New reagent for the introduction of Boc protecting group to amines: Boc-OASUD

Article abstract of DOI:10.1080/00397911.2017.1366525

A new reagent, tert-butyl (2,4-dioxo-3-azaspiro [5,5] undecan-3-yl) carbonate (Boc-OASUD) for the preparation of N-Boc-amino acids is described. The Boc-OASUD reacts with amino acids and their esters at room temperature in the presence of a base and gives N-Boc-amino acids and their esters in good yields and purity. Introduction of the Boc group takes place without racemization. The Boc-OASUD, being a solid and more stable, is a better alternative to di-tert-butyl dicarbonate which is low melting and has to be dispensed in plastic containers than glass because of its poor stability.

Full text of DOI:10.1080/00397911.2017.1366525

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
A new reagent for the introduction of boc
protecting group to amines: Boc-OASUD
B. Leela Maheswara Rao, Shaik Nowshuddin, Anjali Jha, Murali K. Divi & M.
N. A. Rao
To cite this article: B. Leela Maheswara Rao, Shaik Nowshuddin, Anjali Jha, Murali K. Divi & M. N.
A. Rao (2017): A new reagent for the introduction of boc protecting group to amines: Boc-OASUD,
Synthetic Communications, DOI: 10.1080/00397911.2017.1366525
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Date: 29 August 2017, At: 13:58

A new reagent for the introduction of Boc protecting group
to amines: Boc-OASUD
B. Leela Maheswara Rao
Divis Research Center, Divis Laboratories Limited, Hyderabad, India
Department of Chemistry, GIS, GITAM University, Visakhapatnam, India
Shaik Nowshuddin
Divis Research Center, Divis Laboratories Limited, Hyderabad, India
Anjali Jha
Department of Chemistry, GIS, GITAM University, Visakhapatnam, India
Murali K. Divi
Divis Research Center, Divis Laboratories Limited, Hyderabad, India
M. N. A. Rao*
Divis Research Center, Divis Laboratories Limited, Hyderabad, India
*ADDRESS CORRESPONDENCE TO M. N. A. RAO, DIVIS RESEARCH CENTER,
DIVIS LABORATORIES LIMITED, B-34, SANATHNAGAR, HYDERABAD-500018,
INDIA. TEL: +91 40 23816743. E-MAIL:
MNARAO@DIVISLABORATORIES.COMABSTRACT
1

A new reagent, tert-butyl (2, 4-dioxo-3-azaspiro [5,5] undecan-3-yl) carbonate (Boc-
OASUD) for the preparation of N-Boc-amino acids is described. The Boc-OASUD reacts with
amino acids and their esters at room temperature in the presence of a base and gives N-Boc-
amino acids and their esters in good yields and purity. Introduction of the Boc group takes place
without racemization. The Boc-OASUD, being a solid and more stable, is a better alternative to
di-tert-butyl dicarbonate which is low melting and has to be dispensed in plastic containers than
glass because of its poor stability.
GRAPHICAL ABSTRACT
KEYWORDS: amino acids, Boc-OASUD, HO-ASUD, protecting group
Di-tert-butyl dicarbonate (Boc2O) is the most widely used reagent for the introduction of
the tert-butoxycarbonyl (Boc) group for the protection of the amino group in the peptide
synthesis.[1] However, the main drawback of Boc2O is its low melting range of 22-24oC, making
it a liquid in most geographical regions and seasons. It tends to decompose on storage. Bottles of
Boc2O build internal pressure in sealed containers caused by its slow decomposition to tert-
butanol and CO2 in the presence of moisture. For this reason, it is usually sold and stored in
plastic bottles rather than glass ones.[2] The inhalational toxicity of Boc2O is also high and is
comparable to that of phosgene.[2] Thus there is a need for an alternative reagent which is solid
and more stable for the introduction of Boc group.
2

