The impact of pyrrolidine hydroxylation on the conformation of proline-containing peptides

Article abstract of DOI:10.1021/jo0490043

(Chemical Equation Presented) A series of eight dipeptides of the general formula Ac-Phe-Pro*-NHMe was synthesized and the thermodynamics of the cis → trans isomerization about the central amide bond were studied by NMR. Pro* represents the following prol

Full text of DOI:10.1021/jo0490043

The Impact of Pyrrolidine Hydroxylation on the Conformation of
Proline-Containing Peptides
Carol M. Taylor,* Renaud Hardre´, and Patrick J. B. Edwards
Institute of Fundamental Sciences, Massey University, Private Bag 11-222,
Palmerston North, New Zealand
C.M.Taylor@massey.ac.nz
Received June 12, 2004
A series of eight dipeptides of the general formula Ac-Phe-Pro*-NHMe was synthesized and the
thermodynamics of the cis f trans isomerization about the central amide bond were studied by
NMR. Pro* represents the following prolines: L-proline (Pro), L-trans-4-hydroxyproline (Hyp), L-cis-
4-hydroxyproline (hyp), L-cis-4-methoxyproline (hyp[OMe]), L-trans-3-hydroxyproline (3-Hyp), L-cis-
3-hydroxyproline (3-hyp), L-2,3-trans-3,4-cis-3,4-dihydroxyproline (DHP), and L-2,3-cis-3,4-trans-
3,4-dihydroxyproline (dhp). The conformation of the pyrrolidine ring in each case is discussed in
light of previous structural studies, analysis of potential stereoelectronic effects, and NMR data.
Hydroxy substituents at C-4 have a greater impact on cis f trans isomerization than analogous
substituents at C-3 as a result of the intervening bond distances and bridging groups. The position
of the equilibrium and its dependence on temperature are a reflection of both enthalpic and entropic
factors, the latter being complicated in this study by an Ar-Pro interaction in the cis conformation.
The substituents on the pyrrolidine ring determine the conformation of the five-membered ring,
which in turn influences the strength of the Ar-Pro interaction, backbone dihedral angles, and the
relative energy of the cis and trans species. The ultimate position of the equilibrium depends on a
complex blend of steric, electronic, and conformational factors.
1. Background and Introduction
embedded in a peptide or protein, the hydroxy (OH)
substituents on the five-membered pyrrolidine ring pro-
vide subtle electronic control, fine-tuning the bond angles
and orientation of the peptide backbone. Much has been
It has long been recognized that proline plays a unique
and important role in the conformation of peptides and
proteins.1 Early NMR studies contributed to our under-
standing the cis-trans isomerism of prolyl amide bonds.2
Proline residues have been used to good effect in the
design of peptides and peptidomimetics with defined
conformation.3
made of the importance of this role in collagen,5
a
structural protein of immense importance that consists
of repeating Gly-X-Y triads where X is often Pro (1) and
Y is often trans-4-hydroxyproline (2). The importance of
hydroxyproline in stabilizing the triple helical structure
of collagen was recognized many years ago,6 but the
mechanism by which stabilization is achieved is still
evolving and has been the subject of much discussion
recently.5
It was long-believed that stabilization arose from
hydrogen bonds mediated by a network of bridging water
molecules, as supported by X-ray crystallographic evi-
dence.7 More recent structural studies have led to dif-
ferent conclusions.8 In 1998, Raines proposed that sta-
bilization is achieved principally via the “inductive
effect”.9 4-Fluoroprolines (4-6)10 have been widely used
Nature has produced a plethora of modified prolines,4
including the hydroxyprolines. When the latter are
* Corresponding author. Phone: 64-6-356-9099, ext 3571. Fax: 64-
6-350-5682.
(1) (a) Vanhoof, G.; Goossens, F.; De Meester, I.; Hendriks, D.;
Scharpe´, S. FASEB 1995, 9, 736-744. (b) Reiersen, H.; Rees, A. R.
TIBS 2001, 26, 679-684.
(2) For some early examples of NMR analysis of X-Pro peptide bonds,
see: (a) Grathwohl, C.; Wu¨therich, K. Biopolymers 1976, 15, 2025-
2041. (b) Cheng, H. N.; Bovey, F. A. Biopolymers 1977, 16, 1465-1472.
(c) Bovey, F. A.; Brewster, A. I.; Patel, D. J.; Tonelli, A. E.; Torchia, D.
A. Acc. Chem. Res. 1972, 5, 193-200. (d) Brewster, A. I. R.; Hruby, V.
J.; Glasel, J. A.; Tonelli, A. E. Biochemistry 1973, 12, 5294-2304.
(3) For some examples see: (a) Tuchscherer, G.; Mutter, M. Chimia
2001, 55, 306-313. (b) Hinds, M. G.; Welsh, J. H.; Brennand, D. M.;
Fisher, J.; Glennie, M. J.; Richards, N. G. J.; Turner, D. L.; Robinson,
J. A. J. Med. Chem. 1991, 34, 1777-1789. (c) Halab, L.; Lubell, W. D.
J. Org. Chem. 1999, 64, 3312-3321. (d) Fisk, J. D.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 2000, 122, 5443-5447.
(4) Mauger, A. B. J. Nat. Prod. 1996, 59, 1205-1211.
(5) (a) Jenkins, C. L.; Raines, R. T. Nat. Prod. Rep. 2002, 19, 49-
59. (b) Berisio, R.; Vitagliano, L.; Mazzarella, L.; Zagari, A. Protein
Pept. Lett. 2002, 9, 107-116.
10.1021/jo0490043 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/22/2005
1306
J. Org. Chem. 2005, 70, 1306-1315

Prolyl Amides: Pyrrolidine Hydroxylation
to probe the effect of electron-withdrawing groups, since
fluorine is more electronegative than oxygen, but less
capable of hydrogen bonding.11 Raines and co-workers
found that (Gly-Pro-Flp)10 formed a triple helix that was
more stable than (Gly-Pro-Hyp)10, which in turn was
more stable than (Gly-Pro-Pro)10.9 This implied that the
electron-withdrawing nature of the substituent is the
driving force toward stability. trans-4-Aminoproline (Amp,
7) was recently incorporated into a collagenous sequence
and shown to afford more stable triple helices than the
corresponding peptides containing Hyp (2).12 Babu and
Ganesh investigated the amino group because it is both
electronegative and has the potential to form hydrogen
bonds. However, as they discovered, it has the added
complication of being ionizable.
FIGURE 1. Proline and 4-substituted derivatives.
It was observed as early as 1976 that collagen mimetics
incorporating cis-4-hydroxyproline (hyp, 3) did not form
a triple helix.13 Thus, the configuration of the stereogenic
center bearing the hydroxy substituent is important. This
issue has been probed with use of N-acetyl amino acid
methyl esters (Ac-Pro*-OMe, where Pro* is a substituted
proline).14 Bretscher et al. prepared derivatives incorpo-
rating prolines 1 to 514 and Renner et al. prepared
derivatives incorporating prolines 4 to 6.11d Both groups
concluded that electron-withdrawing 4R substituents
stabilize the trans conformation of the preceding amide
bond. [The terms “cis”and “trans” are used to describe
the relative stereochemistry of two substituents on a
pyrrolidine ring. The geometry of the peptide bond is also
described as being “cis” or “trans”. Care should be taken
not to confuse these two applications of the same descrip-
tors.] However, the same substituents in the 4S orienta-
tion shift the equilibrium in the opposite direction.
Interestingly, the compound bearing two fluorines at C4
(incorporating 6) favored the cis conformation slightly
FIGURE 2. Dipeptides synthesized.
more than Pro itself.11d As we have recently shown,
caution needs to be exercised in utilizing esters of single
amino acid residues to draw conclusions about peptide
conformation.15
(6) (a) Gustavson, K. H. Nature 1955, 175, 70-74. (b) Berg, R. A.;
Prockop, D. J. Biochem. Biophys. Res. Commun. 1973, 52, 115-120.
(c) Ramachandran, G. N.; Bansal, M.; Bhatnagar, R. S. Biochim.
Biophys. Acta 1973, 322, 166-171. (d) Sakakibara, S.; Inouye, K.;
Shudo, K.; Kishida, Y.; Kobayashi, Y.; Prockop, D. J. Biochim. Biophys.
Acta 1973, 303, 198-202. (e) Prockop, D. J.; Berg, R. A.; Kivirikko, I.;
Uitto, J. In Biochemistry of Collagen; Ramachandran, G. N., Reddi,
A. H., Eds.; Plenum: New York, 1976; pp 163-273. (f) Chopra, R. K.;
Ananthanarayanan, V. S. Proc. Natl. Acad. Sci., U.S.A. 1982, 79,
7180-7184. (g) Brodsky, B.; Ramshaw, J. A. M. Matrix Biol. 1997,
15, 545-554. (h) Rao, N. V.; Adams, E. Biochem. Biophys. Res.
Commun. 1979, 86, 654-660.
Our interest in hydroxylated prolines, and their role
in nature, began with our bid to synthesize the repeating
decapeptide unit16 of the mussel adhesive protein, Mefp1,17
which contains three proline residues in three different
oxidation states. As a prelude to studying the conforma-
tion of larger peptides containing hydroxylated prolines,
we decided to look first at dipeptides, of the general
formula Ac-L-Phe-Pro*-NHMe, to consider the empirical
effect of pyrrolidine hydroxylation on peptide conforma-
tion. This follows from our foundation analysis of the
factors contributing to the conformation of X-Pro dipep-
tides.15
(7) Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Science 1994,
266, 75-81.
(8) Berisio, R.; Vitagliano, L.; Mazzarella, L.; Zagari, A. Protein Sci.
2002, 11, 262-270.
(9) Holmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T.
Nature 1998, 392, 666-667.
