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Studies on amino acids and peptides XII1
Tcmddron Vol. 42. No. 23. pp. 6555 lo 6564.1986 Printed in GrentBritain.
0040-4020/86 SS.oO+.oO C 1986 Pergamon Journals Ltd.
STUDIES ON AMINO ACIDS AND PEPTIDES XIII SYNTHESISOF THIATEDANALOGUES OF Boc-S-Ala-Aib-S-Ala-OMe AND Ac-S-Ala-Aib-S-Ala-OMs
OCE E.JENSEN AND ALEXANDER SENNINC”
Department of Organic Chemistry, Chemical Institute, University of Aarhus, DK-8000 Aarhus C, Denmark
(Received in UK 6 Ocrober 1986)
Abstract - The two model peptides Boc-S-Ala-Aib-S-Ala-Ofle (la) and Ac-S-Ala-Aib-S-Ala-OMe (lb) and their monothisted analogues Boc-S(R)-AlsY(CSNH)-Aib-S-Ala-OMe (&),Boc-S-Ala-AibY(CSNH)-S-Ala-OMe (%), AcY(CSNH)-S-Ale-Aib-S-Ale-OMe (b), Ac-S-Ala-Aib’t’(CSNH)-S-Ala-OMe (4b), end the dithiated Boc-S-AlsY(CSNH)-Aib’Y(CSNH)-S-Ale-OHe (2) are synthEized.’ Peptide 3a wes obtsined from the coupling of HCl’H-S-Ala-OMe to S-2-(l-(tert-butoxyc&bonylamino)ethyl-4,4-dimethyl-1,3-thiazol-5(4a)-one (11). The thiosmide snalogues 4a (together with 2) and 2b were obtained by resoselective thiation of the respective model peptides E end lb using 2,4-bis(4-methylphenyl)-1,3,2,4dithiadiphosphetane 2,4-dizlfide, Lawesson’s Reagent (LR). Deprotection of the Boc group of 4a,followed by acetylstion of the product,sfforded *. The magnetic nonequivalence of the gem-methyl groups of Aib is discussed.
INTRODUCTION Thioamide tor
analogues
interactions
of physiologically
hanced stability
against
with carboxypeptidase Sin&
enzymetic
in the antibiotic
I.C.1
class
which,
of peptides
pic,
C-terminal
end conformations1
the selective
thiation
to a further
exploration
thiation
of the peptide
the different cerbonyls.
carbonyl In this
Furthermore,
peptides and/or
hydrolyses
are attractive
potent
modifications
than their
can be expected
psrent
on the basis
since
compounds;b of previous
in recepalso
an en-
experience
A.3-5
the discovery
amino alcohol
active
they may be more selective
in 1958 of the natural 13959 6 and later apart group, aspects
from their there
of the selectivity
groups,
paper it
is
of Aib (a-aminoisobutyric
large
contents
Aib-containing
interest
in the synthetic,
of LR in thiation
reactions.
described
A general
a
by sn
spectrosco-
The promising
previously
first 7,8
are characterized
model peptides.c
Boc-Gly-S-Ale-Aib-OMe
acid)
the “peptaibols”,
of Aib residues,
has been considerable
of shorter
of the model peptide
function
occurrence
on in e number of antibiotics,
results ‘*
of
inspired
method for direct
in protected dipeptides demonstrated that LR discriminates between 13,14 i.e. the amide carbonyl is thisted before urethane and ester shown that
we wanted to investigate
LR is able
to distinguish
the possibility
of thiating
between different the extremely
amide groups.
hindered
Aib re-
sidue. H&O&~:~~OCH~
S LR a The nomenclature mission
of the compounds is in accordance with the recommendations Pure Appl. Chem. 2, 595 (1984). on Biochemical Nomenclature,
b For a review see ref.2. ’ For reviews see refs 9-11.
6555
of the IUPAC-IUB Com-
0.
6556
E. JENSENand A. SEINNIN~
a
-0Me -0Me -0Me -0Me -0Me
Scheme
1
Ala
Ala
Aib
(4)
IS) (6)
(3a)
Scheme
2
Ala
Ala
Aib
Boc--
HCI~H--0Me
OH MA
(1)
Boc
19)
Boc
/IO)
OMe S + S
LR OMe NaOH /MeOH
Boc-”
OH S
(11) *
+
COCI
Boc-‘\
HCI*H--0Me
5 S
130)
Boc
OMe
*
Scheme
3
Ala
Aib
(la)
f4a)* H:i=p=g
(12) (4bl *(27%
of &a(X=O)
and 14% of &(X41
Scheme
#
See Scheme 6
4
Ala
6551
Type title studies on amino acids and peptides-XII The model peptides
scopic
and structural
chosen,
comparative
&
and lb,
studies
provided
since
us with the posslbility
they have recently
of direct
spectro-
been examined by G. Jung and co-
workers.15’16 Investigation more general graphic
of the crystal
study
of B-turn
structures
conformation
work on the structures
of 2b,
of the thismide
in ronothiated
4a, s
analogues
Aib peptide
and -5a will
is a continuation
surrogates
be published
elsewhere
It=
b)
of a
l2 and crystallowith C. Toniolo
et
al.
RESULTS
Throughout dride have
AND DISCUSSION
this
coupling
procedures
(MA)
used successfully
been
prepare
work mixed anhy-
dipeptides.
protection
to
The Boc-de-
was achieved
by treat-
ment with 4 N HCl/dioxane. The strategy esis E
for
the synth-
a)
of the model peptides
-la and in Scheme 1. The
is outlined
reason
for
coupling
Boc-S-Ala-
-Aib-OH and HCl*H-S-Ala-OMe stead
of coupling
Boc-S-Ala-OH
and HCl*H-Aib-S-Ala-OMe in the latter reaction risk
case
time,
is that
prolonged
R=CH,
1a.b
: X80.
Y*O.
zmo
2b
: x=s.
Y-0.
z=o
3a
:
x.0.
Y-S.
z=o
Lo.b
:
x.0.
Y.0.
2.s
5a
:
x= 0,
Y-S.
zas
reFig.
and consequently,
of racemization
low yields
in-
t-0.
together
f
with
at the tripeptide
stage
may be expected
due to the notoriously
hindered
amino group of
Aib. The saponification to give turned for
good yields
of Boc-S-Ala-Aib-OHe
of the corresponding
out to be compatible
the reaction.”
derivative
E
with the thiated
with CR and demonstrates reactions.
were found by Jung et al.
The dithioester
(1).
to be optimal
discrimination
between different
carbonyl
route
method
5) was employed in the o-
riginal
strategy
of 2
for
the prepara-
(Scheme 2).
dithioester
(6)
(c).
product
and the dithioester
had totally
before Aib
purity
A of the
LR 8ooc-
;c=o
“I BUO 3 “H-N
S Boc-NH-CHR-8-N
3
CH,l 3
fHF_
showed that
rscemized
reactions
0
Boc-NH-CHR-E-N
during
s
That the dithioester
method is not always thioamidation
Boc -NH-CHR-E-OH
However,
recovered.
dithioester
the reaction.
with
0
was not formed
of the optical
recovered
Cl
The alanine
was treated
HCl*H-Aib-S-Ala-OMe the expected
1.5 h
which also
reliable
Boc-NH-CM-E-SCH,
for
has been
in one case. I9 Here the amino terminal
sites
in
to -2b would be the reaction of methyl dithioacetate with The preparation of methyl dithioacetate, however, is a three-step
(Scheme
crowded
2,
at 40 “C for
conditions,
la-19
synthesis.
seen
These somewhat forcing
analogue
the reagent’s
An alternative
HCl*H-S-Ala-Aib-S-Ala-OMa
it
in (NaOH/MsOH) was performed
of -lb with LR at room temperature in THF afforded the monothiated in 92% yield; this was expected from previous results l2 with specific
thiation
check
acid.
The thiation
exclusively
thiation
tion
(1)
free
of 13 is
Schmw
5
0. E. JFMFN and A. SENMNO
6558
Table
1.
“C
WR Chemical
shifts
of
peptides
Ala
Boc/Ac
and intermdiet’e
fragmnte
Aib
Ala
XHe
Cq
C”3
c=x
ccl
%
C=Y
ca
cB
c=z
1.
