One step synthesis of biotinylated amino acids

Article abstract of DOI:10.3184/030823410X12759895186422

A rapid method is reported for the preparation of biotinylated 4-amino-D-phenylalanine(D-Aph) with Fmoc as the protecting group at the a-amino. Different coupling reagents and conditions were studied to get the biotinylated compound. Coupling reagents like 1-[bis(dimethylamino)methylene]-1H -benzotriazolium 3-oxide hexafluorophosphate and BOP were found to be very efficient. Biotinylated Boc-L-Aph and biotinylated Fmoc-L-lysine were also successfully prepared by this method.

Full text of DOI:10.3184/030823410X12759895186422

3
48 RESEARCH PAPER
JUNE, 348–350
JOURNAL OF CHEMICAL RESEARCH 2010
One step synthesis of biotinylated amino acids
a
a
a,b
a
Siliang Feng , Senhao Li , Xiaoyu Kang and Keliang Liu *
a
Beijing Institute of Pharmacology andToxicology, Beijing 100850, P. R. China
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China
b
A rapid method is reported for the preparation of biotinylated 4-amino-D-phenylalanine(D-Aph) with Fmoc as the
protecting group at the α-amino. Different coupling reagents and conditions were studied to get the biotinylated
compound. Coupling reagents like 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophos-
phate and BOP were found to be very efficient. Biotinylated Boc-L-Aph and biotinylated Fmoc-L-lysine were also
successfully prepared by this method.
Keywords: biotin, 4-aminophenylalanine, lysine, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluoro-
phosphate
Vitamin transporters have been reported to facilitate intestinal
solution after introduction of biotin, which may cause hydroly-
sis of Fmoc. To get the biotinylated amino acid, different
coupling reagents and conditions were studied. 1-[bis(dimethy-
lamino)methylene]-1H-benzotriazolium 3-oxide hexafluoro-
phosphate (HBTU) worked better than others at room
temperature (Scheme 1). The results were presented in Table 1.
Boc-L-Aph(Biotin)-OH (3b) and Fmoc-L-Lys(Biotin)-OH
1,2
peptide uptake, and the bio-formulations using this transport
3
,4
system have shown promising results.
Glucagon-like
peptide-1 was recently modified with biotin (vitamin H),
which actively traverses the intestine membrane via sodium-
5
dependent multivitamin transportation (SMVT). The bio-
tinylated products displayed higher Caco-2 cell monolayer
permeability and higher oral hypoglycemic efficacy than the
corresponding compounds without biotin.
(
3c) were also obtained by this way (Scheme 2 and Table 2)
and the yield of the latter was moderate higher than that
7
1
reported with biotin-OSu method. H NMR and high resolu-
tion mass spectrums were used to identify the structures of the
three biotinylated compounds.
The introduction of biotin to a peptide could be accom-
plished via direct peptide biotinylation at the amino group
5
of the N-terminal or the side chain of Lys, or synthesis of a
peptide with biotinylated amino acids as building blocks. The
latter was more convenient for the preparation of a peptide
compared with the former. 4-aminophenylalanine (Aph) is an
unnatural amino acid with an amino group on its side chain
and is often used in bioactive peptides research. So we believed
that biotinylated 4-aminophenylalanine (Aph (Bio)) should be
another good choice to introduce a biotin into a bioactive pep-
tide from its side chain. Here we reported a one-step-synthesis
of fluorenylmethyloxycarbonyl (Fmoc) and -butyloxycarbonyl
Table 1 Synthesis of biotinylated Fmoc-D-Aph under different
conditions
Reaction CR
no.
T
Reaction HPLC
t
R
Yield
o
/ C time /min /% /min /%
a
a
b
1
2
3
4
Biotin-OSu
25
50
75
25
—
—
—
—
—
—
—
—
—
—
—
—
Biotin-OSu
Biotin-OSu
.
c
Biotin/EDCHCl/
HOBt
90
95.1 9.663 68
(
Boc) protected Aph (Bio) from biotin and α-amino protected
.
70c,d
5
Biotin/EDCHCl/
50
—
—
—
Aph. Fmoc and Boc protected amino acids were mostly used
in solid phase peptide synthesis. Our method provides an effi-
cient way to introduce biotin at desired position in synthesised
peptides.
HOBt
6
7
8
9
Biotin/HBTU/DIEA 25
Biotin/HBTU/DIEA 50
60e
95.4 9.688 76
d,e
40
—
—
—
e
Biotin/BOP/DIEA
Biotin/BOP/DIEA
25
50
60
96.2 9.681 75
d,e
40
—
—
—
Generally, a biotinylated amino acid was prepared by reac-
tion the reactive intermediate, biotin hydroxysuccinimide ester
.
EDCHCl: N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide
hydrochloride; HOBt: 1-hydroxy-1H-benzotriazole; BOP: ben-
zotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoro-
phosphate.
6
,7
(
biotin-OSu), with amino acid bearing a free carboxyl group.
In another way, the carboxyl group of the amino acid was
first protected and then hydrolyzed after biotinylation with a
a
No product observed in 48 hours.
8
coupling reagent like dicyclohexylcarbodiimide. However,
b
Decomposition in 30 minutes.
c
we found that biotin-OSu would not react with Fmoc-D-
Including 30 minutes for preactivating the carboxyl group.
6
,7
d
Aph-OH under reported conditions. The reaction might be
inhibited by the lesser activity of the aromatic amino group
and greater steric hindrance of the phenyl ring on the amino
acid compared with lysine. Moreover, the second method is
very inconvenient and maybe unsuitable since the carboxyl
group must be protected first and then removed with NaOH
New byproduct observed.
e
R
Including 5 minutes for pre-activating the carboxyl group; t :
retention time; HPLC conditions: Phase A: 0.1% trifluoroacetic
acid/water(v/v); Phase B: 0.1% trifluoroacetic acid/70% acetoni-
trile/water(v/v/v). PhaseB:0min, 30%;5min, 100%;15min, 100%;
1
7min, 30%; phase A + Phase B = 100%. The wavelength of
−1
the detector was set at 210 nm. The flow rate was 1mL min .
Scheme 1 Synthesis of Fmoc-D-Aph(Bio) under different conditions. CR represented coupling reagent(s),
either biotin hydroxysuccinimide ester or biotin with a coupling reagent.
*
Correspondent. E-mail: keliangliu@yahoo.com

