Incorporation of achiral peptoid-based trimeric sequences into collagen mimetics

Article abstract of DOI:10.1021/ja9810134

Initial studies of collagen mimetics with achiral peptoid-based trimeric sequences were conducted. The achiral unit Gly-Nleu-Nleu was positioned between Gly-Pro-Hyp trimeric repeats in collagen-like structures in order to examine the effect of an achiral

Full text of DOI:10.1021/ja9810134

7420
J. Am. Chem. Soc. 1998, 120, 7420-7428
Incorporation of Achiral Peptoid-Based Trimeric Sequences into
Collagen Mimetics
Elizabeth A. Jefferson, Elsa Locardi, and Murray Goodman*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of California,
San Diego La Jolla, California 92093-0343
ReceiVed March 25, 1998
Abstract: This report represents initial studies of collagen mimetics with achiral peptoid-based trimeric
sequences. The incorporation of achiral units into collagen-like structures is of considerable interest for the
structural simplification of collagen-like biomaterials. The achiral unit Gly-Nleu-Nleu (where Nleu represents
N-isobutylglycine) was positioned between Gly-Pro-Hyp trimeric repeats in collagen-like structures in order
to examine the effect of an achiral block on triple helicity. A series of single chain structures, Ac-(Gly-Pro-
Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (where n ) 1-3), and a template-assembled structure, KTA-
[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3 (where KTA represents cis,cis-1,3,5-trimethyl-
cyclohexane-1,3,5-tricarboxylic acid), were investigated. Biophysical studies were carried out in both H2O
and ethylene glycol (EG)/H2O (2:1, v/v) solvents, using circular dichroism and optical rotation measurements.
Highly cooperative melting curves from optical rotation determinations were obtained for Ac-(Gly-Pro-Hyp)n-
(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (n ) 2, 3) and KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-
Hyp)2-NH2 ]3, revealing that the achiral trimer can participate in triple helical structures. These results were
also supported by circular dichroism spectroscopy. For the molecules Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-
(Gly-Pro-Hyp)3-NH2 and KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3, the presence of
1
collagen-like structures was also supported by H NMR spectroscopy in H2O. For each structure, a distinct
set of resonances, obtained at low temperature, disappeared once a thermal denaturation temperature was reached.
Furthermore, the analysis of NOE cross-peaks established the close packing of Pro, Hyp, and Nleu. The
spatial proximity of Pro and Nleu residues and of Hyp and Nleu residues belonging to different chains was
confirmed by molecular modeling of triple helical Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2.
Introduction
tures and template assembled structures with (Gly-Pro-
9-11
12,13
Nleu)n
and (Gly-Nleu-Pro)n
repeats. These sequences
The design of synthetic collagen-like structures is important
for the development of novel biomaterials. Such biomaterials
have diverse applications which include drug delivery systems,
ocular devices, and wound healing materials.1 Collagen proteins
have large triple helical domains made up of three polyproline
II-like helices supercoiled in a right-handed manner.2-7 The
primary sequence consists of repeating trimeric units of (Gly-
X-Y)n, in which Gly-Pro-Hyp sequences are the most common
structural repeats.
are capable of forming triple helical structures once a certain
chain length is reached. We decided to take this work a leap
forward and determine if entirely achiral trimeric sequences
could participate in triple helical arrays. The incorporation of
achiral repeats is of considerable interest in order to simplify
the overall synthesis and primary structure of collagen-like
biomaterials. This simplification arises from the ease of
synthesis of the achiral units which do not involve the
protections and racemizations-free activations required to
prepare optically pure sequences.
Collagen-like structures can have enhanced biostability
against enzymatic degradation through the incorporation of
unnatural residues, such as peptoid residues.8 We have dis-
covered that the peptoid residue N-isobutylglycine (Nleu) is an
excellent proline surrogate by investigating single-chain struc-
We report here the first studies exploring the incorporation
of an achiral sequence into collagen mimetics. The collagen
mimetics in this study consist of structures with achiral trimeric
units of Gly-Nleu-Nleu (1), positioned between Gly-Pro-Hyp
(1) Kaufman, H. E.; Steinemann, T. L.; Lehman, E.; Thompson, H. W.;
Varnell, E. D.; Jacob-LaBarre, J. T.; Gebhardt, B. M. J. Ocular Pharmacol.
1994, 10, 17-27.
(8) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.;
Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C.
K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.;
Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367-9371.
(9) Melacini, G.; Feng, Y.; Goodman, M. J. Am. Chem. Soc. 1996, 118,
10725-10732.
(2) Kielty, C. M.; Hopkinson, I.; Grant, M. E. In ConnectiVe Tissue and
its Heritable Disorders; Royce, P. M., Steinmann, B., Eds.; Wiley and Sons
Inc.: New York, 1993; pp 103-148.
(3) Prockop, D. J.; Kivirikko, K. I. Annu. ReV. Biochem. 1995, 64, 403-
434.
(4) Brown, J. C.; Timpl, R. Int. Arch. Allergy Immunol. 1995, 107, 484-
490.
(10) Goodman, M.; Melacini, G.; Feng, Y. J. Am. Chem. Soc. 1996, 118,
10928-10929.
(11) Feng, Y.; Melacini, G.; Taulane, J. P. Biopolymers 1996, 39, 859-
872.
(5) Fraser, R. D. B.; MacRae, T. P. Conformation in Fibrous Proteins;
Academic Press: New York, 1973.
(6) Ramachandran, G. N.; Kartha, C. Nature 1955, 176, 593-595.
(7) Rich, A.; Crick, F. H. C. J. Mol. Biol. 1961, 3, 484-506.
(12) Feng, Y.; Melacini, G.; Goodman, M. Biochemistry 1997, 36, 8716-
8724.
(13) Melacini, G.; Feng, Y.; Goodman, M. Biochemistry 1997, 36, 8725-
8732.
