Peptide bond isosteres: Ester or (E)-alkene in the backbone of the collagen triple helix

Article abstract of DOI:10.1021/ol050780m

(Chemical Equation Presented) Collagen is the most abundant protein in animals. Interstrand N-H...O=C hydrogen bonds between backbone amide groups form a ladder in the middle of the collagen triple helix. Isosteric replacement of the hydrogen-bond-donatin

Full text of DOI:10.1021/ol050780m

ORGANIC
LETTERS
2005
Vol. 7, No. 13
2619-2622
Peptide Bond Isosteres: Ester or
(E)-Alkene in the Backbone of the
Collagen Triple Helix
Cara L. Jenkins,† Melissa M. Vasbinder,‡ Scott J. Miller,‡ and
Ronald T. Raines*,†,§
Departments of Chemistry and Biochemistry, UniVersity of WisconsinsMadison,
Madison, Wisconsin 53706, and Department of Chemistry, Boston College,
Chestnut Hill, Massachusetts 02467
raines@biochem.wisc.edu
Received April 11, 2005
ABSTRACT
Collagen is the most abundant protein in animals. Interstrand N−H‚‚‚OdC hydrogen bonds between backbone amide groups form a ladder
in the middle of the collagen triple helix. Isosteric replacement of the hydrogen-bond-donating amide with an ester or (E)-alkene markedly
decreases the conformational stability of the triple helix. Thus, this recurring hydrogen bond is critical to the structural integrity of collagen.
In this context, an ester isostere confers more stability than does an (E)-alkene.
Hydrogen bonds between main-chain amides are a dominant
feature of folded proteins.1 Perhaps the most recurrent main-
chain-main-chain hydrogen bond is found in collagen, the
most abundant protein in animals.2 Collagen consists of a
triple helix of (XaaYaaGly)n strands, in which Xaa is often
(2S)-proline (Pro), Yaa is often (2S,4R)-4-hydroxyproline
(Hyp), and n is ca. 300. The GlyN-H groups and XaaCdO
groups form a ladder of hydrogen bonds that are buried in
the middle of the triple helix and inaccessible to solvent
(Figure 1).3 The contribution of this prevalent hydrogen bond
to the conformational stability of collagen is unknown.
One approach to estimating the strength of a main-chain-
main-chain hydrogen bond is to replace the amide N-H with
an ester O.4 Substituting an ester for an amide is relatively
conservative because the two functional groups are isosteric
and have similar conformational preferences.5 For example,
replacing an alanine residue with a lactic acid in an R-helical
model peptide has been shown to induce minimal structural
perturbations.6 On the other hand, there is more flexibility
around an ester linkage than around an amide,7 and the two
(3) (a) Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Science 1994,
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† Department of Chemistry, University of WisconsinsMadison.
‡ Department of Chemistry, Boston College.
§ Department of Biochemistry, University of WisconsinsMadison.
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Structures; Springer-Verlag: Berlin, 1991. (b) McDonald, I. K.; Thornton,
J. M. J. Mol. Biol. 1994, 238, 777-793. (c) Myers, J. K.; Pace, C. N.
Biophys. J. 1996, 71, 2033-2039.
(2) For recent reviews, see: (a) Berisio, R.; Vitagliano, L.; Mazzarella,
L.; Zagari, A. Protein Pept. Lett. 2002, 9, 107-116. (b) Jenkins, C. L.;
Raines, R. T. Nat. Prod. Rep. 2002, 19, 49-59.
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5943.
10.1021/ol050780m CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/03/2005

