An improved synthesis of releasable luciferin-CPP conjugates

Article abstract of DOI:10.1016/j.tetlet.2009.06.038

We have improved the synthesis of a previously published luciferin-linker, used in an assay enabling rapid real-time quantification of luciferin-CPP conjugate uptake and cytosolic cargo release. We also present the synthesis of a new luciferin-linker with the same conjugation ability. Both luciferin-linkers are now available via an efficient one-pot procedure.

Full text of DOI:10.1016/j.tetlet.2009.06.038

Tetrahedron Letters 50 (2009) 4731–4733
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
An improved synthesis of releasable luciferin–CPP conjugates
*
Emelía Eiríksdóttir, Ülo Langel, Katri Rosenthal-Aizman
Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
We have improved the synthesis of a previously published luciferin-linker, used in an assay enabling
rapid real-time quantification of luciferin–CPP conjugate uptake and cytosolic cargo release. We also
present the synthesis of a new luciferin-linker with the same conjugation ability. Both luciferin-linkers
are now available via an efficient one-pot procedure.
Received 9 March 2009
Revised 23 May 2009
Accepted 5 June 2009
Available online 11 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Luciferin
Luciferin-linker
Conjugate
Cell-penetrating peptide
Releasable luciferin
Cell-penetrating peptides (CPPs) are a heterogeneous class of
peptides1 which have been used extensively as carriers in biology
and medicine.2 Numerous studies conclusively demonstrate the
potential of CPPs, both in vitro and in vivo, for intracellular delivery
of various cargoes, that otherwise have difficulties crossing the
plasma membrane, such as small molecules, proteins and oligonu-
cleotides.3 Today, as the number of reported CPPs is growing, reli-
able assays are urgently needed for evaluating the comparative
internalization efficacy and further characterization of CPPs as cel-
lular delivery agents.
Frequently used assays in the CPP field are, for example, dye-la-
belled CPPs for uptake and distribution experiments,4 and biologi-
cal read-out strategy, where peptide nucleic acid (PNA)–CPPs
conjugates are used for splice correction.5 In the former assay, CPPs
are bound to the cellular membrane or trapped in intracellular ves-
icles giving rise to false-positive results.6 These methods allow nei-
ther reliable evaluation of transporters in real time, nor give a
direct measure of internalization efficacy.
A recently published assay relying on luminescence from the
luciferin/luciferase reaction enables a real-time quantification of
a cytosolic luciferin–CPP conjugate in cells and in animal models.7,8
In this assay, free luciferin is released immediately upon cytosolic
entry of the luciferin–CPP conjugate due to the high concentration
of cytosolic glutathione. The presence of cytosolic luciferase en-
zymes results in a light-emitting reaction when free luciferin is
converted into oxyluciferin. Due to the luminescent origin of the
emitted light, this assay has the advantage of giving a strong signal
and low background.
The beauty of the releasable luciferin assay7 inspired us to
investigate its application for the real-time quantification of up-
take for a number of CPPs. The synthetic protocol described by
Jones et al.7 (Scheme 1, steps a–c) involves synthesis of activated
disulfide 5 by reacting hydroxyl-thiol 1 with three equivalents of
2,20-dithiodipyridine 2 (20-aldrithiol). After flash-chromatographic
purification, disulfide 3 (97%) was converted into the chlorofor-
mate 5 by reacting with triphosgene 4 at room temperature. The
organic solvent was removed in vacuo and 5 was used without fur-
ther purification in the reaction with the potassium salt of luciferin
6, yielding the carbonate 7, which after purification (58%) was used
for conjugation to an octaarginine transporter.
The synthetic route described to the luciferin conjugate with
activated disulfide is a concise solution for attachment of luciferin
to a transporter. However, in our hands, preparation of the conju-
gate was hampered by synthetic difficulties. The main issues were
(1) time-consuming purification of 3. (2) Reaction with triphos-
gene at room temperature was very problematic and resulted in
a very low yield of 7. (3) Long reaction time for conjugation to a
transporter.
We thought that a one-pot method avoiding isolation of inter-
mediates would improve the utility of this assay. To this end,
and supported by earlier reported investigations,9,10 we made
improvements to the reported synthesis. In addition, we present
a synthetic route to a new luciferin-linker.
20-Aldrithiol (2) is a well-known reagent used for the prepara-
tion of unsymmetrical disulfides.11 Normally, an excess of 2 is re-
acted with the thiol of interest in good yields (above 60-70%).11
* Corresponding author. Tel.: +46 8 161 265; fax: +46 8 161 371.