79.0
20.1
155.4
49.9
17.9
172.0
56.0
24.6
174.6
52.3
9
00.4
20.2
155.6
59.0
21.0
204.0
56.6
24.0
173.2
52.5
2”
70.0
20.2
154.9
49.4
10.4
171.9
54.0
;;.;
175.5
00.0
20.2
155.0
56.0
21.0
204.1
60.4
23.0
176.7
70.1
20.1
151.2
47.0
17.4
172.8
55.6
;;‘;
173.9
40.3
16.9
173.0
-10 la+ 3
.
c,
93
c=o
XC%
47.5
17.0
172.1
51.0
47.8
16.7
160.9
51.0
173.2 172.0
40.4
17.9
173.2
52.3
206.5
50.0
16.4
172.3
52.3
202.4
54.0
16.5
172.4
52.3
24’o
38
00.6
20.2
155.7
57.9
20.9
104.2
60:3 56 0
ii:;
-4a
00.2
20.1
155.7
53.0
17.4
172.2
62.0
:;‘;
-5a
01.2
20.1
156.0
59.5
20.9
204.0
65.1
;;‘;
-lb*
23.0
169.0
49.1
17.0
173.0
55.7
;;*;
173.9
47.7
16.7
172.3
51.0
2b”
32.6
199.0
54.7
17.2
172.9
55.9
g;
173.7
47.7
17.0
170.5
51.0
4b
22.0
170.0
53.0
17.5
172.7
62.5
;;‘;
206.5
49.7
16.4
172.3
52.4
4”
77.0
20.2
154.0
45.7
17.0
170.2
5
79.3
20.3
154.6
52.0
22.1
202.0
a
00.0
20.3
154.6
61.1
19.2
241.5
-7*
78.0
20.1
154.1
-11
00.1
28.2
154.9
All
spectra
were
recorded
Table
27.0
in
50.7
;;.;
102.6
marked
4.05 4.29 3.05 3.95 4.59 4.55 4.27 3.09 4.50 5.00 5.00 4.09 4.51 5.01
1.20 1.30 1.30 1.14 1.47 1.25 1.20 1.32 1.43 1.30 1.53 1.23 1.32 1.36
6.90 0.49 6.00 7.00 0.60
4.41 4.72 4.77 -
1.36 1.36 1.41 1.35
6.06 6.70 5.26 recorded
in CDCl,
47.6
* which
were
and intenediate
Aib NH
F F 5 p
with
peptides
C0H
1.45 1.45 1.43 1.02 2.40 2.00
were
174.2
C,H
5.33 5.26 5.20 6.74 5.49 5.40 7.57 0.31 5.13 5.10 5.15 0.17 10.14 6.00
spectra
of
;;:;
NH
1.30 1.35 1.40 1.36 1.40 1.45 1.30
.
55.5
Ala
1 9 T F 1iT ii -iTa* y 3a G 5 iP K* 6
All
those
ehifts
.
24.0
167.6
except
CDCl,
1H NM0 Chemical
2.
Boc/Ac .C”3
19.1
.
.
1.11 1.19 1.50 except
those
17.1
recorded
in
NH
DMSO-dg
fragnents
XHe/Uli
Ala C6”
51.0
173.0
Co”
C6H
CH3/0H
y.96 0.65 0.30 7.05 0.51 0.17 0.25 7.37
1.39 1.59 1.55 1.33 1.74 1.40 1.35 1.35 1.72 1.69 1.73 1.35 1.35 1.60
7.u4 7.07 6.53 0.05 0.43 7.57 7.43 0.76
3.00 4.25 4.30 4.10 4.34 4.22 4.23 4.40
1.15 1.27 1.30 1.56 1.43 1.16 1.22 1.47
3.62 3.59 3.70 3.73 3.70 3.62 3.60 3.75
6.79
1.29
7.74
4.27
1.24
3.75
marked
with
3.54 3.50 12.23 7.15
l
which
were
recorded
in DMSO-d6
Type title studies on amino a&ds and peptides-XII to be the reason
believed
It is known that feasible.
Instead
sponding tomerism
(Scheme 6).
the C-terminal we decided pivaloyl
to prepare
H-S-Ala-One
in coupling
amino acid
with
not sufficient-
(0)
of the reaction
via their
product
keto-enol
where the achiral
would be expected is
which was isolated
not
are nommlly
of the thiazlactone
(11)
generally
to the corre-
and since
reduced,
tau-
Aib is Aib bears
is not possible
from the reaction
to Scheme 6, with R I CH,. Subsequently, ‘H NMR spectrum
The
racemization
is
is converted
racemized
of Boc-S-AlaY(CSNH)-Aib-OH
via the thiazlactone
-3a.
and are readily
in which the activity
n according
yielded
case
from the C-terminal
thiopeptide
These thiazlsctonea
reactions
no major problems
form, 2
product.’
by coupling
with the amine the activated
In the special
the enol
chloride
of a coupling
of thiopeptides
1g*20’21 (thiazol-5(48)-one).
to participate
no a-hydrogen
the absence
of coupling
thiazlactone
ly reactive
for
elongation
653
of -.10 with of -11 with HCl*
aminolysis
revealed
the presence
of two
I
,*,
Boc -NH-CH-C-HH-TR-COOH -
CHl
CY /
S-
w
I
P,Ch
____,
\
Boc-uH-y-C*N,C,
H,N-fli-COOMe
CY
C’%
S II
SW
‘y
CH,
CH,
Cn,
J!
diastereomerst isolation
via
the ‘H M
thiazlactone
thiazlactone
in the coupling examples
reagents
towards
2o
It is,
derivative
formation
reaction
Several
is
to give
for
the first
but quite
with e smell
Thietion
was assumed to be the easiest
this
analogue.
group were less
sterically
The NM resonances
for
field
with respect
uhich
the change is about due to coupling
the verification Similarly,
the a-,
to the parent
nal of the Aib residue doublets
thietion hindered
of
appears
the 13C signals
of amidation
thiazlactons
by deprotection
has been isolated.
because
2).
as a singlet,
of Boc-Aib-S-Ala-
of the Boc group end coupling
of -la with LR gave a reesoneble
of Ala%=0 the 8-,
site(s)
And product
and shown to be weak acylating
and hence used
amidea.‘3”4”*
for
prolongation.
the lack
have been isoleted
may to a,
with the C protons,
the thiation
example of e thiopeptide
C-terminal for
the
yield
of 4a,
compound Boc-S-AlaY(CSNH)-AibY(CSNH)-S-Ale-C&,
than those
1.7 ppm (Table
indicating
time the postulated
the thiation
amount of the dithieted
We anticipated
as the reasw,
followed
unexpectedly
together
the first
peeks,
of -4a end in turn -4b wes based on thiation
Boc-AibY(CSNH)-S-Ala-Me,
with Boc-S-Ala-OH,
single
23
and peptides.
the preparation
showed only
to our knowledge,
has been suspected
this
amino acids
spectrum
end of any thiopeptide
of Z-phenylthiezol-5(4e)-ones
The strategy -0Me (1)
crystallization
its
CH,
%
of one of the diestereomars.
prolongation although
After
p
Boc -NH-WC-Nli-C-C-NH-%I-COOU.
,s,
of Aib’C-0
it
seamed that
The lergest This observetion in contrast
in a quantitative
the subetituents
attempt
of
the thiosmides are found for
ere shifted
that
spectroscopy
down-
the N-protons
of Ala which are
an efficient
tool
way (Fig.2).
a and B to the thioanide
for
the N-H group sig-
to the N-H group signals lH Ml
of this
gem-methyl groups.
values
and the fact
2.
to prepare
the surroundings
with the two adjacent
and the N-protons
makes simple
in our first
function
exhibit
for
0. E. JENSENand A. &mmo
6560
Fig. (2)
2. in
lH NHR spectra 6DC1,
downfield nyl
shifts
of
Boc-Ala’-Aib2Y(CSNH)-Als3-OMe
1).
(Table
The thiocarbonyl
carbon. ‘3’14’18*24 An interesting
magnetic
ally,
nonequivalence
protected
stereotopic peptides
dipeptides
induction with less
the large stricted
Aib
(HNE)
hindered
the
rotation
occurs
resonates
B-methyl
a smell
centres
groups
13C HNE of
of neighbouring
around
in our series
the
is strongly
twisted
(or
sulfur)
which increasea
for
the methyl group in guestion.16’26
and in
effect
the electrostatic
at 204 ppm YS 173 ppm for
N-Co or
very
large
in the Aib residues
(Table
1).