Recently, we reported N-hydroxy-3-azaspiro[5,5]undecane-2,4-dione (HO-ASUD 1), as
an useful condensing agent, for preparing peptides.[3] Based on 1, we also reported, Fmoc-
OASUD, as a novel reagent for the introduction of Fmoc group.[4]
Here, we describe tert-butyl (2, 4-dioxo-3-azaspiro [5,5] undecan-3-yl) carbonate (Boc-
OASUD 2) as a new Boc reagent for preparing N-Boc-amino acids (Scheme 1). The 2 is a
crystalline solid exhibiting better stability and is a good alternative for Boc2O.
Results and Discussion
The reagent, 2 is prepared conveniently by reacting HO-ASUD[3] with Boc2O in a
suitable solvent such as acetonitrile in the presence of a base such as triethylamine (Scheme 2).
The reaction results in a colorless crystalline 2 in high yields (>85%) and purity (>99.5%
HPLC).The reagent is soluble in most organic solvents such as methanol, ethanol, 2-propanol,
tert-butanol, ethyl acetate, methyl tert-butyl ether, toluene, chloroform, acetonitrile, acetone, 1,4-
dioxane, THF, 2-MeTHF, DMF and DMSO. It is insoluble in petroleum ether, n- Hexane,
cyclohexane and water.
The reagent was found to be stable when stored at room temperature (25±3oC) protected
from air and moisture. After nine months storage, slight degradation (<0.5%) was observed and
the main degradation product was 1. The reagent was also found to be stable when kept at 40 oC
and 70 oC for 24 hours (99.9% and 99.7% HPLC respectively). However at 90 oC, it melted (mp:
85-88C) and decomposed completely to OH-ASUD, 1 (97.8% HPLC).
The reagent 2 has high reactivity towards the aliphatic amino group under mild
conditions. A number of N-Boc-amino acids were prepared by reacting amino acids with 2 in the
presence of a base at room temperature in high yields and purity (Table 1). The amino acid is
3

dissolved in an aqueous solution containing a base such as sodium carbonate and reacted with a
solution of 2 dissolved in a suitable water miscible solvent such as acetone at room temperature.
Depending on amino acid, the reaction is completed in 9 to 18 hours (TLC). After the reaction,
the pH was adjusted to 6 with KHSO4 and washed with an organic solvent such as ethyl acetate
to remove the liberated 1 and unreacted 2. The 1 can be recovered again and converted back to 2
by evaporating the organic layer and by reacting the crude obtained with fresh Boc2O and
triethylamine. The aqueous solution was further acidified using KHSO4 to pH 2 to 4 and
extracted with ethyl acetate. Evaporation of the solvent and recrystallization of the residue from
ethyl acetate and hexane results in N-Boc-amino acid. In the case of Lysine and Valine, the
corresponding Boc amino acids were isolated as their dicyclohexylamine (DCHA) salts. The H-
Lys(Boc)-OH (N€-Boc-lysine, entry 12), was prepared using lysine copper complex to block the
alpha amino group. The copper complex was reacted with the reagent 2, followed by cleaving
the complex using EDTA resulted in the H-Lys (Boc)-OH.
All the N-Boc-derivatives were obtained in good yields and purity. During the reaction,
the chiral integrity is maintained. In addition to sodium carbonate, other bases such as sodium
hydroxide, or organic bases such as triethyl amine can also be used.
o
Apart from room temperature, the reaction was also studied at 50 (±2) C. In the case of
L-Phenyl alanine, at both the temperatures, similar yields and purity were obtained. However in
the case of L-Serine, racemization was observed at 50 (±2) oC (Chiral purity: 92.93 Vs 99.8%).
For comparison, we prepared N-Boc-L-Phe-OH and N-Boc-L-Ala-OH using both the 2
and Boc2O. Reacting L-Phenyl alanine with 2 in acetone using Na2CO3 for 9 hours at 25 (±2) oC
resulted in N-Boc-L-Phe-OH in 95% yield (99.9% HPLC). Reacting with Boc2O under same
conditions resulted in 93% yield (99.9% HPLC). Similarly, reaction of L-Alanine with 2 in
4

