Propargylamine-isothiocyanate reaction: Efficient conjugation chemistry in aqueous media

Article abstract of DOI:10.1039/c4cc00863d

A coupling reaction between secondary propargyl amines and isothiocyanates in aqueous media is described. The reaction is high-yielding and affords cyclized products within 2-24 h. A functionalized ether lipid was synthesized in 8 steps, formulated as liposomes with POPC and conjugated to FITC under mild conditions using this method. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00863d

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Propargylamine–isothiocyanate reaction: efficient
conjugation chemistry in aqueous media†
H. M.-F. Viart,a T. S. Larsen,a C. Tassone,b T. L. Andresenbc and M. H. Clausen*ac
Cite this: Chem. Commun., 2014,
50, 7800
Received 1st February 2014,
Accepted 28th May 2014
DOI: 10.1039/c4cc00863d
www.rsc.org/chemcomm
A coupling reaction between secondary propargyl amines and iso-
thiocyanates in aqueous media is described. The reaction is high-
yielding and affords cyclized products within 2–24 h. A functionalized
ether lipid was synthesized in 8 steps, formulated as liposomes with
POPC and conjugated to FITC under mild conditions using this method.
Scheme 1 Propargyl amine–isothiocyanate reaction to form thiazolidines.
For bioconjugation purposes, and in the development of green
chemistry, it is important to have a toolbox of efficient high-
yielding reactions that can occur in aqueous media.1 It is an
advantage, if these reactions are stoichiometric in nature, as
many bioactive molecules used in conjugation reactions represent a
significant investment, financially and/or in terms of effort. Exam-
ples of such reactions are the Cu-catalyzed2 (or strain promoted)3
Huisgen cycloaddition, the Staudinger ligation,4 thiol addition to
maleimide5 and amide formation from NHS‡ esters and amines.6
Another popular method, which is used frequently for labeling
biomolecules with fluorescent dyes, is the reaction between isothio-
cyanates and amines to afford a thiourea functionality while forging
a covalent linkage between the dye and the molecule containing the
amino group.7 We are interested in expanding the conjugation
chemistries available and came across an old reaction, which
hitherto has only been described in organic solvents, often under
forcing conditions: the reaction between isothiocyanates (ITCs)
and propargyl amines to afford 2-imino-4-methylenethiazolidines
or 2-amino-4-methylenethiazolines, Scheme 1.
with antihypertensive properties and CNS depressant activity. They
tested a variety of different propargyl amine derivatives which in
general yielded the 4-methylenethiazolidine product, but in some
cases a small quantity of endocyclic double bond isomers was
isolated and for two reactions this was the sole product observed.
We were interested in studying whether this transformation could be
used as a conjugation method in aqueous media, since isothio-
cyanates are often used for functionalizing amines and a number of
ITC-functionalized fluorophores are commercially available. Here
we report the successful application of this reaction in water and
demonstrate, as an example of its use, that it is possible to func-
tionalize liposomes with a fluorophore in a very convenient manner
using this conjugation method. We also evaluate the scope of the
reaction, with regards to different buffer systems and a polyfunc-
tional decapeptide carrying a propargyl amine.
Mixing propargyl amine (1) and fluorescein isothiocyanate
(FITC, 3) in water/tert-butanol 3 : 2 and stirring for 4 h afforded the
desired thiazolidine product 4 in 81% isolated yield after column
chromatography (Scheme 2). Due to the modest solubility of FITC
in water, initial investigation of the reaction was done in mixed
This transformation was first disclosed by Dillard and
co-workers in 1964,8 and later studied by Eloy and Deryckere.9
In 1976 the group of Honkan10 published a study on the synthesis of
different thiazolidine derivatives related to clonidine, a compound
a Department of Chemistry, Technical University of Denmark, Building 207,
DK-2800 Kgs. Lyngby, Denmark. E-mail: mhc@kemi.dtu.dk; Tel: +45 4525 2131
b Department of Micro- and Nanotechnology, Technical University of Denmark,
Building 423, DK-2800 Kgs. Lyngby, Denmark
c Center for Nanomedicine and Theranostics, Technical University of Denmark,
Denmark
† Electronic supplementary information (ESI) available: Detailed description of
the synthetic approach to compounds 4–9 and 11–13 and their characterization.