(10) cis-4-Fluoroproline was first incorporated into collagens when
incubated with matrix-free chick embryo tendon cells: Uitto, J.;
Prockop, D. J. Arch. Biochem. Biophys. 1977, 181, 293-299.
(11) (a) Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D.
R.; Raines, R. T. Int. J. Peptide Protein Res. 1994, 44, 262-269. (b)
Eberhardt, E. S.; Panasik, N., Jr.; Raines, R. T. J. Am. Chem. Soc.
1996, 118, 12261-12266. (c) Holmgren, S. K.; Bretscher, L. E.; Taylor,
K. M.; Raines, R. T. Chem. Biol. 1999, 6, 63-70. (d) Renner, C.;
Alefelder, S.; Bae, J. H.; Budisa, N.; Huber, R.; Moroder, L. Angew
Chem., Int. Ed. Engl. 2001, 40, 923-925. (e) De Rider, M. L.; Wilkens,
S. J.; Waddell, M. J.; Bretscher, L. E.; Weinhold, F.; Raines, R. T.;
Markley, J. L. J. Am. Chem. Soc. 2002, 124, 2497-2505. (f) Hodges,
J. A.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 9262-9263.
(12) (a) Babu, I. R.; Ganesh, K. N. J. Am. Chem. Soc. 2001, 123,
2079-2080. (b) Umashankara, M.; Babu, I. R.; Ganesh, K. N. J. Chem.
Soc., Chem. Commun. 2003, 2606-2607.
2. Results and Discussion
Eight dipeptides of the general formula Ac-Phe-Pro*-
NHMe were synthesized (Figure 2). Compound 8, studied
previously by ourselves15 and others,18 can be regarded
(15) Taylor, C. M.; Hardre´, R.; Edwards, P. J. B.; Park, J. H. Org.
Lett. 2003, 5, 4413-4416.
(16) Taylor, C. M.; Weir, C. A. J. Org. Chem. 2000, 65, 1414-1421.
(17) Taylor, S. W.; Waite, J. H.; Ross, M. M.; Shabanowitz, J.; Hunt,
D. F. J. Am. Chem. Soc. 1994, 116, 10803-10804.
(18) (a) Kang, Y. K.; Jhon, J. S.; Han, S. J. J. Peptide Res. 1999, 53,
30-40. (b) Halab, L.; Lubell, W. D. J. Am. Chem. Soc. 2002, 124, 2474-
2484.
(13) Inouye, K.; Sakakibara, S.; Prockop, D. J. Biochim. Biophys.
Acta 1976, 420, 133-141.
(14) Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.;
Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777-778.
J. Org. Chem, Vol. 70, No. 4, 2005 1307

Taylor et al.
FIGURE 3. Van’t Hoff plots for Ac-Phe-Pro-NHMe (8), Ac-
Phe-Hyp-NHMe (9), Ac-Phe-hyp-NHMe (10) and Ac-Phe-hyp-
(OMe)-NHMe (11).
FIGURE 4. Pyrrolidine conformation in dipeptide 9.
as the parent compound of the series. Compounds 9 and
10 both have a hydroxy group in the 4-position of the
proline residue and as such incorporate the much dis-
cussed trans-4-hydroxyproline residue (Hyp, 2) and its
diastereoisomer, cis-4-hydroxyproline (hyp, 3). We de-
cided to consider the effect of hydroxylation at the
3-position (dipeptides 12 and 13), since 3-hydroxyprolines
have also been isolated from natural sources,4 including
collagens.19 We prepared dipeptides 14 and 15 incorpo-
rating L-2,3-trans-3,4-cis-3,4-dihydroxyproline (DHP)17
and L-2,3-cis-3,4-trans-3,4-dihydroxyproline (dhp),20 re-
spectively, which were available in our laboratory via
synthesis.21 The dipeptides were synthesized by standard
techniques, although the approach varied depending on
the availability of each proline derivative and its reactiv-
ity. The synthesis and manipulation of the various
proline derivatives are included as Supporting Informa-
tion. Dipeptides were purified by RP-HPLC.
With the introduction of a trans-4-hydroxy substituent,
there is a significant stabilization of the trans peptide
bond: Kt/c more than doubles, from Kt/c ) 2.1 in Ac-Phe-
Pro-NHMe (8) to Kt/c ) 5.0 in Ac-Phe-Hyp-NHMe (9) in
D2O at 298 K. Conversely, the introduction of a cis-4-
hydroxy substituent leads to little change in Kt/c values
relative to 8, but there appears to be a slight negative
slope of the Van’t Hoff plot for Ac-Phe-hyp-NHMe (10).
It has long been recognized that the trans-4-hydroxy
substituent of Hyp is responsible for stabilizing the
collagen triple helix23 and that its diastereomer does not
have this capacity. The basis for this is slowly emerging
from a number of important new developments.5 Raines’
early work demonstrated that electron-withdrawing groups
in the 4-position inductively withdraw electron density
from the peptide bond, increasing N-pyramidalization,
reducing the bond order of the C-N linkage, and thereby
facilitating the interconversion of the cis and trans
species to favor that which is lower in energy.11a,b In the
case of trans-4-hydroxyproline, the OH group leads to a
strong preference for a Cγ-exo (“up”) conformation of the
pyrrolidine ring (Figure 4),24 and it has been suggested
recently that this conformation is desirable in the Y-
position of Gly-X-Y triads.25 This has been attributed by
Bretscher et al. to the gauche effect26sin the Cγ-exo
conformation, a gauche orientation of the 4-OH group and
the pyrrolidine nitrogen, relative to the Cγ-Cδ bond axis,
is possible (Figure 4). Moreover, recent theoretical studies
suggest that there is a related hyperconjugative inter-
action27 that is the driving force for adopting the Cγ-exo
conformation: donation from the σ(Câ-H)ax and σ(Cδ-H)ax
bonding orbitals into the σ*(Cγ-O) antibonding
While there are three amide bonds in each molecule,
only the central peptide bond (between the Phe and Pro
residues) was expected to exist as discrete cis-trans
conformations on the NMR time scale. As described
elsewhere,15 1H NMR spectra were assigned on the basis
of various 2D experiments, and the equilibrium constants
for the cis f trans interconversion in D2O were deter-
mined over a range of temperatures by integration of
1
well-resolved signals in the H NMR spectra. The con-
formation of the pyrrolidine ring influences the cis-trans
conformational preference of the X-Pro peptide bond and
thereby has an impact on the neighboring peptide
backbone.22 Our results are presented below, along with
potential explanations for the differences in behavior.
Let us first consider the introduction of a hydroxy
group in the 4-position of the proline residue (Figure 3).
(23) Hyp in the X-position destabilizes the triple helix: Inouye, K.;
Kobayashi, Y.; Kyogoku, Y.; Kishida, Y.; Sakakibara, S.; Prockop, D.
J. Arch. Biochem. Biophys. 1982, 219, 198-203.
(19) (a) Ogle, J. D.; Arlinghaus, R. B.; Logan, M. A. J. Biol. Chem.
1962, 237, 3667-3673. (b) Piez, K. A.; Eigner, E. A.; Lewis, M. S.
Biochemistry 1963, 2, 58-66. (c) Gryder, R. M.; Lamon, M.; Adams,
E. J. Biol. Chem. 1975, 250, 2470-2474.
(24) (a) Benzi, C.; Improta, R.; Scalmani, G.; Barone, V. J. Comput.
Chem. 2002, 23, 341-350. (b) Mooney, S. D.; Kollman, P. A.; Klein, T.
E. Biopolymers 2002, 64, 63-71.
(20) Isolated from the acid hydrolysates of the cell walls of dia-
toms: Nakajima, T.; Volcani, B. E. Science 1969, 164, 1400-1401.
(21) (a) Weir, C. A.; Taylor, C. M. J. Org. Chem. 1999, 64, 1554-
1558. (b) Weir, C. A.; Taylor, C. M. Org. Lett. 1999, 1, 787-789. (c)
Taylor, C. M.; Barker, W. D.; Weir, C. A.; Park, J. H. J. Org. Chem.
2002, 67, 4466-4474.
(25) Improta, R.; Benzi, C.; Barone, V. J. Am. Chem. Soc. 2001, 123,
12568-12577.
(26) A tendency to adopt the structure that has the maximum
number of gauche interactions between the adjacent electron pairs and/
or polar bonds (Wolfe, S. Acc. Chem. Res. 1972, 5, 2-111).
(27) (a) Weinhold, F. Nature 2001, 411, 539-541. (b) Pophristic, V.;
Goodman, L. Nature 2001, 411, 565-568. (c) Alabugin, I. V.; Zeidan,
T. A. J. Am. Chem. Soc. 2002, 124, 3175-3185.
(22) Chakrabarti, P.; Pal, D. Prog. Biophys. Mol. Biol. 2001, 76,
1-102.
1308 J. Org. Chem., Vol. 70, No. 4, 2005

Prolyl Amides: Pyrrolidine Hydroxylation
in this conformation afford almost the same stabilizing
interactions as the Cγ-endo conformation (Figure 5).
