Sequentially photocleavable protecting groups in solid-phase synthesis

Article abstract of DOI:10.1021/ol027454g

(Matrix presented) A sequential solid-phase peptide synthesis was developed using both photolabile linker and protecting groups. The chromatic sequential lability between a tert-butyl ketone-derived linker (sensitive to irradiation at 305 nm) and a nitroveratryloxycarbonyl (NVOC) group (sensitive at 360 nm) was exploited to prepare Leu-Enkephalin in a 55% overall yield. This new strategy allows the preparation of peptides in essentially neutral medium, by avoiding the use of common deprotection reagents such as trifluoroacetic acid or piperidine.

Full text of DOI:10.1021/ol027454g

ORGANIC
LETTERS
2003
Vol. 5, No. 8
1179-1181
Sequentially Photocleavable Protecting
Groups in Solid-Phase Synthesis
,†
,‡
Martin Kessler,† Ralf Glatthar,† Bernd Giese,* and Christian G. Bochet*
Department of Chemistry, UniVersity of Basel, 19 St. Johanns-Ring,
CH-4056 Basel, Switzerland, and Department of Organic Chemistry,
UniVersity of GeneVa, 30 quai Ernest-Ansermet, CH-1211 GeneVa 4, Switzerland
bernd.giese@unibas.ch; christian.bochet@chiorg.unige.ch
Received December 12, 2002
ABSTRACT
A sequential solid-phase peptide synthesis was developed using both photolabile linker and protecting groups. The chromatic sequential
lability between a tert-butyl ketone-derived linker (sensitive to irradiation at 305 nm) and a nitroveratryloxycarbonyl (NVOC) group (sensitive
at 360 nm) was exploited to prepare Leu-Enkephalin in a 55% overall yield. This new strategy allows the preparation of peptides in essentially
neutral medium, by avoiding the use of common deprotection reagents such as trifluoroacetic acid or piperidine.
Solid-phase organic synthesis (SPOS) has dramatically
increased in importance in the last four decades.1 It is now
the main strategy in most of the automated synthesis
schemes, for both parallel and combinatorial approaches.2
The relevant substrate is attached to the solid support via a
linker throughout the synthetic process; essentially, this link
should be robust and withstand a large spectrum of reagents.
At the end of the sequence, usually quite harsh conditions,
basic or acidic, trigger the release of the modified compound.
Photorelease is an attractive alternative to aggressive re-
agents.3 Such linkers are usually derived from known
photolabile groups (e.g., 1 or 2).4,5 We recently described 3,
which contains a robust linker with very efficient photo-
cleavage.6 However, the development of a linker cleavable
under very mild conditions is only of interest if all the other
transformations in the sequence are equally mild. For
example, in peptide synthesis, the deprotection of the
N-terminus of the growing chain requires basic (Fmoc
removal) or acidic (Boc removal) conditions. We recently
described a new protecting strategy based on the group
differentiation by light of specific wavelengths (chromatic
orthogonality).7,8 We show here how such a strategy could
be used in peptide synthesis, with both linker and temporal
protecting groups removed photochemically at different
wavelengths. All these transformations can, in principle, be
carried out at neutral pH.
Preliminary Photolysis Experiments. The negligible
absorbance of the photosensitive tert-butyl ketone 2 above
330 nm prompted us to check whether it could be possible
to remove the more sensitive nitroveratrole protecting group
† University of Basel.
‡ University of Geneva.
(1) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (b)
(4) (a) Patchornik, A; Amit, B.; Woodward, R. B. J. Am. Chem. Soc.
1970, 92, 6333-6335. (b) Peukert, S.; Giese, B. J. Org. Chem. 1998, 63,
9045-9051.
Merrifield, R. B. Science 1965, 165, 178-185.
(2) (a) Seneci, P. Solid-Phase Synthesis and Combinatorial Technologies;
John Wiley: New York, 2000. (b) Thompson, L. A.; Ellman, J. A. Chem.
ReV. 1996, 96, 555-600. (c) Balkenhohl, F.; von dem Bussche-Hu¨nnefeld,
C.; Lansky, A.; Zechel, C. Angew Chem., Int. Ed. 1996, 35, 2288-2337.
(3) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Tetrahedron 1993, 49,
11065-11133.
(5) For a recent review: Bochet, C. G. J. Chem. Soc., Perkin Trans. 1
2002, 125-142.
(6) Glatthar, R.; Giese, B. Org. Lett. 2000, 2, 2315-2317.
(7) Bochet, C. G. Angew. Chem., Int. Ed. 2001, 40, 2071-2073.
(8) Blanc, A.; Bochet, C. G. J. Org. Chem. 2002, 67, 5567-5577.
10.1021/ol027454g CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/20/2003