S0002-7863(98)01013-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/21/1998

Achiral Peptoid-Based Trimeric Sequences
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7421
repeats. These compounds include the following acetyl-
Scheme 1
terminated structures as well as a template-assembled stucture:
Ac-(Gly-Pro-Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (where
n ) 1-3) and KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-
(Gly-Pro-Hyp)2-NH2]3 (where KTA represents cis,cis-1,3,5-
trimethylcyclohexane-1,3,5-tricarboxylic acid, the Kemp tri-
acid14). For the single-chain compounds, triple helix formation
would occur as an intermolecular process, while for the
template-assembled compound, triple helix formation would
follow an intramolecular process. From our previous studies,
the KTA-(Gly-OH)3 template (2) is known to enhance triple
helix formation significantly.9-11,15-17
The triple helical propensity of the Gly-Nleu-Nleu-containing
structures was investigated using circular dichroism (CD)
spectroscopy and temperature-dependent optical rotation meas-
urements. The conformational characterization of Ac-(Gly-Pro-
Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (where n ) 1, 2)
and KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-
Hyp)2-NH2]3were studied using 1D and 2D 1H NMR spectros-
copy in H2O. These analyses provided sequence-specific
assignments and NOE connectivities. Molecular modeling
analysis of triple helices of Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-
Nleu)3-(Gly-Pro-Hyp)3-NH2 assembled with the three chains,
staggered by one residue, confirms the interchain proton
connectivities found experimentally.
acetylated with acetic anhydride. The product was removed
from the resin using standard HF cleavage techniques.
The template-assembled structure, KTA-[Gly-(Gly-Pro-Hyp)2-
(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3, was synthesized in a
manner similar to that previously described by us for other KTA
template assembled analogues.17 The template KTA-(Gly-OH)3
was allowed to react with the N-terminus of [Gly-Pro-Hyp-
(OBzl)2]2-(Gly-Nleu-Nleu)2-[Gly-Pro-Hyp(OBzl)]2-resin. Af-
terward, the assembly was cleaved from the resin using standard
HF cleavage methods.
All compounds were purified to >97% homogeneity by
preparative reversed-phase HPLC prior to biophysical charac-
terization. Molecular weights were confirmed by mass spec-
trometry and were uniformly in agreement with expected values.
Denaturation Studies. Triple helical structures can be
distinguished from nonsupercoiled polyproline II-like structures
based on thermal denaturation behavior. Triple helices melt in
a highly cooperative fashion since their structures stabilized by
both intra- and interstrand hydrogen-bonding water networks.21-23
Melting curves for the Gly-Nleu-Nleu-containing structures
were constructed by monitoring the optical rotation at 365 nm,
at various temperatures. Melting curves were obtained in both
H2O and ethylene glycol (EG)/H2O (2:1, v/v) solutions, at
concentrations of 0.2 mg/mL. The solvent system EG/H2O (2:
1, v/v) was chosen since ethylene glycol is known to stabilize
triple helices and therefore can be useful for detecting weak
triple helices.24,25 The thermal melting curves are displayed in
Figures 1 and 2, and the thermal melting temperatures (Tm) are
tabulated in Table 1, along with Tm values for analogous peptides
containing only Gly-Pro-Hyp repeats.17
Results and Discussion
Synthesis. The single-chain peptoid-containing structures,
Ac-(Gly-Pro-Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (n )
1-3), were assembled by solid-phase segment condensation
methods, first introduced by Sakakibara and co-workers.18 The
compounds were synthesized on a 4-methylbenzhydrylamine
(MBHA) resin using Boc (tert-butyloxycarbonyl) synthetic
strategies, according to Scheme 1. For each segment condensa-
tion, 2 equiv of building block, either Boc-Gly-Nleu-Nleu-OH19
or Boc-Gly-Pro-Hyp(OBzl)-OH,17 was used with coupling
reagents diisopropylcarbodiimide (DIC) and N-hydroxybenzo-
triazole (HOBt). Each coupling reaction was monitored to
completion (4-8 h) using the Kaiser ninhydrin test.20 After
the desired chain length was synthesized, the N-terminus was
The analogue Ac-(Gly-Pro-Hyp)-(Gly-Nleu-Nleu)-(Gly-Pro-
Hyp)-NH2 does not exhibit a cooperative melting curve in
neither H2O nor EG/H2O (2:1, v/v) solution since its chain length
(14) Kemp, D. S.; Petrakis, K. S. J. Org. Chem. 1981, 46, 5140-5143.
(15) Goodman, M.; Feng, Y.; Melacini, G.; Taulane, J. P. J. Am. Chem.
Soc. 1996, 118, 5156-5157.
(20) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-598.
(21) Bella, J.; Eaton, M.; Brodsky, N.; Berman, H. M. Science 1994,
266, 75-81.
(22) Bella, J.; Brodsky, B.; Berman, H. M. Structure 1995, 3, 893-906.
(23) Heidemann, E.; Roth, W. AdV. Polym. Soc. 1982, 43, 143-203.
(24) Brown, F. R., III.; Carver, J. P.; Blout, E. R. J. Mol. Biol. 1969, 39,
307-313.
(25) Brown, F. R., III.; Di Corato, A.; Lorenzi, G. P.; Blout, E. R. J.
Mol. Biol. 1972, 63, 85-99.
(16) Melacini, G.; Feng, Y.; Goodman, M. J. Am. Chem. Soc. 1996, 118,
10359-10364.
(17) Feng, Y.; Melacini, G.; Taulane, J. P.; Goodman, M. J. Am. Chem.
Soc. 1996, 118, 10351-10358.
(18) Sakakibara, S.; Kishida, Y.; Kikuchi, Y.; Sakai, R.; Kakiuchi, K.
Bull. Chem. Soc. Jpn. 1968, 41, 1273.
(19) Jefferson, E. A.; Gantzel, P.; Benedetti, E.; Goodman, M. J. Am.