triple helix containing a native backbone. The results provide
new information on the contribution of the prevalent inter-
strand hydrogen bonds to triple helix stability and differenti-
ate between an ester and (E)-alkene as an amide bond isostere
in this context.
In previous work, we determined that the H/D fractionation
factors of the GlyN-H‚‚‚OdCXaa hydrogen bonds in
(ProProGly)10 and (ProHypGly)10 triple helices were not
distinguishable, suggesting that the GlyN-H‚‚‚OdCXaa
hydrogen bonds have a similar strength in these two
contexts.12 Accordingly, we limit our analysis herein to
isosteres of (ProProGly)10. Also in previous work, we
reported a five-step synthesis for FmocProFlpGly (where Flp
refers to (2S,4R)-4-fluoroproline) based on standard Boc
chemistry with an average overall yield of 17%.13 We have
now made this route more efficient by eliminating the need
to switch from a Boc to Fmoc protecting group. Following
the route in Scheme 1 and monitoring the hydrogenolysis
Scheme 1
Figure 1. Interstrand hydrogen bond in the middle of a collagen
triple helix, and ester and (E)-alkene isosteres of the hydrogen bond
donor.
functional groups have distinct electronic properties.8 Ac-
cordingly, hydrogen bond strengths obtained by ester sub-
stitutions are estimates that, nonetheless, have been infor-
mative.4
An alternative approach is to replace the amide NH with
an alkene CH.9 This approach is beyond the realm of
biosynthesis.10 Although alkene isosteres have been incor-
porated into peptides by chemical synthesis,11 we are not
aware of a comparison between an ester and alkene as a
surrogate for an amide that forms a solvent-inaccessible
main-chain-main-chain hydrogen bond.
Here, we synthesize collagen strands in which a single
amide bond is replaced with an ester (depsipeptide 1) or (E)-
alkene (alkenyl peptide 2). We compare the conformational
stability of the resulting triple helices with that of a collagen
(10) Nilsson, B. L.; Soellner, M. B.; Raines, R. T. Annu. ReV. Biophys.
Biomolec. Struct. 2005, 34, 91-118.
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Soc. 1995, 117, 3280-3281. (c) Masse, C. E.; Knight, B. S.; Stavropoulos,
P.; Panek, J. S. J. Am. Chem. Soc. 1997, 119, 6040-6047. (d) Wipf, P.;
Henninger, T. C.; Geib, S. J. J. Org. Chem. 1998, 63, 6088-6089. (e)
Gardner, R. R.; Liang, G.-B.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121,
1806-1816. (f) Oishi, S.; Kamano, T.; Niida, A.; Odagaki, Y.; Hamanaka,
N.; Yamamoto, M.; Ajito, K.; Tamamura, H.; Otaka, A.; Fujii, N. J. Org.
Chem. 2002, 67, 6162-6173. (g) Tamamura, H.; Hiramatsu, K.; Miyamoto,
K.; Omagari, A.; Oishi, S.; Nakashima, H.; Yamamoto, N.; Kuroda, Y.;
Nakagawa, T.; Otaka, A.; Fujii, N. Bioorg. Med. Chem. Lett. 2002, 12,
923-928. (h) Vasbinder, M. M.; Miller, S. J. J. Org. Chem. 2002, 67, 6240-
6242. (i) Tamamura, H.; Koh, Y.; Ueda, S.; Sasaki, Y.; Yamasaki, T.; Aoiki,
M.; Maeda, K.; Watai, Y.; Arikuni, H.; Otaka, A.; Mitsuya, H.; Fujii, N. J.
Med. Chem. 2003, 46, 1764-1768. (j) Wang, X. J.; Hart, S. A.; Xu, B.;
Mason, M. D.; Goodell, J. R.; Etzkorn, F. A. J. Org. Chem. 2003, 68, 2343-
2349. (k) Wipf, P.; Xiao, J. Org. Lett. 2005, 7, 103-106.
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(7) Mammi, S.; Goodman, M. Int. J. Peptide Protein Res. 1986, 28, 29-
44.
(8) For example, an n f σ* interaction between the “ether” O and
“carbonyl” CdO stabilizes the Z conformation of esters. Pawar, D. M.;
Khalil, A. A.; Hooks, D. R.; Collins, K.; Elliott, T.; Stafford, J.; Smith, L.;
Noe, E. A. J. Am. Chem. Soc. 1998, 120, 2108-2112.
(9) (a) Hann, M. M.; Sammes, P. G.; Kennewell, P. D.; Taylor, J. B.
Chem. Commun. 1980, 234-235. (b) Wipf, P.; Fritch, P. C. J. Org. Chem.
1994, 59, 4875-4886.
(12) Danielson, M. A.; Raines, R. T. In Peptides for the New Millen-
nium: Proceedings of the Sixteenth American Peptide Symposium; Fields,
G. B., Tam, J. P., Barany, G., Eds.; Kluwer Academic: Dordrecht, The
Netherlands, 2000; pp 347-348.
(13) (a) Holmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T.
Nature 1998, 392, 666-667. (b) Holmgren, S. K.; Bretscher, L. E.; Taylor,
K. M.; Raines, R. T. Chem. Biol. 1999, 6, 63-70.
2620
Org. Lett., Vol. 7, No. 13, 2005

