Synthesis of proline-modified analogues of the neuroprotective agent glycyl-L-prolyl-glutamic acid (GPE)

Article abstract of DOI:10.1016/j.tet.2005.08.026

The synthesis of ten proline-modified analogues of the neuroprotective tripeptide GPE is described. Five of the analogues incorporate a proline residue with a hydrophobic group at C-2 and two further analogues have this side chain locked into a spirolactam ring system. The pyrrolidine ring was also modified by replacing the γ-CH₂ group with sulfur and/or incorporation of two methyl groups at C-5.

Full text of DOI:10.1016/j.tet.2005.08.026

Tetrahedron 61 (2005) 10018–10035
Synthesis of proline-modified analogues of the neuroprotective
agent glycyl-L-prolyl-glutamic acid (GPE)
*
Paul W. R. Harris, Margaret A. Brimble, Victoria J. Muir, Michelle Y. H. Lai,
Nicholas S. Trotter and David J. Callis
Neuren Pharmaceuticals Medicinal Chemistry Group, Department of Chemistry, University of Auckland, 23 Symonds Street,
Auckland 1000, New Zealand
Received 23 May 2005; revised 15 July 2005; accepted 4 August 2005
Available online 24 August 2005
Abstract—The synthesis of ten proline-modified analogues of the neuroprotective tripeptide GPE is described. Five of the analogues
incorporate a proline residue with a hydrophobic group at C-2 and two further analogues have this side chain locked into a spirolactam ring
system. The pyrrolidine ring was also modified by replacing the g-CH2 group with sulfur and/or incorporation of two methyl groups at C-5.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
order to investigate structure–activity relationships and in
an attempt to improve properties such as metabolic stability
and oral bioavailability.22–24 We herein, report the synthesis
of analogues of GPE modified at the proline residue.
The tripeptide Gly-Pro-Glu (GPE) is a naturally occurring
peptide, which is proteolytically cleaved from insulin-like
growth factor-1 (IGF-1).1–7 IGF-1 is a potent neurotrophic
factor8,9 produced endogenously in damaged regions of the
brain.10 It has been postulated that some of the neuro-
protective actions of IGF-1 are mediated by GPE11 although
the precise mechanism of action remains unclear. GPE has a
different mode of action to IGF-1 as GPE does not bind to
the IGF-1 receptor,12,13 rather GPE has been shown to bind
with low affinity to the N-methyl-D-aspartate (NMDA)
receptor and also elicit a biological response via other
mechanisms. GPE facilitates the release of dopamine
through interaction with the NMDA receptor16 but GPE
stimulated acetyl choline release is via an unknown, non
NMDA pathway.14,16
2. Results and discussion
In order to investigate the importance of the proline residue
in GPE, ten analogues modified at either Pro or at the Pro-
Glu bond were synthesized. In particular conformationally
restricted analogues were prepared in order to gain
insight into the receptor bound conformation. The
general synthetic strategy employed involved the prepa-
ration of several modified proline residues that were then
coupled to a glycine derivative and a glutamic acid di-ester
(Scheme 1).
The presence of (S)-2-methylproline (2-MePro) is known to
stabilize turns25,26 and may also prevent peptidases
recognizing the Pro-Glu amide bond resulting in resistance
to proteolytic degradation.27 Hence, the synthesis of glycyl-
L-2-methylprolyl-L-glutamic acid (G-2MePE) 1 was under-
taken. In order to further explore the influence of
modifications at this position, four other 2-alkylproline
analogues 2–5 were also synthesized (Scheme 2).
It has been demonstrated that GPE can act as a neuronal
rescue agent following hypoxic-ischemic brain injury,11,14
NMDA challenge15 and in animal models of Parkinson’s
and Alzheimer’s disease.16,17 Analogues of GPE are thus of
interest in the development of novel pharmaceutical agents
for the treatment of central nervous system (CNS) injuries
and neurodegenerative disorders.18–21
In our previous work, we have prepared GPE peptido-
mimetics modified at the glycine and glutamate residues in
The 2-alkylproline derivatives were synthesized using
Wang and Germanas’s modification28 of Seebach’s method
of self-reproducing chirality.29,30 Condensation of L-proline
6 with choral (trichloroacetaldehyde) gave oxazolidinone
7,31 which was used for the synthesis of the five
Keywords: Proline; Neuroprotective; Peptide; Peptidomimetic.
*
Corresponding author. Tel.: C64 9 3737599; fax: C64 9 3737422;
e-mail: m.brimble@auckland.ac.nz
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.08.026

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10019
Scheme 1. General retrosynthesis for GP*E analogues.
2-alkylproline modified tripeptides 1–5. Treatment of 7 with
LDA to effect enolate formation followed by alkylation with
iodomethane, iodoethane, allyl bromide or benzyl bromide,
respectively, afforded alkylated oxazolidinones 8–11.
Esterification with thionyl chloride (for 8,9) or acetyl
chloride (for 10,11) in methanol gave the methyl ester
hydrochlorides 12–15, which were coupled with N-benzyl-
oxycarbonyl–glycine 16 (for 12–14) or N-tert-butyloxy-
carbonyl-glycine 17 (for 14 and 15) to give the dipeptides
18–22.
Scheme 2. Reagents, conditions and yields: (i) chloral, CHCl3, reflux, 6 h (77%); (ii) LDA, THF, K78 8C, MeI, EtI, CH3CH2]CH2Br or PhCH2Br, K78–O
K30 8C, 4 h, 8, 63%, 9, 46%, 10, 60%, 11, 2.5 h, 32%; (iii) SOCl2, CH3OH, reflux, 3 h, 12, 100%, 13, 71%, AcCl, CH3OH, reflux, 24 h, 14, 63%, 15, 48%;
(iv) for 18, 19, 20: Et3N, BoPCl, 16, CH2Cl2, rt, 18, 20.5 h, 92%, 19, 19.5 h, 46%, 20, 20 h, 30%; for 21, 22: Et3N, BoPCl, 17, CH2Cl2, rt, 19.5 h, 21, 45%, 22,
18.5 h, 22%; (v) dioxane, 1 M aqueous NaOH, rt, 15–20 h, 23, 90%, 24, 95%, 25, 92%, 26, 83%, 27, 95%; (vi) for 30, 31, 32: Et3N, BoPCl, 28, CH2Cl2, rt, 17 h,
30, 89%, 31, 17.5 h, 70%, 32, 19.5 h, 76%; for 33, 34: Et3N, BoPCl, 29, CH2Cl2, rt, 17.5 h, 33, 77%, 34, 17 h, 68%; (vii) H2, 10% Pd/C, CH3OH/H2O (90:10),
rt, 23 h, 1, 86%, 2, 20 h, 99%, 3, 19 h, 100%; (viii) CF3CO2H, CH2Cl2, rt, 6.5 h, 4, 96%, 5, 3.5 h, 100%.

10020
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
Scheme 3. Reagents, conditions and yields: (i) O3, MeOH/CH2Cl2 (1:1), 15 min then PPh3, 24 h, then silica gel, 63%; (ii) CF3CO2H/Et3SiH/CH2Cl2 (1:1:1), rt,
45 min, 96%; (iii) 10% Pd/C, CH3OH/H2O (88:12), 18 h, 35, 78%, 36, 99%.
The optimal conditions for the amide bond formation
were investigated using the coupling between 12 and 16;
bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl) was
found to be superior (92% yield compared to 66% with
DCC) and was used for all subsequent coupling reactions.
Hydrolysis of the methyl esters 18,19,20 (NaOH in dioxane)
to the carboxylic acids 23,24,25 followed by coupling
(BoPCl) with dibenzyl glutamate 28 afforded benzyl
protected tripeptides 30,31,32. Finally, global deprotection
of the benzyl groups gave tripeptides 132 and 2 whilst
concomitant hydrogenolysis of the allyl group in 25 gave
tripeptide 3.
conformation and different ring systems have been shown
to mimic both b-38 and g-turns.39 A spirocyclic g-lactam
bridge can be formed between the 2-position of the proline
residue and the nitrogen of the glutamate residue in GPE
thus, presenting an opportunity to investigate the effect of
such conformational restriction in GPE analogues.
The synthesis of GP-[5.5]spirolactamE 35 and the corre-
sponding GP-[5.5]hydroxyspirolactamE 36 is summarized
in Scheme 3.
Ozonolysis of alkene 32 followed by treatment with
triphenylphosphine proceeded via the intermediacy of
aldehyde 37 to give alcohols 38 as a 1:1 mixture of
diastereoisomers. Direct hydrogenation of 38 gave the
hydroxyspirolactam 36 whereas initial reduction of the
hydroxyl group (trifluoroacetic acid–triethylsilane–dichloro-
methane) to 39 before the hydrogenolysis step afforded
spirolactam 35. Both spirolactams adopted exclusively the
trans conformation about the proline ring.
For the synthesis of the tripeptides 4 and 5 incorporating a
2-allylproline and a 2-benzylproline unit, respectively, Boc
and t-butyl protecting groups were used. In these cases
coupling of acids 26 and 27 with di-tert-butyl glutamate 29
gave tripeptides 33 and 34 affording tripeptides 4 and 5 as
trifluoroacetate salts after deprotection with TFA.
In contrast to most peptide bonds that adopt exclusively the
trans conformation, the amide bond between Xaa-Pro can
exist as a mixture of cis and trans isomers.33 The nature of
the conformation about this bond can affect the biological
activity of a peptide and there is evidence that some
proteases only recognize the trans peptide bond.34,35 The
existence of specific peptidyl-prolyl cis-trans isomerases
would seem to corroborate this evidence.36 GPE is present
as a 20:80 cis-trans mixture of isomers in D2O solution as
established by NMR analysis.22 When an alkyl group is
substituted at the 2-position of proline the trans confor-
mation is preferred. Compounds 1–4 adopt the all trans
conformation and the trans population also increased in
compound 5 with only 10% adopting the cis conformation.
The prevalence of the trans isomer in 2-methylproline
compounds has been attributed to the bulky methyl group
destabilizing the cis conformation.37
The pyrrolidine ring of proline is capable of adopting two
distinct conformations. These down- and up-puckered
conformations are defined as occurring when Cg and the
carbonyl group of proline lie on the same and opposite sides,
respectively, of the plane defined by Cd, N and Ca. The
presence of a sulfur atom in the proline ring can affect the
bond angles and bond lengths, in some cases altering
the proline ring conformation. Kang found that replacement
of the proline residue in AcProNHMe with 4-thiaproline 40
[(R)-thiozolidine-4-carboxylic acid (Thz)] resulted in a
more puckered conformation.40 Further conformational
changes in the proline ring can be promoted by the addition
of methyl groups at the 5 position of proline or Thz.41–43
The next set of analogues incorporated such pseudo-proline
moieties where the g-CH2 of Pro was replaced with sulfur
and/or with dimethyl substitution at Cd.
Another method of conformationally constraining a peptide
is to synthesize a peptidomimetic containing a spirolactam
ring system. It has been suggested that a spirolactam ring
system may lock a compound into predominantly one
The pseudo-prolines: 4-thiaproline 40 [(R)-thiozolidine-4-
carboxylic acid (Thz)] and 2,2-dimethylthiazolidine-4-
carboxylic acid 41 were easily accessed by the reaction of

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10021
Scheme 4. Reagents, conditions and yields: (i) 37% aqueous HCHO, H2O, rt, 22 h, then pyridine, 56%; (ii) dimethoxypropane, acetone, reflux, 2 h, 58%.
cysteine 42 with formaldehyde44 or 2,2-dimethoxypro-
pane,45,46 respectively (Scheme 4).
Boc-protected methyl D,L-5,5-dimethylprolinate 43 was
prepared from nitrile 44 (Scheme 5). Nitrile 44 was
prepared as described in the literature,47,48 however,
subsequent hydrolysis of the nitrile moiety and hydro-
genation of the intermediate N-oxide as described49 was
concomitant with acid catalysed methyl migration yielding
1
a mixture of 45 and 46. (6:4 ratio, H NMR). Extensive
modification of the hydrolysis reaction could not overcome
Scheme 5. Reagents, conditions and yields: (i) 32% aqueous HCl, 50 8C
5 h; (ii) H2 (44 psi), 10% Pd/C, MeOH/H2O (1:1), 20 h; (iii) SOCl2, MeOH,
0 8C to rt, overnight then Boc2O, N-methylmorpholine, CH2Cl2, reflux, 48 h
[43 (22%) 47 (42%)] over four steps.
the formation of N-methyl compound 45. It is interesting
that this unwanted reaction has not been reported during the
synthesis of 5,5-dimethylproline that has been described by
several research groups.49,50
Scheme 6. Reagents, conditions and yields: (i) 17, i-BuOCOCl, Et3N, THF, 0 8C to rt, then 40, Et3N, H2O, rt, 2 h, 52, 81%; (ii) 51, 41, i-Pr2EtN, DMF, rt, 18 h
then MeOH, Me3SiCl, rt, 15 h, 53, 65%; (iii) 43, CF3CO2H, CH2Cl2, rt, 2 h then 17, BoPCl, i-Pr2EtN, CH2Cl2, rt, 15 h, 54, 52%; (iv) dioxane, 1 M aqueous
NaOH, rt, 21 h, 55, 94%; (v) for 56: EtOCOCl, Et3N, CH2Cl2, 0 8C, 35 min then 29, Et3N, CH2Cl2, 0 8C to rt, 15 h, 56, 54%; for 57, 58: BoPCl, i-Pr2EtN,
CH2Cl2, 28, rt, 7 h, 57, 68%, 58, 24 h, 67%; (vi) CF3CO2H, Et3SiH, CH2Cl2, rt, 4 h, 48, 61%; (vii) H2(42 psi), 10% Pd/C, CH3OH/H2O (80:20), 24 h, 49, 48%;
(viii) CF3CO2H, CH2Cl2, rt, 75 min then H2, 10% Pd/C, CH3OH/H2O (80:20), 15 h, 50, 93%.

10022
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
Protection of both the acid and amine functionalities as a
methyl ester and a tert-butyloxy carbamate (Boc), respect-
ively, allowed facile separation and characterisation of the
protected 5,5-dimethylproline 43 (22% yield over four
steps) and the N-methyl by-product 47 (42% yield, over four
steps). The desired protected 5,5-dimethylproline 43 existed
as a mixture of epimers (55:45) exclusively as the cis
conformer.
(compounds 35 and 36). In contrast, dimethylation at C-5 on
the proline destabilises the trans conformation resulting in
an increased population of the cis conformer (compounds 49
and 50). These GP*E mimetics are valuable tools to provide
information about the influence of the proline residue on the
bioactivity of the parent peptide GPE.
4. Experimental
The tripeptides 48–50 were synthesized in a similar fashion
to the 2-alkylproline analogues (Scheme 6). Coupling of the
4-thia-proline building block 40 to Boc-glycine 17 was
carried out using a mixed anhydride activation procedure
whereas the more hindered 5,5-dimethyl-4-thia-proline 41
required use of the more reactive acid fluoride 51 to afford
53. In the case of the 5,5-dimethylproline 43 the Boc group
was removed with trifluoroacetic acid before BoPCl
coupling with Boc-glycine 17 to afford 54. Hydrolysis of
the methyl ester then afforded acid 55 in preparation for the
second peptide coupling.
4.1. General details
All reactions were conducted in flame-dried or oven-dried
glassware under a dry nitrogen atmosphere unless otherwise
noted. All reagents were used as supplied. Tetrahydrofuran
was dried over sodium/benzophenone and distilled prior to
use. Flash chromatography was performed using Merck
Kieselgel 60 (230–400 mesh) with the indicated solvents.
Thin-layer chromatography (TLC) was carried out on
precoated silica plates (Merck Kieselgel 60F254) and
compounds were visualized by UV fluorescence and heating
of plates dipped in anisaldehyde in ethanolic sulphuric acid
or alkaline potassium permanganate solution. Melting
points in degrees Celsius (8C) were measured on an
Electrothermalw melting point apparatus and are uncor-
rected. Infrared spectra were recorded with a Perkin Elmer
1600 series Fourier-transform infrared spectrometer as thin
films between sodium chloride plates. Absorption maxima
are expressed in wavenumbers (cmK1) with the following
abbreviations: sZstrong, mZmedium, wZweak and brZ
broad nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker AVANCE DRX400 (1H, 400 MHz;
13C, 100 MHz), a Bruker AVANCE 300 (1H, 300 MHz;
13C, 75 MHz) or a Bruker AC200 (1H, 200 MHz; 13C,
50 MHz) spectrometer at 298 K. For 1H NMR data,
chemical shifts are described in parts per million (ppm)
relative to tetramethylsilane (d 0.00), DOH (d 4.75),
CHD2OD (d 3.30) or CHD2S(O)CD3 (d 2.50) and are
reported consecutively as position (dH), relative integral,
multiplicity (sZsinglet, dZdoublet, tZtriplet, qZquartet,
pZpentet, qZquintet, sZsextet, ddZdoublet of doublets,
mZmultiplet, and where brZbroad), coupling constant
(J/Hz) and assignment. For 13C NMR data, chemical shifts
(ppm) are referenced internally to CDCl3 (d 77.0), CD3OD
(d 49.1) and (CD3)2S(O) (d 39.4) or externally to
3-(trimethylsilyl)-1-propanesulfonic acid sodium salt
(DSS) and are reported consecutively as position (dC),
degree of hybridisation and assignment. The asterisk*
denotes resonances assigned to the minor conformer. High
resolution mass spectra were recorded using a VG70-SE
spectrometer operating at nominal accelerating voltage of
70 eV. Chemical ionisation (CI) mass spectra were obtained
with ammonia as the reagent gas. Optical rotations were
measured at 20 8C on a Perkin Elmer 341 polarimeter using
10 cm path length cells and are given in units of 10K1
degrees cm2 gK1. Samples were prepared in the solvent
indicated at the concentration specified (measured in
g/100 cm3).
Coupling of the pseudo dipeptides 52, 53 and 55 with either
dibenzyl glutamate 28 or or di-tert-butyl glutamate 29 using
either a mixed anhydride protocol or BoPCl gave the desired
peptides 56, 57 and 58. The nature of the final deprotection
step depended on the protecting groups employed in the
synthesis thus, for 57 removal of the benzyloxycarbonyl and
benzyl groups by hydrogenolysis provided tripeptide 49.
The Boc and tert-butyl ester groups in 56 were removed
using trifluoroacetic acid to give tripeptide 48 as the
trifluoroacetate salt whereas for the deprotection of 58,
treatment with trifluoroacetic acid followed by hydro-
genolysis afforded tripeptide 50.51
The presence of a sulfur atom at C-4 in the pyrrolidine ring
of proline, by itself did not appear to significantly alter the
conformation of the peptide about the Gly-Pro bond. In
the GPE analogue 48 the cis:trans ratio was established to be
20:80, unchanged from the native peptide. The presence of
the two methyl groups at C-5 had a more dramatic influence
on the conformation with the cis:trans ratio dramatically
shifted to favour the cis conformer. The population of the cis
conformer in 5,5-dimethylated peptide 50 increased to 72%
compared with the 20% seen with GPE. An even greater
effect was observed with analogue 49, which exhibited a
85:15 cis:trans ratio indicating that the presence of a sulphur
atom at C-4 in combination with two methyl groups at C-5
in the proline ring plays a key role in determining the ratio
of cis:trans isomers about the Gly-Pro bond. The high
population of the cis conformer in related 5,5-dimethyl-
prolines has been attributed to the effects of steric hindrance
due to the methyl groups when the compound adopts the
trans conformation.41
3. Conclusions
In summary, we herein report the synthesis of ten analogues
of GPE. In five of these analogues the trans conformation
about the Gly-Pro* bond was stabilized by either the
presence of a hydrophobic alkyl group at C-2 on the proline
(compounds 1–5) or by a spirolactam bridge between the
2-position of the proline and the nitrogen of the glutamate
4.1.1. (2R,5S)-2-Trichloromethyl-1-aza-3-oxabicyclo-
[3.3.0]octan-4-one 7.28,31 A suspension of L-proline
(10.0 g, 86.8 mmol) and chloral hydrate (21.6 g,
130 mmol) were heated under reflux in chloroform

