Enantioselection in peptide bond formation

Article abstract of DOI:10.1039/b211914e

Selectivity in abiotic condensations of amino acids remains controversial and stereochemically little explored. We find that competitive activated couplings of N-acyl derivatives of glycine, alanine, valine, proline and phenylalanine with binary, ternary and quaternary mixtures of amides and esters of the same group of amino acids show little selectivity among the reactants, except with respect to configuration, where a consistent and significant preference for heterochiral outcomes, mostly >80%, is observed. One possible explanation of this selectivity predicts a predisposition to homochiral coupling under conditions that would require the two carboxyl functions to be co-facial in the activated complex.

Full text of DOI:10.1039/b211914e

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Enantioselection in peptide bond formation
Roger R. Hill,*a David Birch,b Graham E. Jeffsa and Michael North†b
a Department of Chemistry, The Open University, Milton Keynes, UK MK7 6AA.
E-mail: r.r.hill@open.ac.uk
b Department of Chemistry, University of Wales, Bangor, Gwynedd, UK LL57 2UW
Received 5th December 2002, Accepted 29th January 2003
First published as an Advance Article on the web 25th February 2003
Selectivity in abiotic condensations of amino acids remains controversial and stereochemically little explored.
We find that competitive activated couplings of N-acyl derivatives of glycine, alanine, valine, proline and
phenylalanine with binary, ternary and quaternary mixtures of amides and esters of the same group of amino
acids show little selectivity among the reactants, except with respect to configuration, where a consistent and
significant preference for heterochiral outcomes, mostly >80%, is observed. One possible explanation of this
selectivity predicts a predisposition to homochiral coupling under conditions that would require the two
carboxyl functions to be co-facial in the activated complex.
peptide elongation. For example, N-phthaloyl (Phth) amino
Introduction
acids are not only convenient for chromophorically-based
detection in product analysis but also preclude oxazolone-
mediated α-CH epimerization, and N,N-dimethylamido
(DMA) derivatives of amino acids afford co-reactants with an
inert C-terminus. In confining our studies to derivatives of
glycine, alanine, phenylalanine, proline and valine, we also
avoided potentially complicating factors with the more reactive
side chains while retaining a stereochemically representative
selection.
Competitive activated couplings involved presenting one type
of substrate with the opportunity to react with two, three or
four of the other type, the latter usually present in considerable
molar excess. We found that stereochemical selectivity not only
is the dominant factor controlling product distribution but,
with N-terminal acyl protection, it also strongly favours
reaction between substrates of opposite configuration.
The results we reported in a preliminary publication11 have
been extended to more complex mixtures of Phth- and DMA-
derivatives and to show the effects of changes in activating
group, solvent, N-protecting group and C-terminal group.
The heterochiral bias remains the major influence on product
distribution in the more complex mixtures and appears to
be independent of activating and C-terminal groups. It is lost,
however, when acyl N-protection is replaced by a urethane.
One possible explanation of these observations, supported by
a solvent effect, implies that a predisposition to homochiral
coupling would occur if the two reactants were linked through
their carboxyl functions.
The formation of peptide bonds in biological protein synthesis
is totally prescribed. Well-known but complex derivatives of
α-amino acids, cofactors and templates ensure that, in each
case, successive couplings involve a unique sequence of homo-
chiral amino acids. Spontaneous abiotic formation of peptides
from mixtures of amino acids have been widely investigated,
however, and prompt much interest within the contexts of
prebiotic chemistry,1,2 peptide synthesis3 and combinatorial
chemistry.4 In principle, some selectivity among amino acids
may be anticipated, leading to crudely ‘informational’ products.
In practice, however, the significance of any selectivity remains
controversial. Competitive condensations in aqueous solutions
of proteinogenic amino acids mediated by a carbodiimide indi-
cate a range of about three-fold in reactivity, too small to allow
any meaningful sequence selection.5 On the other hand, coup-
ling propensities in more recently reported condensations, in
saturated salt solutions containing Cu() and on alumina sur-
faces, appear not only to have significant variation but also to
align broadly with the dipeptide units occurring most fre-
quently in the membrane proteins of archaebacteria and
prokarytic cells.2
Stereochemical selectivity is fundamentally important as
homochirality is considered a prerequisite for, rather than a
consequence of life.6 Moreover, with no selectivity, only one
molecule in 500 would be homochiral in a mixture of deca-
peptides, where secondary structure becomes significant.7
Studies of N-carboxyanhydride elongation indicate some bias
towards a homochiral preference in extending a peptide chain,8
but the limited scope of both models and process prompt the
examination of other systems before the wider significance of
these results can be evaluated. Any such selectivity would be
inconsequential if conditions allowed thermodynamically
driven epimerization to occur, but this would, in any case,
weaken the relevance of the experiments to protobiology.9 In
spite of a great many theories and experimental studies, a plaus-
ible scenario for spontaneous abiotic assembly of a homochiral
peptide remains a severe challenge.10
Results and discussion
The outcomes of 71 competitive coupling experiments are
summarised in Tables 1–5. Table 1 shows, with one exception, a
consistent and significant preference for heterochiral couplings,
irrespective of whether the excess reagent is the Phth or the
DMA component. Evidence for kinetic rather than thermo-
dynamic control in these reactions can be seen from the experi-
ment with equimolar reactants that, on completion, shows a
product ratio close to 1 : 1. The heterochiral preference was
further confirmed by the results from equimolar racemic
reagents (not tabulated). Thus, the percentages of heterochiral
diastereoisomer in reactions between racemic Phth-alanine and
racemic dimethylamides of alanine, valine and phenylalanine
were 75.6, 89.3 and 87.9, respectively. Results from more
complex mixtures also emphasise a heterochiral preference
when that option is available (Table 3). Tables 2 and 3 otherwise
As a contrast to N-carboxyanhydride derivatives, we chose to
investigate competitions between amino acid derivatives that
involve other forms of activation popular in modern peptide
synthesis, together with selective protection that is both con-
venient for analytical purposes and suitable for modelling
† Present address: Department of Chemistry, Kings College, University
of London, The Strand, London, UK WC2R 2LS.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Table 1 Competition outcomes in reactions between Phth--A and
racemic B-DMA using DCC–HOBt coupling in CH2Cl2, except where
indicated
Table 3 Competition outcomes between Phth-A and equimolar mix-
tures of B-DMA, C-DMA and D-DMA using DCC–HOBt couplinga
Product distribution (%)b
[B-DMA]
A
B
Initial [Phth--A]
% -dipeptide derivativea
A
B
C
D
AB
AC
AD
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Phe
Phe
Phe
Phe
Phe
Phe
Val
Val
Val
Ala
Ala
Ala
Phe
Phe
Phe
Val
Val
Val
Val
Phe
Phe
Phe
Ala
Val
Pro
Val
Ala
Phe
9.9
1.0
86
55
85
92
90
85
94
94c
83d
74e
89
87
75
90
94
54
96
95f
83
Gly
Gly
Gly
-Phe
-Phe
-Ala
Gly
Gly
Gly
-Ala
-Ala
-Ala
-Ala
-Val
-Ala
-Phe
-Phe
-Phe
-Val
52
54
54
57
15
14
30
33
39
18
6
18
12
7
0.08b
7.0
-Phe
-Phe
-Val
-Val
-Val
18.0
45.0
8.0
10.7
9.5
24
4() 75()
8() 64()
14
a {[B-DMA] ϩ [C-DMA] ϩ [D-DMA]} / [Phth-A] = 15.0 except for the
first experiment (10.0). b Error < 5%.
9.7
10.0
6.3
0.1b
7.5
10.0
10.0
10.0
10.0
8.3
a Error <
5%. b Racemic Phth-A and -B-DMA. c DCC–HOSu in
CH2Cl2. d DCC–HOSu in DMF. e DCC–HOBt in DMF. f CZE analysis
using a Beckman P/ACE instrument, fused silica capillary (40 cm ×
75 µm id), 40 mmol SDS in 20 mmol borate/phosphate buffer, pH 8.6
adjusted with NaOH, benzyl alcohol as EOF marker, capillary temp.
25 ЊC, applied voltage 20 kV and UV detection at 214 nm.
factors are considered to be stereochemically influential in the
transition states of other reactions; for example, secondary
orbital interactions in the Diels–Alder reaction12 and ring-
based conformational preferences in chelate-assisted aldol con-
densations.13) Some minor trends seen in Tables 1–3 would be
consistent with this hypothesis; for example, the small but con-
sistently progressive effect as the side chains become more
bulky, the lesser selectivity seen when DMF, another amide, is
used as solvent, the apparent insensitivity to a change in activ-
ating group (Table 1) and an additional influence that may be
anticipated with benzyl side chains in close proximity with each
other or with a six-membered ring.14 The exceptional result
in Table 1 with the dimethylamide of proline may reflect
some resistance to the trans ring fusion prescribed by a chair
transition state.
demonstrate that when a choice of amino acid is present, select-
ivity additional to that of stereochemistry is only significant in
respect of the preferred coupling with glycyl residues and of the
slightly disfavoured coupling with phenylalanyl residues.
