
5
6
7
8
Z-Gly-Gly-OEt
Fmoc-Gly-Val-OMe
76
88
80.5
80
74
71
83
69
78
86
72
84
78.6
76.4
75
6020
—
+18.2
—
8921
+18.6 (CHCl3)
ꢀ13.4 (DMF)
ꢀ51.5 (MeOH)
ꢀ43.1 (MeOH)
ꢀ4.2 (EtOH)
ꢀ40.1 (DMF)
ꢀ18.2 (1 N NaOH)
—
ꢀ13.1 (MeOH)
ꢀ121.5 (MeOH)
—
ꢀ8.5 (MeOH)
ꢀ23.9 (EtOH)
—
Z-
L
-Pro-Val-OH
7822
ꢀ12.6
Boc-
Boc-
Boc-
L
L
L
-Ala-Ala-OMe
-Ala-Ala-OH
-Phe-Gly-OEt
66.723a
5723a
7820
ꢀ50.8
ꢀ42.6
ꢀ4.7
Z-
Z-
Fmoc-
L
-Trp-Ala-OH
-Gly-Leu-OH
7723b
ꢀ38.5
L
5620
ꢀ18.3
9
L
-Orn(Boc)-Val-OMea
New
8313
ꢀ29.2 (MeOH)c
ꢀ12.9
10
11
12
13
14
15
Boc-
Boc-
L
-Lys(Z)-Gly-OMe
-Pro-Pro-OH
L
7423c
New
6923d
7123e
New
ꢀ123.2
+34.7 (MeOH)d
ꢀ8.3
Fmoc-
Z- -Glu(OMe)-Dihydroxy-Tyr-OMe
Boc-Gly-Pro-OH
Boc-
-Tyr-Gly-OEta
L
-Cys(trt)-Gly-OEta
L
ꢀ22.4
L
+3.3(MeOH)e
a
b
c
New peptides fully characterized by 1H NMR, 13C NMR, IR and Mass spectra.
Observed values are obtained under the identical conditions of the reported literature conditions. ORD = ½a D20
ꢁ
(c 10 mg/ml, CH3OH).
Chiral HPLC: Chiral PACK-AS-H; n-Hexane/IPA/TFA (50:50:0.1%); %ee: 98.29.
Chiral HPLC: Chiral PACK-AS-H; n-Hexane/IPA/TFA (80:20:0.1%); %ee: 99.78.
Chiral HPLC: Chiral PACK-AD-H; n-Heptane/IPA/TFA (90:10:0.1%); %ee: 99.96.
d
e
formation. The co-product HO-ASUD was washed off with 5%
Na2CO3 solution. A control experiment was performed using the
N-protected amino acid-N-hydroxy succinimide method18 for TLC
analysis and enantiomeric purity. The purity of the N-protected
dipeptides was monitored by HPLC19 and compared with that of
the respective peptides obtained by the N-OSU method. The struc-
ture of all the dipeptides prepared was characterized by micro
analysis, IR, NMR and Mass spectra and compared with the re-
ported literature. The IR spectrum correlated more than 95% with
standard samples. All of the dipeptides prepared by the present
method and NOSU method are listed in Table 1. Entries 9, 12 and
15 in the table are new dipeptides, which are hitherto unreported.
Their structure is established by mass, 1H NMR, 13C NMR and infra
red spectra. The 13C NMR has peaks at 169, 165 and 156 ppm,
respectively for the newly formed peptide carbonyl carbon, respec-
nals at 8.3, 9.07 and 9.04 ppm, respectively representing the newly
formed peptide proton. Similarly the pseudo molecular ions ob-
tained at 689 (M++H2O) and 365 (Mꢀ1) in the ES Mass confirms
the structure of 12 and 15. In the case of entry 9, no molecular
ion peak in the mass spectrum was obtained. However the frag-
ment obtained at m/z 512 is the ion obtained after the loss of an
OMe and the side chain of valine.
The present method of dipeptide synthesis has the following
merits. Preparation of HO-ASUD is simple. It has shelf stability at
room temperature and is not hygroscopic like N-OSU. It is highly
soluble in most of the organic solvents employed in peptide
synthesis. It retains all the advantage of DCC coupled reactions.