Chem. Soc. 1997, 119, 3187-3188.

7422 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
Jefferson et al.
Figure 1. (A) Thermal melting curves for Ac-(Gly-Pro-Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (n ) 1-3) in H2O, at 0.2 mg/mL. (B) Thermal
melting curves for Ac-(Gly-Pro-Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (n ) 1-3) in EG/H2O (2:1, v/v), at 0.2 mg/mL.
Pro-Hyp)9-NH2 by three Gly-Nleu-Nleu repeats causes a
decrease in the thermal melting temperature from 67 to 25 °C,
in H2O. Similarily, Tm values determined in EG/H2O (2:1, v/v)
solution decreased from >95 °C for Ac-(Gly-Pro-Hyp)9-NH2
to 43 °C for Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-
Hyp)3-NH2. A Tm value of 80 °C was obtained for KTA-[Gly-
(Gly-Pro-Hyp)6-NH2]3 while a Tm value of 20 °C was obtained
forKTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-
NH2]3. It is clear from these comparisons that substitution of
Gly-Nleu-Nleu into triple helical structures containing Gly-Pro-
Hyp repeats significantly destabilizes the triple helix. However,
the co-oligomeric structures are still able to form triple helices.
It is probable that the origin of the destabilization effect can
arise from the increase in flexibility of the achiral central
sequences which in turn weakens the interchain hydrogen
bonding.
Circular Dichroism Studies. Natural collagen possesses a
unique CD spectrum with a small positive peak about 220 nm,
Figure 2. Thermal melting curves for KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-
a crossover near 213 nm, and a large negative peak about 200
nm.24,25 Triple helical Ac-(Gly-Pro-Hyp)9-NH2, a synthetic
Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3 in H2O (a) and EG/H2O (2:1, v/v)
(b) solutions.
collagen-like structure, has a CD spectrum in H2O with a
positive peak at 224 nm, a crossover at 218 nm, and a negative
is too short to support a triple helix. This is consistent with
peak at 198 nm.17 The CD spectra of the structures containing
our previous work where we found that Ac-(Gly-Pro-Hyp)3-
the Gly-Nleu-Nleu repeat are displayed in Figures 3 and 4, and
NH2 is not triple helical.17 For Ac-(Gly-Pro-Hyp)2-(Gly-Nleu-
the CD data is tabulated in Table 2. Table 2 also contains the
Nleu)2-(Gly-Pro-Hyp)2-NH2, a cooperative melting curve was
observed in EG/H2O solutions (Tm ) 25 °C) but not in H2O
solution. For Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-
CD data of analogous collagen-like structures containing only
Gly-Pro-Hyp repeats.
In our previous studies, we defined Rpn values as the ratio
of positive peak intensity, at ca. 220 nm, over negative peak
intensity, at ca. 200 nm.17 These values have been useful for
establishing the presence of triple helical conformations in
solution.17
Hyp)3-NH2, a cooperative melting curve was observed in both
H2O (Tm ) 25 °C) and EG/H2O (2:1, v/v) (Tm ) 43 °C)
solutions.
The template-assembled compound exhibited cooperative
melting curves in both H2O (Tm ) 20 °C) and EG/H2O (2:1,
v/v) (Tm ) 43 °C) solutions (Figure 2). The thermal melting
temperature of KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-
(Gly-Pro-Hyp)2-NH2]3 is 17 °C higher than that obtained for
Ac-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2, in
EG/H2O (2:1, v/v). This result highlights the stablizing effect
that the KTA template has on triple helicity.
The effect of Gly-Nleu-Nleu incorporation on triple helical
structures is apparent by comparing Tm values of the Gly-Nleu-
Nleu-containing compounds to the Tm values of similar chain
length Gly-Pro-Hyp-containing compounds17 (Table 1). Re-
placement of three central Gly-Pro-Hyp repeats of Ac-(Gly-
For the single-chain compounds, in H2O solvent, a higher
Rpn value is obtained for Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-
(Gly-Pro-Hyp)3-NH2 (Rpn ) 0.18) compared to both Ac-(Gly-
Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2 (Rpn ) 0.04)
and Ac-(Gly-Pro-Hyp)-(Gly-Nleu-Nleu)-(Gly-Pro-Hyp)-NH2 (Rpn
) 0.03). This comparison of Rpn values suggests that Ac-(Gly-
Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2 contains triple
helical conformations, while the shorter chain analogues do not.
This conclusion is consistent with a previous result where triple
helical Ac-(Gly-Pro-Hyp)9-NH2 was found to have an Rpn value
of 0.14.17

Achiral Peptoid-Based Trimeric Sequences
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7423
Tm values
Table 1. Thermal Melting Temperatures of Collagen-like Structuresa
compound
H2O
EG/H2O (2:1, v/v)
b
Ac-(Gly-Pro-Hyp)-(Gly-Pro-Hyp)-(Gly-Pro-Hyp)-NH2
Ac-(Gly-Pro-Hyp)3-NH2
no transition
no transition
no transition
36 °C
25 °C
67 °C
no transition
no transition
25 °C
59 °C
43 °C
>95 °C
43 °C
>95 °C
c
b
Ac-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2
c
Ac-(Gly-Pro-Hyp)6-NH2
b
Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2
c
Ac-(Gly-Pro-Hyp)9-NH2
b
KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3
20 °C
81 °C
c
KTA-[Gly-(Gly-Pro-Hyp)6-NH2]3
a Concentration ) 0.2 mg/mL. b This work. c Reference 17.
Figure 3. (A) CD spectra of Ac-(Gly-Pro-Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (n ) 1-3) in H2O, at 0.2 mg/mL. (B) CD spectra of
Ac-(Gly-Pro-Hyp)n-(Gly-Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (n ) 1-3) in EG/H2O (2:1, v/v), at 0.2 mg/mL.
comparison suggests that the structures Ac-(Gly-Pro-Hyp)n-(Gly-
Nleu-Nleu)n-(Gly-Pro-Hyp)n-NH2 (n ) 2, 3) have triple helical
character.
