N-terminus FITC labeling of peptides on solid support: the truth behind the spacer

Article abstract of DOI:10.1016/j.tetlet.2008.10.141

Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has wide ranging application in biochemistry. It has been extensively used to label peptides and proteins. However, its use in solid phase peptide synthesis is restr

Full text of DOI:10.1016/j.tetlet.2008.10.141

Tetrahedron Letters 50 (2009) 260–263
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N-terminus FITC labeling of peptides on solid support:
the truth behind the spacer
Magali Jullian b, Anaïs Hernandez b, Amélie Maurras b, Karine Puget b,
Muriel Amblard a, Jean Martinez a, Gilles Subra a,
*
a Institut des Biomolécules Max Mousseron UMR 5247, CNRS, universités Montpellier 1 et 2, 15 Avenue Charles Flahault BP 14491, 34093 Montpellier Cédex 5, France
b Genepep ZA Les Baronnes, 34730 Prades le Lez, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has wide ranging
application in biochemistry. It has been extensively used to label peptides and proteins. However, its use
in solid phase peptide synthesis is restricted. Indeed, in acidic conditions required for linker cleavage, N-
ter FITC-labeled peptides undergo a cyclization leading to the formation of a fluorescein with subsequent
removal of the last amino acid. This can be avoided when a spacer such as amino hexanoic acid is used or
if non-acidic cleavage is operated to release targeted peptide from the resin.
Received 2 October 2008
Revised 28 October 2008
Accepted 29 October 2008
Available online 3 November 2008
Keywords:
FITC
Ó 2008 Elsevier Ltd. All rights reserved.
Fluorescein isothiocyanante
Peptide labeling
SPPS
HMBA linker
Thiohydantoin
Peptide synthesis
Side reaction
Fluorescent dyes are major tools for the labeling of biomole-
cules. Fluorescein derivatives are probably the most widely used
of this kind of reagents displaying a wide range of applications in
fluorescence microscopy,1 flow cytometry,2 and immunofluores-
cence-based3 assays. The fluorescein isothiocyanate (FITC) is more
reactive than the carboxy fluorescein which, moreover, has to be
activated before use. FITC was used extensively to react with sulf-
hydryl4 targeting reduced cysteine side chains and especially ami-
no groups in peptides or proteins.5 For numerous applications, it is
convenient to introduce fluorescent label during the chemical syn-
thesis.6,7 FITC may react during the course of solid phase peptide
synthesis, either with a lysine or an ornithine side chain after selec-
tive unmasking of protecting group8 or with a primary amino
group at the N-terminal side of the growing peptide. In the latter
case, an alkyl spacer such as aminohexanoic acid (Ahx) is intro-
duced between the last amino acid and the thiourea linkage gener-
ated through the reaction of isothiocyanate and amine.9 This is
traditionally justified to take away the bulky fluorescent dye from
the bioactive sequence.
SPPS. FITC was introduced at the N-terminal side of anchored pep-
tides. In this strategy, we believed that a degradation could happen
during TFA by a mechanism similar to Edman degradation induced
by phenylthioisocyanate.10 The proposed mechanism is detailed in
Scheme 1 and yields fluorescein thiohydantoïne (FTH) 4. A recent
patent drew profit from this reaction in solution to propose a
method for analyzing protein adducts.11 It is worth noting that
some FTH were already obtained in the late seventies from unpro-
tected aminoacids or from albumin.12
In order to evaluate the importance of this side reaction on solid
support, we performed a study on five model peptides (10a–e),
which are synthesized on Fmoc-Rink amide PS resin (0.98 mol/g).
After classical Fmoc-SPPS,13 an aliquot (ꢀ10 mg) of each pepti-
dyl-Rink amide PS resin 1a–e was submitted to TFA/TIS/H2O
95:2.5:2.5 for 2 h to check the purity of model peptides. After
diethyl ether precipitation of crude peptides and lyophilization,
LC/MS analyses showed that expected unprotected peptides
10a–e were obtained with good purity (Table 1).
Resins 1a–e were then treated with FITC and DIEA in the dark
for 2 h to yield supported compounds 2a–e. At this stage, only DIEA
and FITC used in excess were detected in coupling solution but no
thiohydantoïne 4a–e. Moreover, Kaiser test14 performed on resin
beads 2a–e was negative, indicating the efficiency of isocyanate
coupling on the primary amine. Then, resins 2a–e were submitted
As part of a project that aimed to design new cell penetrating
peptides, we synthesized a series of fluorescent peptides by Fmoc
* Corresponding author. Tel.: +33 4 67 54 86 55; fax: +33 4 67 54 86 54.
E-mail address: gilles.subra@univ-montpl.fr (G. Subra).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.10.141

