Photolytic Mass Laddering for Fast Characterization of Oligomers on Single Resin Beads

Full text of DOI:10.1021/jo970866w

5662
J . Org. Chem. 1997, 62, 5662-5663
Sch em e 1. Syn th esis of a P h otola bile Am in o Acid
P h otolytic Ma ss La d d er in g for F a st
Ch a r a cter iza tion of Oligom er s on Sin gle
Resin Bea d s
Kevin Burgess,* Carlos I. Martinez,
David H. Russell, Hunwoo Shin, and Alex J . Zhang
Department of Chemistry, Texas A&M University,
College Station, Texas 77843-3255
Received May 15, 1997
The divide/couple/recombine methodologies, also re-
ferred to as “split syntheses”, represent a powerful
combinatorial approach whereby a large number of
supported compounds can be prepared in comparatively
few synthetic operations. Originally, the technique was
demonstrated for peptides,1 and then coupled with a
combinatorial screen.2 Thus a library of approximately
2.47 × 106 different supported pentapeptides was mixed
with a monoclonal antibody (mAb), and those ligands
with highest affinities were identified via a marker on
the mAb. The methodology is not restricted to peptides,
however; split syntheses are conceptually applicable to
any supported oligomers or collections of compounds that
can be transformed via repetitive couplings.
Lead compound characterization is an obstacle that
must be overcome whenever split syntheses are applied
to generate libraries of oligomers that do not consist
solely of the protein amino acids. In such cases, auto-
mated peptide/protein sequencers using Edman’s degra-
dation3 chemistry are unsuitable. Mass spectrometric
methods like matrix-assisted laser desorption/ionization
(MALDI)4 and electrospray ionization (ESI)5 give data
that can sometimes be used to characterize the product,
but time-consuming interpretations are required and
ambiguities can arise. Direct observation of the products
by NMR is impractical unless exceptionally large and
high-loading beads are used. Consequently, several
innovative solutions to the problem of characterizing
compounds on single resin beads have been reported.6-11
This communication describes our own method which is
one that may be described as a “photolytic mass-ladder-
ing technique”. The split synthesis is performed such
that most of the oligomer on any bead will consist of an
unperturbed sequence, but a small fraction will have a
photolabile group X inserted before each of the coupling
steps. On irradiation, an isolated bead generates frag-
ments of incrementally different molecular masses. MS
analyses of the material liberated could be used to deduce
the sequence from the molecular mass differences as
shown below.
Many of our original experiments to test the photolytic
mass-laddering concept were unsuccessful. Ultimately,
it became apparent that upon swelling most polystyrene-
containing beads (e.g. Rink and TentaGel resins) liberate
small polymer fragments of less than approximately 550
Da into solution, and that these obscured low concentra-
tions of any other materials liberated from the bead.
Above 550 Da, however, the spectra are relatively clean.
Consequently, it was necessary to prepare photocleavable
groups that would leave relatively heavy fragments (X*)
on the liberated strand, which would generate mass
ladders consisting of fragments larger than 550 Da.
Scheme 1 describes a synthesis of molecular fragment
X that could be used for photolytic mass laddering.
Quantitative alkylation of acetovanillone with methyl
5-bromopentanecarboxylate, followed by reduction with
sodium borohydride gave 1 in 97% yield after two steps
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Biol. 1996, 3, 679-688.
(3) Edman, P.; Begg, G. Eur. J . Biochem. 1967, 1, 80-91.
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Spectrom. Ion Processes 1987, 78, 53-68.
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Winograd, N. Science 1994, 264, 399-402.
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C. M. Science 1989, 246, 64-71.
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S0022-3263(97)00866-9 CCC: $14.00 © 1997 American Chemical Society