The template-assembled structure, KTA-[Gly-(Gly-Pro-Hyp)2-
(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3, has Rpn values of 0.09
and 0.17 in H2O and EG/H2O (2:1, v/v) solutions, respectively.
As a comparison, triple helical KTA-[Gly-(Gly-Pro-Hyp)6-NH2]3
has Rpn values of 0.12 and 0.17 in H2O and EG/H2O solutions,
respectively. This comparison suggests that KTA-[Gly-(Gly-
Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3 also con-
tains triple helical conformations in both solvents.
NMR Spectroscopy. For the acetyl-terminated analogue
with nine trimeric repeats and the KTA analogue with six
trimeric repeats, 1H NMR spectroscopy was used to verify that
the Gly-Nleu-Nleu repeats were actually taking part in the triple
helical assemblies.
For the interpretation of the NOESY spectra, three polypep-
tide chains were considered to have identical repetitive se-
quences symmetrically and closely packed. Because of the
overlap of spectral resonances arising from the same type of
residue along the sequence or belonging to different peptide
chains, it is not possible directly to characterize triple-helices
Figure 4. CD spectra of KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-
(Gly-Pro-Hyp)2-NH2]3 in H2O (a) and EG/H2O (2:1, v/v) (b) solutions.
using NOE-based distance constraints. A given NOE cross-
peak can result from either interchain or intrachain interactions.
However, Brodsky et al.26 used X-ray fiber diffraction data from
triple helical Gly-Pro-Hyp sequences to identify NOEs that are
predicted to arise uniquely from interchain resonances in a triple
In EG/H2O (2:1, v/v) solvent, Ac-(Gly-Pro-Hyp)-(Gly-Nleu-
Nleu)-(Gly-Pro-Hyp)-NH2 exhibits an Rpn value of 0.07 while
the longer chain structures, Ac-(Gly-Pro-Hyp)n-(Gly-Nleu-
Nleu)n-(Gly-Pro-Hyp)n-NH2 (n ) 2, 3) have Rpn values of 0.11
(n ) 2) and 0.20 (n ) 3). Analogous triple helical structures,
Ac-(Gly-Pro-Hyp)n-NH2 (n ) 6, 9) have Rpn values of 0.16 (n
) 6) and 0.17 (n ) 9), in EG/H2O (2/1) solvent. This
(26) Li, M. H.; Fan, P.; Brodsky, B.; Baum, J. Biochemistry 1993, 32,
7373-7387.

7424 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
Jefferson et al.
Table 2. Circlular Dichroism Data for Collagen-Based Structuresa
H2O
EG/H2O (2:1, v/v)
cross
(nm)
cross
(nm)
compounds
max (nm)
min (nm)
Rpnb
max (nm)
min (nm)
Rpn
Ac-(Gly-Pro-Hyp)-(Gly-Nleu-Nleu)-
(Gly-Pro-Hyp)-NH2
Ac-(Gly-Pro-Hyp)3-NH2c
Ac-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-
(Gly-Pro-Hyp)2-NH2
Ac-(Gly-Pro-Hyp)6-NH2c
Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-
(Gly-Pro-Hyp)3-NH2
224 (7.10 × 102) 219 199 (-2.20 × 104) 0.03 223 (1.33 × 103) 217 200 (-2.01 × 104) 0.07
223 (1.8 × 103)
218 200 (-2.3 × 104)
0.08 223 (2.2 × 103)
217 199 (-2.4 × 104) 0.09
223 (7.50 × 102) 218 199 (-1.90 × 104) 0.04 222 (3.49 × 103) 216 199 (-3.12 × 104) 0.11
224 (3.4 × 103)
218 198 (-2.9 × 104)
0.12 223 (5.3 × 103)
217 198 (-3.4 × 104) 0.16
221 (6.75 × 103) 215 199 (-3.84 × 104) 0.18 222 (8.84 × 103) 215 199 (-4.49 × 104) 0.20
Ac-(Gly-Pro-Hyp)9-NH2c
224 (4.4 × 103)
218 198 (-3.1 × 104)
0.14 223 (6.7 × 103)
217 197 (-4.0 × 104) 0.17
KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2- 223 (3.18 × 103) 216 198 (-3.42 × 104) 0.09 223 (6.94 × 103) 216 199 (-4.06 × 104) 0.17
(Gly-Pro-Hyp)2-NH2]3
KTA-[Gly-(Gly-Pro-Hyp)6-NH2]3c
224 (4.1 × 103)
218 198 (-3.4 × 104)
0.12 224 (3.7 × 103)
218 199 (-2.2 × 104) 0.17
a CD spectra were obtained at 20 °C, at 0.2 mg/mL. The peak intensities are included in parentheses. b Rpn represents the ratio of positive peak
intensity over negative peak intensity. c Reference 17.
The same pattern of interchain NOEs was found for the KTA-
[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3
as reported in Figure 9, confirming its triple-helical conformation
in solution.
A list of the “interunit” NOEs, referring to both the acetyl-
terminated and the KTA-assembled analogues, is reported in
Table 3. Since these connectivities cannot arise from non-
supercoiled polyproline II-like structures (distances > 5 Å),
these data represent clear evidence of assembled structures and
provide insight into the packing of the polypeptide chains.
Specifically, the NOE pattern is consistent with the hypothesis
of assembled structures between the Gly-Pro-Hyp and Gly-Nleu-
Nleu trimeric sequences. Some connectivities between Pro and
Hyp residues anticipated to be interchain from the crystal model
of triple helices composed of Gly-Pro-Hyp repeats are deter-
mined as well.
