Increased polymer length of oligopeptide-substituted polynorbornenes with LiCl

Article abstract of DOI:10.1021/jo0265737

The ring-opening metathesis polymerization (ROMP) reaction is extraordinarily useful for the preparation of a large variety of polymers. We report that the length (n = 25-50) of high-substituent-density oligopeptide polymers synthesized by ROMP is dramati

Full text of DOI:10.1021/jo0265737

SCHEME 1. Rea ction Sequ en ce for ROMP
In cr ea sed P olym er Len gth of
Oligop ep tid e-Su bstitu ted P olyn or bor n en es
w ith LiCl
Kenny S. Roberts and Nicole S. Sampson*
Department of Chemistry, State University of New York,
Stony Brook, New York 11794-3400
nicole.sampson@stonybrook.edu
Received October 16, 2002
the utility of this strategy is limited by the efficiency of
the conjugation that can be obtained. In our system, this
may be due to either steric hindrance or aggregration as
suggested by later experiments described below. In a
third approach, polymerizations are performed with pro-
tected monomers that are organic-soluble under homo-
geneous, nonaqueous conditions. The polymers are sub-
sequently deprotected to yield water-soluble polymers.
This is the approach that has previously been utilized to
prepare oligopeptide polynorbornenes with fully protected
peptides;7 however, the more reactive 2,3-dihydroimid-
azolylidene ruthenium catalysts were required. To date,
the longest high-density oligopeptide polymers reported
are 10-mers.7a We report here that significantly longer
polymers with high-density pendent oligopeptides may
be obtained by using the third synthetic strategy with
either of Grubbs’ catalysts, Cl2(PCy3)2RudCHPh or
Cl2(PCy3)(H2IMes)RudCHPh, by including lithium chlo-
ride in the polymerization reaction to reduce peptide
aggregation. This method appears to be independent of
peptide sequence.
Abstr a ct: The ring-opening metathesis polymerization
(ROMP) reaction is extraordinarily useful for the prepara-
tion of a large variety of polymers. We report that the length
(n ) 25-50) of high-substituent-density oligopeptide poly-
mers synthesized by ROMP is dramatically improved upon
addition of LiCl to reduce polymer and oligopeptide aggrega-
tion. This methodology should significantly expand the
variety of polymers that may be prepared by ROMP and be
of general use with norbornyl oligopeptides of any sequence.
Ring-opening metathesis polymerization (ROMP) has
emerged as a truly remarkable and versatile synthetic
strategy in polymer and organic chemistry. Its popularity
is due in part to the versatile and robust nature of
alkylidene molybdenum and ruthenium catalysts de-
signed by Schrock1 and Grubbs.2 These catalysts show
either exceptional functional group tolerance or stereo-
selectivity and allow the synthesis of polymers with
narrowly defined molecular weights.
