SPOCC: A resin for solid-phase organic chemistry and enzymatic reactions on solid phase

Article abstract of DOI:10.1021/ja984355i

SPOCC resin 1, a novel, highly permeable, polar support for chemical and enzymatic solid-phase methods, is presented. The synthesis of SPOCC resin is based on the cross-linking of long-chain poly(ethylene glycol) (PEG) terminally substituted with oxetane by cationic ring-opening polymerization, affording a polymer containing only primary ether and alcohol C-O bonds. The polymer was prepared using Et₂O-BF₃ as initiator either via bulk polymerization in solution or via suspension polymerization in silicon oil, the latter yielding a beaded resin. The polymerization reaction was investigated with respect to the effects of PEG chain length, the fraction of bisoxetanylated PEG, initiator amount, and temperature in order to vary the swelling, loading, and mechanical stability of the resin. Furthermore, the resin was derivatized with various functional groups and subsequently applied to peptide synthesis and organic reactions in both organic solvents and water. An N-terminal peptide aldehyde was generated on the solid phase and employed to synthesize peptide isosteres by nucleophilic addition of various ylides. Solid-phase glycosylation of peptides and enzymatic reactions were successfully performed with SPOCC resin. Enzymatic proteolytic cleavage of a resin-bound decapeptide on treatment with the 27 kDa protease subtilisin BNP' demonstrated the accessibility of the interior of the SPOCC resin for enzymes.

Full text of DOI:10.1021/ja984355i

J. Am. Chem. Soc. 1999, 121, 5459-5466
5459
SPOCC: A Resin for Solid-Phase Organic Chemistry and Enzymatic
Reactions on Solid Phase†
J o1 rg Rademann, Morten Grøtli, Morten Meldal,* and Klaus Bock
Contribution from the Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10,
DK-2500 Valby, Denmark
ReceiVed December 15, 1998
Abstract: SPOCC resin 1, a novel, highly permeable, polar support for chemical and enzymatic solid-phase
methods, is presented. The synthesis of SPOCC resin is based on the cross-linking of long-chain poly(ethylene
glycol) (PEG) terminally substituted with oxetane by cationic ring-opening polymerization, affording a polymer
containing only primary ether and alcohol C-O bonds. The polymer was prepared using Et2O‚BF3 as initiator
either via bulk polymerization in solution or via suspension polymerization in silicon oil, the latter yielding a
beaded resin. The polymerization reaction was investigated with respect to the effects of PEG chain length,
the fraction of bisoxetanylated PEG, initiator amount, and temperature in order to vary the swelling, loading,
and mechanical stability of the resin. Furthermore, the resin was derivatized with various functional groups
and subsequently applied to peptide synthesis and organic reactions in both organic solvents and water. An
N-terminal peptide aldehyde was generated on the solid phase and employed to synthesize peptide isosteres
by nucleophilic addition of various ylides. Solid-phase glycosylation of peptides and enzymatic reactions were
successfully performed with SPOCC resin. Enzymatic proteolytic cleavage of a resin-bound decapeptide on
treatment with the 27 kDa protease subtilisin BNP′ demonstrated the accessibility of the interior of the SPOCC
resin for enzymes.
Introduction
aqueous solvents and for enzymatic chemistry.11 In contrast, a
resin constructed with PEG as macromonomer can be fully
compatible with water as first demonstrated by the synthesis of
PEGA resin using radical polymerization of acrylamide-
substituted PEG. Characterized by high swelling volumes
in both nonpolar solvents and water, PEGA has been success-
Solid-phase organic chemistry has evolved rapidly during the
past few years mainly due to the enormous potential of peptide
1,2
3
and non-peptide libraries in medicinal chemistry and chemical
biology. The success of solid-phase organic chemistry depends
crucially on the properties of the solid support.4,5 Resins are
preferred which are chemically inert to a broad range of reaction
conditions, mechanically stable, and applicable in many solvents
1
2,13
1
2
fully applied in the synthesis of difficult peptide sequences
1
4
as well as solid-phase enzyme reactions. The favorable
swelling properties of PEGA and other resins constructed by
the cross-linking of PEG chains can be attributed to the stretched
helical superstructures adopted by PEG in aqueous solution.15
However, many organic reactions are not compatible with PEGA
resin because of its rich abundance of amide functionality. For
5
of different polarity. In particular, resins swelling in water are
attractive for use in enzymatic reactions and on-bead enzymatic
assays.6 The resins presently used for solid-phase organic
chemistry are constructed predominantly from cross-linked
7
polystyrene as polymer. Although grafting of the polystyrene
example, solid-phase glycosylation was hampered by the
core with polar linear polymers such as poly(ethylene glycol)
presence of the amide groups in the solid support1
6,17
which
PEG) may improve the swelling in polar solvents,8 the PEG-
-10
(
interacted with both the oxocarbenium ion intermediate from
the carbohydrate donor and the Lewis acids employed for
activation. Similarly, strong bases cannot be used with this type
of resin because they may readily deprotonate the amide
nitrogen. Consequently, a PEG resin was developed based on
polyoxyethylene/polyoxypropylene copolymer (POEPOP), which
grafted resins have shown limitations regarding their use in
*
To whom correspondence should be addressed.
SPOCC resin ) superpermeable organic combinatorial chemistry resin.
†
(1) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski,
W. M.; Knapp, R. J. Nature 1991, 354, 82-84.
2) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley,
C. T.; Cuervo, J. H. Nature 1991, 354, 84-86.
3) Balkenhohl, F.; Bussche-H u¨ nnefeld, C. v. d.; Lansky, A.; Zechel, C.
Angew. Chem., Int. Ed. Engl. 1996, 35, 2288-2337.
4) Hodge, P. C.; Sherrington, D. C. Polymer-supported reactions in
organic chemistry; Wiley: Chichester, 1988.
5) Meldal, M. In Solid-Phase Peptide Synthesis; Fields, G., Ed.;
Academic Press: New York, 1998; pp 83-104.
6) Meldal, M.; Svendsen, I.; Juliano, L.; Juliano, M. A.; Del Nery, E.;
(
18
contained only ether bonds, and this has been successfully
(
applied in the solid-phase organic synthesis of peptide iso-
1
9
steres. POEPOP could be prepared effectively by anionic
(
(
(11) Burger, M. T.; Bartlett, P. A. J. Am. Chem. Soc. 1997, 119, 9,
12697-12698.
(12) Meldal, M. Tetrahedron Lett. 1992, 33, 3077-3080.
(13) Kempe, M.; Barany, G. J. Am. Chem. Soc. 1996, 118, 7083-7093.
(14) Meldal, M.; Auzanneau, F.-I.; Hindsgaul, O.; Palcic, M. M. J. Chem.
Soc., Chem. Commun. 1994, 1849-1850.
(15) Tasaki, K. J. Am. Chem. Soc. 1996, 118, 8459-8469.
(16) Rademann, J. Solid-phase syntheses with carbohydrates. Dissertation,
University of Konstanz, Germany, 1997.
(17) Schleyer, A.; Meldal, M.; Renil, M.; Paulsen, H.; Bock, K. Angew.
Chem., Int. Ed. Engl. 1997, 109, 22064-2067.
(
Scharfstein, J. J. Pept. Sci. 1998, 4, 83-91.