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10023
(100 cm3) for 6 h with a reverse Dean-Stark trap. The
solution was washed with water (2!30 cm3) and the water
washings were extracted with chloroform (50 cm3). The
combined organic layers were dried (MgSO4), filtered and
the solvent removed in vacuo to afford a light brown solid
(19.8 g). The crude product was recrystallised from ethanol
(80 cm3) at 40 8C to form oxazolidinone 7 (16.1 g, 77%) as a
white solid: mp 107–109 8C (lit.31 ethanol, 107.6 8C): [a]D
C34.2 (c 2 in C6H6), lit.28 [a]D C33 (c 2.0 in C6H6); dH
(200 MHz; CDCl3) 1.67–2.29 (4H, m, Prob-H2 and Prog-
H2), 3.08–3.20 (1H, m, Prob-HAHB), 3.37–3.49 (1H, m,
Prob-HAHB), 4.09–4.15 (1H, m, Proa-H) and 5.17 (1H, s,
NCH); dC (50 MHz; CDCl3) 25.3 (CH2, Prog-C), 29.9
(CH2, Prob-C), 57.9 (CH2, Prod-C), 62.4 (CH, Proa-C),
100.6 [quat., C(Cl3)], 103.6 (CH, NCH) and 175.5 (quat.,
CO); m/z (EIC) 244 (MHC244).
preparation of oxazolidinone 8 using n-butyllithium
(1.31 M, 9.93 cm3, 13.0 mmol), diisopropylamine (1.82 cm3,
13.0 mmol), oxazolidinone 7 (2.10 g, 8.7 mmol) and allyl
bromide (2.25 cm3, 26.0 mmol) to afford oxazolidinone 10
(1.48 g, 60%) as a light orange oil for which the NMR data
were in agreement with the literature.28
4.1.5. (2R,5R)-5-Benzyl-2-trichloromethyl-1-aza-3-oxa-
bicyclo[3.3.0]octan-4-one 11.28 The reaction was carried
out following a similar procedure to that described for the
preparation of oxazolidinone 8 using n-butyllithium
(1.31 M, 5.53 cm3, 7.2 mmol), diisopropylamine
(1.01 cm3, 7.24 mmol), oxazolidinone
7
(1.18 g,
4.8 mmol) and benzyl bromide (1.72 cm3, 14.5 mmol) to
afford oxazolidinone 11 (0.52 g, 32%) as a colourless
crystalline solid: mp 75–77 8C (lit.28 72–77); dH (400 MHz;
CDCl3) 1.32–1.55 (2H, m, Prog-H2), 1.93–2.13 (2H, m,
Prob-H2), 2.58–2.65 (1H, m, Prod-H2), 2.92 (1H, d, JZ
13.6 Hz, PhCHAHB), 2.98–3.03 (1H, m, Prod-H2), 3.32
(1H, d, JZ13.6 Hz, PhCHAHB), 4.99 (1H, s, NCH) and
7.21–7.35 (5H, m, PhH); dC (100 MHz; CDCl3) 24.8 (CH2,
Prog-C), 34.6 (CH2, Prob-C), 41.6 (CH2, Prod-C), 58.4
(CH2, PhCH2), 72.3 (quat., Proa-C), 100.6 (quat., CCl3),
102.8 (CH, NCH), 127.0 (CH, Ph), 128.2 (CH, Ph), 130.9
(CH, Ph), 135.5 (quat., Ph) and 176.6 (quat., C]O); m/z
4.1.2. (2R,5S)-5-Methyl-2-trichloromethyl-1-aza-3-oxa-
bicyclo[3.3.0]octan-4-one 8. n-Butyllithium (1.31 M,
4.68 cm3, 6.14 mmol) was added dropwise to a stirred
solution of diisopropylamine (0.86 cm3, 6.14 mmol) in dry
tetrahydrofuran (10 cm3) at K78 8C under an atmosphere of
nitrogen. The solution was stirred for 5 min, warmed to 0 8C
and stirred for 15 min. The solution was added dropwise to a
solution of oxazolidinone 7 (1.00 g, 4.09 mmol) in dry
tetrahydrofuran (20 cm3) at K78 8C over 20 min (reaction
mixture turned dark), stirred for a further 30 min then
iodomethane (0.76 cm3, 12.3 mmol) added dropwise over
5 min. The solution was warmed to K50 8C over 2 h. Water
(15 cm3) was added, the solution warmed to room
temperature and extracted with chloroform (3!40 cm3).
The combined organic extracts were dried (MgSO4), filtered
and evaporated to dryness in vacuo to give a dark brown
semi-solid. Purification of the residue by flash column
chromatography (15% ethyl acetate–hexane) afforded
oxazolidinone 8 (0.67 g, 63%) as a pale yellow solid: mp
55–57 8C (lit.28 57–60 8C); dH (300 MHz; CDCl3) 1.53 (3H,
s, CH3), 1.72–2.02 (3H, m, Prob-H and Prog-H2), 2.18–2.26
(1H, m, Prob-H), 3.15–3.22 (1H, m, Prod-H), 3.35–3.44
(1H, m, Prod-H) and 4.99 (1H, s, NCH).
(EIC) 333.0081 [(MCH)C C14H Cl3NO2 requires
35
14
333.0090], 335.0069 [(MCH)C C14H Cl237ClNO2 requires
35
14
35
14
335.0061], 337.0014 [(MCH)C C14H Cl 37Cl2NO2
requires 337.0031] and 339.0009 [(MCH)C C14H14
37Cl3NO2 requires 339.0002].
4.1.6. Methyl L-2-methylprolinate hydrochloride 12.
Thionyl chloride (4.30 mL, 58.9 mmol) was added dropwise
cautiously to a stirred solution of 8 (7.57 g, 29.4 mmol) at
0 8C under a nitrogen atmosphere. The cooling bath was
removed and mixture stirred at room temperature for 20 min
then heated to reflux for 3 h. The volatiles were removed in
vacuo, the residue suspended in toluene (20 mL) and
concentrated at 50 8C to remove traces of thionyl chloride.
Trituration with dry ether yielded a brown solid. The
yellow/orange ether was decanted and the solid was shaken
with dry ether, decanted and the procedure repeated until the
ether was colourless. Removal of traces of ether in vacuo at
50 8C afforded 12 (ca. 5.0 g, 100%) as a free flowing,
hygroscopic brown solid that was used without any further
purification: mp 107–109 8C (lit.52 106–108 8C).
4.1.3. (2R,5S)-5-Ethyl-2-trichloromethyl-1-aza-3-oxabi-
cyclo[3.3.0]octan-4-one 9. The reaction was carried out
following a similar procedure to that described for the
preparation of oxazolidinone 8 using n-butyllithium
(1.31 M, 28.3 cm3, 37.1 mmol), diisopropylamine
(5.2 cm3, 37.1 mmol), oxazolidinone 7 (6.0 g, 24.7 mmol)
and iodoethane (5.9 cm3, 73.8 mmol) to afford oxazolidi-
none 9 (3.05 g, 46%) as a light red oil that solidified on
standing to a pale brown solid: mp 76–77 8C: [a]D C18.5 (c
0.25 in CHCl3); dH (300 MHz; CDCl3) 1.04 (3H, t, JZ
7.5 Hz, CH3), 1.60–1.80 (1H, m, CHAHBCH3), 1.72–1.99
(4H, m, CHAHBCH3, Prob-HAHB and Prog-H2), 2.20–2.30
(1H, m, Prob-HAHB), 3.22–3.29 (2H, m, Prod-H2) and 5.00
(1H, s, NCH); dC (75 MHz; CDCl3) 8.4 (CH3, CH3), 25.5
(CH2, CH2CH3), 30.9 (CH2, Prog-C), 35.6 (CH2, Prob-C),
58.6 (CH2, Prod-C), 72.5 (quat., Proa-C), 100.9 [quat.,
C(Cl3)], 102.5 (CH, NCH) and 176.9 (quat., CO); m/z (EIC)
4.1.7. Methyl L-2-ethylprolinate hydrochloride 13. An
ice-cooled solution of oxazolidinone 9 (2.86 g, 10.6 mmol)
in dry methanol (35 cm3) under an atmosphere of nitrogen
was treated dropwise with a solution of thionyl chloride
(2.3 cm3, 31.5 mmol). The solution was heated under reflux
for 3 h, cooled and the solvent removed under reduced
pressure. The resultant brown oil was purified by flash
column chromatography (10% methanol–dichloromethane)
to afford hydrochloride 13 (1.45 g, 71%) as a light brown
semi-solid: [a]D K61.1 (c 0.3 in CHCl3); dH (300 MHz;
CDCl3) 1.07 (3H, t, JZ7.3 Hz, CH3), 1.95–2.33 (5H, m,
CH2CH3, Prog-H2 and Prob-HAHB), 2.43–2.47 (1H, Prob-
HAHB), 3.63 (3H, s, OCH3) and 6.98–7.35 (2H, br s, NH2);
dC (75 MHz; CDCl3) 9.9 (CH3, CH3), 22.8 (CH2, CH2CH3),
28.9 (CH2, Prog-C), 35.1 (CH2, Prob-C), 45.7 (CH2, Prod-
C), 53.8 (CH3, OCH3), 73.9 (quat., Proa-C) and 171.0
272.0014 (MHC C9H ClN3O2 requires 272.0012).
35
13
4.1.4. (2R,5R)-5-Allyl-2-trichloromethyl-1-aza-3-oxabi-
cyclo[3.3.0]octan-4-one 10.28 The reaction was carried
out following a similar procedure to that described for the

10024
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
(quat., CO); m/z (EIC) 158.1181 (MHC C8H16NO2 requires
158.1181).
1329, 1310, 1284, 1257, 1220, 1195, 1172, 1135, 1107,
1082, 1052, 1029, 986, 965, 907, 876, 829, 775, 738 and
699; dH (300 MHz; CDCl3) 1.49 (3H, s, CH3), 1.77–2.11
(4H, m, Prob-H2 and Prog-H2), 3.43–3.48 (2H, m, Prod-H2),
3.61 (3H, s, OCH3), 3.85–3.89 (2H, m, Glya-H2), 5.04 (2H,
s, PhCH2), 5.76 (1H, br s, N–H) and 7.21–7.28 (5H, s, ArH);
dC (75 MHz; CDCl3) 21.1 (CH3, Proa-CH3), 23.5 (CH2,
Prog-C), 38.0 (CH2, Prob-C), 43.3 (CH2, Glya-C), 46.6
(CH2, Prod-C), 52.1 (CH3, OCH3), 66.0 (quat., Proa-C),
66.3 (CH2, PhCH2), 127.5 (CH, Ph), 127.6 (CH, Ph), 128.1
(CH, Ph), 136.2 (quat., Ph), 155.9 (quat., NCO2), 166.0
(quat., Gly-CON) and 173.6 (quat., CO2CH3); m/z (EIC)
334.1535 (MC C17H22N2O5 requires 334.1529).
4.1.8. Methyl L-2-allylprolinate hydrochloride 14.28,53 An
ice-cooled solution of oxazolidinone 10 (0.64 g, 2.24 mmol)
in dry methanol (15 cm3) was treated dropwise with a
solution of acetyl chloride (0.36 cm3, 5.0 mmol) in
methanol (5 cm3). The solution was heated under reflux
for 24 h, cooled and the solvent removed under reduced
pressure. The resultant brown oil was dissolved in toluene
(40 cm3) and concentrated to dryness to remove residual
thionyl chloride and methanol, then purified by flash column
chromatography (5–10% CH3OH–CH2Cl2; gradient
elution) to afford hydrochloride 14 (0.29 g, 63%) as a
solid for which the NMR data were in agreement with that
reported in the literature.28,53 dH (300 MHz; CDCl3) 1.72–
2.25 (3H, m, Prob-HAHB and Prog-H2), 2.32–2.52 (1H, m,
Prob-HAHB), 2.72–3.10 (2H, m, Prod-H2), 3.31–3.78 (2H,
m, CH2CH]CH2), 3.84 (3H, s, CO2CH3), 5.20–5.33 (2H,
m, CH]CH2), 5.75–5.98 (1H, m, CH]CH2) and 8.06 (1H,
br s, N–H); m/z (CIC) 170.1183 [(MCH)C C9H16NO2
requires 170.1181].
4.1.11. Methyl N-benzyloxycarbonyl-glycyl-L-2-ethyl-
prolinate 19. Dry triethylamine (2.88 cm3, 20.7 mmol)
was added dropwise to a solution of hydrochloride 13
(1.14 g, 5.9 mmol) and N-benzyloxycarbonylglycine 16
(2.47 g, 11.8 mmol) in dry dichloromethane (100 cm3)
under an atmosphere of nitrogen at 0 8C, and the reaction
mixture stirred for 10 min. Bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride (3.00 g, 11.8 mmol) was added and
the solution was stirred for 2 h, warmed to room
temperature and further stirred for 17.5 h. Dichloromethane
(50 cm3) was added and the solution washed successively
with 0.5 M aqueous hydrochloric acid (2!50 cm3) and
saturated aqueous sodium hydrogen carbonate (2!50 cm3),
dried (MgSO4), filtered and evaporated in vacuo to give a
light orange gum. Purification of the resultant residue by
flash column chromatography (40% ethyl acetate/hexane)
yielded ester 19 (0.95 g, 46%) as a clear oil: [a]D K9.2
(c 0.13 in CHCl3); dH (300 MHz; CDCl3) 0.81 (3H, t, JZ
7.5 Hz, CH3), 1.85–2.09 (5H, m, CH2CH3, Prog-H2 and
Prob-HAHB), 2.38 (1H, sextet, JZ7.5 Hz, Prob-HAHB),
3.43–3.47 (1H, m, Prod-HAHB), 3.61–3.67 (1H, m, Prod-
HAHB), 3.70 (3H, s, OCH3), 4.10–4.13 (2H, m, Glya-H2)
5.11 (2H, s, OCH2Ph), 5.71 (1H, br s, Gly-NH) and 7.27–
7.35 (5H, m, Ph); dC (75 MHz; CDCl3) 8.3 (CH3, CH3), 24.1
(CH2, CH2CH3), 26.5 (CH2, Prog-C), 35.3 (CH2, Prob-C),
44.1 (CH2, Glya-C), 48.2 (CH2, Prod-C), 52.9 (CH3,
OCH3), 67.0 (CH2, OCH2Ph), 70.2 (quat., Proa-C), 128.2
(CH, Ph), 128.3 (CH, Ph), 128.7 (CH, Ph), 136.8 (quat., Ph),
156.5 (quat., NCO), 166.8 (quat., Gly-CON) and 174.5
(quat., CO2CH3); m/z (EIC) 348.1688 (MHC C18H24N2O5
requires 348.1685).
4.1.9. Methyl L-2-benzylprolinate hydrochloride 15.54 An
ice-cooled solution of oxazolidinone 11 (1.03 g, 3.07 mmol)
in dry methanol (10 cm3) was treated dropwise with a
solution of acetyl chloride (0.71 cm3, 10.0 mmol) in
methanol (10 cm3). The solution was heated under reflux
for 24 h, cooled and the solvents removed under reduced
pressure. The resultant brown oil was dissolved in toluene
(80 cm3), concentrated to dryness to remove residual thionyl
chloride and methanol, then purified by flash column
chromatography (5% CH3OH–CH2Cl2) to afford hydro-
chloride 1528,53 (0.38 g, 48%) as a beige solid; dH
(400 MHz; D2O) 1.92–2.01 (1H, m, Prog-HAHB), 2.11–
2.23 (2H, m, Prob-HAHB and Prog-HAHB), 2.52–2.60 (1H,
m, Prob-HAHB), 3.19 (1H, d, JZ14.3 Hz, PhCHAHB), 3.24–
3.31 (1H, m, Prod-HAHB), 3.37–3.43 (1H, m, Prod-HAHB),
3.53 (1H, d, JZ14.3 Hz, PhCHAHB), 3.83 (3H, s, CO2CH3)
and 7.26–7.47 (5H, m, PhH); dC (100 MHz; D2O) 24.4
(CH2, Prog-C), 36.8 (CH2, Prob-C), 43.8 (CH2, PhCH2),
47.6 (CH2, Prod-C), 56.0 (CH3, OCH3), 75.9 (quat., Proa-
C), 130.4 (CH, Ph), 131.5 (CH, Ph), 131.7 (CH, Ph), 137.1
(quat., Ph) and 175.8 (quat., C]O); m/z (CIC) 220.1340
[(MCH)C C13H18NO2 requires 220.1338].
4.1.10. Methyl N-benzyloxycarbonyl-glycyl-L-2-methyl-
prolinate 18. Dry triethylamine (0.27 cm3, 1.96 mmol) was
added dropwise to a solution of hydrochloride 12 (0.11 g,
0.61 mmol) and N-benzyloxycarbonylglycine 16 (0.17 g,
0.79 mmol) in dry dichloromethane (35 cm3) under an
atmosphere of nitrogen at room temperature and the
reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazolidi-
nyl)phosphinic chloride (0.196 g, 0.77 mmol) was added
and the resultant colourless solution was stirred for 20.5 h.
The solution was washed successively with 10% aqueous
hydrochloric acid (30 cm3) and saturated aqueous sodium
hydrogen carbonate (30 cm3), dried (MgSO4), filtered and
evaporated to dryness in vacuo. Purification of the resultant
residue by flash column chromatography (50–80% ethyl
acetate–hexane; gradient elution) yielded ester 18 (0.18 g,
92%) as a colourless oil: [a]D K33.0 (c 1.0 in MeOH); nmax
(film)/cmK1 3406, 2952, 1732, 1651, 1521, 1434, 1373,
4.1.12. Methyl N-benzyloxycarbonyl-glycyl-L-2-allyl-
prolinate 20. Dry triethylamine (1.07 cm3, 7.70 mmol)
was added dropwise to a solution of hydrochloride 14
(0.50 g, 2.41 mmol) and N-benzyloxycarbonyl-glycine 16
(0.65 g, 3.13 mmol) in dry dichloromethane (80 cm3) under
an atmosphere of nitrogen at room temperature, and the
reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazo-
lidinyl)phosphinic chloride (0.772 g, 3.03 mmol) was added
and the solution stirred for 20 h, then washed successively
with 10% aqueous hydrochloric acid (80 cm3) and saturated
aqueous sodium hydrogen carbonate (80 cm3), dried
(MgSO4), filtered and evaporated to dryness in vacuo.
Purification of the resultant residue by flash column
chromatography (60% ethyl acetate–hexane, seven drops
of Et3N for every 200 cm3) yielded ester 20 (0.26 g, 30%) as
a yellow oil: [a]D C46.0 (c 0.50 in CH2Cl2); nmax (film)/
cmK1 3405, 3066, 3032, 2953, 2877, 1723, 1655, 1586,