The presence of one reactant in excess allows the approxim-
ation that product ratios in competing reactions correspond
with ratios of rate constants. Thus, the rate of peptide bond
formation in these reactions is largely independent of β-substi-
tution but significantly and uniformly faster between amino
acid derivatives of opposite configuration. The probable activ-
ated complex for the hydroxybenzotriazole (HOBt)-mediated
condensations, 1, is generally consistent with this observation,
for the α-carbon atoms are separated by two other atoms
whereas any β-carbon atoms would be less sterically significant.
The consistent preference for heterochiral outcomes is less
readily anticipated, but would follow if 1 gained secondary
stabilization by interactions between the terminal groups in a
six-membered chair conformation. If such were the case, the
more favoured diequatorial disposition of side chains might be
expected, and would lead to the observed results (2). (Similar
Table 2 Competition outcomes in reactions between Phth-X and equimolar mixtures of Y-DMA and Z-DMA using DCC–HOBt coupling
[PhthXYDMA]
[PhthXYDMA]
X
Y
Z
Initial[Y-DMA] ϩ [Z-DMA]
Final
%  dipeptide derivativea
[Phth-X]
-Ala
-Ala
-Ala
-Ala
-Ala
-Ala
-Ala
Gly
-Ala
-Ala
-Ala
-Ala
-Ala
-Ala
-Ala
Gly
Gly
Gly
Gly
Gly
-Phe
-Phe
-Phe
-Phe
-Val
-Val
-Val
-Ala
-Phe
-Val
-Ala
-Val
-Phe
-Val
-Val
-Val
6.0
0.17
13.0
10.2
10.0
11.0
10.0
9.3
1.0
0.8
0.3
3.5
1.4
0.2
9.0
2.7
7.3
4.2
2.1
2.3
9.0
0.07
1.06
7.3
—
—
78
78
—
85
90
—
—
—
—
—
—
93
—
88
Gly
Gly
8.1
10.0
10.0
8.0
-Val
-Val
-Val
-Phe
-Phe
-Phe
Gly
8.8
-Phe
-Phe
-Phe
10.0
10.0
10.0
a Error < 5%.
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Table 4 Competition outcomes in reactions between X--A-OH and racemic H-B-Y using DCC–HOBt coupling in CH2Cl2, except where indicated
[B-Y]
X
A
B
Y
Initial [X--A]
% -dipeptide derivativea
Succ
Ac
Fmoc
Fmoc
Fmoc
Z
Phe
Pro
Ala
Phe
Pheb
Ala
Phe
Val
Phe
Phe
Ala
Phe
Phe
Ala
Phe
Val
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
10.0
10.0
10.0
10.0
10.0
9.2
88
52
40
49
39
37
53
44
Z
Z
10.8
10.0
Z
Z
Z
Z
Fmoc
Fmoc
Ala
Ala
Phe
Val
Ala
Ala
Phe
Val
Ala
Val
OMe
OMe
OMe
OMe
OMe
OMe
10.2
11.0
11.0
10.5
11.0
10.0
40c
38
59
54
37
55
Ala
Valb
Fmoc
Fmoc
Fmoc
Fmoc
Phth
Phth
Phth
Phth
Phth
Phth
Ala
Ala
Val
Val
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Val
Val
Ala
Ala
Val
Val
Phe
Phe
O-resind
O-resin
O-resin
O-resin
O-resin
O-resin
O-resin
O-resin
O-resin
O-resin
0.1
1.0
0.1
1.0
0.1
1.0
0.1
1.0
0.1
1.0
44
49
51
50
70
52
69
56
66
56
Phth
Succ
Phth
Fmoc
Val
Phe
Ala
Ala
Val
Val
Ala
Ala
NHMe
NHMe
Ala-O-resin
Ala-O-resin
10.0
10.0
0.1
90
87
96
47
0.1
a Error < 5%. b Pfp (perfluorophenate) activated. c DCC–HOSu in CH2Cl2. d (CH2C6H4OC6H4CHCH2)n; all experiments with resin based reactants
used racemic X-A-OPfp and -B-Oresin, with products converted to NMe2 derivative for analysis.
Table 5 Enantioselectivitya in the reactions between P1NHCH(R1)COX and H2NCH(R2)COP2
P2
P1
NMe2
NHMe
AlaOresin
Oresin
OMe
X
Phth
Phth
Phth
Phth
Phth
Sucnmd
Acb
Fmoc
Fmoc
Z
OBt–DCM
OBt–DMF
OSu–DCM
OSu–DMF
Pfp
OBt–DCM
OBt–DCM
OBt–DCM
Pfp
ϩϩ
ϩ
ϩϩ
ϩ
ϩϩ
ϩϩ
ϩ (ϩϩ)
0 (Ϫ)
ϩϩ
0
Ϫ(0)
ϩϩ
0
Ϫ
0 (Ϫ)
0
OBt–DCM
0 (Ϫ)
a Summarising outcomes of 74 reactions with derivatives of alanine, valine, phenylalanine and proline: ϩϩ >80% ; ϩ60–80% ; 0 <60% ;
Ϫ60–80% ; minority outcome in parentheses. b N-acetylproline.
Table 4 shows outcomes with further structural variation in
reactants. Thus, perfluorophenate activation, replacing the
DMA group by a secondary amido function or the ester linkage
of a Wang resin, and replacing phthalimido by succinimido all
retain the heterochiral bias. Reactions with N-acetyl proline
(another ‘oxazolone-safe’ reagent), however, show no selectivity
but as before, one may anticipate a distinctive conformational
influence on the outcome in this case.15 Urethane protection,
even with DMA co-reactants, also leads to little or no selectiv-
ity, implying that the stabilising interaction proposed in 2 would
need a significant electrophilic contribution from the N-protect-
ing group. Table 5 summarises all the outcomes and suggests
that selectivity requires an amide function at the N-terminus
of the carboxy reactant but allows both amide and ester at the
C-terminus of the amino reactant.
generally be too small to direct side-chain sequences.5 At the
same time, however, they demonstrate a clear possibility of
stereochemical selectivity in abiotic condensations, which could
be further enhanced with more closely interacting secondary
features and lower temperatures.16 In the absence of epimeriz-
ation equilibria, reactions analogous to those we have exam-
ined, for example, would convey a prospect of sequences in
extendable systems having alternate configurations at Cα until
interrupted by glycyl.
As mentioned earlier, a contrasting but weaker homochiral
preference has been observed in chain extension mediated by
N-carboxyanhydrides.8 A transition state that simultaneously
minimises steric hindrance between side chains and allows
secondary stabilizing interactions with the anhydride ring (3)
predicts this outcome. We are thus prompted to consider what
features might promote homochiral selection in the more gen-
eral case, such as ours, of activated open-chain reactants. If the
The results support Dose’s contention that rate differences
in condensations in mixtures of activated amino acids would
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stabilized activated complex was constrained to adopt a
boat-like conformation similar to 3, then the more favoured
diequatorial disposition of side chains would correspond with
homochiral configurations. Such conformations (4) would be
prescribed if the two carboxyl derivatives were linked.
HPLC analyses were performed using a Waters Millennium
System comprising a ‘616’ quaterniary gradient pump, a ‘996’
photodiode array and a ‘717ϩ’ autosampler. Conditions were
selected for optimum resolution of Phth-DMA dipeptide
derivatives, including diastereoisomers, for each competition
experiment using the synthesised standards, one of three
columns (Phenomenex Luna C18 5 µm 250 × 4.6 mm id;
Spherisorb S5 ODS1 250 × 4.6 mm id; Hypercarb 5 µm 100 ×
4.6 mm id) and aqueous acetonitrile gradients.19 Peak purities
for Phth-DMA derivatives in competition mixtures were
checked using protocols available from the system software,
including visual scrutiny of superimposed UV spectra.
Wang resin and Fmoc- and ester derivatives of amino acids
were obtained from Novabiochem or synthesized by standard
procedures.20 FmocOSu was kindly donated by Eastman
Chemicals, Llangefni and the Sigma or Avocado Chemical
Companies supplied all other chemicals.
General procedure for the synthesis of N-phthaloyl and
N-succinimido amino acids21
Amino acid (0.02 mol) and the appropriate anhydride
(0.02 mol) were refluxed in toluene (20 cm3) for 20 h.22 The
protected amino acid crystallised on cooling and was isolated
by filtration. Yields 62–83%.