(
7) Merrifield, B. J. Am. Chem. Soc. 1963, 85, 2149-2153.
(8) Becker, H.; Lucas, H.-W.; Maul, J.; Pillai, V. N. R.; Anzinger, H.;
Mutter, M. Makromol. Chem., Rapid Commun. 1982, 3, 217-223.
9) Hellermann, H.; Lucas, H.-W.; Maul, J.; Pillai, V. N. R.; Mutter, M.
Macromol. Chem. 1983, 184, 2603-2617.
10) Rapp, W.; Zhang, L.; H a¨ bish, R.; Bayer, E. In Peptides 1988,
(
(
Proceedings of the European Peptide Symposium; Jung, G., Bayer, E., Eds.;
Walter de Gruyter: Berlin, 1989; pp 199-201.
(18) Renil, M.; Meldal, M. Tetrahedron Lett. 1996, 37, 6185-6188.
1
0.1021/ja984355i CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/29/1999

5460 J. Am. Chem. Soc., Vol. 121, No. 23, 1999
Rademann et al.
Scheme 1. Synthesis of SPOCC-400 and -1500 Resins 1
Synthesis of PEG-Oxetane Macromonomers. Starting
macromonomers for the polymerization were prepared by
alkylation of PEG with the oxetane moiety. 3-Methyl-3-
(alkoxymethyl)oxetanes have been reported to be favorable
starting materials for cationic ring-opening polymerization
because the dialkyl-substituted 3-position adjacent to the ring
oxygen stabilizes the cationic intermediates formed during
20,23
polymerization.
Furthermore, the presence of only secondary
and quaternary carbon atoms in the polymerization product was
expected to provide increased stability of the polymer. Therefore,
3
-methyl-3-[[(4-tolylsulfonyl)oxy]methyl]oxetane (3)24 was se-
lected as the alkylating agent. This sterically demanding
isopentyl building block cannot eliminate toluenesulfonic acid
under the harsh, basic conditions required to alkylate PEG chains
due to the absence of â-hydrogens. Macromonomers 2a were
synthesized from PEG chains of different lengths (PEG-400 and
PEG-1500) to obtain resins with a wide range of swelling and
loading properties. Macromonomers 2a were metalated employ-
ing potassium bis(trimethylsilyl) amide (KHMDS). The hex-
amethyldisilazane (HMDS) formed in the metalation step was
removed by coevaporation with DMF at elevated temperature.
Alkylation was subsequently conducted with tosylate 3 at 75
°
C. In the alkylation step potassium was a more favorable
counterion than sodium, which gave a low yield of alkylated
PEG monomer. Component 2a was obtained with g95%
conversion to bisoxetanylated PEG. The amount of free hydroxy
groups after alkylation was assessed by acetylation using acetic
anhydride/pyridine, yielding acetylated monomer 2b, and
subsequent comparison in the NMR spectra of the acetylated
samples using the integration of the signals at 2.03, 4.31, and
4
.47 ppm of acetyl CH3 and ring CH2 protons, respectively.
The Polymerization Reaction. Macromonomers (2a,b) were
first polymerized in CH2Cl2 with boron trifluoride diethyl
etherate (Et2O‚BF3) as a catalyst. Initially general conditions
for cationic ring-opening polymerization of 2 were investigated.
Previously, oxetanes had been polymerized at low temperature,
using a minimum of Lewis acid catalyst in a nonpolar solvent.20
Thus, reaction parameters such as temperature and the amount
of Lewis acid were investigated to define the mildest conditions
for effecting polymerization of macromonomers 2. Acetylated
macromonomer 2b-1500 was dissolved in an equal volume of
CH2Cl2 and treated with increasing amounts of Et2O‚BF3 at
augmenting temperatures to determine the onset of polymeri-
zation. A temperature of at least +4 °C and 0.15 equiv of Et2O‚
BF3 were found to be required for effecting polymerization.
Subsequently, conditions were established to minimize the time
needed for polymer formation as indicated by the inhibition of
magnetic stirring. Shortest sticky times of less than 1 min were
realized when 0.4-0.6 equiv of Et2O‚BF3 was used in diglyme
polymerization of oxirane-derivatized PEG; however, this
process produced a resin having functional groups comprised
of an equal mixture of secondary and primary alcohols. A more
homogeneous resin was preferred to ensure equal reactivity at
all sites. Furthermore, POEPOP was chemically labile under
extremely strong acidic or basic conditions due to the presence
of secondary ether bonds formed during the polymerization
process.
Results and Discussion
A novel cross-linked polymer was designed that contained
exclusively primary ethers and alcohols: the SPOCC resin 1
(
Scheme 1). The first resin synthesized by cationic ring-opening
20,21
polymerization
for solid-phase organic chemistry, SPOCC
resin 1 was prepared by Lewis acid-catalyzed polymerization
of PEG chains of various length functionalized with four-
membered oxetane rings. Oxetanes possess high reactivity for
polymerization under acid catalysis combined with high stability
toward basic and nucleophilic conditions.22 Thus, basic condi-
tions could be used for the alkylation reaction followed by acidic
reaction conditions in the following polymerization step. High
ring strain makes the oxetanes more reactive under cationic ring-
opening polymerization than the corresponding five-membered
tetrahydrofurans. On the other hand, oxetanes are less susceptible
to nucleophilic ring-opening than the corresponding three-
membered oxiranes.
(diethylene glycol dimethyl ether) at room temperature or at
slightly elevated temperatures.
After optimal conditions were defined for the cross-linking
polymerization of 2, resins were synthesized for examination
in solid-phase chemical and enzymatic reactions. The properties
of the polymerization product could be varied by controlling
the following parameters (Table 1): the PEG chain length, the
amount and the type of Lewis acid catalyst added, the fraction
of bisoxetanylated PEG, the presence of O-acetyl or hydroxy
groups in 2, and the temperature. After extensive curing, the
bulk polymer was granulated, washed, and dried to yield resins
1, which were characterized with respect to loading, swelling,
(
(
(
19) Rademann, J.; Meldal, M.; Bock, K. Chem. Eur. J., in press.
20) Farthing, A. C. J. Chem. Soc. 1955, 3648-3654.
21) Motoi, M.; Nagahara, S.; Yokoyama, M.; Saito, E.; Nishimura, O.;
(23) Motoi, M.; Suda, H.; Shimamura, K.; Nagahara, S.; Takei, M.;
Kanoh, S. Bull. Chem. Soc. Jpn. 1988, 61, 1653-1659.
(24) Dale, J.; Fredriksen, S. B. Acta Chem. Scand., B 1992, 46, 271-
277.
Kanoh, S.; Suda, H. Bull. Chem. Soc. Jpn. 1989, 62, 1572-1581.
22) Reiff, H.; Dieterich, D.; Braden, R.; Ziemann, H. Liebigs Ann. Chem.
973, 365-374.