Interestingly, the Van’t Hoff plot for dipeptide 10
demonstrates a slight negative gradient. We speculated
that this might be attributable to a hydrogen bond that
can form on the lower face of the pyrrolidine ring,
between the hydroxy group and the proline amide car-
bonyl (OH‚‚‚OdC). This hydrogen bond has been invoked
by Bretscher et al. and has been estimated to contribute
1.5 kcal mol-1 of stabilization to the endo conformation
in Ac-hyp-OMe,14 although Barone and co-workers cau-
tion that such intramolecular hydrogen bonds are in
competition for intermolecular bonds in polar, protic
solvents.25 To test for the importance of such an intramo-
lecular hydrogen bond in dipeptide 10, we synthesized
dipeptide 15 in which the hydroxyl group is capped as a
methyl ether. We need to bear in mind that we are also
increasing the size of the substituent, reducing slightly
the inductive electron withdrawing nature,33 and altering
the energy of the σ*(C-O) orbital. Comparison of the
Van’t Hoff plots for compounds 10 and 11 (Figure 3)
indicates lower Kt/c values for compound 11. Such a shift
in favor of the cis amide bond might be construed as a
stronger preference for the Cγ-endo conformation in
compound 11. This downplays the role of the hydrogen
bond and the superficial inductive effect in stabilizing
the Cγ-endo conformation. A recent paper by Alabugin
and Zeidan suggests that a σ(C-H)fσ*(C-OCH3) hy-
perconjugative interaction is stronger than the corre-
sponding σ(C-H)fσ*(C-OH) interaction.27c They also
revealed an inverse correlation in the hyperconjugative
acceptor ability of C-X bonds and the electronegativity
of X. It would therefore be consistent to propose that
there is a stronger σ(C-H)fσ*(C-OCH3) interaction in
compound 11 (relative to the hyperconjugative interac-
tion in dipeptide 10), which leads to a stronger preference
for the Cγ-endo conformation and inter alia the cis amide
bond.
We next sought to explore the regiochemistry of
the pyrrolidine hydroxylation. trans-3-Hydroxyproline
(3-Hyp) has been indentified in the X-position in collag-
enous sequences.19 Recent studies by Jenkins et al.
involved the substitution of one modified triad (Gly-trans-
3-Hyp-trans-4-Hyp or Gly-Pro-trans-3-Hyp) at the center
of 21-mer collagen mimics.34 Both oligopeptides formed
less stable helices than the parent compound containing
only Gly-Pro-trans-4-Hyp triads, although the effect was
less with trans-3-Hyp in the X-position.
Jenkins et al. have reported X-ray data for N-(13C2-
acetyl)-3(S)-hydroxyproline methyl ester, which showed
the conformation of the pyrrolidine ring is such that the
CR, Cγ, and N atoms lie within a plane and Cδ deviates
only a little.34 The carbon bearing the substituent, Câ, is
the “flap” atom of the five membered ringsit points in
the opposite direction to the CONHMe group at CR. In
simplified terms, we might refer to this conformation as
Câ-exo. Dipeptide 12, incorporating trans-3-hydroxypro-
FIGURE 5. Pyrrolidine conformation in dipeptides 10 and
11.
orbital.25,27c Evidence for this stereoelectronic effect is
found in the short Câ-Cγ and Cγ-Cδ bonds (1.512 and
1.510 Å, respectively) and the relatively long C-O bond
(1.425 Å) in Ac-Hyp-OMe.11a Conversely, a Cγ-endo
conformation does not allow for such stabilizing effects
(Figure 4). A strong preference for the Cγ-exo conforma-
tion aligns the neighboring peptide backbone28 for an
effective n f π* electrostatic interaction between the
carbonyl groups of the i - 1 and i residues when the
peptide bond adopts a trans conformation.14 It has been
demonstrated in a recent report by Hinderaker and
Raines that this stereoelectronic effect is more important
than steric effects which have traditionally been held
responsible for the energetic preference for trans peptide
bonds.29
On the other hand, it has been strongly suggested that
introduction of a cis-4-hydroxy substituent leads to a
preference for the Cγ-endo conformation of the pyrroli-
dine ring (Figure 5).25 Indeed, Beausoleil et al. have
reported structural evidence for a Cγ-endo conformation
for the pyrrolidine ring of (2S,4S)-N-acetyl-3,3-dimethyl-
4-hydroxy-L-proline N-methylamide.30 The endo confor-
mation for hyp provides an appealing opposite to the
trans-4-hydroxy diastereoisomer in terms of stereoelec-
tronic interactions. As illustrated in Figure 5, a gauche
effect is achieved in the Cγ-endo conformation, along with
stabilizing hyperconjugative interactions. However, ac-
cording to X-ray crystallographic data for “allo-4-hydroxy-
L-proline dihydrate”, it is the Câ atom that deviates from
the plane of the other four atoms of the pyrrolidine ring
in the free amino acid, giving rise to a Câ-exo conforma-
tion.31 It was shown early on that pyrrolidine conforma-
3
tions determined by analysis of JHH coupling constants
from NMR data agree remarkably well with those
determined from crystal structures.32 The dihedral angles
(28) (a) Vitagliano, L.; Berisio, R.; Mastrangelo, A.; Mazzarella, L.;
Zagari, A. Protein Sci. 2001, 10, 2627-2632. (b) Vitagliano, L.; Berisio,
R.; Mazzarella, L.; Zagari, A. Biopolymers 2001, 58, 459-464.
(29) Hinderaker, M. P.; Raines, R. T. Protein Sci. 2003, 12, 1188-
1194.
(30) Beausoleil, E.; Sharma, R.; Michnick, S. W.; Lubell, W. D. J.
Org. Chem. 1998, 63, 6572-6578.
(31) Shamala, N.; Row, T. N. G.; Venkatesan, K. Acta Crystallogr.
B 1976, B32, 3267-3270.
(32) Haasnoot, C. A. G.; De Leeuw, F. A. A. M.; De Leeuw, H. P.
M.; Altona, C. Biopolymers 1981, 20, 1211-12425.
(33) (a) Janesko, B. J.; Gallek, C. J.; Yaron, D. J. Phys. Chem. A
2003, 107, 1655-1663. (b) Swain, C. G.; Lupton, E. C., Jr. J. Am. Chem.
Soc. 1968, 90, 4328-4337.
(34) Jenkins, C. L.; Bretscher, L. E.; Guzei, I. A.; Raines, R. T. J.
Am. Chem. Soc. 2003, 125, 6422-6427.
J. Org. Chem, Vol. 70, No. 4, 2005 1309

Taylor et al.
FIGURE 6. Pyrrolidine conformation in dipeptide 12.
FIGURE 8. Van’t Hoff plots for Ac-Phe-Pro-NHMe (8), Ac-
Phe-trans-3-Hyp-NHMe (12), and Ac-Phe-cis-3-Hyp-NHMe
(13).
structure of N-(13C2-acetyl)-3(S)-hydroxyproline methyl
ester shows that the CR-Câ bond is 1.536 Å, a typical
single bond. Consistent with the σ(Cγ-H)axfσ*(Câ-O)
hyperconjugative interaction depicted in Figure 6, the
Câ-Cγ bond length is relatively short (1.512 Å).34 While
both diastereoisomers of the dipeptides containing a
3-hydroxyproline favor the trans rotamer about the Phe-
Pro peptide bond relative to the parent compound, the
inductive effect is not as strong as in the 4-hydroxypro-
line-containing compounds. Perhaps the Câ-exo confor-
mation (more than the Câ-endo conformation) of the
pyrrolidine ring influences φ and ψ dihedral angles in
such a way as to strengthen the backbone stereoelectronic
interaction in the trans conformation. This would explain
the consistently higher Kt/c values in dipeptide 13 relative
to dipeptide 12.
To test our developing understanding of the conforma-
tion of peptides containing hydroxylated prolines we
finally considered dipeptides 14 and 15, which incorpo-
rate 3,4-dihydroxylated prolines.37 Both have the same
relative stereochemistry between C2 and C4, viz. a trans
relationship between the CONHMe and 4-OH substitu-
ents on the pyrrolidine ring. As such, they share the
features of trans-4-hydroxyproline which stabilize the
Cγ-exo pyrrolidine conformation. On the basis of our
results for the 3-hydroxyprolines, we might expect the
incorporation of a second hydroxy group at C3 to have
little influence of the cis f trans equilibrium. This turns
out not to be the case, as illustrated by the Van’t Hoff
plots in Figure 9, and we need to carefully consider how
the two hydroxy groups work together to determine the
dominant conformation of the pyrrolidine ring and the
position of the cis f trans equilibrium.
FIGURE 7. Pyrrolidine conformation in dipeptide 13.
line, is likely to adopt this Câ-exo conformation as a result
of a gauche interaction and a stabilizing σ(Cγ-H)f
σ*(Câ-O) interaction (Figure 6).
For completeness, we also looked at dipeptide 13
containing a cis-3-hydroxyproline (3-hyp). This amino
acid has not been identified in collagens, but does occur
in the cyclic peptide antibiotic telomycin.35 The N-methyl
derivative has been isolated in significant quantities from
a South Australian Dendrilla sponge.36 Assuming a
pyrrolidine conformation in which Câ is the “flap atom”
as discussed above, analysis of the potential stereoelec-
tronic effects predicts that dipeptide 13 would contain a
pyrrolidine ring in a Câ-endo conformation (Figure 7).
Van’t Hoff plots for dipeptides 12 and 13 alongside the
parent compound 8 are given in Figure 8. We are forced
to conclude that hydroxylation in the 3-position, regard-
less of stereochemistry, has a small effect on the confor-
mation of the preceding peptide bond. Why do 3-OH
groups have less effect on the cis f trans equilibrium
than 4-OH groups? The hydroxy groups are the same
number of bonds away from the peptide bond, although
the intervening atoms are different. In the case of the
4-OH there is the -CδH2- unit and in the case of the
3-OH there is the -CRH(CONHMe)- bridge. There is
structural evidence for a relatively short Cγ-Cδ bond in
N-acetyl-trans-4-hydroxyproline methyl ester;11a the
shorter distance and the inherent partial double bond
character likely account for a strong inductive effect by
substituents at C4. On the other hand, the X-ray crystal
Van’t Hoff plots for dipeptides 14 and 15 are given in
Figure 9, along with those of Ac-Phe-Pro-NHMe (8) and
Ac-Phe-Hyp-NHMe (9), which can now both be regarded
as “parent compounds.” One of the dihydroxyprolines
(DHP in dipeptide 14) displayed similar behavior to
trans-4-Hyp, giving rise to the largest equilibrium con-
stants in the series (Kt/c ) 5.6 at 298 K). The diastere-
oisomer, varying only in configuration at Câ of the proline
(35) (a) Misiek, M.; Fardig, O. B.; Gourevich, A.; Johnson, D. L.;
Hooper, I. R.; Lein, J. Antibiot. Annu. 1957-1958, 852-855. (b)
Sheehan, J. C.; Mania, D.; Nakamura, S.; Stock, J. A.; Maeda, K. J.