o
acetonitrile using Na2CO3 for 10 hours at 25 (±2) C resulted in N-Boc-L-Ala-OH in 92% yield
(99.2% HPLC) and with Boc2O the yield was 96% (98.6% HPLC). Thus both reagents gave
similar yields and purity.
The reagent was found to be specific to the amino group without reacting with either
phenolic or aliphatic hydroxyl group when one equivalent of 2 was used. When L-Ser was
reacted with 1.5 and two equivalents of 2, about 0.39% and 1.98% of N-Boc-Ser (OBoc)-OH
was observed respectively. Similarly, about 0.52% and 1.92% of N-Boc-Tyr (OBoc)-OH was
observed respectively.
It is always a challenge to prepare N-Boc amino acid ester from the corresponding ester
because of the possibility of ester hydrolysis in an alkaline aqueous medium. However, by using
triethylamine as a base and reacting the ester with reagent 2, in a non-aqueous organic solvent
such as dichloromethane, a number of N-Boc-amino acid esters have been prepared (Table 2).
Here also, in all the cases, good yields have been obtained without any racemization.
Conclusion
In conclusion, we have described tert-butyl (2, 4-dioxo-3-azaspiro [5,5] undecan-3-yl)
carbonate (Boc-OASUD 2) as a novel reagent for the preparation of N-Boc protected amino
acids and their esters. The new reagent is a stable crystalline material and reacts with the amino
group in a facile manner under mild conditions without causing racemization.
Experimental
Analytical methods
5

All chemicals used were obtained from commercial sources and used without further
purification. TLC was performed on Merck 60 F254 Silicagel plates Visualization was
accomplished with UV light (254 nm) and ninhydrin. Melting points were determined in open
capillaries on a Polmon melting point apparatus (Model No: MP-96) and are uncorrected. Optical
rotations were measured with a Jasco DIP-1000 polarimeter. FTIR spectra were obtained from
1
13
Perkin-Elmer Spectrum one, Spectrophotometer. H and C NMR spectra were recorded on a
Bruker instrument at 300 and 75 MHz, respectively, with CDCl3, CD3OD or DMSO-d6 as
solvent and relative to TMS as internal standard. Mass spectra were recorded on a Thermo
scientific LCQ Fleet spectrometer with ion trap mass spectrometer. Column chromatography was
conducted over silica gel (200-400 mesh).
Preparation of tert-butyl (2, 4-dioxo-3-azaspiro [5.5] undecan-3-yl) carbonate
(Boc-OASUD, 2)
N-Hydroxy-3-azaspiro[5,5]undecane-2,4-dione (HO-ASUD) (5.0 g, 25.3 mmol), and
triethyl amine (3.08 g, 30.4 mmol) were dissolved in acetonitrile (50 mL). To this solution, was
added Boc2O (6.63 g, 30.4 mmol) at 0-5C and stirred at 25-30C for 5h. The reaction mixture
was concentrated under reduced pressure and the crude material was dissolved in ethyl acetate
(50 mL), washed with water (25 mL) and brine solution (25mL). The ethyl acetate layer was
dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting material
was precipitated with hexane (50 mL) to yield Boc-OASUD as a color less solid (6.50 g, 86.3%
yield). mp: 85-88C; Purity: 99.86% (by HPLC analysis: XTerra RP 18 (250 x 4.6 mm) column,
5 µm; eluent: water/acetonitrile (30:70); flow rate 1.0 mL/min; Temp 27C, detection at 210
nm]. IR (KBr, cm-1): vmax 3445, 3008, 2980, 2927, 2848, 2860, 1790, 1754, 1713, 1454, 1428,
6