While the overall complexity of ribosomal protein syn-
thesis17 obscures relevance of these ideas to the present day
system, far less complex and more directly linked carboxyl
derivatives, including mineral surface complexes,6,18 could apply
in the prebiotic context, and a study of model compounds
further exploring such concepts is in progress.
General procedure for the synthesis of amino acid
dimethylamides and methylamides
Z-protected amino acids were synthesised using N-(benzyloxy-
carbonyloxy) succinimide.23 Z-protected amino acid (0.017
mol) was dissolved in DCM (20 cm3) and HOBt24 (0.018 mol)
was added, followed by DCC (0.018 mol). To this solution, a
mixture of dimethylamine or methylamine HCl (0.018 mol) and
triethylamine (0.018 mol), suspended in DCM (10 cm3), was
added and the mixture stirred at room temperature for 24 h.
Precipitated DCHU was removed by filtration, DCM removed
by rotary evaporation and subsequent trituration with cold
EtOAC removed more DCHU. The EtOAc solution was
washed with 0.5 M aq HCl (3 × 15 cm3), 0.5 M aq KHCO3
(3 × 15 cm3), water (2 × 15 cm3) and dried (anh. MgSO4).
Solvent removal left the Z-amino acid dimethylamide or
methylamide as a crystalline solid in yields of 64–72%. These
were treated with HBr–HOAc (45%, 5 cm3) for 2 h at room
temperature. Addition of ice-cold Et2O precipitated the amino
acid dimethylamide or methylamide HBr salt which was
recovered by filtration in yields of 42–53%.
Conclusions
Significant enantioselection in abiotic peptide bond formation
is demonstrable between N-acyl α-amino acids and α-amino
acid amides or esters, in which heterochiral coupling predomin-
ates. This would be consistent with the tetrahedral intermediate
being formed via an activated complex that can access a stabilis-
ing non-bonded interaction in the chair conformation of a
quasi six-membered ring, thereby placing the side chains in the
more favoured diequatorial positions. Such positions in the
analogous boat-like conformation, expected when the two
carboxyl functions are constrained to be on the same side of the
ring, would lead to homochiral coupling. While sharing some
similarities with those of the peptide transfer step of protein
synthesis, these features are perhaps more indicative of one way
homochiral peptides could have arisen prebiotically, given the
probable availability of suitable carboxyl-binding templates.
General procedure for the synthesis of dipeptide derivatives
N-Protected amino acid (0.01 mol) and amino acid di-
methylamide or methylamideؒHBr (0.01 mol) were coupled
using the above procedure to give crystalline solids which were
purified by column chromatography on silica with Et2O–MeOH
as eluent; yields 10–24%. Methyl esters were prepared in a simi-
lar manner, coupling the N-protected amino acid with the
amino acid methyl ester hydrochloride salt; yields 20–25%.
Experimental section
General
Melting temperatures were measured using a Gallenkamp
instrument. Optical rotations given in 10Ϫ1 deg cm2 gϪ1 were
recorded on an Optical Activity Polar 2001 polarimeter, at 25
ЊC and a concentration of 0.01 g cmϪ3 in chloroform. Infrared
spectra were recorded as films evaporated from CHCl3 solu-
General procedure for competitive coupling experiments
N-Protected--amino acid (0.1 mmol) and racemic amino acid
amide or ester halide salt (1.0 mmol) were allowed to react
using the above coupling procedure for 48 h. The DCM was
removed by rotary evaporation, the residue dissolved in the
initial HPLC eluent (30% aq MeCN) and a homogenous
sample analysed by the area ratio of peaks identified by stand-
ards as the   and   diastereoisomers of the corresponding
derivatized peptides.
1
tions on a Perkin-Elmer 1600 series FTIR spectrometer. H
NMR spectra were recorded at 250 MHz on a Bruker AM250
spectrometer fitted with a 1H–13C dual probe, at 293 K. CDCl3
was used as solvent and the spectra were internally referenced
to the residual solvent peak. 13C NMR spectra were recorded at
62.9 MHz on the same spectrometer, at 293 K, using CDCl3
and spectra were referenced to the solvent peak. Peak assign-
ments were assisted by DEPT editing of the spectra. Elemental
analyses were performed on a Carlo Erba Model 1106 or 1108
analyser. High resolution mass measurements were made on a
VG ZAB-E spectrometer.
General procedure for resin coupling experiments
Experiments were conducted with freshly distilled solvents in a
25-cm3 vessel designed for solid-state synthesis.25 In a typical
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experiment, Fmoc--Ala-Wang resin (Novabiochem, 100 mg)
was treated with piperidine (20% v/v in DMF) for 2 h,
washed with DCM (3 × 10 cm3) and DMF (3 × 10 cm3), and the
deprotected amine allowed to react with Fmoc-rac-Ala-Pfp
(Novabiochem, 37 mg in 10 cm3 DMF) for 24 h. The dipeptide
derivative was released from the resin with TFA–DCM (1 : 1)
and converted to the methyl ester with 9 : 1 MeOH–AcCl over
24 h in an overall yield of 57%.
13.09%); νmax/cmϪ1 (selected bands) 3370, 2924, 1715, 1630,
1385, 1049, 881; δH 1.34 (d, 3H), 1.71 (d, 3H), 2.91 (s, 3H), 3.01
(s, 3H), 4.81–4.98 (m, 2H), 7.04 (br d, 1H), 7.68 (m, 2H), 7.82
(m, 2H); δC 15.2, 18.3, 35.7, 36.9, 45.8, 48.9, 123.5, 132.0, 134.1,
167.9, 168.2, 172.1.
Phth-L-Ala-L-Val-DMA
Mp: 152–154 ЊC. [α]D: ϩ34.3. (Found: C, 61.94; H, 6.63;
N, 12.07. C18H23N3O4 requires: C, 61.95; H, 6.76; N, 12.04%);
νmax/cmϪ1 (selected bands) 3316, 2931, 1716, 1634, 1386, 1216,
756; δH 0.81 (d, 3H), 0.84 (d, 3H), 1.62 (d, 3H), 1.90–2.01 (m,
1H), 2.90 (s, 3H), 3.07 (s, 3H), 4.71 (m, 1H), 4.81 (m, 1H), 7.30
(br d, 1H), 7.69 (m, 2H), 7.82 (m, 2H); δC 15.3, 17.4, 19.6, 31.6,
35.7, 37.4, 49.11, 53.9, 123.5, 131.9, 134.2, 167.7, 169.0, 171.5.
Phth-Gly-Gly-DMA
Mp: 165 ЊC. (Found: C, 57.62; H, 5.23; N, 14.73. C14H15-
N3O4ؒ0.2 H2O requires: C, 57.41; H, 5.29; N, 14.35%); νmax/cmϪ1
(selected bands) 3314, 1721, 1650, 1418, 952, 714; δH 2.91 (s,
3H), 3.00 (s, 3H), 4.08 (d, 2H), 4.49 (s, 2H), 7.00 (br, s, 1H), 7.73
(m, 2H), 7.78 (m, 2H); δC 35.6, 35.9, 40.5, 41.4, 123.5, 123.6,
134.2, 166.0, 167.7.
Phth-L-Ala-D-Val-DMA
Mp: 118–120 ЊC. [α]D: Ϫ3.1. (Found: C, 60.59; H, 6.58; N,
11.45. C18H23N3O4ؒ2H2O requires: C, 60.49; H, 6.86; N,
11.76%); νmax/cmϪ1 (selected bands) 3314, 2932, 1716, 1634,
1388, 1216, 757; δH 0.90 (d, 3H), 0.93 (d, 3H), 1.71 (d, 3H),
1.94–2.13 (m, 1H), 2.91 (s, 3H), 3.10 (s, 3H), 4.82 (q, 1H), 4.91
(q, 1H), 6.75 (br d, 1H), 7.69 (d, 2H), 7.80 (d, 2H); δC 15.3, 17.4,
19.5, 31.6, 35.6, 37.3, 49.0, 53.9, 123.4, 131.9, 134.7, 167.7,
168.8, 171.4.
Phth-Gly-L-Ala-DMA
Mp: 177 ЊC. [α]D: Ϫ204.0. (Found: C, 60.08; H, 5.99; N, 13.96.
C15H17N3O4 requires: C, 59.40; H, 5.68; N, 13.85%); νmax/cmϪ1
(selected bands) 3315, 1723, 1638, 1417, 953; δH 1.29 (d, 3H),
2.91 (s, 3H), 3.09 (s, 3H), 4.91 (q, 1H), 4.35 (s, 2H), 7.02 (br, s,
1H), 7.25 (m, 2H), 7.88 (m, 2H); δC 18.5, 35.8, 36.9, 40.5, 49.2,
123.5, 132.0, 134.1, 165.2, 167.8, 172.0; m/z (FABϩ) 304.1294.