The ensemble of interchain NOEs is useful to check modeled
collagen-like triple helices of the Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-
Nleu)3-(Gly-Pro-Hyp)3-NH2 derivative. The lowest energy
Figure 5. (A) Ac-(Gly-Pro-Hyp)-(Gly-Nleu-Nleu)-(Gly-Pro-Hyp)-NH2.
(B) Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2. 1H NMR
spectral regions including the assembled (boxed peaks) and the
unassembled Nleu methyl resonances at 1.02 ppm and at 0.87 ppm,
respectively. All spectra were acquired in D2O at 275 K.
structure arising from the molecular modeling simulations is
reported in Figure 10. Only the central region of the modeled
triple helical structures is highlighted. All experimental inter-
chain NOEs between Nleu and Pro and between Nleu and Hyp
residues are observed. Therefore, this modeled structure
represents the most reasonable triple helical pattern and can be
used to provide an insight into the close packing of the Pro,
Hyp, and Nleu residues.
helical model. These connectivities provide a critical test for
the triple helical tendency of modified sequences and give
insight into triple helical packing.
The two acetyl analogues (n ) 1, 3) were found to be
distinguishable by NMR spectroscopy in aqueous solution at
low temperature since an additional set of resonances appear
for the Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-
NH2 compound. Resonances arising from the methyl protons
of the Nleu residues are displayed in Figure 5. A similar high
field set of resonances has been shown to correspond to
collagen-like triple helical structures and therefore can be
denoted as the assembled set of resonances.9,13
The triple helical character of Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-
Nleu)3-(Gly-Nleu-Nleu)3-NH2 is supported by three independent
experimental observations: (1) the new set of resonances is
absent in the derivative with only one repeat for each unit; (2)
as the temperature increases, the intensities of the new reso-
nances decrease and disappear at temperatures above the Tm
value determined by optical rotation measurements (Figure 6);
(3) the new resonances are involved in “interunit” NOEs
between Nleu and Pro, and Nleu and Hyp residues which are
not observed in the NOESY spectrum acquired for the analogue
with the shorter sequence (Figure 7, 8).
Conclusion
A major goal of this report focuses on co-oligomer collagen
mimetic structures. We explored the effect of the achiral
trimeric sequence, Gly-Nleu-Nleu, on triple helical structures.
We found that the achiral sequences can participate in the
formation of an overall chiral, triple helical array where the
achiral sequence is sandwiched between Gly-Pro-Hyp repeats.
The Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2
and the KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-
Hyp)2-NH2]3 structures were found to be triple helical in both
H2O and EG/H2O (2:1, v/v) solvents while the Ac-(Gly-Pro-
Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2 compound was
found to be triple helical only in EG/H2O (2:1, v/v) solution
(concentrations ) 0.2 mg/mL). Both optical rotation measure-
ments and CD spectroscopy support these conclusions. NMR
spectroscopy also provides support for the triple-helical arrays
since the NOE patterns are consistent with the proximity of Nleu
to Pro and Nleu to Hyp residues which implies packing among
different trimeric sequences of different chains. Furthermore,

Achiral Peptoid-Based Trimeric Sequences
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7425
and HPLC-grade solvent were purchased from Fisher Scientific.
Palladium on activated carbon (10% palladium) was purchased from
Acros. HOBt, DIC, TFA (HPLC-grade), and BOP were purchased from
Chem-Impex International. THF solvent was dried by refluxing over
Na in the presence of benzophenone.
General Procedures. All reactions carried out in solution were
monitored by thin-layer chromatography on precoated silica gel 60F-
54 plates (Merck). Compounds were visualized by UV or ninhydrin.
Preparative reversed-phase HPLC purification and analysis was
carried out on a MILLENNIUM 2010 system consisting of a Waters
715 Ultra WISP sample processor, a Waters TM 996 photodiode array
detector, two Waters 510 pumps, and a NEC Power Mate 486/33I
computer. Solvents used in HPLC purification were as follows: solvent
A, 0.1% trifluoroacetic acid (TFA)/H2O; solvent B, 0.1% TFA/CH3CN.
The flow rate was 8 mL/min for the preparatory column (Vydac C-18,
25 × 2.2 cm) and 1.0 mL/min for the analytical column (Vydac C-18,
25 × 0.46 cm).
Mass spectra were determined at the Scripps Research Institute using
fast atom bombardment, electrospray ionization, and MALDI mass
spectrometric techniques.
Circular dichroism (CD) measurements were carried out on a
modified Cary-61 spectropolarimeter as described previously.17 Spectra
were obtained using a 0.05 cm path length cell by signal averaging 10
scans from 190 to 300 nm with a scan speed of 1.0 nm/s.
Optical rotations were measured with a Perkin-Elmer 241 polarimeter
equipped with a Model 900 isotemp refrigerator circulator (Fisher
Scientific), and data were collected at 365 nm (Hg). At each
temperature point, the sample was allowed to equilibrate until the optical
rotation was time independent. To allow for equilibration of triple helix
formation, sample solutions were stored at 4 °C, for at least 1 week
prior to the optical rotation determinations.
The NMR samples were prepared in H2O/D2O (9:1) and D2O
(purchased from Isotec, Inc.) with a peptide concentration of about 1.5
mg/mL. The two linear mimetics were dissolved in H2O and kept at
5 °C for at least 2 weeks prior to measurements. The KTA-assembled
mimetic was dissolved in H2O and kept at 5 °C, overnight. All NMR
experiments were carried out on an AMX-500 Bruker spectrometer.
1D spectra of 32K data points were acquired at temperatures between
275 and 315 K in order to follow the denaturation of the triple helical
structure. The 2D NMR spectra were acquired at 275 K to improve
the nuclear Overhauser effect (NOE). Depending on the experimental
sensitivity, a variable number of scans in the range between 64 and
144 was acquired for each t1 increment, and 450-512 t1 increments
were collected of 2K data points each. Acquisition was performed in
the phase-sensitive mode using the time proportional phase increment
method (TPPI). Free induction decays were multiplied by appropriate
windows functions in both time domains and zero filled to 2K×2K
real points.
