The aberrance of the 4 S diastereomer of 4-hydroxyproline

Article abstract of DOI:10.1021/ja103082y

Prolyl 4-hydroxylases install a hydroxyl group in the 4R configuration on the γ-carbon atom of certain (2S)-proline (Pro) residues in tropocollagen, elastin, and other proteins to form (2S,4R)-4-hydroxyproline (Hyp). The gauche effect arising from this prevalent post-translational modification enforces a C^γ-exo ring pucker and stabilizes the collagen triple helix. The Hyp diastereomer (2S,4S)-4-hydroxyproline (hyp) has not been observed in a protein, despite the ability of electronegative 4S substituents to enforce the more common C^γ-endo ring pucker of Pro. Here, we use density functional theory, spectroscopy, crystallography, and calorimetry to explore the consequences of hyp incorporation on protein stability using a collagen model system. We find that the 4S-hydroxylation of Pro to form hyp does indeed enforce a C^γ-endo ring pucker but a transannular hydrogen bond between the hydroxyl moiety and the carbonyl of hyp distorts the main-chain torsion angles that typically accompany a C^γ-endo ring pucker. This same transannular hydrogen bond enhances an nφ* interaction that stabilizes the trans conformation of the peptide bond preceding hyp, endowing hyp with the unusual combination of a C^γ-endo ring pucker and high trans/cis ratio. O-Methylation of hyp to form (2S,4S)-4-methoxyproline (mop) eliminates the transannular hydrogen bond and restores a prototypical C^γ-endo pucker. mop residues endow the collagen triple helix with much more conformational stability than do hyp residues. These findings highlight the critical importance of the configuration of the hydroxyl group installed on C^γ of proline residues.

Full text of DOI:10.1021/ja103082y

Published on Web 07/16/2010
The Aberrance of the 4S Diastereomer of 4-Hydroxyproline
Matthew D. Shoulders,†,‡ Frank W. Kotch,†,§ Amit Choudhary,⊥ Ilia A. Guzei,† and
Ronald T. Raines*,†,|
Departments of Chemistry and Biochemistry, and the Graduate Program in Biophysics,
UniVersity of WisconsinsMadison, Madison, Wisconsin 53706
Received April 16, 2010; E-mail: rtraines@wisc.edu
Abstract: Prolyl 4-hydroxylases install a hydroxyl group in the 4R configuration on the γ-carbon atom of
certain (2S)-proline (Pro) residues in tropocollagen, elastin, and other proteins to form (2S,4R)-4-
hydroxyproline (Hyp). The gauche effect arising from this prevalent post-translational modification enforces
a Cγ-exo ring pucker and stabilizes the collagen triple helix. The Hyp diastereomer (2S,4S)-4-hydroxyproline
(hyp) has not been observed in a protein, despite the ability of electronegative 4S substituents to enforce
the more common Cγ-endo ring pucker of Pro. Here, we use density functional theory, spectroscopy,
crystallography, and calorimetry to explore the consequences of hyp incorporation on protein stability using
a collagen model system. We find that the 4S-hydroxylation of Pro to form hyp does indeed enforce a
Cγ-endo ring pucker but a transannular hydrogen bond between the hydroxyl moiety and the carbonyl of
hyp distorts the main-chain torsion angles that typically accompany a Cγ-endo ring pucker. This same
transannular hydrogen bond enhances an nfπ* interaction that stabilizes the trans conformation of the
peptide bond preceding hyp, endowing hyp with the unusual combination of a Cγ-endo ring pucker and
high trans/cis ratio. O-Methylation of hyp to form (2S,4S)-4-methoxyproline (mop) eliminates the transannular
hydrogen bond and restores a prototypical Cγ-endo pucker. mop residues endow the collagen triple helix
with much more conformational stability than do hyp residues. These findings highlight the critical importance
of the configuration of the hydroxyl group installed on Cγ of proline residues.
1. Introduction
The hydroxylation of proline residues by prolyl 4-hydroxylase
to form (2S,4R)-4-hydroxyproline (Hyp) is the most common
post-translational modification in animals.1 All known prolyl
4-hydroxylases install the hydroxyl group in the R configuration,
that is, on the face of the pyrrolidine ring opposite from the
carboxyl group.2 The ensuing gauche effect stabilizes the Cγ-
exo ring pucker, which enables an nfπ* interaction that
stabilizes the trans peptide bond (Figure 1A).3 The preorgani-
zation that arises from these stereoelectronic effects has
important consequences for the conformational stability of
collagen and other proteins.4
In marked contrast to Hyp, its 4S diasteromer, (2S,4S)-4-
hydroxyproline (hyp), has not been observed in a natural protein.
This absence is intriguing given the recent discovery that
nonnatural analogues of hyp can enhance the conformational
stability of proteins. For example, (2S,4S)-4-fluoroproline (flp)
has a demonstrated ability to modulate the stability of structural
Figure 1. Attributes of Pro derivatives. (A) The pucker of the pyrrolidine
ring can be enforced by the gauche effect arising from an electronegative
Cγ substituent (R1 or R2), which adopts the pseudoaxial position. Hyp: R1
) OH, R2 ) H; hyp: R1 ) H, R2 ) OH; Mop: R1 ) OMe, R2 ) H; mop:
R1 ) H, R2 ) OMe. (B) Intramolecular hydrogen bond in hyp. (C)
Interstrand hydrogen bonds in a collagen triple helix. Oi of a hyp residue
in the Xaa position is red in panels B and C.
† Department of Chemistry.
motifs (e.g., polyproline I- and II-type helices5 and ꢀ-turns6)
and whole proteins (e.g., triple-helical collagen,7 barstar,8 and
green fluorescent protein9). The flp residue prefers a Cγ-endo
ring pucker, as expected from a gauche effect, and has a low
trans/cis ratio.3d
Although Hyp is especially abundant in collagen, its use is
judicious. Structures of collagen triple helices reveal that proline
and its derivatives in the Yaa position of the Xaa-Yaa-Gly repeat
have a Cγ-exo ring pucker, whereas those in the Xaa position
‡ Present address: Department of Chemistry, Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037.
§ Present address: Small-Molecule Screening & Medicinal Chemistry
Facility, University of WisconsinsMadison, Madison, Wisconsin 53706.
⊥ Graduate Program in Biophysics.
| Department of Biochemistry.
(1) Gorres, K. L.; Raines, R. T. Crit. ReV. Biochem. Mol. Biol. 2010, 45,
106–124.
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Field, R. A.; Lawrence, C. C.; Merritt, K. D.; Schofield, C. J.
Tetrahedron Lett. 1993, 34, 7489–7492.
9
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J. AM. CHEM. SOC. 2010, 132, 10857–10865 10857

A R T I C L E S
Shoulders et al.
have a Cγ-endo ring pucker.4,10 Hyp is prevalent in the Yaa
position,11 consistent with its preferred pucker. Seminal studies
of synthetic collagen triple helices indicate that hyp in the Yaa
position severely destabilizes the triple helix.12 Surprisingly, hyp
in the Xaa position is also severely destabilizing,12,13 despite
its preference for the Cγ-endo ring pucker that is desirable in
the Xaa position.14
Here, we explore the consequence of incorporating hyp into
a protein using collagen as a model system. We were driven
by the results of a recent gas-phase microwave spectroscopic
analysis of the free amino acid, hypOH, which revealed a
transannular hydrogen bond between the hydroxyl and carboxyl
groups.14 We use O-methylation to form (2S,4S)-4-methox-
yproline (mop) as a probe, because a methoxy group retains
the electron-withdrawing15 and hyperconjugative16 ability of a
hydroxyl group but loses the ability to donate a hydrogen bond.17
Our theoretical, spectroscopic, crystallographic, and calorimetric
analyses of hyp and mop in a small molecule model and in a
collagen triple helix reveal that hydrogen bonding can com-
promise the potential benefits of an underlying stereoelectronic
effect to the conformational stability of a protein.