(C15H18N3O4 requires 304.1297.)
Phth-L-Ala-L-Phe-DMA
Phth-Gly-L-Val-DMA
Mp: 108–112 ЊC. [α]D: ϩ18.1. (Found: C, 65.99; H, 5.86; N,
10.41. C22H23N3O4ؒ0.5 H2O requires: C, 66.15; H, 5.97; N,
10.52%); νmax/cmϪ1 (selected bands) 3317, 2928, 1712, 1631,
1135, 1175, 721; δH 1.78 (d, 3H), 2.57 (s, 3H), 2.86 (s, 3H),
2.86–3.09 (m, 2H), 4.99 (m, 1H), 5.04 (m, 1H), 6.95 (br d, 1H),
7.08–7.18 (m, 5H), 7.67 (m, 2H), 7.78 (m, 2H); δC 15.2, 35.6,
36.7, 39.4, 49.1, 50.7, 123.5, 127.0, 128.4, 129.5, 131.5, 131.9,
134.2, 136.0, 167.7, 168.3, 170.7.
Mp: 154 ЊC. [α]D: Ϫ145.5. (Found: C, 61.85; H, 6.67; N, 12.52.
C17H21N3O4 requires: C, 61.62; H, 6.39; N, 12.68%); νmax/cmϪ1
(selected bands) 3296, 1719, 1630, 1420, 954, 714; δH 0.85
(d, 3H), 0.91 (d, 3H), 2.0 (q, 1H), 2.97 (s, 3H), 2.98 (s, 3H), 4.41
(d, 2H), 4.82 (q, 1H), 6.75 (br d, 1H), 7.69 (m, 2H), 7.88 (m,
2H); δC 17.4, 19.6, 24.9, 31.8, 33.9, 40.5, 53.8, 123.6, 132.11,
134.1, 165.9, 167.7, 171.4.
Phth-Gly-L-Phe-DMA
Phth-L-Ala-D-Phe-DMA
Mp: 96–98 ЊC. [α]D: Ϫ125.0. (Found: C, 65.77; H, 5.63; N,
Mp: 104–107 ЊC. [α]D: Ϫ33.3. (Found: C, 64.91; H, 5.83; N,
10.16. C22H23N3O4ؒ3H2O requires: C, 64.93; H, 6.07; N,
10.33%); νmax/cmϪ1 (selected bands) 3318, 2928, 1714, 1634,
1386, 1257, 723; δH 1.79 (d, 3H), 2.60 (s, 3H), 2.85 (s, 3H),
2.87–3.09 (m, 2H), 4.90 (m, 1H), 5.05 (m, 1H), 6.90 (br d, 1H),
7.09–7.19 (m, 5H), 7.68 (m, 2H), 7.79 (m, 2H); δC 15.2, 35.6,
36.8, 39.3, 49.1, 50.6, 123.4, 124.0, 127.0, 128.4, 129.5, 131.9,
134.1, 136.0, 167.7, 168.8, 171.4.
11.34. C21H21N3O4 requires: C, 65.85; H, 6.04; N, 10.97%); νmax
/
cmϪ1 (selected bands) 3407, 1718, 1635, 1419; δH 2.50 (s, 3H),
2.71 (s, 3H), 2.95 (m, 2H), 4.39 (s, 2H), 5.08 (q, 1H), 7.1 (m,
5H), 7.48 (br d, 1H), 7.68 (m, 2H), 7.84 (m, 2H); δC 24.9, 25.6,
39.1, 40.4, 50.5, 123.5, 127.0, 128.4, 129.4, 132.1, 134.1, 136.0,
165.6, 167.8, 171.0; m/z (FABϩ) 380.1615. (C15H18N3O4
requires 380.1610.)
Phth-L-Val-Gly-DMA
Phth-L-Val-L-Ala-DMA
Mp: 85–87 ЊC. [α]D: Ϫ2.0. (Found: C, 61.43; H, 6.12; N, 12.60.
C17H21N3O4 requires: C, 61.62; H, 6.39; N, 12.68%); νmax/cmϪ1
(selected bands) 3334, 1715, 1649, 1384, 1070, 718; δH 0.85
(d, 3H), 1.12 (d, 3H), 2.90 (m, 1H), 2.95 (s, 3H), 3.0 (s, 3H), 4.02
(d, 2H), 4.51 (d, 1H), 7.70 (m, 2H), 7.86 (m, 3H); δC 19.5, 20.1,
27.5, 35.5, 35.8, 41.7, 62.4, 123.7, 131.5, 134.3, 167.5, 168.2,
168.7.
Mp: 135–137 ЊC. [α]D: ϩ27.2. (Found: C, 62.42; H, 6.92; N,
12.02. C18H23N3O4 requires: C, 62.59; H, 6.71; N, 12.16%);
νmax/cmϪ1 (selected bands) 3392, 2965, 2932, 1716, 1636, 1384,
1070, 730; δH 0.85 (d, 3H), 1.11 (d, 3H), 1.32 (d, 3H), 2.85 (m,
1H), 2.91 (s, 3H), 3.02 (s, 3H), 4.41 (d, 1H), 4.88 (m, 1H), 7.70
(m, 3H), 7.88 (m, 3H); δC 18.3, 19.5, 20.0, 27.4, 35.7, 36.9, 45.7,
62.3, 123.6, 131.4, 134.2, 167.7, 168.1, 171.8.
Phth-L-Ala-L-Ala-DMA
Phth-L-Val-D-Ala-DMA
Mp: 166–168 ЊC. [α]D: ϩ8.6. (Found: C, 61.11; H, 6.04; N,
Mp: 123–125 ЊC. [α]D: Ϫ12.0. (Found: C, 61.21; H, 6.57; N,
11.79. C18H23N3O4ؒH2O requires: C, 61.00; H, 6.83; N, 11.86%);
νmax/cmϪ1 (selected bands) 3419, 2932, 1716, 1636, 1384, 1070,
730; δH 0.87 (d, 3H), 1.12 (d, 3H), 1.34 (d, 3H), 2.90 (m, 1H),
2.98 (s, 3H), 3.08 (s, 3H), 4.41 (d, 1H), 4.87 (m, 1H), 7.72 (m,
2H), 7.75 (br d, 1H), 7.89 (m, 2H); δC 18.3, 19.5, 20.1, 27.5, 35.7,
36.9, 45.7, 62.3, 123.7, 131.5, 134.3, 167.7, 168.2, 172.0.
13.28. C16H19N3O4 requires: C, 60.56; H, 6.03; N, 13.24%); νmax
/
cmϪ1 (selected bands) 3395, 2934, 1714, 1635, 1387, 1215, 754;
δH 1.30 (d, 3H), 1.70 (d, 3H), 2.955 (s, 3H), 3.08 (s, 3H), 4.80–
5.00 (m, 2H), 7.05 (br d, 1H), 7.70 (m, 2H), 7.84 (m, 2H);
δC 14.8, 17.9, 35.2, 36.4, 45.4, 48.5, 123.1, 131.9, 133.7, 167.7,
168.0, 171.9; m/z (FABϩ) 318.1450. (C16H20N3O4 requires
318.1454.)
Phth-L-Val-L-Val-DMA
Phth-L-Ala-D-Ala-DMA
Mp: 170–171 ЊC. [α]D: ϩ0.7. (Found: C, 64.45; H, 7.10; N,
Mp: 148–150 ЊC. [α]D: Ϫ2.0. (Found: C, 60.14; H, 6.17; N,
13.29. C16H19N3O4. 0.25 H2O requires C, 59.88; H, 6.09; N,
11.70. C20H27N3O4 requires: C, 64.32; H, 7.29; N, 11.25%); νmax/
cmϪ1 (selected bands) 3344, 2967, 2932, 1716, 1637, 1385, 1216,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 9 6 5 – 9 7 2
969

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754; δH 0.86 (d, 3H), 0.92 (d, 3H), 0.98 (d, 3H), 1.12 (d, 3H),
2.02 (m, 1H), 2.89–2.91 (m, 1H), 2.91, 3.09 (s, 3H), 4.41 (d, 1H),
4.81 (m, 1H), 7.34 (br d, 1H), 7.72 (m, 2H), 7.89 (m, 2H);
δC 17.5, 19.4, 19.6, 20.1, 27.4, 31.1, 35.7, 37.4, 53.9, 62.2, 123.6,
131.4, 134.3, 168.1, 168.5, 171.3.