E-mail address: katri@neurochem.su.se (K. Rosenthal-Aizman).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.06.038

4732
E. Eiríksdóttir et al. / Tetrahedron Letters 50 (2009) 4731–4733
Scheme 1. Synthetic route to luciferin-linker 7.
However, the excess of 2 and the formation of mercaptopyridine
10 as a by-product makes laborious chromatographic purification
unavoidable. In order to achieve clean conversion into 3 we treated
1 with an equimolar amount of methoxycarbonylsulfenyl chloride
8 and thereafter with an equimolar amount of 2-mercaptopyridine
10 (Scheme 1, steps a0–a00), relying on the reactivity of the S-sulfe-
nylcarbomethoxy functionality in compound 9.9,10,12 Gratifyingly,
we obtained product 3 in nearly quantitative yield and of sufficient
purity to use without isolation (Fig. 1, Supplementary data).
In this very efficient route to 3, the starting materials were com-
pletely consumed and the only side-products formed were car-
bonyl sulfide gas and methanol.13 This reaction is driven by the
release of gaseous carbonyl sulfide and the reaction course is easily
monitored by the disappearance of the yellow colour of 10.
In the next step (Scheme 1, step b), we experienced great diffi-
culties in obtaining chloroformate derivative 5 using triphosgene
at room temperature. The main product was a symmetrical dialky-
lated carbonate side-product, which is in agreement with the fact
that it is often essential to keep phosgene/triphosgene reactions
at low temperature to minimize the formation of symmetrical dial-
kyl carbonate side-products.14 In our hands, decreasing the tem-
perature from room temperature to ꢀ10 °C was a crucial factor
in the synthesis of 5. Another important factor that had to be con-
sidered was the ratio of the alcohol and triphosgene reactants. We
found that a 1:1 ratio of 3-(2-pyridinyldithio)-1-propanol (3) and
triphosgene (4) resulted in a very clean synthesis of 3-(2-pyr-
idinyldithio)-1-propanyl chloroformate (5) while a 3:1 ratio gave
a 1:1 mixture of product 5 and a symmetrical dialkyl carbonate
side-product (Fig. 2, Supplementary data).
according to Jones.7 Also, employing conditions routinely used in
our laboratory for similar conjugation reactions, we were able to
demonstrate that all the reactions were complete within 30 min
(Fig. 3, Supplementary data). This short reaction time allows this
procedure to be carried out without an inert gas atmosphere,15
which further simplifies this step. We also found that a 1:1 molar
ratio of luciferin-linker to transporter CPP was as effective as the
1:2 ratio employed by Jones.7
In an attempt to further advance our study, we synthesized the
novel activated carbonate derivative 12 (Scheme 2).16 Here, S-sul-
fenylcarbomethoxy-functionalized thiopropanol 9 was directly
converted into the chloroformate 11, which was then reacted with
salt 6 to give the desired product 12 in high purity. Both reaction
steps a and c go to completion, however, our analysis showed that
conversion of 9 into 11 (step b) was not complete (ca. 60% yield).
We believe that increasing the reaction time or raising the temper-
ature to ꢀ5 °C could improve the yield. Crude product 12 was ob-
tained in 48% yield. The high purity of crude 12 synthesized in one
pot (Fig. 4, Supplementary data) is in sharp contrast with that of
crude 7, which is also prepared in one pot (Fig. 5, Supplementary
data), despite several attempts to improve the purity. This high
purity potentially allows direct conjugation of crude 12 to the
carrier.
Pure 12 was equally effective as pure 7 in the conjugation
reaction with the three CPPs, giving clean and complete conver-
sion into luciferin-transporters in 30 min (Fig. 6, Supplementary
data).17
In summary, we have presented an improved and practical pro-
tocol for the synthesis of luciferin-linker 7. We have also described
a simple procedure allowing the one-pot preparation of a novel
luciferin-linker 12. Our synthetic route to the activated luciferin
carbonate is very effective in terms of time and purity, allowing
the total synthesis of the desired luciferin-transporter in two to
three days.
