Advances in Ma terials Physics and Che mist ry, 2012, 2, 226-228
doi:10.4236/ampc.2012.24B057 Published Online December 2012 (htt p://www.SciRP.org/journal/ampc)
A Novel Method for the Protection and Activation of Histidine*
Yinan Zh a o1,2, Shubiao Zha ng1, Shao hui C ui1, Huiying Chen1, B ing Wang1, Shufen Zh an g 2
1Key Laboratory of Bio-chemistry Engineering, The State Ethnic Affairs Commission-Ministry of Ed ucation,
Dalian Nationalities University, Dalian, China
2State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, China
Received 2012
ABSTRACT
The yield and purity of synthetic peptides were greatly related to the amino acid protection and activation during the synthesis
process. Therefore, the amino acid protection and activation are the most important steps in peptide synthesis. By using tetrahyd r o-
furan as the solvent, 9-fluorenylmethoxycarbonyl as protection group, 2-(7-azobenzotri- azo l-1-yl ) -N, N, N',N'-tetramethyluronium
he xaflu- orophosphate (HATU) as condensation reagent an amino protected histidine ester was given. In this article a novel synthesis
method for N-(9- fluorenylmethoxycarbonyl)-hist idine active ester was establ ished. Th e reaction con ditions for preparing t his active
ester were optimized. The experimen tal result s indicat ed that sol vents and act ive reagents h ad remarkab le effects o n the yield of ac-
tive ester. The best conditions for preparin g t he active ester was a ratio of n (Fmoc-His-OH): n (HATU) = 1:1.2 with THF used as the
solvent at room temperature. The yield of the final product was about 80% with a purity of over 85%. This simple method would
provide fundamentals for the synthesis of other pr otected amin o acid active esters.
Keywords: Histidine; Protection; Activa tion; Peptide
1. Introduction
Proteins and peptides are important classes of bioactive ma-
cromolecules that play key roles in controlling biological func-
tions. Pep tides o ften h ave a specific bio logical signal; and they
can significantly improve the adhesion of cells on the surfac e
[1]. Indeed, there are numerous preclinical and clinical trials
using proteins or peptides. These macromolecules are also
widely used as biochemical o r pharmacological tools, especial-
ly peptides which are available at high purity grade by
large-scale chemical synthesis. With the development of poly-
peptide drugs, various fields of scientist from chemistry, biolo-
gy and medicine are paying more and more attention to the
research of this kind of new drugs. Recent years peptides and
their derivatives have been used for restraining cancer cell mi-
gration, curing anti-thrombosis, treating acute renal failure,
reducing anti-inflammation and promoting skin regeneration,
etc [2,3]. Especially people found that the introduction of pep-
tide groups into cationic lipids can prolong their half-life in vivo
and enhance targeting thereof. The value of cationic liposomes
with peptides for the delivery of genetic materials has been
realized gradually by researchers [4,5], as they have many ad-
vantages, including numerous free active functional groups on
their surface, aviru lence, u sed either in vitro or in vivo [6-8], no
obvious limits for materials contained in size, no inflammati on,
and the control of the amount of the materials into the cells [8].
Moreover, they maintain their physiological concentration ad-
vantages, most amino groups have been protonated in the
process of carrying genes, and thereby with positive charge
they could combine with negatively charged plasmid DNA to
form liposomes/DNA complexes by electrostatic attraction [6].
All the applications will depend on the artificial peptide synthe-
sis through the controlled connection of different amino acids.
The key difficulty is that the reagents used for the peptide con-
nection can easily react with other groups such as the amino
groups at the N-terminal of residues, carboxylic groups at the
C-ter- minal of residues, reactive groups on the side chains and
especially much more active SH groups. Therefore, in order to
obtain the synthetic peptide with specific order, people can only
use the method of stereospecific synthesis step by step. Before
the connection of different amino acids was performed, these
reactive groups must be blocked or protected to avoid the side
reaction with the activated reagents. In this paper, we eluci-
dated a novel method for the simple synthesis and purification
of protected histidine active ester, which would lay a founda-
tion of the large scale synthesis of peptides. The results herein
also showed much improvement over our previous study [9].
2. Materials and Methods
2.1. Materials
All the reagents employed in the synthesis of active ester were
of analytical purity. Methylene dichloride (CH2Cl2), tetrahy-
drofuran (THF), ethyl acetate and ethyl ether were purchased
from Shanghai Chemical Industry Co., Ltd. (Shanghai, China).
Histidine，N-(9-Fluorenylmethoxycarbony loxy) succinimide
(Fmoc -OSu ), Boc-Gly-OH, O-(7-Azabenzotriazole-1-yl ) - N,N,
N′,N′-tetramethyl uranium (HATU) were purchased from
Shanghai Medpep Co., Ltd. (Shanghai, China).
2.2. Instruments
Rotary evaporator, Shanghai Yarong Biochemistry Instrment
*The study was supported by the National Natural Science Foundation of China
(20876027 and 21176046) and the Fundamental Research Funds for the Central
Universities(DC12010104).