νmax/cmϪ1 (selected bands) 3318, 2932, 1716, 1634, 1387, 1216,
752; δH 0.87 (d, 3H), 1.02 (d, 3H), 1.95–2.12 (m, 1H), 2.94 (s,
3H), 3.00 (s, 3H), 3.55 (m, 2H), 4.86 (m, 1H), 5.18 (m, 1H), 6.98
(br d, 1H), 7.10 (m, 5H), 7.63 (m, 2H), 7.78 (m, 2H); δC 17.5,
19.6, 31.6, 34.7, 35.7, 37.4, 54.0, 55.4, 123.4, 126.9, 128.6, 128.9,
131.5, 134.1, 136.7, 167.8, 168.1, 171.3.
Phth-L-Val-D-Val-DMA
Phth-L-Phe-L-Phe-DMA
Mp: 110–111 ЊC. [α]D: Ϫ0.8. (Found: C, 63.16; H, 7.04; N,
11.52. C20H27N3O4ؒ0.5H2O requires: C, 63.31; H, 7.35; N,
11.07%); νmax/cmϪ1 (selected bands): 3351, 2967, 2930, 1716,
1638, 1384, 1216, 756; δH 0.81 (d, 3H), 0.90 (d, 3H), 0.98 (d,
3H), 1.12 (d, 3H), 2.00 (m, 1H). 2.84–2.88 (m, 1H), 2.89 (s, 3H),
3.17 (s, 3H), 4.38 (d, 1H), 4.81 (m, 1H), 7.34 (br d, 1H), 7.72 (m,
2H), 7.85 (m, 2H); δC 17.6, 19.4, 19.6, 20.1, 27.4, 31.1, 35.7,
37.4, 54.0, 62.1, 123.6, 131.4, 134.3, 168.2, 168.6, 171.4.
Mp: 128–130 ЊC. [α]D: Ϫ76.0. (Found: C, 71.71; H, 5.82;
N, 9.17. C28H27N3O4 requires: C, 71.65; H, 5.76; N, 8.95%);
νmax/cmϪ1 (selected bands) 3377, 2929, 1716, 1635, 1384, 1104,
753; δH 2.60 (s, 3H), 2.85 (s, 3H), 2.90–3.10 (m, 2H), 3.51 (m,
2H), 5.15 (m, 2H), 6.90 (br d, 1H), 7.10 (m, 10H), 7.61 (m, 2H),
7.75 (m, 2H); δC 34.6, 35.5, 36.8, 39.5, 55.3, 59.7, 123.5, 126.8,
127.0, 128.4, 128.5, 128.9, 129.4, 131.5, 134.1, 136.1, 136.7,
167.6, 167.7, 170.7.
Phth-L-Val-L-Phe-DMA
Phth-L-Phe-D-Phe-DMA
Mp: 144–145 ЊC. [α]D: ϩ5.2. (Found: C, 67.88; H, 6.78; N,
10.06. C24H27N3O4 requires: C, 68.07; H, 6.88; N, 9.92%);
νmax/cmϪ1 (selected bands) 3332, 2928, 1716, 1636, 1384, 1070,
730; δH 0.85 (d, 3H), 1.00 (d, 3H), 2.81–2.85 (m, 1H), 2.66 (s,
3H), 2.84 (s, 3H), 3.01 (m, 2H), 4.41 (d, 1H), 5.30 (m, 1H), 7.30
(br d, 1H), 7.70 (m, 2H), 7.81 (m, 2H); δC 19.5, 19.8, 27.4, 35.6,
36.8, 39.4, 50.3, 62.2, 123.7, 126.9, 128.3, 129.3, 131.5, 134.3,
136.2, 168.0, 168.2, 171.0.
Mp: 111–113 ЊC. [α]D: Ϫ46.9. (Found: C, 70.01; H, 5.88; N,
9.04. C28H27N3O4ؒ0.6 H2O requires: C, 69.98; H, 5.88; N,
8.75%); νmax/cmϪ1 (selected bands) 3318, 2926, 1715, 1636,
1384, 1215, 756; δH 2.63 (d, 3H), 2.85 (d, 3H), 2.91–3.20 (m,
2H), 3.55 (m, 2H), 5.13 (m, 2H), 7.03 (br d, 1H), 7.12 (m, 10H),
7.65 (m, 2H), 7.78 (m, 2H); δC 34.6, 35.5, 36.8, 39.4, 50.6, 55.6,
123.5, 126.8, 127.0, 128.4, 128.6, 128.8, 129.4, 131.5, 134.1,
136.0, 136.6, 167.6, 167.8, 170.6.
Phth-L-Val-D-Phe-DMA
Phth-L-Phe-L-Pro-DMA
Mp: 103–105 ЊC. [α]D: Ϫ2.3. (Found: C, 67.46; H, 6.77; N,
10.08. C24H27N3O4ؒ0.5H2O requires: C, 67.43; H, 6.52; N,
9.83%); νmax/cmϪ1 (selected bands) 3339, 2932, 1716, 1636,
1384, 1070, 730; δH 0.81 (d, 3H), 1.06 (d, 3H), 2.71 (s, 3H), 2.89
(s, 3H), 2.77–2.91 (m, 1H), 2.98 (m, 2H), 4.39 (d, 1H), 5.10 (q,
1H), 6.90 (m, 5H), 7.51 (br d, 1H), 7.70 (m, 2H), 7.79 (m, 2H);
δC 19.5, 19.9, 27.3, 35.6, 36.8, 38.9, 50.4, 62.7, 123.6, 131.4,
134.3, 168.1, 168.5, 171.3.
Mp: 180–181 ЊC. [α]D: Ϫ148.8. (Found: C, 68.77; H, 6.53;
N, 10.05. C24H25N3O4 requires: C, 68.72; H, 6.01; N, 10.02%);
νmax/cmϪ1 (selected bands) 3331, 2929, 1716, 1648, 1382, 1101,
750, 721; δH 1.85–1.97 (m, 2H), 2.01–2.11 (m, 2H), 2.90 (s, 3H),
3.06 (s, 3H), 3.28–3.41 (m, 2H), 3.61–3.65 (m, 2H), 4.48–4.92
(m, 1H), 5.20–5.24 (m, 1H), 7.08–7.27 (m, 5H), 7.64 (m, 2H),
7.75 (m, 2H); δC 26.3, 26.4, 29.9, 35.5, 37.1, 46.8, 55.6, 58.5,
124.7, 124.9, 128.1, 129.8, 129.9, 130.0, 130.3, 130.5, 130.8,
135.4, 135.5, 166.9, 167.6, 171.1, 171.6.
Phth-L-Phe-L-Ala-DMA
Mp: 159–161 ЊC. [α]D: Ϫ9.7. (Found: C, 63.88; H, 6.17; N,
10.38. C22H23N3O4ؒH2O requires: C, 64.22; H, 6.12; N, 10.21%);
νmax/cmϪ1 (selected bands) 3422, 2922, 1715, 1635, 1384, 1104,
721; δH 1.25 (d, 3H), 2.87 (s, 3H), 3.02 (s, 3H) 3.51 (m, 2H), 4.85
(t, 1H), 5.15 (t, 1H), 7.05 (m, 5H), 7.12 (br d, 1H), 7.51 (m, 2H),
7.71 (m, 2H); δC 18.3, 35.7, 36.9, 45.9, 55.3, 123.5, 126.8, 128.5,
128.9, 131.5, 134.1, 134.7, 136.7, 167.3, 167.7, 171.8.
Phth-L-Phe-LD-Pro-DMA (diastereomeric mixture)
Mp: 130–134 ЊC. [α]D: Ϫ103.0. (Found: C, 67.07; H, 6.29; N,
9.47. C24H25N3O4ؒ0.5H2O requires: C, 67.27; H, 6.12; N,
1
9.81%); distinctive H NMR features, δ: 2.97 (s, 3H), 3.11 (s,
3H), 3.69–3.79 (m, 2H); distinctive 13C NMR features, δ: 36.0,
38.5, 48.6, 56.9, 57.1, 166.3.
Phth-L-Phe-D-Ala-DMA
Z-L-Val-L-Val-OMe
Mp: 116–117 ЊC. [α]D: Ϫ72.7. (Found: C, 66.59; H, 5.84; N,
10.45. C22H23N3O4ؒ0.25 H2O requires: C, 66.55; H, 5.94; N,
10.58%); νmax/cmϪ1 (selected bands) 3417, 1714, 1636, 1385,
1104, 720; δH 1.30 (d, 3H), 2.92 (s, 3H), 3.09 (s, 3H), 3.59 (m,
2H), 4.90 (t, 1H), 5.15 (t, 1H), 7.10–7.14 (m, 6H), 7.65 (m, 2H),
7.75 (m, 2H); δC 18.4, 35.6, 36.9, 45.9, 55.3, 123.5, 126.9, 128.5,
128.9, 131.5, 134.1, 134.7, 136.7, 167.3, 167.8, 171.8.
