N-Fmoc-α-sulfo-β-alanine: A versatile building block for the water solubilisation of chromophores and fluorophores by solid-phase strategy

Article abstract of DOI:10.1039/c1ob05730h

An easy and efficient solid-phase synthesis strategy to obtain rapidly water-soluble chromophores/fluorophores in highly pure form has been developed. This first successful use of N-Fmoc-α-sulfo-β-alanine as a SPPS building block opens the way to the futu

Full text of DOI:10.1039/c1ob05730h

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N-Fmoc-a-sulfo-b-alanine: a versatile building block for the water
solubilisation of chromophores and fluorophores by solid-phase strategy†
Anthony Romieu,*a,b Thomas Bruckdorfer,d Guillaume Clave´,a Virgile Grandclaude,a Ce´drik Massifa and
Pierre-Yves Renarda,b,c
Received 9th May 2011, Accepted 27th May 2011
DOI: 10.1039/c1ob05730h
An easy and efficient solid-phase synthesis strategy to obtain
rapidly water-soluble chromophores/fluorophores in highly
pure form has been developed. This first successful use of N-
Fmoc-a-sulfo-b-alanine as a SPPS building block opens the
way to the future development of promising direct “on-resin”
peptide labelling and water-solubilising methods.
valuable properties. In this context, we have recently reported a
straightforward method to enhance the water solubility of organic
dyes by a post-synthetic chemical derivatisation of their carboxylic
acid functionality (or equivalent) with a poly-sulfonated peptide-
based linker (i.e., a-sulfo-b-alanine di- or tripeptide) through
a Schotten–Baumann reaction performed in aq. or organic
media.9–11 Despite the efficiency of this “solution-phase” synthetic
strategy, the laborious and time-consuming handling and purifi-
cation of highly polar sulfonated intermediates prevents the rapid
generation of libraries of bio-conjugatable dyes in high purity.
This major drawback could be circumvented by using a more
practical approach based on a solid-phase methodology. Indeed,
Cardenas-Maestre and Sanchez-Martin have recently reported the
synthesis of naphthofluorescein derivatives bearing various bio-
conjugatable moieties (i.e., amino, carboxylic acid and sulfhydryl
groups) in high yield and purity, through an efficient solid-phase
strategy involving the use of 2-chlorotrityl resin.12 Furthermore,
the “catch-and-release” method is now well established as the
preferred method for the synthesis of unsymmetrical cyanine dyes
in good yields.13 However, a solid-phase synthesis approach has
never been applied to the derivatisation of fluorophores with
water-solubilising moieties whereas this latter methodology would
be helpful to get rapidly these valuable fluorescent markers in
high yield and with a purity which does not necessarily require
chromatographic purification.
Herein, we report the implementation of this practical approach
for the water solubilisation of inexpensive commercially available
chromophores/fluorophores (i.e., anthracene, benzophenone, 7-
hydroxycoumarin and naphthalene) and more functionalised “in
house” fluorescent organic dyes (i.e., rhodamine 6G (R6G) and
non-symmetrical pentamethine cyanine dye analogues)14 covering
a broad spectral range from the UV to the near-infrared (NIR)
region. Furthermore, the choice of these polycyclic aromatic
compounds has been also guided by their well-known stability
under harsh anhydrous acidic conditions (i.e., TFA) currently
used for releasing products from standard resins involved in solid-
phase peptide synthesis (SPPS). The successful use of N-Fmoc
protected a-sulfo-b-alanine 1 both in manual and automated SPPS
constitutes the cornerstone of this synthetic strategy.15 The optical
properties of the resulting water-soluble fluorophores were then
evaluated under physiological conditions to demonstrate their
potential utility as bio-labelling reagents.