Figure 6. 1H NMR spectral regions for Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-
Nleu)3-(Gly-Pro-Hyp)3-NH2 including the assembled (boxed peaks) and
the unassembled Nleu methyl resonances at 1.02 ppm and at 0.87 ppm,
respectively. All the spectra were acquired in D2O.
The water signal was suppressed by preirradiation with a selective
gated pulse during the relaxation delay. For nuclear Overhauser effect
spectroscopy (NOESY), preirradiation was also applied during the
mixing time. The carrier frequency was placed at the frequency of
water resonance spanning a SW of 6004 Hz. Chemical shift assign-
ments were obtained in a straightforward manner27 by the combined
use of DQF-COSY28-30 and NOESY31 experiments. The TOCSY32
experiments were acquired using the MLEV-17 spin-lock sequence
typically at a field strength of 10 kHz for a total mixing time of 75-
80 ms. NOESY spectra were carried out with a mixing time of 200
and 300 ms.
some NOE cross-peaks between Pro and Hyp residues predicted
to be unique interchain connectivities based on the molecular
26
modeling of the triple helical (Pro-Hyp-Gly)10 confirm the
close packing of these residues and their involvement in a coiled-
coil collagen-like structure.
Our results demonstrate that the achiral trimeric sequence,
Gly-Nleu-Nleu, can participate in triple helical structures. This
is of importance since much effort in the field of biomaterials
is directed toward structural simplifications. Other oligomeric
achiral sequences are now under investigation in our laborato-
ries.
(27) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley and
Sons: New York, 1986.
(28) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64,
2229-2246.
(29) Bax, A.; Freeman, R. J. Magn. Reson. 1981, 44, 542-561.
(30) Rance, M.; Sorensen, O. W.; Bodenhausen, G.; Wagner, G.; Ernst,
R. R.; Wu¨thrich, K. Biochem. Biophys. Res. Commun. 1984, 117, 479-
485.
Experimental Section
(31) Kumar, A.; Ernst, R. R.; Wu¨thrich, K. Biochem. Biophys. Res.
Commun. 1980, 95, 1-6.
(32) Davis, D.; Bax, A. J. Am. Chem. Soc. 1985, 107, 2820-2821.
Materials. All amino acids are of the L-configuration. Protected
amino acids and EDC were purchased from Bachem. Reagent grade

7426 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
Jefferson et al.
Figure 7. Aliphatic regions of the NOESY spectra of Ac-(Gly-Pro-Hyp)-(Gly-Nleu-Nleu)-(Gly-Pro-Hyp)-NH2 (A) and Ac-(Gly-Pro-Hyp)3-(Gly-
Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2 (B) in D2O at 275 K. The ensemble interchain NOE cross-peaks are indicated by rectangles.
Figure 9. Aliphatic region of the NOESY spectrum of KTA-[Gly-
(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3 in D2O solu-
tion at 275 K. The expected unique interchain NOEs based on the triple
helical model of (Pro-Hyp-Gly)10 are indicated by circles and
“interunit” NOEs between Nleu and Pro, Hyp residues are indicated
Figure 8. Expanded regions of a TOCSY (top) and NOESY (bottom)
spectra of Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2
in D2O at 275 K. In the latter spectrum, interchain connectivities
between Nleu and Pro/Hyp residues are indicated.
26
by rectangles.
Molecular modeled structures were built using Insight II.33 Energy
minimizations were carried out using the Discover program34 and the
force field CFF91 which provides high-quality parameters for all of
the Nleu internal coordinates. A distant dependent dielectric constant
was used to approximate the solvent effect. For the molecular modeling
of triple helices which include the Gly-Nleu-Nleu trimeric sequence,
we focused on the linear analogue Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-
Nleu)3-(Gly-Pro-Hyp)3-NH2. The three chains staggered by one residue
were constrained to have the same backbone torsions and interchain
hydrogen-bond pattern as the structure proposed by Miller, Nemethy,
and Sheraga35 for (Gly-Pro-Hyp)n. This structure was shown not to
be sensitive toward substitutions of other amino acids in place of Pro
(33) Biosym Technologies, Inc., San Diego, CA.
(34) Discover 95.0/3.00 User Guide, Biosym/MSI, San Diego, CA, 1995.
(35) Miller, M. H.; Nemethy, G.; Scheraga, H. A. Macromolecules 1980,
13, 470-478.

Achiral Peptoid-Based Trimeric Sequences
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7427
Table 3. Expected Unique Interchain NOEs (distances smaller
than 4.5 Å) Based on the Model of Triple Helical (Pro-Hyp-Gly)10
and Observed “Interunit” NOEsa
were achieved fixing the backbone torsions (φ, ψ, and ω) at their
original values and searching energy minima in the ø1 (CR-N-Câ-
Cγ), ø2 (N-Câ-Cγ-Cδ) torsional space of the Nleu residues as it has
13
been done in our laboratories for (Gly-Pro-Nleu)n9 and (Gly-Nleu-Pro)n
exptl
NOEs
sequences. The ensemble interchain NOEs provide a critical test for
the modeled structures.
expected NOEs
interunit NOEs
Hyp CδH2-Pro CδH1
Hyp CR,γH-Pro CδH1
w
m
m
Hyp CR,γH-Nleu CâH
Hyp CδH-Nleu CâH
Hyp CδH-Nleu CγH n.r.
w
w
Syntheses of Trimeric Building Blocks. Boc-Gly-Pro-Hyp(OBzl)-
OH was synthesized by coupling Boc-Gly-Pro-OH37 with Hyp(OBzl)-
OBzl38 using EDC and HOBt as coupling reagents. Hydrolysis of Boc-
Gly-Pro-Hyp(OBzl)-OBzl with KOH, in THF/H2O (1:1, v/v) solution
afforded Boc-Gly-Pro-Hyp(OBzl)-OH.39
Pro CγH1-Hyp CR,γ
H
Pro CδH1-Hyp CâH1
Pro CδH1-Hyp CâH2
Pro CδH2-Hyp CâH2
n.d. Hyp CR,γH-Nleu CγH n.r.