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10025
1507, 1434, 1373, 1333, 1309, 1248, 1169, 1121, 1083,
1047, 1027, 1002, 919, 866, 827, 776, 737 and 699; dH
(300 MHz; CDCl3) 1.92–2.17 (4H, m, Prob-H2 and Prog-
H2), 2.60–2.67 (1H, m, CHAHBCH]CH2), 3.09–3.16 (1H,
m, CHAHBCH]CH2), 3.35–3.42 (1H, m, Prod-HAHB),
3.56–3.63 (1H, m, Prod-HAHB), 3.70 (3H, s, OCH3), 3.96
(2H, d, JZ4.4 Hz, Glya-H2), 5.07–5.12 (4H, m, PhCH2 and
CH]CH2), 5.58–5.70 (1H, m, CH]CH2) and 7.27–7.35
(5H, s, PhH); dC (75 MHz; CDCl3) 23.6 (CH2, Prog-C), 34.9
(CH2, Prob-C), 37.6 (CH2, CH2CH]CH2), 43.6 (CH2,
Glya-C), 47.5 (CH2, Prod-C), 52.5 (CH3, OCH3), 66.7
(CH2, PhCH2), 68.8 (quat., Proa-C), 119.4 (CH2, CH]H2),
127.9 (CH, Ph), 128.0 (CH, Ph), 128.4 (CH, Ph), 132.8 (CH,
CH]CH2), 136.4 (quat., Ph), 156.1 (quat., NCO2), 166.4
(quat., Gly-CON) and 173.7 (quat., CO2CH3); m/z (EIC)
360.1682 (MC C19H24N2O5 requires 360.1685).
chromatography (50% ethyl acetate–hexane) yielded ester
22 (0.11 g, 22%) as a pale yellow oil: [a]D C105.3 (c 0.99
in CH2Cl2); nmax (film)/cmK1 3419, 3061, 3028, 2977,
2873, 1799, 1739, 1715, 1655, 1582, 1497, 1454, 1432,
1392, 1366, 1330, 1250, 1167, 1121, 1093, 1049, 1026, 948,
915, 865, 819, 765, 736, 706 and 653; dH (300 MHz; CDCl3)
1.08–1.12 (1H, m, Prog-HAHB), 1.47 [9H, s, C(CH3)3],
1.67–1.72 (1H, m, Prog-HAHB), 2.01–2.17 (2H, m, Prob-
H2), 2.86–2.92 (1H, m, Prod-HAHB), 3.08 (1H, d, JZ
13.8 Hz, PhCHAHB), 3.36–3.42 (1H, m, Prod-HAHB), 3.75
(3H, s, OCH3), 3.83 (1H, m, PhCHAHB), 3.90 (2H, d, JZ
3.6 Hz, Glya-CH2), 5.54 (1H, br s, N–H) and 7.06–7.30
(5H, m, PhH); dC (100 MHz; CDCl3) 23.4 (CH2, Prog-C),
28.4 [CH3, C(CH3)3], 34.7 (CH2, Prob-C), 37.8 (CH2,
PhCH2), 43.6 (CH2, Glya-C), 47.4 (CH2, Prod-C), 52.6
(CH3, OCH3), 69.6 (quat., Proa-C), 79.6 [quat., C(CH3)3],
126.8 (CH, Ph), 128.3 (CH, Ph), 130.5 (CH, Ph), 136.7
(quat., Ph), 155.8 (quat., NCO2), 167.2 (quat., Gly-CON)
and 174.0 (quat., CO2CH3); m/z (EIC) 376.2001 (MC
C20H28N2O5 requires 376.1998).
4.1.13. Methyl N-tert-butyloxycarbonyl-glycyl-L-2-allyl-
prolinate 21. Dry triethylamine (0.28 cm3, 2.02 mmol) was
added dropwise to a solution of hydrochloride 14 (0.13 g,
0.63 mmol) and N-tert-butyloxycarbonylglycine 17 (0.14 g,
0.82 mmol) in dry dichloromethane (35 cm3) under an
atmosphere of nitrogen at room temperature, and the
reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazolidi-
nyl)phosphinic chloride (0.20 g, 0.80 mmol) was added and
the solution stirred for 19.5 h, then washed successively
with 10% aqueous hydrochloric acid (35 cm3) and saturated
aqueous sodium hydrogen carbonate (35 cm3), dried
(MgSO4), filtered and evaporated to dryness in vacuo.
Purification of the resultant residue by flash column
chromatography (40% ethyl acetate–hexane) yielded ester
21 (0.09 g, 45%) as a light yellow oil: [a]D C33.8 (c 0.83 in
CH2Cl2); nmax (film)/cmK1 3419, 3075, 2977, 2930, 2874,
1739, 1715, 1656, 1499, 1434, 1392, 1366, 1332, 1268,
1248, 1212, 1168, 1122, 1051, 1026, 1003, 943, 919, 867,
830, 779, 739, 699 and 679; dH (300 MHz; CDCl3) 1.42
[9H, s, C(CH3)3], 1.93–2.08 (4H, m, Prob-H2 and Prog-H2),
2.59–2.67 (1H, m, CHAHBCH]CH2), 3.09–3.16 (1H, m,
CHAHBCH]CH2), 3.35–3.44 (1H, m, Prod-HAHB), 3.56–
3.62 (1H, m, Prod-HAHB), 3.70 (3H, s, OCH3), 3.89 (2H, d,
JZ4.2 Hz, Glya-H2), 5.06–5.11 (2H, m, CH]CH2), 5.42
(1H, br s, Gly-NH) and 5.58–5.72 (1H, m, CH]CH2); dC
(75 MHz; CDCl3) 23.7 (CH2, Prog-C), 28.3 [CH3,
C(CH3)3], 35.0 (CH2, Prob-C), 37.6 (CH2, CH2CH]CH2),
43.3 (CH2, Glya-C), 47.5 (CH2, Prod-C), 52.5 (CH3,
OCH3), 68.8 (quat., Proa-C), 79.5 [quat., C(CH3)3], 119.4
(CH2, CH]CH2), 132.9 (CH, CH]CH2), 155.7 (quat.,
NCO2), 166.9 (quat., Gly-CON) and 173.8 (quat., CO2CH3);
m/z (EIC) 326.1845 (MC C16H26N2O5 requires 326.1842).
4.1.15. N-Benzyloxycarbonyl-glycyl-L-2-methylproline
23. To a solution of mthyl ester 18 (0.56 g, ca.
1.67 mmol) in 1,4-dioxane (33 cm3) was added dropwise
1 M aqueous sodium hydroxide (10 cm3, 10 mmol) and the
mixture was stirred for 19 h at room temperature.
Dichloromethane (100 cm3) was then added and the organic
layer extracted with saturated aqueous sodium hydrogen
carbonate (2!100 cm3). The combined aqueous layers
were carefully acidified with dilute hydrochloric acid,
extracted with dichloromethane (2!100 cm3), and the
combined organic layers dried (MgSO4), filtered, and
concentrated to dryness in vacuo. Purification of the ensuing
residue (0.47 g) by flash column chromatography (50%
ethyl acetate–hexane to 30% methanol–dichloromethane;
gradient elution) gave acid 23 (0.68 g, 90%) as a white
foam: [a]D K62.3 (c 0.20 in CH2Cl2); nmax (film)/cmK1
3583, 3324 br, 2980, 2942, 1722, 1649, 1529, 1454, 1432,
1373, 1337, 1251, 1219, 1179, 1053, 1027, 965, 912, 735
and 698; dH (300 MHz; CDCl3) 1.59 (3H, s, Proa-CH3),
1.89 (1H, 6 lines, JZ18.8, 6.2, 6.2 Hz, Prob-HAHB), 2.01
(2H, dtt, JZ18.7, 6.2, 6.2 Hz, Prog-H2), 2.25–2.40 (1H, m,
Prob-HAHB), 3.54 (2H, t, JZ6.6 Hz, Prod-H2), 3.89 (1H,
dd, JZ17.1, 3.9 Hz, Glya-HAHB), 4.04 (1H, dd, JZ17.2,
5.3 Hz, Glya-HAHB), 5.11 (2H, s, OCH2Ph), 5.84 (1H, br t,
JZ4.2 Hz, N–H), 7.22–7.43 (5H, m, Ph) and 7.89 (1H, br s,
–COOH); dC (75 MHz; CDCl3) 21.3 (CH3, Proa-CH3), 23.8
(CH2, Prog-C), 38.2 (CH2, Prob-C), 43.6 (CH2, Glya-C),
47.2 (CH2, Prod-C), 66.7 (quat., Proa-C), 66.8 (CH2,
OCH2Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4, (CH,
Ph), 136.4 (quat., Ph), 156.4 (quat., NCO2), 167.5 (quat.,
Gly-CON) and 176.7 (quat., CO); m/z (EIC) 320.1368 (MC
C16H20N2O5 requires 320.1372).
4.1.14. Methyl N-tert-butyloxycarbonyl-glycyl-L-2-ben-
zylprolinate 22. Dry triethylamine (0.59 cm3, 4.22 mmol)
was added dropwise to a solution of hydrochloride 15
(0.34 g, 1.32 mmol) and N-tert-butyloxycarbonylglycine 17
(0.30 g, 1.71 mmol) in dry dichloromethane (70 cm3) under
an atmosphere of nitrogen at room temperature, and the
reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazo-
lidinyl)phosphinic chloride (0.42 g, 1.66 mmol) was added
and the solution stirred for 18.5 h, then washed successively
with 10% aqueous hydrochloric acid (70 cm3) and saturated
aqueous sodium hydrogen carbonate (70 cm3), dried
(MgSO4), filtered and evaporated to dryness in vacuo.
Purification of the resultant residue by flash column
4.1.16. N-Benzyloxycarbonyl-glycyl-L-2-ethylproline 24.
To a solution of methyl ester 19 (0.39 g, 1.11 mmol) in
dioxane (15 cm3) was added dropwise 1 M NaOH (7.5 cm3,
7.50 mmol) and the mixture was stirred for 20 h at room
temperature. The solution was acidified with 1 M HCl and
evaporated in vacuo. The resulting aqueous layer was
extracted with dichloromethane (2!30 cm3), dried
(MgSO4), filtered and evaporated in vacuo to form a clear
gum, which solidified on standing to acid 24 (0.35 g, 95%)

10026
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
as a colourless solid, which was used without further
purification: [a]D K9.9 (c 0.11 in MeOH); dH (300 MHz;
CDCl3) 0.83 (3H, t, JZ7.4 Hz, CH3), 1.93–2.22 (5H, m,
CH2CH3, Prog-H2 and Pro-bHAHB), 2.35–2.40 (1H, m, Pro-
bHAHB), 3.43–3.46 (1H, m, Prod-HAHB), 3.57–3.62 (1H, m,
Prod-HAHB), 4.01 (2H, dd, JZ4.3, 17.3 Hz, Glya-H2), 5.11
(2H, s, OCH2Ph), 5.82 (1H, br s, Gly-NH), 7.26–7.36 (5H,
m, Ph) and 7.70 (1H, br s, CO2H); dC (75 MHz; CDCl3) 8.5
(CH3, CH3), 23.9 (CH2, CH2CH3), 26.7 (CH2, Prog-C), 34.8
(CH2, Prob-C), 44.1 (CH2, Glya-C), 48.7 (CH2, Prod-C),
67.3 (CH2, OCH2Ph), 71.9 (quat., Proa-C), 127.3 (CH, Ph),
127.9 (CH, Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 128.9 (CH,
Ph), 136.7 (quat., Ph), 156.7 (quat., NCO2), 168.9 (quat.,
Gly-CON) and 175.8 (quat., CO2H); m/z (EIC) 334.1523
(MC C17H22N2O5 requires 334.1529).
Prob-HAHB), 2.63–2.70 (1H, m, CHAHBCH]CH2), 3.04–
3.11 (1H, m, CHAHBCH]CH2), 3.35–3.47 (1H, m, Prod-
HAHB), 3.53–3.62 (1H, m, Prod-HAHB), 3.92 (2H, m, Glya-
H2), 5.08–5.13 (2H, m, CH]CH2), 5.52 (1H, br s, Gly-NH),
5.56–5.71 (1H, m, CH]CH2) and 8.31 (1H, br s, OH); dC
(75 MHz; CDCl3) 23.5 (CH2, Prog-C), 28.3 [CH3,
C(CH3)3], 34.6 (CH2, Prob-C), 37.5 (CH2, CH2CH]CH2),
43.4 (CH2, Glya-C), 48.1 (CH2, Prod-C), 69.8 (quat., Proa-
C), 79.8 [quat., C(CH3)3], 119.8 (CH2, CH]CH2), 132.3
(CH, CH]CH2), 155.8 (quat., NCO2), 168.5 (quat., Gly-
CON) and 175.9 (quat., CO2H); m/z (EIC) 312.1692 (MC
C15H24N2O5 requires 312.1685).
4.1.19. N-tert-Butyloxycarbonyl-glycyl-L-2-benzyl-
proline 27. To a solution of ester 22 (0.098 g, 0.26 mmol)
in dioxane (5.2 cm3) was added dropwise 1 M aqueous
NaOH (1.56 cm3, 1.56 mmol) and the mixture was stirred
for 15 h at room temperature. Dichloromethane (20 cm3)
was then added and the organic layer extracted with
saturated aqueous sodium bicarbonate (3!20 cm3). Careful
acidification of the combined aqueous layers with dilute
hydrochloric acid, extraction with dichloromethane (3!
20 cm3), drying of the combined organic layers (MgSO4),
filtration and concentration to dryness gave the acid 27
(0.09 g, 95%) as a colourless glass-like solid: mp 74–77 8C:
[a]D C69.8 (c 0.99 in CH2Cl2); nmax (CH2Cl2)/cmK1 3415,
3060, 3028, 2978, 2879, 2621, 1711, 1655, 1497, 1454,
1392, 1367, 1252, 1167, 1120, 1092, 1081, 1049, 1029, 988,
942, 915, 887, 872, 814, 764, 736, 705 and 653; dH
(400 MHz; CDCl3) 1.14–1.21 (1H, m, Prog-HAHB), 1.50
[9H, s, C(CH3)3], 1.72–1.78 (1H, m, Prog-HAHB), 2.11–
2.29 (2H, m, Prob-H2), 2.94–3.00 (1H, m, Prod-HAHB),
3.13 (1H, d, JZ13.9 Hz, PhCHAHB), 3.42–3.48 (1H, m,
Prod-HAHB), 3.83 (1H, d, JZ13.9 Hz, PhCHAHB), 4.13
(2H, m, Glya-H2), 5.69 (1H, br s, Gly-NH), 7.10–7.33 (5H,
m, PhH) and 8.37 (1H, br s, OH); dC (100 MHz; CDCl3)
23.3 (CH2, Prog-C), 28.3 [CH3, C(CH3)3], 34.5 (CH2, Prob-
C), 37.8 (CH2, PhCH2), 43.7 (CH2, Glya-C), 47.8 (CH2,
Prod-C), 70.3 (quat., Proa-C), 79.8 [quat., C(CH3)3], 126.9
(CH, Ph), 128.4 (CH, Ph), 130.5 (CH, Ph), 136.3 (quat., Ph),
156.0 (quat., NCO2), 168.6 (quat., Gly-CON) and 176.9
(quat., CO2H); m/z (EIC) 362.1834 (MC C19H26N2O5
requires 362.1842).
4.1.17. N-Benzyloxycarbonyl-glycyl-L-2-allylproline 25.
To a solution of ester 20 (0.12 g, 0.34 mmol) in dioxane
(7 cm3) was added dropwise 1 M aqueous NaOH (2.06 cm3,
2.06 mmol) and the mixture stirred for 20 h at room
temperature. Dichloromethane (25 cm3) was then added
and the organic layer extracted with saturated aqueous
sodium bicarbonate (3!25 cm3). Careful acidification of
the combined aqueous layers with dilute hydrochloric acid,
extraction with dichloromethane (3!25 cm3), drying of the
combined organic layers (MgSO4), filtration and concen-
tration to dryness gave the acid 25 (0.11 g, 92%) as a yellow
oil: [a]D K3.16 (c 0.95 in CH2Cl2); nmax (film)/cmK1 3408,
2957, 2923, 2850, 2622, 1715, 1650, 1531, 1453, 1375,
1333, 1259, 1214, 1173, 1121, 1083, 1056, 1028, 1002, 916,
823, 737 and 698; dH (300 MHz; CDCl3) 1.93–2.07 (3H, m,
Prob-HAHB and Prog-H2), 2.22–2.26 (1H, m, Prob-HAHB),
2.62–2.69 (1H, m, CHAHBCH]CH2), 3.04–3.11 (1H, m,
CHAHBCH]CH2), 3.31–3.62 (2H, m, Prod-H2), 3.91–4.05
(2H, m, Glya-H2), 5.08–5.13 (3H, m, PhCH2 and
CH]CHAHB), 5.55–5.72 (1H, m, CH]CHAHB), 5.89
(1H, m, CH]CH2), 7.29–7.45 (5H, m, PhH) and 8.12 (1H,
br s, N–H); dC (75 MHz; CDCl3) 23.5 (CH2, Prog-C), 34.6
(CH2, Prob-C), 37.5 (CH2, CH2CH]CH2), 43.7 (CH2,
Glya-C), 48.1 (CH2, Prod-C), 66.9 (CH2, PhCH2), 69.9
(quat., Proa-C), 119.8 (CH2, CH]CH2), 127.97 (CH, Ph),
128.04 (CH, Ph), 128.4 (CH, Ph), 132.3 (CH, CH]CH2),
136.4 (quat., Ph), 156.4 (quat., NCO2), 168.2 (quat., Gly-
CON) and 176.1 (quat., CO2H); m/z (EIC) 346.1526 (MC
C18H22N2O5 requires 346.1529).
4.1.20. Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-
methylprolyl-L-glutamate 30. Triethylamine (0.50 cm3,
3.59 mmol) was added dropwise to a solution of dipeptide
23 (0.36 g, 1.12 mmol) and L-glutamic acid dibenzyl ester
p-toluenesulphonate 28 (0.73 g, 1.46 mmol) in dichloro-
methane (60 cm3) under nitrogen at room temperature, and
the reaction mixture stirred for 10 min. Bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (0.37 g, 1.41 mmol) was
added and the colourless solution stirred for 17 h. The
dichloromethane solution was washed successively with
10% aqueous hydrochloric acid (50 cm3) and saturated
aqueous sodium hydrogen carbonate (50 cm3), dried
(MgSO4), filtered, and evaporated to dryness in vacuo.
Purification of the resultant residue by repeated flash
column chromatography (30–70% ethyl acetate–hexane;
gradient elution) yielded protected tripeptide 30 (0.63 g,
89%) as a colourless oil. Tripeptide 30 was shown to adopt
exclusively the trans conformer by NMR: Rf 0.55 (EtOAc):
[a]D K41.9 (c 0.29 in CH2Cl2); nmax (film)/cmK1 3583,
4.1.18. N-tert-Butyloxycarbonyl-glycyl-L-2-allylproline
26. To a solution of ester 21 (0.039 g, 0.12 mmol) in
dioxane (2.4 cm3) was added dropwise 1 M aqueous NaOH
(0.72 cm3, 0.72 mmol) and the mixture was stirred for 16 h
at room temperature. Dichloromethane (10 cm3) was then
added and the organic layer extracted with saturated
aqueous sodium bicarbonate (3!10 cm3). Careful acidifi-
cation of the combined aqueous layers with dilute
hydrochloric acid, extraction with dichloromethane (3!
10 cm3), drying of the combined organic layers (MgSO4),
filtration and concentration to dryness gave acid 26
(0.031 g, 83%) as a yellow oil: [a]D K15.8 (c 0.89 in
CH2Cl2); nmax (film)/cmK1 3414, 3076, 2978, 2931, 2620,
1713, 1654, 1510, 1454, 1434, 1392, 1367, 1268, 1250,
1213, 1169, 1121, 1056, 1028, 920, 869, 779, 736 and 701;
dH (300 MHz; CDCl3) 1.43 [9H, s, C(CH3)3], 1.95–2.15
(3H, m, Prob-HAHB and Prog-CH2), 2.21–2.35 (1H, m,