1412, 1397, 1369, 1349, 1339, 1273, 1244, 1196, 1158, 1142, 1115, 1080, 1052, 982, 963, 944,
924, 904, 894, 874, 805, 768, 718, 642, 608, 566, 542; 1H NMR (300 MHz, CDCl3): δ 2.75-2.66
(m, 4H), 1.57-1.49 (m, 19H); 13C NMR (75 MHz, CDCl3): δ 166.01, 150.19, 86.63, 44.10, 36.78,
34.88, 32.53, 27.48, 25.55, 21.45; MS: m/z 319.75 [M+Na]+.
Preparation of N-(tert-Butoxycarbonyl)-L-phenylalanine (Table 1, entry 1)
L-Phenylalanine (1.0 g, 6.05 mmol) and sodium carbonate (0.71 g, 6.70 mmol) in water
(10 mL) was reacted with a solution of 2 (1.89 g, 6.36 mmol) in acetone (10 mL). The reaction
mixture was stirred at room temperature till the reaction completes, as monitored by TLC. The
resulting solution was concentrated, to the residue was added water (10 mL) and ethyl acetate
(10 mL), pH adjusted to 6.0 with 10% KHSO4 and stirred for 5 minutes. The organic layer was
removed and the aqueous layer was acidified to pH 2.0 with 10% KHSO4 at 0-5C and extracted
with ethyl acetate (2 x 10 mL). The combined organic layers were washed with 5% NaHCO3
solution, water, brine and dried over anhydrous Na2SO4. The ethyl acetate layer was
concentrated and the residue was crystallized from EtOAc/hexane (2:8) mixture to give the
20
product as a white solid (1.53g, 95% yield). mp: 85-87C (lit.[5] 86-88C); [α]D = +25.90 (c 1
20
in EtOH) {lit.[5] [α]D = +25.50 (c 1 in EtOH)}; 99.88% purity by HPLC [Method: XTerra RP
18 (250 x 4.6 mm) column, 5 µm; eluent: water/acetonitrile/glacial AcOH (65:35:0.1%); flow
rate 1.0 mL/min; Temp 27C, detection at 210 nm]; % ee: 99.76% (D-isomer Rt = 6.26 min; L-
isomer Rt = 12.40 min) [Method: Chiralpak IA, 250 x 4.6 mm, 5 µm; eluent: n-Hexane: IPA:
TFA (90:10:0.1%); flow rate 1.2 mL/min; Temp 27C, detection: 215 nm]; IR (KBr, cm-1): vmax
3373, 3028, 2984, 2935, 1724, 1708, 1692, 1525, 1442, 1421, 1392, 1366, 1342, 1297, 1269,
1
1252, 1168, 1082, 1053, 1028, 1003, 966, 949, 888, 853, 789, 758, 748, 701; H NMR (300
7

MHz, DMSO-d6): δ 12.59 (br, 1H), 7.30-7.16 (m, 5H), 7.11-7.09 (d, 1H), 4.12-4.04 (m, 1H),
3.04-2.98 (dd, 1H), 2.85-2.77 (m, 1H), 1.31 (s, 6H) and 1.25 (s, 3H); 13C NMR (75 MHz,
DMSO-d6): δ 173.55, 155.39, 137.97, 129.03, 128.06, 126.25, 77.98, 55.08, 36.45, 28.01,27.78;
MS: m/z 288.88 [M+Na]+.
The other amino acids given in Table 1 were prepared and characterized in a similar
manner.
Preparation of methyl (tert-butoxycarbonyl)-L-phenylalaninate (Table 2, entry 1)
L-Phenylalanine methyl ester hydrochloride (5.0 g, 23.2 mmol), triethyl amine (2.47 g,
24.4 mmol) and 2 (7.24 g, 24.4 mmol) were added to dichloromethane (50 mL) and stirred at
reflux temp (38-40C) for 5h. After completion of the reaction, filtered to remove salts and the
filtrate was washed with 5% KHSO4 (20 mL), water (25 mL), brine (25 mL), and dried over
sodium sulfate. The solvent was evaporated under reduced pressure to obtain a pale yellow oil.
The oil was purified by column chromatography (silica gel, n-hexane/ ethyl acetate, 8:2) to
afford 5.96 g (92%) Methyl (tert-butoxycarbonyl)-L-phenylalaninate as a colorless oil. Rf 0.5 (n-
25
25
Hexane: EtOAc-4:1); [α]D = -4.50 (c 1 in MeOH) {lit.[6] [α]D = -6.00 (c 2.5, MeOH)}; IR
(neat, cm-1): vmax 3437, 3020, 2980, 2954, 1735, 1719, 1604, 1507, 1492, 1454, 1444, 1437,
1
1391, 1364, 1250, 1177, 1154, 1079, 1053, 1015, 951, 932, 858, 817, 779, 739, 701, 668; H
NMR (300 MHz, CDCl3): δ 7.32-7.11 (m, 5H), 4.98-4.95 (d, 1H), 4.62-4.55 (q, 1H), 3.71 (s,
13
3H), 3.15-3.01 (m, 2H), 1.41 (s, 9H); C NMR (75 MHz, CDCl3): δ 172.33, 155.08, 136.05,
129.28, 128.53, 127.00, 79.86, 54.44, 52.15, 38.34, 28.28; MS: m/z 302.90 [M+Na]+.
The other N-Boc- amino acid esters given in Table 2 were prepared and characterized in
a similar manner.
8