n.d. Hyp CδH-Nleu CδH
n.d. Hyp CR,γH-Nleu CδH
Hyp CâH-Nleu CδH
s
s
m
Boc-Gly-Nleu-Nleu-OH19 was synthesized by coupling the peptoid
containing dimer Boc-Gly-Nleu-OH12 with Nleu-OEt40 using BOP as
the activating reagent to afford Boc-Gly-Nleu-Nleu-OEt. The trimer
was then saponified with KOH in H2O/THF (1:1, v/v) to give Boc-
Gly-Nleu-Nleu-OH. The detailed procedures for the synthesis of Boc-
Gly-Nleu-Nleu-OEt and Boc-Gly-Nleu-Nleu-OH are outlined below.
(A) Boc-Gly-Nleu-Nleu-OEt. A solution of Boc-Gly-Nleu-OH (26
g, 92 mmol) and HCl‚Nleu-OEt (18 g, 92 mmol) was cooled to 0 °C.
The coupling reagent, BOP (41 g, 92 mmol), was then added
portionwise to the solution, followed by the addition of triethylamine
(26 mL, 183 mmol). The ice bath was removed, and the solution was
left stirring overnight. The DMF was removed by rotary evaporation
under reduced pressure, and H2O (800 mL) and EtOAc (800 mL) were
added. The EtOAc layer was washed with H2O (800 mL), NaHCO3
(2 × 800 mL), brine (800 mL), 2 N NaHSO4 (2 × 800 mL), and brine
(3 × 800 mL). The organic layer was dried over anhydrous Na2SO4,
the volatiles were removed in vacuo, and the residue was purified by
flash chromatography (SiO2, 1/1, hexanes/EtOAc) to give 28 g (72%
yield) of Boc-Gly-Nleu-Nleu-OEt: Rf ) 0.2 (1/1 hexanes/EtOAc); 1H
NMR (300 MHz, CDCl3) δ 5.49 (br s, 1H), 4.22-4.01 (m, 8 H), 3.19-
3.06 (m, 4 H), 1.93-1.82 (m, 2 H), 1.40 (s, 9 H), 1.24 (t, 3 H) 0.85-
0.98 (m, 12 H).
Pro CRH-Nleu CâH
Pro CδH-Nleu CâH
w
w (only for the
KTA-analog)
Pro CRH-Nleu CγH
Pro CRH-Nleu CδH
Pro CδH-Nleu CδH
n.r.
s
w
a Connectivities which cannot be identified because of spectral
overlap are not included in the table. Observed NOEs are indicated
with w (weak), m (medium), and s (strong) based upon their intensities.
The hydrogens are named according to ref 33. NOE cross-peaks refer
to the Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2 and
the KTA-[(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3 un-
less otherwise indicated in parentheses. The n.d. stands for not
determined NOE. The n.r. stands for not well resolved for integration.
FAB-MS: m/z ) 430 (M + H) calcd for C21H39N3O6 + H 430,
obsd 430.
(B) Boc-Gly-Nleu-Nleu-OH. A solution of Boc-Gly-Nleu-Nleu-
OEt (4.7 g, 11 mmol), H2O (20 mL), and THF (20 mL) was cooled to
0 °C. KOH was dissolved in H2O (6 mL) and was then added slowly
to the solution. The ice bath was removed, and the solution was left
stirring at room temperature for 30 min. The THF was then removed
in vacuo, and the water was then cooled to 0 °C in an ice bath.
Concentrated HCl was added dropwise until the solution had a pH )
2. A white solid precipitated which was extracted with EtOAc. The
EtOAc extracts were washed with brine (3 × 30 mL) and then dried
over Na2SO4. After filtration, the solvent was removed in vacuo and
the residual solid was recrystallized from CH3CN to afford Boc-Gly-
Nleu-Nleu-OH (3.4 g, 78% yield): 1H NMR (300 MHz, CDCl3) δ
0.05-0.98 (m, 12 H), 1.42 (s, 9 H), 1.80-2.00 (m, 2 H), 3.10-3.19
(m, 4 H), 4.04-4.21 (m, 6 H), 5.73 (br s, 1 H).
MS-MALDI: m/z ) 402 calcd for C19H35N3O6 + H 402, obsd 402;
m/z ) 424 calcd for C19H35N3O6 + Na 424, obsd 424.
General Solid-Phase Synthetic Methods. Solid-phase segment
condensations were carried out using Boc synthetic strategies. The
MBHA resin‚HCl (200-400 mesh, 1% DVB, 0.45 mmol/g substitution,
0.2 mmol scale) was swollen in DCM for 1 h and washed with 10%
TEA in DCM (10 mL, 2×, 2 min) followed by DCM (10 mL, 4×). A
solution of 25% DMF in DCM was used as the solvent for the
couplings. DIC (2 equiv) and HOBt (2 equiv) were used as the coupling
reagents with either Boc-Gly-Pro-Hyp(OBzl)-OH (1.2-1.5 equiv) or
Boc-Gly-Nleu-Nleu-OH (1.2-1.5 equiv). The reactions were moni-
tored by the Kaiser ninhydrin test.20 After completion of each coupling
step, the solution was filtered and the resin was washed with MeOH
(10 mL, 5 min) and DCM (10 mL, 1 min, 4×). The N-terminal Boc
Figure 10. Central region of modeled triple helical structures of Ac-
(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2. The two tri-
meric sequences (Gly-Pro-Hyp) and (Gly-Nleu-Nleu) are highlighted
in red and green, respectively. On the left side the backbone ribbon
diagram is shown for each of the three chains which are staggered by
one residue. On the right side enlarged views of the N-terminus (bottom)
and the C-terminus (top) of the (Gly-Nleu-Nleu)3 sequence are reported
showing the interchain proximities between Nleu and Hyp residues and
between Pro and Nleu residues.