(
1

SPOCC Resin
J. Am. Chem. Soc., Vol. 121, No. 23, 1999 5461
Table 1. Influence of PEG Chain Length, Derivatization of
Macromonomer, and Reaction Protocol on the Swelling of SPOCC
Polymers
PEG
chain
swelling, mL/g
O DMF CH Cl
2
length, oxetane, protection reaction loading,
Da
%
-OR
-H
protocol mmol/g
H
2
2
4
4
4
500
500
00
00
00
>95
>95
70
>95
>95
A
B
B
A
C
0.6
0.6
1.1
0.4
0.4
2.2 3.9
2.0 3.1
2.8 4.3
8.4 8.5
5.1 5.4
5.2
4.0
6.3
12.4
7.9
-Ac/-H
-Ac/-H
-H
1
1
-Ac/-H
and physical stability. As previously observed with POEPOP
resin, the PEG chain length had a strong influence on the
swelling of the SPOCC resin polymer. The PEG-1500-based
resins attained much larger swelling volumes than PEG-400
resins. The amount of Lewis acid initiator exhibited a major
influence on the resin properties. Polymers obtained with use
of the minimum amount of Et2O‚BF3 (0.15 equiv) did not form
physically stable resins; instead swelling and granulation gave
a clear, translucent gel from which removal of solvent by
filtration was problematic. When more than 0.7 equiv of Lewis
acid was employed, initiation of polymerization was ac-
companied by an increase in viscosity; however, even after a
prolonged reaction time, the resin never stabilized and remained
in a highly viscous liquid state. Resins useful for synthesis were
obtained by using Lewis acid in amounts ranging from 0.3 to
Figure 1. Beads of SPOCC-400 obtained by suspension polymeriza-
tion.
stirring during emulsion formation. Stirring at 200 rpm for 1
min appeared to be very efficient and afforded beads with good
uniformity and reproducibility in the size range between 300
and 500 µm (Figure 1). Stirring at 2000 rpm yielded beads in
the size range between 5 and 50 µm. Temperature influenced
the outcome of the polymerization. Emulsion formation at 45
°C resulted in large amounts of aggregation, while at 0 °C hardly
any polymerization occurred. When the silicon oil was main-
tained at 25 °C, the polymerization proceeded in a controlled
manner to give the optimally beaded polymer.
Chemical Stability of the Resulting Polymer. Chemical
stability was examined by exposing the resins to different
reaction conditions and reaction times, and the results with
SPOCC resin were compared with those of other resins used
for solid-phase organic chemistry. No change of the SPOCC
resin was observed under the reaction conditions 37% aqueous
HCl (4 weeks), neat anhydrous hydrogen fluoride (45 min and
0.5 equiv. In the preferred method to yield a mechanically stable
resin, Lewis acid was added at 0 °C and polymerization was
initiated by warming to 25 °C and continued by a 48 h curing
period at 50-70 °C. Furthermore, the loading of SPOCC resin
1
could be significantly enhanced by incomplete oxetanylation
and subsequent acetylation of the starting monomers followed
by polymerization and deacetylation (entry 5 in Table 1). At
present, the maximum loading obtained by this method has been
1
.2 mmol/g for SPOCC-400. In summary, SPOCC resin can
24 h), and BuLi (10 equiv in comparison to the determined resin
be synthesized by variation of the reaction parameters to yield
transparent resins with well-defined properties designed for
different chemical and biochemical purposes.
loading), nor did heating the SPOCC resin with 20 equiv of
thionyl chloride (SOCl2) in toluene at reflux temperature for
7
2 h affect the resin stability. Under the same conditions the
Synthesis of Beaded SPOCC Resin. Suspension and disper-
POEPOP resin was previously shown to dissolve after 10 min.
Finally, SPOCC resin samples were exposed to 35% HBr in
glacial acetic acid and to a mixture of 5% trimethylsilyl
trifluoromethanesulfonate (TMSOTf) in 1:1 acetic anhydride/
CH2Cl2, and the time for complete dissolution was measured
and compared with values previously determined for the
POEPS- and POEPOP resins. After 10 min POEPS was
completely dissolved under both conditions. Similarly, POEPOP
dissolved after 30 min in the HBr solution and after 3 min in
the TMSOTf/Ac2O solution. In contrast, SPOCC resin was much
more stable and was dissolved by the HBr/HOAc and TMSOTf/
Ac2O solutions after 36 and 12 h, respectively. These results
indicate that cleavage of the C-O ether bonds of the PEG chains
in SPOCC resin occurs during extended treatment with these
reagents.
Functionalization of the SPOCC Resin. After synthesis and
workup the SPOCC resin was obtained in the form of a hydroxy-
functionalized polymer. The conversion of the hydroxyl groups
into amine and thiol functionalities was conducted to provide
resins to be used for different types of chemistry. Initially,
halogenated resin 4 was prepared by bromination of SPOCC
resin 1 by a triphenylphoshine/imidazole/bromine system (Scheme
2. Three methods were then evaluated for introducing amino
groups to yield 5. Tritylamine and H u¨ nigs base were reacted
with the brominated resin 4 at 60 °C for 18 h to afford a resin
with a loading of only 0.12 mmol/g as determined by analysis
sion polymerization methods for producing beads from acrylic
monomers are well established.2
5-27
These approaches offer a
very attractive alternative to bulk polymerizations because they
provide a means to produce beads of controlled and uniform
size. Although water and highly polar organic solvents are
usually used as the continuous phase for the polymerizations
of hydrophobic monomers such as styrene, such solvents are
incompatible with cationic ring-opening polymerization. An
alternative approach was thus developed that avoided the use
of dispersing agents which interfered with the cationic polym-
erization. This suspension polymerization technique employed
emulsions of the monomers formed in silicon oil, which was
immiscible with the PEG derivatives. The polymerization was
carried out in acetonitrile to maintain the droplets floating in
the silicon oil. Other solvents such as chloroform, dichloro-
methane, and tetrahydofuran did not yield droplets with optimal
gravitational properties. The Lewis acid was added to the
reaction mixture at -20 °C, and then the mixture was suspended
in the silicon oil at room temperature. Attempts to add the Lewis
acid directly to the emulsion resulted in bead aggregation. The
size of the beads could be controlled by varying the rate of
(25) Munger, M.; Trommsdorff, E. In Polymerization Processes; Schild-
knecht, C. E., Skeist, I., Eds.; Wiley-Interscience: New York, 1998; pp
1
06-142.
(
26) Arshady, R.; Ledwith, A. React. Polym. 1983, 1, 159-174.
27) Arshady, R. J. Chromatogr. 1991, 586, 181-197.
(

5462 J. Am. Chem. Soc., Vol. 121, No. 23, 1999
Rademann et al.
Scheme 2. Functionalization of SPOCC Resins 1
Scheme 3. Generation of N-Terminal Peptide Aldehyde 8
on the SPOCC Resin
after acid-mediated trityl cleavage. Potassium phthalimide
reacted with 4 at 60 °C for 18 h to yield an amine loading of
0
.38 mmol/g after deprotection with hydrazine. The highest
loading (0.45 mmol/g) was obtained by displacement bromine
4
with azide followed by reduction with dithiothreitol and
28
DBU.
Thiol resin 6 was also prepared by the substitution of bromide
4
with trityl thiol in the presence of sodium hydride, followed
Scheme 4. Nucleophilic Reactions on the SPOCC Resin
by acidic trityl cleavage to yield a resin with a thiol loading of
0.53 mmol/g.