Introduction
Over the last decade, fluorescence reporter technologies (especially
fluorescent bio-probes, polypeptide tags and reporter proteins)
have become very powerful and useful tools in life science research,
especially in the context of challenging bio-analytical and medical
applications.1 Thus, the covalent labelling of natural biological
molecules or related biopolymers with fluorescent organic dyes
(i.e., fluorescent bio-labelling) has progressively emerged as a
convenient alternative to radioisotope labelling and is now widely
adopted to generate molecular probes of biological interest.2
Among the myriad of commercial and/or published fluorophores,
some of them exhibit interesting photophysical properties (yet
mainly in organic solvents) but few of them fulfil all the require-
ments for their routine use as bio-labelling reagents: good water
solubility, resistance to aggregation, high fluorescence emission
in aq. media and the presence of a functional group readily and
selectively derivatisable under mild bio-compatible conditions.
In recent years, some academic groups and private biotech
companies have revisited the chemistry of well-known fluorescent
cores such as BODIPY,3,4 coumarin,5 cyanine,6 oxazine7 and
xanthene dyes,8 to synthesise new analogues with these latter
aEquipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038,
rue Lucien Tesnie`re, 76131 Mont-Saint-Aignan, France. E-mail: anthony.
romieu@univ-rouen.fr; Fax: +33 2-35-52-29-71; Tel: +33 2-35-52-24-27;
Web: http://ircof.crihan.fr
bUniversite´ de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan,
France
cInstitut Universitaire de France, 103 Boulevard Saint-Michel, 75005, Paris,
France
dIris Biotech GmbH, Waldershofer Str. 49-51, 95615 Marktredwitz, Ger-
many; Web: www.iris-biotech.de
† Electronic supplementary information (ESI) available: Detailed synthetic
procedures and characterisation/spectral data for compounds 1–14 and
10-BSA conjugate. See DOI: 10.1039/c1ob05730h
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moiety of the Wang resin (effect of the free sulfonate group?).
Such hypothesis was confirmed by a solution-phase BOP/DIEA-
mediated coupling reaction between 1 and 4-methoxybenzyl
alcohol, which gave only the benzotriazole ester (Bt) 2 as the
main isolated product. Indeed, this latter compound is formally
the reduced derivative of HOBt esters and its formation is known
to be favoured by the absence of strong enough nucleophiles in the
coupling reaction mixture.22
Results and discussion
Since SPPS protocols require the use of protected amino acid
building blocks in large excess (generally, a 4- to 10-fold molar
excess is used), we first revisited the synthesis of 1 previously
reported by us,9 in order to produce this N-Fmoc building block
in large scale (10 g scale synthesis) and in a very cheap and
simple manner. Thus, we found that the use of 9-fluorenylmethyl
chloroformate (Fmoc-Cl) instead of the succinimidyl carbonate
derivative (FmocOSu) enables us to get 1 in quantitative yield
and high purity (>95%) from the corresponding a-sulfo-b-
alanine through simple washing with CH3CN and subsequent
recrystallisation in water and lyophilisation of the crude product.
Next, the anchoring of this unusual N-Fmoc sulfonated amino
acid to the Wang resin was studied (Scheme 1).16 Surprisingly, use
of standard esterification conditions (i.e., DIC/DMAP) allowing
the in situ formation of the corresponding symmetrical anhydride
of 1 completely failed.17 Three other immobilisation methods,
namely: (1) BOP/DIEA (in situ formation of HOBt active esters
of 1),18 (2) Boc2O/pyridine/DMAP (in situ formation of mixed
carbonic carboxylic anhydride of 1)19 and (3) SN2 reaction of
the Wang chloride resin with 1 in the presence of KI/DIEA,20
were attempted but failed too except for the SN2-alkylation which
gave the resin-bound product in a very poor and unusable yield
(5%). At this stage of the study, we do not have definitive relevant
explanation(s) for these surprising negative results because N-
Fmoc b-amino acids such as b-alanine are known to behave like
the corresponding a-amino acids in SPPS.21 However, we suspect
that the active N- or O-acyl derivatives of 1 (i.e., HOBt esters
or anhydrides) are unreactive toward the 4-alkoxybenzyl alcohol
Concerning the disappointing result obtained with the SN2
approach, this hypothesis (vide supra) cannot be invoked because
the nucleophilic character of the carboxylate anion of 1 is the
key factor influencing the rate of this latter reaction. Thus, the
alternative hypothesis that the free sulfonate group could have a
deleterious effect on the stability of the ester linkage connecting the
sulfonated amino acid to the solid support seems also judicious.