Mp: 94–95 ЊC. [α]D: ϩ1.5. (Found: C, 62.26; H, 7.64; N, 7.76.
C19H28N2O5ؒ0.2 H2O requires: C, 62.01; H, 7.78; N, 7.61%);
νmax/cmϪ1 (selected bands) 3306, 2962, 1730, 1658, 1538, 1212,
1027; δH 0.89–1.00 (m, 12H), 2.15 (m, 2H), 3.72 (s, 3H), 4.05 (t,
1H), 4.52 (q, 1H), 5.10 (s, 2H), 5.45 (br d, 1H), 6.45 (br d, 1H),
7.38 (s, 5H); δC 17.7, 17.8, 18.9, 19.1, 31.2, 52.1, 57.1, 60.3, 66.9,
127.9, 128.1, 128.5, 171.5, 172.2.
Phth-L-Phe-L-Val-DMA
Z-L-Val-D-Val-OMe
Mp: 138–139 ЊC. [α]D: Ϫ40.6. (Found: C, 67.51; H, 6.54; N,
9.53. C24H27N3O4ؒ0.5 H2O requires C, 67.43; H, 6.52; N,
9.83%); νmax/cmϪ1 (selected bands) 3308, 2929, 1714, 1635,
1384, 1215, 1103, 756; δH 0.85 (d, 3H), 1.00 (d, 3H), 1.90–1.98
(m, 1H), 2.94 (s, 3H), 3.01 (s, 3H), 3.55 (m, 2H), 4.86 (m, 1H),
5.15 (m, 1H), 7.01 (br d, 1H), 7.10 (m, 5H), 7.61 (m, 2H), 7.75
(m, 2H); δC 17.4, 19.5, 31.6, 34.7, 35.5, 37.2, 54.0, 55.3, 123.4,
126.9, 128.6, 128.9, 131.5, 134.1, 136.7, 167.8, 168.1, 171.3.
Mp: 142–143 ЊC. [α]D: Ϫ5.9. (Found: C, 62.01; H, 7.55; N, 7.83.
C19H28N2O5ؒ0.1 H2O requires: C, 62.31; H, 7.76; N, 7.65%);
νmax/cmϪ1 (selected bands) 3386, 2958, 1731, 1688, 1646, 1540,
1250; δH 0.82–0.98 (m, 12H), 2.16 (m, 2H), 3.74 (s, 3H), 4.10 (t,
1H), 4.56 (q, 1H), 5.12 (s, 2H), 5.45 (br d, 1H), 6.44 (br d, 1H),
7.43 (s, 5H); δC 17.6, 17.8, 18.9, 19.3, 31.1, 52.2, 57.2, 60.4, 67.1,
128.0, 128.2, 171.5, 172.2.
Phth-L-Phe-D-Val-DMA
Z-L-Phe-L-Phe-OMe
Mp: 138–140 ЊC. [α]D: Ϫ108. (Found: C, 68.02; H, 6.74; N,
10.20. C24H27N3O4ؒH2O requires: C, 68.07; H, 6.88; N, 9.91%);
Mp: 134–136 ЊC. [α]D: Ϫ21.9. νmax/cmϪ1 (selected bands) 3306,
2928, 1652, 1538, 1454, 1216, 749, 699; δH 2.96–3.07 (m, 4H),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 9 6 5 – 9 7 2
970

View Online
3.68 (s, 3H), 4.45 (m, 2H), 5.08 (s, 2H), 5.46 (br s, 1H), 6.45 (br
s, 1H), 7.20–7.41 (m, 15H); δC 37.8, 52.4, 53.3, 127.1, 128.0,
128.2, 128.6, 128.7, 129.2, 129.3, 135.5, 136.1, 136.2, 170.5,
171.3; m/z (FABϩ) 461.2070. (C26H29N2O5 requires 461.2076.)
2.91 (s, 3H), 3.00 (s, 3H), 4.15 (q, 1H), 4.18 (m, 2H), 4.32 (d,
2H), 5.40 (br d, 1H), 6.95 (br d, 1H), 7.20–7.39 (m, 4H), 7.37 (d,
2H), 7.57 (d, 2H); δC 18.5, 19.2, 35.8, 36.9, 45.4, 47.1, 52.1, 67.1,
120.0, 125.1, 127.0, 127.7; m/z (FABϩ) 410.2075. (C23H28N3O4
requires 410.2080).
Z-L-Phe-D-Phe-OMe
Fmoc-L-Phe-L-Phe-DMA
Mp: 132–134 ЊC. [α]D: Ϫ28.1. νmax/cmϪ1 (selected bands) 3378,
1658, 1539, 1216, 698; δH 2.96–3.02 (m, 4H), 3.66 (s, 3H), 4.49
(br d, 1H), 4.85 (q, 1H), 5.06 (s, 2H), 5.51 (br s, 1H), 6.50 (br s,
1H), 7.20–7.31 (m, 15H); δC 37.8, 38.5, 52.3, 53.0, 67.1, 127.1,
127.2, 128.0, 128.1, 128.2, 128.5, 128.6, 128.7, 129.2, 129.3,
135.5, 136.1, 136.2, 170.5, 171.5; m/z (FABϩ) 461.2072.
(C26H29N2O5 requires 461.2076.)
Mp: 116–117 ЊC. [α]D: ϩ1.2. (Found: C, 71.80; H, 6.45; N, 7.23.
C35H35N3O4ؒ1.25 H2O requires: C, 71.53; H, 6.43; N, 7.15%);
νmax/cmϪ1 (selected bands) 3280, 3063, 2926, 1720, 1631, 1537,
1449, 1252, 1043, 740, 700; δH 2.59 (s, 3H), 2.86 (s, 3H), 2.99 (d,
2H), 3.09 (d, 2H), 4.09–4.30 (m, 3H), 4.45 (q, 1H), 4.62 (br d,
1H), 5.12 (q, 1H), 5.65 (br s, 1H), 7.18–7.44 (m, 14H), 7.55 (t,
2H), 7.77 (d, 2H); δC 35.5, 36.8, 38.6, 39.7, 47.1, 50.3, 55.9, 67.0,
112.0, 125.1, 127.0, 127.7, 128.4, 128.6, 136.1, 136.3, 141.3,
143.8, 170.1, 170.6.
Z-L-Ala-L-Ala-OMe and Z-L-Ala-D-Ala-OMe
Were synthesised by the above general procedure and showed
characterisation data consistent with that reported in the
literature.26
Fmoc-L-Phe-D-Phe-DMA
Mp: 126–129 ЊC. [α]D: Ϫ18.2. (Found: C, 72.52; H, 6.52;
N, 7.32. C35H35N3O4 requires: C, 72.77; H, 6.11; N, 7.27%);
νmax/cmϪ1 (selected bands) 3279, 3062, 2922, 1720, 1633, 1496,
1450, 1250, 1046, 906, 737, 700; δH 2.57 (s, 3H), 2.82 (s, 3H),
3.08 (d, 4H), 4.17–4.43 (m, 3H), 4.53 (q, 1H), 5.07 (q, 1H), 5.52
(br d, 1H), 6.94 (br s, 1H), 7.15–7.43 (m, 14H), 7.53 (d, 2H),
7.65 (m, 2H); δC 35.6, 36.8, 39.0, 39.6, 47.1, 50.1, 56.0, 67.0,
112.0, 125.1, 127.1, 127.7, 128.5, 128.7, 129.4, 136.0, 136.3,
141.2, 143.8, 170.1, 170.7.
Z-L-Ala-L-Ala-DMA
Mp: 126–128 ЊC. [α]D: Ϫ1.9. (Found: C, 60.99; H, 7.34; N,
13.06. C16H23N3O4ؒ0.2 H2O requires: C, 59.14; H, 7.26; N,
12.89%); νmax/cmϪ1 (selected bands) 3323, 2927, 1626, 1535,
1453, 1245, 1070; δH 1.24 (d, 3H), 1.48 (d, 3H), 2.91 (s, 3H), 3.01
(s, 3H), 4.23 (br q, 1H), 4.80 (q, 1H), 5.06 (s, 2H), 5.54 (br s,
1H), 7.30 (s, 5H), 7.40 (br s, 1H); δC 18.5, 19.2, 35.8, 36.9, 49.3,
50.6, 66.9, 128.0, 128.5, 171.5, 172.0.