These catalysts have been applied in a wide variety of
systems ranging from polymer composites3 to neoglyco-
polymers.4 For use in our studies to dissect the roles of
sperm ADAM proteins in mammalian fertilization, we
required oligopeptide-substituted polynorbornenes of vary-
ing lengths. Various strategies have been utilized to
prepare high-density polynorbornenes substituted with
bioactive, water-soluble molecules with use of Grubbs’
catalyst, ruthenium carbene Cl2(PCy3)2RudCHPh. In the
first approach, polymerizations are performed with mono-
mers that are themselves water-soluble. For example,
homogeneous mixtures of polar solvents have been used
to prepare neoglycopolymers. However, emulsion condi-
tions that utilize a phase-transfer reagent are often
required for preparing longer (n ) 100) polymers that
have lower solubility in nonaqueous polar solvents.5 A
second strategy employed in the preparation of neoglyco-
polymers was polymerization of a norbornene active ester
under homogeneous nonaqueous conditions followed by
conjugation of the biomolecule.6 However, we found that
We initially explored all three strategies in our studies
to prepare polymers in which the fertilization inhibition
peptide ECDVT derived from fertilinâ, or a scrambled
control, is incorporated into a polymer. We found that
polymerization of side-chain protected peptides with
norbornene at the N-terminus, e.g., 1, in homogeneous
solvent conditions (CH2Cl2/MeOH, 3:1) with alkylidene
5a and a catalytic amount of acid,8 gave the most
reproducible results and the cleanest product (Scheme 1
and Table 1, entries 1 and 2). The average DP determined
1
by H NMR of 8 corresponded to the [M]o/[C]o of 10/1 in
the reaction mixture. However, upon increasing the [M]o/
[C]o to 20/1 or 50/1, we again obtained short polymers
with DP of 10 (Table 1, entries 3 and 4). We obtained
early termination products accompanied by precipitation,
regardless of peptide sequence (Table 1, entry 5). Several
factors may influence the size and length of the polymers
formed. These factors include activation of the catalyst,
aggregation of the growing polymer chain, or steric
interactions. We employed the more active and temper-
ature stable, second generation dihydroimidazolylidene
catalyst 5b and found no improvement (Table 1, entry
6). Early chain termination still occurred. Our observa-
tions combined with earlier reports of the difficulties of
(1) Schrock, R. R. Acc. Chem. Res. 1990, 23, 158-165.
(2) Trnka, T. M.; Grubbs, R. H. Acc. Chem. Res. 2001, 34, 18-29.
(3) White, S. R.; Scotlos, N. R.; Geubelle, P. H.; Moore, J . S.; Kessler,
M. R.; Sriram, S. R.; Brown, E. N.; Viswanatha, S. Nature 2001, 409,
794-797.
(4) Kiessling, L. L.; Gestwicki, J . E.; Strong, L. E. Curr. Opin. Chem.
Biol. 2000, 4, 696-703.
(7) (a) Maynard, H. D.; Okada, S. Y.; Grubbs, R. H. Macromolecules
2000, 33, 6239-6248. (b) Maynard, H. D.; Okada, S. Y.; Grubbs, R. H.
J . Am. Chem. Soc. 2001, 123, 1275-1279.
(8) Lynn, D. M.; Mohr, B.; Grubbs, R. H. J . Am. Chem. Soc. 1998,
120, 1627-1628.
(5) Kanai, M.; Mortell, K. H.; Kiessling, L. L. J . Am. Chem. Soc.
1997, 119, 9931-9932.
(6) Strong, L. E.; Kiessling, L. L. J . Am. Chem. Soc. 1999, 121,
6193-6196.
10.1021/jo0265737 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/25/2003
2020
J . Org. Chem. 2003, 68, 2020-2023

TABLE 1. Solven t Con d ition s Su r veyed for ROMP of Nor bor n yl Oligop ep tid esa
b
entry
monomer
catalyst
solvent
CH2Cl2/MeOH (3:1)
[M]o/[C]o
av n (DP)c
% yieldd
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
1
1
1
2
1
3
2
2
2
2
2
2
2
5a
5a
5a
5a
5a
5b
5a
5a
5a
5a
5a
5a
5a
5a
10/1
10/1
25/1
50/1
10/1
50/1
25/1
25/1
25/1
25/1
25/1
25/1
50/1
50/1
NRe
8
0
40
9
38
0
35
40
54
0
CH2Cl2/MeOH (3:1), 1 N HClf
CH2Cl2/MeOH (3:1), 1 N HCl
CH2Cl2/MeOH (3:1), 1 N HCl
CH2Cl2/MeOH (3:1), 1 N HCl
CH2Cl2/MeOH (3:1)
10
10
NR
10
10
24
NR
NR
8
CH2Cl2/MeOH (3:1), 1 N HCl
CH2Cl2/MeOH (3:1), 1 N HCl, 6 M LiCl
TFE
TFE, 1 N HCl
TFE, 1 N HCl, 6 M LiCl
CH2Cl2/MeOH (3:1), 6 M LiCl
CH2Cl2/MeOH (3:1), 6 M LiCl
CH2Cl2/MeOH (3:1), 1 N HCl, 6 M LiCl
0
20
68
52
47
25
50
40
a
b
d
General reaction conditions: rt, 3-4 h, [M]o ) 0.2-0.3 M. Initial feed ratio. c Calculated from 1H NMR spectrum. Isolated yield
after purification and deprotection. e NR: no reaction or less than 5% product formed as monitored by TLC. f [HCl] ) [C]o.