Figure 2. Ac-hyp-OMe conformations optimized at the B3LYP/6-
311+G(2d,p) level of theory and depicted with the program PyMOL (Delano
Scientific, Palo Alto, CA). (A) Cγ-endo ring pucker, trans peptide bond,
and intramolecular hydrogen bond. (B) Cγ-endo ring pucker, cis peptide
bond, and intramolecular hydrogen bond. (C) Cγ-endo ring pucker, trans
peptide bond. (D) Cγ-endo ring pucker, cis peptide bond.
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2. Results and Discussion
2.1. Computational Analyses of Ac-hyp-OMe. We began by
exploring the conformational preferences of Ac-hyp-OMe with
density functional theory (DFT) calculations at the B3LYP/6-
311+G(2d,p) level. We chose Ac-hyp-OMe as a model to avoid
the γ-turn formation that has been observed in Ac-Xaa-NHMe.18
First, gas-phase geometry optimization and frequency calcula-
tions were performed on six conformations of Ac-hyp-OMe.
We found that a transannular hydrogen bond, formed between
the 4S hydroxyl group and the ester carbonyl group of Ac-hyp-
OMe (Figure 1B), was an important conformational determinant.
The lowest energy conformations of Ac-hyp-OMe have this
hydrogen bond in both cis and trans conformers (Figure 2). The
conformation of Ac-hyp-OMe with a hydrogen bond, Cγ-endo
ring pucker, and trans amide bond (Figure 2A) is >3 kcal/mol
more stable than any non-hydrogen bonded conformation
(Figure 2C and 2D). Gas-phase calculations can overestimate
the stabilization conferred on a particular conformation by
hydrogen bonding, and we were interested in understanding how
hyp would behave both in a protein environment and in aqueous
solution. Therefore, we also performed complete geometry
optimizations of six conformations of Ac-hyp-OMe using the
conductor-like polarizable continuum (CPCM) model at the
B3LYP/6-311+G*(2d,p) level of theory.19 Analysis of the
resulting self-consistent field (SCF) energies showed that the
most stable conformation in chloroform is the internally
hydrogen bonded conformation depicted in Figure 2A, whereas
in water the non-hydrogen bonded trans Cγ-endo conformation
in Figure 2C is preferred. These results are consistent with the
hypothesis that in nonpolar or aprotic solvents (or in a protein
environment) hyp prefers a Cγ-endo ring pucker with an
intramolecular transannular hydrogen bond; that preference is
obviated by aqueous solvation. The transannular hydrogen bond
within Ac-hyp-OMe alters the main-chain torsion angles φ
(C′i-1-Ni-CR -C′i) and ψ (Ni-CR -C′i-Ni+1) from those
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Aberrance of the 4S Diastereomer of 4-Hydroxyproline
A R T I C L E S
Table 1. Values of Ktrans/cis and υester for Ac-Pro-OMe, Ac-hyp-OMe,
and Ac-mop-OMe
Ac-Xaa-OMe
Ktrans/cis (CDCl3)a
υester (CHCl3; cm-1
)
Ktrans/cis (D2O)a
Ac-Pro-OMe
Ac-hyp-OMe
Ac-mop-OMe
4.0b
5.0
1.9
1743c
1725c
1752
5.3b
2.7
2.0
a Values were determined by 1H NMR spectroscopy. b From ref 3e.
c From ref 3c.
observed in standard Cγ-endo ring puckers (φ ≈ -70°, ψ ≈
152°).3b,d The value of ψ ) 140° in Ac-hyp-OMe (Figure 2A)
is closer to that of a typical Cγ-exo ring pucker (ψ ≈ 143°).3b,d
Similarly, the value of φ ) -65° in Ac-hyp-OMe is intermediate
between that of a typical Cγ-exo ring pucker (which has φ ≈
-59°) and a typical Cγ-endo ring pucker (φ ≈ -70°).3b,d
2.2. Spectroscopic Analyses of Ac-hyp-OMe and Ac-mop-
OMe. Next, we synthesized Ac-hyp-OMe and Ac-mop-OMe
1
and explored their solution conformations by H NMR spec-
troscopy. In aqueous solution, both Ac-hyp-OMe3c and Ac-mop-
OMe have particularly low values of Ktrans/cis (Table 1),
suggesting that they adopt a Cγ-endo ring pucker. In CDCl3,
however, Ac-hyp-OMe but not Ac-mop-OMe has a high value
of Ktrans/cis. The chemical shift of the hydroxyl proton of
Ac-hyp-OMe decreases with temperature (δ ) 3.16 ppm,
T ) 17.0 °C; 3.12, 22.8; 3.06, 29.6; 3.01, 36.4), as expected
from its participation in a hydrogen bond (Figure 1B).20 Because
the stability of that hydrogen bond should be enhanced in an
aprotic solvent (as predicted by the CPCM calculations), we
propose that the high Ktrans/cis value of Ac-hyp-OMe in CDCl3
occurs because the transannular hydrogen bond distorts the φ
and ψ angles of Ac-hyp-OMe in CDCl3 to values similar to
those typically observed in a Cγ-exo ring pucker, as observed
in our computational analysis. These φ and ψ angles enable an
Figure 3. Crystal structures of hyp and mop derivatives. (A) Molecular
drawing of Ac-hyp-OMe with 50% probability ellipsoids. (B) Structure of
Ac-hyp-OMe depicted with the program PyMOL. (C) Molecular drawing
of Boc-mop-OH (8) with 50% probability ellipsoids. (D) Structure of Boc-
mop-OH (8) depicted with the program PyMOL.
nfπ* interaction,3d,h which increases the value of Ktrans/cis
.
Indeed, second-order perturbation theory using natural bond
orbital analysis21 indicates that the nfπ* interaction in the
lowest energy hydrogen-bonded, Cγ-endo conformation of Ac-
hyp-OMe (Figure 2A) could be worth as much as 1.0 kcal/
mol. This same interaction is worth <0.05 kcal/mol in the lowest
energy non-hydrogen bonded conformation, the Cγ-endo con-
formation of Ac-hyp-OMe (Figure 2C).
(see: Table S1 in the Supporting Information), which are
indicative of a predominantly Cγ-endo conformation.
2.3. Crystallographic Analyses of hyp and mop. Next, we
crystallized Ac-hyp-OMe to explore its conformation in the solid
state. The crystal structure of Ac-hyp-OMe (Figures 3A and
3B) displayed a cis peptide bond and the expected Cγ-endo ring
pucker. In the solid state, the hydroxyl group formed a hydrogen
bond with the amide oxygen of an adjacent molecule in the
crystal lattice. A crystal structure of Ac[S]-hyp-OMe (which is
the thioamide analog of Ac-hyp-OMe) was similar to that of
Ac-hyp-OMe, displaying a Cγ-endo ring pucker but with a trans
peptide bond.23
Despite an extensive effort, we were unable to obtain a crystal
structure of Ac-mop-OMe. Nonetheless, we did obtain a
structure of a related molecule, Boc-mop-OH (8) (Figure 3C
and 3D). As expected, Boc-mop-OH (8) displayed the Cγ-endo
ring pucker in the crystalline state. The methoxy group was
oriented away from the ring, perhaps to avoid unfavorable steric
interactions with the carboxylate group. A crystal structure of
Ac[S]-mop-OMe (which is the thioamide analogue of Ac-mop-
OMe) reiterates these results, displaying a Cγ-endo ring pucker
and main-chain torsion angles typical for that pucker.23
We also used infrared spectroscopy to examine the proline
derivatives. An ester carbonyl stretching vibration (υester
)
decreases with decreasing CdO bond order. In CHCl3, Ac-hyp-
OMe has a much lower υester than does either Ac-Pro-OMe or
Ac-mop-OMe (Table 1; Figure S1 in the Supporting Informa-
tion). The nfπ* interaction in Ac-hyp-OMe could contribute
up to 6 cm-1 to this difference.3c The unusually large magnitude
of the shift in υester for Ac-hyp-OMe provides additional support
for the existence of a transannular hydrogen bond, which
decreases the CdO bond order substantially.