Am. Chem. Soc. 1968, 90, 462-470.
(36) Capon, R. J.; Ovenden, S. P. B.; Dargaville, T. Aust. J. Chem.
1998, 51, 169-170.
(37) Taylor, S. W.; Waite, J. H. In Prolyl Hydroxylase, Protein
Disulfide Isomerase, and Other Structurally Related Proteins; Guzman,
N. A., Ed.; Marcel Dekker: New York, 1998.
1310 J. Org. Chem., Vol. 70, No. 4, 2005

Prolyl Amides: Pyrrolidine Hydroxylation
FIGURE 9. Van’t Hoff plots for Ac-Phe-Pro-NHMe (8), Ac-
Phe-Hyp-NHMe (9), Ac-Phe-DHP-NHMe (14), and Ac-Phe-
dhp-NHMe (15).
TABLE 1. 1H NMR Data for the Pyrrolidine Ring of
Dipeptide 14 in CD3OD at 298 K and 400 MHz
FIGURE 10. Pyrrolidine conformation in dipeptide 14.
proton δ (ppm) multiplicity
coupling constant/s
JR,â ) 5.2 Hz
JR,â ) 5.2 Hz, Jâ,γ ) 4.1 Hz
Jâ,γ ≈ Jγ,δ ≈ Jγ,δ′ ) 4.5 Hz
Jδ,δ′ ) 10.6 Hz, Jγ,δ′ ) 5.2 Hz
Jδ,δ′ ) 10.6 Hz, Jγ,δ ) 4.5 Hz
HR
Hâ
Hγ
Hδ′
Hδ
4.14
4.08
4.19
3.62
3.72
d
dd
app q
dd
dd
residue, led to a significant reduction in Kt/c (∼2.5 at 298
K) and the Van’t Hoff plot displayed a significant tem-
perature dependence.
Dipeptide 14, incorporating 2,3-trans-3,4-cis-3,4-dihy-
droxyproline (DHP), demonstrates similar behavior to
dipeptide 9 (containing trans-4-hydroxyproline). Indeed,
the augmented equilibrium constants are consistent with
this dihydroxyproline having a stronger preferance for a
Cγ-exo conformation than Hyp itself, and therefore a
superior alignment of backbone dihedral angles to sta-
bilize the trans peptide bond. No crystallographic data
exist for this dihydroxyproline, or derivatives thereof.
However, analysis of 1H NMR coupling constants around
the pyrrolidine ring in dipeptide 14 supports a Cγ-exo
conformation. Most significantly, Hγ appears as an
apparent quartet, as detailed in Table 1, since it makes
approximately the same dihedral angle with Hâ and each
of the δ-protons.
Analysis of potential stereoelectronic effects (Figure 10)
shows that two additional gauche effects are possible in
this conformation of the pyrrolidine ring of 14, along with
the same σ(Cδ-H)axfσ*(Cγ-O) and σ(Câ-H)axfσ*(Cγ-
O) stabilizing interactions as discussed for dipeptide 9.
These additional gauche interaction and the extra induc-
tive electron-withdrawing capabilities of the 3-OH ac-
count for the similar but enhanced behavior of 14 relative
to 9.
FIGURE 11. Pyrrolidine conformation in dipeptide 15.
deviation of 0.009 Å. The Câ atom lies 0.60 Å out of the
plane, on the same side of the pyrrolidine ring as the
carboxyl group.38 This means the two hydroxy groups are
axial and this conformation can be justified in terms
of the kinds of stereoelectronic effects discussed earlier:
two gauche interactions are possible and a single
σ(CR-H)axfσ*(Câ-O) stabilizing interaction (Figure 11).
This gives the expectation of a short CR-Câ bond, but
in fact it is 1.537 Å long in the crystal structure of the
free amino acid.38 The short Câ-Cγ and Cγ-Cδ bonds
(1.518 and 1.517 Å, respectively) are also puzzling.
Thermodynamic parameters for the cis f trans equi-
libria are presented in Table 2. All compounds demon-
strate a small, positive entropy, since the trans species
represents a more random conformation. The Ar-Pro
interaction and perhaps more extensive hydrogen bond-
ing with solvent by a more exposed amide bond render
the cis species more “ordered”. The small but positive
enthalpies of isomerization for all dipeptides except 9 and
14 indicate that the cis conformation is energetically
favored in these cases, presumably by an Ar-Pro interac-
X-ray crystallographic studies of 2,3-cis-3,4-trans-3,4-
dihydroxy-L-proline (dhp) demonstrated that the pyrro-
lidine ring exhibited a “Câ-endo” conformation in which
CR, Cγ, Cδ, and N lie in a plane, with a maximum
(38) (a) Karle, I. L.; Daly, J. W.; Witkop, B. Science 1969, 164, 1401-
1402. (b) Karle, I. L. Acta Crystallogr. B 1970, B26, 765-770.
J. Org. Chem, Vol. 70, No. 4, 2005 1311

Taylor et al.
TABLE 2. Thermodynamic Parameters for Compounds 8-15
Kt/ca D2O,
298 K
∆G,b 298 K
∆δHR,c
298 K
∆Hd
∆Sd
compd
(kcal mol-1 K-1
)
(kcal mol-1
)
(cal mol-1 K-1
)
Ac-Phe-Pro-NHMe (8)
2.1
5.0
1.9
1.4
2.4
2.2
5.6
2.5
-0.44
-0.90
-0.40
-0.18
-0.51
-0.47
-1.01
-0.57
-0.73
-0.53
-0.15
-0.81
-0.44
-0.81
-0.27
-0.63
+0.25
-0.24
+1.21
+1.40
+0.65
+0.48
-0.41
+0.80
+2.3
+2.2
+5.4
+5.3
+3.9
+3.2
+2.0
+4.6
Ac-Phe-Hyp-NHMe (9)
Ac-Phe-hyp-NHMe (10)
Ac-Phe-hyp(OMe)-NHMe (11)
Ac-Phe-3Hyp-NHMe (12)
Ac-Phe3hyp-NHMe (13)
Ac-Phe-DHP-NHMe (14)
Ac-Phe-dhp-NHMe (15)
1
a Determined by integration of well-resolved signals in the H NMR spectrum. b Calculated by using ∆G ) -RT ln K. c δHR(trans) -
δHR(cis). d Calculated by using the equation ∆G ) ∆H - T∆S for the line of best fit for the Van’t Hoff plot.
tion. Dipeptides 9 and 14 (incorporating trans-4-Hyp and
2,3-trans-3,4-cis-DHP, respectively) exhibit a negative
enthalpy; in these two cases the enthalpic and entropic
terms work together to produce a significant free energy
difference (∆G) between the cis and trans species.
Wu and Raleigh demonstrated a linear relationship
between the equilibrium constant and ∆δHR,39 the
distance by which HR of the cis conformation is shifted
upfield relative to its trans counterpart, as a consequence
of the shielding effect of the aromatic ring. While this is
undoubtedly valid in the comparison of X-Pro peptide
bonds in which a Cγ-endo conformation is the norm, there
is no direct relationship between Kt/c and ∆δHR for the
series of compounds discussed herein. Not surprisingly,
chemical shift differences are affected by the substitution
pattern of the pyrrolidine ring and its conformation.
substituents and pyrrolidine conformation have an influ-
ence on this.
In summary, the regiochemistry, stereochemistry and
degree of hydroxylation of proline residues has a marked
impact on peptide conformation. This work represents
the first systematic study in this regard and has produced
some surprising results which we have attempted to
rationalize in light of other experimental, theoretical, and
structural information.
4. Experimental Section
General. See refs 16 and 21 for details. Standard conditions
for RP-HPLC purification of dipeptides utilized an Econosil
C-18 column (21 mm diameter; 250 mm long) and a flow rate
of 12 mL min-1. A gradient method was employed as follows
(% acetonitrile in water): 20-30% over 8 min; 30-95% over
2 min; 95% for 5 min; 95-20% over 5 min. The compounds
absorbed approximately 10-fold less strongly at 259 nm than
they did at 218 nm. NMR assignments are based on the
3. Conclusions
analysis of H-1H COSY, HMQC, and HMBC spectra.
The placement of a secondary alcohol functional group
at the â- and γ-positions of a Pro residue has a strong
influence of the conformation of the five-membered
pyrrolidine ring. The preferred conformation is one in
which there are a maximum number of gauche effects
and the underlying σ(C-H)axfσ*(C-O)ax stabilizing
interactions.
The pyrrolidine conformation that has the strongest
influence on the position of the cis f trans equilibrium
for the preceding X-Pro peptide bond is Cγ-exo. This
conformation favorably predisposes the peptide backbone
to a n(On-1)fπ*((CdO)n) Coulombic interaction. The
importance of this stereoelectronic effect on peptide
conformation is emerging strongly29 and our results
support this. For the first time we have identified another
hydroxylated proline that stabilizes the trans conforma-
tion of the peptide bond better than trans-4-Hyp. 2,3-
trans-3,4-cis-3,4-Dihydroxyproline (DHP) has a stronger
preference for the Cγ-exo conformation and the additional
inductive effect of the 3-OH is likely to enhance the
pyramidalization of the nitrogen and the electrophilicity
of the Pro CdO group.