Chemical Synthesis on the SPOCC Resin. Organic syn-
thesis and peptide chemistry were performed on resin 5 by using
various linker strategies and reaction conditions to demonstrate
the versatility of the SPOCC resin. Reactions of peptide
aldehydes to introduce C-C bonds into the backbone as amide
isosteres are of particular interest for generating potential
protease inhibitors. This concept for the synthesis of peptide
isosteres has been demonstrated recently on the POEPOP-400
1
9
resin. However, the base-labile HMBA linker employed
limited the use of strongly basic nucleophiles. To extend the
scope, the acid-labile Rink linker was attached to SPOCC-400
resin 5 with a loading of 0.45 mmol/g by reaction with an Fmoc-
protected Rink linker using O-(1H-benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium tetrafluoroborate (TBTU)29 and N-ethyl-
morpholine (NEM) (Scheme 3). After deprotection of the linker,
tetrapeptide 7 was assembled on the resin using Fmoc-amino
acids and the same acylation conditions. The N-terminal serine
residue was transformed into resin-bound peptide aldehyde 8
using periodate cleavage in water buffered with 0.01 M sodium
3
0
phosphate at pH 7 to avoid cleavage of the acid-labile linker.
Aldehyde 8 was successfully reacted with ylides in Wittig-type
and Horner-Wadsworth-Emmons-type reactions (Scheme 4).
For example, triethyl phosphonoacetate deprotonated with less
than 1 equiv of n-butyllithium added to the aldehyde resin to
produce exclusively the trans isomer as determined by HPLC,
MS, and NMR spectroscopy after treatment of the resin with
colored solution that was added to the resin at -40 °C. Cleavage
with TFA/water yielded a 1:1 mixture of the 2- and 3-hydroxy
products 10a and 10b resulting from hydration of the product
acrylamido functionality.
9
5% TFA/water to furnish compound 9. The Wittig reaction
between aldehyde 8 and the nonactivated methyltriphenylphos-
phonium iodide was performed as an example involving strongly
basic conditions. The ylide was generated with less than 1 equiv
of n-butyllithium at -78 °C to provide the characteristically
In addition to the olefination chemistry to yield peptide
isosteres, electrophilic reactions promoted by Lewis acids were
investigated on the SPOCC resin. For example, glycopeptide
1
7
(
28) Meldal, M.; Juliano, M. A.; Jansson, A. M. Tetrahedron Lett. 1997,
8, 2531-2534.
29) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron
Lett. 1989, 30, 1927-1930.
30) Pallin, T. D.; Tam, J. P. J. Chem. Soc., Chem. Commun. 1995,
021-2022.
13 was prepared via solid-phase glycosylation of the pen-
tapeptide Fmoc-ASFLG-SPOCC resin (11) that was synthesized
directly on the resin using the Fmoc-amino acids and TBTU/
NEM conditions (Scheme 5). Serine was incorporated without
side-chain protection. Tetra-O-acetylgalactopyranosyl trichlo-
3
(
(
2

SPOCC Resin
J. Am. Chem. Soc., Vol. 121, No. 23, 1999 5463
Scheme 5. Synthesis of Glycopeptides by Solid-Phase
Glycosylation
Scheme 6. Enzymatic Reactions on SPOCC-1500 Resin:
Cleavage of Decapeptide Substrate 14 by Proteases
degradation and MALDI-TOF-MS. The resin contained only
the pentapeptide H-ARK(Abz)GG-OH.
roacetimidate31 was added to the Fmoc-protected peptide resin
and activated with TMSOTf to effect glycosylation. No linker
was used, and the first amino acid, Fmoc-glycine, was directly
attached to the resin by an ester bond. Therefore, after two cycles
of glycosylation (each with 5 equiv) the glycopeptide was
cleaved from resin 12 with hydrazine to afford galactopyranosyl
peptide 13 as the C-terminal peptide hydrazide in good yield.
On the other hand, the reaction of resin 14 bearing a substrate
for the matrix metalloprotease MMP-9 (a considerably larger
protease of 72 kDa) was not cleaved as shown by the absence
of fluorescence and a complete recovery of the decapeptide after
cleavage and analysis by HPLC-MS. The enzyme was highly
active with the substrate in solution. Presumably, the enzymatic
reaction on the solid phase was impeded by the resin excluding
the larger protease, indicating the importance of the permeability
of the polymer in solid-phase enzyme reactions. Furthermore,
the size limit for enzyme reactions on the new SPOCC resins
may be further expanded by the use of longer PEG chains as
starting materials as recently demonstrated by the complete
cleavage of the identical substrate with MMP-9 employing a
PEGA-6000 resin (unpublished results).
Enzyme Reactions with SPOCC-1500 Resin. Enzymatically
catalyzed reactions on solid supports deserve particular interest
because of their potential use in enzyme-assisted synthesis and
on-bead screening of substrate and inhibitor libraries. The
feasibility of enzyme reactions with SPOCC-1500 resin was
examined with a decapeptide protease substrate that was
synthesized on the solid support. Hydroxyl-functionalized
SPOCC resin 1 was acylated with Fmoc-Gly-OH using 1-(2-
mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) and N-
Conclusions
3
2
methylimidazole (MeIm) in CH2Cl2 for activation. After
deprotection of the amino group, a fully protected nonapeptide
fragment (AY(3-NO2)GPLGLYARK(Abz)G) was coupled to
A polar polymeric resin (SPOCC) equally suited for general
organic chemistry and for enzyme reactions has been presented.
Cationic ring-opening polymerization was demonstrated as the
preferred method for SPOCC resin preparation. It enabled
various parameters such as swelling, loading, and mechanical
stability to be modified in the resulting polymer. Oxetanes
served as the reactive moieties, allowing both macromonomer
alkylation without formation of elimination byproducts and
Lewis acid-catalyzed polymerization. Acetylation of residual
hydroxy groups facilitated the polymerization. SPOCC resins
possess only primary ether and alcohol functionalities. The resin
is therefore inert to most reaction conditions required in organic
chemistry, even to strongly acidic or basic conditions. SPOCC
polymers proved to be susceptible only to PEG backbone
decomposition under conditions effecting cleavage of primary
ether bonds and after reaction times substantially longer than
those destroying other PEG-based resins. Prepared as beaded
polymers with hydroxyl, amino, and thiol functionalities, the
novel resins were used for solid-phase peptide synthesis and
for organic chemistry. The syntheses of glycopeptide derivatives
and peptide isosteres were demonstrated. Furthermore, complete
enzymatic digestion of a peptide substrate bound to the solid
support was achieved. As indicated by these experiments, the
ꢀ
yield resin 14. The 3-nitrotyrosine and N -(2-aminobenzoyl)-
lysine residues were incorporated to serve as a resonance energy
transfer pair of chromophores for visual detection of substrate
cleavage (Scheme 6).33 The substrate containing resin 14 was
treated with a solution of the 27 kDa protease subtilisin (0.1
µM in a 0.01 M pH 7 phosphate buffer). After 10 min, bright
fluorescence was observed using a fluorescence microscope.
Furthermore, the emission of light was uniform from the entire
volume of the resin particles, indicating the reaction to be
complete after a reaction time of 30 min. To elucidate the nature
of the enzyme reaction, Edman degradation, peptide cleavage,
and HPLC analysis were conducted and proved the digestion
of the starting substrate to be complete with no detection of
residual decapeptide. Residual fragments were cleaved off the
resin and purified by HPLC. They were identified by Edman
(
(
31) Schmidt, R. R.; Stumpp, M. Liebigs Ann. Chem. 1983, 1249-1256.
32) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron Lett.
1
990, 31, 1701-1704.
33) Meldal, M.; Svendsen, I.; Breddam, K.; Auzanneau, F. I. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 3314-3318.
(

5464 J. Am. Chem. Soc., Vol. 121, No. 23, 1999
Rademann et al.
novel resin should be suitable for the generation of peptide
isostere libraries and for identification of new protease inhibitors
on the solid support.
the volatiles including the HMDS were removed in a 50 °C water bath
on a rotary evaporator. The residue was dissolved in DMF (15 mL),
treated with portions of tosylated oxetane 3 (24 mmol) at room
temperature, and heated for 12 h at 75 °C. After cooling to ambient
temperature, water (2 mL) was added, and the mixture was stirred for
Experimental Section
1
5 min to hydrolyze unreacted alkylating agent. The solvents were
removed at 40 °C under reduced pressure. The remaining slurry was
resuspended in CH Cl (10 mL), filtered through a 2 cm layer of
kieselguhr (Celite, prewetted with CH Cl and compressed) on a fritted
glass filter, and washed with CH Cl (50 mL). Evaporation gave a yield
General Procedures. All solvents were stored over molecular sieves.
DMF was distilled under reduced pressure prior to use. Solid-phase
peptide chemistry and solid-phase organic chemistry were performed
in plastic syringes. Flat-bottom PE syringes were equipped with sintered
Teflon filters (50 µm pores), Teflon tubing, and valves for applying
2
2
2
2
2
2
of 90% of 2a. NMR spectroscopy on the acetylated product indicated
the alkylation of >95% of the PEG hydroxyl groups with oxetane rings;
NMR spectroscopy on the product indicated less than 5% of remaining
tosylate salts. The product obtained by the described filtration method
was of higher purity than that obtained by a precipitation procedure.
When less alkylating reagent 3 was employed (15 and 18 mmol), the
3
4
suction to the syringes from below. For moisture-sensitive and low-
temperature reactions such as the Wittig olefination, commercially
available reaction vials with septa and screw caps were employed
(Reacti-Vial, Pierce, Rockford, IL). Resin loadings were determined
by Fmoc cleavage and optical density measurement at 290 nm and
were calculated employing a calibration curve. In the case of the starting
hydroxyl resin the loading was measured after esterification with Fmoc-
Gly using MSNT as condensing agent and MeIm as base.32 Starting
loadings were calculated from the measured loadings and the molar
percentage of oxetanyl groups decreased (66%, 80%).
1
Data for 2a-400. H NMR (250 MHz, CDCl
3
): δ ) 1.28 (s, 3 H,
2
3
)
(
(
-Me), 3.52 (s, 2 H, 3-CH
5.7 Hz, 2 H, 2-CH ), 4.47 (d, J ) 5.7 Hz, 2 H, 2-CH
MHz, CDCl ): δ ) 21.33 (3-methyl), 70.5-70.95 (PEG-CH
2 2
O), 3.62 (br s, 16 H, PEG-CH ), 4.31 (d, J
2
13
2
2
). C NMR
), 76.56
mass increase (∆M) employing the formula loadingstart ) loadingmeasd
/
3
2
(
1 - loadingmeasd∆M/1000) and expressed in millimoles per gram (the
3-CH
2
O-), 80.12 (2-CH
Data for 2a-1500. H NMR (250 MHz, CDCl
2
).
division by 1000 is required since the molar mass increase is used as
grams per mole).
1
3
): δ ) 1.28 (s, 3 H,
2
3
)
-Me), 3.52 (s, 2 H, 3-CH
5.7 Hz, 2 H, 2-CH ), 4.47 (d, J ) 5.7 Hz, 2 H, 2-CH
Acetylation of Mixtures of Mono- and Bisoxetanylated PEG (2b).
2
O), 3.62 (br s, 34 H, PEG-CH
2
), 4.31 (d, J
Analysis of all solid-phase reactions was performed after product
cleavage from a resin sample. When the Rink linker was used, a small
portion of the resin (1-2 mg) was weighed in an Eppendorf tube and
treated with TFA/water (95%, 50 µL, 1 h). The resin was dried in a
centrifuge in vacuo for 15 min and resuspended in acetonitrile/water
2
2
2
).
Residue 2a (10 g) was dissolved in pyridine (20 mL), treated with acetic
anhydride (10 mL), and stirred at room temperature for 24 h. Solvents
were removed under reduced pressure to give the product in a
(1:1, 50 µL). A sample of this solution (10-20 µL) was examined on
1
quantitative yield. The H NMR spectrum of the product was recorded,
analytical HPLC (8 × 200 mm C-18 column, Millipore Delta PAK 15
and the degree of acetylation was quantified by using the integration
of the signals at 2.03, 4.31, and 4.47 ppm of acetyl CH and ring CH
3 2
protons, respectively.
µm) with detection at 215 and 280 nm using a photodiode array detector
(Waters M 991). Eluents A (1% TFA in water) and B (10% of A with
1
% TFA in acetonitrile) were used in a linear gradient (0% B f 100%
1
Data for 2b-400. H NMR (250 MHz, CDCl
3
): δ ) 1.28 (s, 3 H,
B in 50 min). Preparative HPLC was performed using a 25 × 200 mm
semipreparative RP-18 column (Millipore Delta Pak 15 µ), employing
a linear gradient starting with 85% A and 15% B, a slope of 0.5% per
min, and a flow rate of 10 mL/min. Collected fractions were analyzed
by ES/MS (positive mode on a Fisons VG Quattro Instrument).
Alternatively a MALDI-TOF mass spectrum (Finnigan MAT 2000)
was recorded employing R-cyano-4-hydroxycinnamic acid or 1,4-
dihydroxybenzoic acid as matrix. The calculated molecular weights of
all products are the averaged molecular masses. Functionalized resins
were investigated with MAS solid-phase NMR on a 3-5 mg sample
placed using a nanoprobe tube using a Varian unity INOVA 500 MHz
instrument and different deuterated solvents. Solution-phase NMR was
performed on a Bruker DRX 250 MHz instrument or a Varian 500
MHz instrument. Chemical shifts were calibrated relative to the signal
of tetramethylsilane (0 ppm) or internal solvent signals (3.30 and 36.067
3
-Me), 2.03 (s, Ac), 3.52 (s, 2 H, 3-CH
2
O), 3.62 (br s, 16 H, PEG-
), 4.47 (d, J ) 5.7 Hz, 2 H,
2
2
CH
2
), 4.31 (d, J ) 5.7 Hz, 2 H, 2-CH
2
2
-CH ).