In addition to extracting Ktrans/cis values from 1H NMR
spectroscopy, we discerned the dominant ring pucker of Ac-
hyp-OMe in solution from the coupling constants between
hydrogen atoms on its pyrrolidine ring.22 We could not evaluate
its conformation in CDCl3 because of overlapping peaks in its
1H NMR spectrum. In D2O, however, we observed small
coupling constants (∼2.0 Hz) for HR-Hꢀ′, Hꢀ′-Hγ, and Hꢀ′-Hδ′
(22) (a) Gerig, J. T.; McLeod, R. S. J. Am. Chem. Soc. 1973, 95, 5725–
5729. (b) Sonntag, L.-S.; Schweizer, S.; Ochsenfeld, C.; Wennemers,
H. J. Am. Chem. Soc. 2006, 128, 14697–14703. (c) Shoulders, M. D.;
Kamer, K. J.; Raines, R. T. Bioorg. Med. Chem. Lett. 2009, 19, 3859–
3862.
(20) Ohnishi, M.; Urry, D. W. Biochem. Biophys. Res. Commun. 1969,
36, 194–202.
(21) Glendening, E. D.; Badenhoop, J. K.; Reed, A. E.; Carpenter, J. E.;
Bohmann, J. A.; Morales, C. M.; Weinhold, F. NBO 5.0; Theoretical
Chemistry Institute, University of Wisconsin: Madison, WI, 2001.
(23) Choudhary, A.; Raines, R. T. Unpublished results.
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Shoulders et al.
Scheme 1. Synthesis of Fmoc-hyp(tBu)ProGly-OH (5)
Scheme 2. Synthesis of Fmoc-mopProGly-OH (11)
2.4. Summary of Data on Ac-hyp-OMe and Ac-mop-OMe.
Taken together, our computational, spectroscopic, and crystal-
lographic data suggest that a transannular hydrogen bond in hyp
has a strong impact on its conformation. Distortion of the main-
chain torsion angles typically adopted by a Pro derivative with
a Cγ-endo ring pucker results in an anomalously high Ktrans/cis
value due to a strengthened nfπ* interaction. In contrast,
elimination of the transannular hydrogen bond by O-methylation
to form mop results in a strong preference for a prototypical
Cγ-endo ring pucker with standard main-chain torsion angles.
Hence, the O-methylation of hyp residues in proteins could
revert the unusual consequences of the transannular hydrogen
bond in hyp.
2.5. Effects of hyp and mop Residues on the Conforma-
tional Stability of a Protein. Collagen provides an appealing
system to explore the effects of hyp and mop on protein
structure. As described above, collagen triple helices are usually
stabilized by Cγ-endo puckered Pro derivatives in the Xaa
position, though hyp is known to destabilize the triple helix
when in that position.12,13 Collagen-related peptides (CRPs) are
widely employed to study the effects of specific amino acid
residues on triple-helix structure and stability.24 Hence, we
expected that a set of CRPs with hyp, mop, and Pro in the Xaa
position could provide valuable insight on the ability of these
residues to modulate protein stability.
2.5.1. Synthesis of (hypProGly)15, (mopProGly)15, and (Pro-
ProGly)15. Collagen triple helices with hyp in the Xaa position
are notoriously unstable and have not been characterized to date.
Because triple-helix stability is length-dependent, we believed
that the 45-residue peptide (hypProGly)15 might form a triple
helix of stability sufficient to allow for its detailed characteriza-
tion.13 Hence, we synthesized (hypProGly)15, (mopProGly)15,
and (ProProGly)15.
The peptide (hypProGly)15 was synthesized by the condensa-
tion of Fmoc-hyp(tBu)ProGly-OH (5) segments on a solid phase.
Tripeptide 5 was synthesized by the route shown in Scheme 1.
Briefly, Fmoc-hyp-OBn (1)25 was converted into its O-tert-
butoxy derivative Fmoc-hyp(tBu)-OBn (2) by treatment with
isobutylene and then into the free carboxylic acid Fmoc-
hyp(tBu)-OH (3) by hydrogenolysis. PyBroP-mediated coupling
with the hydrochloride salt of H-ProGly-OBn26 and hydro-
genolysis yielded Fmoc-hyp(tBu)ProGly-OH (5) in 67% overall
yield.
The peptide (mopProGly)15 was prepared by segment con-
densation of Fmoc-mopProGly-OH (11) on a solid phase.
Tripeptide 11 was synthesized by the route shown in Scheme
2. Briefly, Boc-hyp-OBn (6)27 was converted to Boc-mop-OBn
(7) by treatment with methyl iodide in the presence of Ag(I)
oxide.28 Hydrogenolysis yielded Boc-mop-OH (8). Boc depro-
tection under acidic conditions followed by treatment with
Fmoc-OSu under basic conditions provided Fmoc-mop-OH (9).
PyBroP-mediated coupling of 9 and the hydrochloride salt of
H-ProGly-OBn and hydrogenolysis of the benzyl ester yielded
Fmoc-mopProGly-OH (11) in 25% overall yield.
The peptide (ProProGly)15 was synthesized by the segment
condensation of Fmoc-ProProGly-OH26 on a solid phase.
2.5.2. Conformational Analyses of (hypProGly)15, (mopPro-
Gly)15, and (ProProGly)15. We used circular dichroism (CD)
spectroscopy to analyze the conformations of (hypProGly)15,
(mopProGly)15, and (ProProGly)15 in solution. The signature CD
spectrum of the collagen triple helix has a maximum near 225
nm and a minimum near 210 nm. By this criterion, (hypPro-
Gly)15, (mopProGly)15, and (ProProGly)15 form a triple helix at
4 °C (Figure 4A). Upon heating, a cooperative transition of the
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Aberrance of the 4S Diastereomer of 4-Hydroxyproline
A R T I C L E S
Figure 5. DSC scans of (hypProGly)15 (122 µM), (mopProGly)15 (122 µM),
and (ProProGly)15 (96 µM) in 50 mM HOAc (pH 3.0; scan rate ) 6 °C/h).
removes the transannular hydrogen bond, thereby strengthening
the interstrand hydrogen bond. Second, our computational
analysis of Ac-hyp-OMe suggests that its transannular hydrogen
bond results in peptide main-chain torsion angles inconsistent
with the Xaa position of the collagen triple helix. This
deleterious preorganization substantially reduces the stability
of a (hypProGly)15 triple helix. In contrast, O-methylation results
in proper preorganization.
2.5.3. Thermodynamic Analyses of (hypProGly)15, (mopPro-
Gly)15, and (ProProGly)15. Given the seemingly favorable
consequences of hyp O-methylation for triple-helix stabilization,
we were surprised to observe that a (mopProGly)15 triple helix
is less stable than a (ProProGly)15 triple helix. To determine
the thermodynamic basis for the relative stabilities of (hypPro-
Gly)15, (mopProGly)15, and (ProProGly)15 triple helices, we
analyzed their thermal denaturation by differential scanning
calorimetry (DSC) (Figure 5). Thermodynamic parameters were
obtained as described in the Experimental Section and are listed
in Table 2.
Figure 4. Conformational analysis of (hypProGly)15, (mopProGly)15, and
(ProProGly)15 by CD spectroscopy. (A) Spectra of peptide solutions (0.2
mM in 50 mM acetic acid) incubated at e4 °C for g24 h. (B) Effect of
temperature on the molar ellipticity at 226 nm. Data were recorded at
intervals of 3 °C after equilibration for g5 min.
Table 2. Thermodynamic Parameters for Folding of Triple-Helical
Collagen-Related Peptides
a
Tm
peptide
∆H (kcal/mol)b
-T∆S (kcal/mol)b
∆G (kcal/mol)b
(hypProGly)15
(mopProGly)15
(ProProGly)15
25
47
65
-29.9
-24.7
-32.8
20.5
13.1
19.0
-9.4
-11.6
-13.8
Triple-helix formation is made difficult by the need to
assemble three strands and the absence of a substantial
hydrophobic core to stabilize the resulting assembly.22c These
factors account for the strongly unfavorable ∆S value observed
for all three triple helices (Table 2). It is noteworthy that the
most stable triple helix, which is formed by (ProProGly)15, also
has the most favorable ∆H value. The entropic cost for the
folding of (ProProGly)15 is between those for the folding of
(hypProGly)15 and (mopProGly)15.