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10027
3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257,
1214, 1188, 1166, 1051, 911, 737 and 697; dH (400 MHz;
CDCl3; Me4Si) 1.64 (3H, s, Proa-CH3), 1.72 (1H, dt, JZ
12.8, 7.6, 7.6 Hz, Prob-HAHB), 1.92 (2H, 5 lines, JZ6.7 Hz,
Prog-H2), 2.04 (1H, 6 lines, JZ7.3 Hz Glub-HAHB), 2.17–
2.27 (1H, m, Glub-HAHB), 2.35–2.51 (3H, m, Prob-HAHB
and Glug-H2), 3.37–3.57 (2H, m, Prod-H2), 3.90 (1H, dd,
JZ17.0, 3.6 Hz, Glya-HAHB), 4.00 (1H, dd, JZ17.1,
5.1 Hz, Glya-HAHB), 4.56 (1H, td, JZ7.7, 4.9 Hz, Glua-
H), 5.05–5.20 (6H, m, 3!OCH2Ph), 5.66–5.72 (1H, br m,
Gly-NH), 7.26–7.37 (15H, m, 3!Ph) and 7.44 (1H, d, JZ
7.2 Hz, Glu-NH); dC (100 MHz; CDCl3) 21.9 (CH3, Proa-
CH3), 23.4 (CH2, Prog-C), 26.6 (CH2, Glub-C), 30.1 (CH2,
Glug-C), 38.3 (CH2, Prob-C), 43.9 (CH2, Glya-C), 47.6
(CH2, Prod-C), 52.2 (CH, Glua-C), 66.4 (CH2, OCH2Ph),
66.8 (CH2, OCH2Ph), 67.1 (CH2, OCH2Ph), 68.2 (quat.,
Proa-C), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.1, (CH, Ph),
128.2, (CH, Ph), 128.2, (CH, Ph), 128.3, (CH, Ph), 128.4,
(CH, Ph), 128.5, (CH, Ph), 128.5, (CH, Ph), 135.2 (quat.,
Ph), 135.7 (quat., Ph), 136.4 (quat., Ph), 156.1 (quat.,
NCO2), 167.3 (quat., Gly-CO), 171.4 (quat., CO), 172.9
(quat., CO) and 173.4 (quat., CO); m/z (FABC) 630.2809
(MHC C35H40N3O8 requires 630.2815).
0.49 mmol) was added dropwise to a solution of acid 25
(0.05 g, 0.15 mmol) and L-glutamic acid dibenzyl ester
p-toluenesulphonate 28 (0.10 g, 0.20 mmol) in dry dichloro-
methane (8.2 cm3) under an atmosphere of nitrogen at room
temperature, and the reaction mixture stirred for 10 min.
Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.05 g,
0.19 mmol) was added and the solution was stirred for
19.5 h. The solution was washed successively with 10%
aqueous hydrochloric acid (7 cm3) and saturated aqueous
sodium hydrogen carbonate (7 cm3), dried (MgSO4),
filtered and evaporated to dryness in vacuo. Purification
of the resultant residue by flash column chromatography
(50–80% ethyl acetate–hexane; gradient elution) yielded
protected tripeptide 32 (0.08 g, 76%) as a colourless oil:
[a]D K27.8 (c 0.79 in CH2Cl2); nmax (film)/cmK1 3943,
3583, 3413, 3055, 3032, 2982, 2956, 2880, 2685, 2411,
2305, 1955, 1732, 1668, 1586, 1499, 1454, 1423, 1388,
1329, 1265, 1214, 1169, 1170, 1081, 1058, 1028, 994, 924,
897, 824, 737 and 701; dH (400 MHz; CDCl3) 1.85 (3H, m,
Prob-HAHB and Prog-H2), 1.99–2.08 (1H, m, Glub-HAHB),
2.17–2.25 (1H, m, Glub-HAHB), 2.32–2.49 (3H, m, Prob-
HAHB and Glug-H2), 2.71–2.76 (1H, m, CHAHBCH]CH2),
3.05–3.10 (1H, m, CHAHBCH]CH2), 3.33 (1H, m, Prod-
HAHB), 3.51 (1H, m, Prod-HAHB), 3.96 (2H, d, JZ3.9 Hz,
Glya-H2), 4.55 (1H, dd, JZ7.6, 5.1 Hz, Glua-H), 5.07–5.19
(8H, m, 3!PhCH2 and CH]CH2), 5.53–5.63 (1H, m,
CH]CH2), 5.71 (1H, br s, Gly-NH), 7.32–7.36 (15H, m,
3!Ph) and 7.90 (1H, d, JZ7.2 Hz, Glu-NH); dC (100 MHz;
CDCl3) 23.1 (CH2, Prog-C), 26.7 (CH2, Glub-C), 30.2
(CH2, Glug-C), 34.2 (CH2, Prob-C), 37.9 (CH2, CH2-
CH]CH2), 44.1 (CH2, Glya-C), 48.5 (CH2, Prod-C), 52.2
(CH, Glua-C), 66.4 (CH2, PhCH2), 66.9 (CH2, PhCH2), 67.2
(CH2, PhCH2), 71.9 (quat., Proa-C), 119.9 (CH2,
CH]CH2), 127.9 (CH, Ph), 128.05 (CH, Ph), 128.1 (CH,
Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 128.45
(CH, Ph), 128.5 (CH, Ph), 132.1 (CH, CH]CH2), 135.3
(quat., Ph), 135.7 (quat., Ph), 136.4 (quat., Ph), 156.2 (quat.,
NCO2), 168.1 (quat., Gly-CO), 171.3 (quat., Glua-CO),
172.7 (quat., Glug-CO) and 173.0 (quat., Pro-CON); m/z
(FABC) 656.2970 (MHC C37H42N3O8 requires 656.2992).
4.1.21. Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-ethyl-
prolyl-L-glutamate 31. Dry triethylamine (0.44 cm3,
3.16 mmol) was added to a solution of acid 24 (0.30 g,
0.91 mmol) and L-glutamic acid dibenzyl ester p-toluene
sulphonate 28 (0.57 g, 1.13 mmol) in dry dichloromethane
(50 cm3) under an atmosphere of nitrogen at 0 8C, and the
reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazolidi-
nyl)phosphinic chloride (0.29 g, 1.14 mmol) was added and
the solution stirred for 2 h, warmed to room temperature
and further stirred for 17.5 h. The solution was washed
successively with 0.5 M aqueous hydrochloric acid
(10 cm3) and saturated aqueous sodium hydrogen carbonate
(10 cm3), dried (MgSO4), filtered and evaporated in vacuo
to form a light orange gum. Purification of the resultant
residue by flash column chromatography (30% ethyl
acetate/hexane) yielded protected tripeptide 31 (0.41 g,
70%) as a clear oil: [a]D K52.7 (c 0.16 in MeOH); dH
(300 MHz; CDCl3) 0.78 (3H, t, JZ7.4 Hz, CH3), 1.25–2.24
(7H, m, CH2CH3, Glub-H2, Prog-H2 and Prob-HAHB),
2.34–2.40 (2H, m, Glug-H2), 2.50–2.60 (1H, m, Prob-
HAHB), 3.34 (1H, q, JZ9.4 Hz, Prod-HAHB), 3.49–3.53
(1H, m, Prod-HAHB), 3.96 (2H, ddd, JZ4.9, 17.3 Hz, Glya-
H2), 4.51–4.54 (1H, td, JZ5.4, 7.8 Hz, Glua-H), 5.06–5.19
(6H, m, 3!OCH2Ph), 5.70 (1H, br s, Gly-NH), 7.26–7.36
(15H, 3!Ph) and 8.09 (1H, d, JZ7.3 Hz, Glu-NH); dC
(75 MHz; CDCl3) 8.8 (CH3, CH3), 23.6 (CH2, CH2CH3),
27.2 (CH2, Prog-C), 27.7 (CH2, Glub-C), 30.6 (CH2, Glug-
C), 34.3 (CH2, Prob-C), 44.5 (CH2, Glya-C), 49.0 (CH2,
Prod-C), 52.6 (CH, Glua-C), 66.9 (CH2, OCH2Ph), 67.3
(CH2, OCH2Ph), 67.5 (CH2, OCH2Ph), 73.9 (quat., Proa-C),
128.4 (CH, Ph), 128.5 (CH, Ph), 128.6 (CH, Ph), 128.7 (CH,
Ph), 128.8 (CH, Ph), 128.9 (CH, Ph), 135.7 (quat., Ph),
136.1 (quat., Ph), 136.8 (quat., Ph), 156.6 (quat., NCO2),
168.7 (quat., Gly-CON), 171.8 (quat., Pro-CON), 172.9
(quat., Glua-CO) and 173.6 (quat., Glug-CO); m/z (FABC)
644.2981 (MHC C36H42N3O8 requires 644.2972).
4.1.23. Di-tert-butyl N-tert-butyloxycarbonyl-glycyl-L-2-
allylprolyl-L-glutamate 33. Dry triethylamine (0.044 cm3,
0.32 mmol) was added dropwise to a solution of acid 26
(0.031 g, 0.10 mmol) and L-glutamic acid di-tert-butyl ester
hydrochloride 29 (0.038 g, 0.129 mmol) in dry dichloro-
methane (5.30 cm3) under an atmosphere of nitrogen at
room temperature, and the reaction mixture stirred for
10 min. Bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(0.032 g, 0.13 mmol) was added and the solution stirred
for 17.5 h. The solution was washed successively with 10%
aqueous hydrochloric acid (5 cm3) and saturated aqueous
sodium hydrogen carbonate (5 cm3), dried (MgSO4),
filtered and evaporated to dryness in vacuo. Purification of
the resultant residue by flash column chromatography (50%
ethyl acetate–hexane) yielded protected tripeptide 33
(0.43 g, 77%) as a light yellow oil: [a]D K29.9 (c 0.90 in
CH2Cl2); nmax (film)/cmK1 3583, 3417, 3076, 2978, 2931,
1728, 1664, 1522, 1453, 1426, 1392, 1367, 1329, 1251,
1158, 1056, 1028, 949, 919, 846 and 735; dH (300 MHz;
CDCl3) 1.26 [9H, s, C(CH3)3], 1.42 [9H, s, C(CH3)3],
1.43 [9H, s, C(CH3)3], 1.85–1.94 (4H, m, Prob-HAHB,
Glub-HAHB and Prog-H2), 2.02–2.12 (1H, m, Glub-HAHB),
4.1.22. Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-allyl-
prolyl-L-glutamate 32. Dry triethylamine (0.07 cm3,

10028
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
2.16–2.33 (2H, m, Glug-H2), 2.48–2.53 (1H, m, Prob-
HAHB), 2.69–2.77 (1H, m, 0.5 CHAHBCH]CH2), 3.08–
3.15 (1H, m, CHAHBCH]CH2), 3.29–3.38 (1H, m, Prod-
HAHB), 3.53–3.56 (1H, m, Prod-HAHB), 3.91 (2H, d, JZ
4.0 Hz, Glya-H2), 4.33 (1H, dd, JZ7.5, 5.2 Hz, Glua-H),
5.08–5.14 (2H, m, CH]CH2), 5.47 (1H, br s, Gly-NH),
5.52–5.66 (1H, m, CH]CH2) and 7.77 (1H, d, JZ7.4 Hz,
Glu-NH); dC (75 MHz; CDCl3) 23.1 (CH2, Prog-C), 27.4
(CH2, Glub-C), 27.9 [CH3, C(CH3)3], 28.0 [CH3, C(CH3)3],
28.3 [CH3, C(CH3)3], 31.5 (CH2, Glug-C), 34.2 (CH2, Prob-
C), 38.0 (CH2, CH2CH]CH2), 43.7 (CH2, Glya-C), 48.4
(CH2, Prod-C), 52.7 (CH, Glua-C), 71.8 (quat., Proa-C),
79.5 [quat., C(CH3)3], 80.6 [quat., C(CH3)3], 81.9 [quat.,
C(CH3)3], 119.8 (CH2, CH]CH2), 132.3 (CH, CH]CH2),
155.7 (quat., NCO2), 168.4 (quat., Gly-CO), 170.8 (quat.,
Glua-CO), 172.3 (quat., Glug-CO) and 172.8 (quat., Pro-
CON); m/z (EIC) 553.3359 (MC C28H47N3O8 requires
553.3363).
in 90:10 methanol–water (22 cm3) was stirred under an
atmosphere of hydrogen at room temperature, protected
from light, for 23 h. The reaction mixture was filtered
through a Celitee pad and the pad washed with 75:25
methanol–water (200 cm3). The filtrate was concentrated to
dryness under reduced pressure and the residue triturated
with anhydrous diethyl ether to afford tripeptide 1 (0.27 g,
86%) as an hygroscopic colourless solid. Tripeptide 1 was
shown to adopt the trans conformation by NMR analysis:
mp 144 8: [a]D K52.4 (c 0.19 in H2O); dH (400 MHz; D2O)
1.62 (3H, s, Proa-CH3), 1.97–2.25 (6H, m, Prob-H2, Prog-
H2 and Glub-H2), 2.45 (2H, t, JZ7.3 Hz, Glug-H2), 3.62–
3.70 (2H, m, Prod-H2), 3.96 (1H, d, JZ16.5 Hz, Glya-
HAHB), 4.02 (1H, d, JZ16.4 Hz, Glya-HAHB) and 4.28
(1H, dd, JZ8.4, 4.7 Hz, Glua-H); dC (100 MHz; D2O) 19.9
(CH3, Proa-CH3), 23.0 (CH2, Prog-C), 26.9 (CH2, Glub-C),
30.9 (CH2, Glug-C), 38.8 (CH2, Prob-C), 40.7 (CH2, Glya-
C), 47.5 (CH2, Prod-C), 54.4 (CH, Glua-C), 67.8 (quat.,
Proa-C), 164.6 (quat., Gly-CO), 175.3 (quat., Pro-CON),
177.2 (quat., Glua-CO), and 178.5 (quat., Glug-CO); m/z
(FABC) 316.1508 (MHC C13H22N3O6 requires 316.1509).
4.1.24. Di-tert-butyl N-tert-butyloxycarbonyl-glycyl-L-2-
benzylprolyl-L-glutamate 34. Dry triethylamine (0.11 cm3,
0.77 mmol) was added dropwise to a solution of acid 27
(0.09 g, 0.24 mmol) L-glutamic acid di-tert-butyl ester
hydrochloride 29 (0.09 g, 0.31 mmol) in dry dichloro-
methane (13 cm3) under an atmosphere of nitrogen at room
temperature, and the reaction mixture stirred for 10 min.
Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.08 g,
0.30 mmol) was added and the solution stirred for 17 h,
then washed successively with 10% aqueous hydrochloric
acid (12 cm3) and saturated aqueous sodium hydrogen
carbonate (12 cm3), dried (MgSO4), filtered and evaporated
to dryness in vacuo. Purification of the resultant residue by
flash column chromatography (40% ethyl acetate–hexane)
yielded protected tripeptide 34 (0.10 g, 68%) as a colourless
oil: [a]D C15.7 (c 1.15 in CH2Cl2); nmax (film)/cmK1 3418,
3357, 3060, 3028, 2978, 2933, 2875, 1727, 1663, 1582,
1519, 1497, 1454, 1426, 1392, 1367, 1330, 1251, 1158,
1093, 1051, 1029, 949, 914, 846, 736, 705 and 651; dH
(400 MHz; CDCl3) 1.28–1.39 (1H, m, Prog-HAHB), 1.43
[27H, s, C(CH3)3], 1.47 [27H, s, C(CH3)3], 1.48 [27H, s,
C(CH3)3], 1.69–1.74 (1H, m, Prog-HAHB), 1.89–1.99 (2H,
m, Prob-H2), 2.08–2.17 (1H, m, Glub-HAHB), 2.23–2.41
(3H, m, Glub-HAHB and Glug-H2), 2.89–2.95 (1H, m, Prod-
HAHB), 3.13 (1H, d, JZ13.4 Hz, PhCHAHB), 3.40–3.46
(1H, m, Prod-HAHB), 3.85 (1H, d, JZ13.4 Hz, PhCHAHB),
3.92 (2H, d, JZ3.8 Hz, Glya-H2), 4.33 (1H, td, JZ7.5,
5.2 Hz, Glua-H), 5.58 (1H, br s, Gly-NH), 7.07–7.28 (5H,
m, PhH) and 7.71 (1H, d, JZ7.2 Hz, Glu-NH); dC
(100 MHz; CDCl3) 23.0 (CH2, Prog-C), 27.4 (CH2, Glub-
C), 28.0 [CH3, C(CH3)3], 28.1 [CH3, C(CH3)3], 28.3 [CH3,
C(CH3)3], 31.5 (CH2, Glug-C), 34.2 (CH2, Prob-C), 38.0
(CH2, PhCH2), 44.1 (CH2, Glya-C), 48.2 (CH2, Prod-C),
52.9 (CH, Glua-C), 72.4 (quat., Proa-C), 79.6 [quat.,
C(CH3)3], 80.7 [quat., C(CH3)3], 82.1 [quat., C(CH3)3],
126.8 (CH, Ph), 128.3 (CH, Ph), 130.4 (CH, Ph), 136.3
(quat., Ph), 155.8 (quat., NCO2), 168.6 (quat., Gly-CO),
171.0 (quat., Glua-CO), 172.5 (quat., Glug-CO) and 173.1
(quat., Pro-CON); m/z (FABC) 604.3592 (MHC
C32H50N3O8 requires 604.3598).
4.1.26. Glycyl-L-2-ethylprolyl-L-glutamic acid 2. A
mixture of protected tripeptide 31 (0.51 g, 0.80 mmol) and
10% palladium on activated carbon (0.09 g, 0.08 mmol) in
90:10 methanol–water (50 cm3) was stirred under an
atmosphere of hydrogen at room temperature for 20 h.
The solution was filtered through a Celitee pad, the pad
washed with methanol (2!30 cm3) and the filtrate
evaporated to dryness to give a clear gum. The gum was
placed under vacuum for 30 min then triturated with
anhydrous diethyl ether to tripeptide 2 (0.26 g, 99%) as an
hygroscopic colourless solid. Tripeptide 2 was shown to be
exclusively the trans conformer by 1H and 13C NMR
analysis: mp 82–85 8C: [a]D K43.8 (c 0.1 in MeOH); dH
(400 MHz; D2O) 0.86 (3H, t, JZ7.4 Hz, CH3), 1.94–2.40
(8H, m, CH2CH3, Glub-H2, Prob-H2 and Pro-gH2), 2.52–
2.56 (2H, m, Glug-H2), 3.55–3.61 (1H, td, JZ6.9, 9.7 Hz,
Prod-HAHB), 3.75–3.80 (1H, m, Prod-HAHB), 4.08 (2H, q,
JZ16.6 Hz, Glya-H2) and 4.44 (1H, q, JZ4.9 Hz, Glua-H);
dC (100 MHz; CDCl3) 6.9 (CH3, CH3), 22.8 (CH2,
CH2CH3), 25.3 (CH2, Prog-C), 25.5 (CH2, Glub-C), 30.11
(CH2, Glug-C), 35.0 (CH2, Prob-C), 40.7 (CH2, Glya-C),
48.6 (CH2, Prod-C), 52.6 (CH, Glua-C), 71.7 (quat., Proa-
C), 164.9 (quat., Gly-CON), 175.2 (quat., Pro-CON) 176.0
(quat., Glua-CO) and 177.3 (quat., Glug-CO); m/z (FABC)
330.1667 (MHC C14H24N3O6 requires 330.1665).
4.1.27. Glycyl-L-2-propylprolyl-L-glutamic acid 3. A
mixture of protected tripeptide 32 (64 mg, 0.097 mmol)
and 10% palladium on activated carbon (20 mg, 0.19 mmol)
in 90:10 methanol–water (9.8 cm3) was stirred under an
atmosphere of hydrogen at room temperature, protected
from light, for 19 h. The reaction mixture was filtered
through a Celitee pad and the pad washed with 75:25
methanol–water (50 cm3). The filtrate was concentrated to
dryness under reduced pressure to afford tripeptide 3
(33 mg, 100%) as a colourless solid. Tripeptide 3 was
shown to be exclusively the trans conformer by NMR
analysis: mp 278–280 8C (dec.): [a]D K16.7 (c 0.18 in
H2O); dH (300 MHz; D2O) 0.98 (3H, t, CH2CH3), 1.09
(1H, m, CHAHBCH3), 1.44 (1H, m, CHAHBCH3), 1.82–2.31
(8H, m, Prob-H2, Prog-H2, Glub-H2 and CH2CH2CH3),
4.1.25. Glycyl-L-2-methylprolyl-L-glutamic acid 1. A
mixture of the protected tripeptide 30 (0.63 g, 1.00 mmol)
and 10% palladium on activated carbon (0.32 g, 0.30 mmol)