Communications
J . Org. Chem., Vol. 62, No. 17, 1997 5663
(no purification necessary). Conversion of the alcohol to
the azide was then performed using a Mitsunobu type12
displacement. The resulting azide 2 was nitrated with
copper(2+) nitrate and subsequently reduced to the
intermediate free amine 3. This oxygenated nitrobenzyl
system is desirable since such compounds cleave ef-
ficiently when irradiated at 360 nm.13 In the second
branch of this convergent approach to X, phenylalanine
ethyl ester was coupled with Cbz-Lys(BOC). Hydro-
genolysis of the Cbz group, reaction with bromobenzene-
sulfonyl chloride, and hydrolysis of the methyl ester
yielded brominated, and relatively heavy, intermediate
4. Finally, the free amine 3 was coupled to dipeptide 4
and hydrolyzed to give the photolabile amino acid 5 (X).
The photocleavable amino acid X was incorporated into
a series of representative tetra- and pentapeptides of
known sequence to provide substrates to demonstrate
photolytic mass laddering. Thus, six different peptides
were constructed on TentaGel amino resin (80-100 pmol/
bead). The peptide syntheses were performed in such a
way that a small mole fraction of X (5 mol %, except the
initial example attempted, FFMF, 10 mol %) was coupled
prior to each regular coupling step.
Single beads of each of the six test sequences were
placed in small cuvettes with 5 µL of a 2:1 water/
acetonitrile mixture and irradiated at a distance of
approximately 1 cm from an 8 W 360 UV lamp. The rate
of photochemical cleavage is proportional to the flux of
radiation inputted to X. An irradiation time of 3 h was
routinely used in these experiments, but the most recent
studies have shown that this was longer than was
actually necessary; for instance, only 5 min exposure of
the sequence FFMF to a 8 W UV source was sufficient
(data not shown).
MALDI spectra of the samples obtained from single
beads are shown in Figure 1. Isotopic distribution of
bromine isomers in the heavy part of the linker, X*, gives
1:1 molecular mass distributions for peaks containing
this fragment, allowing them to be easily differentiated
from background signals (unfortunately these doublets
do not show up well in this photoreduced figure, but they
are clear on normal size spectra, see Supporting Informa-
tion). Oxidation of methionine was observed for peptide
f. This is probably due to the presence of radicals
produced by the photolytic reaction.14 However, this
oxidation did not generate any problems with the as-
signment process.
In summary, this work demonstrates that photolytic
mass laddering can be used for efficient sequence deter-
mination of peptides generated in split syntheses. The
technique could also be used for other oligomer types by
modifying the functionality of the photocleavable group
X to accommodate the preferred coupling scheme. Iso-
meric residues could be encoded by appropriate use of
two X groups of different masses. There is a concern that
activity in the binding assays may be observed solely as
a result of the linker system, but only a small fraction
(e.g. 4 × 5% if 5 mol % of linker is used for a tetramer)
of the sequences displayed on any given bead will
contain a linker unit. Consequently, this phenomenom
will probably not occur frequently. However, if it does
occur, the false positives that result should be easily
identified by further screens. Photolytic mass laddering
has some advantages over other MS methodologies
F igu r e 1. MALDI-MS of peptide samples/photocleavable tag
sequences, liberated from single beads. The known peptide
sequences were (a) GALF, (b) GGALF, (c) FFRF, (d) GAR(Mtr)F,
(e) GAYF, and (f) FFMF. Matrix: R-cyano-4-hydroxycinnamic
acid. Instrument: Voyager-Elite XL MALDI mass spectrom-
eter in the linear mode with a stainless steel sample plate.
developed for the same purpose. For instance, it is
compatible with methionine, whereas methods that
involve cyanogen bromide cleavage are not.11 It is
appropriate for split syntheses involving more than three
coupling cycles whereas isotopic encoding methodologies15
would give very complicated mass spectra for libraries
of the same composition. In conclusion, this paper
demonstrates proof of concept for photolytic mass lad-
dering of oligomers formed in split syntheses, a charac-
terization technique which has potential advantages over
some of the methods reported to date.
Ack n ow led gm en t. Financial support was provided
by the NIH (DA09358) and The Robert A. Welch
Foundation, and K.B. thanks the NIH for a Research
Career Development Award and the Alfred P. Sloan
Foundation for a fellowship. C.I.M. was supported by
an NIH fellowship (GMS-17567). NMR facilities were
provided by the NSF via the chemical instrumentation
program, and the MALDI facility was funded by The
U.S. Department of Energy.
Su p p or tin g In for m a tion Ava ila ble: Procedures for syn-
thesis of the photolabile linker, protocol for the peptide
syntheses, method for analyte preparation and MALDI analy-
sis, full size copies of the MALDI-MS spectra that were reduced
to form Figure 1 (14 pages).
(13) Holmes, C. P.; Chinn, J . P.; Look, G. C.; Gordon, E. M.; Gallop,
M. A. J . Org. Chem. 1995, 60, 7328-33.
(14) Holmes, C. P. J . Org. Chem. 1997, 62, 2370-80.
(15) J urczak, J .; Golebiowski, A.; Raczko, J . J . Org. Chem. 1989,
54, 2495.
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