Fmoc-L-Val-LD-Val-OMe (diastereomeric mixture)
Z-L-Ala-D-Ala-DMA
Mp: 138–140 ЊC. [α]D: ϩ33.2. (Found: C, 68.19; H, 7.52; N,
8.82. C26H32N2O5ؒ0.5 H2O requires: C, 68.33; H, 7.64; N,
8.85%); νmax/cmϪ1 (selected bands) 3373, 2964, 1717, 1542,
1451, 1218, 1100, 1027; δH 0.88 (d, 3H), 0.98 (d, 3H), 1.20 (d,
3H), 1.26 (d, 3H), 2.18 (m, 1H), 2.46 (m, 1H), 3.74 (s, 3H), 3.75
(s, 3H), 4.25 (m, 1H), 4.33 (m, 1H), 4.53 (d, 2H), 4.96 (t, 1H),
5.53 (d, 1H), 7.40 (m, 4H), 7.60 (d, 2H), 7.77 (d, 2H,) 8.08 (d,
1H); δC 17.8, 18.9, 19.0, 19.3, 31.2, 47.1, 52.1, 52.2, 57.1, 68.1,
120.7, 120.9, 121.0, 121.2, 122.0, 126.1, 128.1, 129.0, 130.0,
142.3, 145.0, 172.2, 172.3, 173.2. HPLC (cf below): rt, 24.67 and
25.10 min; λmax(eluent)/nm 220.0, 270.5, 292.2, 302.0.
Mp: 133–135 ЊC. [α]D: Ϫ7.1. (Found: C, 60.99; H, 7.34; N,
13.06. C16H23N3O4ؒ0.2 H2O requires: C, 59.14; H, 7.26; N,
12.89%); νmax/cmϪ1 (selected bands) 3280, 2926, 1631, 1538,
1450, 1252, 1043, 741, 700; δH 1.29 (d, 3H), 1.36 (d, 3H), 2.86 (s,
3H), 3.03 (s, 3H), 4.25 (br q, 1H), 4.80 (q, 1H), 5.02 (d, 2H),
5.54 (br s, 1H), 7.21 (s, 5H), 7.30 (br s, 1H); δC 18.0, 18.6, 35.9,
37.1, 49.3, 50.7, 66.7, 125.8, 126.3, 126.6, 127.9, 128.0, 172.3,
172.7; m/z (FABϩ) 322.1767. (C16H24N3O4 requires 322.1767.)
Z-L-Phe-L-Phe-DMA
Mp: 115–117 ЊC. [α]D: Ϫ12.1. (Found: C, 69.68; H, 6.56; N,
9.03. C28H31N3O4ؒ0.5 H2O requires: C, 69.69; H, 6.68; N,
8.71%); νmax/cmϪ1 (selected bands) 3392, 1719, 1632, 1496,
1453, 1256, 1047, 743, 698; δH 2.57 (s, 3H), 3.00 (s, 3H), 3.01–
3.16 (br q, 2H), 4.69 (q, 1H), 5.03 (s, 2H), 5.20 (q, 1H), 5.91 (br
s, 1H), 7.05–7.34 (m, 15H), 8.11 (br s, 1H); δC 35.6, 35.8, 38.8,
39.6, 49.0, 55.9, 66.7, 126.8, 127.0, 127.9, 128.0, 128.4, 128.5,
129.4, 129.5, 129.8, 170.7, 170.9, 171.1.
Fmoc-L-Val-L-Val-OMe
Was isolated as a yellow oil without further purification.
νmax/cmϪ1 (selected bands) 3372, 2931, 1713, 1537, 1450, 1216,
1101, 1025; δH 0.89 (d, 3H), 0.91 (d, 3H), 0.94 (d, 3H), 0.98 (d,
3H), 2.18 (m, 2H), 3.74 (s, 3H), 4.23 (m, 1H), 4.43 (m, 1H), 4.57
(d, 2H), 4.94 (t, 1H), 5.51 (br s, 1H), 7.40 (m, 4H), 7.62 (d, 2H),
7.78 (d, 2H,) 8.05 (d, 1H). HPLC: rt, 24.67 min; λmax(eluent)/nm
220.0, 270.5, 292.2, 302.0.
Z-L-Phe-LD-Phe-DMA (diastereomeric mixture)
Mp: 115–118 ЊC. [α]D: ϩ0.5. (Found: C, 70.81; H, 6.60; N, 8.95.
C28H31N3O4 requires: C, 71.02; H, 6.60; N, 8.87%); νmax/cmϪ1
(selected bands) 3279, 1049, 746. Distinctive 1H NMR features,
δ: 2.82 (s, 3H), 3.05 (d, 2H), 4.49 (q, 1H), 5.01–5.03 (m, 3H),
5.35 (br s, 1H), 6.96 (br s, 1H). Distinctive 13C NMR features,
δ: 38.8, 50.1, 50.3.
Fmoc-L-Ala-L-Ala-OMe and Fmoc-L-Ala-D-Ala-OMe
Were synthesised by the above general procedure and showed
characterisation data consistent with that reported in the
literature.27
Suc-L-Phe-L-Phe-DMA
Fmoc-L-Ala-L-Ala-DMA
Mp: 145–148 ЊC. [α]D: Ϫ0.4. (Found: C, 67.13; H, 6.85; N,
Mp: 198–199 ЊC. [α]D: Ϫ1.4. νmax/cmϪ1 (selected bands) 3294,
2928, 1718, 1636, 1508, 1449, 1248, 1117, 758; δH 1.29 (d, 3H),
1.35 (d, 3H), 2.92 (s, 3H), 3.05 (s, 3H), 4.20 (d, 2H), 4.82 (q,
1H), 5.45 (br d, 1H), 6.95 (br s, 1H), 7.20–7.40 (m, 4H), 7.57 (d
2H), 7.74 (d 2H); δC 18.6, 19.0, 35.8, 47.1, 47.2, 52.2, 67.1,
120.0, 125.2, 127.1, 127.2; m/z (FABϩ) 410.2081. (C23H28N3O4
requires 410.2080.)
10.36. C24H27N3O4 requires: C, 67.46; H, 6.67; N, 10.26%); νmax
/
cmϪ1 (selected bands) bands) 3315, 2930, 2855, 1775, 1708,
1622, 1561, 1393, 1255, 1164, 751; δH 2.51 (d, 4H), 2.62 (s, 3H),
2.87 (s, 3H), 2.90–3.04 (m, 2H), 3.46–3.51 (m, 2H), 4.98 (q, 1H),
5.11 (q, 1H), 7.03 (br s, 1H), 7.12–7.29 (m, 10H); δC 27.9, 33.7,
35.6, 36.8, 39.4, 50.7, 55.4, 127.0, 127.1, 128.4, 128.6, 128.9,
129.4, 136.0, 136.4, 167.1, 176.7, 186.1.
Fmoc-L-Ala-D-Ala-DMA
Suc-L-Phe-LD-Phe-DMA (diastereomeric mixture)
Mp: 145 ЊC. [α]D: ϩ11.4. νmax/cmϪ1 (selected bands) 3410, 1718,
1636, 1521, 1449, 1248, 1118, 740; δH 1.29 (d, 3H), 1.35 (d, 3H),
Mp: 156–158 ЊC. [α]D: Ϫ76.8. (Found: C, 68.10; H, 6.90; N,
10.00. C24H27N3O4ؒ0.7 H2O requires: C, 68.17; H, 6.76; N,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 9 6 5 – 9 7 2
971

View Online
9.94%); Distinctive IR bands (see above)/cmϪ1: 1635, 1533,
1387, 751; distinctive 1H NMR features, δ: 2.45 (d, 4H), 2.59 (d,
3H), 4.90 (q, 1H), 5.05 (q, 1H), 7.03 (br s, 1H); distinctive 13C
NMR features, δ: 24.7, 25.4, 36.0, 38.7, 49.6, 53.5, 171.6, 182.4.
Prog. Biophys. Mol. Biol., 2001, 75, 75; B. M. Rode, J. Phys. Chem.,
1992, 96, 4170; A. Brack, Origins Life Evol. Biosphere, 1987, 17, 367;
K. Dose, Naturwissenschaften, 1983, 70, 378.
2 B. M. Rode, Peptides, 1999, 20, 773; B. M. Rode and Y.
Suwannachot, Coord. Chem. Rev., 1999, 192, 1085; J. Budjak and
B. M. Rode, J. Inorg. Biochem., 2002, 90, 1–7.
Suc-L-Phe-L-Val-DMA
3 M. Mutter, Int. J. Pept. Protein Res., 1979, 13, 274.
4 N. K. Terret, Combinatorial Chemistry, OUP, Oxford, 1998, p. 19,
55; S. R. Wilson and A. W. Czarnik, Combinatorial Chemistry,
Wiley, New York, 1997, p. 225; A. Whiting, Chem. Br., 1999, 31.