Supplemental Material
Complete experimental and spectral details are available online in the Supplemental
Material.
Acknowledgement
The authors are thankful to Dr. P. Gundu Rao, former Director, Research &
Development, for his suggestions and discussions.
References
[1] Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 4th ed.; Johan Wiley
& Sons: New York, 2007.
[2] (a) Wakselman, M. In Encyclopedia of Reagents for Organic Synthesis, Paquette, L. A. (Ed.);
John Wiley and Sons, Inc.: New York, 1995, Vol. 3, 1602–1608 and references therein.
(b) Di-tert-butyl dicarbonate, Wikipedia the free encyclopedia, 27 May 2017.
https://en.wikipedia.org/wiki/Di-tert-butyl_dicarbonate.
[3] Rao, B. L. M.; Nowshuddin, S.; Jha, A.; Divi, M. K.; Rao, M. N. A. Tetrahedron: Asymmetry
2016, 27, 487–491.
[4] Rao, B. L. M.; Nowshuddin, S.; Jha, A.; Divi, M. K.; Rao, M. N. A. Tetrahedron Lett. 2016,
57, 4220–4223.
[5] Keller, O.; Keller, W. E.; Look, G. V.; Wersin, G. Org. Synth. 1985, 63, 160.
[6] Ouchi, H.; Saito, Y.; Yamamoto, Y.; Takahata, H. Org. Lett. 2002, 4, 585–587.
9

Table 1. Synthesis of N-Boc protected amino acids with 2a.
Entry N-Boc Amino acid
L/D Base
Solvent
Time
(h)
Yield
(%)
1
Boc-Phe-OH
L
L
–
Na2CO3
acetone
9
95
92
94
88
91
86
91
81
90
90
85
78
2
Boc-Ala-OH
Na2CO3
acetonitrile
acetone
10
12
15
15
12
9
3
Boc-Gly-OH
NaOH
4
Boc-Leu-OH
L
L
L
L
L
L
D
L
L
Na2CO3
1, 4-dioxane
acetonitrile
acetone
5
Boc-Trp-OH
Triethyl amine
Triethyl amine
Na2CO3
6
Boc-Pro-OH
7
Boc-Tyr-OH
acetonitrile
1, 4-dioxane
acetone
8
Boc-Lys (Boc)-OHb
Boc-Val-OHb
4-Cl-Boc-Phe-OH
3-F-Boc-Phe-OH
H-Lys (Boc)-OHc
NaOH
18
14
14
12
18
9
Na2CO3
10
11
12
Triethyl amine
Na2CO3
acetone
acetonitrile
acetone
Na2CO3
10

13
14
15
Boc-Asp-OH
Boc-Phg-OH
Boc-Ser-OH
L
NaOH
acetonitrile
1, 4-dioxane
acetone
15
12
11
80
89
89
D
D
Na2CO3
Triethyl amine
aExperimental conditions: The amino acid is dissolved in an aqueous solution containing a base and reacted with a solution of 2
dissolved in a suitable solvent at room temperature. After the reaction, the base was neutralized, extracted with an organic
solvent. Evaporation followed by crystallization from EtOAc/hexane gives the required N-Boc amino acids. All products were
identified by comparison with their spectral data (FTIR, 1H & 13CNMR and MASS).
bThe Boc-Lys (Boc)-OH (entry.8) and Boc-Val-OH (entry. 9) were isolated as a DCHA salt.
cThe H-Lys (Boc)-OH (entry. 12) was prepared using copper complex method.
11

Table 2. Synthesis of N-Boc amino acid esters with 2*.
Entry
R
R1
Yield (%)
1
2
3
4
5
6
7
8
9
10
PhCH2
CH3
Me
Me
Et
92
89
85
93
91
90
81
86
87
85
CH3
H
Me
Et
H
H
Bn
Me
Bn
Me
Me
CH2OH
CH2OH
(CH3)2CH
11
12
4-OH-PhCH2
4-OH-PhCH2
Me
Et
82
84
12

*L-amino acid ester hydrochloride (23.2 mmol), triethyl amine (24.4 mmol) and 2 (24.4 mmol) were added to dichloromethane
(50 mL). The resulting mixture was refluxed for 3–5 h. The reaction mixture was filtered and concentrated under reduced
pressure. The obtained residue was purified using column chromatography. All products were identified by comparison with their
spectral data (FTIR, 1H & 13CNMR and MASS).
13

Scheme 1. Synthesis of N-Boc protected amino acids and esters using Boc-OASUD.
14

Scheme 2. Synthesis of Boc-OASUD.
15