Gener-
0.5
ppm which
is explained
whereas in schirel
Ca-C+O bonds FINE is
analoguee
lacking.25
is brought
about
thus,
group close e large
Obviously, by s very re-
to the csrbonyl downfield
by die-
of chiral
between Aib Co and Aib carbonyl
moves one methyl
interaction;
csrbo-
is the
about
&
the
end 2
residues,
of thiosmide
in which the torsion’angle
conformation
(CG-Co-C-O)
and Boc-Als’Y(CSNH)-Aib2-Als3-OMe
of the two model peptides
geminal
exhibit
from chirsl
MNE which also backbone
of
carbon
property
(&)
shift
oxygen oxygen is caused
6561
Type title studier on amino acids and pcptides-XII The changs carbonyl
to a greater
ence *as seen,
probably
in the regions
3450-3230
1670-1630
(amide I),
cm-l
I bands fall stic
value.
stretch tive
magnetic
group was expected
because
cm”
a large
stretching),
(N-H
of
stronger
2760-1745 I),
absorptiona
26 in the Aib
groups,
cm-l (eater),
1720-1680
cm-’ (urethane),
and 1165 cm-1 (dithioeater).
fran the C-O stretching
The thioamide
and ere of less
diagno-
The absorptions
for the N-H stretch below 3400 cm-’ and erethane and/or amide C=O -1 at 1690 and 1660 cm , respectively, of the target compounds (5 and b series) seem indicsof hydrogen
group is a characteristic
in the rsnge
260-307
The mass spectra [M-isobutene]?
spectra
oxygen by sulfur
but no unambigous differ19 are observed The IR absorptiona
dependme.
cm-l (thioamide
The thiocarbonyl
ments
on substituting
solvent
of the existence
lengths
nm with
cases
= 241 nm (methanol)
state.
UV chrcmophore
log E values
fron.3.0
[H}? or [~+l]+,
Occasional
and [M-tertBuO)]+.
of the thioamide
2,4_disubstituted
bonds in the solid
l9 show in most
loss
l9 which absorbs
strongly
at rave-
to 4.1. together
of H,S and/or
with
the
characteristic
HS wsa observed
frag-
in the mass
analogues.
The IR and UV absorptiona
&x
of
1170-1130
in the.region
anisotropy
to enhance the IWE of the gem-methyl
of 11,
1630 (C-N),
ars in accordance
1710-1720
with those
cm-’ (urethane,
found in the literature
thiazlsctone for
the keto
C-0)
and
forms of
thiazolin-5rones.27
+I(a&ratim is believedto cam at the N-tennlnP1 Ala resi&e via ruita B in sdnm 6, giving rise to two diastereanars of the final probct 2. After completim of this work, use of catalysts tilch mimt suppress this epinarlzatim has appearedin the 1itcrature.~ EXPERIMENTAL Instruments ‘H NMRspectra were recorded at 60 MHz on Varisn EM-360 or at 80 MHz on Vsrian CFT-20 spectrometers with CDCl, as solvent. Samples with CMSO-ds as solvent were recorded et 300 HHz on a Varian XL-300 spectrometer. 13C NMRspectra were recorded at 25 MHz on Varisn XL-100-15 or at 75.426 MHz on Varian XL-300 spectrometers with CDCls or DMSO-ds as solvents. Chemical shifts are reported as parts per million on the 6 scale, with reference to TM’Sas 0 ppm (for ‘H spectra), COCl, as 76.95 ppm and DMSO-d6 as 39.5 ppm. Spectra with E#%O-d6 as solvent were recorded at the Department of Chemistry, University of Louisville, Louisville, KY, USA. UV absorption spectra were recorded on Perkin Elmer 402 and Uvikon 860 (compounds 4s and Se) spectrophotometers. IR absorption spectra mere obtained with a Beckman IR-18 apectroph~omete~ Mass spectra and precise msas measurements were recorded on a Micromass 7070F spectrometer operating at 70 eV with direct inlet. Elemental analyses were carried out by Lavena Kemisk Fabrik, DK-2750 Ballerup (Microanalytical Lsboratory). Optical rotstions were measured in a 1 ckn cell in a Perkin Elmer 241 polarimeter. Silica gel 60 (Merck) 63-200 pm was used for column chromatography and silica gel 60 (Merck) 40-63 um for flash chromatography. The dimensions for the flash column were 30 x 200 mn, and the flow rate was 2.54 cm/min (Nr gas). The following systems were used for TLC monitoring (I): butanol/scetic acid/ water (4:1:1), (II): chloroform/methanol/acetic acid (B5:10:5), (III): butanol/acetic acid/water (3:1:1), (IV): 2_butanol/acetic acid/water (67:10:23). The TLC plates were prepared as described earlier.12 The ninhydrin spray solution consisted of 0.25% ninhydrin in butanol. For LTVmonitoring the wavelengths 254 mu and 350 mu were used to detect thioamides and amides, respectively. For detection of ninhydrin negative impurities the TLC plates were exposed to iodine vapour. The methyl 29 derivstives ofS-alanine and a-aminoisobutyric eater hydrochlorides 28 and N-tert-butyloxycarbonyl acid were prepared by standard methods although for Aib prolonged resction times were allowed. N-tart-Butyloxycarbonyl-S-alanyl-a-aminoisobutyric acid methyl ester (Boc-S-Ala-Aib-One, 1).17~30 Boc-S-Ala-OH; 37.84 g (0.200 mol), was dissolved in a mixture of 20.23 g (0.200 mol) N-meFhylmorpholine and 182 ml dry tetrahydrofuran. After cooling to - 20 “C, 25.45 g (0.19 mol) isobutyl chloroformate was added dropwise, keeping the temperature below - 10 OC. The mixed anhydride was alloued to form in 15 min of activation time; then a precooled mixture (- 10 “C) of 72.7 g (0.180 mol) HCl-H-Aib-We, 18.2 g (O.lBO mol) N-methylmorpholine, and 113 ml tetrahydrofuran was added over e period of I5 min. The reaction proceeded for 0.5 hr at - 10 PC, then for 1 hr at 0 OC and finally 1 hr at rocm temperature, after which the temperature again was lowered to 0 OC and 70 ml of saturated KHCO3solution was added to destroy any unreacted mixed anhydride. After 0.5 hr the organic solvent was removed in vacua. The aqueous slurry was extracted with 100 ml snd 2 x 50 ml ethyl acetate. The combined organic phases were washed with 1 N HCl (3 x 50 ml), water (3 x 50 ml), 5% KHCO, (3 x 50 ml) end 10 ml water, then dried over MgSOr. The solvent was evaporated under vacuum to yield 49.79 g of 1 as a colourless oil. Rf = 0.6 in ethyl acetate [al;’ = - 33.6O (c = 1 in methanol). The spectrosco$c data correspond well with those found in the literature. N-tert-Butyloxycarbonyl-S-thioalanyld-aminoisobutyric acid methyl ester tBoc-S-AlaY(CSNHI-Aib-One, 9j.12 Boc-S-Ala-Aib-We (l), (7.55 g, 0.0262 mol) and LR (5.82 g, 0.0144 mol) was refluxed in dry toluene (15 ml) for 0.5 hr. The purification follows the same procedure as applied for 5. The dimensions of the column were 64 x 240 mn. The yield of 2 waa 0.8 g as a slightly yellGil. Rf I 0.52 in 10% diethyl ether/dichlorcxnethane. All data correspond well with those previously found. N-tert-Eutyloxycarbonyl-S-alanyl-a-aminofsobutyric
acid (Boc-S-AlUa-Aib-OH,2).17*M Boc-S-Ala-Aib&He
6564
0.