HO-ASUD is more reactive than N-OSU and gave comparable yields
in less time. The overall yield of dipeptide with the present method
and with the N-OSU as activating group under identical conditions
is shown in Table 1. It can be noticed from Table 1 that
HOASUD activation is comparable with the N-OSU method. The
tively. The proton NMR spectra of Fmoc-L-Orn(Boc)-Val-OMe,
Fmoc- -Cys(trt)-Gly-OEt and Boc- -Tyr-Gly-OEt exhibited new sig-
L
L

24
S. Nowshuddin, A. Ram Reddy / Tetrahedron: Asymmetry 22 (2011) 22–25
enantiomeric purity was measured by optical rotatory dispersion
(ORD) and compared with standard samples.18 It has been found
that the dipeptide formation occurred with complete preservation
of stereochemistry. HO-ASUD activation of protected amino acids
is not sensitive to any particular protecting group or amino acid
and can be widely employed in peptide synthesis.
trated to give a colourless viscous oily liquid of Fmoc-
ASUD ester. Yield 1.3 g (100%).
L-Orn(Boc)-
4.5. Preparation of Fmoc-L-Orn(Boc)-Val-OMe 9
L
-Valine methyl ester 0.27 g (0.002 mol) and Na2CO3 0.63 g
(0.006 mol) were dissolved in 16 ml of water at room temperature
in a 100 ml three necked round bottomed flask. Next, Fmoc- -Orn(-
L
3. Conclusion
Boc)-ASUD ester 1.3 g (0.002 mol) dissolved in 16 ml of THF was
added to the above reaction mixture at room temperature over a
period of 5 min. After the addition was complete, the reaction mix-
ture was stirred for 10 h at room temperature. The reaction mix-
ture was acidified with 5% KHSO4 to reach a pH of 2–3. The
product was extracted into ethyl acetate. The ethyl acetate extract
was washed with 5% Na2CO3 solution, water, brine solution and
dried over anhydrous Na2SO4. The ethyl acetate was removed at re-
duced pressure. The oily crude product obtained was crystallized
using ethyl acetate and n-hexane. The yield of pale yellow crystal-
Synthesis of N-protected dipeptides and N-protected dipeptide
methyl esters via N-protected L-amino acid activated ASUD ester
intermediates is simple and can be carried out not only in THF
but also in other organic solvents such as acetone, dichlorometh-
ane, toluene and acetonitrile. The overall yield of the dipeptides
obtained is high and comparable to that of N-OSU.12 We have pre-
pared three new dipeptides involving these activated ASUD ester
intermediates.
line Fmoc-
-Orn(Boc)-Val-OMe is 0.9 g (78%). Mp: 155–157 °C, 1H
L
4. Experimental
NMR (300 MHz, CDCl3-d): d 7.1–7.7 (m, 8H), d 5.92 (d, 1H), d 5.5
(dd, 1H), d 4.1–4.8 (m, 2H), d 3.37 (s, 3H), d 3.0 (d, 1H), d 2.5–2.8
(m, 2H), d 1.9 (m, 2H), d 1.7 (m, 2H), d 1.50–1.59 (s, 9H), d 0.77–
0.98 (dd, 6H); 13C NMR (75 MHz,CDCl3-d): d 168.93, 168.69,
157.45, 156.43, 144.04, 141.22, 127.69, 127.08, 125.17, 119.9,
79.1, 77.5, 76.66, 67.2, 54.6, 49.2, 47.1, 44.0, 39.8, 36.6, 35.8,
33.7, 29.8, 28, 26.9, 18.1; IR (cmꢀ1): 3332.56, 2930.63, 2853.36,
164.71, 1627.82; MS m/z 512.2 (MꢀOMe and side chain of valine).
4.1. Preparation of Boc-
L-alanine–ASUD ester
Boc- -alanine 1.0 g (0.0052 mol), OH-ASUD 1.04 g (0.00529
L
mol), DCC 1.19 g (0.0058 mol) and THF (25 ml) were stirred in a
100 ml RB flask at room temperature for 12 h. The dicyclohexyl-
urea was filtered and the filtrate was concentrated to give the
a 2D0
ꢁ
¼ ꢀ29:2 (c 1, CH3OH); Chiral HPLC: Chiral PACK-AS-H;
Boc-L-alanine-ASUD ester. Yield 1.9 g (100%).
½
n-Hexane/IPA/TFA (50:50:0.1%); %ee: 98.29; Elemental analysis;
observed (theoretical): C = 65.13 (65.59); H = 7.21 (7.28); N = 7.35
(7.4).