reaction with care, we achieved an overall yield of 54% over
three steps for the synthesis of FmocProProGly from BocPro.
One particular advantage of the route in Scheme 1 is the
facile purification of the product in the final step.
To minimize diketopiperazine formation, we synthesized
depsipeptide 1 by condensing two dipeptide segments,
FmocProOGly (6, Scheme 2) and FmocGlyPro, to install the
Scheme 2
Figure 2. Effect of trimethylamine N-oxide concentration on the
value of Tm for triple helical (ProProGly)10, depsipeptide 1, and
alkenyl peptide 2 in PBS. Lines were obtained by linear least-
squares analysis of the data in PBS containing TMAO (0.5-4.0
M).
residues near the labile ester linkage. This strategy (Scheme
3) avoided the presence of an unprotected (and thus nucleo-
characteristic of triple helix content, with increasing tem-
perature. In contrast, (ProProGly)10 had a Tm value (which
is the temperature at the midpoint of the thermal transition
between native and unfolded states) of 41 °C in 50 mM
AcOH.13 The failure of depsipeptide 1 to assemble into triple
helices indicates that each interstrand hydrogen bond con-
tributes substantially to triple helix stability.
Scheme 3
The natural osmolyte trimethylamine N-oxide (TMAO)
enhances the conformational stability of collagen14 and other
proteins,15 presumably by enhancing water structure and
thereby discouraging backbone-water interactions.16 Incu-
bating depsipeptide 1 with various concentrations of TMAO
in phosphate-buffered saline (PBS) at 4 °C for 24 h led to
an increase in ellipticity at 225 nm with increasing TMAO
concentration. Triple helices of depsipeptide 1 began to show
a thermal transition in solutions containing 2 M TMAO or
higher. MALDI-TOF mass spectrometric analysis showed
negligible decomposition of depsipeptide 1 after thermal
denaturation experiments.
The Tm value of triple helical (ProProGly)10 and depsipep-
tide 1 showed a similar linear dependence on TMAO
concentration (Figure 2). For triple helical (ProProGly)10, the
extrapolated Tm value at 0 M TMAO is 32.8 °C, which is in
gratifying agreement to the Tm value of 33 °C measured in
PBS. For triple helical depsipeptide 1, the extrapolated Tm
value is 10.7 °C. With a ∆Sm value of 0.21 kcal/mol,13b this
∆Tm ) 22.1 °C corresponds to ∆∆Gm ) ∆Tm∆Sm ) 4.2
philic) amino group two residues from the labile ester group
during the synthesis. Cleavage from the resin and purification
by HPLC yielded depsipeptide 1.
A solution of depsipeptide 1 (0.2 mM) in 50 mM AcOH
was incubated at 4 °C for 24 h and then analyzed by circular
dichroism (CD) spectroscopy. Wavelength scans from 200
to 260 nm indicated that the peptide had not assembled into
a triple helix. The absence of the triple helix was confirmed
by the linear decrease in ellipticity at 225 nm, which is
(14) Beck, K.; Chan, V. C.; Shenoy, N.; Kirkpatrick, A.; Ramshaw, J.
A. M.; Brodsky, B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4273-4278.
(15) Baskakov, I.; Bolen, D. W. J. Biol. Chem. 1998, 273, 4831-4834.
(16) Zou, Q.; Bennion, B. J.; Daggett, V.; Murphy, K. P. J. Am. Chem.
Soc. 2002, 124, 1192-1202.
Org. Lett., Vol. 7, No. 13, 2005
2621