1
Ac-Phe-trans-4-Hyp-NHMe (9). I. Fmoc-Phe-trans-4-
Hyp-NHMe. N-Hydroxysuccinimide (30 mg, 0.258 mmol, 1.0
equiv), followed by DCC (53 mg, 0.258 mmol, 1.0 equiv), was
added to a solution of Fmoc-Phe-OH (100 mg, 0.258 mmol, 1.0
equiv) in CH2Cl2 (3 mL) at 0 °C under N2. The solution was
stirred at 0 °C for 20 min and then warmed to room temper-
ature and stirred for a further 4 h. The suspension was filtered
through a plug of cotton in a Pasteur pipet. The filtrate was
concentrated to 2 mL and then refrigerated for 2 h. The
suspension was filtered again and the residue was evaporated
to give a colorless foam that was dissolved in DMF (1.5 mL)
and cooled to 0 °C under N2. L-trans-4-Hydroxyproline (34 mg,
0.258 mmol, 1.0 equiv) was added as a solid in one portion,
followed by the dropwise addition of diisopropylethylamine (45
µL, 33 mg, 0.258 mmol, 1.0 equiv). The solution was gradually
warmed to room temperature and left to stir overnight. The
mixture was diluted with ethyl acetate (20 mL) and washed
with 2 M HCl (20 mL). The acidic aqueous layer was back-
extracted with ethyl acetate (20 mL). The organic layers were
combined, washed with water (40 mL) and brine (40 mL),
filtered through MgSO4, and concentrated to give Fmoc-Phe-
trans-4-Hyp-OH. This crude acid was dissolved in CH2Cl2 (3
mL) and cooled to 0 °C under N2. Methylamine hydrochloride
(17 mg, 0.258 mg, 1.0 equiv) was added, followed by triethyl-
amine (83 µL, 60 mg, 0.595 mmol, 2.4 equiv) and finally BOP
reagent (110 mg, 0.258 mmol, 1.0 equiv). The mixture was
stirred at room temperature for 16 h, diluted with CH2Cl2 (25
mL), washed with 2 M HCl (30 mL), water (30 mL), and brine
(30 mL), filtered through MgSO4, and concentrated. The
product was isolated by flash chromatography, eluting with
2-5% MeOH in CH2Cl2 to give Fmoc-Phe-trans-4-Hyp-NHMe
The results have been difficult to interpret as a result
of the Ar-Pro interaction. The importance of this local
interaction and its role in protein-folding are also emerg-
ing and our results present some new elements to
consider. We tentatively suggest that the strength of this
interaction depends on how easily the aromatic ring can
approach the CR atom of the Pro residue, and that
1
(63 mg, 49%). Rf 0.38 (9:1 CH2Cl2-MeOH; H NMR (CDCl3,
400 MHz) δ 2.14-3.31 (m, 1H), 2.69 (d, J ) 4.8 Hz, 3H), 2.60-
2.67 (m, 1H), 3.69 (d, J ) 11.0 Hz, 1H), 4.08-4.42 (m, 5H),
(39) Wu, W.-J.; Raleigh, D. P. Biopolymers 1998, 45, 381-394.
1312 J. Org. Chem., Vol. 70, No. 4, 2005

Prolyl Amides: Pyrrolidine Hydroxylation
4.57 (t, J ) 7.7 Hz, 1H), 4.69 (dd, J ) 14.3, 7.3 Hz, 1H), 5.89
(d, J ) 7.9 Hz, 1H), 6.62 (d, J ) 4.8 Hz, 1H), 7.11-7.30 (m,
8H), 7.37 (t, J ) 7.5 Hz, 2H), 7.60 (t, J ) 7.5 Hz, 2H), 7.73 (d,
J ) 7.5 Hz, 2H); 13C NMR (CDCl3, 67.5 MHz) δ 26.3, 36.4,
38.5, 47.0, 53.7, 55.3, 58.8, 67.0, 69.9, 119.9, 124.9, 125.0, 127.0,
127.6, 129.2, 129.3, 135.5, 141.1, 143.5, 155.8, 171.0, 171.2;
HRMS (FAB+, NBA, MeOH) calcd for (MH)+ C30H32N3O5
514.2342, obsd 514.2326.
amine was isolated by flash chromatography, eluting first with
2:1 EtOAc-hexanes to elute the Fmoc byproducts and then
with 9:1 CH2Cl2-MeOH to isolate the ninhydrin-active amine
(Rf 0.37 in 9:1 CH2Cl2-MeOH). The relevant fractions were
combined and evaporated down to give a straw-colored oil (58.8
mg). This was dissolved in a mixture of pyridine (1 mL) and
acetic anhydride (1 mL) and the berry-red mixture was stirred
at room temperature under N2 overnight. The mixture was
concentrated and then the residue dissolved in a mixture of
CH2Cl2 (1 mL) and TFA (0.5 mL) and stirred for 4 h at room
temperature under N2. The mixture was concentrated and the
product isolated by RP-HPLC under the standard conditions
(RT ) 9.00 min) to give 10 (38.3 mg; 53% over 3 steps). 1H
NMR (400 MHz, D2O Kt/c ) 1.9 at 298 K) δ 1.47 (ddd, J ) 13.6,
II. Ac-Phe-trans-4-Hyp-NHMe (9). Diethylamine (4 mL)
was added to a solution of Fmoc-Phe-Hyp-NHMe (266 mg, 0.52
mmol, 1.0 equiv) in acetonitrile (10 mL). The solution was
stirred at room temperature for 1 h, concentrated, and then
concentrated twice more from acetonitrile. The residue was
suspended in dichloromethane (8 mL) and the mixture was
cooled to 0 °C. Triethylamine (145 µL, 105 mg, 1.04 mmol, 2.0
equiv) was added followed by acetyl chloride (37 µL, 41 mg,
0.52 mmol, 1.0 equiv). The solution was warmed to room
temperature, stirred for 16 h, and concentrated. The residue
was filtered through a plug of silica gel washing with ethyl
acetate (200 mL), followed by a mixture of 9:1 EtOAc-
methanol (300 mL). Finally, 8:2 EtOAc-methanol (200 mL)
was passed through the column and the eluent concentrated
to give a colorless foam (230 mg) that was further purified by
RP-HPLC under the standard conditions (RT ) 4 min) to give
Ac-Phe-Hyp-NHMe (39 mg, 23%). 1H NMR (D2O, 400 MHz,
Kt/c ) 5.0 at 298 K) δ 1.71-1.78 (m, 1Hcis, Proâ′cis), 1.87 (s,
9.1, 4.1 Hz, 1Hcis, hypâ′cis), 1.74-1.93 (m, 1Htrans/1Hcis, hypâ′trans
,
hypâcis), 1.81 (s, 3Htrans, Actrans), 1.90 (s, 3Hcis, Accis), 2.17 (ddd,
J ) 12.7, 9.3, 4.3 Hz, 1Htrans, hypâtrans), 2.55 (s, 3Hcis, NHMecis),
2.58 (s, 3Htrans, NHMetrans), 2.83 (dd, J ) 12.6, 10.7 Hz, 1Hcis
,
Pheâ′cis), 2.91-2.94 (m, 1Hcis, Pheâcis), 2.93 (d, J ) 7.7 Hz,
2Htrans, Pheâ′trans, Pheâtrans), 3.19 (d, J ) 11.2 Hz, 1Htrans
,
hypδ′trans), 3.28 (d, J ) 13.3 Hz, 1Hcis, hypδ′cis), 3.42 (dd, J )
13.3, 4.7 Hz, 1Hcis, hypδcis), 3.48 (d, J ) 8.9 Hz, 1Hcis, hypγcis),
3.78 (dd, J ) 11.2, 4.4 Hz, 1Htrans, hypδtrans), 4.14 (t, J ) 3.5
Hz, 1Hcis, hypRcis), 4.25-4.30 (m, 2Htrans, hypγtrans, hypRtrans),
4.46 (dd, J ) 9.3, 6.1 Hz, 1Hcis, PheRcis), 4.69 (t, J ) 6.2 Hz,
1Htrans, PheRtrans), 7.10-7.30 (Ar, 5H); 13C NMR (D2O, 100
MHz) δ 24.2, 28.8, 38.8 and 39.6, (40.8 and 41.2), 55.7 (56.0),
58.3 (57.6), 62.7 (62.3), 72.5 (70.4), 130.2, 130.5, 131.6, 131.9,
132.1, 138.1, 138.7, 175.6, (175.5), 176.3 and 176.6 (175.9 and
176.0); HRMS calcd for (MH2)+ C17H24N3O4 334.1767, obsd
334.1778.
3Htrans, Actrans), 1.91 (s, 3Hcis, Accis), 1.97-2.08 (m, 1Htrans/1Hcis
Proâ′trans, Proâcis), 2.11-2.19 (m, 1Htrans, Proâtrans), 2.70 (s, 3Hcis
,
,
NHMecis), 2.73 (s, 3Htrans, NHMetrans), 2.84 (dd, J ) 14.0, 9.0
Hz, 1Htrans, Pheâ′trans), 2.90 (dd, J ) 13.0, 7.2 Hz, 1Hcis, Pheâ′cis),
2.98 (dd, J ) 13.0, 7.6 Hz, 1Hcis, Pheâcis), 3.12 (dd, J ) 14.0,
6.7 Hz, 1Htrans, Pheâtrans), 3.36 (dd, J ) 12.0, 4.4 Hz, 1Hcis
,
Ac-Phe-hyp(OMe)-NHMe (11). Boc-hyp(OMe)NHMe (17
mg, 0.0625 mmol, 1.0 equiv) was dissolved in a 1:1 (v/v)
solution of TFA and dichloromethane (2 mL). The solution was
stirred at room temperature for 45 min then concentrated. The
residue was left on the pump for 16 h and then dissolved in
dry acetonitrile (2 mL). Diphenylphosphoryl azide (18 µL, 23
mg, 0.081 mmol, 1.3 equiv) was added, followed by N-
acetylphenylalanine (17 mg, 0.081 mmol, 1.3 equiv) and
triethylamine (31 µL, 23 mg, 0.220 mmol, 3.5 equiv). The
solution was stirred at room temperature under nitrogen for
18 h and then concentrated. The residue was purified by flash
column chromatography, eluting 0-20% MeOH in ethyl
acetate. The relevant fractions were combined and concen-
trated and the final product was further purified by RP-HPLC
Proδ′cis), 3.56-3.64 (m, 1Hcis, Proδcis), 3.59 (dd, J ) 10.8, 6.6
Hz, 1Htrans, Proδ′trans), 3.77 (d, J ) 10.8 Hz, 1Htrans, Proδtrans),
3.93 (dd, J ) 8.4, 4.7 Hz, 1Hcis, Proγcis), 4.25 (dt, J ) 11.1, 5.5
Hz, 1Hcis, ProRcis), 4.41-4.49 (m, 2Htrans, Proγtrans, ProRtrans),
4.70 (t, J ) 7.5 Hz, 1Hcis, PheRcis); 4.84 (dd, J ) 9.0, 5.5 Hz,
1Htrans, PheRtrans), 7.12-7.32 (m, 5H); 13C NMR (CD3OD, 100
MHz) δ 22.2 (22.2), 26.4 (26.5), 38.3 (39.8), 38.8 (40.4), 54.2
(53.8), 56.5 (55.0), 60.7 (60.8), 70.9 (68.5), 127.8 (128.1), 129.5
(129.4), 129.5 (129.6), 130.4 (129.8), 138.4 (137.6), 172.7, 172.9,
174.6; HRMS (FAB+, NBA, MeOH) calcd for (MH)+ C17H24N3O4
334.1767, obsd 334.1773.