Chloroformate 5 was used without purification in accordance
with Jones’ method.7 It was treated with luciferin potassium salt
6 in water to form the luciferin-linker 7 (Scheme 1, step c). The
luciferin-linker 7 was purified by RP-HPLC and conjugated to three
different cysteine-containing CPPs (Table 1, Supplementary data)

E. Eiríksdóttir et al. / Tetrahedron Letters 50 (2009) 4731–4733
4733
Scheme 2. Synthetic route to luciferin-linker 12.
added dropwise over 20 min to triphosgene 4 (22.1 mg, 0.0745 mmol) and
dissolved in 650
5 h at ꢀ10 °C (ice/NaCl bath), and the CH2Cl2 was evaporated to give crude 3-(2-
pyridinyldithio)-1-propanyl chloroformate 5. A solution containing luciferin
potassium salt 6 (23.7 mg, 0.0745 mmol) dissolved in 2.37 ml of ice-cold water
Acknowledgements
l
l of cold (ꢀ10 °C) CH2Cl2. The reaction mixture was stirred for
The work presented in this article was supported by the Swed-
ish Research Council (VR-NT); by the Center for Biomembrane Re-
search, Stockholm; by Knut and Alice Wallenberg’s Foundation and
by the Swedish Governmental Agency for Innovation Systems
(VINNOVA-SAMBIO 2006). We thank Dr. Heiner Eckert for valuable
advice regarding the triphosgene reactions.
and149 llof ice-coldsodiumhydroxide solution(0.500 M inH2O, 0.0745 mmol)
was added dropwise to crude 5 according to Jones’ method7 to give luciferin-
linker 7 after 4 h reaction on ice. Compound 7 was obtained as a white powder
after RP-HPLC purification. The molecular weight of 7 was verified by MALDI-
TOF: calcd monoisotopic mass for [M+H+] is 508.01, found [M+H+]
507.99.Scheme 1, steps a–c: These reaction steps were carried out according to
Ref. 7 with one modification; product 3 (step a) was washed repeatedly with
Na2CO3 solution (1 g in 20 ml of H2O) to remove the side-product 10 instead of
using flash-chromatographic purification.
Supplementary data
13. Brois, S. J.; Pilot, J. F.; Barnum, H. W. J. Am. Chem. Soc. 1970, 92, 7629.
14. Matzner, M.; Kurkjy, R. P.; Cotter, R. J. Chem. Rev. 1964, 64, 645.
15. Tam, J. P.; Wu, C. R.; Liu, W.; Zhang, J. W. J. Am. Chem. Soc. 1991, 113, 6657.
16. Synthesis of luciferin-linker 12 (Scheme 2): steps a–c: 3-mercapto-1-propanol 1
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.06.038.
(21.7 mg, 0.235 mmol), dissolved in 160
dropwise over 10 min to methoxycarbonylsulfenyl chloride
0.259 mmol), dissolved in 500 l of ice-cold CH2Cl2. The reaction mixture was
stirred for 30 min on ice to give crude 3-[(methoxycarbonyl)disulfanyl]-1-
propanol 9. Pyridine (18.6 mg, 0.235 mmol) in 340
was added to a CH2Cl2 solution of crude compound 9 which was then added
dropwise over 10 min to triphosgene 4 (69.7 mg, 0.235 mmol), dissolved in 2 ml
of cold (ꢀ10 °C) CH2Cl2. The reaction mixture was stirred for 5 h at ꢀ10 °C (ice/
NaCl bath), and the CH2Cl2 was evaporated to give crude 3-[(methoxy-
carbonyl)disulfanyl]-1-propanyl chloroformate 11. Luciferin potassium salt 6
l
l of ice-cold CH2Cl2, was added
References and notes
8
(32.7 mg,
l
1. Langel, Ü. Handbook of Cell-Penetrating Peptides, 2nd ed.; CRC Press: Boca Raton,
2006.
2. Stewart, K. M.; Horton, K. L.; Kelley, S. O. Org. Biomol. Chem. 2008, 6, 2242.
3. Mäe, M.; Langel, Ü. Curr. Opin. Pharmacol. 2006, 6, 509.
4. Holm, T.; Johansson, H.; Lundberg, P.; Pooga, M.; Lindgren, M.; Langel, Ü. Nat.
Prot. 2006, 1, 1001.
5. El Andaloussi, S.; G0075terstam, P.; Langel, Ü. Nat. Prot. 2007, 2, 2043.
6. Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.; Verbeure, B.; Gait, M. J.;
Chernomordik, L. V.; Lebleu, B. J. Biol. Chem. 2003, 278, 585.
7. Jones, L. R.; Goun, E. A.; Shinde, R.; Rothbard, J. B.; Contag, C. H.; Wender, P. A. J.
Am. Chem. Soc. 2006, 128, 6526.
8. Wender, P. A.; Goun, E. A.; Jones, L. R.; Pillow, T. H.; Rothbard, J. B.; Shinde, R.;
Contag, C. H. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10340.