We next focused on the possibility that aggregation of
the growing polymer chain was the predominant reason
for early termination. It is well-known in solid-phase
peptide synthesis that secondary structures and/or ag-
gregates form on the solid support during the synthesis
of â-sheet forming peptides. Aggregation reduces the
efficiency of coupling the next amino acid by limiting
access to the nucleophile. We reasoned that similar types
of aggregation could be occurring during polymerization
of our norbornene pendent peptide monomers and be
limiting access of the monomer to the catalyst reaction
center. Although the peptide sequences we utilized do not
show a propensity for â-sheet formation in solution,
tethering of the peptides to the polymer backbone might
induce â-sheet formation between neighboring peptides
in the growing polymer chain. In turn, this sheet forma-
tion would lead to insolubility and precipitation, thus
removing the catalyst from solution and further reaction.
This phenomenon would explain why introduction of a
triglycine linker between the norbornene and monomer
did not improve polymerization efficiency, as the tri-
glycine is also able to form â-sheet with a neighboring
oligopeptide
The issues of secondary structure and aggregation may
be resolved by the use of structure-altering solvents such
as trifluoroethanol,9 and/or the addition of lithium salts.
Seebach10 and co-workers have shown that LiCl and other
forms of inorganic salts can influence the solubility of
peptides in nonpolar organic solvents. Lansbury11 and
Hilvert12 have shown that this idea can be applied to
improve coupling reactions and cleavage reactions, re-
spectively, in peptide chemistry.
F IGURE 1. Monomers prepared for ROMP.
Peptide 2 was chosen for optimization of the reaction
conditions. This peptide and peptides 1, 3, and 4 were
synthesized in good overall yields by solution-phase
synthesizing short (DP ) 10) polymers with pendent
peptide chemistry, using TBTU, HOBt, and DIEA in
CH2Cl2. The monomers were purified by flash column
oligopeptide and no reports of longer polymers with 100%
pendent oligopeptide suggested to us that either aggrega-
tion of the oligopeptide chains during polymerization or
steric interactions at the catalyst center could be the
cause of the early termination of our polymerizations. To
test whether steric interactions at the catalyst reaction
center were the cause, we synthesized a new monomer,
3, that incorporated a triglycine linker between the
norbornene and the ECD. However, polymerizations with
this monomer also terminated early with a DP of 10
regardless of the [M]o/[C]o used (Table 1, entry 7).
1
chromatography and characterized by H and 13C NMR
spectroscopy.
Polymerization reactions were carried out in different
solvent conditions that included TFE, LiCl, and 1 N HCl,
(9) Buck, M. Q. Rev. Biophys. 1998, 31, 297-355.
(10) Seebach, D.; Thales, A.; Beck, A. K. Helv. Chim. Acta 1989,
72, 857-867.
(11) Hendrix, J . C.; Malverson, K. J .; J arrett, J . T.; Lansbury, P. T.
J Org Chem. 1990, 55, 4514-4518.
(12) Quaderer, R.; Hilvert, D. Org. Lett. 2001, 3, 3181-3184.
J . Org. Chem, Vol. 68, No. 5, 2003 2021

TABLE 2. Nor bor n yl Oligop ep tid es Syn th esized w ith
LiCla
that catalyst stability plays a minor although important
role in these polymerizations.
In summary, we have demonstrated that the addition
of 6 M LiCl to ROMP reactions using norbornyl mono-
mers with pendent oligopeptides that are catalyzed by
Ru catalysts significantly improves the catalytic reaction.