Scheme 1. Photolabile Linkers
Scheme 3. Photolysis of Resin-Bound Amino Acids
1 photolytically at a longer wavelength. Hence, we prepared
the diester 5, by first esterifying glutaric acid with alcohol 1
under acidic catalysis (24%)9 and then coupling 4 with 2
using N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide me-
thyl-p-toluenesulfonate (CMC) (Scheme 2). The photolysis
Scheme 2. Sequential Photolysis of Diesters
Application to the Sequential Peptide Synthesis. All the
requirements for sequential solid-phase peptide synthesis
were now fulfilled. We chose Leu-Enkephalin as a test
substrate for its well-known characterization data and our
prior experience.5 In addition, the presence of UV-absorbing
amino acids (Tyr) would be a test for the scope of our
strategy (an authentic sample of Leu-Enkephalin showed less
than 1% degradation after irradiation at 305 nm for 60 min,
as determined by HPLC). Leu-Enkephalin (H-Tyr-Gly-Gly-
Phe-Leu-OH) is an endorphin pentapeptide isolated from
brain, with morphin-like activity.11
Preparation of N-Protected Amino Acids. The com-
mercially available amino acid hydrotosylates 12a-d were
protected with the nitroveratryloxycarbonyl group (NVOC)
according to our previously published procedure (Scheme
1).12 Thus, neutralization of the salts with sodium hydride
in dichloromethane (25 °C, 30 min) was followed by
acylation with a mixture of 2-nitroveratrole 1 and N,N′-
carbonyldiimidazole (CDI) to give carbamates 13a-d. The
of the diester 5 at 360 nm smoothly gave the monomethyl-
ester 6 (87%) after treatment with diazomethane (together
with 2% totally photolyzed 7). A second photolysis at 300
nm gave the dimethyl glutarate 7 in quantitative yield after
methyl ester formation.
With this encouraging modulated lability,10 we confirmed
that the same strategy could also be applied to the solid
phase, by using the glutamic acid derivative 8, attached to
the TentaGel-bound photosensitive linker 3. Hence, N,N′-
diisopropylcarbodiimide-mediated (DIC) coupling of 8 to the
resin was followed by the cleavage of the Fmoc group with
piperidine (in DMF, 25 °C, 30 min); titration of the resulting
dibenzofulvene by UV spectroscopy indicated a resin loading
of 0.3 mmol/g. As expected, the photolysis at 360 nm of 9
followed by esterification with a solution of trimethylsilyl
diazomethane in hexane smoothly led to methylester 10. Final
release from the resin by irradiation at 335 nm gave 11 in a
45% overall yield (from initial compound 8) (Scheme 3).
The identity was determined by HPLC and electrospray mass
spectrometry. Clearly, the presence of the resin does not have
a detrimental effect on the photolytic processes.
Scheme 4. Preparation of N-Protected Amino Acids
(9) Kern, W.; Spiteller, G. Liebigs Ann. Chem. 1985, 1168-1174.
(10) For a good overview, see: Schelhaas, M.; Waldmann, H. Angew.
Chem., Int. Ed. Engl. 1996, 35, 2056-2083.
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Org. Lett., Vol. 5, No. 8, 2003

reaction of the released amine and the aldehyde photoproduct
of NVOC.4a To circumvent this side-reaction, we trapped in
situ the aldehyde as soon as it was produced, by using
solvents containing 0.5% semicarbazide hydrochloride for
all our photolyses at 360 nm. After 20 min of irradiation,
the solvent was filtered and analyzed for the presence of
side products. The process was repeated until completion of
the deprotection. The same sequence was repeated four times
using NVOC-amino acids 14c, 14a, 14a, and 14b.15 The
concentration of free Leu-Enkephalin in the last photodepro-
tection sample was below the detection limit of the electro-
spray mass spectrometer. After each coupling step, complete
acylation was checked by picric acid monitoring.16 Final
cleavage photolysis was performed by irradiation at higher
energy (Osram 500W high-pressure mercury lamp, 305 nm
cut off filter) in a 4:1 THF-water mixture (16 °C, 30 min).
Leu-Enkephalin was released in a yield of 55% and in a
purity of 92%. Shorter peptides were formed in quantities
of less than 1%. This absence of deletion in the sequence is
very important, since it proves the near quantitative yield
for each individual step.
Scheme 5. Preparation of Leu-Enkephalin
free acids were obtained by the Rh-catalyzed hydrolysis of
the allyl esters (EtOH-H2O).13,14
In summary, we were able to synthesize a pentapeptide
in an iterative scheme, without the need for chemical
deprotection steps in both the substrate elaboration and final
release phases. This allowed the handling of sensitive side
chains, since the whole protocol operates under essentially
neutral conditions.
Sequential Synthesis of Leu-Enkephalin. The TentaGel-
bound linker 3 was coupled with NVOC-Leu-OH 14d (DIC,
HOBt, DMAP cat., rt, 20 h). Exposure to 360 nm light
(Osram 500 W high-pressure mercury lamp with a 360 nm
cutoff filter) for 3-4 h led to a significant darkening of the
solution and mediocre deprotection. Such poor results have
already been observed and were attributed to the subsequent
Acknowledgment. This work was supported by the Swiss
National Science Foundation and Novartis.
Supporting Information Available: Typical experimen-
tal procedures and characterization data for selected com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) (a) Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.;
Morgan, B. A.; Morris, H. R. Nature 1975, 258, 577-579. (b) Goldstein,
A. Science 1976, 193, 1081-1086. (c) Bower, J. D.; Guest, K. P.; Morgan,
B. A. J. Chem. Soc., Perkin Trans. 1 1976, 2488-2492. (d) Vilkas, E.;
Vilkas, M.; Sainton, J. Int. J. Pept. Protein Res. 1980, 15, 29-31.
(12) D’Addona, D.; Bochet, C. G. Tetrahedron Lett. 2001, 42, 5227-
5229.
OL027454G
(13) Waldmann, H.; Kunz, H. Liebigs Ann. Chem. 1983, 10, 1712-1725.
(14) A similar route was also used, with the tbutyl ester hydrochlorides
as starting materials, replacing the final saponification by acidic hydrolysis
(CF3COOH). Overall yields were comparable.
(15) Experimental details on the synthesis on a solid support and
photorelease of Leu-Enkephalin are available as Supporting Information.
(16) Arad, O.; Houghton, R. A. Pept. Res. 1990, 3, 42-50.
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