2
SPOCC Resin 1. Procedure A. Under argon, oxetanylated PEG-
500 or -400 (2a; 1-20 mmol) or acetylated derivative 2b was
1
dissolved in CH
2 2
Cl (equal volume), and the solution was cooled to
-20 °C, stirred with a magnetic stirbar, and treated with boron
trifluoride diethyl etherate (0.15-0.3 equiv). The reaction mixture was
warmed gradually to determine at which temperature polymerization
occurred (-10 °C, 2 h; 0 °C, 2 h; 4 °C, 2 h). Finally, the viscosity of
the solution increased and magnetic stirring stopped (sticky point) after
the solution was kept at 4 °C for 30 min. The polymer was kept at this
temperature for 2 days and an additional day at room temperature. For
workup the polymer was cut into pieces, swollen (CH
then granulated through a metal sieve (1 mm pore size) employing a
pestle. The granulated resin was washed thoroughly (CH Cl , THF,
DMF, water, 3 M HCl, water, 0.5 M NaOH, water, DMF, THF, CH
Cl ) and dried in vacuo. Resin loading was determined as described in
2 2
Cl , 2 h), and
4 6
ppm for d -MeOD, 2.49 ppm for d -DMSO).
Preparation of SPOCC Resins. (3-Methyloxetan-3-yl)methyl
2
2
2
-
4
-Toluenesulfonate (3). 4-Toluenesulfonyl chloride (20 g, 105 mmol)
2
2 2
was dissolved in CH Cl (50 mL) and pyridine (50 mL). With cooling
in an ice bath, 3-(hydroxymethyl)-3-methyloxetane (100 mmol, 9.9 mL)
was added dropwise. The temperature was increased to 20 °C overnight,
2 2
the solution was diluted with CH Cl (100 mL), and the solvent was
extracted with water. The organic phase was dried with magnesium
sulfate and filtered, and the volatile solvents were removed by
evaporation. The remaining material was concentrated several times
with toluene to remove the residual pyridine and with chloroform to
remove the toluene. The crude product was sufficiently pure for further
the General Procedures, and the swelling volumes in different solvents
were determined following a literature procedure35 (see Table 1). In
the case of partially acetylated macromonomers (2b) the resin was
treated with 0.5 M NaOH overnight; nanoprobe MAS-NMR of the dried
1
resin proved the disappearance of any acetyl signals in the H spectrum.
Procedure B. A solution of oxetanylated PEG-1500 (2a) in CH
2
-
Cl
2
(1 mL/g monomer) was cooled to 0 °C, treated with Et O‚BF (0.4
2
3
equiv), warmed to room temperature, and stirred until the sticky point
was reached after 10 min, as indicated by the increased viscosity halting
the magnetic stirrer. After stirring stopped the reaction was maintained
at an oil bath temperature of 60 °C for 2 d, cooled to room temperature,
and worked-up as described in Procedure A.
use. Yield: 22 g of a white crystalline solid (92%). TLC: R
f
(petroleum
ether/ethyl acetate, 1:1) 0.56. The spectroscopic data were in accordance
2
4
with those in the literature.
Bis[(3-methyloxetan-3-yl)methyl]PEG (2a). Poly(ethylene glycol)
Procedure C. A solution of oxetanylated PEG-400 (2a,b) in diglyme
(10 mmol; -400 or -1500) was dried carefully by removal of water
(1 mL/g monomer) was stirred at room temperature and treated slowly
through coevaporation with toluene and then dissolved in a 1:1 toluene/
DMF solution (30 mL). With stirring, potassium hexamethyldisilazane
with Et (0.4 equiv). Stirring stopped after 1 min. The reaction
2
O‚BF
3
was warmed to 70 °C for 2 d, cooled to room temperature, and worked-
(KHMDS; 22 mmol) was added at room temperature, and after 15 min,
(
34) Christiansen-Brams, I.; Meldal, M.; Bock, K. J. Chem. Soc., Perkin
(35) Auzanneau, F.-I.; Meldal, M.; Bock, K. J. Pept. Sci. 1995, 1, 31-
44.
Trans. 1 1993, 1461-1471.

SPOCC Resin
J. Am. Chem. Soc., Vol. 121, No. 23, 1999 5465
up as described in Procedure A. The same procedure was employed
for PEG-1500 monomers (2a,b) dissolved in 1,2-dichloroethane (1 mL/g
monomer), reaching the sticky point after 5 min.
Suspension Polymerization. Under argon, a solution of macro-
monomer 2b-400 (600 mg, 1.21 mmol) in dry acetonitrile (0.3 mL)
pH 7). The resin was filtered, washed with water, DMF, THF, and
CH
2 2
Cl , dried, and analyzed after resin cleavage as described above.
HPLC: retention time 25.6 min. MALDI-MS: calcd (M ) C19
26 4 5
H N O )
+
+
2
390.44, found (MNa , MH ONa ) m/z 413.3, 431.4.
p-[r-[[N-(Ethylfumaroyl)-L-phenylalanyl]-L-leucylglycylamido]-
,4-dimethoxybenzyl]phenoxyacetylamido-SPOCC Resin (9). Ly-
was cooled to -20 °C, treated with Et
2
3
O‚BF (0.06 mL, 0.48 mmol),
2
and then added to silicon oil (75 mL) at room temperature with
emulsification by stirring at 200 rpm for 1 min. Stirring was slowed to
a minimum, and the reaction was allowed to proceed overnight. The
slurry of beads was filtered off and washed with 5 mL volumes of
each of the following solutions: 1 M aqueous HCl, water, MeOH, DMF,
and CH Cl . The beads were air-dried for 1 h and then dried under
2 2
vacuum to yield 490 mg (83%) of material having a loading of 0.5
mmol/g, exhibiting swelling volumes of 3.3 mL/g (water), 3.2 mL/g
ophilized resin 8 (90 mg, 0.032 mmol) was treated with toluene (1
mL) and triethyl orthoformate (0.5 mL) for 2 h and washed with dry
toluene (6 × 2 mL). In a separate round-bottom flask triethyl
phosphonoacetate (32 µL, 5 equiv) was dissolved in toluene (1 mL),
the solution was cooled to 0 °C, and n-butyllithium (4.5 equiv, 1.6 M
solution in hexane) was added. After 10 min the solution was added to
the resin and allowed to react at ambient temperature for 90 min. After
2 2
being washed (DMF, THF, CH Cl ) and dried, the resin was treated
(
DMF), and 4.6 mL/g (CH
Functionalization of SPOCC Resins. Bromo-SPOCC Resin (4).
Resin 1 (1 g, 0.6 mmol) was suspended in CH Cl (10 mL) and treated
2 2
Cl ).
with NaOH (0.1 M) for a period of 2 h, washed, and dried again.
Cleavage of an analytical sample and HPLC analysis were conducted
as described in the General Procedures. Preparative cleavage of 30 mg
of resin yielded 3.3 mg (64%). HPLC: retention time 27.0 min.
2
2
with triphenylphosphine (787 mg, 5 equiv) and imidazole (204 mg, 5
equiv). After the reagents had dissolved, the suspension was cooled to
+
MALDI-MS: calcd (M ) C H N O ) 432.48, found (MH
2
1
28
4
6
) m/z 433.4.
H NMR (250 MHz, d -MeOD): δ ) 0.88-0.98 (2 d, 6 H, Leu-Me),
1
0 °C in a water bath and treated dropwise with bromine (155 µL, 5
1
4
equiv). The water bath was removed, and after being stirred overnight
at room temperature, the resin was filtered and washed with DMF,
1.6-1.75 (m, 3 H, Leu), 3.0, 3.25 (2 dd, 2 H, Phe-CH ), 3.8-4.0 (2 d,
2
2
2 H,
J ) 17.8 Hz, Gly-R), 4.4-4.5 (dd, 1 H, Leu-R), 4.85 (dd, 1 H,
Jtrans ) 15.5 Hz, olefinic protons), 7.15-
water, 10% sodium thiosulfate solution, water, DMF, THF, and CH
Cl
(3 × 20 mL each). The bromine content was 0.59 mmol/g as
determined by elemental analysis.