JOURNAL OF CHEMICAL RESEARCH 2010 349
Scheme 2 Synthesis of biotinylated amino acids with HBTU method.
Table 2 Synthesis of biotinylated amino acids with HBTU
method
Preparation of 3a–c with HBTU method at room temperature; general
procedure
Diisopropylethylamine (DIEA, 0.17 mL, 1.0 mmol) was added to
N-protected amino acid, 1a–c, (1.0 mmol) in a dimethylformamide
Compd Reaction
HPLC
/%
t
R
Yield M.p.
o
no.
time /min
/min
/%
/ C
(
DMF 5 mL) solution and this was stirred at room temperature for
a
5 minutes. DIEA (0.33 mL, 2.0 mmol) was added to D-Biotin
(244 mg, 1.0 mmol) DMF (10 mL) solution in the same way. The
solutions were mixed and the reaction was monitored by TLC. After
about 50 minutes, the reaction was completed and DMF was removed
3a
3b
3c
3
60
95.4
93.3
94.6
NA
9.688
8.494
9.163
NA
76
82
85
72
201–203 (dec.)
136–138 (dec.)
178–180
a
60
a
60
b
c
>600
181–182
under vacuum. Saturated NaHCO (50 mL) solution was poured into
a
3
Including 5 minutes for pre-activating the carboxyl group.
Data reported in ref. 7 with biotin-OSu method; NA: not
the reaction bottle and the mixture was stirred vigorously. A white
precipitate appeared in about 1 hour. The solid was collected by suc-
tion filtration and washed first with water, and then with cold metha-
nol. The powder was suspended in water(50 mL) and acidified with
saturated solution of citric acid solution. The precipitate was collected
by filtration under vacuum and washed with water and cold methanol
sequentially and was dried. The crude product was recrystallised
from methanol-DMF to give a pure product. The structures of the
b
R
available; t : retention time; HPLC conditions: Phase A: 0.1%
trifluoroacetic acid/water(v/v); Phase B: 0.1% trifluoroacetic
acid/70% acetonitrile/water(v/v/v). Phase B: 0min, 30%; 5min,
1
00%; 10min, 100%; 17min, 30%; phase A + Phase B = 100%.
The wavelength of the detector was set at 210nm. The flow rate
−1
was 1mL min .
1
compounds 3a–c were identified by H NMR and MS.
In conclusion, a one-step-synthesis for rapid preparation
of biotinylated amino acids was reported. Fmoc-D-Aph
(
Fmoc-D-Aph(Biotin)-OH (3a):Yield: 76%; m.p. 201–203 °C (dec.);
1
H NMR (DMSO-d ): δ 1.28–1.72 (m, 6H), 2.28 (t, J = 7.4 Hz, 2H),
6
Biotin)-OH(3a), Boc-L-Aph(Biotin)-OH(3b) and Fmoc-L-
2
4
2
1
.58 (d, J = 12.4 Hz, lH), 2.76–2.85 (m, 2H), 2.96–3.16 (m, 2H),
.06–4.32 (m, 6H), 6.35 (s, 1H), 6.43 (s, 1H), 7.10–7.20 (d, J = 8.4 Hz,
H), 7.22–7.72 (m, 7H), 7.82–7.92 (m, 2H), 9.81 (s, 1H), 12.72 (s,
Lys(Biotin)-OH (3c) were successfully synthesised in this
manner. To the best of our knowledge, the preparation both
of Fmoc-D-Aph(Biotin)-OH(3a) and Boc-L-Aph(Biotin)-
OH(3b) have not been reported previously. We found that
biotin-OSu did not work on preparing Fmoc-D-Aph(Biotin)-
OH(3a) due to the less activity of the aromatic amino group.
The synthesis of Fmoc-L-Lys(Biotin)-OH (3c) by the way
described above was much faster and gave moderate higher
yield than that reported method using biotin-OSu method.
Our work provide a rapid and convenient method for the
preparation of biotinylated amino acids.
25
H); M-1: 627.22892, exact masscal.: 628.23556; [α]D +24.0 (c=1.0,
DMSO).
Boc-L-Aph(Biotin)-OH (3b): Yield 82%; m.p. 136–138 °C (dec.);
1
H NMR (DMSO-d
2.58 (d, J = 12.4 Hz, lH), 2.76–2.85 (m, 2H), 2.96–3.16 (m, 2H),
.95–4.32 (m, 3H), 6.39 (s, 1H), 6.48 (s, 1H), 7.04–7.20 (m, 3H), 7.48
d, 2H), 9.84 (s, 1H), 12.57 (s, 1H). M-1: 505.21256; exact masscal.:
): δ 1.23–1.72 (m, 15H), 2.28 (t, J = 7.4 Hz, 2H),
6
3
(
5
25
06.21991; [α]D +31.6 (c=1.0, DMSO).
Fmoc-L-Lys(Biotin)-OH (3c):Yield 85%; m.p. 178–180 °C (Ref[8]:
1
1
81–182 °C) ; H NMR (DMSO-d ): δ 1.25–1.36 (m, 6H), 1.37–1.50
6
(
m, 3H), 1.55–1.66 (m, 2H), 1.65–1.72 (m, 1H), 2.04 (t, J = 7.6 Hz,
Experimental
2H), 2.56 (d, J = 12.4 Hz, lH), 2.81 (dd, J = 12.2, 5.2 Hz, 1H, ), 2.96–
3.04 (m, 2H), 3.04–3.11 (m, 1H), 3.86–3.94 (m, 1H), 4.08–4.14 (m,
1H), 4.18–4.32 (m, 4H), 6.35 (s, 1H), 6.41 (s, 1H),7.29–7.45 (m, 4H),
Melting points were measured on a type YRT-3 melting point
1
apparatus. The H NMR (400 MHz) spectra were recorded on a JNM-
25
ECA-400 spectrometer in DMSO-d with tetramethylsilane as internal
7.58–7.92 (m, 6H), 12.55 (s, 1H); [α]
D
+15.6 (c=1.0, DMSO).
6
standard. Mass spectra were performed on a type JMS-700 instru-
ment. HPLC was performed on a Shimadzu LC-10AT VP Plus liquid
chromatograph system with a Wondasil 4.6 mm × 150 mm C18
column. Optical rotations were measued by a Polaar 3005 polarimeter
We thank the National Key Technologies R&G Program
for New Drugs of China (No. 2009ZX09301-002 and
2
009ZX09503-015) for financial support.
(
Optical Activity Limited). Fmoc-D-Aph and Boc-L-Aph were
prepared according to ref. 9 from D-Phe and L-Phe respectively.
Fmoc-L-Lys were purchased from Chengnuo Biochem (Chengdu)
Ltd.. HBTU, BOP, DCC, HOBt, EDCHCl and DIEA were purchased
Received 9 April 2010; accepted 18 May 2010
Paper 1000061 doi: 10.3184/030823410X12759895186422
Published online: 2 July 2010
.
from GL Biochem (Shanghai) Ltd.