Extended polymers with high substituent density and of
the expected length are obtained (n ) 25-50). This
methodology should significantly expand the variety of
polymers that may be prepared by ROMP and be of
general use with norbornyl oligopeptides of any sequence.
av n
%
entry monomer catalyst [M]o/[C]o (DP)b yieldc polymer
b
1
2
3
4
5
6
1
2
2
3
4
1
5a
5a
5a
5a
5a
5b
50/1
25/1
50/1
50/1
50/1
50/1
50
25
50
45
48
50
79
68
79
64
73
88
6
7a
7b
8
9
6
a
General reaction conditions: CH2Cl2:MeOH (3:1), 6 M LiCl,
rt, 4 h, [M]o/[C]o ) 0.3 M. Calculated from 1H NMR spectrum.
b
c Isolated yield after purification and deprotection.
Exp er im en ta l Meth od s
Ma ter ia ls a n d Meth od s. CH2Cl2 was freshly distilled from
CaH; CF3CH2OH, CH3OH, and Et2O were used without further
purification. LiCl was oven-dried and stored over P2O5 before
use. All reactions were carried out under an N2 or Ar atmosphere
in oven-dried glassware. Moisture and oxygen-sensitive reagents
were handled in an N2-filled drybox. 5-Norbornene-exo-carboxylic
acid was synthesized according to the literature.13
as well as the original solvent used, CH2Cl2/MeOH (3:1).
Upon completion of the polymerization reactions, the
solvent was removed and the product was washed with
water if LiCl was used in the reaction. Polymers were
redissolved in CH2Cl2 and isolated by precipitation with
cold Et2O. Unreacted monomer was removed by repeated
washing with Et2O. The oligopeptide polymers were then
deprotected with TFA, using water and triisopropylsilane
as scavengers. The polymers were fully reduced by the
addition of TCEP and precipitated with 1 N HCl for
storage. The [M]o/[C]o ratios ranged from 25/1 to 50/1
(Table 1, entries 8-14). The results of the polymerization
reactions clearly indicate that aggregation must have
been the cause of early chain termination as the addition
of LiCl leads to the formation of polymers with DPs that
correlate to the [M]o/[C]o in the reaction mixture. Poly-
merization in TFE did not occur, because of the low
solubility of the monomer in the solvent. As the LiCl
results were promising, the use of other solvent mixtures
that included TFE was not investigated.
Analytical thin-layer chromatography (TLC) was performed
on precoated silica gel plates (60F254), and flash chromatography
on silica gel-60 (230-400 mesh). TLC spots were detected by
UV light and by staining with phosphomolybdic acid (PMA).
Peptides were purified by flash column chromatography on silica
gel-60. Spectra were recorded in CDCl3 unless otherwise noted.
Chemical shifts are quoted in parts per million (ppm) and 1H
NMR data are assumed to be first order. The purity of the
peptides was assessed by reversed-phase HPLC (C18, 2 column
volume linear gradient of 30% CH3CN/H2O to 40% CH3CN/H2O,
followd by an 8 column volume linear gradient of 40% CH3CN/
H2O to 95% CH3CN/H2O). The purity of polymers was assessed
by aqueous-phase gel-filtration chromatography (BioSep-SEC-S
2000), using 50 mM potassium phosphate, pH 7. The identity
of the peptides was confirmed by MALDI/TOF mass spectroscopy
with use of R-hydroxycinnamic acid as a matrix. Peptides were
analyzed by analytical HPLC and polymers were analyzed by
analytical GFC immediately before use to confirm that the
cysteines had not oxidized to form disulfide dimer.
P ep tid e Syn th esis. (a ) Gen er a l P r oced u r e for Am in o
Acid Cou p lin g. A typical amino acid coupling was carried out
in dry CH2Cl2 with TBTU/HOBt (1.1 equiv/0.37 equiv) and DIEA
(1.6 equiv). Each reaction was carried out under Ar at a final
concentration of 0.7 M in the amine compound, and a 1.1-fold
excess of the carboxylic acid component. Upon completion of the
reaction, the mixture was diluted with CH2Cl2 and washed with
1 N HCl and 5% NaHCO3, and the organic layer was dried with
Na2SO4. The solvent was rotary evaporated. The peptide was
purified by flash chromatography eluting with 10% acetone/
CH2Cl2 or 20% EtOAc/CH2Cl2.