E. Jmsa~ and A. .%WING
with added silica gel, and chromatographed on a column (20 ~200 mm) with dichloromethane until the “P,S” by-product (Rp = 0.88 in 30% diethyl ether/dichloromsthane) was eluted. The thiopiperidide was then eluted with 30% diethyl ether/dichloromsthane. Evaporation of the collected fractions yieldsd 2.74 g (63%) of 5 as a colourless powder with m.p. 85.0-85.5 OC. Rf = 0.73 in’30% diethyl ether/ dichloromethane. [av= + l9.2O (c I 0.5 in ethyl acetate). IR (KEr)r 3340 (N-H stretch),1710 UI?-~(Wthane). UV (diethyl ether): X (log E) = 280 nm (4.1>. MS: m/z = 272 [fl]‘$ 239 [H-H’S]‘, 216 ‘t_ isobutene]+, 199 [M-tert-BUOY, 183 [M-isobutene-HS] 155 WN(C~H~~)HS] 128 [I+-CSN(CSHI~) 5 Elemental anslysis: C~~HZ~NZO~S (272.2) Calc.: C 5;.32, H 8.88, N 10.28,‘s 11.75 Found: C 57.08, H 8.87, N 9.85, S 11.68 N-tert-Butyloxycarbonyl-S-dithioalanine methyl ester (Boc-S-Ala Y(C.Sl.SMe,6). The thiopiperidide 5 (2.55 g, 0.0094 m01) and iodomethane (3.0 ml, 0.0094 mol) in 8 ml of tetraFydrofursn were sllouedto react under enhydrous conditions for 24 hrs, wheresfter the mixture wss evaporated in VXIIO. The remaining slurry was taken up in 5 ml of methanol and a stream of HrS gas was bubbled through the solution for 20 min. The solution was allowed to stand for another 20 min before evaporation. The oily product was purified on a column with diethyl ether yielding 1.40 g (60%) of 5 as an oil which was crystallized from pentane to give yellow crystals. M.p. 73.0-73.5 OC. Rf = 0.79 in diethyl ether, [al?? = -52.3” (c = 0.2 in ethyl acetate). IR (KfJr): 3310 (N-H stretch), 1700 (ufethane 1165 cm-’ MS: m/z I 235 [M] , 179 ? M-isobu(dithjoester. (log cl = 300 nm (4.0). UV (diethyl ether): tene] , 120 [H-tert-BUO] , 1.44 ~Boc- +m H(Mee)]+. Precise mass measurement: Calc. m/z 235.0700, found m/z I 235.0700 [Ml’. N-tert-Butoxycarbonyl-a-sminoisobutyryl-S-alsnine methyl ester IBoc-Aib-S-Ala-OMe, 1). Boc-Aib-OH (8.12 g, 0.04 mol) and HCl*H-S-Ala-Me (5.03 g, 0.036 mol) were coupled by the mixed anhydride method described for 1 except for the activation time which wss 20 min. The yield was 6.2 g (74%) of a colourless solid which was recrystallired from ethyl acetste/ ‘ght petroleum ether (b.p.40 OC), m.p. [a]# = -18.7O 95.0-95.5 OC. Rf = 0.55 in diethyl ether/dichloromethane. (c = 0.4 in methanol). IR (KBr): 3410-3280 (N-H stretch), 1745 (ester), 1680 (urethane), 1670 cm-’ (amide I). UV (ethanol): %ex (log E) = 201 nm (3.2). MS: m/z = 289 [M+l]‘, 233 [M+l-isobutene]+, 189 [M+Z-tert-BuCCO]*, 158 [Boc-NHC(Me)z]‘, 102 [M-(Ale-We)]+. Elemental analysis: CirHr*N205 (288.3) Calc.: C 54.15, H 8.39, N 9.72 Found: C 54.46, H 8.23, N 9.77 a-Aminoisobutyryl-S-alanine methyl ester hydrochloride IECl'Ii-Aib-S-Ala-OMe, 0). Boc-Aib-S-Ala-We, 1 (3.00 g, 0.0104 mol) was deprotected with 52 ml of 1 N HCl/dioxane for 40 mTn. Evsporation of the solvent yielded 2.03 g (89%) of g as a hygroscopic foam. Rf = 0.50 in III, Rp = 0.45 in IV. [aIF= - 23.3O (c = 2.6 in methanol). UV (ethanol): A,, (log E) = 201 nm (3.2). Acknowledgements - Part of this work was done at the Department of Chemistry, University of Louisville, KY, USA, and Professor A. F. Spatola’s cooperation and grant are gratefully acknowledged for fscilitating the stay for O.E.J. Ha wish also to thank Or. R. A. Porter for NHR recordings at the University of Louisville.
1. PmtxI, see T.P.k-&rsen and A.Semiq, Liebigsm.olsn. (submitted). 2. A.F.wtola in chsnis@ an3 tiafisnistnlof &ukw &ids, E~ptidasa& Fz~4~in5,(Ed. B.YinStein), Ckkker,Vo1.7, Pp. 267-357(1983). 3. w.L.W, J.-T.&m, J.Y.Tsang,aioorPm.Bi~s.R?.s.CZmI.02 589 (19Bl). 4. P.Ca@all ti N.T.t&kd, J.An.Ckm.scC. 104 5221 (1982). 5. P.A.Bartlett,K.L.Spearand N.E.Jaccbsan, ~&mistry~ 1608 (1982). 6. G.Y.Kemr and R.C.heppard, Nature (London)& 48 (1958). 7. E.Buwdatti, A.Bavoso,B.Oi Blasio,V.Pwona, C.Pedrms, C.Tmiolo, and G.kBmOrf~, I'LtZ.~~.~.=i.t=~ 7951 (1982). 8. H.BrUdwr and H.Graf,l?qerimtiaE 528 (1983). Fqtidss, (Eds C.Voelterand G.Msitzel,da Gruy9. G.&ng, H.Br+cknsrand H.Sdnitt in stn&unz acd ZctivityofWxr& ter, Berlin, 1981, pp. 75-114. lo. B.v.vankataram Rasad and P.Balarm, CRCQit.kc.Bic&3x 16 307 (1%). 11. C.Tmiolo, G.H.&xmra, A.Bwoso, E.Banedstti,B.01 Blasio,V.Wvaw and C.psdmn, BWl~ners 22 205 (198%. Tl&badm 41 5595 (1985) 12. 0.E.Jens.q S.-O.Le*essm, Mardi, A.M.Piauesl,ad C.Tmiolo, 7 3635 (1961);14. I&, Qwx.9~. 20 14 (1982);See also F. 13. K.Clausen,M.Tl-mr.sen, and S.-O.Lauessm, lWr&&m rapine., D&s.Akstr.Int.B 1986.46 (111, 3849 (C.A.E 24625 (19B6). 15. R.&xch, G.m, K.-P.Vogesand Y.Wintar,LiebigsIhn.Ck???. 1117 (19W). 10% (1983). 16. G.Jung,n.Briickner, R.Borh, knintar, ~.Schalland J.Str&hle,LiebipSRn.m. G.m, Liebigsrtn.chsn.1151 (1979). 17. R.Oskommopr10s & 18. K.Clausm, H.llwrsm, S.-O.Lawssson and A.F.Spatola,J.Uxm.*.. *&in m. 1785 (1984). 19. W.Thorsan,B.Vde, U.Padsrs.sn, K.Clausm,and S.-O.Lak@eSsOn, ~~&s&tn 39 3429 (1%3). 20. G.Lajoie, F.Lapine,L.l+aziak and B.&llaau, ZWrdpdrm L&t. 24 3815 (1983).See also G.Lajoia,Dk.w.fit.Bm 45(10) 3225; (c.A.~cn 204285 (19B5)).2oe.P.Wlpf md H.Hslnpartw, flelV.olim.ti69 1153 (19B6). 21. u.Staglich,GMiPle md L.Ullsdwitz, ZMrakdrm I.&t. 169 (1970). fi 39 (1960). 22. M.T.Lepley, O.S.JMes, G.Y.KmH?r and R.C.Sheppard,lb23. G.C.Barrett,J.CYBTI..~X.(C~ 1380 (1971).- 24. I.O.Rae,mt.J.Olsn. n 561 (1979). 2 2165 (1982). R.OekonanopllosandG.Jng,*~ 25. O.Laibfritz,E.-t, N.tilschar, H.lxhmm, 26. P.L.ScuWwicic,J.A.Fittgerald md G.E.Millirran, Zktrahedra,L&t. E 1247 (1%5). 27. J.H.W~S, ~.~.~evie~and R.A.G.Carringtm,J.Qrm.*.. @&in ~zxs.~ 1983 (1972). verlag,StuttWt, 1974. 28. nxix0nsy1,wulxknderorg.o1Em., Bd. XV/l, p.316, G?Org l-bie!na 29. L.kwockr, A.Hellatt, E.Wu&, O.Kallerand G.krsin, WqlIer's WM.-. Z 1651 (1976). 30. R.Kagsrajand P.Balarm, Zktrdmdron r 1263 (1981). 31. K.Clausm and S.-O.Lawsson,W.J.a. 9 43 (1980). 32. S.Sctmibye,B.S.Pe&rsm and S.-O.LawSSm, ~l..5k.~.6+7. 3 229 (1978).