The striking instability of (hypProGly)15 relative to (ProPro-
Gly)15 derives from both enthalpic and entropic effects. The
less favorable ∆H value of (hypProGly)15 could result from the
transannular hydrogen bond in hyp (Figure 1B) reducing the
favorable enthalpy from the interstrand hydrogen bond in the
triple helix (Figure 1C). This reduction in the value of ∆H upon
4S-hydroxylation of Pro in the Xaa position of (ProProGly)n is
in stark contrast to the highly enthalpically favorable effect of
4R-hydroxylation of Pro in the Yaa position of (ProProGly)n.4,17,30
The latter result is due to the enthalpically favorable (but
entropically disfavorable) hydration of the hydroxyl moiety of
Hyp in the Yaa position of folded triple helices. We might
anticipate an even greater reduction in the value of ∆H for
(hypProGly)15 relative to (ProProGly)15 due to disruption of the
interstrand hydrogen bond, but similar hydration of the hydroxyl
moiety in hyp could compensate for a reduced value of ∆H
a Values ((1 °C) were determined in triplicate by CD spectroscopy.
b Values were estimated by DSC and reported here at 46 °C. The largest
source of error ((5%) was in the determination of [peptide].
molar ellipticity at 226 nm, another hallmark of the collagen
triple helix, was observed for all three CRPs (Figure 4B). Values
of Tm, which is the temperature at the midpoint of the thermal
transition between folded and unfolded states, were determined
by fitting the data to a two-state model (Table 2).29 We observed
that (ProProGly)15 forms a highly stable triple helix with Tm )
65 °C. In contrast, (hypProGly)15 forms a weakly stable triple
helix, with Tm ) 25 °C. O-Methylation of hyp rescues much of
the instability of (hypProGly)15, yielding a triple helix with Tm
) 47 °C. Sedimentation equilibrium experiments confirmed the
self-assembly of (hypProGly)15, (mopProGly)15, and (ProPro-
Gly)15 at 4 °C and the complete disassembly of (hypProGly)15
at 40 °C (see Figure S2 in the Supporting Information).
The increased stability of (mopProGly)15 triple helices relative
to (hypProGly)15 triple helices is likely due to two factors: First,
the carbonyl moiety of hyp required to form the essential
interstrand hydrogen bond in the collagen triple helix (Figure
1C) is distracted by an intramolecular transannular hydrogen
bond with the hydroxyl moiety of hyp (Figure 1B). This
hydrogen bond is less accessible to solvent than is the
transannular hydrogen bond in Ac-hyp-OMe. O-Methylation
(30) Nishi, Y.; Uchiyama, S.; Doi, M.; Nishiuchi, Y.; Nakazawa, T.;
(29) Becktel, W. J.; Schellman, J. A. Biopolymers 1987, 26, 1859–1877.
Ohkubo, T.; Kobayashi, Y. Biochemistry 2005, 44, 6034–6042.
9
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A R T I C L E S
Shoulders et al.
derivatives. hyp is the only known Pro derivative that can adopt
a Cγ-endo ring pucker concomitant with a high trans/cis ratio.
As such, hyp or related Pro derivatives that can engage in a
transannular hydrogen bond could be the only Cγ-endo puckered
Pro derivatives that form stable polyproline II-type helices,
which usually require Pro derivatives with a Cγ-exo ring pucker.
In contrast, mop, like flp and clp, adopts a Cγ-endo ring pucker
with prototypical main-chain torsion angles and a low trans/cis
ratio.
4R-Hydroxylation inverts the inherent preference of Pro for
a Cγ-endo ring pucker3d and preorganizes the main-chain torsion
angles that accompany a Cγ-exo ring pucker. In contrast, 4S-
hydroxylation results in a derivative with the main-chain torsion
angles mandated by 4R-hydroxylation but a diminished capacity
to accept a hydrogen bond (Figure 1B). It is interesting to
speculate that the evolution of prolyl 3S-hydroxylases was
favored by their ability to enforce the Cγ-endo ring pucker in
proline residues.32 These enzymes contain the key active-site
residues of prolyl 4R-hydroxylases33 and likewise install a
hydroxyl group on the face of the pyrrolidine ring opposite from
the carboxyl group, averting transannular hydrogen-bond forma-
tion. Collagen prolyl 3R-hydroxylases, like collagen prolyl 4S-
hydroxylases, have not been identified in natural proteomes.
Finally, we note a general implication of our data. In the Yaa
position, a Mop residue is more beneficial than a Hyp residue
to triple-helix stability.17 Likewise, in the Xaa position, mop is
more beneficial than hyp (Figures 4 and 5; Table 2). These
parallel findings have a common origin with important
ramificationsshydrogen bonding can compromise the benefits
of an underlying stereoelectronic effect to the conformational
stability of a protein.
Figure 6. Space-filling models of a segment of a (ProMopGly)n and
(mopProGly)n triple helix. Models were constructed from the three-
dimensional structure of a (ProProGly)n triple helix (PDB entry 1k6f10) by
replacing the appropriate hydrogen with a methoxy group in its least
hindered conformation using the program SYBYL (Tripos, St. Louis, MO).
Models were depicted with the program PyMOL.
(but, once again, at the expense of entropy). The instability of
(hypProGly)15 triple helices is also due, in part, to another
increase in the entropic cost for their formation. The hyp residues
are preorganized poorly for the Xaa position of the triple helix,
as the transannular hydrogen bond disrupts the main-chain
torsion angles that accompany a normal Cγ-endo ring pucker.
Previously, we showed that (ProMopGly)n strands form much
more stable triple helices than do (ProProGly)n strands.17 This
hyperstability is the result of an entropic advantage that likely
arises from preorganization. Like (ProMopGly)n triple helices,
(mopProGly)n triple helices have a more favorable entropy of
formation than do (ProProGly)n triple helices (Table 2). Yet,
(mopProGly)n triple helices are less stable than (ProProGly)n
triple helices due to a less favorable enthalpy of formation. To
understand the dichotomy between the relative stabilities of
(mopProGly)n and (ProMopGly)n triple helices, we resorted to
molecular modeling. Our models show that the 4R-methoxy
group of a Mop residue in the Yaa position does not interact
unfavorably with any neighboring atoms in a folded triple helix
(Figure 6A). In contrast, the 4S-methoxy group of a mop residue
in the Xaa position is sterically hindered (Figure 6B). In a triple
helix, the carbonyl group of the residue in the Xaa position
engages the N-H of a Gly residue in another strand (Figure
1C). The unfavorable steric interaction of the methoxy residue
in a (mopProGly)n triple helix with the neighboring strand
(Figure 6B) could weaken this essential interstrand hydrogen
bond, leading to the reduced enthalpy of triple-helix formation
for (mopProGly)15 relative to (hypProGly)15 and (ProProGly)15.
This competing enthalpic effect is likely to be the reason that
triple helices formed from (mopProGly)15 are less stable than
(ProProGly)15 triple helices, despite their apparently high degree
of preorganization.
3. Experimental Section
3.1. General. Commercial chemicals were of reagent grade or
better and were used without further purification. Anhydrous CH2Cl2
was obtained from a CYCLE-TAINER solvent delivery system
(J. T. Baker, Phillipsburg, NJ). In all reactions involving anhydrous
solvents, glassware was either oven- or flame-dried. NaHCO3(aq)
and brine (NaCl) refer to saturated aqueous solutions. Flash
chromatography was performed with columns of silica gel 60,
230-400 mesh (Silicycle, Que´bec City, Canada). Semipreparative
HPLC was performed at 60 °C with a Macherey-Nagel C-8
reversed-phase column after preheating peptides to 70 °C for g30
min to disassemble any oligomers. Analytical HPLC was performed
with a Varian C-18 reversed-phase column. HPLC purifications and
analyses employed linear gradients of solvent A (H2O containing
0.1% v/v TFA) and solvent B (CH3CN containing 0.1% v/v TFA).