(b) Gen er a l P r oced u r e for Cbz Hyd r ogen a tion . A metha-
nolic solution of Cbz-protected peptide (0.3 M) and 10% Pd-C
(0.05 equiv) was stirred under an H2 atmosphere for 2 h. The
catalyst was removed by filtration and the amine used without
further purification.
(c) Gen er a l P r oced u r e for F m oc Rem ova l. A solution of
Fmoc-protected peptide in dry CH2Cl2 (0.5 M) was treated with
octanethiol (0.1 equiv) and a catalytic amount of DBU (0.001
equiv).14 The reaction was stirred at rt for 16 h. The solvent was
concentrated and the product was purified by flash column
chromatography eluting with a step gradient ranging from 2%
to 50% EtOAc/CH2Cl2.
(d ) Nor bor n en e-E(tBu )C(Tr t)D(tBu )VT(tBu )-OMe (1).
Peptide 1 was prepared in 5 coupling steps to yield 0.75 g (76%
overall yield). 1H NMR (250 MHz) δ 7.38 (m, 6H), 7.20 (m, 11H),
6.98 (dd, J ) 8.1 and 6.3 Hz, 1H), 6.72 (dd, J ) 7.2 and 3.3 Hz,
1H), 6.47 (dd, J ) 9.0 and 2.7 Hz, 1H), 6.0 (m, 2H), 4.74 (m,
We then explored whether the addition of LiCl to
polymerization reactions was of general utility. We
performed ROMP of three other norbornyl oligopeptides,
1, 3, and 4. Polymers of the expected lengths (n ) 25-
50) were obtained in excellent isolated yields ranging
from 64 to 79% (Table 2). We compared catalyst 5b to
5a and found that polymer chain extension was equally
efficient and proceeded in slightly higher yield (Table 2,
entries 1 and 6). Thus, our optimized reaction conditions
work with norbornyl oligopeptides with high functionality
to give polymers of high substituent density, regardless
of sequence or catalyst.
Initially, we found it necessary to add 1 equiv (to [C]o)
of HCl to the polymerization reaction to activate catalyst
5a 8 or to employ the more active dihydroimidazolylidene
catalyst 5b.7 However, with the LiCl modification, the
HCl was not necessary, and in fact, slowed the reaction
rate, in addition to decreasing the polymerization ef-
ficiency (Table 1, entries 12 and 13). This reduction is
presumably due to decomposition of the catalyst. More-
over, faster initiation and higher catalyst stability alone
were insufficent for production of longer high-density
polymers; LiCl was again required for 5b-catalyzed
polymerization (Table 1, entry 6; Table 2, entry 6). Both
catalysts 5a and 5b yielded polymers of the desired
lengths; however, the dihydroimidazolylidene catalyst
provided a slightly higher yield. These results suggest
that disrupting aggregation is the predominant factor in
achieving polymerization of norbornyl oligopeptides and
(13) Manning, D. D.; Strong, L. E.; Hu, X.; Beck, P. J .; Kiessling, L.
L. Tetrahedron 1997, 53, 11937-11952.
(14) Sheppek, J . E.; Kar, H.; Hong, H. Tetrahedron Lett. 2000, 41,
5329-5333.