To overcome this major synthetic difficulty, we decided to first
derivatise the Wang resin with a glycine spacer by using the
standard symmetrical anhydride method (i.e., DIC/DMAP), and
after removal of the Fmoc group, make an efficient peptide
coupling reaction with N-Fmoc a-sulfo-b-alanine 1 to give resin
3 (Scheme 2). BOP reagent in the presence of DIEA was found
to be effective in this reaction performed either automatically or
manually. Following Fmoc removal with 20% piperidine in NMP,
benzophenone-4-carboxylic acid 4 and R6G carboxylic acid 5
readily reacted with aminosulfonate-resin through BOP/DIEA
activation.23 The same protocol was used for the solid-phase
immobilisation of other fluorophores but to counterbalance
their higher hydrophobic character, the resin bearing (a-sulfo-
b-alaninyl)2-glycinyl spacer arm was preferred (Scheme 2). It is
interesting to note that these coupling reactions, especially those
involving sterically hindered dyes such as 5 and 8, are more efficient
Scheme 1 Reagents and conditions: (a) 1 (4 equiv.), DIC (2 equiv.), DMAP (0.15 equiv.), NMP, rt, overnight; (b) 1 (4 equiv.), BOP (4 equiv.), DIEA (12
equiv.), NMP, rt, overnight; (c) 1 (5 equiv.), Boc2O (5 equiv.), pyridine (10 equiv.), DMAP (0.3 equiv.), DMF, rt, 19 h; (d) SOCl2 (5 equiv.), CH2Cl2, 4 ◦C,
45 min; (e) 1 (3 equiv.), KI (3 equiv.), DIEA (6 equiv.), DMF, rt, 24 h, 5% (overall yield for the steps d–e).
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Scheme 2 Reagents and conditions: (a) Fmoc-Gly-OH (4 equiv.), DIC (2 equiv.), DMAP (0.15 equiv), CH2Cl2–NMP (6 : 4), rt, overnight; (b)
piperidine–NMP (2 : 8), rt, 10 min; (c) 1 (4 equiv.), BOP (4 equiv.), DIEA (12 equiv.), NMP–DMF (55 : 45), rt, 30 min; (d) chromophore or fluorophore
4, 5, 6, 7 or 8 (5 equiv.), BOP (5 equiv.), DIEA (20 equiv.), NMP, rt, overnight; (e) TFA–CH2Cl2 (1 : 1), 4 ◦C to rt, 1 h followed by azeotropic evaporation,
lyophilisation and RP-HPLC purification.
when the carboxylic acid is converted into the corresponding
HOBt esters rather than the conventional N-hydroxysuccinimidyl
(NHS) ester (in situ formation with TSTU/DIEA)24 After cleav-
age from the resin using TFA–CH2Cl2 (1 : 1) and subsequent
azeotropic evaporation and lyophilisation, the desired sulfonated
chromophores/fluorophores 9–13 were obtained in satisfactory
yields (10–50% isolated overall yield from the underivatised Wang
resin and after RP-HPLC purification and lyophilisation) and
relatively good purity (70–80%), suitable for applications of these
dyes as bio-labelling reagents. However, additional reversed-phase
HPLC (RP-HPLC) purifications were carried out to provide
these compounds in a pure form (purity >95%) suitable for both
detailed characterisation (in particular NMR and mass spectrom-
etry data, see ESI†) and accurate measurement of their optical
properties.
In order to expand the scope of this water-solubilising method
to fluorogenic phenol based dyes for which the presence of a free
carboxylic acid moiety on the fluorophore core is not required for
bioconjugation, and with applications in mind based on the pro-
fluorescence concept,25 we have also investigated the derivatisation
of Rink Amide MBHA resin and its subsequent use in the sulfona-
tion of 7-hydroxycoumarin-4-acetic acid.26 Thus, the same peptide
coupling conditions used for the immobilisation of fluorophores
4–8 were applied to 7-hydroxycoumarin-4-acetic acid, and TFA
cleavage provided the desired water-soluble derivative 14 in good
overall yield (41%) and a high purity (see ESI†).