M. Jullian et al. / Tetrahedron Letters 50 (2009) 260–263
261
HO
1a-e
O
H+, TFA cleavage
Rp
O
O
FITC
H
N
O
peptide-NH-Rink-
TFA,TIS,water
H2N
H2N
HO
O
S
Rp
O
H
O
O
R'p
N
peptide-NH-Rink-
O
O
N
H
N
H
HO
O
R'p
R
O
2a-e
N
H+
H
N
C
S
HO
peptide-NH2
1'a-e
R'
O
H
R
O
S
O
H2N
H
peptide-NH2
N
peptide-NH2
R'
HN
R'
3a-e
O
truncated peptide
N
R
O
HO
O
HN
N
S
O
O
S
O
O
NH
O
N
R
HO
OH
HO
O
O
O
4a-e
OH
OH
fluorescein thiohydantoin
fluorescein thiazolinone
Scheme 1.
Table 1
Model peptides 10a–e in Scheme 1
Compound
Sequence and MW calc.
Ions detected (ESI+)
Puritya (%)
10a
10b
10c
10d
10e
Neurokine A H-HKTDSFVGLM-NH21132.6 Da
Neurokine B H-DMHDFFVGLM-NH2 1209.5 Da
Fulicine H-FNEFV-NH2 653.3 Da
Nle CCK4 H-W Nle DF-NH2 578.4
FLPAG peptide H-FLPAG-NH2 502.3
[M+H]+ m/z = 1133.3; [M+2H]2+ m/z = 516.7
[M+H]+ m/z = 1210.5; [M+2H]2+ m/z = 605.8
[M+H]+ m/z = 654.3; [M+2H]2+ m/z = 319.3
[M+H]+ m/z = 579.2
99
90b
96
96
97
[M+H]+ m/z = 503.2; [2M+H]+ m/z = 1005.6
a
Purity percent was estimated by integration of peak area in LC/MS chromatogram recorded at 214 nm.
10% of oxidation of methionine was detected.
b
Table 2
Cleavage products of resin 2a–e
Truncated peptide 3
MW calc.
Ions detected (ESI+)
Puritya (%)
MW calc.
Ions detected (ESI+)
S
O
NH
N
R
O
HO
O
O
OH
Hydantoïne 4
[M+H]+ m/z = 527.2
H
N
3a, H-KTDSFVGLM-NH2
995.5
[M+H]+ m/z = 996.7 [M+2H]2+
m/z = 498.9
63b
4a, R =
526.1
[M+2H]2+ m/z = 264.1
N
O
3b, H-MHDFFVGLM-NH2
3c, H-NEFV-NH2
1094.5
506.3
392.2
355.2
[M+H]+ m/z = 1095.4 [M+2H]2+
m/z = 584.3
71b
96
62
75
4b, R =
4c, R =
4d, R =
4e, R =
504.5
536.1
575.2
536.1
[M+H]+ m/z = 505.1
[M+H]+ m/z = 537.2
[M+H]+ m/z = 576.2
[M+H]+ m/z = 537.1
HO
[M+H]+ m/z = 507.2 [2M+H]+
m/z = 1013.7
3d, H-Nle DF-NH2
[M+H]+ m/z = 393.1 [2M+H]+
m/z = 785.5
HN
3e, H-LPAG-NH2
[M+H]+ m/z = 356.3
a
Purity percent was estimated by integration of peak area in LC/MS chromatogram recorded at 214 nm. Yield ranged from 49% to 55% calculated on the basis of theoretical
resin loading.
b
10–15% of oxidation of methionine was detected.