Y. A. ZHAO ET AL.
227
Factor y (Shanghai, China); Vacuum drying oven, DHG-9070A,
Shanghai Jing Hong Laboratory Instrument Co., Ltd. (Shanghai,
China); Infrared Spectrometer, NICOLET370, Thermo Electron
Co., Ltd. (United States); HPLC-MS, SHIMADZU Co., Ltd.
(Japan ) .
2.3. Synthesis of
9-Fluorenylmethoxycarbonyl- L-His-OH
N-(9-Fluorenylmethoxycarbonyloxy) succinimide was used to
protect the amino group of Histidine to obtain
9-Fluorenylmethoxycarbonyl-L-His-OH (Fmoc-His-OH) dur-
ing a one-step process which is depicted in Scheme 1. In this
one-step process, histidine was dissolved in 25mL distilled
water, F moc -OSu in 20mL THF. Then Histidine solution was
added to Fmoc-OSu solution slowly, following the adjustment
of pH of 8-9 by using 10% sodium hydroxide and reaction at
room temperature for 2h. After the reaction, the mixture fluid
was extracted with ethyl ether for three times, and then 20mL
distilled water was added following the adjustment of pH to 3
with 10% hydrochloride. After extracted with ethyl acetate for
three times, the organic phase was washed with the 5% citric
acid, saturated salt water and distilled water, respectively. The
solution was dried with anhydrous magnesium sulfate and the
solvent was removed by rotary evaporation. Finally the residual
powder was subject to recrystallization through ethyl ether .
2.4. Synthesis of
9-Fluorenylmethoxycarbonyl-L-Histidine
Anhydrid
9-Fluorenylmethoxycarbonyl-L-histidine ester, whose synthesis
process is depicted in Scheme 2. In this synthesis process, 9-
Fluorenylmethoxycarbonyl-L-His-OH (3mmol) was dissolved
in 20mL THF, HATU (3.6mmol) was dissolved in 20mL ace-
tonitrile, the two solutions were dropwise mixed slowly at roo m
temperature and kept reaction for 16h. After the reaction was
completed, the solution was washed with CH2Cl2 and solvent
was removed by rotary evaporation. The final product with the
yield of 80% or so and the purity of over 85% was obtained
through recrystallization from ethyl a cetate for three ti mes.
O
OO N
O
O
+
HN
N
OH
NH
2
O
NaOH
O
O
NH H CCH 2
OOH N
NH
Scheme 1. Synthesis of 9-Fluorenylmethoxycarbonyl-L-His-OH.
O
O
NH H CCH
2
OOH N
NH
N
NN
ON
N
+
+O
O
NH H CCH
2
OON
NH
P
F
F
F
F
F
F
N
N
N
Scheme 2. Synthesis of 9-Fluorenylmethoxycarbonyl-L-Histidine
ester.
3. Results
Many studies have shown that the amino acid protection and
activation have great influence on yield and purity of 9-Fluo-
renylmethoxycarbonyl-L-Histidine ester. Solvent and other
reagents used have influences on protection and activation.
Through the study of protection and activation of histidine, the
optimum conditions of amino acid activation were n (Fmoc -
His-OH): n (HATU) = 1:1.2 by using tetrahydrofuran as the
solvent at the temperature of room temperature. The analytical
result s of chemical s tructure o f the active est er are list ed below
as shown in Figures 1 and 2 of IR, and Figures 3 and 4 of MS.
408 29
507. 83
548. 02
744. 16
1053. 21
1252. 06
1304. 96
1412. 28
1445. 32
1618. 16
1696. 46
1767. 94
2930. 17
3275. 13
5. 0
5. 2
5. 4
5. 6
5. 8
6. 0
6. 2
6. 4
6. 6
6. 8
7. 0
7. 2
%Transmittance
1000
2000
3000 Wavenumber s ( cm-1)
Figure 1. The infrared spectrum of 9-Fluorenylmethoxycarbony
l-L-His-OH.
747. 46
1015. 85
1250. 11
1292. 01
1405. 59
1448. 371488. 86
1545. 42
1616. 541696.48
1767. 03
2923. 72
3059. 24
3328. 11
3415. 57
3464. 98
3543. 78
13. 0
13. 5
14. 0
14. 5
15. 0
15. 5
16. 0
16. 5
17. 0
17. 5
18. 0
18. 5
19. 0
19. 5
20. 0
20. 5
21. 0
21. 5
22. 0
22. 5
%Transmittance
1000
2000
3000
4000 Wavenumber s ( cm-1)
Figure 2. The infrared spectrum of 9-Fluorenylmethoxycarbonyl-
L-Histidine ester.
25050075010001250 m/z
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000Inten.
378.2
400.1
514.5
277.0 693.7
Figure 3. The mass spectrum of 9-Fluorenylmethoxycarbonyl-L-
His-OH.