Ac-Phe-cis-4-hyp-NHMe (10). I. Fmoc-Phe-cis-4-Hyp-
(OtBu)-NHMe. A solution of Fmoc-cis-4-Hyp(OtBu)-NHMe (80
mg, 0.189 mmol, 1.0 equiv) in acetonitrile (1.5 mL) and
diethylamine (1.5 mL) was stirred at room temperature under
N2 for 30 min and then concentrated. The residue was
concentrated twice more from acetonitrile and then suspended
in CH2Cl2 (3 mL) under N2. To this was added Fmoc-Phe-OH
(77 mg, 0.199 mmol, 1.15 equiv), diisopropylethylamine (82
µL, 61 mg, 0.473 mmol, 2.5 equiv), and BroP reagent (77 mg,
0.199 mmol, 1.05 equiv). The flask was flushed with N2,
stoppered, and left to stir at room temperature overnight. The
mixture was concentrated and the residue applied to a flash
column in a minimum volume of CH2Cl2. The column was
eluted with 3:1 EtOAc-hexanes to afford Fmoc-Phe-cis-4-Hyp-
(OtBu)-NHMe (106 mg, 98%). Rf 0.20 (2:1 EtOAc-hexanes);
1H NMR (CDCl3, 270 MHz) δ 1.34 (s, 9H), 1.76-1.90 (m, 1H),
2.07-1.25 (m, 1H), 2.78 (d, J ) 4.8 Hz, 3H), 3.26-3.65 (m,
3H), 4.09-4.47 (m, 7H), 6.42 (br d, 1H), 7.29-7.42 (m, 9H),
7.58 (d, J ) 7.3 Hz, 2H), 7.76 (d, J ) 7.3 Hz, 2H); 13C NMR
(CDCl3, 67.5 MHz) δ 26.3, 28.0, 36.8, 39.5 (39.1), 47.1, 53.6
(53.9), 55.6 (55.3), 60.2 (60.0), 67.2 (67.1), 69.5, 74.2, 119.9,
125.0, 125.1, 127.0, 127.3, 127.6, 128.6, 128.7, 129.3, 135.9,
141.1, 143.6, 155.6, 171.3, 171.5; HRMS calcd for (MH)+
C34H40N3O5 570.2968, obsd 570.2944.
1
to give Ac-Phe-hyp(OMe)-NHMe (13) (14 mg, 58%). H NMR
(D2O, 400 MHz, Kt/c ≈ 1.4 at 298 K) δ 1.20 (ddd, J ) 14.0, 9.2,
4.0 Hz, 1Hcis, hypâ′cis), 1.77 (s, 3Htrans, Actrans), 1.82 (s, 3Hcis
,
Accis), 1.93 (br d, J ) 14.2 Hz, 1Htrans, hypâ′trans), 2.03 (d, J )
14.0 Hz, 1Hcis, hypâcis), 2.14 (ddd, J ) 14.2, 9.6, 4.7 Hz, 1Htrans
hypâtrans), 2.49 (s, 3Htrans/3Hcis, NHMetrans, NHMecis), 2.77 (dd,
J ) 12.5, 10.4 Hz, 1Hcis, Pheâ′cis), 2.75-2.84 (m, 2Htrans/1Hcis
Pheâ′trans, Pheâtrans, Pheâcis), 2.97 (s, 3Hcis, OMecis), 3.00 (s,
3Htrans, OMetrans), 3.29-3.38 (m, 1Htrans/2Hcis, hypδ′trans, hypδ′cis
,
,
,
hypγcis), 3.41 (d, J ) 8.8 Hz, 1Hcis, hypRcis), 3.69-3.76 (m,
1Htrans/1Hcis, hypδtrans, hypδcis), 3.93 (br s, 1Htrans, hypγtrans), 4.22
(br d, J ) 9.8 Hz, 1Htrans, hypRtrans), 4.38 (dd, J ) 10.0, 5.9 Hz,
1Hcis, PheRcis), 4.65-4.71 (m, 1Htrans, PheRtrans), 7.04-7.31 (m,
5H, Ar); 13C NMR (D2O, 100 MHz) δ 24.1 (24.1), 28.8, 36.4
(37.2), 36.4 (37.2), 39.4 (40.7), 55.1 (55.1), 55.7 (56.1), 58.6
(58.1), 62.2 (62.4), 81.7 (79.9), 130.3 (130.5), 131.7 (131.9), 132.0
(132.0), 138.8 (138.1), 176.0, 176.4, 176.5 (175.6, 175.8, 176.0);
HRMS (FAB+, glycerol, MeOH) calcd for (MH)+ C18H26N3O4
348.192332, obsd 348.191533.
Ac-Phe-trans-3-Hyp-NHMe (12). I. Fmoc-Phe-trans-3-
Hyp-NHMe. N-Hydroxysuccinimide (148.5 mg, 1.29 mmol, 1.0
equiv), followed by DCC (266.2 mg, 1.29 mmol, 1.0 equiv), was
added to a solution of Fmoc-Phe-OH (500 mg, 1.29 mmol, 1.0
equiv) in CH2Cl2 (15 mL) at 0 °C under N2. The solution was
stirred at 0 °C for 20 min and then warmed to room temper-
ature and stirred for a further 4 h. The suspension was filtered
through a plug of cotton in a Pasteur pipet. The filtrate was
II. Ac-Phe-cis-4-Hyp-NHMe (10). A solution of Fmoc-Phe-
cis-4-Hyp(OtBu)-NHMe (106 mg, 0.176 mmol) in acetonitrile
(2 mL) and diethylamine (2 mL) was stirred at room temper-
ature under N2 for 30 min and then concentrated. The residue
was concentrated twice more from acetonitrile. The primary
J. Org. Chem, Vol. 70, No. 4, 2005 1313

Taylor et al.
concentrated to 5 mL and then refrigerated for 2 h. The
suspension was filtered again and the residue evaporated to
give a colorless foam that was dissolved in DMF (8 mL) and
cooled to 0 °C under N2. L-trans-3-Hydroxyproline (169.2 mg,
1.29 mmol, 1.0 equiv) was added as a solid in one portion,
followed by the dropwise addition of diisopropylethylamine
(225 µL, 166.8 mg, 1.29 mmol, 1.0 equiv). The solution was
gradually warmed to room temperature and left to stir
overnight. The mixture was diluted with ethyl acetate (100
mL) and washed with 2 M HCl (100 mL). The acidic aqueous
layer was back-extracted with ethyl acetate (100 mL). The
organic layers were combined, washed with water (200 mL)
and brine (200 mL), filtered through MgSO4, and concentrated
to give Fmoc-Phe-trans-3-Hyp-OH (568 mg, 1.13 mmol; 88%);
Rf 0.57 (6:4:1 CHCl3-MeOH-H2O). This residue was dissolved
in CH2Cl2 (15 mL) and cooled to 0 °C under N2. Methylamine
hydrochloride (77 mg, 1.13 mmol, 1.0 equiv) was added,
followed by triethylamine (380 µL, 276 mg, 2.72 mmol, 2.4
equiv) and finally BOP reagent (502 mg, 1.13 mmol, 1.0 equiv).
The mixture was stirred at room temperature for 16 h and
then diluted with EtOAc (100 mL), washed with 2 M HCl (100
mL), water (100 mL), and brine (100 mL), filtered through
MgSO4, and concentrated. The product was isolated by flash
chromatography, eluting with 2-5% MeOH in CH2Cl2 to give
Fmoc-Phe-trans-3-Hyp-NHMe (420 mg, 63% over 2 steps). Rf
0.36 (9:1 CH2Cl2-MeOH); 1H NMR (CDCl3, 270 MHz) δ 1.70-
2.05 (m, 2H), 2.64 (d, J ) 7.4 Hz, 3H), 3.01-3.21 (m, 3H), 3.81
(m, 1H), 4.09-4.56 (m, 4H), 4.66-4.85 (m, 1H), 5.96 (d, J )
7.9 Hz, 1H), 6.36 (d, J ) 4.8 Hz, 1H), 6.86 (br s, 1H), 7.18-
7.30 (m, 7H), 7.37 (t, J ) 7.3 Hz, 2H), 7.54 (t, J ) 6.4 Hz, 2H),
7.73 (d, J ) 7.3 Hz, 2H); 13C NMR (CDCl3, 67.5 MHz) δ 26.2,
32.8, 38.9, 45.5, 47.1, 53.6, 67.0, 68.6, 72.4, 119.8, 125.0, 126.9,
127.1, 127.6, 128.5, 129.3, 135.8, 141.1, 143.5, 14.6, 155.7,
169.5, 171.7; HRMS (FAB+, NBA, CH2Cl2) calcd for (MH)+
C30H32N3O5 514.2342, obsd 514.2339.
at room temperature for 45 min and concentrated. The residue
was dried in vacuo then dissolved in dry acetonitrile (2 mL).