2022 J . Org. Chem., Vol. 68, No. 5, 2003

1H), 4.43 (dd, J ) 8.7 and 1.8 Hz, 1H), 4.19 (m, 3H), 3.92 (m,
1H), 3.65 (s, 3H), 2.78 (m, 5H), 2.49 (m, 2H), 2.27 (m, 1H), 2.02
(m, 3H), 1.83 (m, 2H), 1.60 (m, 2H), 1.41 (d, J ) 4.5 Hz, 9H),
1.39 (s, 9H), 1.24 (m, 2H), 1.11 (d, J ) 6.3 Hz, 3H), 1.07 (s, 9H),
0.90 (d, J ) 6.6 Hz, 3H), 0.87 (d, J ) 6.9 Hz, 3H). 13C NMR (250
MHz) δ 18.04, 19.01, 20.82, 26.41, 27.97, 28.26, 30.44, 30.54,
30.77, 32.22, 32.96, 33.05, 36.72, 41.48, 44.39, 46.33, 46.98, 49.72,
51.97, 52.51, 52.61, 54.15, 57.73, 58.86, 73.95, 81.19, 81.31,
126.83, 128.02, 129.45, 135.87, 138.07, 144.15, 169.33, 169.96,
170.56, 170.76, 171.00, 171.36, 173.66, 176.73. MALDI, calcd for
(MNa+) 1132.89, found 1133.96. HPLC purity was 98%.
Nor b or n yl Oligop ep t id e P olym er s. (a ) Gen er a l P oly-
m er iza tion P r oced u r e. Catalyst 5a or 5b was weighed in an
N2-filled drybox and dissolved in CH2Cl2/CH3OH (3/1) to give a
typical concentration of 0.03 M. Monomers were each dissolved
in a minimum amount of CH2Cl2/CH3OH (3/1) and LiCl (6 M)
was added to the mixture. The desired portion of catalyst was
added via syringe to the reaction bottle under an inert atmo-
sphere. A typical reaction was carried out at an initial monomer
concentration of 0.2 to 0.3 M. The reaction was stirred at rt for
3 to 4 h before quenching with ethyl vinyl ether and stirring for
an additional 30 min. The solvent was removed and the product
was washed with H2O. Polymers were dissolved in CH2Cl2 and
precipitated with cold Et2O. Product was isolated by centrifuga-
tion and dried under vacuum in the presence of P2O5.
(b) Gen er a l Dep r otection a n d Red u ction P r oced u r e.
Polymers were deprotected in a cocktail containing H2O, TIS,
and TFA (2.5, 2.5, 95) for 5 h. The reaction mixtures were
concentrated with N2 and precipitated in cold Et2O and centri-
fuged. Polymers were dissolved in H2O at pH 6 and reduced with
excess TCEP for 5 h with stirring at 37 °C. Pure deprotected
product was isolated by precipitation with 1 N HCl. Excess
TCEP was removed by repeated washing with H2O. A gray white
solid was collected, dried, and stored at -20 °C.
(e) Nor bor n en e-C(Tr t)VD(tBu )E(tBu )T(tBu )-OMe (2).
Peptide 2 was prepared in 5 coupling steps to yield 0.92 g (78%
overall yield). 1H NMR (600 MHz) δ 7.43 (m, 7H), 7.23 (m, 12H),
6.81 (d, J ) 9.6 Hz, 1H), 6.57 (dd, J ) 7.8 and 8.4 Hz, 1H), 6.07
(m, 2H), 4.80 (m, 1H), 4.54 (dd, J ) 7.8 and 13.8 Hz, 1H), 4.46
(m, 1H), 4.19 (ddd, J ) 2.1 and 6.3 Hz, 1H), 4.06 (m, 2H), 3.37
(s, 3H), 2.87 (d, J ) 16.8 Hz, 1H), 2.78 (m, 1H), 2.75 (m, 1H),
2.63 (m, 3H), 2.34 (t, J ) 7.5 Hz, 2H), 2.17 (m, 2H), 1.92 (m,
2H), 1.86 (dt, J ) 3.9 and 11.4 Hz, 1H), 1.79 (dt, J ) 4.0 and
12.0 Hz, 1H), 1.58 (dd, J ) 7.8 and 8.4 Hz, 1H), 1.41 (d, J ) 1.8
Hz, 9H), 1.39 (d, J ) 3.0 Hz, 9H), 1.26 (m, 2H), 1.13 (d, J ) 6.6
Hz, 3H), 1.10 (s, 9H), 0.91 (dd, J ) 7.2 and 7.8 Hz, 3H), 0.84
(dd, J ) 3.0 and 3.0 Hz, 3H). 13C NMR (250 MHz,) δ 17.4, 19.3,
20.7, 27.9, 28.2, 29.9, 30.1, 30.4, 31.5, 32.6, 37.0, 41.5, 44.1, 44.2,
46.1, 47.0, 47.4, 49.4, 52.0, 52.5, 52.8, 57.9, 59.4, 67.2, 67.3, 74.0,
80.4, 81.4, 126.9, 128.1, 129.4, 135.7, 135.8, 138.4, 144.3, 170.1,
170.2, 170.7, 170.9, 171.0, 172.5, 176.4, 176.4. MALDI, calcd for
(MNa+) 1132.89, found 1134.87. HPLC purity was 97%.