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10029
2.40 (1H, m, Glug-H2), 3.55–3.63 (1H, m, Prod-HAHB),
3.80 (1H, m, Prod-HAHB), 4.03 (1H, d, JZ16.6 Hz, Glya-
HAHB), 4.15 (1H, d, JZ16.6 Hz, Glya-HAHB) and 4.27
(1H, dd, JZ8.2, 4.8 Hz, Glua-H); dC (100 MHz; D2O) 16.0
(CH3, CH2CH3), 19.0 (CH2, CH2CH3), 25.4 (CH2, Prog-C),
30.9 (CH2, Glub-C), 36.3 (CH2, Glug-C), 37.4 (CH2,
CH2CH2CH3), 38.3 (CH2, Prob-C), 43.3 (CH2, Glya-C),
51.2 (CH2, Prod-C), 58.1 (CH, Glua-C), 74.1 (quat., Proa-
C), 167.7 (quat., NCO), 177.8 (quat., Pro-CON), 180.9
(quat., Glua-CO) and 184.7 (quat., Glug-CO); m/z (FABC)
344.1827 (MHC C15H26N3O6 requires 344.1822).
Glub-C), 32.5 (CH2, Glug-C), 37.7 (CH2, Prob-C), 39.2
(CH2, PhCH2), 43.5 (CH2, Glya-C), 50.6 (CH2, Prod-C),
54.9 (CH, Glua-C), 56.0* (CH, Glua-C), 73.8 (quat., Proa-
C), 117.0 (quat., CF3), 129.6 (CH, Ph), 131.1 (CH, Ph),
132.9 (CH, Ph), 138.3 (quat., Ph), 165.4 (quat., CF3CO2),
167.7 (quat., Gly-CO), 177.2 (quat., Pro-CON), 178.0
(quat., Glua-CO) and 179.7 (quat., Glug-CO); m/z
(FABC) 392.1817 (MCKCF3CO2 C19H26N3O6 requires
392.1822).
4.1.30. Dibenzyl (2S,50R,80R)- and (2S,50R,80S)-[10-(200-
benzyloxycarbonylamino-acetyl)-80-hydroxy-60-oxo-10,
70-diazaspiro[4.4]non-70-yl]-1,5-pentanedioate
38.
4.1.28. Glycyl-L-2-allylprolyl-L-glutamic acid trifluoro-
acetate 4. To a solution of the protected tripeptide 33
(41 mg, 0.073 mmol) in dichloromethane (4.5 cm3) at room
temperature was added trifluoroacetic acid (0.75 cm3,
9.74 mmol) dropwise and the reaction mixture was stirred
for 6.5 h. The solution was evaporated under reduced
pressure to form tripeptide 4 (32 mg, 96%) as a pale yellow
solid. Tripeptide 4 was shown to exist exclusively as the
trans-conformer by NMR analysis: mp 105–108 8C: [a]D
K7.64 (c 0.39 in H2O); dH (400 MHz; D2O) 1.90–2.02 (1H,
m, Prog-HAHB), 2.03–2.14 (2H, m, Prog-HAHB and Glub-
HAHB), 2.19–2.32 (3H, m, Prob-H2 and Glub-HAHB), 2.54
(2H, ddd, JZ8.1, 7.3, 2.0 Hz, Glug-H2), 2.74 (1H, dd, JZ
13.7, 7.3 Hz, CHAHBCH]CH2), 3.12 (1H, dd, JZ13.7, 7.3,
0.5 Hz CHAHBCH]CH2), 3.48–3.55 (1H, m, Prod-HAHB),
3.72–3.77 (1H, m, Prod-HAHB), 3.98 (1H, d, JZ16.7 Hz,
Glya-HAHB), 4.10 (1H, d, JZ16.7 Hz, Glya-HAHB), 4.46
(1H, dd, JZ4.9, 9.5 Hz, Glua-H) 5.20–5.26 (2H, m,
CH]CH2) and 5.73–5.82 (1H, m, CH]CH2); dC
(100 MHz; D2O) 25.4 (CH2, Prog-C), 28.1 (CH2, Glub-
C), 32.7 (CH2, Glug-C), 38.1 (CH2, Prob-C), 39.1 (CH2,
CH2CH]CH2), 43.3 (CH2, Glya-C), 51.0 (CH2, Prod-C),
55.1 (CH, Glua-C), 73.2 (quat., Proa-C), 120.3 (quat., CF3),
122.6 (CH2, CH]CH2), 134.4 (CH, CH]CH2), 165.4
(quat., CF3CO2), 167.5 (quat., Gly-CO), 177.5 (quat., Pro-
CON), 178.0 (quat., Glya-CO) and 179.8 (quat., Glug-CO);
m/z (EIC) 342.1653 (MCKCF3CO2 C15H24N3O6 requires
342.1665).
Alkene 32 (96 mg, 0.15 mmol) was dissolved in dry
dichloromethane–methanol (6 cm3, 1/1) and the solution
cooled to K78 8C. A slow stream of ozone was bubbled
through the solution for 15 min, followed by O2 to remove
excess ozone. Triphenylphosphine (57.5 mg, 0.22 mmol)
was added and the resulting mixture vigorously stirred for
24 h, then a small amount of silica was added to the reaction
mixture (containing aldehyde 37) and the solvent removed
under reduced pressure. The resulting residue was purified
by flash column chromatography (100% ethyl acetate) to
afford hydroxyspirolactam 38 (61 mg, 63%) as a pale
yellow oil. Hydroxyspirolactam 38 was shown to be a 7:3
1
mixture of diastereomers by H NMR analysis. The ratio
was estimated from the relative intensities of the multiplet at
d 4.76–4.79 and the doublet of doublets at d 5.00, assigned
to the 2-H protons of the minor and major isomers,
respectively) with the isomers being inseparable: [a]D
K44.4 (c 0.90 in MeOH); nmax (film)/cmK1 3410, 3064,
3034, 2953, 2881, 2083, 1718, 1649, 1498, 1454, 1332,
1267, 1215, 1170, 1121, 1082, 1048, 1028, 1003, 984, 909,
776, 736 and 698; dH (400 MHz; CDCl3) 1.74–1.771 (0.3H,
m, 90-HAHB), 1.93–2.04 (1.3H, m, 90-HAHB and 30-HA*HB),
2.11–2.39 [4.4H, m, 40-HAHB, 30-HAHB, 3-HAHB and
3-HAHB(major)], 2.42–2.66 (3.3H, m, 3-HAHB*, 4-H2
and 40-HAHB), 2.76 (0.7H, dd, JZ12.9, 6.1 Hz, 90-HAHB),
3.46–3.58 (2H, m, 20-H2), 3.80–3.85* (0.3H, obscured,
200-HAHB), 3.87 (0.7H, dd, JZ17.5, 3.3 Hz, 200-HAHB), 4.02
(0.7H, dd, JZ17.5, 5.1 Hz, 200-HAHB), 3.99–4.05* (0.3H,
obscured, 200-HAHB), 4.69 (1H, s, OH), 4.76–4.79* (0.3H,
m, 2-H), 5.00 (0.7H, dd, JZ10.8, 4.8 Hz, 2-H), 5.09–5.21
(6H, m, 3!OCH2Ph), 5.25* (0.3H, dd, JZ12.3, 7.1 Hz,
80-H), 5.42 (0.7H, dd, JZ6.0, 2.4 Hz, 80-H), 5.48* (0.3H, m,
NH), 5.58–5.59 (0.7H, m, NH) and 7.27–7.39 (15H, m, 3!
Ph); dC (100 MHz; CDCl3) 24.1* (CH2, 3-C), 24.2 (CH2,
3-C), 24.5 (CH2, 30-C), 25.3* (CH2, 30-C), 30.2 (CH2, 40-C),
30.7* (CH2, 40-C), 37.9 (CH2, 4-C), 39.4 (CH2, 9-C), 39.7*
(CH2, 9-C), 43.0* (CH2, 200-C), 43.7 (CH2, 200-C), 47.0
(CH2, 2-C), 47.7* (CH2, 2-C), 54.3 (CH, 20-C), 54.7* (CH,
20-C), 65.6* (quat., 5-C), 66.2* (CH2, OCH2Ph), 66.4 (CH2,
OCH2Ph), 66.8 (CH2, OCH2Ph), 67.0* (CH2, OCH2Ph),
67.2* (CH2, OCH2Ph), 67.3 (CH2, OCH2Ph), 68.1 (quat.,
5-C), 77.7 (CH, 8-C), 80.6* (CH, 8-C), 128.05 (CH, Ph),
128.09 (CH, Ph), 128.2 (CH, Ph), 128.4 (CH, Ph), 128.5
(CH, Ph), 128.6 (CH, Ph), 128.7 (CH, Ph), 134.5 (quat., Ph),
135.3* (quat., Ph), 135.8 (quat., Ph), 136.0* (quat.,00Ph),
136.4 (quat., Ph), 156.1 (quat., NCO2), 166.0 (quat., 1 -C),
167.1* (quat., 100-C), 170.1* (quat., 10-C), 172.5 (quat.,
10-C), 172.7* (quat., 50-C), 173.6 (quat., 50-C), 173.8*
(quat., 6-C) and 175.1 (quat., 6-C); m/z (FABC) 658.2749
(MHC C36H40N3O9 requires 658.2765).
4.1.29. Glycyl-L-2-benzylprolyl-L-glutamic acid trifluoro-
acetate 5. To a solution of the protected tripeptide 34
(98 mg, 0.16 mmol) in dichloromethane (4 cm3) at room
temperature was added trifluoroacetic acid (0.75 cm3)
dropwise and the reaction mixture was stirred for 3.5 h.
The solution was evaporated under reduced pressure to give
tripeptide 5 (82 mg, 100%) as an hygroscopic colourless
solid. Tripeptide 5 was shown to be a 90:10 trans:cis
1
mixture of conformers by H NMR analysis (the ratio was
estimated from the relative intensities of the double doublets
and multiplet at d 4.51 and 4.33, assigned to the Glua-H
protons of the major and minor conformers, respectively):
mp 73–82 8C: [a]D C41.0 (c 1.61 in MeOH); dH (300 MHz;
D2O) 1.27–1.39 (1H, m, Prog-HAHB), 1.68–1.83 (1H, m,
Prog-HAHB), 2.07–2.42 (4H, m, Prob-H2 and Glub-H2),
2.57 (2H, t, JZ7.1 Hz, Glug-H2), 2.82–2.92 (1H, m, Prod-
HAHB), 3.24 (1H, d, JZ13.3 Hz, PhCHAHB), 3.50–3.59
(1H, m, Prod-HAHB), 3.70 (1H, d, JZ13.3 Hz, PhCHAHB),
3.91 (1H, d, JZ16.7 Hz, Glya-HAHB), 4.09 (1H, d, JZ
16.7 Hz, Glya-HAHB), 4.33* (0.1H, m, Glua-H), 4.51
(0.9H, dd, JZ4.8, 9.6 Hz, Glua-H) and 7.21–7.44 (5H, m,
PhH); dC (100 MHz; D2O) 25.0 (CH2, Prog-C), 27.9 (CH2,