See DOI: 10.1039/c4cc00863d
Scheme 2 Initial reactions with FITC.
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Table 1 Competition experiments in aqueous solventsa
Entry
Solvent
Conversion FITC 24 h (%)
Ratio 5 : 6
1
2
3
4
H2O
72b
96
498
498
2 : 1
2 : 1
3 : 1
9 : 1
HEPES, pH 7.0
Borate, pH 8.3
PBS, pH 7.4
a
Mixtures of ethanolamine (1 equiv.), propargyl amine 2 (1 equiv.) and
FITC (0.5 equiv.) in four different aqueous solutions (0.5 mM FITC)
were reacted at 20 1C and monitored by HPLC and LC-MS (see ESI).
Fig. 1 Minor thiourea product formed from ethanolamine.
b
Conversion 498% after 48 h.
organic–aqueous solvent systems. The thiourea product was
not formed and no evidence of alkene isomerization to the
iminothiazole was observed.
Having established the feasibility of this transformation in the
presence of water, we next tested the performance of a secondary
amine, N-propargyl ethanolamine (2).11 Under identical reaction
conditions, the reaction was done in 2 h and 5 was subsequently
isolated in 78% yield.
Based on these initial promising results we decided to inves-
tigate whether the thiazolidene formation was competitive with
the formation of thioureas in the presence of a primary amine as a
competing nucleophile. Incubation of FITC with a 1 : 1 mixture of
ethanolamine and propargyl amine 2 afforded a 1 : 10 mixture of
thiourea 6 (Fig. 1) and thiazolidine 5, demonstrating that the
thiazolidene formation was the dominant reaction pathway.
We also subjected 2-(but-2-ynylamino)ethanol 7 (ref. 12) to
our standard reaction conditions and after 3 h isolated a 2 : 3
mixture of the thiourea 8 and the thiazolidine 9 (Scheme 3).
Increasing the reaction time to 24 h led to complete formation
of the thiazolidine with no thiourea product detected, which
strongly indicates that the reaction takes place in a step-wise
fashion where the thiazolidene is formed from an initial thiourea
intermediate.
We next wanted to test whether this reaction could be used
for bioconjugation under mild, aqueous conditions at ambient
temperature. Liposome surface functionalization was chosen as
model system since it assesses both the performance in buffer
and the feasibility of conjugation to nanoparticles. We decided
to synthesize the propargyl amine functionalized lipid 13 that
could be incorporated in a lipid bilayer and subsequently conju-
gated to FITC. The synthesis can be seen in Scheme 4 and started
from tetraethylene glycol 10, which was monobenzylated and
mesylated to afford 11 in 89% yield, an improvement over a
published method.13 Reaction of isopropylidene glycerol and 11
followed by deprotection and alkylation with 1-bromooctadecane
yielded the sn-3 PEGylated dietherlipid 12. Hydrogenolysis,
mesylation and nucleophilic displacement with propargyl amine
afforded the desired lipid 13.
The propargyl amine functionalized lipid 13 (1%) was mixed
with POPC (99%) and formulated as liposomes by extrusion in
PBS buffer using the dry lipid film technique,14 yielding clear
dispersions. The particle size of the formulated lipids was
measured by DLS. The formed liposomes had a mean diameter
of 145 nm (PDI: 0.245). The functionalized liposomes (c = 12.5 mM)
were incubated with FITC (31.25 mM; 0.5 equiv.) and samples were
taken for HPLC analysis after 5, 30, 60, 120, 240 and 1440 minutes.
The analysis showed that after 5 min, more than 50% of the FITC
(3, RT = 6.0 min) had been converted to a major (RT = 18.6 min)
and a minor (RT = 17.0 min) product. After 4 h, all the FITC had
been converted to the two products and after 24 h, only the fast
eluting (RT = 17.0 min) product was detected, see Fig. 2. We
performed the reaction between the propargyl amine functionalized
The performance of the reaction in a range of aqueous buffers
was also evaluated. The results are summarized in Table 1 and
reveal some interesting differences.
The reaction is generally complete after 24 h, but in non-buffered
water, it is slower (entry 1). The selectivity between the thiazolidine
product 5 and the thiourea 6 ranged between 2 : 1 (entries 1 and 2)
and 9 : 1 (entry 4), performing best in PBS buffer.