Diphenylphosphoryl azide (23 µL, 29 mg, 0.103 mmol, 1.1
equiv) was added, followed by N-acetylphenylalanine (22 mg,
0.103 mmol, 1.1 equiv) and triethylamine (40 µL, 29 mg, 0.28
mmol, 3 equiv). The solution was stirred at room temperature
under nitrogen for 18 h, then concentrated. The residue was
purified by flash column chromatography, eluting with 0-10%
MeOH in ethyl acetate. Relevant fractions were combined and
concentrated and the final product was isolated from the
residue by RP-HPLC to give Ac-Phe-3-cis-hypNHMe (13) (8
mg, 26%). 1H NMR (D2O, 400 MHz, Kt/c ) 2.19 at 298 K) δ
1.54 (m, 2Hcis, hypγ′cis, hypγcis), 1.74 (s, 3Htrans, Actrans), 1.80
(s, 3Hcis, Accis), 1.80-1.92 (m, 1Htrans, hypγ′trans), 1.94-2.08 (m,
1Htrans, hypγtrans), 2.53 (s, 3Hcis, NHMecis), 2.56 (s, 3Htrans
NHMetrans), 2.75-2.83 (m, 1Htrans/2Hcis, Pheâ′trans, Pheâ′cis
,
,
Pheâcis), 3.00 (dd, J ) 13.9, 5.5 Hz, 1Htrans, Pheâtrans), 3.19-
3.31 (m, 1Hcis, hypδ′cis), 3.33-3.44 (m, 2Hcis, hypδcis, hypRcis),
3.53 (q, J ) 7.6 Hz, 1Htrans, hypδ′trans), 3.76 (br q, J ) 7.6 Hz,
1Htrans, hypδtrans), 3.88 (q, J ) 6.4 Hz, 1Hcis, hypâcis), 4.23 (d, J
) 6.1 Hz, 1Htrans, hypRtrans), 4.35-4.50 (m, 1Hcis, PheRcis), 4.43
(q, J ) 5.5 Hz, 1Htrans, hypâtrans), 4.65-4.71 (m, 1Htrans
,
PheRtrans), 7.04-7.35 (m, 5H′ Ar); 13C NMR (D2O, 100 MHz) δ
24.2 (24.4), 28.6 (28.6), 35.3 (32.6), 39.2 (41.4), 48.4 (47.3), 55.5
(55.6), 67.9 (67.1), 72.8 (74.1), 130.1 (130.3), 131.6 (131.8), 132.0
(132.0), 139.2 (138.5), 173.0 (173.4), 175.3, 176.6 (175.0, 175.6);
HRMS (FAB+, NBA, MeOH) calcd for C17H23O4N3 (MH)+
334.1752, obsd. 334.1780.
Ac-Phe-(2,3-trans-3,4-cis-3,4-dihydroxy)-l-Pro-NHMe
(14). I. Fmoc-Phe-(3,4-O-isopropylidine-DHP)-NHMe (DHP
) 2,3-trans-3,4-cis-3,4-dihydroxy-L-proline). Diethylamine
(2 mL) was added to a solution of Fmoc-(3,4-di-O-isopropy-
lidine)DHP-NHMe (63.9 mg, 0.152 mmol, 1.0 equiv) in aceto-
nitrile (2 mL). The solution was stirred at room temperature
under N2 for 30 min and then concentrated, then concentrated
two further times from acetonitrile. The residue was dissolved
in CH2Cl2 (4 mL) under N2 and cooled to 0 °C. Fmoc-Phe-OH
(64.6 mg, 0.167 mmol, 1.1 equiv) was added, followed by
diisopropylethylamine (66 µL, 49 mg, 0.379 mmol, 2.5 equiv)
and finally BrOP reagent (64.7 mg, 0.167 mmol, 1.1 equiv).
The mixture was gradually allowed to warm to room temper-
ature and stirred for 2 d. The mixture was concentrated and
the residue applied to a flash column in a minimum volume
of CH2Cl2. The column was eluted with 3:1 EtOAc-hex to
isolate Fmoc-Phe-(3,4-O-isopropylidine)DHP-NHMe (65.2 mg;
75%). Rf 0.25 (2:1 EtOAc-hexanes); 1H NMR (CDCl3, 400
MHz) δ 1.26 (s, 3H), 1.35 (s, 3H), 2.76 (s, 3H), 2.93 (dd, J )
12.3, 4.8 Hz, 1H), 3.04 (d, J ) 6.7 Hz, 1H), 3.83 (d, J ) 12.4
Hz, 1H), 4.21 (t, J ) 7.1 Hz, 1H), 4.34 (dd, J ) 10.9, 3.6 Hz,
1H), 4.46 (dd, J ) 10.6, 7.1 Hz, 1H), 4.73 (t, J ) 5.3 Hz, 1H),
4.80 (dd, J ) 14.9, 6.6 Hz, 1H), 5.04 (d, J ) 6.0 Hz, 1H), 5.67
(d, J ) 8.2 Hz, 1H), 6.29 (d, J ) 4.6 Hz, 1H), 7.09 (dd, J ) 7.7,
1.8 Hz, 2H), 7.18-7.43 (m, 7H), 7.58 (d, J ) 7.5 Hz, 2H), 7.77
(d, J ) 7.5 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 24.7, 26.1,
26.6, 38.9, 47.1, 52.7, 53.1, 65.4, 67.0, 79.3, 80.0, 111.9, 120.0,
125.0, 125.1, 127.0, 127.2, 127.7, 128.6, 129.4, 135.5, 141.3,
143.7, 143.8, 155.3, 168.5, 170.7; HRMS (FAB+, glycerol,
MeOH) calcd for (MH)+ C33H34N3O6 570.2604, obsd 570.2613.
II. Ac-Phe-DHP-NHMe (14). Diethylamine (1.5 mL) was
added to a solution of Fmoc-Phe-(3,4-O-isopropylidine)DHP-
NHMe (34.7 mg, 0.061 mmol) in acetonitrile (1.5 mL). The
mixture was stirred under N2 for 30 min, concentrated, and
then concentrated twice more from acetonitrile. The residue
was applied to a flash column in a minimum volume of CH2-
Cl2 and then eluted with 2:1 EtOAc-hexanes, to elute the
Fmoc-piperidine adduct, then the polarity increased to 9:1 CH2-
Cl2-MeOH to elute the primary amine (Rf 0.41, 9:1 CH2Cl2-
MeOH) (17.4 mg). The amine was dissolved in pyridine (1 mL)
and acetic anhydride (1 mL) and stirred at room temperature
under N2 overnight. The mixture was concentrated then
dissolved in CH2Cl2 (2 mL) and TFA (1 mL). The mixture was
stirred under N2 at room temperature for 4 h and then
II. Ac-Phe-trans-3-Hyp-NHMe (12). Diethylamine (1 mL)
was added to a solution of Fmoc-Phe-trans-3-Hyp-NHMe (31.4
mg, 0.061 mmol) in acetonitrile (1 mL). The mixture was
stirred under N2 at room temperature, concentrated, and then
concentrated twice more from acetonitrile. The residue was
dissolved in a mixture of pyridine (1 mL) and acetic anhydride
(1 mL) and left to stir at room temperature under N2 for 48 h.
The mixture was evaporated down and then suspended in a
mixture of methanol (2 mL) and water (1 mL). Potassium
carbonate (15 mg) was added and the mixture was stirred for
2 h, after which time HPLC analysis of the reaction mixture
indicated that hydrolysis of the acetate ester was complete.
The reaction mixture was freeze-dried to give a beige powder
(39.2 mg) that was subjected to RP-HPLC under the standard
conditions to isolate the product (RT ) 10.32 min), Ac-Phe-
trans-3-Hyp-NHMe (14.2 mg; 70% over 3 steps). 1H NMR (400
MHz, D2O, Kt/c ) 2.37 at 298 K) δ 1.67 (ddt, J ) 14.0, 7.5, 2.6
Hz, 1Hcis, Hypγ′cis), 1.79-1.93 (m, 1Htrans/1Hcis, Hypγ′trans
Hypγcis), 1.79 (s, 3Hcis, Accis), 1.81 (s, 3Htrans, Actrans), 2.03 (dddd,
,
J ) 17.8, 13.4, 8.4, 4.9 Hz, 1Htrans, Hypγtrans), 2.59 (s, 3Hcis
,
NHMecis), 2.62 (s, 3Htrans, NHMetrans), 2.82 (dd, J ) 14.0, 8.6
Hz, 1Htrans, Pheâ′trans), 2.86 (dd, J ) 13.4, 6.9 Hz, 1Hcis, Pheâ′cis),
2.90 (dd, J ) 13.4, 7.9 Hz, 1Hcis, Pheâcis), 3.03 (dd, J ) 14.0,
5.9 Hz, 1Htrans, Pheâtrans), 3.40 (dt, J ) 10.8, 7.6 Hz, 1Hcis
,
Hypδ′cis), 3.46-3.55 (m, 1Htrans/1Hcis, Hypδ′trans, Hypδcis), 3.69
(m, 1Hcis, HypRcis), 3.79 (ddd, J ) 8.7, 8.7, 8.2 Hz, 1Htrans
Hypδtrans), 4.13 (m, 1Htrans, HypRtrans), 4.18-4.30 (m, 1Htrans
1Hcis, Hypâtrans, Hypâcis), 4.51 (dd, J ) 7.9, 6.9 Hz, 1Hcis
,
/
,
PheRcis), 4.78 (dd, J ) 8.6, 6.2 Hz, 1Htrans, PheRtrans), 7.13-
7.31 (Ar, 5H); 13C NMR (CD3OD, 67.5 MHz) δ 22.2, 26.5, 34.1,
38.4 (39.0), 46.5 (46.2), 53.8 (54.2), 70.2 (70.8), 74.4 (76.4),
127.7, 129.4, 130.1, 130.3, 138.3, 172.3, 172.6, 172.8; HRMS
(FAB+, glycerol) calcd for (MH)+ C17H23N3O4 334.177466, obsd
334.177466.