5 K. Dose, Interdiscip. Sci. Rev., 1988, 13, 348.
6 P. Cintas, Angew. Chem., Int. Ed., 2002, 41, 1139; A. Brack, in
The Molecular Origin of Life, ed. A. Brack, Cambridge Univ. Press.,
Cambridge, 1998, pp. 2–8.
7 I. A. Kozlov, L. E. Orgel and P. E. Nielsen, Angew. Chem., Int. Ed.,
2000, 39, 4292; S. Roberts and J. Skidmore, Chem. Br., 2000, 31.
8 S. I. Goldberg, J. M. Crosby, N. D. Iusem and U. E. Yonnes,
J. Am. Chem. Soc., 1987, 109, 823; K. E. Wen and L. E. Orgel,
Origins Life Evol. Biosphere, 2001, 31, 241; T. Hitz, M. Blocher,
P. Walde and P. L. Luisi, Macromolecules, 2001, 34, 2443.
9 M. Bengtson and E. D. Edstrom, in Advances in Biochirality,
eds. G. Palyi, C. Zucchi and L. Caglioti, Elsevier, Amsterdam, 1999,
p. 115; V. A. Basiuk, Adv. Space Res., 2001, 27, 335.
10 A. Saghatelian, Y. Yokobayashi, K. Soltani and M. R. Ghadiri,
Nature, 2001, 409, 797; T. Munegumi and A. Shimoyama, Origins
Life Evol. Biosphere, 2000, 30, 216; R. Shapiro, Origins Life Evol.
Biosphere, 2000, 30, 243.
Colourless oil. [α]D: Ϫ37.2. νmax/cmϪ1 (selected bands) 3315,
2932, 1708, 1648, 1534, 1388, 1153, 747; δH 0.85 (d, 3H), 0.93 (d,
3H), 2.09 (m, 1H), 2.58 (br s, 4H), 2.80 (d, 3H), 3.43 (m, 2H),
4.21 (t, 1H), 5.02 (q, 1H), 6.50 (br s, 1H), 6.82 (br s, 1H), 7.16–
7.28 (m, 5H); δC 17.9, 19.2, 26.2, 27.8, 33.7, 34.8, 55.4, 59.2,
125.2, 127.2, 128.8, 129,2, 136.1, 168.1, 171.3, 176.8, 176.9;
m/z (FABϩ) 360.1921. (C19H26N3O4 requires 360.1923).
Suc-L-Phe-LD-Val-DMA (diastereomeric mixture)
Colourless oil. [α]D: Ϫ13.4. Distinctive IR bands (see above)/
cmϪ1: 3326, 1654, 1216, 1163, 751; distinctive 1H NMR features,
δ: 2.73 (d, 4H), 2.80 (d, 3H); distinctive 13C NMR features,
δ: 18.1, 18.3, 30.6, 34.0, 126.3, 126.7, 137.0, 182.4; m/z (FABϩ)
360.1920. (C19H26N3O4 requires 360.1923).
N-Acetyl-L-Pro-L-Phe-DMA
11 Preliminary results reported in D. Birch, R. R. Hill, G. E. Jeffs and
M. North, Chem. Commun., 1999, 941.
12 (a) E. Eliel and S. H. Wilen, Stereochemistry of Organic Compounds,
Wiley Interscience, New York, 1994, p. 921; (b) G. Mehta and
R. Uma, Acc. Chem. Res., 2000, 33, 278; P. Caramella, P. Quadrelli
and L. Toma, J. Am. Chem. Soc., 2002, 124, 1130.
13 Ref. 12a p. 899. For recently cited examples, see T. D. Machajewski
and C. H. Wong, Angew. Chem., Int. Ed., 2000, 39, 1352.
14 S. M. Butterfield, P. R. Patel and M. L. Waters, J. Am. Chem. Soc.,
2002, 124, 9751; C. D. Tatko and M. L. Waters, J. Am. Chem. Soc.,
2002, 124, 9372; Y. Imanishi, in Advances in Polymer Science, 20,
ed. H. J. Cantow, Springer Verlag, Berlin, 1976, p. 1; M. J. O.
Anteunis, Bull. Soc. Chim. Belg., 1978, 87, 627.
Mp: 59–160 ЊC. [α]D: Ϫ70.4. (Found: C, 64.21; H, 8.02; N,
12.64. C18H25N3O5ؒ0.25 H2O requires: C, 64.36; H, 7.95; N,
12.50%); νmax/cmϪ1 (selected bands) 3448, 2926, 1627, 1531,
1443, 1420, 1249, 702; δH 1.16–1.22 (m, 2H), 1.86–1.92 (m 2H),
2.06 (s, 3H), 2.67 (s, 3H), 2.80 (d, 1H), 2.85 (s, 3H), 2.89 (d, 1H),
3.36–3.49 (m, 2H), 4.47 (d, 1H), 5.12 (q, 1H), 7.15–7.27 (m,
5H), 7.71 (dd, 1H); δC 22.4, 22.6, 35.6, 36.8, 38.9, 39.2, 49.1,
50.0, 126.8, 128.2, 128.5, 129.2, 129.4, 136.0, 136.4, 170.5,
170.8.
N-Acetyl-L-Pro-LD-Phe-DMA (diastereomeric mixture)
15 D. A. Block, D. Yu, D. A. Armstrong and A. Rauk, Can. J. Chem.,
1998, 76, 1042; G. B. Laing, C. J. Rito and S. H. Gellman,
Biopolymers, 1992, 32, 293; A. M. Sapse, L. Mallahlevy, S. B.
Daniels and B. W. Erikson, J. Am. Chem. Soc., 1987, 109, 3526.
16 J. L. Bada and A. Lazcano, Science, 2002, 296, 1982.
17 L. Stryer, Biochemistry, 3rd edn., W. H. Freeman, New York,
1988, p. 757; S. Joseph, M. L. Whirl, D. Kondo, H. F. Noller and
R. B. Altman, RNA, 2000, 6, 220.
18 J. P. Ferris, A. R. Hill, R. Liu and L. E. Orgel, Nature, 1996, 381, 20;
J. M. Bailey, FASEB J., 1998, 12, 503; J. Bujdak and B. M. Rode,
Origins Life Evol. Biosphere, 1999, 29, 451; V. A. Basiuk and J. Sainz-
Rojas, Adv. Space Res., 2001, 27, 225.
Mp: 162 ЊC. [α]D: Ϫ99.4. (Found: C, 64.23; H, 7.73; N, 12.20.
C18H25N3O5ؒ0.25 H2O requires: C, 64.36; H, 7.95; N, 12.50%);
Distinctive IR bands (see above)/cmϪ1: 1633, 1542, 1451; dis-
1
tinctive H NMR features, δ: 1.16–1.79 (m, 2H), 2.09 (s, 3H),
2.79 (s, 3H), 2.85 (s, 3H), 2.89 (d, 1H), 2.97 (dd, 1H), 5.01–5.06
(m, 1H), 7.37 (dd, 1H); distinctive 13C NMR features, δ: 39.4,
48.1, 50.0, 59.7.
Acknowledgements
19 Specific details for each separation are available from the authors.
20 M. Bodansky and A. Bodansky, The Practice of Peptide Synthesis,
Springer-Verlag, Berlin, 1984.
21 G. H. L. Nefkens, G. I. Tesser and R. I. F. Nivard, Recl. Trav. Chim.
Pays-Bas, 1960, 79, 688.
22 Valine required 50 h reflux.
23 A. Paquet, Can. J. Chem., 1976, 54, 733.
24 G. C. Windridge and E. C. Jorgensen, J. Am. Chem. Soc., 1971, 93,
6318.
We are grateful for facilities and helpful comments provided by
Dr P J Murphy and Dr M Fletcher of the University of Wales,
Bangor. We also thank the EPSRC Mass Spectrometry Service
at the University of Wales, Swansea for low and high resolution
mass spectra, and Jane Davies and Glyn O’Connolly for other
analytical services.
25 Novabiochem, The Fine Art of Solid-State Synthesis, (Catalogue
2002–3) Synthesis Notes, p. 3.37.
26 M. Kempe and K. Mosbach, Tetrahedron Lett., 1995, 36, 3563.
27 J. S. Davies and E. Hakeem, J. Chem. Soc. Perkin Trans. 2, 1984,
1387.
References
1 S. A. Kaufmann, The Origins of Order, OUP, Oxford, 1993, p. 295;
F. G. Mosqueira, S. Ramos-Bernal and A. Negron-Mendoza,
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