(c) P olym er 6. Yield 41 mg (79%); 1H NMR (D2O, 600 MHz)
δ 7.20 (m), 5.28 (br s), 4.72-3.98 (with max at 4.62, 4.51, 4.40,
4.22, 4.15), 3.69, 3.62 (s), 2.92-2.31 (with max at 2.68, 2.49,
2.43), 2.22-1.66 (with max at 1.78, 1.83, 2.19), 1.60 (m), 1.17
(m), 0.91 (br s). GFC purity was 97%.
(d ) P olym er 7a . Yield 26 mg (68%); 1H NMR (D2O, 600 MHz)
δ 7.21 (m), 5.30 (br s), 4.69 (br s), 4.36 (br s), 4.29 (br s), 4.19
(m), 4.28-3.99 (with max at 4.18, 4.15, 4.13), 3.70-3.51 (with
max at 3.64, 3.61, 3.50), 3.20 (s), 3.14 (m), 2.81 (br s), 2.63-2.29
(with max at 2.52, 2.40, 2.31), 2.10-1.45 (with max at 2.17, 1.91,
1.78, 1.60), 1.40-1.00 (with max at 1.30, 1.18, 1.11), 0.89 (s).
GFC purity was 98%.
(f) Nor bor n en e-GGGE(tBu )C(Tr t)D(tBu )-OMe (3). Pep-
tide 3 was prepared in 4 coupling steps to yield 0.20 g (68%
1
overall yield). H NMR (DMSO, 500 MHz) δ 8.06 (m, 6H), 7.28
(m, 10H), 7.21 (m, 4H), 6.08 (m, 2H), 5.66 (m, 1H), 4.54 (dd, J )
6.6 and 6.6 Hz, 1H), 4.26 (m, 2H), 3.74 (m, 2H), 3.72 (m, 2H),
3.71 (m, 2H), 3.52 (s, 3H), 3.16 (s, 2H), 2.83 (s, 1H), 2.80 (s, 1H),
2.63 (dd, J ) 6.6 and 6.0 Hz, 1H), 2.52 (d, J ) 6.0 Hz, 1H), 2.48
(s, 1H), 2.36 (m, 2H), 2.18 (m, 2H), 2.11 (dt, J ) 8.4 and 4.2 Hz,
1H), 2.03 (m, 2H), 1.83 (m, 1H), 1.77 (m, 1H), 1.71 (m, 1), 1.59
(d, J ) 7.8 Hz, 1H), 1.34 (s, H), 1.33 (s, 9H), 1.16 (m, 2H). 13C
NMR (DMSO, 250 MHz) δ 27.30, 27.51, 29.76, 30.51, 31.06,
33.30, 36.76, 40.93, 41.93, 42.09, 42.35, 42.85, 45.58, 46.63, 48.56,
51.44, 51.82, 54.44, 65.80, 168.67, 168.77, 169.12, 169.27, 169.59,
170.51, 170.64, 171.49, 175.27. MALDI: calcd for (MK+) 1063.80,
found 1063.73. HPLC purity was 98%.