Y. A. ZHAO ET AL.
228
2505007501000 1250 m/z
0
50000
100000
150000
200000
250000Inten.
521.0
376.0
248.6 384.8 657.2
693.5
570.9 874.0 1155.0 1485.8
112.5
179.2 965.0 1287.1
Figure 4. The mass spectrum of 9-Fluorenylmethoxycarbonyl-L-
Histidine ester.
F mo c -Hi s-OH: ESI-MS m/z 377.53[M], 378.53 [M+H]+,
400.53 [M+Na]+. IR ν/cm-1: 3300-3400 (νNH), 3275.13(OH),
2950.27-2853.67(νCH), 1767.94(νC=O), 1252.06 and 1053.21
(Fmoc νC-O-C), 744(δCH).
9-Fluorenylmethoxycarbonyl-L-Histidine ester: ESI-MS
m/z, 493.18[ M]，248.6 [M+2H]+，376, 384.8(segment peaks).
IR ν/cm-1: 3328.11~3464.98(νNH), 2950.27-2923.72 (νCH),
1767 .03~1696. 48(νC=O), 1292.01 and 1250.11(νOCOCO).
4. Discussion
The results showed that the solvent system and dosage of acti-
vation reagent HATU have great influence on yield and purity
of 9-Fluorenylmethoxycarbonyl-L-Histidine ester. The yield of
active ester could be greatly improved by using infirm polarity
of tetrahydrofuran as the solvent system. The light excess of
HATU was also beneficial to the yield. During the process of
9-Fluorenylmethoxycarbonyl-L-His-OH synthesis, the pH of
reaction system must be kept between 9~10, other wise, activity
of Fmoc-OSu could be greatly lowered. On the other hand, the
rapid dropping speed could cause inadequate reaction. In the
purification process, we have tried many other solvents includ-
ing ethanol, acetone, toluene, and so on. It was finally found
that acet ate ester was the best solvent fo r the recrystallization of
both of 9-Fluorenylmethoxycarbonyl-L-His-OH, and
9-Fluore nyl- methoxycarbonyl-L-His ester. This study provided
a novel method for the simple synthesis and purification of
protected histi dine acti ve ester. This yield was highly improved
compared with the previous experiment [9], and the purity of
product was also well increased. Base on the study of the pro-
tection and activation of histidine, we have built up a method
for the synthesis and purification of other amino acids, which
could forward the synthesis of peptides for the drugs and drug
deli very systems.
5. Acknowledgements
The study was supported by the National Natural Science
Foundation of China (20876027 and 21176046) and the Fun-
damental Research Funds for the Central Universities
(DC12010104).
REFERENCES
[1] S. B. Zhang, Y. N. Zhao, and B. D. Zhao. “Hybrids of nonviral
vectors for gene delivery,” Bioconjugate Chem. vol. 21, pp.
1003-1009, June 2010.
[2] D. J. Coles, A. Esposito, and H. T. Chuah. “The synthesis and
characterization of lipophilic peptide-based carriers for gene de-
li v e ry, ” Tetrahedron. vol. 66, pp.5435-5441, July 2010.
[3] Y. T. Ko, C. Falcao, and V. P. Torchilin. “Cationic liposomes
loaded with proapoptotic peptide D-(KLAKLAK)2 and Bcl-2
antisense oligodeoxynucleotide G3139 for enhanced anticancer
therapy,”Molecular Pharmaceutics, vol.6,pp.971-977, March
2009.
[4] D. L. McKenzie, K. Y. Kwok, and K. G. Rice. “A potent new
class of reductively activated peptide gene delivery agents,” J.
Biol. Chem., vol. 275, pp. 9970-9977, April 2000.
[5] M. A. Mintzer, E. E. Simanek. “Nonviral vectors for gene deli-
very,” Chem Rev., vol. 109, pp.259-302, Ap ril 2009.
[6] M. Nishikawa, M. Yamauchi, and K. Morimoto. “Hepto-
cyte -targeted in vivo gene expression by intraveneous injection
of pla smid DNA com plexed wit h synt heti c multi -funct iona l gen e
delivery system,” G ene Th er .,vol. 7, pp.548-555, J uly 2000.
[7] M. J. Schuster, G. Y. Wu, and C. M.Wa lt on. “Multicomponent
DNA carrier with a vesicular stomatitis virus G-peptide greatly
enhaances liver-targeted gene expression in mice,”Bioconjugate
Chem.,vol.10, pp.1075-1083, O ctober 1999.
[8] S. B. Zhang, Y. M. Xu, and B.Wang. “Cationic compounds used
in lipoplexes and polyplexes for gene delivery,” J.Control Re-
lease, vol. 100, pp.165-180, No v ember 2004.
[9] Y. N. Zhao,S. B. Zhang, and S. H. Cui. “Preparation of amino
aci d act ive est er i n pep tid e inter medi ate, ” Chemical Word, vol.2,
pp. 105-109, February 2012.