10030
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
4.1.31. Dibenzyl (2S,50R)-[10-(200-benzyloxycarbonyl-
amino-acetyl)-60-oxo-10,70-diazaspiro[4.4]non-70-yl]-1,5-
pentanedioate 39. A solution of hydroxyspirolactam 38
(33 mg, 0.05 mmol) in trifluoroacetic acid–triethylsilane–
dichloromethane (1.0 cm3, 1/1/1), at room temperature, was
stirred for 45 min then concentrated in vacuo to give an
opaque white oil, which was purified by flash column
chromatography (100% ethyl acetate) to afford spirolactam
39 (31 mg, 96%) as a colourless oil: [a]D K18.0 (c 0.67 in
CH2Cl2); nmax (film)/cmK1 3583, 3412, 3032, 2952, 1734,
1654, 1497, 1454, 1432, 1289, 1259, 1215, 1171, 1048,
982, 738 and 698; dH (300 MHz; CDCl3) 1.76–2.19 (6H, m,
90-HAHB, 40-H2, 30-HAHB and 3-H2), 2.31–2.63 (4H, m,
90-HAHB, 30-HAHB and 4-H2), 3.31 (1H, dd, JZ16.8,
8.1 Hz, 80-HAHB), 3.40–3.47 (1H, m, 80-HAHB), 3.51–3.55
(2H, m, 20-H2), 3.86 (1H, dd, JZ17.1, 3.3 Hz, 200-HAHB),
4.05 (1H, dd, JZ17.1, 5.4 Hz, 200-HAHB), 4.90 (1H, dd, JZ
11.3, 4.5 Hz, 2-H), 5.07–5.18 (6H, m, 3!OCH2Ph), 5.63
(1H, br s, N–H) and 7.27–7.34 (15H, m, 3Ph); dC (75 MHz;
CDCl3) 23.4 (CH2, 30-C), 24.0 (CH2, 3-C), 29.8 (CH2, 90-C),
30.4 (CH2, 4-C), 36.2 (CH2, 40-C), 39.8 (CH2, 80-C), 43.7
(CH2, 200-C), 47.1 (CH2, 20-C), 53.8 (CH, 2-C), 66.3 (CH2,
OCH2Ph), 66.9 (CH2, OCH2Ph), 67.2 (CH2, OCH2Ph), 68.2
(quat., 5-C), 128.0 (CH, Ph), 128.05 (CH, Ph), 128.1 (CH,
Ph), 128.3 (CH, Ph), 128.49 (CH, Ph), 128.5 (CH, Ph), 128.7
(CH, Ph), 135.2 (quat., Ph), 136.0 (quat.,00Ph), 136.5 (quat.,
Ph), 156.2 (quat., NCO2), 166.1 (quat., 1 -C), 170.1 (quat.,
1-C), 172.7 (quat., 5-C) and 174.4 (quat., 60-C); m/z (FABC)
642.2802 (MHC C36H40N3O8 requires 642.2815).
pressure to afford spirolactam 36 (14 mg, 99%) as a
colourless solid. Spirolactam 36 was shown to be a 7:3
mixture of two diastereomers by 1H NMR analysis (the ratio
was estimated from the relative intensities of the doublet of
doublets at d 4.47 and 4.53, assigned to 2-H of the minor and
major isomers, respectively): mp 216–218 8C (dec.): [a]D
C0.86 [c 0.35 in MeOH–H2O (1:1)]; dH (400 MHz; D2O)
2.10–2.46 (9H, m, 90-HAHB, 40-H2, 30-H2, 3-H2 and 4-H2),
2.65* (0.3H, dd, JZ13.8, 7.0 Hz, 90-HAHB), 2.75 (0.7H, dd,
JZ13.6, 6.2 Hz, 90-HAHB), 3.55–3.61 (1H, m, 20-HAHB),
3.69–3.73 (1H, m, 20-HAHB), 3.94–4.09 (2H, d, JZ16.5 Hz,
200-H2), 4.47* (0.3H, dd, JZ9.9, 5.8 Hz, 2-H), 4.53 (0.7H,
dd, JZ10.4, 5.0 Hz, 2-H), 5.49* (0.3H, dd, JZ6.8, 5.1 Hz,
80-H) and 5.59 (0.7H, d, JZ6.1 Hz, 80-H); dC (100 MHz;
D2O) 26.0* (CH2, 30-C), 26.1 (CH2, 30-C), 27.4 (CH2, 3-C),
29.6* (CH2, 3-C), 39.3* (CH2, 4-C), 40.1 (CH2, 4-C), 40.7
(2!CH2, 40-C and 90-C), 42.2* (CH2, 90-C), 43.1 (CH2,
200-C), 49.8 (CH2, 20-C), 49.9* (CH2, 20-C), 60.2* (CH,
2-C), 60.7 (CH, 2-C), 70.1* (quat., 50-C), 70.9 (quat., 50-C),
80.6 (CH, 80-C0)0, 83.2* (CH, 80-C),0 166.9 (quat., 100-C),
167.1* (quat., 1 -C), 178.2* (quat., 6 -C), 179.7 (2!quat.,
1-C and 60-C), 180.4 (quat., 1-C), 182.5 (quat., 5-C) and
182.8* (quat., 5-C); m/z (FABC) 344.1467 (MHC
C14H22N3O7 requires 344.1458).
4.1.34. Methyl N-tert-butyloxycarbonyl-(D,L)-5,5-
dimethylprolinate 43.49 Nitrile 4449,55 (2 g, 14.3 mmol)
was dissolved in 32% hydrochloric acid (6 cm3) and heated
to 50 8C for 5 h. Evaporation of the solvent afforded a
residue that was dissolved in methanol–water (1/1, 30 cm3)
and hydrogenated over 10% palladium on activated carbon
(0.3 g) under 44 psi of hydrogen for 20 h. The catalyst was
removed by filtration through Celitee and the solvent
removed in vacuo to yield a 6:4 mixture of N-methyl-5-
methylproline* 45, and the desired 5,5-dimethylproline 46;
dH (300 MHz; D2O) 1.34–1.40 (8.6H, m, 4!CH3), 1.67–
1.74 (1.3H, m) 1.91 (1.6H, t, JZ12.2 Hz), 2.07–2.32 (3.5H,
m), 2.40–2.54 (2H, m), 2.90* (3.6H, s, N-CH3), 3.45–3.53
(1.3H, m), 4.23* (1.3H, dd, JZ9.7, 7.5 Hz, Proa-H) and
4.47 (1H, d, JZ8.4 Hz, Proa-H); dC (75 MHz; D2O) 14.6*
(CH3), 24.5 (CH3), 24.7 (CH3), 25.9* (CH2), 27.2 (CH2),
29.9* (CH2), 37.0 (CH2), 39.1* (CH3, N-CH3), 58.9* (CH,
Prod-C), 67.1 (quat., Prod-C), 67.6* (CH, Proa-C), 69.2
(CH, Proa-C), 171.5* (quat., Proa-CO) and 172.4 (quat.,
Proa-CO). The mixture was subsequently dissolved in dry
methanol (60 cm3), cooled to 0 8C and thionyl chloride
(4.2 cm3, 57.2 mmol) was added dropwise over 5 min. The
reaction was allowed to warm to room temperature and
stirred overnight. The solvent was removed, the residue was
dissolved in saturated sodium hydrogen carbonate solution
and the products extracted with chloroform to yield a
mixture of inseparable methyl esters (1.53 g), which were
dissolved in dry dichloromethane (20 cm3). N-Methyl-
morpholine (1.56 cm3, 14.2 mmol) and di-tert-butyldi-
carbonate (3.1 g, 14.2 mmol) were added and the reaction
was heated at reflux under nitrogen for 48 h. After cooling to
room temperature the reaction mixture was washed with
water, 1 M aqueous hydrochloric acid (2!30 cm3), brine
and dried (MgSO4). The aqueous layer was concentrated in
vacuo to give methyl N-methyl-5-methylprolinate 47
(1.093 g, 42%, four steps) as its hydrochloride salt.
This was neutralized with saturated sodium hydrogen
carbonate and purified by chromatography (silica gel, 4:1,
4.1.32. (2S,50R)-[10-(200-amino-acetyl)-60-oxo-10,70-di-
azaspiro[4.4]non-70-yl]-1,5-pentanedioic acid 35. A mix-
ture of protected spirolactam 39 (30 mg, 0.047 mmol) and
10% palladium on activated carbon (9.6 mg, 0.09 mmol) in
88:12 methanol–water (6.6 cm3) was stirred under an
atmosphere of hydrogen at room temperature, protected
from light, for 18 h. The reaction mixture was filtered
through a Celitee pad with 75:25 methanol–water (30 cm3),
and the filtrate concentrated to dryness under reduced
pressure to give a yellow solid that was purified by reverse-
phase C18 flash column chromatography (H2O) to afford
spirolactam 35 (12 mg, 78%) as a colourless solid: mp 238–
239 8C (dec.): [a]D K23.5 [c 1.15 in MeOH–H2O (1/1)]; dH
(400 MHz; D2O) 1.97–2.61 (10H, m, 90-H2, 40-H2, 30-H2,
3-H2 and 4-H2), 3.44–3.78 (4H, br m, 80-H2 and 20-H2)
3.81–4.12 (2H, br m, 200-H2) and 4.41 (1H, m, 2-H); dC
(100 MHz; D2O) 25.8 (CH2, 30-C), 27.8 (CH2, 3-C), 31.4
(CH2, 90-C), 37.3 (2!CH2, 40-C and 4-C), 43.1 (2!CH2,
80-C and 200-C), 50.1 (CH2, 20-C), 60.2 (CH, 2-C), 72.5
(quat., 50-C), 173.6 (quat., 100-C), 178.9 (quat., 60-C) 179.1
(quat., 1-C) and 184.9 (quat., 5-C); m/z (FABC) 328.1521
(MHC C14H21N3O6 requires 328.1509).
4.1.33. (2S,50R,80R)- and (2S,50R,80S)-[10-(200-amino-
acetyl)-80-hydroxy-60-oxo-10,70-diazaspiro[4.4]non-70-
yl]-1,5-pentanedioic acid 36. A mixture of protected
hydroxyspirolactam 38 (27 mg, 0.041 mmol) and 10%
palladium on activated carbon (8.4 mg, 0.079 mmol) in
88:12 methanol–water (5.8 cm3) was stirred under an
atmosphere of hydrogen at room temperature, protected
from light, for 18 h. The reaction mixture was filtered
through a Celitee pad with 75:25 methanol–water (30 cm3)
and the filtrate concentrated to dryness under reduced

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10031
dichloromethane/ethyl acetate); dH (200 MHz; CDCl3;
Me4Si) 1.06 (3H, d, JZ6.0 Hz, Prod-CH3), 1.35–1.55
(1H, m), 1.70–2.01 (3H, m), 2.17–2.27 (1H, m), 2.24 (3H,
s, N-CH3), 2.90 (1H, t, JZ7.8 Hz, Proa-H) and 3.65 (3H, s,
2-CO2CH3); dC (75 MHz; CDCl3) 18.2 (CH3, Prod-CH3),
26.4 (CH2), 31.3 (CH2), 38.9 (CH3, N-CH3), 51.3 (CH3,
Proa-CO2CH3) 61.8 (CH, Prod-C), 68.4 (CH, Proa-C) and
173.6 (quat., Proa-CO); m/z (CIC) 158.1176 (MHC.
C8H16NO2 requires 158.1181); The organic layer was
concentrated in vacuo and purified by chromatography
(silica gel, dichloromethane then chloroform) to give
methyl N-tert-butyloxycarbonyl-(D,L)-5,5-dimethylpro-
linate 43 (0.795 g, 22%, four steps) as a yellow oil. This
compound existed as a mixture of epimers (55:45) almost
exclusively as the cis conformer; dH (300 MHz; CDCl3)
1.26–1.53 [15H, m, C(CH3)2, C(CH3)3], 1.60–1.90 (4H, m,
Prob-H2 and Prog-H2), 3.66 (3H, CO2CH3), 4.24 (0.55H, dd
JZ8.9, 3.5 Hz, Proa-H) and 4.35 (0.45H, dd JZ8.5, 2.6 Hz,
Proa-H); dC (75 MHz; CDCl3) 25.7 (CH3, Prod-CH3), 25.9
(CH2, Prog-C), 26.0 (CH3, Prod-CH3), 26.5 (CH2, Prog-C),
26.6 (CH3, Prod-CH3), 27.2 (CH3, Prod-CH3), 28.2 [CH3,
C(CH3)3], 28.3 [CH3, C(CH3)3], 39.8 (CH2, Prob-C), 40.7
(CH2, Prob-C), 51.6 (CH3, Proa-CO2CH3), 51.7 (CH3,
Proa-CO2CH3), 60.5 (quat., Prod-C), 61.16 (CH, Proa-C),
61.2 (CH, Proa-C), 61.3 (quat., Prod-C), 79.1 [quat.,
C(CH3)3], 79.6 [quat., C(CH3)3], 152.4 (quat., NCO2),
154.0 (quat., NCO2), 173.4 (quat., Proa-CO) and 173.8
(quat., Proa-CO); m/z (EIC) 257.1624 (MC C13H23NO4
requires 257.1627).
168.0* (quat., Gly-CO), 171.6 (quat., 2-CO) and 172.0*
(quat., 2-CO); m/z (EIC) 290.0938 (MC C11H18N2O5S
requires 290.0936).
4.1.36. Di-tert-Butyl N-tert-butyloxycarbonylglycyl-L-4-
thiaprolyl-L-glutamate 56. Ethyl chlorofomate (0.048 g,
0.445 mmol) was added dropwise to a solution of acid 52
(0.129 g, 0.445 mmol) and triethylamine (0.050 g,
0.49 mmol) in dichloromethane (3 cm3) at 0 8C. The
solution was stirred for 35 min at 0 8C then a solution of
glutamic acid di-tert-butyl ester hydrochloride 29 (0.132 g,
0.445 mmol) and triethylamine (0.050 g, 0.49 mmol) in
dichloromethane (3 cm3) was added. The mixture was
stirred overnight, washed successively with 2 M aqueous
hydrochloric acid, saturated sodium hydrogen carbonate
solution, dried (Na2SO4), filtered and the solvent removed.
Purification by flash chromatography (dichloromethane/
ethyl acetate, 3:1) afforded protected tripeptide 56 (0.128 g,
54%) as a colourless solid. 56 was shown to be a 66:34
1
trans:cis mixture of conformers by H NMR analysis (the
ratio was estimated from the integration of the thiaProN–H
protons at d 7.20 and 7.43 assigned to major and minor
conformers, respectively): mp 99–100 8C: [a]D K70 (c 0.6
in CH2Cl2); dH (400 MHz; CDCl3) 1.25–1.45 [27H, m,
C(CH3)3], 1.80–1.95 (1H, m, Glub-HAHB), 1.97–2.14 (1H,
m, Glub-HAHB), 2.18–2.35 (2H, m, Glug-H2), 3.14 (0.66H,
dd, JZ11.2, 7.0 Hz, 3-HAHB), 3.22–3.353 (0.34H, br m,
3-HAHB), 3.38 (1H, dd, JZ12.3, 3.6 Hz, 3-HAHB), 3.85–
4.05 (2H, m, Glya-H2), 4.38 (1H, br t, JZ4.8 Hz, Glua-H),
4.49–4.57 (1.66H, 5-HAHB and *5-HAHB), 4.69–4.77*
(0.72H, *5-HAHB and *2-H), 4.98 (0.66H, br s, 2-H), 5.51
(1H, br s, GlyN–H), 7.20 (0.66H, d, JZ7.2 Hz, thiaProN–
H), 7.20* (0.34H, br s, thiaProN–H); dC (100 MHz; CDCl3)
26.4* (CH2, Glub-C), 27.1 (CH2, Glub-C), 27.8 [CH3,
C(CH3)3], 27.9 [CH3, C(CH3)3], 28.2 [CH3, C(CH3)3], 31.3
(CH2, Glug-C), 32.2 (CH2, 3-C), 35.4* (CH2, 3-C), 43.2
(CH2, Glya-C), 43.5* (CH2, Glya-C), 48.2 (CH2, 5-C),
49.7* (CH2, 5-C), 52.5 (CH, Glua-C), 62.3* (CH, 2-C), 62.5
(CH, 2-C), 79.7 [quat., C(CH3)3], 80.6 [quat., C(CH3)3],
80.9* [quat., C(CH3)3], 82.1 [quat., C(CH3)3], 82.2* [quat.,
C(CH3)3], 155.7 (quat., NCO2), 167.9 (quat., Gly-CO),
168.6* (quat., 2-CO), 168.9 (quat., 2-CO), 170.4 (quat.,
Glua-CO) and 172.4 (quat., Glug-CO); m/z (FABC)
532.2628 (MHC C24H42N3O8S requires 532.2693).
4.1.35. N-tert-Butyloxyglycyl-L-4-thiaproline 52. iso-
Butyl chloroformate (0.154 g, 1.12 mmol) was added to a
stirred solution of N-tert-butyloxycarbonylglycine 17 and
triethylamine (0.215 g, 1.15 mmol) in tetrahydrofuran
(6 cm3) at 0 8C. A white precipitate was observed, the
cooling bath was removed and the mixture stirred at room
temperature for 10 min. A solution of 4-thiaproline
hydrochloride 4044 (0.150 g, 1.12 mmol) and triethylamine
(0.215 g, 1.15 mmol) in water (2 cm3) was added and the
resultant solution was stirred for 1 h. The mixture was
acidified with 2 M HCl and extracted with ethyl acetate. The
combined organic extracts were dried (Na2SO4), filtered and
concentrated in vacuo to yield an oil (0.39 g) that was
purified by flash chromatography (hexane/ethyl acetate/
acetic acid 2:1:0.3 then 1:1:0.2) gave dipeptide 52 (0.264 g,
81%) as a hygroscopic white foam. 52 was shown to be a
62:38 trans:cis mixture of conformers by 1H NMR analysis
(the ratio was estimated from the integration of the GlyN–H
protons at d 5.65 and 5.75 assigned to major and minor
conformers, respectively): [a]D K93.5 (c 0.25 in CH2Cl2);
dH (400 MHz; CDCl3) 1.432 [3.4H, s, C(CH3)3], 1.44 [5.6H,
s, C(CH3)3], 3.31 (1.24H, d, JZ5.1 Hz, 3-HAHB), 3.37*
(0.38H, dd, JZ11.8, 5.2 Hz, 3-HAHB), 3.49* (0.38H, dd,
JZ11.7, 1.4 Hz, 3-HAHB), 3.90–4.20 (2H, m, Glya-H2),
4.55–4.62 (1.62H, m 5-H2, *5-HAHB), 4.79* (0.38H, d, JZ
9.7 Hz, 5-HAHB), 4.86* (0.38H, d, JZ5.7 Hz, 2-H), 5.11
(0.62H, t, JZ4.9 Hz, 2-H), 5.65 (0.62H, br s, GlyN–H) and
5.75* (0.38H, br s, GlyN–H); dC (100 MHz; CDCl3) 28.2
[CH3, C(CH3)3], 32.3 (CH2, 3-C), 34.4* (CH2, 3-C), 43.1
(CH2, Glya-C), 43.4* (CH2, Glya-C), 47.6 (CH2, 5-C),
48.9* (CH2, 5-C), 60.7* (CH, 2-C), 61.6 (CH, 2-C), 80.3*
[quat., C(CH3)3], 80.7 [quat., C(CH3)3], 156.1 (quat.,
NCO2), 156.4* (quat., NCO2), 167.8 (quat., Gly-CO),
4.1.37. Glycyl-L-4-thiaprolyl-L-glutamic acid trifluoro-
acetate 48. Trifluoroacetic acid (1 cm3) was added to a
stirred solution of protected tripeptide 56 (0.128 g, 0.241)
and triethylsilane (0.084 g, 0.723 mmol) in dichloro-
methane (3 cm3). The resultant solution was stirred for 4 h
at room temperature and the volatiles removed in vacuo.
Purification of the residue by chromatography (reverse
phase C18, water, 10–20% acetonitrile/water) and lyophili-
sation gave 48 (0.066 g, 61%) as a hygroscopic off white
solid. 48 was shown to be a 80:20 trans:cis mixture of
1
conformers by H NMR analysis (the ratio was estimated
from the integration of the Glya protons at d 3.80–3.92 and
3.58 assigned to major and minor conformers, respectively):
no mp due to hygroscopic sample: [a]D K88.6 (c 0.203 in
H2O); dH (400 MHz; D2O) 1.73–1.82 (1H, m, Glub-HAHB),
1.96–2.06 (1H, m, Glub-HAHB), 2.28 (2H, t, JZ6.9 Hz,
Glug-H2), 2.96 (0.8H, dd, JZ12.4, 3.7 Hz, 3-HAHB), 3.13*
(0.2H, d, 12.2, 3-HAHB), 3.20 (0.8H, dd, JZ12.4, 7.3 Hz,