(e) P olym er 7b. Yield 20 mg (79%); 1H NMR (D2O, 600 MHz)
δ 7.22 (m), 6.14 (br s), 5.60-5.14 (with max at 5.55, 5.34), 4.60
(br s), 3.72 (m), 3.40 (br s), 2.60 (m) 2.22-1.41(with max at 2.16,
1.97, 1.78, 1.60), 1.40-0.99 (with max at 1.23, 1.10), 0.80 (br s).
GFC purity was 96%.
(f) P olym er 8. Yield 15 mg (64%); 1H NMR (D2O, 600 MHz)
δ 5.18 (m), 4.56 (m), 4.40 (m), 4.21 (br s), 3.89 (m), 3.60 (m),
3.60 (m), 3.15 (m), 3.01-2.53 (with max at 2.86, 2.6), 2.45 (s),
2.33 (s), 2.16 (m), 1.91 (m), 1.84 (m), 1.68 (m), 1.59 (m), 1.39-
1.03 (with max 1.38, 1.24, 1.20, 1.11). GFC purity was 96%.
(g) P olym er 9. Yield 10 mg (73%) 1H NMR (D2O, 600 MHz)
δ 7 0.20 (m), 5.30 (br s), 5.01 (br s), 4.51 (s), 4.39 (m), 4.28 (br s),
4.09 (m), 3.70 (br s), 3.64 (s), 3.00-2.38 (with max at 2.73, 2.61,
2.55), 2.23-1.72 (with max at 2.18, 1.93, 1.80), 1.59 (m), 1.40-
0.91 (with max at 1.09, 1.17), 0.89 (m), 0.79 (br s). GFC purity
was 97%.
(g) Nor bor n en e-C(Tr t)T(tBu )E(tBu )VD(tBu )-OMe (4).
Peptide 4 was prepared in 5 coupling steps to yield 0.40 g (76%
1
overall yield). H NMR (600 MHz) δ 7.42 (m, 6H), 7.30 (m, 6H),
7.25 (m, 4H), 6.92 (m, 2H), 6.89 (d, J ) 6.6 Hz, 1H), 6.13 (m,
2H), 5.67 (d, J ) 4.8 Hz, 1H), 4.82 (m, 1H), 4.37 (m, 1H), 4.27
(m, 2H), 4.13 (m, 1H), 4.00 (m, 1H), 3.72 (s, 3H), 2.87(m, 2H),
2.76 (m, 2H), 2.66 (m, 1H), 2.33 (m, 3H), 2.14 (m, 1H), 1.92 (m,
2H), 1.83 (d, J ) 12.0 and 3.3 Hz, 1H), 1.43 (s, 9H), 1.42 (s, 9H),
1.27 (m, 2H), 1.17 (s, 9H), 1.07 (d, J ) 6.6 Hz, 3H), 0.95 (d, J )
6.6 Hz, 3H), 0.93 (d, J ) 6.6 Hz, 3H). 13C NMR (250 MHz) δ
17.68, 19.05, 19.87, 26.11, 26.51, 27.27, 27.99, 28.44, 30.45, 30.67,
31.90, 33.96, 37.18, 38.54, 41.54, 44.39, 46.21, 47.06, 47.67, 48.53,
49.26, 51.02, 52.47, 52.59, 58.55, 65.39, 66.67, 67.65, 68.05, 74.32,
80.77, 81.73, 126.79, 127.92, 129.39, 135.84, 138.24, 144.23,
169.98, 170.44, 170.59, 170.98, 171.12, 172.58, 175.02, 175.05.
MALDI: calcd (MNa+) 1132.89, found 1133.96. HPLC purity was
96%.
Ack n ow led gm en t. We thank Dr. Mark J anik for
initial help with this project. Funding for this work was
provided by grant HD38519 from the NIH to N.S.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for precursors to compounds 1-4 and 6-9 and their
spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O0265737
J . Org. Chem, Vol. 68, No. 5, 2003 2023