3
50 JOURNAL OF CHEMICAL RESEARCH 2010
5
6
7
Y.S. Youn, S.Y. Chae, S. Lee, M.J. Kwon, H.J. Shin and K.C. Lee, Eur.
J. Pharm. Biopharm., 2008, 68, 667.
References
1
2
3
4
G.J. Russell-Jones, S.W. Westwood, P.G. Farnworth, J.K. Findlay and
H.G. Burger, Bioconjug. Chem., 1995, 6, 34.
G.J. Russell-Jones, S.W. Westwood and A.D. Habberfield, Proc. Int. Symp.
Control. Release Bioact. Mater., 1996, 23, 49.
K.B. Chalasani, G.J. Russell-Jones, S.K. Yandrapu, P.V. Diwan and S.K.
Jain, J. Control Rel., 2007, 117, 421.
M.F. Francis, M. Cristea and F.M. Winnik, Biomacromol., 2005, 6, 2462.
M. Hashimoto, S. Okamoto, K. Nabeta and Y. Hatanaka, Bioorg. Med.
Chem. Lett., 2004, 14, 2447.
Z.X. Shen, Y.W. Zhang and C.G. Zhou, Chin. J. Syn. Chem., 2002, 10,
3
59.
8
9
S. Bhuniya, S.M. Park and B.H. Kim, Org. Lett., 2005, 7, 1741.
P. Singh, C.R. Hurrell, J.B.C. Findlay and C.W.G. Fishwick, Bioorg. Med.
Chem. Lett., 1997, 7, 715.

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