0040-4020/86 SS.oO+.oO C 1986 Pergamon Journals Ltd.
STUDIES ON AMINO ACIDS AND PEPTIDES XIII SYNTHESISOF THIATEDANALOGUES OF Boc-S-Ala-Aib-S-Ala-OMe AND Ac-S-Ala-Aib-S-Ala-OMs
OCE E.JENSEN AND ALEXANDER SENNINC”
Department of Organic Chemistry, Chemical Institute, University of Aarhus, DK-8000 Aarhus C, Denmark
(Received in UK 6 Ocrober 1986)
Abstract - The two model peptides Boc-S-Ala-Aib-S-Ala-Ofle (la) and Ac-S-Ala-Aib-S-Ala-OMe (lb) and their monothisted analogues Boc-S(R)-AlsY(CSNH)-Aib-S-Ala-OMe (&),Boc-S-Ala-AibY(CSNH)-S-Ala-OMe (%), AcY(CSNH)-S-Ale-Aib-S-Ale-OMe (b), Ac-S-Ala-Aib’t’(CSNH)-S-Ala-OMe (4b), end the dithiated Boc-S-AlsY(CSNH)-Aib’Y(CSNH)-S-Ale-OHe (2) are synthEized.’ Peptide 3a wes obtsined from the coupling of HCl’H-S-Ala-OMe to S-2-(l-(tert-butoxyc&bonylamino)ethyl-4,4-dimethyl-1,3-thiazol-5(4a)-one (11). The thiosmide snalogues 4a (together with 2) and 2b were obtained by resoselective thiation of the respective model peptides E end lb using 2,4-bis(4-methylphenyl)-1,3,2,4dithiadiphosphetane 2,4-dizlfide, Lawesson’s Reagent (LR). Deprotection of the Boc group of 4a,followed by acetylstion of the product,sfforded *. The magnetic nonequivalence of the gem-methyl groups of Aib is discussed.
INTRODUCTION Thioamide tor
analogues
interactions
of physiologically
hanced stability
against
with carboxypeptidase Sin&
enzymetic
in the antibiotic
I.C.1
class
which,
of peptides
pic,
C-terminal
end conformations1
the selective
thiation
to a further
exploration
thiation
of the peptide
the different cerbonyls.
carbonyl In this
Furthermore,
peptides and/or
hydrolyses
are attractive
potent
modifications
than their
can be expected
psrent
on the basis
since
compounds;b of previous
in recepalso
an en-
experience
A.3-5
the discovery
amino alcohol
active
they may be more selective
in 1958 of the natural 13959 6 and later apart group, aspects
from their there
of the selectivity
groups,
paper it
is
of Aib (a-aminoisobutyric
large
contents
Aib-containing
interest
in the synthetic,
of LR in thiation
reactions.
described
A general
a
by sn
spectrosco-
The promising
previously
first 7,8
are characterized
model peptides.c
Boc-Gly-S-Ale-Aib-OMe
acid)
the “peptaibols”,
of Aib residues,
has been considerable
of shorter
of the model peptide
function
occurrence
on in e number of antibiotics,
results ‘*
of
inspired
method for direct
in protected dipeptides demonstrated that LR discriminates between 13,14 i.e. the amide carbonyl is thisted before urethane and ester shown that
we wanted to investigate
LR is able
to distinguish
the possibility
of thiating
between different the extremely
amide groups.
hindered
Aib re-
sidue. H&O&~:~~OCH~
S LR a The nomenclature mission
of the compounds is in accordance with the recommendations Pure Appl. Chem. 2, 595 (1984). on Biochemical Nomenclature,
b For a review see ref.2. ’ For reviews see refs 9-11.
6555
of the IUPAC-IUB Com-
0.
6556
E. JENSENand A. SEINNIN~
a
-0Me -0Me -0Me -0Me -0Me
Scheme
1
Ala
Ala
Aib
(4)
IS) (6)
(3a)
Scheme
2
Ala
Ala
Aib
Boc--
HCI~H--0Me
OH MA
(1)
Boc
19)
Boc
/IO)
OMe S + S
LR OMe NaOH /MeOH
Boc-”
OH S
(11) *
+
COCI
Boc-‘\
HCI*H--0Me
5 S
130)
Boc
OMe
*
Scheme
3
Ala
Aib
(la)
f4a)* H:i=p=g
(12) (4bl *(27%
of &a(X=O)
and 14% of &(X41
Scheme
#
See Scheme 6
4
Ala
6551
Type title studies on amino acids and peptides-XII The model peptides
scopic
and structural
chosen,
comparative
&
and lb,
studies
provided
since
us with the posslbility
they have recently
of direct
spectro-
been examined by G. Jung and co-
workers.15’16 Investigation more general graphic
of the crystal
study
of B-turn
structures
conformation
work on the structures
of 2b,
of the thismide
in ronothiated
4a, s
analogues
Aib peptide
and -5a will
is a continuation
surrogates
be published
elsewhere
It=
b)
of a
l2 and crystallowith C. Toniolo
et
al.
RESULTS
Throughout dride have
AND DISCUSSION
this
coupling
procedures
(MA)
used successfully
been
prepare
work mixed anhy-
dipeptides.
protection
to
The Boc-de-
was achieved
by treat-
ment with 4 N HCl/dioxane. The strategy esis E
for
the synth-
a)
of the model peptides
-la and in Scheme 1. The
is outlined
reason
for
coupling
Boc-S-Ala-
-Aib-OH and HCl*H-S-Ala-OMe stead
of coupling
Boc-S-Ala-OH
and HCl*H-Aib-S-Ala-OMe in the latter reaction risk
case
time,
is that
prolonged
R=CH,
1a.b
: X80.
Y*O.
zmo
2b
: x=s.
Y-0.
z=o
3a
:
x.0.
Y-S.
z=o
Lo.b
:
x.0.
Y.0.
2.s
5a
:
x= 0,
Y-S.
zas
reFig.
and consequently,
of racemization
low yields
in-
t-0.
together
f
with
at the tripeptide
stage
may be expected
due to the notoriously
hindered
amino group of
Aib. The saponification to give turned for
good yields
of Boc-S-Ala-Aib-OHe
of the corresponding
out to be compatible
the reaction.”
derivative
E
with the thiated
with CR and demonstrates reactions.
were found by Jung et al.
The dithioester
(1).
to be optimal
discrimination
between different
carbonyl
route
method
5) was employed in the o-
riginal
strategy
of 2
for
the prepara-
(Scheme 2).
dithioester
(6)
(c).
product
and the dithioester
had totally
before Aib
purity
A of the
LR 8ooc-
;c=o
“I BUO 3 “H-N
S Boc-NH-CHR-8-N
3
CH,l 3
fHF_
showed that
rscemized
reactions
0
Boc-NH-CHR-E-N
during
s
That the dithioester
method is not always thioamidation
Boc -NH-CHR-E-OH
However,
recovered.
dithioester
the reaction.
with
0
was not formed
of the optical
recovered
Cl
The alanine
was treated
HCl*H-Aib-S-Ala-OMe the expected
1.5 h
which also
reliable
Boc-NH-CM-E-SCH,
for
has been
in one case. I9 Here the amino terminal
sites
in
to -2b would be the reaction of methyl dithioacetate with The preparation of methyl dithioacetate, however, is a three-step
(Scheme
crowded
2,
at 40 “C for
conditions,
la-19
synthesis.
seen
These somewhat forcing
analogue
the reagent’s
An alternative
HCl*H-S-Ala-Aib-S-Ala-OMa
it
in (NaOH/MsOH) was performed
of -lb with LR at room temperature in THF afforded the monothiated in 92% yield; this was expected from previous results l2 with specific
thiation
check
acid.
The thiation
exclusively
thiation
tion
(1)
free
of 13 is
Schmw
5
0. E. JFMFN and A. SENMNO
6558
Table
1.
“C
WR Chemical
shifts
of
peptides
Ala
Boc/Ac
and intermdiet’e
fragmnte
Aib
Ala
XHe
Cq
C”3
c=x
ccl
%
C=Y
ca
cB
c=z
1.