10032
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
3-HAHB), 3.27* (0.2H, dd, JZ12.4, 6.9 Hz, 3-HAHB), 3.58
(0.2H, d, JZ16.4 Hz, Glya-HAHB), 3.80–3.92 (1.8H, m,
Glya-H2 and *Glya-HAHB), 4.25 (0.8H, dd, 9.4 and 4.8,
Glua-H), 4.30* (0.2H, dd, 10.0 and 4.8, Glua-H), 4.35–4.49
(2H, m, 5-H2) and 4.67 (1H, dd, JZ6.9 Hz, 3.8 Hz, 2-H); dC
(100 MHz; CDCl3) 24.8* (CH2, Glub-C), 25.3 (CH2, Glub-
C), 29.5 (CH2, Glug-C), 29.8* (CH2, Glug-C), 32.8 (CH2,
3-C), 34.9* (CH2, 3-C), 40.3 (CH2, Glya-C), 40.5* (CH2,
Glya-C), 48.4 (CH2, 5-C), 49.6* (CH2, 5-C), 51.7 (CH,
Glua-C), 51.9* (CH, Glua-C), 61.6* (CH, 2-C), 62.6 (CH,
2-C), 115.7 (quat., q, JZ290.7 Hz, CF3CO2H), 161.9 (quat.,
q, JZ36.2 Hz, CF3CO2H), 165.0 (quat., Gly-CO), 165.6*
(quat., Gly-CO), 171.0* (quat., 2-CO), 171.5 (quat., 2-CO),
174.0* (quat., Glua-CO), 174.1* (quat., Glua-CO) and
176.7 (quat., Glug-CO); m/z (FABC) 320.0921 [M(free
base)HC C11H18N3O6S requires 320.0916].
the solvent removed. The residue was suspended in ethyl
acetate, washed with 10% citric acid solution, saturated
sodium hydrogen carbonate, brine and dried (MgSO4).
Removal of the solvent and subsequent chromatography
(2:1, hexane/ethyl acetate, then 1:1) gave protected
tripeptide 57 (0.5 g, 68%) as a colourless oil. Protected
tripeptide 57 was shown to be a 90:10 cis:trans mixture of
1
conformers by H NMR analysis (the ratio was estimated
from the integration of the broad singlets at d 5.69 and 5.80
and assigned to the GlyN–H protons of the major and
minor conformers, respectively): [a]D K35.6 (c 0.399 in
dichloromethane); dH (300 MHz; CDCl3) 1.85 (3H,
Pd-CH3) 1.97 (3H, Pd-CH3), 2.08–2.15 (1H, m, Glub-
HAHB), 2.29–2.38 (1H, m, Glub-HAHB), 2.47 (2H, t, JZ
5.4 Hz, Glug-H2), 3.32 (2H, m, Pb-H2), 3.85 (1H, d, JZ
16.6 Hz, Glya-HAHB), 3.91 (1H, dd, JZ17.0, 8.0 Hz, Glya-
HAHB), 4.72 (2H, br s, Pa-H, Glua-H), 5.20–5.08 (6H, m,
3!OCH2Ph), 5.69 (0.9H, br s, Gly-NH), 5.80* (0.1H, br s,
Gly-NH) and 7.33–7.39 (15H, m, Ph); dC (75 MHz; CDCl3)
26.3 (CH2, Glub-C), 26.45* (CH2, Glub-C), 27.21* (CH3,
Pd-CH3), 27.43 (CH3, Pd-CH3), 28.4 (CH3, Pd-CH3), 29.91
(CH2, Glug-C), 30.09* (CH2, Glug-C), 32.3 (CH2, Pb-C),
44.7 (CH2, Glya-C), 45.1* (CH2, Glya-C), 52.1 (CH, Glua-
C), 52.3* (CH, Glua-C), 65.8 (CH, Pa-H), 66.4 (CH2,
OCH2Ph), 66.8 (CH2, OCH2Ph), 67.4 (CH2, OCH2Ph), 74.5
(quat., Pd-C), 127.8 (CH, Ph), 127.9 (CH, Ph), 128.1, (CH,
Ph), 128.14 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.5
(CH, Ph), 134.8 (quat., Ph), 135.5 (quat., Ph), 136.2 (quat.,
Ph), 156.4 (quat., NCO2), 167.1 (quat., Gly-CO), 169.6
(quat., P-CON), 170.9 (quat., Glua-CO) and 172.7 (quat.,
Glug-CO); m/z (FABC) 662.2536 (MHC C35H40N3O8S
requires 662.2536).
4.1.38. N-Benzyloxycarbonylglycyl-L-thia-5,5-dimethyl-
proline 53. To a stirred solution of 5,5,-dimethyl-4-
thiaproline hydrochloride 4145,46 (0.354 g, 1.79 mmol)
under nitrogen in dry dimethylformamide (35 cm3) was
added diisopropylethylamine (0.594 cm3, 1.9 mmol) and
acid fluoride 5156,57(0.341 g, 1.61 mmol). The solution was
stirred for 18 h, the solvent was removed in vacuo and the
residue was redissolved in ethyl acetate, washed with 10%
citric acid solution, brine and dried (MgSO4). The solvent
was removed and the residue purified by flash chromato-
graphy (4:1:0.5, ethyl acetate/hexane/acetic acid, then
3:1.0.4) to give the desired compound contaminated with
N-benzyloxycarbonylglycine 16 (14%, 1H NMR). This
mixture was dissolved in methanol, trimethylsilyl chloride
(0.07 cm3) added and the solution stirred overnight.
Removal of the solvent in vacuo and subsequent flash
chromatography (3:1, ethyl acetate/hexane [to remove
N-benzyloxycarbonylglycine methyl ester] then 3:1.0.4
ethyl acetate/hexane/acetic acid) gave dipeptide 53
(0.320 g, 65%, based on amount of acid fluoride reacted)
as a white solid. This compound existed purely as the cis
conformer: mp 130–132 8C: [a]D K51.7 (c 0.116 in
4.1.40. Glycyl-L-thia-5,5-dimethylprolyl-L-glutamic acid
49. Protected tripeptide 57 (0.44 g, 0.66 mmol) was
dissolved in methanol–water (4/1, 50 cm3), placed in a
Parr bottle. The vessel was flushed with nitrogen, 10%
palladium on activated carbon (70 mg, 0.066 mmol) was
added and the mixture was pressurized to 40 psi with
hydrogen and shaken for 3 h. Further 10% palladium on
activated carbon (70 mg) was added and the reaction
continued for 21 h. The reaction mixture was filtered
through Celite,e washed with methanol–water (4/1) and
†
dichloromethane); dH (300 MHz; CDCl3) 1.85 (3H, s, Pd-
CH3) 1.91 (3H, s, Pd-CH3), 3.30 (1H, dd, JZ12.1, 5.6 Hz,
Pb-HAHB), 3.40 (1H, d, JZ12.1 Hz, Pb-HAHB), 3.87 (1H,
dd, JZ16.6, 3.7 Hz, Glya-HAHB), 4.09 (1H, dd, JZ16.6,
3.7 Hz, Glya-HAHB), 4.82 (1H, d, JZ5.2 Hz, Pa-H), 5.13
(2H, s, OCH2Ph), 6.10 (1H, br t, JZ3.8 Hz, GlyN–H) and
7.29–7.39 (5H, m, Ph); dC (75 MHz; CDCl3) 27.0 (CH3, Pd-
CH3) 29.4 (CH3, Pd-CH3), 31.6 (CH2, Pb-C), 44.8 (CH2,
Glya-C), 64.3 (CH, Pa-C), 67.3 (CH2, OCH2Ph), 74.2
(quat., Pd-C), 127.9 (CH, Ph), 128.1 (CH, Ph), 128.4 (CH,
Ph), 135.8 (quat., Ph), 156.8 (quat., NCO2), 166.6 (quat.,
Gly-CO) and 172.0 (quat., Pa-CO); m/z (EIC) 352.1088
(MC C16H20N2O5S requires 352.1093).
1
the solvent removed to yield an oil, which by TLC and H
NMR analysis contained the desired product and products
containing varying amounts of debenzylation. The mixture
was dissolved in water and passed through a C18 column
eluting with water, then 10% methanol–water. The relevant
fractions were combined, the solvent removed and the
residue was triturated with dry ether to yield tripeptide 49
(0.110 g, 48%) as a white solid. Tripeptide 49 was shown to
1
be a 85:15 cis:trans mixture of conformers by H NMR
analysis (the ratio was estimated from the integration of the
doublets at d 4.95 and 5.02 and assigned to the Pa-H protons
of the major and minor conformers, respectively): mp 145–
150 8C: [a]D K75 (c 0.064 in water); dH (300 MHz; D2O)
1.84 (2.55H, s, Pd-CH3). 1.90* (0.45H, s, Pd-CH3) 1.93 (3H,
s, Pd-CH3), 1.96–2.05 (1H, m, Glub-HAHB), 2.18–2.27 (1H,
m, Glub–HAHB), 2.42 (2H, t, JZ7.5 Hz, Glug-H2), 3.36
(1H, d, JZ12.7 Hz, Pb-HAHB) 3.59 (1H, dd, JZ12.8,
6.4 Hz, Pb-HAHB),, 3.70 (1H, d, JZ16.2 Hz, Glya-HAHB),
4.01 (1H, d, JZ16.3 Hz, Glya-CHAHB), 4.23* (0.15H, dd,
JZ9.1, 4.9 Hz, Glua-H), 4.32 (0.85H, dd, JZ9.1, 4.9 Hz,
4.1.39. Dibenzyl N-benzyloxycarbonyl-glycyl-L-thia-5,5-
dimethylprolyl-L-glutamate 57. To a stirred solution of
dipeptide 53 (0.398 g, 1.11 mol), L-glutamic acid dibenzyl
ester p-toluenesulfonate 28 (0.670 g, 1.34 mol) and diiso-
propylethylamine (0.51 cm3, 2.90 mmol) in dry dichloro-
methane (40 cm3) was added bis(2-oxo-3-oxazo-
lidinyl)phosphinic chloride (0.370 g, 1.45 mmol) in one
portion. The solution was stirred for 7 h under nitrogen and
† P refers to the proline analogue portion in question.

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10033
Glua-H), 4.95 (0.85H, d, JZ6.2 Hz, Pa-H) and 5.02*
(0.15H, d, JZ6.0 Hz, Pa-H); dC (75 MHz; D2O) 26.1*
(CH3, Pd-CH3), 26.3 (CH3, Pd-CH3), 26.56 (CH2, Glub-C),
27.6 (CH3, Pd-CH3), 27.9* (CH3, Pd-CH3), 31.1 (CH2,
Glug-C), 30.3* (CH2, Glug-C), 32.1 (CH2, Pb-C), 32.3*
(CH2, Pb-C), 41.1* (CH2, Glya-C), 41.6 (CH2, Glya-C),
54.4 (CH, Glua-C), 55.0* (CH, Glua-C), 65.3* (CH, Pa-H),
65.5 (CH, Pa-H), 74.3* (quat., Pd-C), 74.6 (quat., Pd-C),
164.4* (quat., Gly-CO), 164.6 (quat., Gly-CO), 170.5 (quat.,
P-CON), 170.8* (quat., P-CON), 176.9 (quat., Glua-CO)
and 178.3 (quat., Glug-CO); m/z (FABC) 348.1249 (MHC
C12H22N3O6S requires 348.1229).
sodium hydroxide (5.91 cm3, 5.91 mmol) and the mixture
stirred for 21 h. The reaction was acidified with solid citric
acid and the product was extracted with ethyl acetate. The
combined organic extracts were washed with brine, dried
(MgSO4) and the solvent removed in vacuo to yield an oil,
which was purified by chromatography (silica gel, hexane/
ethyl acetate, 2:1, 1:1, 4:6), to give acid 55 (0.324 g, 94%)
as a white foam, which liquified rapidly. Acid 55 was shown
to be an 80:20 cis:trans mixture of conformers by 1H NMR
analysis (the ratio was estimated from the integration of the
broad singlets at d 5.81 and 5.66 assigned to the GlyN–H
protons of the major and minor conformers, respectively);
dH (300 MHz; CDCl3) 1.40 (2.4H, s, Prod-CH3), 1.43 [7.2H,
s, C(CH3)3], 1.44* [1.8H, s, C(CH3)3], 1.47* (0.6H, s, Prod-
CH3), 1.58* (0.6H, s, Prod-CH3), 1.61 (2.4H, s, Prod-CH3),
1.74–2.33 (4H, m, Prob-H2, Prog-H2), 3.37 (0.8H, dd, JZ
16.7, 3.3 Hz, Glya-HAHB), 3.65 (0.8H, dd, JZ16.8, 3.6 Hz,
Glya-HAHB), 3.96 (1H, dd, JZ16.9, 3.9 Hz, Glya-HAHB,
Glya-HAHB* partially obscured), 4.21* (0.2H, dd, JZ17.0,
5.9 Hz, Gya-HAHB), 4.42 (0.8H, d, JZ7.2 Hz, Proa-H),
4.67* (0.2H, d, JZ7.9 Hz Proa-H), 5.66* (0.2H, br s, Gly-
NH), 5.81 (0.8H, br s, Gly-NH) and 6.2 (1H, br s, OH); dH
(75 MHz; CDCl3) 24.8 (CH3, Prod-CH3), 25.1* (CH2, Prob-
C), 26.3 (CH3, Prod-CH3), 27.1* (CH3, Prod-CH3), 27.6*
(CH3, Prod-CH3), 28.0 (CH2, Prob-C), 28.2 [CH3,
C(CH3)3], 39.2 (CH2, Prog-C), 42.0* (CH2, Prog-C),
43.0* (CH2, Glya-C), 43.5 (CH2, Glya-C), 60.5 (CH,
Proa-C), 61.8* (quat., Prod-C), 62.2* (CH, Proa-C), 64.3
(CH, Proa-C), 79.7* [quat., C(CH3)3], 80.2 [quat.,
C(CH3)3], 156.0* (quat., NCO2), 156.4 (quat., NCO2),
166.8 (quat., Gly-CO), 169.5* (quat., Gly-CO), 173.9 (quat.,
Proa-CO) and 174.5* (quat., Proa-CO); m/z (CIC)
315.1927 (MHC C15H27N2O5 requires 315.1920).
4.1.41. Methyl N-tert-buyltoxycarbonylglycyl-(D,L)-5,5-
dimethylprolinate 54. Trifluoroacetic acid (1 cm3) was
added to a stirred solution of carbamate 43 (0.584 g,
2.27 mmol) in dichloromethane (6 cm3). The solution was
stirred for 2 h after which time the volatiles were removed in
vacuo and traces of trifluoroacetic acid were removed by
placing the sample on an oil pump for 2 h. The salt was then
dissolved in dichloromethane (20 cm3) and diisopropyl-
ethylamine (1.3 cm3, 7.49 mmol) was added (white fumes)
followed by N-tert-butyloxycarbonylglycine 17 (0.477 g,
2.73 mmol) and bis(2-oxo-3-oxazolidinyl)phosphinic chlor-
ide (0.691 g, 2.28 mmol). Additional dichloromethane
(5 cm3) was added and the solution was stirred overnight
under nitrogen. The solvent was then removed in vacuo, the
residue was dissolved in ethyl acetate and washed
sequentially with 2 M aqueous hydrochloric acid, saturated
sodium hydrogen carbonate and dried (MgSO4). Removal
of the solvent gave an oil (0.440 g) that was purified by
chromatography (silica gel, hexane/ethyl acetate, 2:1, then
1:1) to give dipeptide 54 (0.368 g, 52%) as a colourless oil.
Dipeptide 54 was shown to be 80:20 cis:trans mixture of
1
conformers by H NMR analysis (the ratio was estimated
4.1.43. Dibenzyl N-tert-butoxycarbonylglycyl-(D,L)-5,5-
dimethylprolyl-L-glutamate 58. To a stirred solution of
acid 55 (0.298 g, 1 mmol) in dry dichloromethane (40 cm3)
was added successively diisopropylethylamine (0.453 cm3,
2.6 mmol), L-glutamic acid dibenzyl ester p-toluenesulpho-
nate 28 (0.648 g, 1.3 mmol) and bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride (0.330 g, 1.3 mmol). The resultant
solution was stirred at room temperature under nitrogen
overnight and the solvent removed. The residue was
dissolved in ethyl acetate, washed with 10% aqueous citric
acid solution, saturated sodium hydrogen carbonate, brine
and dried (MgSO4). The solvent was evaporated and the
product purified by chromatography (silica gel, hexane/
ethyl acetate, 1:1) to give protected tripeptide 58 (0.408 g,
67%) as a yellow oil (1:1 mixture of Proa-C epimers).
Tripeptide 58 was shown to be 85:15 cis:trans mixture of
from the integration of the chemical shifts at d 4.28 and 4.46
assigned to the Proa-H protons of the major and minor
conformers, respectively); dH (300 MHz; CDCl3) 1.26
(2.4H, s, Prod-CH3), 1.28 [9H, s, C(CH3)3], 1.315 (0.6H,
s, Prod-CH3), 1.44* (0.6H, s, Prod-CH3), 1.46 (2.4H, s,
Prod-CH3), 1.59–2.15 (4H, m, Prob-H2, Prog-H2), 3.37
(0.8H, dd, JZ16.7, 3.3 Hz, Glya-HAHB), 3.57* (0.6H, s,
Proa-CO2CH3), 3.61 (2.4H, s, Proa-CO2CH3), 3.74 (0.8H,
dd, JZ16.7, 3.3 Hz, Glya-HAHB), 3.84* (0.2H, dd, JZ
16.9, 3.9 Hz, Glya-HAHB), 3.39–4.01* (0.2H, m, Glya-
HAHB), 4.28 (0.8H, d, JZ8.3 Hz, Proa-H), 4.46* (0.2H, dd,
JZ8.0, 2.2 Hz, Proa-H) and 5.40 (1H, br s, 1H, Gly-NH);
dH (75 MHz; CDCl3) 24.5 (CH3, Prod-CH3), 25.0* (CH2,
Prob-C), 26.1 (CH3, Prod-CH3), 26.9* (CH3, Prod-CH3),
27.2* (CH3, Prod-CH3), 27.4 (CH2, Prob-C), 27.9 [CH3,
C(CH3)3], 38.9 (CH2, Prog-C), 41.7* (CH2, Prog-C), 42.8*
(CH2, Glya-C), 43.1 (CH2, Glya-C), 51.7* (CH3, Proa-
CO2CH3), 52.3 (CH3, Proa-CO2CH3), 60.1 (CH, Proa-C),
60.9* (quat., Prod-C), 61.7* (CH, Proa-C), 63.8 (CH, Proa-
C), 78.0 [quat., C(CH3)3], 155.3 (quat., NCO2), 155.4*
(quat., NCO2), 166.5 (quat., Gly-CO), 167.7* (quat., Gly-
CO), 171.9 (quat., Proa-CO) and 172.4* (quat., Proa-CO);
m/z (CIC) 315.1927 (MHC C15H27N2O5 requires
315.1920).
1
conformers by H NMR analysis (the ratio was estimated
from the integration of the chemical shifts at d 5.31–5.38
and 5.48 assigned to the Gly-NH protons of the major and
minor conformers, respectively); dH (300 MHz; CDCl3)
1.41 [9H, s, C(CH3)3], 1.44 (2.55H, s, Prod-CH3), 1.52*
(0.45H, s, Prod-CH3), 1.54* (0.45H, s, Prod-CH3), 1.64
(2.55H, s, Prod-CH3), 1.67 (2.55H, s, Prod-CH3), 1.70–2.24
(6H, m, Prob-H2, Prog-H2, Glub-H2), 2.35–2.45 (2H, m,
Glug-H2), 3.58–3.69 (0.85H, m, Glya-HAHB), 3.85 (0.85H,
m, Glya-HAHB), 3.88–3.98* (0.15H, m, Glya-HAHB),
4.12–4.17* (0.15H, m, Glya-HAHB), 4.20–4.31 (1H, m,
Proa-H), 4.59–4.71 (1H, m, Glua-H), 5.10–5.22 (m, 4H,
2!OCH2Ph), 5.31–5.38 (0.85H, m, Gly-NH) and 5.48*
4.1.42. N-tert-Butoxycarbonylglycyl-(D,L)-5,5-dimethyl-
proline 55. To a solution of dipeptide 54 (0.362 g,
1.16 mmol) in dioxane (12 cm3) was added 1 M aqueous