As expected, these novel sulfonated dyes were found to be per-
fectly soluble in water and related aq. buffers in the concentration
range (1.0 mM to 10 mM) suitable for bio-labelling applications,
except the N-protected Cy 5.5 amino acid 13 which requires the
use of CH3CN as a co-solvent to prepare a homogeneous 1.0 mM
stock solution (CH3CH–H2O, 2 : 1). All compounds bearing a
glycine tether are potentially useful for covalent labelling of bi-
ological material through the formation of carboxamide linkages.
As an illustrative example, water-soluble R6G 10 was converted
into the corresponding NHS ester whose the ability to readily react
with NH2 groups of lysine residues of proteins was demonstrated
through the fluorescent labelling of bovine serum albumin (BSA).
The attached fluorophore to protein molar (F/P) ratio and
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Table 1 Spectral properties of water-soluble chromophores/fluorophores 9–14 in PBS at 25 ◦
Compound max, abs/nm
C
a
l
e/dm3 mol-1 cm-1
l
max, em/nm
UF
b
b
263
23 250
—
—
528
65 000
554
0.70
235.5, 281.5
39 900, 5 500
124 400
368
0.23
0.12
254c
395, 416
632, 680
698d
65 860, 89 700
157 500d
700
0.05
712d
0.21d
338, 364
7800, 6800
467
0.77
a See ESI for the experimental details related to these measurements (standards, l ex/nm, . . . ). b Non fluorescent. c Other maxima were observed at longer
wavelengths but their e are very low (see ESI). d In PBS + 5% BSA.
quantum yield of the resulting fluorescent bio-conjugate were
found to be 0.5 and 0.24 respectively (see ESI† for the experimental
protocol and the corresponding spectra). Furthermore, water-
soluble and non-fluorescent chromophores such as benzophenone
9 are highly valuable (but seldom reported in the literature),
because these aryl ketones are key photoreactive components of
activity-based probes currently used in the identification of enzyme
activities in complex proteomes.27 The photophysical properties
of these novel water-soluble chromophore/fluorophores 9–14
were then determined under simulated physiological conditions
(i.e., phosphate buffered saline (PBS), pH 7.5) by using UV-vis
spectroscopy and spectrofluorimetric analysis (Table 1 and see
ESI† for the corresponding spectra). These results confirm that the
newly synthesised sulfonated derivatives behaved similarly to the
parent hydrophobic dyes. Indeed, molar absorption coefficients
(e) are comparable, and quantum yields in aq. buffers (UF) are
good especially for the water-soluble coumarin and rhodamine
10 and 14. Due to its amphiphilic character, cyanine 13 shows
a tendency to aggregate in aq. solution, which caused a severe
quenching of its fluorescence (Table 1, entry 5). The observed blue
shift in the absorption maximum at 632 nm is in keeping with
the formation of H-dimers. This aggregation is persistent even
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at concentrations as low as 1 mM in PBS. The free fluorescent
monomer of 13 is obtained in PBS using 5% (w/v) BSA, an
additive often used in buffers for mimicking body fluids. The
strong enhancement in emission in conjunction with the red shift
in the absorption maximum in the presence of BSA observed for
13 points to dye–BSA interactions. Such a beneficial effect of dye–
protein interactions on quantum yield has been already reported
for series of BODIPY, cyanine and rhodamine labels.4,11,28
Tetrahedron Lett., 2010, 51, 1269–1272; (k) S. Atilgan, T. Ozdemir and
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Anal. Chem., 2011, 83, 2852–2857; (b) A. R. Katritzky, J. Cusido and
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Renard, Bioconjugate Chem., 2008, 19, 279–289.