9. Kaneko, T.; Willner, D.; Monkovic, I.; Knipe, J. O.; Braslawsky, G. R.; Greenfield,
R. S.; Vyas, D. M. Bioconjug. Chem. 1991, 2, 133.
10. Vlahov, I. R.; Santhapuram, H. K.; Kleindl, P. J.; Howard, S. J.; Stanford, K. M.;
Leamon, C. P. Bioorg. Med. Chem. Lett. 2006, 16, 5093.
l
l of cold (ꢀ10 °C) CH2Cl2,
(74.8 mg, 0.235 mmol), dissolved in ice-cold water (3 ml), was added to 470 ll
of ice-cold sodium hydroxide solution (0.500 M in H2O, 0.235 mmol). The
luciferin/NaOH solution was added dropwise over 10 min to crude compound
11 and the reaction mixture was stirred for 4 h on ice. The reaction was
quenched with 15 ml of 1% TFA and extracted with CH2Cl2 (3x15 ml). The
CH2Cl2 phase was dried over MgSO4 and evaporated to yield luciferin-linker 12,
which after RP-HPLC purification was obtained as
a white powder. The
molecular weight of 12 was verified by MALDI-TOF: calcd monoisotopic mass
for [M+H+] is 488.99, found [M+H+] 488.96. 1H NMR (CDCl3, 500 MHz, 25 °C): d
8.12 (d, 1H, J = 8.4 Hz, aromatic), 7.79 (s, 1H, aromatic), 7.35 (d, 1H, J = 8.4 Hz,
aromatic), 5.43 (m, 1H, NCH), 4.43 (t, 2H, J = 6.1 Hz, OCH2), 3.90 (s, 3H, CH3), 3.80
11. Witt, D.; Klajn, R.; Barski, P.; Grzybowski, B. A. Curr. Org. Chem. 2004, 8, 1763.
12. The peptides were synthesized in a stepwise manner on an automated peptide
synthesizer (Applied Biosystems model 433A, USA) by tert-butoxycarbonyl (t-
Boc) chemistry and purified on a semi-preparative reverse-phase (RP)-HPLC
column. Their purity was checked by RP-HPLC and the correct molecular weight
was verified by MALDI-TOF mass spectrometry. Synthesis of luciferin-linker 7
(Scheme 1): Steps a0–c: 3-mercapto-1-propanol 1 (1.018 g, 11.05 mmol) was
added dropwise to methoxycarbonylsulfenyl chloride 8 (1.398 g, 11.05 mmol) in
ice-cold CH2Cl2 (10 ml), and stirred for 30 min on ice, followed by the addition of
2-mercaptopyridine 10 (1.228 g, 11.05 mmol). The reaction mixture was stirred
for 1 h on ice. After washing with (NH4)2CO3 solution (1 g in 20 ml of H2O), the
CH2Cl2 phase was dried over MgSO4 and evaporated to give crude 3-(2-
pyridinyldithio)-1-propanol 3 as a colourless oil. The molecular weight of 3
was verified by MALDI-TOF: calcd monoisotopic mass for [M+H+] is 202.04,
(m, 2H, SCH2), 2.94 (t, 2H, J = 7.0 Hz, SSCH2), 2.16 (q, 2H, J = 6.5 Hz, CH2) ppm. 13
C
NMR (CDCl3, 125 MHz, 50 °C): d 171.8, 170.1, 167.6, 160.6, 153.3, 151.2, 150.2,
136.9, 125.5 (aromatic CH), 121.1 (aromatic CH), 114.2 (aromatic CH), 78.3
(NCH), 67.1 (OCH2), 55.5 (CH3), 35.4 (SCH2 and SSCH2), 27.9 (CH2) ppm.
17. Conjugation of luciferin-linkers to peptides. Luciferin-linker 7 and a cysteine-
containing peptide (Table 1, Supplementary data) were mixed in a 1:1 or a 2:1
ratio at a final peptide concentration of 0.88 mM in either DMF or DMF/acetic
acid buffer (pH 5, 50 mM) at room temperature, under nitrogen. Samples taken
after 30 min, 2 h, 4 h and 24 h were analyzed by RP-HPLC, the major
components collected and their molecular weights verified by MALDI-TOF:
calcd monoisotopic mass for luciferin-TP10 [M+H+] is 2680.42, found [M+H+]
2680.42; calcd monoisotopic mass for luciferin-pVEC [M+H+] is 2707.42, found
[M+H+] 2707.42; calcd monoisotopic mass for luciferin-M918 [M+H+] is
3133.62, found [M+H+] 3133.61. Luciferin-linker 12 and the peptides were
mixed in a 1:1 ratio under the same conditions as above.
found [M+H+] 202.02. Pyridine (5.89 mg, 0.0745 mmol) in 300
CH2Cl2, wasadded to crudecompound3 (15.0 mg, 0.0745 mmol)whichwas then
ll of cold (ꢀ10 °C)