4.2. Preparation of Boc-
L-Ala-ala-OH 5
At first,
(0.01548 mol) were dissolved in 16 ml of water at room tempera-
ture in a 100 ml three necked round bottomed flask. Boc- -alanine-
L-alanine 0.45 g (0.00516 mol) and Na2CO3 1.64 g
4.6. Preparation of Fmoc-L-Cys(trt)- ASUD ester
L
ASUD ester 1.9 g (0.00516 mol) dissolved in 16 ml of THF was
added to the above reaction mixture at room temperature over a
period of 5 min. After the addition was complete, the reaction mix-
ture was stirred for 10 h at room temperature. The reaction mix-
ture was acidified with 5% KHSO4 to reach a pH of 2–3.The
product was extracted into ethyl acetate. The ethyl acetate extract
was washed with 5% Na2CO3 solution, water, brine solution and
dried over anhydrous Na2SO4. The ethyl acetate was removed at re-
duced pressure. The oily crude product obtained was crystallized
using ethyl acetate and n-hexane. The yield of white crystalline
Fmoc- -Cys(trt)-OH 1.5 g (0.0025 mol), OH-ASUD 0.5 g
L
(0.0025 mol), DCC 0.52 g (0.0025 mol) and THF (15 ml) were stir-
red in a 50 ml three neck round bottomed flask at room tempera-
ture for 12 h. The dicyclohexylurea was filtered and the filtrate
concentrated to give Fmoc-L-Cys(trt)-ASUD ester. Yield 1.71 g
(100%).
4.7. Preparation of Fmoc-L-Cys(trt)-Gly-OEt 12
Boc-
4.3. Preparation of Boc-
At first, Boc- -alanine-ASUD ester 1.9 g (0.00516 mol), L-alanine
methyl ester hydrochloride 0.71 g (0.00516 mol), triethylamine
1.30 g (0.0129 mol) and THF 20 ml were charged into a 100 ml
three necked round bottomed flask at room temperature. The reac-
tion mixture was stirred over night at room temperature. The tri-
ethylamine hydrochloride salt was filtered and the filtrate was
concentrated under vacuum to give an oily crude. The oily crude
was dissolved in ethyl acetate and washed with 0.5% NaHCO3 solu-
tion and water. The ethyl acetate layer was dried over Na2SO4 and
distilled under vacuum to give an oily crude product. The yield of
L
-Ala-Ala-OH is 0.98 g (74%).
Glycine ethyl ester 0.21 g (0.0022 mol) and Na2CO3 0.7 g
(0.0066 mol) were dissolved in 20 ml of water at room tempera-
ture in a 100 ml three neck round bottomed flask. Fmoc-L-
L-Ala-Ala-OMe 4
Cys(trt)-ASUD ester 1.71 g (0.0022 mol) dissolved in 20 ml of THF
was added to the above reaction mixture at room temperature over
a period of 5 min. After the addition was complete, the reaction
mixture was stirred for 10 h at room temperature. The reaction
mixture was acidified with 5% KHSO4 to reach a pH to 2–3. The
product was extracted into ethyl acetate. The ethyl acetate extract
was washed with 5% Na2CO3 solution, water, brine solution and
dried over anhydrous Na2SO4. The ethyl acetate was removed at re-
duced pressure. The oily crude product obtained was crystallized
using ethyl acetate and n-hexane. The yield of white crystalline
L
Fmoc-
-Cys(trt)-Gly-OEt is 1.25 g (84%). Mp: 78–82 °C, 1H NMR
L
Boc-
4.4. Preparation of Fmoc-
At first, Fmoc- -Orn(Boc)-OH 1.0 g (0.0022 mol), OH-ASUD
0.43 g (0.0022 mol), DCC 0.45 g (0.0022 mol) and THF 10 ml were
stirred in a 50 ml three necked round bottomed flask at room tem-
perature for 12 h. The dicyclohexylurea was filtered and concen-
L
-Ala-Ala-OMe is 1.1 g (80%).