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Conclusions and future work
In conclusion, we have developed an easy and efficient method for
the sulfonation of inexpensive commercially available or readily
synthetically accessible chromophore and/or fluorescent cores, in
order to get rapidly and with high efficacy novel water-soluble
and bio-conjugatable optical markers. Thus, the full-compatibility
of new commercial N-Fmoc a-sulfo-b-alanine building block 1
with standard SPPS protocols was demonstrated for the first time
and we found that the use of a protecting group for the sulfonic
acid moiety is not required. The exemplification of this promising
strategy to more sophisticated and fragile fluorophores by using
an hyper acid-labile resin such as 2-chlorotrityl, SASRINTM or
Sieber resin is currently under investigation. Further efforts are
also devoted to the synthesis and SPPS applications of enzyme-
labile sulfonate esters29 derived from N-Fmoc a-sulfo-b-alanine in
order to produce cell-permeable (fluorescently-labelled) peptides
of biological interest.
Acknowledgements
This work was supported by the Agence Nationale de la
Recherche (Programme Blanc 2009, ANR-09-BLAN-0081-01)
especially for a PhD grant to Ce´drik Massif, La Re´gion Haute-
Normandie via the CRUNCh program (CPER 2007–2013) and
Institut Universitaire de France (IUF). Quidd biotech company
(http://www.quidd.com) is greatly acknowledged for the PhD
grant of Virgile Grandclaude whose contribution to this work is
out of the field of his PhD work. We thank Camille Leroux (bache-
lor of chemistry, Universite´ de Rouen, training period April–June
2010) for the total synthesis of symmetrical sulfobenzindocyanine
dye Cy 5.5 (Cy5.205) used as a standard for quantum yield
measurements.
15 For a review, see: G. B. Fields and R. L. Noble, Int. J. Pept. Protein
Res., 1990, 35, 161–214.
16 The solubility of 1 in DMF (or NMP) was found to be ca. 100 mg cm-3
which is a value compatible with automated SPPS protocols.
17 The loading of the N-Fmoc sulfonated amino acid on the resin was
determined by spectrophotometry through UV absorbance measure-
ments of piperidine–fulvene adduct at 301 nm (e/dm3 mol-1 cm-1 7100).
18 For a recent and comprehensive review on peptide coupling agents, see:
E. Valeur and M. Bradley, Chem. Soc. Rev., 2009, 38, 606–631.
19 M. d. l. A. Laborde, P. Bermejo, D. B. Boggian and E. G. Mata,
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21 For a selected example, see: L. D. Tilley, O. S. Hine, J. A. Kellogg, J. N.
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Agents Chemother., 2006, 50, 2789–2796.
22 J. Coste and J.-M. Campagne, Tetrahedron Lett., 1995, 36, 4253–4256.
23 In our case, ESI-MS analyses of the crude reaction mixtures did not
reveal the presence of the carboxamide derivative resulting from the
coupling reaction between chromophore/fluorophore and piperidine
which is theoretically the counter-ion of sulfonic acid moiety after
Fmoc removal. However, it is also possible to include an additional
Notes and references
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washing step with 10% DIEA in NMP before this coupling step or to
perform the Fmoc deprotection with a non-nucleophile base such as
DBU.
26 A glycine spacer is not needed because this amino resin reacts in a
similar manner to amino acid pre-loaded Wang resins.
27 For selected examples, see: (a) Y. Nakamura, S. Inomata, M. Ebine, Y.
Manabe, I. Iwakura and M. Ueda, Org. Biomol. Chem., 2011, 9, 83–85;
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Chem., 2004, 69, 7344–7347.
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Resch-Genger and I. Hilger, Eur. J. Med. Chem., 2009, 44, 3496–
3503.
24 The acyl transfer agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydro-
qunoline (EEDQ) could be also effective for such amidification
reactions. For an example of a difficult peptide-type coupling mediated
by this reagent, see: I. Villanueva, B. Hernandez, V. Chang and M. D.
Heagy, Synthesis, 2000, 1435–1438.
25 For a review, see: X. Chen, M. Sun and H. Ma, Curr. Org. Chem., 2006,
10, 477–489.
29 L. Rusha and S. C. Miller, Chem. Commun., 2011, 47, 2038–2040.
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