The term “concentrated under reduced pressure” refers to the
removal of solvents and other volatile materials using a rotary
evaporator at water aspirator pressure (<20 Torr) while maintaining
the water-bath temperature below 40 °C. Residual solvent was
removed from samples at high vacuum (<0.1 Torr). The term “high
vacuum” refers to vacuum achieved by a mechanical belt-drive oil
pump.
Further evidence that sterics underlie the reduced conforma-
tional stability of (mopProGly)15 triple helices relative to
(ProProGly)15 triple helices derives from earlier findings for
triple helices with (2S,4S)-4-chloroproline (clp) in the Xaa
position of the triple helix.31 Despite the tendency of clp to adopt
the Cγ-endo ring pucker, a (clpProGly)10 triple helix is slightly
less stable than a (ProProGly)10 triple helix. The destabilization
arising from the bulky 4S-chloro group is consistent with a steric
basis for the slightly reduced stability of (mopProGly)15 triple
helices relative to (ProProGly)15.
NMR spectra were acquired with a Bruker DMX-400 Avance
spectrometer (1H, 400 MHz; 13C, 100.6 MHz) at the National
Magnetic Resonance Facility at Madison (NMRFAM). NMR
spectra were obtained at ambient temperatures on samples dissolved
in CDCl3, unless noted otherwise. Mass spectrometry was performed
with either a Micromass LCT (electrospray ionization, ESI) in the
2.6. Implications for Other Proteins. Our findings have
important implications for the burgeoning application of Pro
(32) Jenkins, C. L.; Bretscher, L. E.; Guzei, I. A.; Raines, R. T. J. Am.
Chem. Soc. 2003, 125, 6422–6427.
(31) Shoulders, M. D.; Guzei, I. A.; Raines, R. T. Biopolymers 2008, 89,
(33) Vranka, J. A.; Sakai, L. Y.; Ba¨chinger, H. P. J. Biol. Chem. 2004,
279, 23615–23621.
443–454.
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Aberrance of the 4S Diastereomer of 4-Hydroxyproline
A R T I C L E S
Mass Spectrometry Facility in the Department of Chemistry or an
Applied Biosystems Voyager DE-Pro (matrix-assisted laser de-
sorption/ionization, MALDI) mass spectrometer in the University
of Wisconsin Biophysics Instrumentation Facility (BIF). The
infrared spectrum of Ac-hyp-OMe was acquired with a Bruker
Equinox 55 FTIR.
TLC was necessary to prevent hydrogenolysis of the Fmoc group.
The suspension was filtered through a pad of Celite and concentrated
under reduced pressure. The crude product was purified by flash
chromatography over silica gel (CH2Cl2 to elute byproducts, then
3% v/v MeOH in CH2Cl2 containing 0.1% v/v formic acid). The
fractions containing the reaction product were concentrated under
reduced pressure, and the formic acid was removed by dissolving
the residue in 10% v/v MeOH in toluene and concentrating under
reduced pressure to afford Fmoc-hyp(tBu)ProGly-OH (5) (1.27 g,
84%, two steps) as a white solid. 1H NMR (MeOH-d4) δ: 1.16 and
1.19 (s, 9H), 1.61-2.23 (m, 5H), 2.33-2.58 (m, 1H), 2.93-3.10
(m, 1.4H), 3.33-3.42 (m, 0.5H) 3.47-3.67 (m, 1.3H), 3.68-3.86
(m, 1.7H), 3.90-4.03 (m, 1H), 4.04-4.53 (m, 5.5H), 4.66-4.74
(m, 0.5H), 7.25-7.45 (m, 4H), 7.53-7.66 (m, 2H), 7.75-7.85 (m,
2H); 13C NMR (MeOH-d4) δ: 22.9, 23.1, 25.7, 25.8, 28.5, 28.6,
30.1, 30.2, 32.5, 33.2, 37.5, 37.7, 38.3, 41.7, 42.1, 47.8, 53.4, 53.7,
47.3, 57.8, 58.1, 67.5, 68.5, 68.7, 69.7, 70.3, 75.0, 75.1, 75.3, 120.9,
125.7, 126.1, 128.2, 128.2, 128.3, 128.8, 142.6, 142.7, 144.9, 145.1,
145.5, 145.7, 156.0, 156.5, 172.6, 172.9, 173.0, 173.3, 174.0, 174.5,
174.7; ESI-EMM (m/z): [M - H]- calcd for C31H36N3O7 562.2558;
found 562.2556.
3.6. N-tert-Butoxycarbonyl-(2S,4S)-4-methoxyproline Ben-
zyl Ester (7). Boc-hyp-OBn (6) (6.94 g, 21.6 mmol), prepared as
described previously,27 was dissolved in anhydrous acetone (150
mL) under Ar(g). MeI (10.73 g, 75.6 mmol) was added, followed
by Ag2O (16.01 g, 69.1 mmol). The resulting suspension was stirred
at room temperature for 24 h. The suspension was filtered and
concentrated under reduced pressure. The residue was dissolved
again in anhydrous acetone (150 mL), MeI (10.73 g, 75.6 mmol)
and Ag2O (16.01 g, 69.1 mmol) were added, and the resulting
suspension was stirred at room temperature for 24 h. The suspension
was filtered and concentrated under reduced pressure. Flash
chromatography over silica gel (50% v/v EtOAc in hexanes)
afforded Boc-mop-OBn (7) (4.13 g, 57%) as a colorless oil.
Unreacted starting material Boc-hyp-OBn (6) was also recovered.
1H NMR δ: 1.35 and 1.46 (s, 9H), 1.57-1.72 (m, 0.3H), 2.11-2.40
(m, 1.7H), 3.16 and 3.20 (s, 3H), 3.26-3.74 (m, 2H), 3.85-3.94
(m, 1H), 4.31-4.52 (m, 1H), 5.03-5.32 (m, 2H), 7.28-7.39 (m,
5H); 13C NMR δ: 28.4, 28.5, 34.7, 35.9, 51.2, 52.0, 56.5, 56.6,
57.5, 57.9, 66.8, 78.1, 79.1, 80.1, 80.2, 128.2, 128.3, 128.4, 128.5,
128.7, 135.9, 136.1, 154.0, 172.0, 172.3; ESI-EMM (m/z): [M +
H]+ calcd for C18H26NO5 336.1806; found 336.1800.
3.7. N-9-tert-Butoxycarbonyl-(2S,4S)-4-methoxyproline (8).
MeOH (100 mL) was added carefully to a mixture of Boc-mop-
OBn (7) (1.80 g, 5.4 mmol) and Pd/C (10% w/w, 1.00 g) under
Ar(g), and the resulting black suspension was stirred under H2(g)
for 14 h. The suspension was filtered through a pad of Celite and
concentrated under reduced pressure to afford Boc-mop-OH (8)
(1.32 g, quant.) as a white solid. 1H NMR (DMSO-d6) δ: 1.34 and
1.40 (s, 9H), 1.93-2.03 (m, 1H), 2.21-2.42 (m, 1H), 3.17 (s, 3H),
3.17-3.24 (m, 1H), 3.47-3.59 (m, 1H), 3.85-3.96 (m, 1H),
4.08-4.20 (m, 1H); 13C NMR δ: 28.4, 32.3, 35.4, 51.5, 53.2, 56.5,
56.8, 78.7, 81.1, 82.1, 154.2, 157.0, 173.1, 175.1; ESI-EMM
(m/z): [M + Na]+ calcd for C11H19NO5Na 268.1161; found
268.1161.