79.0
20.1
155.4
49.9
17.9
172.0
56.0
24.6
174.6
52.3
9
00.4
20.2
155.6
59.0
21.0
204.0
56.6
24.0
173.2
52.5
2”
70.0
20.2
154.9
49.4
10.4
171.9
54.0
;;.;
175.5
00.0
20.2
155.0
56.0
21.0
204.1
60.4
23.0
176.7
70.1
20.1
151.2
47.0
17.4
172.8
55.6
;;‘;
173.9
40.3
16.9
173.0
-10 la+ 3
.
c,
93
c=o
XC%
47.5
17.0
172.1
51.0
47.8
16.7
160.9
51.0
173.2 172.0
40.4
17.9
173.2
52.3
206.5
50.0
16.4
172.3
52.3
202.4
54.0
16.5
172.4
52.3
24’o
38
00.6
20.2
155.7
57.9
20.9
104.2
60:3 56 0
ii:;
-4a
00.2
20.1
155.7
53.0
17.4
172.2
62.0
:;‘;
-5a
01.2
20.1
156.0
59.5
20.9
204.0
65.1
;;‘;
-lb*
23.0
169.0
49.1
17.0
173.0
55.7
;;*;
173.9
47.7
16.7
172.3
51.0
2b”
32.6
199.0
54.7
17.2
172.9
55.9
g;
173.7
47.7
17.0
170.5
51.0
4b
22.0
170.0
53.0
17.5
172.7
62.5
;;‘;
206.5
49.7
16.4
172.3
52.4
4”
77.0
20.2
154.0
45.7
17.0
170.2
5
79.3
20.3
154.6
52.0
22.1
202.0
a
00.0
20.3
154.6
61.1
19.2
241.5
-7*
78.0
20.1
154.1
-11
00.1
28.2
154.9
All
spectra
were
recorded
Table
27.0
in
50.7
;;.;
102.6
marked
4.05 4.29 3.05 3.95 4.59 4.55 4.27 3.09 4.50 5.00 5.00 4.09 4.51 5.01
1.20 1.30 1.30 1.14 1.47 1.25 1.20 1.32 1.43 1.30 1.53 1.23 1.32 1.36
6.90 0.49 6.00 7.00 0.60
4.41 4.72 4.77 -
1.36 1.36 1.41 1.35
6.06 6.70 5.26 recorded
in CDCl,
47.6
* which
were
and intenediate
Aib NH
F F 5 p
with
peptides
C0H
1.45 1.45 1.43 1.02 2.40 2.00
were
174.2
C,H
5.33 5.26 5.20 6.74 5.49 5.40 7.57 0.31 5.13 5.10 5.15 0.17 10.14 6.00
spectra
of
;;:;
NH
1.30 1.35 1.40 1.36 1.40 1.45 1.30
.
55.5
Ala
1 9 T F 1iT ii -iTa* y 3a G 5 iP K* 6
All
those
ehifts
.
24.0
167.6
except
CDCl,
1H NM0 Chemical
2.
Boc/Ac .C”3
19.1
.
.
1.11 1.19 1.50 except
those
17.1
recorded
in
NH
DMSO-dg
fragnents
XHe/Uli
Ala C6”
51.0
173.0
Co”
C6H
CH3/0H
y.96 0.65 0.30 7.05 0.51 0.17 0.25 7.37
1.39 1.59 1.55 1.33 1.74 1.40 1.35 1.35 1.72 1.69 1.73 1.35 1.35 1.60
7.u4 7.07 6.53 0.05 0.43 7.57 7.43 0.76
3.00 4.25 4.30 4.10 4.34 4.22 4.23 4.40
1.15 1.27 1.30 1.56 1.43 1.16 1.22 1.47
3.62 3.59 3.70 3.73 3.70 3.62 3.60 3.75
6.79
1.29
7.74
4.27
1.24
3.75
marked
with
3.54 3.50 12.23 7.15
l
which
were
recorded
in DMSO-d6
Type title studies on amino a&ds and peptides-XII to be the reason
believed
It is known that feasible.
Instead
sponding tomerism
(Scheme 6).
the C-terminal we decided pivaloyl
to prepare
H-S-Ala-One
in coupling
amino acid
with
not sufficient-
(0)
of the reaction
via their
product
keto-enol
where the achiral
would be expected is
which was isolated
not
are nommlly
of the thiazlactone
(11)
generally
to the corre-
and since
reduced,
tau-
Aib is Aib bears
is not possible
from the reaction
to Scheme 6, with R I CH,. Subsequently, ‘H NMR spectrum
The
racemization
is
is converted
racemized
of Boc-S-AlaY(CSNH)-Aib-OH
via the thiazlactone
-3a.
and are readily
in which the activity
n according
yielded
case
from the C-terminal
thiopeptide
These thiazlsctonea
reactions
no major problems
form, 2
product.’
by coupling
with the amine the activated
In the special
the enol
chloride
of a coupling
of thiopeptides
1g*20’21 (thiazol-5(48)-one).
to participate
no a-hydrogen
the absence
of coupling
thiazlactone
ly reactive
for
elongation
653
of -.10 with of -11 with HCl*
aminolysis
revealed
the presence
of two
I
,*,
Boc -NH-CH-C-HH-TR-COOH -
CHl
CY /
S-
w
I
P,Ch
____,
\
Boc-uH-y-C*N,C,
H,N-fli-COOMe
CY
C’%
S II
SW
‘y
CH,
CH,
Cn,
J!
diastereomerst isolation
via
the ‘H M
thiazlactone
thiazlactone
in the coupling examples
reagents
towards
2o
It is,
derivative
formation
reaction
Several
is
to give
for
the first
but quite
with e smell
Thietion
was assumed to be the easiest
this
analogue.
group were less
sterically
The NM resonances
for
field
with respect
uhich
the change is about due to coupling
the verification Similarly,
the a-,
to the parent
nal of the Aib residue doublets
thietion hindered
of
appears
the 13C signals
of amidation
thiazlactons
by deprotection
has been isolated.
because
2).
as a singlet,
of Boc-Aib-S-Ala-
of the Boc group end coupling
of -la with LR gave a reesoneble
of Ala%=0 the 8-,
site(s)
And product
and shown to be weak acylating
and hence used
amidea.‘3”4”*
for
prolongation.
the lack
have been isoleted
may to a,
with the C protons,
the thiation
example of e thiopeptide
C-terminal for
the
yield
of 4a,
compound Boc-S-AlaY(CSNH)-AibY(CSNH)-S-Ale-C&,
than those
1.7 ppm (Table
indicating
time the postulated
the thiation
amount of the dithieted
We anticipated
as the reasw,
followed
unexpectedly
together
the first
peeks,
of -4a end in turn -4b wes based on thiation
Boc-AibY(CSNH)-S-Ala-Me,
with Boc-S-Ala-OH,
single
23
and peptides.
the preparation
showed only
to our knowledge,
has been suspected
this
amino acids
spectrum
end of any thiopeptide
of Z-phenylthiezol-5(4e)-ones
The strategy -0Me (1)
crystallization
its
CH,
%
of one of the diestereomars.
prolongation although
After
p
Boc -NH-WC-Nli-C-C-NH-%I-COOU.
,s,
of Aib’C-0
it
seamed that
The lergest This observetion in contrast
in a quantitative
the subetituents
attempt
of
the thiosmides are found for
ere shifted
that
spectroscopy
down-
the N-protons
of Ala which are
an efficient
tool
way (Fig.2).
a and B to the thioanide
for
the N-H group sig-
to the N-H group signals lH Ml
of this
gem-methyl groups.
values
and the fact
2.
to prepare
the surroundings
with the two adjacent
and the N-protons
makes simple
in our first
function
exhibit
for
0. E. JENSENand A. &mmo
6560
Fig. (2)
2. in
lH NHR spectra 6DC1,
downfield nyl
shifts
of
Boc-Ala’-Aib2Y(CSNH)-Als3-OMe
1).
(Table
The thiocarbonyl
carbon. ‘3’14’18*24 An interesting
magnetic
ally,
nonequivalence
protected
stereotopic peptides
dipeptides
induction with less
the large stricted
Aib
(HNE)
hindered
the
rotation
occurs
resonates
B-methyl
a smell
centres
groups
13C HNE of
of neighbouring
around
in our series
the
is strongly
twisted
(or
sulfur)
which increasea
for
the methyl group in guestion.16’26
and in
effect
the electrostatic
at 204 ppm YS 173 ppm for
N-Co or
very
large
in the Aib residues
(Table
1).