Ac-Phe-cis-3-Hyp-NHMe. (13). Boc-3-cis-hyp-NHMe (23
mg, 0.094 mmol, 1.0 equiv) was dissolved in a 1:1 (v/v) solution
of TFA and dichloromethane (3 mL). The solution was stirred
1314 J. Org. Chem., Vol. 70, No. 4, 2005

Prolyl Amides: Pyrrolidine Hydroxylation
concentrated. The brown residue was subjected to RP-HPLC
according to the standard conditions (RT ) 7.52 min) to isolate
Ac-Phe-DHP-NHMe (12.9 mg, 61% over 3 steps). 1H NMR (400
II. Ac-L-Phe-2,3-cis-3,4-trans-3,4-dihydroxy-L-proline
methyl amide (15). A solution of Fmoc-Phe-dhp(OTBS)2-
NHMe (4.8 mg, 0.006 mmol) in acetonitrile (0.8 mL) and
diethylamine (0.8 mL) was stirred at room temperature under
N2 for 30 min and then concentrated. The residue was
concentrated twice more from acetonitrile. The residue was
suspended in pyridine (0.5 mL) and acetic anhydride (0.5 mL)
and left to stir under an atmosphere of N2 overnight. The
reaction mixture was concentrated and the residue dissolved
in THF (2 mL) and a solution of TBAF (20 µL, 1 M in THF)
was added. After 100 min, the mixture was concentrated and
the product isolated by RP-HPLC under standard conditions
(RT ) 9.0 min) to give compound 15 (2 mg, 91% over 3 steps).
MHz, D2O, Kt/c ) 5.58 at 298 K) δ 1.80 (s, 3Htrans/3Hcis, Accis
,
Actrans), 2.63 (s, 3Hcis, NHMecis), 2.76 (s, 3Htrans, NHMetrans), 2.80
(dd, J ) 13.5, 7.2 Hz, 1Hcis, Pheâ′cis), 2.79 (dd, J ) 13.9, 8.6
Hz, 1Htrans, Pheâ′trans), 2.89 (dd, J ) 13.5, 7.6, 1Hcis, Pheâcis),
2.99 (dd, J ) 13.9, 5.9 Hz, 1Htrans, Pheâtrans), 3.34 (dd, J ) 12.5,
4.5 Hz, 1Hcis, DHPδ′cis), 3.52-3.58 (m, 1Hcis, DHPδcis), 3.54 (dd,
J ) 11.6, 4.4 Hz, 1Htrans, DHPδ′trans), 3.72 (dd, J ) 11.5, 2.9
Hz, 1Htrans, DHPδtrans), 3.74 (d, J ) 4.9 Hz, 1Hcis, DHPRcis),
4.01 (d, J ) 6.7 Hz, 1Htrans, DHPRtrans), 4.05-4.15 (m, 1Htrans
/
2Hcis, DHPγtrans, DHPâcis, DHPγcis), 4.19 (ddd, J ) 7.1, 6.7, 3.5
Hz, 1Htrans, DHPâtrans), 4.50 (dd, J ) 7.6, 7.2 Hz, 1Hcis, PheRcis),
4.73 (dd, J ) 8.6, 5.9 Hz, 1Htrans, PheRtrans), 7.09-7.33 (Ar, 5H);
13C NMR (D2O, 100 MHz) δ 24.2 (24.3), 28.8 (28.9), 39.3 (41.0),
54.9, 55.1, (53.6, 53.9), 67.6 (68.5), 73.1 (71.1), 76.8 (78.4),
130.0, 131.5, 131.7, 131.9, 132.2, 139.0, 138.7), 174.8, 175.2,
176.5.
1H NMR (400 MHz, D2O, Kt/c ) 2.81 at 298 K) δ 1.80 (s, 3Htrans
,
Actrans), 1.85 (s, 3Hcis, Accis), 2.59 (s, 3Hcis, NHMecis), 2.62 (s,
3Htrans, NHMetrans), 2.78-2.86 (m, 1Hcis, Pheâ′cis), 2.82 (dd, J
) 14.1, 9.0 Hz, 1Htrans, Pheâ′trans), 2.98-3.08 (m, 1Hcis, Pheâcis),
3.03 (dd, J ) 14.1, 5.9 Hz, 1Htrans, Pheâtrans), 3.19 (dd, J ) 12.6,
4.4 Hz, 1Hcis, dhpδ′cis), 3.60 (dd, J ) 12.6, 5.7 Hz, 1Hcis, dhpδcis),
3.63-3.78 (m, 2Htrans/2Hcis, dhpδ′trans, dhpδtrans, dhpRcis, dhpγcis),
3.99 (dd, J ) 9.9, 4.7 Hz, 1Hcis, dhpâcis), 4.14 (dd, J ) 7.9, 4.0
Ac-Phe-(2,3-cis-3,4-trans-3,4-dihydroxy-l-Pro)-NHMe
(15). I. Fmoc-L-Phe-dhp(OTBDMS)2-NHMe (dhp ) 2,3-cis-
3,4-trans-3,4-dihydroxy-L-proline). Fmoc-dhp(OTBDMS)2-
NHMe (41.8 mg, 0.068 mmol, 1.0 equiv) was stirred in a
mixture of acetonitrile (1 mL) and diethylamine (1 mL) for 30
min at room temperature under N2. The mixture was concen-
trated, then concentrated from acetonitrile two more times.
The residue was suspended in CH2Cl2 (2.5 mL). To this
mixture was added Fmoc-Phe-OH (29.2 mg, 0.075 mmol, 1.1
equiv), diisopropylethylamine (30 µL, 22 mg. 0.171 mmol, 2.5
equiv), and BrOP reagent (29.2 mg, 0.075 mmol, 1.1 equiv).
The mixture was stirred at room temperature under N2 for
20 h and then concentrated. The residue was applied to a flash
column in a minimum volume of CH2Cl2 and then eluted with
2:1 hexanes-EtOAc, increasing the polarity to 1:1 hexanes-
EtOAc to isolate Fmoc-Phe-dhp(OTBS)2-NHMe (48.8 mg, 94%).
Rf 0.39 (1:1 hexanes-EtOAc); 1H NMR (CDCl3, 400 MHz,
major rotamer) δ -0.02-0.09 (m, 12H), 0.78 (s, 15H), 0.86 (s,
3H), 2.57 (d, J ) 4.9 Hz, 3H), 3.11 (dd, J ) 13.5, 9.0 Hz, 1H),
3.17 (dd, J ) 13.5, 5.3 Hz, 1H), 3.41 (dd, J ) 10.2, 3.0 Hz,
1H), 3.90 (br s, 1H), 4.14-4.31 (m, 3H), 4.33-4.43 (m, 2H),
4.72 (app q, J ) 4.8 Hz, 1H), 4.91 (td, J ) 8.5, 5.3 Hz, 1H),
5.75 (d, J ) 9.4 Hz, 1H), 7.18-7.42 (m, 9H), 5.57-7.60 (m,
2H), 7.73-7.78 (m, 2H); 13C NMR (CDCl3, 67.5 MHz) δ -5.1,
-4.9, -4.6, 17.7, 17.8, 25.5, 25.6, 26.1, 39.5, 47.1, 53.4, 54.4,
65.7, 67.2, 74.8, 76.4, 119.9, 125.0, 127.0, 127.1, 127.3, 127.5,
127.6, 128.6, 129.0, 129.1, 136.2, 141.1, 143.6, 143.7, 155.3,
167.4, 171.7; HRMS calcd for M+ (C42H59N3O6Si2) 758.4021,
obsd 758.4045.
Hz, 1Htrans, dhpγ trans), 4.21 (dd, J ) 6.2, 4.0 Hz, 1Htrans
dhpâtrans), 4.39 (d, J ) 6.2 Hz, 1Htrans, dhpRtrans), 4.48 (dd, J )
,
8.5, 6.5 Hz, 1Hcis, PheRcis), 4.75 (dd, J ) 9.0, 5.9 Hz, 1Htrans
,
PheRtrans), 7.09-7.31 (Ar, 5H); 13C NMR (D2O, 100 MHz) δ 24.2
(22.8), 28.5, 39.3 (41.3), 54.7 (53.5), 55.6 (55.3), 66.5 (71.3), 76.6
(74.5), 77.0 (78.3), 130.0, 130.2, 131.5, 131.8, 131.9, 132.0,
138.4, 139.1, 172.8, 175.5, 176.5.
Acknowledgment. This research was supported by
the Marsden Fund, administered by the Royal Society
of New Zealand (Grant No. 00-MAU-007).
Supporting Information Available: Tables of thermo-
dynamic data and selected NMR spectra for compounds 9-15
and intermediates in their synthesis; procedures and charac-
terization data for the preparation of the following proline
building blocks: NR-Fmoc-cis-4-tert-butyloxy-L-proline meth-
yl amide, NR-Boc-cis-4-methoxy-L-proline methyl amide,
(2S,3R,4S)-NR-Fmoc-3,4-isopropylidine-3,4-dihydroxy-L-pro-
line methyl amide, and (2S,3S,4S)-NR-Fmoc-3,4-tert-butyldim-
ethylsilyloxy-L-proline methyl amide. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO0490043
J. Org. Chem, Vol. 70, No. 4, 2005 1315