Scheme 3 Experiment with an internal triple bond.
Scheme 4 Synthesis of the propargyl amine functionalized lipid 13.
Chem. Commun., 2014, 50, 7800--7802 | 7801
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was observed, which could be confirmed by MALDI-TOF-MS
(see ESI†). However, it was also clear from these experiments
that the reaction did not have a high selectivity for the propargyl
amine. Three main products could be detected by LC-MS in an
approximate ratio of 1 : 1 : 2. Trypsin digestion of the product
mixture confirmed that the N-terminal thiazolidine was formed
as a major product (see ESI†).
In summary, the results described here offer a new way of
conjugating isothiocyanates, exemplified by FITC, to propargyl
amine functionalities. The reaction is atom economic, takes
place under mild reaction conditions in aqueous solutions
without the need for additives or catalysts and is compatible
with complex structures as seen for the functionalized lipid 13
incorporated in the bilayer of liposomes. Our investigations
also show that in the presence of several competing nucleo-
philes, such as the primary amine of lysine side-chains, the
selectivity of the reaction is limited. Nonetheless, we believe
this convenient reaction can find use in conjugation chemistry
where mild reaction conditions are required.
Fig. 2 Example of the monitoring of a conjugation reaction over time by
HPLC at 254 nm. The decreasing peak with RT = 6.0 min belongs to the free
fluorescein isothiocyanate (FITC, 3), while the peaks with RT = 17.0 min and
RT = 18.6 min are the 2-imino-4-methylenethiazolidine and the intermediate
thiourea, respectively.
lipid 13 and FITC in solution (see ESI†), which enabled us to
identify the faster eluting compound (RT = 17.0 min) as the
desired product. HPLC-ESI-MS showed that both peaks had
the same mass, 1198.8, which allowed us to assign the peak at
RT = 18.6 min as the intermediate thiourea. The integrity of the
liposomes after 24 h incubation with FITC were verified by DLS
and showed no significant change in the average size or poly-
dispersity of the particles (mean diameter: 148 nm, PDI: 0.231).
Fluorescent microscopy of FITC-functionalized liposomes can
be seen in Fig. S1 in ESI.†
For conjugation purposes it is common that several functional
groups are present and we wanted to evaluate the scope and
limitation of the approach reported here. Therefore, we prepared
the protected resin-bound decapeptide 14, see Scheme 5 (side-chain
protection groups omitted for clarity). This was functionalized
through reaction with bromoacetic acid and propargyl amine15
and cleaved from the resin with Reagent K16 to afford decapeptide
15. The eight amino acid residues from the N-terminal end are
identical to residues 7–14 in histone class H3 tails, known for a high
number of lysines.17 The C-terminal tyrosines were added to ease
detection by HPLC-DAD.
We thank Professor Christian A. Olsen and Dr Jonas S. Laursen,
University of Copenhagen, for very helpful discussion about
the peptide design and synthesis. We thank Martin Bak, DTU
Nanotech, for MALDI-TOF-MS analyses.
Notes and references
‡ Abbreviations: CNS, central nervous system; DAD, diode-array detector;
DIPEA, diisopropylethylamine; DLS, dynamic light scattering; ESI-MS,
electrospray-ionization mass spectrometry; FITC, fluorescein isothio-
cyanate; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);
HPLC, high-performance liquid chromatography; ITC, isothiocyanate;
MALDI-TOF-MS, matrix-assisted laser-desorption mass spectrometry;
NHS, N-hydroxysuccinimide; PBS, phosphate buffered saline; PDI, poly-
dispersity index; PEG, polyethylene glycol; POPC, 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine; RT, retention time, TFA, trifluoroacetic acid.
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When peptide 15 was reacted with an excess of FITC in PBS
buffer, full conversion of the peptide was observed and products
of both mono- and difunctionalization was observed. In contrast,
when FITC was the limiting reagent, only monofunctionalization
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Scheme 5 Synthesis of the propargyl amine functionalized peptide 15.
7802 | Chem. Commun., 2014, 50, 7800--7802
This journal is ©The Royal Society of Chemistry 2014