Gener-
0.5
ppm which
is explained
whereas in schirel
Ca-C+O bonds FINE is
analoguee
lacking.25
is brought
about
thus,
group close e large
Obviously, by s very re-
to the csrbonyl downfield
by die-
of chiral
between Aib Co and Aib carbonyl
moves one methyl
interaction;
csrbo-
is the
about
&
the
end 2
residues,
of thiosmide
in which the torsion’angle
conformation
(CG-Co-C-O)
and Boc-Als’Y(CSNH)-Aib2-Als3-OMe
of the two model peptides
geminal
exhibit
from chirsl
MNE which also backbone
of
carbon
property
(&)
shift
oxygen oxygen is caused
6561
Type title studier on amino acids and pcptides-XII The changs carbonyl
to a greater
ence *as seen,
probably
in the regions
3450-3230
1670-1630
(amide I),
cm-l
I bands fall stic
value.
stretch tive
magnetic
group was expected
because
cm”
a large
stretching),
(N-H
of
stronger
2760-1745 I),
absorptiona
26 in the Aib
groups,
cm-l (eater),
1720-1680
cm-’ (urethane),
and 1165 cm-1 (dithioeater).
fran the C-O stretching
The thioamide
and ere of less
diagno-
The absorptions
for the N-H stretch below 3400 cm-’ and erethane and/or amide C=O -1 at 1690 and 1660 cm , respectively, of the target compounds (5 and b series) seem indicsof hydrogen
group is a characteristic
in the rsnge
260-307
The mass spectra [M-isobutene]?
spectra
oxygen by sulfur
but no unambigous differ19 are observed The IR absorptiona
dependme.
cm-l (thioamide
The thiocarbonyl
ments
on substituting
solvent
of the existence
lengths
nm with
cases
= 241 nm (methanol)
state.
UV chrcmophore
log E values
fron.3.0
[H}? or [~+l]+,
Occasional
and [M-tertBuO)]+.
of the thioamide
2,4_disubstituted
bonds in the solid
l9 show in most
loss
l9 which absorbs
strongly
at rave-
to 4.1. together
of H,S and/or
with
the
characteristic
HS wsa observed
frag-
in the mass
analogues.
The IR and UV absorptiona
&x
of
1170-1130
in the.region
anisotropy
to enhance the IWE of the gem-methyl
of 11,
1630 (C-N),
ars in accordance
1710-1720
with those
cm-’ (urethane,
found in the literature
thiazlsctone for
the keto
C-0)
and
forms of
thiazolin-5rones.27
+I(a&ratim is believedto cam at the N-tennlnP1 Ala resi&e via ruita B in sdnm 6, giving rise to two diastereanars of the final probct 2. After completim of this work, use of catalysts tilch mimt suppress this epinarlzatim has appearedin the 1itcrature.~ EXPERIMENTAL Instruments ‘H NMRspectra were recorded at 60 MHz on Varisn EM-360 or at 80 MHz on Vsrian CFT-20 spectrometers with CDCl, as solvent. Samples with CMSO-ds as solvent were recorded et 300 HHz on a Varian XL-300 spectrometer. 13C NMRspectra were recorded at 25 MHz on Varisn XL-100-15 or at 75.426 MHz on Varian XL-300 spectrometers with CDCls or DMSO-ds as solvents. Chemical shifts are reported as parts per million on the 6 scale, with reference to TM’Sas 0 ppm (for ‘H spectra), COCl, as 76.95 ppm and DMSO-d6 as 39.5 ppm. Spectra with E#%O-d6 as solvent were recorded at the Department of Chemistry, University of Louisville, Louisville, KY, USA. UV absorption spectra were recorded on Perkin Elmer 402 and Uvikon 860 (compounds 4s and Se) spectrophotometers. IR absorption spectra mere obtained with a Beckman IR-18 apectroph~omete~ Mass spectra and precise msas measurements were recorded on a Micromass 7070F spectrometer operating at 70 eV with direct inlet. Elemental analyses were carried out by Lavena Kemisk Fabrik, DK-2750 Ballerup (Microanalytical Lsboratory). Optical rotstions were measured in a 1 ckn cell in a Perkin Elmer 241 polarimeter. Silica gel 60 (Merck) 63-200 pm was used for column chromatography and silica gel 60 (Merck) 40-63 um for flash chromatography. The dimensions for the flash column were 30 x 200 mn, and the flow rate was 2.54 cm/min (Nr gas). The following systems were used for TLC monitoring (I): butanol/scetic acid/ water (4:1:1), (II): chloroform/methanol/acetic acid (B5:10:5), (III): butanol/acetic acid/water (3:1:1), (IV): 2_butanol/acetic acid/water (67:10:23). The TLC plates were prepared as described earlier.12 The ninhydrin spray solution consisted of 0.25% ninhydrin in butanol. For LTVmonitoring the wavelengths 254 mu and 350 mu were used to detect thioamides and amides, respectively. For detection of ninhydrin negative impurities the TLC plates were exposed to iodine vapour. The methyl 29 derivstives ofS-alanine and a-aminoisobutyric eater hydrochlorides 28 and N-tert-butyloxycarbonyl acid were prepared by standard methods although for Aib prolonged resction times were allowed. N-tart-Butyloxycarbonyl-S-alanyl-a-aminoisobutyric acid methyl ester (Boc-S-Ala-Aib-One, 1).17~30 Boc-S-Ala-OH; 37.84 g (0.200 mol), was dissolved in a mixture of 20.23 g (0.200 mol) N-meFhylmorpholine and 182 ml dry tetrahydrofuran. After cooling to - 20 “C, 25.45 g (0.19 mol) isobutyl chloroformate was added dropwise, keeping the temperature below - 10 OC. The mixed anhydride was alloued to form in 15 min of activation time; then a precooled mixture (- 10 “C) of 72.7 g (0.180 mol) HCl-H-Aib-We, 18.2 g (O.lBO mol) N-methylmorpholine, and 113 ml tetrahydrofuran was added over e period of I5 min. The reaction proceeded for 0.5 hr at - 10 PC, then for 1 hr at 0 OC and finally 1 hr at rocm temperature, after which the temperature again was lowered to 0 OC and 70 ml of saturated KHCO3solution was added to destroy any unreacted mixed anhydride. After 0.5 hr the organic solvent was removed in vacua. The aqueous slurry was extracted with 100 ml snd 2 x 50 ml ethyl acetate. The combined organic phases were washed with 1 N HCl (3 x 50 ml), water (3 x 50 ml), 5% KHCO, (3 x 50 ml) end 10 ml water, then dried over MgSOr. The solvent was evaporated under vacuum to yield 49.79 g of 1 as a colourless oil. Rf = 0.6 in ethyl acetate [al;’ = - 33.6O (c = 1 in methanol). The spectrosco$c data correspond well with those found in the literature. N-tert-Butyloxycarbonyl-S-thioalanyld-aminoisobutyric acid methyl ester tBoc-S-AlaY(CSNHI-Aib-One, 9j.12 Boc-S-Ala-Aib-We (l), (7.55 g, 0.0262 mol) and LR (5.82 g, 0.0144 mol) was refluxed in dry toluene (15 ml) for 0.5 hr. The purification follows the same procedure as applied for 5. The dimensions of the column were 64 x 240 mn. The yield of 2 waa 0.8 g as a slightly yellGil. Rf I 0.52 in 10% diethyl ether/dichlorcxnethane. All data correspond well with those previously found. N-tert-Eutyloxycarbonyl-S-alanyl-a-aminofsobutyric
acid (Boc-S-AlUa-Aib-OH,2).17*M Boc-S-Ala-Aib&He
6564
0.