(37) Wolfe, A.; Bowers, R. J.; Shin, H.-S.; Sohn, C.-K.; Weaver, D. F.;
Yang, K. Can. J. Chem. 1988, 66, 2751-2762.
(38) Rubini, E.; Gilon, C.; Selinger, Z.; Chorev, M. Tetrahedron 1986,
42, 6039-6045.
(39) Feng, Y. Ph.D. Thesis, University of California, San Diego, CA,
1996.
and Hyp residues36 and therefore represents a good starting point as
backbone conformation for molecular modeling. Energy minimizations
(40) Kruijtzer, J. A. W.; Liskamp, R. M. J. Tetrahedron Lett. 1995, 36,
6969-6972.
(36) Nemethy, G. Biochimie 1981, 63, 125-130.

7428 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
Jefferson et al.
protection was removed with 30% TFA in DCM (10 mL, 30 min).
After the deprotection, the solution was filtered and the resin washed
with MeOH (10 mL, 5 min) and DCM (10 mL, 1 min, 6×). The resin
was then neutralized with 10% TEA/DCM (10 mL, 2 min, 2×) followed
by washing with DCM (10 mL, 1 min, 6×). The desired peptide chain
was assembled by consecutive coupling reactions. To prepare the
acetyl-terminated compounds, the N-terminal amines were treated with
acetic anhydride (0.24 mL) and TEA (0.35 mL) in a solution of DMF
(8 mL). The resin was then washed with MeOH (10 mL, 2×) and
DCM (10 mL, 2×) and then dried under vacuum overnight. The
peptide-peptoid conjugates were cleaved from the resin by standard
HF cleavage methods. The HF cleavage reactions were allowed to
proceed for 1 h at -5 to 0 °C. The resin was then dried under vacuum
for 2 h in a desiccator containing KOH. The resin and product was
washed with Et2O (anhydrous) several times. The product was then
separated from the resin by extracting with a mixture of H2O and
CH3CN. The crude product was lyophillized.
Ac-(Gly-Pro-Hyp)-(Gly-Nleu-Nleu)-(Gly-Pro-Hyp)-NH2. Ac-
(Gly-Pro-Hyp)-(Gly-Nleu-Nleu)-(Gly-Pro-Hyp)-NH2 was prepared fol-
lowing the procedures described above in the General Solid-Phase
Synthetic Methods section. Preparatory RP-HPLC was used to purify
Ac-Gly-Pro-Hyp-Gly-Nleu-Nleu-Gly-Pro-Hyp-NH2 as a white powder
(30% yield based on resin substitution). The analytical HPLC gave a
single peak, tR ) 10.5 min (15-40% CH3CN in 0.1% TFA over 20
min at 1 mL/min).
Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2. Ac-
(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2 was prepared
following the procedures described above in the General Solid-Phase
Synthetic Methods section. Preparatory RP-HPLC was used to purify
Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2 as a white
powder (11% yield based on resin substitution). The analytical HPLC
gave a single peak, tR ) 15.3 min (25-55% CH3CN in 0.1% TFA
over 20 min at 1 mL/min).
MALDI-MS: m/z ) 2550 calcd for C116H182N28O34 + K 2550, obsd
2550.
KTA-[Gly-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-
NH2]3. Boc-[Gly-[(Gly-Pro-Hyp(OBzl)]2-(Gly-Nleu-Nleu)2-[Gly-Pro-
Hyp(OBzl)]2-MBHA (0.27 mmol based on resin substitution) was
prepared following the procedures described above in the General Solid-
Phase Synthetic Methods section. The Boc group was removed with
30% TFA/DCM (15 mL, 1.0 mL of anisol was added as scavenger).
The resin was washed with MeOH (1 × 10 mL, 1 min) and DCM (4
× 10 mL, 1 min), followed by 10% TEA in DCM (2 × 15 mL, 2 min)
and DCM (4 × 10 mL, 1 min). KTA-(Gly-OH)3 (35 mg, 0.08 mmol)
and HOBt (50 mg) were added to the reaction vessel, and a solution of
25% DMF/DCM (15 mL) was added. After 3 d, the resin was washed
with MeOH (2 × 15 mL) and DCM (2 × 15 mL), and the resin was
then dried overnight under vacuum. HF cleavage methods were used
to cleave the material from the resin. The resin was then washed with
Et2O, and the peptide-petoid conjugate was extracted with H2O.
Preparatory RP-HPLC was carried out to obtain the product KTA-[Gly-
(Gly-ProHyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2]3 as a white
powder (4% yield based on resin substitution). The analytical HPLC
gave a single peak, tR ) 14.1 min (27-55% CH3CN in 0.1% TFA
over 20 min at 1 mL/min).
FAB-MS: m/z ) 1009 calcd for C40H64N10O12 + Cs 1009, obsd
1009.
Ac-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2. Ac-
(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2 was prepared
following the procedures described above in the General Solid-Phase
Synthetic Methods section. Preparatory RP-HPLC was used to purify
Ac-(Gly-Pro-Hyp)2-(Gly-Nleu-Nleu)2-(Gly-Pro-Hyp)2-NH2 as a white
powder (20% yield based on resin substitution). The analytical HPLC
gave a single peak, tR ) 20.1 min (15-40% CH3CN in 0.1% TFA
over 20 min at 1 mL/min).
MALDI-MS: m/z ) 5357 calcd for C246H384N60O72 + Na 5357, obsd
5357.
Acknowledgment. This research was funded by a grant from
Ciba Vision Inc.
FAB-MS: m/z ) 1717 calcd for C78H123N19O23 + Na 1717, obsd
1717.
JA9810134