10034
P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
(0.15H, m, Gly-NH); dC (75 MHz; CDCl3) 24.38 (CH3,
Prod-CH3), 24.46 (CH3, Prod-CH3), 25.1* (CH2, Prob-C),
25.2* (CH2, Prob-C), 26.2 (CH2, Glub-C), 26.3 (CH2, Glub-
C), 26.6 (CH3, Prod-CH3), 26.8* (CH2, Prob-C), 27.0*
(CH3, Prod-CH3), 27.1* (CH3, Prod-CH3), 27.9* (CH3,
Prod-CH3), 28.2 [CH3, C(CH3)3], 28.5 (CH2, Prob-C), 28.8
(CH2, Prob-C), 30.05* (CH2, Glug-C), 30.1* (CH2, Glug-
C), 30.2 (CH2, Glug-C), 30.25 (CH2, Glug-C), 38.9 (CH2,
Prog-C), 39.1 (CH2, Prog-C), 42.4** (CH2, Prog-C), 43.1*
(CH2, Glya-C), 43.3* (CH2, Glya-C), 43.5 (CH2, Glya-C),
43.6 (CH2, Glya-C), 51.7* (CH, Glua-C), 52.0 (CH, Glua-
C), 52.1 (CH, Glua-C), 61.5* (quat., Prod-C), 61.6* (quat.,
Prod-C), 61.9 (CH, Proa-C), 62.0 (CH, Proa-C), 63.0* (CH,
Proa-C), 63.05* (CH, Proa-C), 64.3 (quat., Prod-C), 64.3
(quat., Prod-C), 66.3* (CH2, OCH2Ph), 66.4* (CH2,
OCH2Ph), 66.41 (CH2, OCH2Ph), 66.5 (CH2, OCH2Ph),
67.0* (CH2, OCH2Ph), 67.1* (CH2, OCH2Ph), 67.21 (CH2,
OCH2Ph), 67.24 (CH2, OCH2Ph), 79.3 [quat., C(CH3)3],
128.1 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4*
(CH, Ph), 128.43 (CH, Ph), 128.5 (CH, Ph), 135.03 (quat.,
Ph), 135.07, (quat., Ph), 153.13* (quat., Ph), 135.18* (quat.,
Ph), 135.6 (quat., Ph), 135.7* (quat., Ph), 155.8* (quat.,
NCO2), 155.9 (quat., NCO2), 156.0 (quat., NCO2), 167.6
(quat., Gly-CO), 167.7 (quat., Gly-CO), 168.8* (quat., Gly-
CO), 169.3* (quat., Gly-CO), 171.1 (quat., Pro-CON),
171.3* (quat., Pro-CON), 171.4* (quat., Pro-CON), 171.6
(quat., Glua-CO), 171.8 (quat., Glua-CO), 172.4 (quat.,
Glug-CO), 172.5* (quat., Glug-CO) and 172.6 (quat., Glug-
CO); m/z (EIC) 609.3035 (MC C33H43N3O8 requires
609.3050).
JZ10.1 Hz, Proa-H) and 4.68* (0.28H, dd, JZ13.5,
4.3 Hz, Proa-H); dC (400 MHz; D2O) 23.56 (CH3, Prod-
CH3), 23.76 (CH3, Prod-CH3), 24.1** (CH3, Prod-CH3),
25.0 (CH2, Glub-C), 25.2 (CH3, Prod-CH3), 25.6 (CH2,
Glub-C), 25.7** (CH3, Prod-CH3), 26.0 (CH2, Glub-C),
26.1* (CH3, Prod-CH3), 26.2* (CH3, Prod-CH3), 26.4 (CH2,
Glub-C), 26.8* (CH2, Glub-C), 28.4, (CH2, Prob-C), 28.7,
(CH2, Prob-C), 30.8* (CH2, Glug-C), 31.0* (CH2, Glug-C),
31.2 (CH2, Glug-C), 38.7 (CH2, Prog-C), 38.8 (CH2, Prog-
C), 40.6* (CH2, Glya-C), 40.7 (CH2, Glya-C), 40.8 (CH2,
Glya-C), 41.1* (CH2, Prog-C), 41.2* (CH2, Prog-C),
46.1** (CH2, Glya-C), 54.1 (CH, Glua-C), 54.5 (CH,
Glua-C), 59.7* (CH, Proa-C), 61.7 (CH, Proa-C), 61.8
(CH, Proa-C), 62.6** (quat., Prod-C), 62.7* (quat., Prod-
C), 63.4* (CH, Proa-C), 63.9** (quat., Prod-C), 65.0 (quat.,
Prod-C), 65.1 (quat., Prod-C), 164.8 (quat., Gly-CO), 164.9
(quat., Gly-CO), 165.6* (quat., Gly-CO), 165.8* (quat.,
Gly-CO), 166.0** (quat., Gly-CO), 172.3** (quat., Pro-
CON), 172.8 (quat., Pro-CON), 173.1 (quat., Pro-CON),
173.3* (quat., Pro-CON), 173.6* (quat., Pro-CON), 176.9
(quat., Glua-CO), 177.3 (quat., Glua-CO) and 178.1 (quat.,
Glug-CO); m/z (FABC) 330.1666 (MHC C14H24N3O6
requires 330.1666).
Acknowledgements
The authors thank Neuren Pharmaceuticals Ltd. for financial
support of this work.
4.1.44. Glycyl-(D,L)-5,5-dimethylprolyl-L-glutamic acid
50. To a stirred solution of protected tripeptide 58
(0.276 g, 0.454 mmol) in dichloromethane (10 cm3) was
added trifluoroacetic acid (1 cm3) and the mixture stirred for
75 min. The solvent was removed in vacuo, the residue was
dissolved in saturated sodium hydrogen carbonate and the
product extracted with ethyl acetate. The combined organic
layers were washed with brine, dried (MgSO4) and the
solvent removed to yield an oil (0.244 g), which was
dissolved in methanol–water (4/1, 50 cm3). The flask was
flushed with nitrogen, 10% palladium on activated carbon
(0.048 mg, 0.454 mmol) was added and the mixture was
then stirred overnight under one atmosphere of hydrogen.
Filtration through Celitee followed by removal of the
solvent yielded an oil that was triturated with dry diethyl
ether to yield tripeptide 50 (0.139 g, 93%) as an off white
solid. Tripeptide 50 was shown to be a 72:28 cis:trans
References and notes
1. Sara, V. R.; Carlsson-Sdwirut, C.; Bergman, T.; Jornvall, H.;
Roberts, P. J.; Crawford, M.; Hakansson, L. N.; Civalero, I.;
Nordberg, A. Biochem. Biophys. Res. Commun. 1989, 165,
766.
2. Bourguignon, J. P.; Gerard, A. Brain Res. 1999, 847, 247.
3. Yamamoto, H.; Murphy, L. J. Endocrinology 1994, 135, 2432.
4. Yamamoto, H.; Murphy J. Endocrinol. 1995, 146, 141.
5. Sara, V. R.; Carlsson-Skwirut, C.; Andersson, C.; Hall, E.;
Sjogren, B.; Holmgren, A.; Jornvall, H. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 4904.
6. Sara, V. R.; Carlsson-Skwirut, C.; Drakenberg, K.; Giacobini,
M. B.; Hakansson, L.; Mirmiran, M.; Nordberg, A.; Olson, L.;
Reinecke, M.; Stahlbom, P. A.; Sandberg Nordqvist, A. C.
Ann. N.Y. Acad. Sci. 1993, 692, 183.
1
mixture of conformers by H NMR analysis (the ratio was
estimated from the integration of the chemical shifts at d
3.57 and 4.15–4.16 assigned to the Glya-H protons of the
major and minor conformers, respectively). Approximately
10% of the final product was tentatively assigned as the
hydrochloride salt**: mp 145–150 8C; dH (400 MHz; D2O)
1.43 (2.16H, s, Prod-CH3), 1.49* (0.84H, s, Prod-CH3),
1.57* (0.84H, s, Prod-CH3), 1.58* (0.84H, s, Prod-CH3),
1.60 (2.16H, s, Prod-CH3), 1.61 (2.16H, s, Prod-CH3), 1.90–
2.48 (8H, m, Prob-H2, Prog-H2, Glub-H2, Glug-H2), 3.57
(0.72H, dd, JZ16.1, 2.8 Hz, Glya-HAHB), 3.75–3.82**
(0.2H, m, Glya-HAHB, Glua-H), 3.94 (0.72H, dd, JZ16.1,
6.5 Hz, Glya-HAHB), 4.11** (0.1H, d, JZ2.5 Hz, Glya-
HAHB), 4.15–4.16* (0.56H, m, Glya-H2), 4.24–4.30 (1H,
m, Glua-H), 4.46–4.51** (0.1H, m, Proa-H), 4.60 (0.72H, t,
7. Carlsson-Skwirut, C.; Jornvall, H.; Holmgren, A.; Andersson,
C.; Bergman, T.; Lindquist, G.; Sjogren, B.; Sara, V. R. FEBS
Lett. 1986, 201, 46.
8. Sara, V. R.; Carlsson-Skwirut, C. Prog. Brain Res. 1988, 73,
87.
9. De Pablo, F.; De La Rosa, E. J. Trends Neurosci. 1995, 18,
143.
10. Gluckman, P. D.; Klempt, N. D.; Guan, J.; Mallard, C. E.;
Sirimanne, E. S.; Dragunow, M.; Klempt, M.; Singh, K.;
Williams, C. E.; Nijolics, K. Biochem. Biophys. Res. Commun.
1992, 182, 593.
11. Sizonenko, S. V.; Sirimanne, E. S.; Williams, C. E.;
Gluckman, P. D. Brain Res. 2001, 922, 42.
12. Sara, V. R.; Carlsson-Sdwirut, C.; Bergman, T.; Jornvall, H.;

P. W. R. Harris et al. / Tetrahedron 61 (2005) 10018–10035
10035
Roberts, P. J.; Crawford, M.; Hakansson, L. N.; Civalero, I.;
Nordberg, A. Biochem. Biophys. Res. Commun. 1989, 165,
766.
35. Lin, L.-N.; Brandts, J. F. Biochemistry 1983, 22, 4480.
36. Fanghanel, J. Angew. Chem., Int. Ed. 2003, 42, 490.
37. Delaney, N. G.; Madison, V. J. Am. Chem. Soc. 1982, 104,
6635.
13. Sara, V. R.; Carlsson-Skwirut, C.; Drakenberg, K.; Giacobini,
M. B.; Hakansson, L.; Mirmiran, M.; Nordberg, A.; Olson, L.;
Reinecke, M.; Stahlbom, P. A.; Sandberg Nordqvist, A. C.
Ann. N.Y. Acad. Sci. 1993, 692, 183.
38. 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, 43, 1777.
14. Guan, J.; Waldvogel, H. J.; Faull, R. L. M.; Gluckman, P. D.;
Williams, C. E. Neuroscience 1999, 89, 649.
39. Fernandez, M. M.; Diez, A. D.; Rubiralta, M.; Montenegro, E.;
Casamitjana, N.; Kogan, M. J.; Giralt, E. J. Org. Chem. 2002,
67, 7587.
15. Saura, J.; Curatolo, L.; Williams, C. E.; Gatti, S.; Benatti, L.;
Peters, C.; Guan, J.; Dragunow, M.; Post, C.; Faull, R. L. M.;
Gluckman, P. D.; Skinner, S. J. M. Neuroreport 1999, 10, 161.
16. Guan, J.; Krishnamurthi, R.; Waldvogel, H. J.; Faull, R. L. M.;
Clark, R.; Gluckman, P. Brain Res. 2000, 859, 286.
17. Alexi, T.; Hughes, P. E.; Van Roon-Mom, W. M. C.; Faull,
R. L. M.; Willaims, C. E.; Clark, R.; Gluckman, P. D. Exp.
Neurol. 1999, 159, 84.
40. Kang, Y. K. J. Phys. Chem. 2002, 106, 2074.
41. Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.; Fischer,
G. S.; Mutter, M. J. Am. Chem. Soc. 1998, 120, 2714.
42. Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wohr, T.;
Mutter, M. M. J. Am. Chem. Soc. 1997, 119, 918.
43. An, S. S. A.; Lester, C. C.; Peng, J.-L.; Li, Y.-J.; Rothwarf,
E. W.; Thannhauser, T. W.; Zhang, L. S.; Tam, J. P.; Scheraga,
H. A. M. J. Am. Chem. Soc. 1999, 121, 11558.
18. Abood, N. A.; Brimble, M. A. PCT Int. Appl. 0,294,856, 2002.
19. Gluckman, P. D.; Williams, C. E.; Guan, J.; Krishnamurthi, R.
V. M. U.S. Patent Appl. Publ. 20020035066, 2002.
20. Gluckman, P.; Alexi, T. PCT Int. Appl. 0,216,408, 2002.
21. Gluckman, P. D.; Sirimanne, E. S.; Krissansen, G. W.;
Kanwar, J. R. U.S. Patent Appl. Publ. 2,003,027,760, 2003.
22. Trotter, N. S.; Brimble, M. A.; Callis, D. J.; Harris, P. W. R.;
Sieg, F. Bioorg. Med. Chem. 2003, 13, 501.
44. Pellegrini, N.; Refouvelet, G.; Crini, G.; Blacque, O.; Kubicki,
M. M.; Robert, J.-F. Chem. Pharm. Bull. 1999, 47, 950.
45. Lewis, N. J.; Inloes, R. L.; Hes, J.; Matthews, R. H.; Milo, G.
J. Med. Chem. 1978, 21, 1070.
46. Kemp, D. S.; Carey, R. I. J. Org. Chem. 1989, 54, 3640.
47. Ono, N.; Kamimura, A.; Miyake, H.; Hamamoyo, I.; Kaji, A.
J. Org. Chem. 1985, 50, 3692.
23. Lai, M. Y. H.; Brimble, M. A.; Callis, D. J.; Harris, P. W. R.;
Levi, M. S.; Sieg, F. Bioorg. Med. Chem. 2003, 13, 533.
24. Brimble, M. A.; Trotter, N. S.; Harris, P. W. R.; Sieg, F.
Bioorg. Med. Chem. 2003, 13, 519.
48. Haire, D. L.; Hilborn, J. W.; Janzen, E. G. J. Org. Chem. 1986,
51, 4298.
49. Magaard, V. W.; Sachez, R. M.; Bean, J. W.; Moore, M. L.
Tetrahedron Lett. 1993, 34, 381.
25. Bisang, C.; Weber, C.; Inglis, J.; Schiffer, C. A.; Gunsteren,
W. F.; Jelesaov, I.; Bosshard, H. R.; Robinson, J. A. M. J. Am.
Chem. Soc. 1995, 117, 7904.
50. An, S. A.; Lester, C. C.; Peng, J.-L.; Li, Y.-J.; Rothwarf, D. M.;
Welker, E.; Thannhauser, T. W.; Zhang, L. S.; Tam, J. P.;
Scheraga, H. A. J. Am. Chem Soc. 1999, 121, 11588.
51. During the preparation of this manuscript another group
reported the synthesis of compounds 48, 49 and 50: De Diego,
S. A. A.; Munoz, P.; Gonzalez-Muniz, R.; Herranz, R.; Martin-
Martinez, M.; Cenarruzabeita, E.; Frechilla, D.; Del Rio, J.;
Jimeno, M. L.; Garcia-Lopez, M. T. Bioorg. Med. Chem. 2005,
15, 2279. Full experimental details were not provided.
52. Lewis, A.; Wilkie, J.; Rutherford, T. J.; Gani, D. J. Chem. Soc.,
Perkin Trans. 1 1998, 3777.
26. Welsh, J. H.; Zerbe, P.; von Phillipborn, W.; Robinsoon, J. A.
FEBS Lett. 1992, 297, 216.
27. Thaisrivongs, S.; Pals, D. T.; Lawson, J. A.; Turner, S. R.;
Harris, D. W. J. Med. Chem. 1987, 30, 536.
28. Wang, H.; Germanas, J. P. Synlett 1999, 1, 33.
29. Seebach, D.; Boes, M.; Naef, R.; Schweizer, W. B. J. Am.
Chem. Soc. 1983, 105, 5390.
30. Beck, A. K.; Blank, S.; Job, K.; Seebach, D. E.; Sommerfeld,
T. J. Org Synth. 1992, 72, 62.
31. Amedjkouh, M.; Ahlberg, P. Tetrahedron: Asymmetry 2002,
13, 2229.
53. Hoffmann, T.; Lanig, H.; Waibel, R.; Gmeiner, P. Angew.
Chem., Int. Ed. 2001, 40, 3361.
32. During the preparation of this manuscript another group
reported the synthesis of G-2MePE: De Diego, S. A. A.;
Munoz, P.; Gonzalez-Muniz, R.; Herranz, R.; Martin-
Martinez, M.; Cenarruzabeita, E.; Frechilla, D.; Del Rio, J.;
Jimeno, M. L.; Garcia-Lopez, M. T. Bioorg. Med. Chem. 2005,
15, 2279. Full experimental details were not provided.
33. Dugave, C.; Demange, L. Chem. Rev. 2003, 103, 2475.
34. Vanhoof, G.; Goosens, F.; De Meester, I.; Hendricks, D.;
Scharpe, S. FASEB J. 1995, 9, 736.
54. Yasuo, H.; Suzuki, M.; Yoneda, N. Chem. Pharm. Bull. 1979,
27, 1931.
55. Bonnet, R.; Brown, R. F. C.; Clark, V. M.; Sutherland, I. O.;
Todd, A. J. Chem. Soc. 1959, 34, 381.
56. Bertho, J.-N.; Loffet, A.; Pinel, C.; Reuther, F.; Sennyey, G.
Tetrahedron Lett. 1991, 32, 1303.
57. Carpino, L. A.; Mansour, El.-S. M. E.; Sadat-Aalaee, D.
J. Org. Chem. 1991, 56, 2611.