(300 MHz, CDCl3-d): 7.1–7.4 (m, 24H), d 4.6 (s, 1H), d 4.2 (s, 2H),
d 4.1 (d, 1H), d 3.5 (m, 4H), d 2.8–3.1 (dd, 2H), d 1.7 (s, 3H); 13C
NMR (75 MHz, CDCl3-d): d 178.0, 165.47, 156.99, 144.49, 144.10,
129.49, 128.16, 127.98, 127.89, 127.05, 126.87, 77.5, 77.10, 76.67,
67.53, 53.19, 49.05,36.1, 29.03, 26.4, 14.1; IR (cmꢀ1): 3327.94,
3056.65, 2928.03, 2851.32, 1681.75, 1627.14; MS m/z 689
L-Orn(Boc)-ASUD ester
L
(M+H2O). ½a 2D0
¼ þ34:7 (c 1, CH3OH); Chiral HPLC: Chiral PACK-
ꢁ
AS-H; n-Hexane/IPA/TFA (80:20:0.1%); %ee: 99.78; Elemental

S. Nowshuddin, A. Ram Reddy / Tetrahedron: Asymmetry 22 (2011) 22–25
25
analysis; observed (theoretical): C = 72.97 (73.41); H = 5.64 (5.71);
Acknowledgements
N = 4.15 (4.18).
Shaik Nowshuddin would like to thank Dr. Murali K. Divi, Chair-
man and Managing Director, Divis Laboratories Limited, Dr. P. Gun-
du Rao and Dr. M. N. A. Rao for their encouragement.
4.8. Preparation of Boc-L-Tyr- ASUD ester
Boc-L-tyrosine 2.0 g (0.0071 mol), OH-ASUD 1.4 g (0.0071 mol),
DCC 1.46 g (0.0071 mol) and THF 25 ml were stirred in a 100 ml
three neck round bottomed flask at room temperature for 12 h.
The dicyclohexylurea was filtered and the filtrate concentrated to
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L
-tyrosine-ASUD ester. Yield 3.2 g (100%).
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4.9. Preparation of Boc-L-Tyr-Gly-OEt 15
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ASUD ester 3.2 g (0.00516 mol) dissolved in 25 ml of THF was
added to the above reaction mixture at room temperature over
a period of 5 min. After the addition was complete, the reaction
mixture was stirred for 10 h at room temperature. The reaction
mixture was acidified with 5% KHSO4 to reach a pH of 2–3.The
product was extracted into ethyl acetate. The ethyl acetate extract
was washed with 5% Na2CO3 solution, water, brine solution and
dried over anhydrous Na2SO4. The ethyl acetate was removed at
reduced pressure. The oily crude product obtained was crystal-
lized using ethyl acetate and n-hexane. The yield of off-white
16. Sureshbabu, V. V.; Narendra, N.; Kantharaju Indian J. Chem., Sect. B 2008, 47,
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17. Warner–Lambert pharmaceutical company, GB Patent. 898,692, 1962.
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crystalline Boc-
-Tyr-Gly-OEt is 1.9 g (75%). Mp: 122–125 °C, 1H
L
NMR (300 MHz, CDCl3-d): d 7.04–7.06 (dd, 2H), d 6.74–6.77 (dd,
2H), d 6.51–6.54 (t, 1H), d 5.1 (m, 1H), d 4.4 (d, 1H), d 4.17–4.24
(q, 2H), d 3.95–4.02 (d, 2H), 3.00–3.02 (d, 2H), d 1.38–1.46 (s,
9H), d 1.26–1.28 (t, 3H); 13C NMR (75 MHz,CDCl3-d): d 172.28,
169.62, 157.40, 155.69, 155.53, 130.32, 127.48, 115.64, 80.44,
77.5, 77.0, 76.65, 61.60, 55.85, 49.18, 41.35,37.61, 33.82, 28.24,
25.53, 24.56, 14.061; IR (cmꢀ1): 3335.50, 2982.98, 2928.95,
1743.08, 1690.05, 1638.92, 1595.19; MS m/z 365.13 (Mꢀ1).
19. HPLC method: column: Purosphere Star RP-18e, 55 ꢂ 4.0 mm, injection
volume, 20 ll, flow rate: 0.05 ml/min (gradient).
20. Paul, R.; Anderson, G. W. J. Am. Chem. Soc. 1960, 82, 4596–4600.
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½
a 2D0
ꢁ
¼ þ3:3 (c 1, CH3OH); Chiral HPLC: Chiral PACK-AD-H;
n-Heptane/IPA/TFA (90:10:0.1%); %ee: 99.96; Elemental analysis;
observed (theoretical): C = 58.82 (59.0); H = 7.11 (7.15); N = 7.60
(7.65).