2
-
Phe-R), 6.68, 7.06 (2 d, 2 H,
7.4 (m, 5 H, aromatic protons).
3
2
r-[N-(2-hydroxypropionyl)-L-phenylalanyl]-L-leucylglycyl-
amide (10a). A suspension of methyltriphenylphosphonium iodide (69
mg, 0.171 mmol) in THF (2 mL) was cooled to -50 °C and treated
with a 1.6 M solution of n-butyllithium (0.154 mmol) to furnish a
yellow-orange solution. After 20 min of stirring under argon, the
solution was warmed to -10 °C, treated with resin 6 (96 mg, 0.034
mmol), and allowed to react for 2 h. Washing and drying the resin
followed by cleavage of the product (95% TFA, 2 h) afforded a mixture
of 2- and 3-hydroxypropionyl products 10a and 10b (5.3 mg, 40%).
Amino-SPOCC Resin (5). Resin 4 (1 g, 0.59 mmol) was suspended
in a solution of sodium azide in DMSO (390 mmol, 10 equiv, 10 mL).
The mixture was warmed to 60 °C for 18 h, cooled to room temperature,
filtered, and washed thoroughly with DMF, water, and DMF. Reduction
was effected employing a 0.5 M solution of 1,4-dithiothreitol (DTT)
in DMF (10 mL), containing 0.1 M 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU). The resin was filtered and washed with DMF, THF, and
2 2
CH Cl . The resin loading was determined by spectrophotometric
measurement of Fmoc cleavage at 290 nm after functionalization of a
resin sample with Fmoc-succinimide (10 equiv, 4 h). The measured
loading was 0.44 mmol/g, corresponding to a loading of 0.48 mmol/g
for the resin employed.
Thiol-SPOCC Resin (6). Triphenylmethylthiol (77 mg, 10 equiv)
in THF (1 mL) was cooled to 4 °C, treated with sodium hydride (9 mg
of 60% NaH, 8 equiv), stirred for 15 min, and added under argon to
bromo-SPOCC resin 4 (44 mg, 0.028 mmol) in a reaction vessel
HPLC: retention time 25.6 min. ESI-MS: calcd (M ) C20
06.22 Da, found (MH , MNa ) m/z 407.4, 429.3.
Data for 10a (2-Hydroxy Product). 1H NMR (250 MHz, d
H
30
N
4
O
5
)
+
+
4
4
-
MeOD): δ ) 0.88-0.98 (m, 6 H, Leu-Me), 1.28 (m, 3 H, Me-CHOH),
2
1
.6-1.75 (m, 3 H, Leu), 3.0-3.2 (2dd, 2 H, Phe-â), 3.82 (dd, 2 H, J
)
17.9 Hz, Gly-R), 3.90 (m, 1 H, CHOH), 4.4-4.5 (dd, 1 H, Leu-R),
4
.6-4.75 (dd, 1 H, Phe-R).
Data for 10b (3-Hydroxy Product). 1H NMR (250 MHz, d
-
4
(
ReactiVial, 0.6 mL). After the solution was stirred for 16 h at room
temperature, the resin was filtered, washed with THF, DMF, water,
DMF, THF, and CH Cl , and dried in vacuo. The trityl protecting group
was cleaved by washing with 10% TFA in CH Cl . TFA washings were
MeOD): δ ) 0.88-0.98 (m, 6 H, Leu-Me), 1.6-1.75 (m, 3 H, Leu),
.0-2.2 (m, 2 H, CH -CH OH), 3.0-3.2 (2 dd, 2 H, Phe-â), 3.82
dd, 2 H, J ) 17.9 Hz, Gly-R), 3.30 (m, 1 H, CH OH), 4.4-4.5 (dd,
H, Leu-R), 4.6-4.75 (dd, 1 H, Phe-R).
[N-(9-Fluorenylmethoxycarbonyl)-L-alanyl]-L-seryl-L-phenyl-
2
(
2
2
2
2
2
2
2
2
1
collected until yellow color was no longer detectable (82 mL) and
measured spectrophotometrically at 410 nm. The loading was calculated
from the amount of cleaved trityl cation by employing a calibration
alanyl-L-leucylglycyl-SPOCC Resin (11). SPOCC-400 resin 1 (326
mg, 0.58 mmol/g loading, 0.19 mmol) was reacted twice with a solution
of Fmoc-Gly-OH (339 mg, 3 equiv), MSNT (338 mg, 3 equiv), and
curve. Finally the resin was washed with DMF, THF, and CH
2 2
Cl , and
dried to yield 42 mg of resin 6 with a loading of 0.54 mmol/g.
2 2
N-methylimidazole (MeIm; 68 µL, 2.25 equiv) in CH Cl (4 mL) each
Organic Synthesis with SPOCC Resins. p-[r-(L-Seryl-L-phenyl-
alanyl-L-leucylglycylamido)-2,4-dimethoxybenzyl]phenoxyacetylamido-
SPOCC Resin (7). Fmoc-protected Rink linker (208 mg, 0.4 mmol),
TBTU (122 mg, 0.38 mmol), and 4-ethylmorpholine (NEM) (83 µL,
time for 45 min. After Fmoc deprotection with treatments using 20%
piperidine in DMF for 2 and 16 min, the glycinyl residue was elongated
by couplings, deprotections, and coupling 3 equiv of the Fmoc-amino
acids Leu, Phe, Ser, and Ala, which were preactivated for 15 min with
TBTU (2.9 equiv, 177 mg) and NEM (4 equiv, 127 µL) in DMF (4
mL) and reacted with the resin for 3 h. A small resin sample (2 mg)
was cleaved with 0.1 M NaOH solution (50 µL, 2 h) and analyzed by
0
.5 mmol) were dissolved in DMF (3 mL). After an activation period
of 10 min, this solution was added to resin 5 (210 mg, 0.1 mmol) and
reacted for 3 h. After the reaction solution was washed with DMF (5
×
10 mL), the Fmoc group was cleaved with 20% piperidine in DMF
2 and 16 min treatments), and the resin was washed again. The
HPLC and MALDI-MS. HPLC: retention time 28 min. MALDI-MS:
(
+
calcd (M ) C23
H
35
N
5
O
7
) 493.6 Da, found (MNa ) m/z 517.7.
deprotected amine was coupled, deprotected, and coupled with 3 equiv
of the Fmoc-amino acids Gly, Leu, Phe, and Ser, using TBTU (93 mg,
[N-(9-Fluorenylmethoxycarbonyl)-L-alanyl][O-(2,3,4,6-tetra-O-
acetyl-â-D-galactopyranosyl)-L-seryl]-L-phenylalanyl-L-leucylglycyl-
SPOCC Resin (12). Resin 11 (100 mg, 0.04 mmol) was lyophilized
from dry toluene (3 mL) in a speed vac overnight and treated with a
0
.29 mmol) and NEM (66 µL, 0.4 mmol) for activation as described
for the linker above. After final Fmoc deprotection the product was
cleaved of a resin sample (2 mg) using 95% TFA in water for 2 h, and
analyzed by HPLC (retention time 24.0 min) and MALDI-MS [calcd
3
1
solution of tetra-O-acetyl-R-D-galactopyranosyl trichloroacetimidate
(100 mg, 5 equiv) in CH Cl (1.5 mL). Under argon, trimethylsilyl
trifluoromethanesulfonate (TMSOTf; 120 µL of a 1 M solution in CH2-
Cl ) was added, and the mixture was allowed to react for 1 h. The
resin was filtered, washed with CH Cl , THF, DMF, THF, and CH
Cl , and dried in vacuo. The glycosylation procedure was repeated,
and analysis was conducted with HPLC and MALDI-MS after cleavage
+
+
+
(
M ) C20
H
31
N
5
O
5
), found (MH , MNa , MK ) m/z 422, 444, 460].