3.8. N - 9 - Fluorenylmethoxycarbonyl - (2S,4S) - 4 - methoxy-
proline (9). Boc-mop-OH (8) (2.15 g, 8.8 mmol) was dissolved in
4 N HCl in dioxane (150 mL), and the resulting solution was stirred
at room temperature under Ar(g) for 2.5 h. The reaction mixture
was concentrated under reduced pressure and dried briefly under
high vacuum. The resultant white solid was dissolved in saturated
NaHCO3(aq) (75 mL). A solution of Fmoc-OSu (2.96 g, 8.8 mmol)
in dioxane (75 mL) was added, and the resulting white suspension
was stirred for 20 h. The reaction mixture was concentrated under
reduced pressure, and the residue was diluted with water (150 mL)
and washed with ether (3 × 150 mL). The aqueous layer was
acidified to pH 1.5 with 2 N HCl, extracted with ether (3 × 150
mL), dried over anhydrous MgSO4(s), and concentrated under
reduced pressure to afford Fmoc-mop-OH (9) (2.50 g, 77%) as a
3.2. N-9-Fluorenylmethoxycarbonyl-(2S,4S)-4-tert-butoxy-
proline Benzyl Ester (2). Fmoc-hyp-OBn (1) (1.25 g, 2.8 mmol),
described previously,25 was dissolved in anhydrous CH2Cl2 (20 mL)
and cooled to -78 °C. Concentrated H2SO4 (30 µL) was added
followed by isobutylene (∼20 mL, ∼220 mmol, condensed at -78
°C). The resulting solution was capped with a rubber septum, sealed
with copper wire around the neck of the round-bottom flask, and
allowed to warm slowly to room temperature with stirring. After
5 d, the solution was cooled to -78 °C, and the septum was
removed to allow slow evaporation of isobutylene. Upon warming
to room temperature, the solution was concentrated under reduced
pressure. The crude product was purified by flash chromatography
over silica gel (40% v/v EtOAc in hexanes) to afford Fmoc-
1
hyp(tBu)-OBn (2) (1.18 g, 84%) as a colorless oil. H NMR δ:
1.14 and 1.17 (s, 9H), 2.03-2.13 (m, 1H), 2.33-2.48 (m, 1H),
3.33-3.44 (m, 1H), 3.71-3.82 (m, 1H), 3.98-4.55 (m, 5H),
4.97-5.29 (m, 2H), 7.19-7.44 (m, 9H), 7.47-7.64 (m, 2H),
7.70-7.79 (m, 2H); 13C NMR δ: 28.3, 37.9, 38.9, 47.3, 53.5, 53.9,
57.7, 57.9, 66.9, 66.9, 67.6, 68.7, 69.6, 74.2, 120.0, 120.1, 125.1,
125.3, 125.5, 127.2, 127.8, 128.2, 128.3, 128.4, 128.6, 135.8, 135.9,
141.4, 141.4, 141.5, 143.8, 144.4, 154.6, 155.0, 171.8, 172.0; ESI-
EMM (m/z): [M + Na]+ calcd for C31H33NO5Na 522.2256; found
522.2231.
3.3. N-9-Fluorenylmethoxycarbonyl-(2S,4S)-4-tert-butoxy-
proline (3). MeOH (100 mL) was added carefully to a mixture of
Fmoc-hyp(tBu)-OBn (2) (1.79 g, 3.6 mmol) and Pd/C (10% w/w,
0.38 g) under Ar(g), and the resulting black suspension was stirred
under H2(g) for 45 min. Careful monitoring by thin-layer chroma-
tography (TLC) was necessary to prevent hydrogenolysis of the
Fmoc group. The suspension was filtered through a pad of Celite
and concentrated under reduced pressure. The crude product was
purified by flash chromatography over silica gel (95% v/v CH2Cl2
in MeOH) to afford Fmoc-hyp(tBu)-OH (3) (1.39 g, 95%) as a
1
white solid. H NMR (MeOH-d4) δ: 1.18 (s, 9H), 1.90-2.08 (m,
1H), 2.35-2.50 (m, 1H), 3.17 and 3.25 (dd, J ) 4.5, 11.0 Hz, 1H),
4.14-4.46 (m, 5H), 7.26-7.43 (m, 4H), 7.57-7.68 (m, 2H),
7.75-7.83 (m, 2H); 13C NMR (MeOH-d4) δ: 28.5, 38.8, 39.7, 48.4,
54.5, 55.0, 58.8, 68.6, 69.0, 69.9, 70.6, 75.2, 120.9, 126.1, 126.2,
128.1, 128.8, 142.5, 142.6, 142.7, 145.1, 145.4, 145.4, 156.5, 156.7,
175.5, 175.7; ESI-EMM (m/z): [M + Na]+ calcd for C24H27NO5Na
432.1787; found 432.1800.
3.4. N-9-Fluorenylmethoxycarbonyl-(2S,4S)-4-tert-butoxy-
prolyl-(2S)-prolylglycine Benzyl Ester (4). A solution of Fmoc-
hyp(tBu)-OH (3) (1.31 g, 3.2 mmol) and the hydrochloride salt of
H-ProGly-OBn (3.60 g, 12.0 mmol, prepared as described previ-
ously26) in anhydrous CH2Cl2 (160 mL) was cooled to 0 °C. PyBroP
(1.49 g, 3.2 mmol) and DIEA (3.31 g, 25.6 mmol) were added,
and the resulting solution was allowed to warm slowly to room
temperature and then stirred for 15 h. The reaction mixture was
washed with 10% w/v aqueous citric acid (60 mL), NaHCO3(aq)
(60 mL), and brine (60 mL). The organic layer was dried over
anhydrous MgSO4(s) and concentrated under reduced pressure. The
crude residue was purified by flash chromatography over silica gel
(gradient: 30% v/v EtOAc in hexanes to 100% v/v EtOAc) to afford
Fmoc-hyp(tBu)ProGly-OBn (4) (1.74 g) as a white solid containing
a slight impurity that was removed after the succeeding step. ESI-
EMM (m/z): [M + Na]+ calcd for C38H43N3O7Na 676.2999; found
676.2982.
3.5. N-9-Fluorenylmethoxycarbonyl-(2S,4S)-4-tert-butoxy-
prolyl-(2S)-prolylglycine (5). MeOH (100 mL) was added carefully
to a mixture of Fmoc-hyp(tBu)ProGly-OBn (4) (1.74 g, 2.7 mmol)
and Pd/C (10% w/w, 0.32 g) under Ar(g), and the resulting black
suspension was stirred under H2(g) for 2 h. Careful monitoring by
9
J. AM. CHEM. SOC. VOL. 132, NO. 31, 2010 10863

A R T I C L E S
Shoulders et al.
white solid. 1H NMR (DMSO-d6) δ: 2.05-2.43 (m, 2H), 3.30-3.37
(m, 1H), 3.55-3.62 (m, 1H), 3.92-4.01 (m, 1H), 4.14-4.41 (m,
4H), 7.28-7.47 (m, 4H), 7.61-7.71 (m, 2H), 7.86-7.94 (m, 2H);
13C NMR (DMSO-d6) δ: 34.00, 35.1, 46.6, 46.7, 50.9, 51.3, 55.9,
57.2, 57.4, 66.6, 66.9, 77.5, 78.4, 120.1, 125.2, 125.3, 127.2, 127.7,
140.7, 140.8, 143.8, 154.0, 172.6, 173.0; ESI-EMM (m/z): [M +
Na]+ calcd for C21H21NO5Na 390.1317; found 390.1134.
3.9. N - 9 - Fluorenylmethoxycarbonyl - (2S,4S) - 4 - methoxy-
prolyl-(2S)-prolylglycine Benzyl Ester (10). A solution of Fmoc-
mop-OH (9) (2.45 g, 6.7 mmol) and the hydrochloride salt of
H-ProGly-OBn (3.59 g, 12.0 mmol, prepared as described previ-
ously26) in anhydrous CH2Cl2 (160 mL) was cooled to 0 °C. PyBroP
(3.12 g, 6.7 mmol) and DIEA (3.46 g, 26.8 mmol) were added.