kcal/mol.17 This value does not take into account the
solvation of amides being greater than that of esters.
Differences in solvation between amides and esters can
be estimated by the ∆∆G° of octanol f water partitioning
of analogous small-molecule amides and esters. For example,
∆∆G°octanolfwater ) -1.7 kcal/mol for methyl acetate versus
N-methylacetamide.18 Then according to the analysis of
Schultz and co-workers,4b each GlyN-H‚‚‚OdCXaa hydro-
isostere 7 results in a triple helix that is much less stable
than one in which the ProGly segment is replaced with an
isosteric ester.
The conformational stability conferred by the two peptide
bond isosteres examined herein decreases in the order: ester
> (E)-alkene (Figure 2). This order could arise from the (E)-
alkene isostere not adopting φ or ψ torsion angles amenable
to triple helix formation20 or having a low dipole moment.21
A disruption in the solvation of the triple helix22 by the
alkenyl group could also play a role. It is noteworthy that
formation of a C-H‚‚‚OdC hydrogen bond23 in a triple helix
of alkenyl peptide 2 (Figure 1) is apparently not able to
compensate for the loss of an N-H‚‚‚OdC hydrogen bond
and other deleterious effects.
Our analysis of collagen triple helices containing backbone
isosteres has demonstrated that the prevalent interstrand
hydrogen bonds make a substantial contribution to the
conformational stability of the collagen triple helix. From
our data with an ester isostere, we estimate the strength of
each hydrogen bond to be 2.0 kcal/mol. Interestingly, an (E)-
alkene isostere confers much less stability than does an ester
isostere and is thus a less well-suited surrogate for a peptide
bond, at least in the context of the collagen triple helix.
gen bond contributes approximately ∆∆G°GlyN-H‚‚‚OdCXaa
)
(∆∆G°octanolfwater - ∆∆Gm)/3 ) -2.0 kcal/mol to the
conformational stability of the collagen triple helix. This
value is typical for a solvent-inaccessible main-chain-main-
chain hydrogen bond.1,4
The central ProGly unit in (ProProGly)10 was also replaced
with alkene 7, which was synthesized by olefin cross-
metathesis as described previously,11h to yield alkenyl peptide
2. As with depsipeptide 1, incubation of alkenyl peptide 2
for 24 h in 50 mM AcOH yielded no evidence for triple
helix formation, as probed by CD spectroscopy. The CD
spectrum from 200 to 260 nm exhibited only a small
maximum near 225 nm, similar to the signature spectra of a
polyproline type II helix.19 The molar ellipticity at 227 nm
decreased linearly with temperature, consistent with the
absence of a triple helix.
Acknowledgment. We are grateful to S. H. Gellman and
F. W. Kotch for contributive discussions. This work was
supported by grants AR44276 and GM44783 (NIH to R.T.R.)
and CHE-0236591 (NSF to S.J.M.) and instrumentation
grants S10 RR04981 (NIH) and CHE-9629688 (NSF).
Supporting Information Available: Procedures for the
preparation of compounds 1 and 2, and related analytical
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
Repeating the thermal denaturation experiments on alkenyl
peptide 2 in the presence of TMAO revealed that a transition
could be observed at g3.5 M TMAO. The Tm value of triple
helical 2 in 3.5 M TMAO was 14.0 °C and that in 4.0 M
TMAO was 19.5 °C. Extrapolation of these data gives a Tm
value of -24.7 °C at 0 M TMAO. Thus, in the context of a
collagen triple helix, replacing a ProGly segment with alkene
OL050780M
(20) In a crystalline (ProProGly)10 triple helix, the average φ torsion angle
of Gly is -72° and the average ψ torsion angle of Pro in the Yaa position
is 152°. Berisio, R.; Vitagliano, L.; Mazzarella, L.; Zagari, A. Protein Sci.
2002, 11, 262-270.
(21) Relevant dipole moments have been measured to be the following:
N-methyl acetamide, µ ) 3.5-3.6 D (CCl4, 25.5 °C); methyl acetate, µ )
1.7 D (CCl4, 25 °C), trans-2-butene, µ ) 0 D (gas, 25-95 °C) McClellan,
A. L. Tables of Experimental Dipole Moments; W. H. Freeman: San
Francisco, 1963.
(17) Pace, C. N.; Scholtz, J. M. In Measuring the Conformational Stability
of a Protein; Creighton, T. E., Ed.; Oxford University: New York, 1997;
pp 299-321.
(18) Leo, A.; Hansch, C.; Elkins, D. Chem. ReV. 1971, 71, 525-555.
(19) Feng, Y.; Melacini, G.; Taulane, J. P.; Goodman, M. Biopolymers
1996, 36, 8716-8724.
(22) Bella, J.; Brodsky, B.; Berman, H. M. Structure 1995, 3, 893-906.
(23) Desiraju, G. R.; Steiner, T. The Weak Hydrogen Bond in Structural
Chemistry and Biology; Oxford University Press: Oxford, 1999.
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