262
M. Jullian et al. / Tetrahedron Letters 50 (2009) 260–263
Rp
O
H
N
peptide-NH-Rink-
H2N
O
R'p
1a-e
1) Fmoc-Ahx-OH/DIC/HOBt
2) DMF/pip 80/20
1) Fmoc-betaAla-OH/DIC/HOBt
2) DMF/pip 80/20
Rp
O
O
Rp
O
O
H
H
N
peptide-NH-Rink-
N
peptide-NH-Rink-
H2N
N
H2N
N
H
H
R'p
O
R'p
O
1) FITC/DIEA
2) TFA/TIS/H2O
1) FITC/DIEA
2) TFA/TIS/H2O
HO
HO
O
O
O
O
O
O
S
O
R
S
O
R
O
O
H
H
peptide-NH2
N
N
peptide-NH2
N
N
N
N
N
H
N
H
H
H
H
H
HO
HO
O
R'
O
R'
6a-e
5a-e
Scheme 2.
to TFA/TIS/H2O treatment for 2 h. Thiohydantoins 4a–e and trun-
cated peptides 3a–e were detected by LC/MS analysis of an aliquot
of TFA cleavage solution (Scheme 1). We noticed that diethyl ether
addition led to precipitation of truncated peptides 3a–e (Table 2).
Thiohydantoins 4a–e were soluble in ether and after evaporation
of the discarded diethylether phase, their presence was confirmed
by LC/MS analysis.
and average yield of 55%. The alkylene spacer has no incidence
on the coupling efficiency of FITC on the free amino group at the
N terminus of supported peptides.
According to our observations, the removal of the last amino
acid by thiohydantoin formation occurs during final TFA treatment.
As an evidence, model peptide 100e (identical to 10e, previously ob-
tained on Rink amide linker) was synthesized on hydroxymethyl
benzamido-PS-resin (HMBA-PS 1.1 mmol/g) using Fmoc SPPS
(Scheme 3). The use of the base-labile linker HMBA15 enabled to
cleave the peptide from resin 7 using ammonia-saturated metha-
nol solution for 4 h (purity = 81%, yield = 40%).16 Resin 7 reacted
as usual with FITC and DIEA in the dark for 2 h to yield fluores-
cent-immobilized peptidyl-resin 8.
The thiazolinone mechanism is driven by the formation of the
five-membered cycle only possible when the last residue in the
peptide sequence is an
a-amino acid. To complete this study, pep-
tidyl-resins 1a–e were coupled with a
e
-amino or a b-amino acid
(Fmoc-Ahx-OH and Fmoc-bAla-OH, respectively) and were reacted
with FITC (Scheme 2). TFA-mediated cleavage afforded the ex-
pected fluorescent peptides 5a–e and 6a–e, respectively. After
LC/MS analyses, no truncated peptides 3a–e were detected, and
the labeled peptides were obtained with high purity (Table 3)
O
MeOH/NH3
H-FLPAG-NH2
HN
O
Leu Pro Ala Gly
HN
1''e
HO
Table 3
O
7
FITC-labeled peptides 5 and 6 (Scheme 2)
FITC, DIEA
2 hours
Compound
MW calc.
1593.6
Ions detected (ESI+)
Puritya (%)
99
O
O
5a
[M+2H]2+ m/z = 797.4
[M+3H]3+ m/z = 532.17
[M+2H]2+ m/z = 836.0
[M+3H]3+ m/z = 557.7
[M+H]+ m/z = 1114.5
[M+2H]2+ m/z = 557.8
[M+H]+ m/z = 1039.5
[M+2H]2+ m/z = 520.4
[M+H]+ m/z = 963.5
[M+2H]2+ m/z = 482.3
[M+2H]2+ m/z = 818.7
[M+3H]3+ m/z = 546.1
[M+H]+ m/z = 857.0
[M+2H]2+ m/z = 571.7
[M+H]+ m/z = 1156.65
[M+2H]2+ m/z = 578.9
[M+H]+ m/z = 1081.5
[M+2H]2+ m/z = 541.4
[M+H]+ m/z = 1005.6
[M+2H]2+ m/z = 503.4
O
O
S
5b
5c
5d
5e
6a
6b
6c
6d
6e
1669.6
1113.4
1038.4
962.4
63
95
95
96
99
99
97
95
96
N
H
N
Leu Pro Ala Gly
O
HN
H
HO
O
8
S
O
NH
N
O
TFA
HO
O
MeOH/NH3
9
1634.6
1711.6
1155.4
1080.4
1004.4
O
OH
HO
O
O
O
S
N
N
Leu Pro Ala Gly NH2
H
H
HO
O
10
a
Purity percent was estimated by integration of peak area in LC/MS chromato-
gram recorded at 214 nm.
Scheme 3.

M. Jullian et al. / Tetrahedron Letters 50 (2009) 260–263
263
TFA treatment performed on an aliquot (10 mg) of resin 8 led to
References and notes
a yellow TFA solution and white resin beads. After TFA evaporation
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the main product and no peptide was released from the resin as ex-
pected. Ammonolysis of the same resin afforded expected fluores-
cent peptide 10 with 86% purity and 71% yield.
In conclusion, we highlighted a side reaction occurring upon
TFA-mediated cleavage of N-terminal FITC-modified peptides on
solid support. This reaction follows the Edman degradation path-
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way affording a fluorescent thiohydantoin (FTH) from the last
amino acid of the peptide sequence. As expected, this reaction
can be avoided by introduction of a non- amino acid spacer be-
a-
a
tween thiourea bond and the peptide or by using basic conditions
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reason why FITC should not be coupled directly on peptide.
Ahx or bAla were used successfully as spacers to generate
FITC-labeled peptides. We also demonstrated that fluorescein-
NH-CS-peptides can be obtained using base-labile linker HMBA.
It is obvious that this last method suffers consistent limitations
as appropriate non-acid labile side chain protecting groups are
required to avoid TFA treatment of FITC-labeled peptide.
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16. 9% C-ter free acid peptide was detected by LC/MS as a result of hydrolysis of
HMBA linker.