The resulting solution was allowed to warm slowly to room
temperature and then stirred for 15 h. The reaction mixture was
washed with 10% w/v aqueous citric acid (60 mL), NaHCO3(aq)
(60 mL), and brine (60 mL). The organic layer was dried over
anhydrous MgSO4(s) and concentrated under reduced pressure. The
crude residue was purified by flash chromatography over silica gel
(gradient: 50% v/v EtOAc in hexanes to 100% v/v EtOAc) to afford
Fmoc-mopProGly-OBn (10) (3.23 g) as a white solid containing a
slight impurity that was removed after the succeeding step. ESI-
EMM (m/z): [M + Na]+ calcd for C35H37N3O7Na 634.2529; found
634.2527.
3.12. Attachment of Fmoc-hyp(tBu)ProGly-OH (5) to
2-Chlorotrityl Resin. Under Ar(g), 80 mg of 2-chlorotrityl chloride
resin (loading: 1.7 mmol/g) were swelled in anhydrous CH2Cl2 (0.7
mL) for 5 min. A solution of compound 5 (75 mg, 0.13 mmol) and
DIEA (32 mg, 0.25 mmol) in anhydrous CH2Cl2 (1.3 mL) was
added by syringe. Additional anhydrous CH2Cl2 (1.3 mL) was used
to ensure complete transfer of 5. After 2 h, anhydrous MeOH (0.6
mL) was added to cap any remaining active sites on the resin. The
resin-bound peptide was isolated by gravity filtration, washed with
several portions of anhydrous CH2Cl2 (∼25 mL), and dried under
high vacuum. The mass of the resin-bound peptide was 90 mg.
Loading was measured by ultraviolet spectroscopy34 to be 0.36
mmol/g.
3.13. Attachment of Fmoc-mopProGly-OH (11) and Fmoc-
ProProGly-OH to 2-Chlorotrityl Resin. Fmoc-tripeptide 11 and
Fmoc-ProProGly-OH were loaded onto 2-chlorotrityl resin in similar
fashion to that described for 5. Loadings were measured by
ultraviolet spectroscopy to be 0.32 mmol/g for 11 and 0.35 mmol/g
for Fmoc-ProProGly-OH.34
3.14. Synthesis of (hypProGly)15, (mopProGly)15, and (Pro-
ProGly)15. The three 45-mer peptides were synthesized by the
segment condensation of their corresponding Fmoc-tripeptides (5,
11, and Fmoc-ProProGly-OH) on a solid phase using an Applied
Biosystems Synergy 432A Peptide Synthesizer at the University
of WisonsinsMadison Biotechnology Center. The first trimer was
loaded onto the resin as described above. Fmoc-deprotection was
achieved by treatment with 20% (v/v) piperidine in DMF. The
trimers (3 equiv) were converted to active esters by treatment with
HBTU, DIEA, and HOBt. Double extended couplings (60-90 min)
were employed at room temperature for all couplings except the
first three couplings in the synthesis of (hypProGly)15, in which
single extended couplings were employed. Peptides were cleaved
from the resin in 95:3:2 TFA/triisopropylsilane/H2O (1.5 mL),
precipitated from methyl tert-butyl ether at 0 °C, and isolated by
centrifugation. Semipreparative HPLC was used to purify the
peptides (hypProGly)15 (10% B for 5 min, increasing to 35% B
over 45 min), (mopProGly)15 (gradient: 10% B to 70% B over 50
min), and (ProProGly)15 (gradient: 10% B to 70% B over 50 min).
All three peptides were >90% pure by analytical HPLC and
MALDI-TOF mass spectrometry (m/z) [M + Na]+ calcd for
3.10. N-9-Fluorenylmethoxycarbonyl-(2S,4S)-4-methoxy-
prolyl-(2S)-prolylglycine (11). MeOH (100 mL) was added
carefully to a mixture of Fmoc-mopProGly-OBn (10) (3.23 g, 5.3
mmol) and Pd/C (10% w/w, 0.63 g) under Ar(g), and the resulting
black suspension was stirred under H2(g) for 45 min. Careful
monitoring by TLC was necessary to prevent hydrogenolysis of
the Fmoc group. The suspension was filtered through a pad of Celite
and concentrated under reduced pressure. The crude product was
purified by flash chromatography over silica gel (CH2Cl2 to elute
byproducts, then 5% v/v MeOH in CH2Cl2 containing 0.1% v/v
formic acid). The fractions containing the reaction product were
concentrated under reduced pressure, and the formic acid was
removed by dissolving the residue in 10% v/v MeOH in toluene
and concentrating under reduced pressure to afford Fmoc-mopPro-
1
Gly-OH (11) (1.98 g, 57%, two steps) as a white solid. H NMR
C
180H257N45O61Na 4048.8, found 4048.6 for (hypProGly)15, calcd
(DMSO-d6) δ: 1.58-2.04 (m, 4H), 2.55-2.70 (m, 1H), 2.99-3.15
(m, 1H), 3.20 and 3.23 (s, 3H), 3.23-3.26 (m, 1H), 3.31-4.02
(m, 7.7H), 4.09-4.17 (m, 0.3H), 4.19-4.66 (m, 4H), 7.26-7.47
(m, 4H), 7.51-7.70 (m, 2H), 7.84-8.07 (m, 2H), 12.53 (bs, 1H);
13C NMR (MeOH-d4) δ: 25.8, 25.9, 30.0, 30.1, 35.2, 35.8, 41.8,
47.9, 49.9, 52.5, 52.8, 57.3, 57.4, 58.1, 58.5, 61.3, 61.6, 67.6, 68.8,
78.9, 79.6, 120.9, 125.7, 125.9, 126.2, 128.2, 128.9, 142.6, 142.7,
145.0, 145.1, 145.4, 145.7, 156.2, 156.7, 172.6, 172.7, 172.8, 174.5,
174.7; ESI-EMM (m/z): [M + Na]+ calcd for C28H31N3O7Na
544.2060; found 544.2065.
3.11. N-Acetyl-(2S,4S)-4-methoxyproline Methyl Ester. Ac-
hyp-OMe (0.34 g, 1.8 mmol), described previously,3c was dissolved
in anhydrous acetone (50 mL) under Ar(g). MeI (1.14 g, 8.0 mmol)
was added, followed by Ag2O (0.94 g, 7.4 mmol). The resulting
suspension was stirred at room temperature for 24 h. The suspension
was filtered and evaporated under reduced pressure. The resulting
residue was dissolved in EtOAc (100 mL), washed with water (2
× 100 mL), dried over anhydrous MgSO4(s), and concentrated
under reduced pressure. Flash chromatography over silica gel
(gradient: 0% v/v MeOH in CH2Cl2 to 10% v/v MeOH in CH2Cl2)
afforded Ac-mop-OH (0.25 g, 69%) as a colorless oil. 1H NMR δ:
2.04 and 2.10 (s, 3H), 2.19-2.28 (m, 1H), 2.30-2.36 (m, 0.65H),
2.56-2.63 (m, 0.35H), 3.25 and 3.30 (s, 3H), 3.55-3.71 (m, 2H),
3.72 and 3.76 (s, 3H), 3.91-4.05 (m, 1H), 4.37-4.41 (m, 0.35H),
4.65 (dd, J ) 3.5, 8.7 Hz, 0.65H); 13C NMR δ: 22.2, 22.4, 34.2,
36.3, 51.7, 52.5, 52.8, 53.1, 56.3, 56.8, 57.0, 59.0, 77.8, 79.2, 105.2,
169.7, 170.3, 172.0; ESI-EMM (m/z): [M + H]+ calcd for
C9H16NO4 202.1074; found 202.1070.
for C195H287N45O61Na 4260.1, found 4258.0 for (mopProGly)15;
calcd for C180H257N45O46Na 3809.9, found 3809.5 for (ProProGly)15.