E. Jmsa~ and A. .%WING
with added silica gel, and chromatographed on a column (20 ~200 mm) with dichloromethane until the “P,S” by-product (Rp = 0.88 in 30% diethyl ether/dichloromsthane) was eluted. The thiopiperidide was then eluted with 30% diethyl ether/dichloromsthane. Evaporation of the collected fractions yieldsd 2.74 g (63%) of 5 as a colourless powder with m.p. 85.0-85.5 OC. Rf = 0.73 in’30% diethyl ether/ dichloromethane. [av= + l9.2O (c I 0.5 in ethyl acetate). IR (KEr)r 3340 (N-H stretch),1710 UI?-~(Wthane). UV (diethyl ether): X (log E) = 280 nm (4.1>. MS: m/z = 272 [fl]‘$ 239 [H-H’S]‘, 216 ‘t_ isobutene]+, 199 [M-tert-BUOY, 183 [M-isobutene-HS] 155 WN(C~H~~)HS] 128 [I+-CSN(CSHI~) 5 Elemental anslysis: C~~HZ~NZO~S (272.2) Calc.: C 5;.32, H 8.88, N 10.28,‘s 11.75 Found: C 57.08, H 8.87, N 9.85, S 11.68 N-tert-Butyloxycarbonyl-S-dithioalanine methyl ester (Boc-S-Ala Y(C.Sl.SMe,6). The thiopiperidide 5 (2.55 g, 0.0094 m01) and iodomethane (3.0 ml, 0.0094 mol) in 8 ml of tetraFydrofursn were sllouedto react under enhydrous conditions for 24 hrs, wheresfter the mixture wss evaporated in VXIIO. The remaining slurry was taken up in 5 ml of methanol and a stream of HrS gas was bubbled through the solution for 20 min. The solution was allowed to stand for another 20 min before evaporation. The oily product was purified on a column with diethyl ether yielding 1.40 g (60%) of 5 as an oil which was crystallized from pentane to give yellow crystals. M.p. 73.0-73.5 OC. Rf = 0.79 in diethyl ether, [al?? = -52.3” (c = 0.2 in ethyl acetate). IR (KfJr): 3310 (N-H stretch), 1700 (ufethane 1165 cm-’ MS: m/z I 235 [M] , 179 ? M-isobu(dithjoester. (log cl = 300 nm (4.0). UV (diethyl ether): tene] , 120 [H-tert-BUO] , 1.44 ~Boc- +m H(Mee)]+. Precise mass measurement: Calc. m/z 235.0700, found m/z I 235.0700 [Ml’. N-tert-Butoxycarbonyl-a-sminoisobutyryl-S-alsnine methyl ester IBoc-Aib-S-Ala-OMe, 1). Boc-Aib-OH (8.12 g, 0.04 mol) and HCl*H-S-Ala-Me (5.03 g, 0.036 mol) were coupled by the mixed anhydride method described for 1 except for the activation time which wss 20 min. The yield was 6.2 g (74%) of a colourless solid which was recrystallired from ethyl acetste/ ‘ght petroleum ether (b.p.40 OC), m.p. [a]# = -18.7O 95.0-95.5 OC. Rf = 0.55 in diethyl ether/dichloromethane. (c = 0.4 in methanol). IR (KBr): 3410-3280 (N-H stretch), 1745 (ester), 1680 (urethane), 1670 cm-’ (amide I). UV (ethanol): %ex (log E) = 201 nm (3.2). MS: m/z = 289 [M+l]‘, 233 [M+l-isobutene]+, 189 [M+Z-tert-BuCCO]*, 158 [Boc-NHC(Me)z]‘, 102 [M-(Ale-We)]+. Elemental analysis: CirHr*N205 (288.3) Calc.: C 54.15, H 8.39, N 9.72 Found: C 54.46, H 8.23, N 9.77 a-Aminoisobutyryl-S-alanine methyl ester hydrochloride IECl'Ii-Aib-S-Ala-OMe, 0). Boc-Aib-S-Ala-We, 1 (3.00 g, 0.0104 mol) was deprotected with 52 ml of 1 N HCl/dioxane for 40 mTn. Evsporation of the solvent yielded 2.03 g (89%) of g as a hygroscopic foam. Rf = 0.50 in III, Rp = 0.45 in IV. [aIF= - 23.3O (c = 2.6 in methanol). UV (ethanol): A,, (log E) = 201 nm (3.2). Acknowledgements - Part of this work was done at the Department of Chemistry, University of Louisville, KY, USA, and Professor A. F. Spatola’s cooperation and grant are gratefully acknowledged for fscilitating the stay for O.E.J. Ha wish also to thank Or. R. A. Porter for NHR recordings at the University of Louisville.
1. PmtxI, see T.P.k-&rsen and A.Semiq, Liebigsm.olsn. (submitted). 2. A.F.wtola in chsnis@ an3 tiafisnistnlof &ukw &ids, E~ptidasa& Fz~4~in5,(Ed. B.YinStein), Ckkker,Vo1.7, Pp. 267-357(1983). 3. w.L.W, J.-T.&m, J.Y.Tsang,aioorPm.Bi~s.R?.s.CZmI.02 589 (19Bl). 4. P.Ca@all ti N.T.t&kd, J.An.Ckm.scC. 104 5221 (1982). 5. P.A.Bartlett,K.L.Spearand N.E.Jaccbsan, ~&mistry~ 1608 (1982). 6. G.Y.Kemr and R.C.heppard, Nature (London)& 48 (1958). 7. E.Buwdatti, A.Bavoso,B.Oi Blasio,V.Pwona, C.Pedrms, C.Tmiolo, and G.kBmOrf~, I'LtZ.~~.~.=i.t=~ 7951 (1982). 8. H.BrUdwr and H.Graf,l?qerimtiaE 528 (1983). Fqtidss, (Eds C.Voelterand G.Msitzel,da Gruy9. G.&ng, H.Br+cknsrand H.Sdnitt in stn&unz acd ZctivityofWxr& ter, Berlin, 1981, pp. 75-114. lo. B.v.vankataram Rasad and P.Balarm, CRCQit.kc.Bic&3x 16 307 (1%). 11. C.Tmiolo, G.H.&xmra, A.Bwoso, E.Banedstti,B.01 Blasio,V.Wvaw and C.psdmn, BWl~ners 22 205 (198%. Tl&badm 41 5595 (1985) 12. 0.E.Jens.q S.-O.Le*essm, Mardi, A.M.Piauesl,ad C.Tmiolo, 7 3635 (1961);14. I&, Qwx.9~. 20 14 (1982);See also F. 13. K.Clausen,M.Tl-mr.sen, and S.-O.Lauessm, lWr&&m rapine., D&s.Akstr.Int.B 1986.46 (111, 3849 (C.A.E 24625 (19B6). 15. R.&xch, G.m, K.-P.Vogesand Y.Wintar,LiebigsIhn.Ck???. 1117 (19W). 10% (1983). 16. G.Jung,n.Briickner, R.Borh, knintar, ~.Schalland J.Str&hle,LiebipSRn.m. G.m, Liebigsrtn.chsn.1151 (1979). 17. R.Oskommopr10s & 18. K.Clausm, H.llwrsm, S.-O.Lawssson and A.F.Spatola,J.Uxm.*.. *&in m. 1785 (1984). 19. W.Thorsan,B.Vde, U.Padsrs.sn, K.Clausm,and S.-O.Lak@eSsOn, ~~&s&tn 39 3429 (1%3). 20. G.Lajoie, F.Lapine,L.l+aziak and B.&llaau, ZWrdpdrm L&t. 24 3815 (1983).See also G.Lajoia,Dk.w.fit.Bm 45(10) 3225; (c.A.~cn 204285 (19B5)).2oe.P.Wlpf md H.Hslnpartw, flelV.olim.ti69 1153 (19B6). 21. u.Staglich,GMiPle md L.Ullsdwitz, ZMrakdrm I.&t. 169 (1970). fi 39 (1960). 22. M.T.Lepley, O.S.JMes, G.Y.KmH?r and R.C.Sheppard,lb23. G.C.Barrett,J.CYBTI..~X.(C~ 1380 (1971).- 24. I.O.Rae,mt.J.Olsn. n 561 (1979). 2 2165 (1982). R.OekonanopllosandG.Jng,*~ 25. O.Laibfritz,E.-t, N.tilschar, H.lxhmm, 26. P.L.ScuWwicic,J.A.Fittgerald md G.E.Millirran, Zktrahedra,L&t. E 1247 (1%5). 27. J.H.W~S, ~.~.~evie~and R.A.G.Carringtm,J.Qrm.*.. @&in ~zxs.~ 1983 (1972). verlag,StuttWt, 1974. 28. nxix0nsy1,wulxknderorg.o1Em., Bd. XV/l, p.316, G?Org l-bie!na 29. L.kwockr, A.Hellatt, E.Wu&, O.Kallerand G.krsin, WqlIer's WM.-. Z 1651 (1976). 30. R.Kagsrajand P.Balarm, Zktrdmdron r 1263 (1981). 31. K.Clausm and S.-O.Lawsson,W.J.a. 9 43 (1980). 32. S.Sctmibye,B.S.Pe&rsm and S.-O.LawSSm, ~l..5k.~.6+7. 3 229 (1978).