2
2
The final peptide loading was 0.36 mmol/g.
p-[r-[(N-Glyoxalyl-L-phenylalanyl)-L-leucylglycylamido]-2,4-
dimethoxybenzyl]phenoxyacetylamido-SPOCC Resin (8). Resin 7
2
2
2
2
-
(200 mg, 0.072 mmol) was treated for 1 h with an aqueous solution of
2
NaIO (46 mg, 3 equiv) in sodium phoshate buffer (2.5 mL of 0.01 M,
4

5466 J. Am. Chem. Soc., Vol. 121, No. 23, 1999
Rademann et al.
with NaOMe in MeOH (0.02 M, 2 h). HPLC: retention time 20.1 min.
I. Substrate Cleavage with Subtilisin. Resin 14 (2 mg) was treated
with a solution of subtilisin BNP′ (Novo Nordisk, Bagsvaerd, Denmark;
+
MALDI-MS: calcd (M ) C29
56.
L-Alanyl[O-(â-D-galactopyranosyl)-L-seryl]-L-phenylalanyl-L-
H N
45 5
O12) 655.7 Da, found (MNa ) m/z
-
7
6
10 M ) 100 nM of the 27 kDa protein) in pH 7 phosphate buffer
(50 mmol of NaH PO in H O). After 15 min strong fluorescence was
observed under UV irradiation. After 3 h the resin was washed with
water, DMF, THF, and CH Cl and dried. One portion of the enzyme-
2
4
2
leucylglycine Hydrazide (13). Resin 12 (35 mg, 4 µmol) was treated
for 3 h with a 10% hydrazine solution in water. Yield: 4.8 mg of
compound 13 (55%). HPLC: retention time 22.0 min. MALDI-MS:
2
2
treated resin (1 mg) was cleaved with NaOH (50 µL of a 0.1 M solution,
2 h) and analyzed by HPLC and MALDI-MS. The other portion of the
resin (1 mg) was subjected to Edman degradation. The HPLC
chromatogram indicated complete cleavage of the starting peptide
substrate and yielded one peptide product. HPLC: retention time 21.0
+
1
calcd (M ) C29
47 7
H N O11) 669.7 Da, found (MNa ) m/z 694. H NMR,
3
2
50 MHz, d
4
-MeOD): δ ) 0.88-0.98 (2 d, 6 H, Leu-Me), 1.45 (d, J
7 Hz, 3 H, Ala-Me), 1.6-1.75 (m, 3 H, Leu), 3.0, 3.25 (2 dd, 2 H,
)
Phe-CH
2
), 3.4-3.6 (dd, m, 2 H, Gal-2-H, Gal-5-H), 3.7-4.1 (m, 8 H,
3
Ala-R, 2 Ser-â, 2 Gly-R, Gal-3-H, Gal-4-H, 2 Gal-6-H), 4.29 (d, J )
42 10 7
min. ES-MS: calcd (M ) C26H N O ) 606.7 Da, found m/z 607.6.
Edman degradation (three cycles): A, Abz; R; K.
7
.2 Hz, 1H, â-Gal-H-1), 4.3-4.4 (dd, 1 H, Leu-R), 4.7 (2 dd, 2 H,
Phe-R, Ser-R), 7.15-7.4 (m, 5 H, aromatic protons).
II. Substrate Cleavage with Matrix Metalloprotease-9 (MMP-
9). Resin 14 (2 mg) was treated with a solution of MMP-9 (recombinant,
72 kDa enzyme, obtained from the Center for Clinical and Basic
Research (CCBR), Ballerup, Denmark) (100 and 275 nM protease) in
pH 7.72 buffer (buffer 17, obtained from CCBR, Ballerup, Denmark)
for 24 h. In neither case was any significant fluorescence observed.
Cleavage and HPLC analysis as described in (I) yielded exclusively
the starting peptide substrate.
Enzymatic Reactions on the SPOCC Resin. L-Alanyl-(3-nitro-
L-tyrosinyl)-L-glycyl-L-prolinyl-L-leucylglycyl-L-leucyl-L-tyrosinyl-
E
L-alanyl-L-arginyl-[N -(2-aminobenzoyl)-L-lysinyl]glycylglycyl-
SPOCC Resin (14). SPOCC-1500 1 (prepared by procedure C, 65 mg,
0
.027 mmol) was treated twice with a solution of Fmoc-Gly-OH (41
mg, 5 equiv), MSNT (40 mg, 5 equiv), and MeIm (8 µL, 3.75 equiv)
in CH Cl (4 mL) for 45 min, filtered, and washed with CH Cl and
2
2
2
2
DMF. The Fmoc group was cleaved (20% piperidine in DMF for 2
and 16 min), and the resin was washed with DMF. Protected
Acknowledgment. We thank Rikke Sindet, Anita M. Jan-
sson, and Pia Breddam for technical assistance. Professor
Christian Pedersen kindly conducted treatment of resins with
HF. This work was carried out in the SPOCC center and was
supported by the Danish National Research Foundation.
t
nonapeptide Fmoc-AY(3-NO
G-OH (43 mg, 3 equiv) in DMF (4 mL) was preactivated with TBTU
6.8 mg, 2.9 equiv) and NEM (3.7 µL, 4 equiv) for 15 min, added to
the resin, and reacted for 3 h. The resin was washed with DMF, THF,
and CH Cl , dried, and treated with 95% TFA for 10 min and for 2.5
h to remove side chain protecting groups. The resin was washed with
5% acetic acid (four times, 5 min), 5% triethylamine in DMF (three
times, 2 min), DMF (two times, 2 min), THF, and CH Cl and dried in
2
)GPLGLY( Bu)AR(Pmc)K(N-Boc-Abz)-
(
2
2
Supporting Information Available: 1H and 13C NMR
1
9
spectra (250 MHz) of macromonomers 2a and 2b, H MAS
2
2
NMR spectra (500 MHz) of resins 1, and HPLC chromatograms
of the enzymatic cleavage of compound 14 (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
vacuo. Cleavage of a resin sample (2 mg, 0.1 M NaOH, 2 h) afforded
material for analysis. HPLC: retention time 32.0 min. MALDI-MS:
+
+
calcd (M ) C68
H
N
99 19
O
2
19) 1486.7 Da, found (MH , MNa - H O)
m/z 1487, 1493.
JA984355I