3.15. Circular Dichroism Spectroscopy of (hypProGly)15,
(mopProGly)15, and (ProProGly)15. Peptides were dried under high
vacuum for at least 24 h before being weighed and dissolved to
0.2 mM in 50 mM HOAc(aq) (pH 2.9). The solutions were
incubated at e4 °C for g24 h before CD spectra were acquired
with an Aviv Associates (Lakewood, NJ) 202SF CD spectropho-
tometer. Spectra were measured with a 1-nm band-pass in cuvettes
with a 0.1-cm path length. The signal was averaged for 5 s during
wavelength scans and denaturation experiments using a 0.6 °C
temperature dead band. During denaturation experiments, CD
spectra were acquired at intervals of 3 °C. At each temperature,
solutions were equilibrated for 5 min before data acquisition. Values
of Tm were determined in triplicate by fitting the molar ellipticity
at 225 nm to a two-state model.29
3.16. Differential Scanning Calorimetry and Thermo-
dynamic Analyses of (hypProGly)15, (mopProGly)15, and
(ProProGly)15. DSC measurements were made with a VP-DSC
instrument (MicroCal, LLC, Northampton, MA). Instrument base-
lines were established by filling both the sample and the reference
cells with degassed 50 mM HOAc(aq) and scanning from 5 to 98
°C at a scan rate of 6 °C/h with a 10-h pre-equilibration period,
until the baseline was reproducible. The final buffer-buffer scan
(34) AppliedBiosystems. http://www3.appliedbiosystems.com/cms/groups/
psm_marketing/documents/generaldocuments/cms_040640.pdf (ac-
cessed April 12, 2010).
9
10864 J. AM. CHEM. SOC. VOL. 132, NO. 31, 2010

Aberrance of the 4S Diastereomer of 4-Hydroxyproline
A R T I C L E S
was used as the baseline for subsequent peptide scans. Peptide
solutions (∼100 µM in 50 mM HOAc(aq)) were incubated at 4 °C
for g24 h prior to degassing and loading in the sample cell (the
reference cell solution was not replaced) during the cooldown from
the previous buffer-buffer scan (at ∼10 °C). Samples were scanned
from 5 to 98 °C at a scan rate of 6 °C/h with a 10-h pre-equilibration
period; the first scan of each sample was used in the analysis.
Subsequent scans of (mopProGly)15 and (ProProGly)15 showed high
reversibility with >90% recovery. (hypProGly)15 folds significantly
more slowly than (mopProGly)15 and (ProProGly)15, but data could
be reproduced reliably by reincubating the sample at 4 °C for g24
h, loading the solution in the sample cell, and rescanning from 5
to 98 °C. After DSC measurements, peptide concentrations were
determined by quantitative amino acid analysis (Protein Chemistry
Core, Biomolecular Resource Facility, University of Texas Medical
Branch). Peptide concentrations of 122, 122, and 96 µM were
obtained for (hypProGly)15, (mopProGly)15, and (ProProGly)15,
respectively.
Data were processed using the MicroCal software in the Origin
7 program (OriginLab, Northampton, MA). The appropriate refer-
ence scan was subtracted from the first sample scan, and the data
were normalized to monomer concentrations. For (hypProGly)15
and (ProProGly)15, a progress baseline was subtracted from the data,
yielding the traces shown in Figure 5. Although its transition was
fully reversible, (mopProGly)15 exhibited a strong exotherm likely
due to peptide aggregation beginning at ∼50 °C, which prevented
determination of a high-temperature baseline (see Figure S3 in the
Supporting Information). A linear baseline based on the low
temperature data was therefore subtracted from the data, and only
the first half of the endotherm was used to extract thermodynamic
parameters. We also evaluated the transition of (mopProGly)15 at
a second concentration, which produced equivalent results and
confirmed the validity of our approach.
Values of ∆H (per mole of monomer) were obtained by direct
integration of the DSC endotherms for (hypProGly)15 and (Pro-
ProGly)15 and by calculating twice the integral of the first half of
the DSC endotherm for (mopProGly)15 (under the reasonable
assumption based on the endotherms for the other two CRPs that
the endotherm was symmetric; see simulated data in Figure 6). The
value of ∆S at the Tm was calculated as Tm ) ∆H/(∆S +
R·ln(0.75c2)), where c is the concentration of monomeric peptide
determined from amino acid analysis and Tm is the maximum of
the DSC endotherm.35 Because our model assumes ∆CP ) 0 for
triple-helix unfolding (an approximation commonly employed for
triple helices formed from collagen-related peptides36,17), ∆H and
∆S are independent of temperature. Values of ∆G at T ) 46 °C
were calculated as ∆G ) ∆H - T∆S, where T ) 46 °C (Table 2),
which is the average of the Tm values for the three peptides
determined by DSC. The thermodynamic parameters are most
accurate at temperatures near the Tm.
3.17. Measurement of Ktrans/cis Values for Ac-hyp-OMe and
Ac-mop-OMe. Ac-hyp-OMe and Ac-mop-OMe (5-10 mg) were
1
dissolved in D2O or CDCl3 (∼0.8 mL). H NMR spectra were
acquired and worked up using NUTS software.37 Values of Ktrans/
cis were determined from the relative areas of the trans and cis peaks.
NOE difference experiments were performed to confirm the peak
assignments.
3.18. Computational Methodology. The conformational prefer-
ences of Ac-hyp-OMe were examined by hybrid DFT as imple-
mented in Gaussian 03.38 Geometry optimizations and frequency
calculations at the B3LYP/6-311+G(2d,p) level of theory were
performed to obtain intramolecularly hydrogen-bonded conformers
of Ac-hyp-OMe in both the trans and cis geometries, as well as on
cis and trans Cγ-endo Ac-hyp-OMe conformers lacking the in-
tramolecular hydrogen bond (Figure 2). Frequency calculations on
the optimized structures yielded no imaginary frequencies, indicat-
ing true stationary points on the potential energy surface. The
resulting SCF energies were corrected by the zero-point vibrational
energy (ZPVE) determined in the frequency calculations (see Table
S2 in the Supporting Information). CPCM calculations were
performed at the B3LYP/6-311+G(2d,p) level of theory using
Gaussian 0338 with OFac )0.8 and RMin )0.5 and dielectric
constants of ε ) 4.9 for chloroform and ε ) 80 for water. The
error on total polarization charges was <0.05 for all cases. Optimized
geometries were analyzed by NBO 5.0 at the B3LYP/6-311+G(2d,p)
level of theory to estimate the energetic stabilization attributable
to the intramolecular hydrogen bond and the nfπ* interaction (see
Table S2 in the Supporting Information).21
Acknowledgment. We are grateful to D. R. McCaslin for
contributive discussions and to C. D. Brown for technical contribu-
tions during the early stages of this work. M.D.S. was supported
by graduate fellowships from the U.S. Department of Homeland
Security and the ACS Division of Medicinal Chemistry. F.W.K.
was supported by NIH Postdoctoral Fellowship AR50881. The BIF
was established with Grants BIR-9512577 (NSF) and S10 RR13790
(NIH). NMRFAM is supported by Grant P41RR02301 (NIH). This
work was supported by Grant R01 AR044276 (NIH).
Note Added after ASAP Publication. The nomenclature for
Hyp was corrected in the Abstract on July 20, 2010.
Supporting Information Available: Detailed procedures and
data for AUC experiments, computational analyses, and X-ray
diffraction analyses of Ac-hyp-OMe and Boc-mop-OH (8), IR
spectrum of Ac-mop-OMe, 1H and 13C NMR spectra for novel
compounds, and complete ref 38. This material is available free
of charge via the Internet at http://pubs.acs.org.
(35) Engel, J.; Ba¨chinger, H. P. Top. Curr. Chem. 2005, 247, 7–33.
(36) (a) Engel, J.; Chen, H.-T.; Prockop, D. J.; Klump, H. Biopolymers
1977, 16, 601–622. (b) Mizuno, K.; Hayashi, T.; Peyton, D. H.;
Ba¨chinger, H. P. J. Biol. Chem. 2004, 279, 38072–38078.
(37) NUTS-NMR Utility Transform Software; Acorn NMR: Livermore, CA.
(38) Frisch, M. J. Gaussian 03, revision C.02 ed.; Gaussian, Inc.:
Wallingford, CT, 2004.
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