Synthesis, bioassay and nmr study of methyleneoxy isosters of oxytocin and vasopressin

Article abstract of DOI:10.1135/cccc20020373

Syntheses of pseudodipeptides H-Tyrψ[CH₂O]Ile-OH and H-Tyrψ[CH₂O]Phe-OH were carried out using the intramolecular Williamson reaction of O-benzyltyrosinol with ethyl chloroacetate followed by N-protection and aldol reaction of the co

Full text of DOI:10.1135/cccc20020373

Methyleneoxy Isosters of Oxytocin and Vasopressin
373
SYNTHESIS, BIOASSAY AND NMR STUDY OF METHYLENEOXY
ISOSTERS OF OXYTOCIN AND VASOPRESSIN
1
2
3
4,
Jan MAŘÍK , Miloš BUDĚŠÍNSKÝ , Jiřina SLANINOVÁ and Jan HLAVÁČEK *
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
2
166 10 Prague 6, Czech Republic; e-mail: marik@uochb.cas.cz, budesinsky@uochb.cas.cz,
3
4
slan@uochb.cas.cz, honzah@uochb.cas.cz
Received Novem ber 21, 2001
Accepted Jan uary 18, 2002
Syn th eses of pseudodipeptides H-Tyrψ[CH2O]Ile-OH an d H-Tyrψ[CH2O]Ph e-OH were carried
out usin g th e in tram olecular William son reaction of O-ben zyltyrosin ol with eth yl ch loro-
acetate followed by N-protection an d aldol reaction of th e correspon din g m orph olin -3-on e
in position C2 with butan on e or ben zaldeh yde, elim in ation of th e h ydroxy group to give de-
rivatives with a double bon d eith er as th e E/Z (1 : 1) diastereom eric m ixture in th e case of
th e form er derivative or as th e Z-isom er on ly in th e case of th e latter on e. Stereoselective
h ydrogen ation an d h ydrolysis of both th e lactam s yielded th e correspon din g pseudo-
dipeptides lackin g th e carbon yl group as a h ydrogen bon d don or. Th e in troduction of th e
pseudodipeptides in to position s 2 an d 3 of oxytocin an d vasopressin caused total absen ce of
all biological activities in th e form ed an alogues. Th e results of th e bioassay an d NMR study
con firm ed th e im portan ce of th e H-bon d between th e backbon e carbon yl of th e Tyr2 an d
NH proton of th e Asn 5 residues for stabilization of th e β-turn in th e cyclic h exapeptide part
of both th e h orm on es an d for th eir biological activity.
Keyw ord s: Peptides; Peptidom im etics; Meth ylen eoxy isosters; Solid ph ase syn th esis; Biologi-
cal activity; 1H an d 13C NMR spectroscopy.
Isosteric bon d in troduction in to peptides is m otivated by th e lim ited usage
of peptides as poten tial th erapeutic agen ts because of th eir fast en zym atic
degradation an d a poor tran sport th rough biom em bran es. Th e replacem en t
of a peptide bon d with a suitable isostere (surrogate) can in crease th e stabil-
ity of a peptide toward en zym atic degradation , prolon g th e h alf-tim e of
peptide action an d im prove peptides tran sport in to a cell1. On th e oth er
h an d, th e substitution with surrogate of larger flexibility or rigidity can in -
fluen ce n on covalen t in teraction s in a given peptide m olecule an d, con se-
quen tly, its space structure an d in teraction with a correspon din g receptor
an d fin ally lead to a ch an ged biological poten cy.
We h ave been in terested in CH2O an d CH2S surrogates for several years2–6
sin ce th ey offer polar an d flexible structures resistan t to proteolytic en -
Collect. Czech. Chem. Commun. (Vol. 67) (2002)
doi:10.1135/cccc20020373

374
Mařík, Buděšínský, Slaninová, Hlaváček:
zym es. Con trary to th e CH2S, th e geom etry of th e CH2O surrogate is very
sim ilar to trans-am ide bon d an d resem bles th e peptide bon d in exten ded
con form ation 7,8. In addition , th e CH2O surrogate exh ibits n egligible
n ucleoph ilicity an d resistan ce to oxidation .
An in troduction of both th e surrogates in to bioactive peptide m olecules
can th us in fluen ce biological activities in a differen t way. For exam ple, th e
CH2O surrogate sign ifican tly accelerated an d in creased biological activity of
oostatic peptides after th eir application in th e flesh fly Neobellieria bullata
con trary to th e CH2S surrogate, wh ich evoked a m oderate effect on ly6. Th e
replacem en t of th e Leu-Gly peptide bon d in oxytocin (1a) eith er m ain -
tain ed (CH2O) or partially dissociated (CH2S) its biological activities2,4 an d
th e in troduction of th e CH2O surrogate in to m olecules of substan ce
P-(6-11)-peptide an d [Leu8]en keph alin am ide resulted in agon ists with af-
fin ity to correspon din g receptors7 an d with selective activity.
H-Cys-Tyr-X-Gln -Asn -Cys-Pro-Y-Gly-NH2
1a, X = Ile, Y = Leu; 1b, X = Ph e, Y = Arg
H-Cys-Tyrψ[CH2O]X-Gln -Asn -Cys-Pro-Y-Gly-NH2
2a, X = Ile, Y = Leu; 2b, X = Ph e, Y = Arg
Fm oc-Tyrψ[CH2O]X-OH
3a, X = Ile; 3b, X = Ph e
In th e presen t study we were in terested in syn th esis of oxytocin (1a) an d
vasopressin (1b) an alogues 2a an d 2b con tain in g th e CH2O surrogate as a
substitute for Tyr2-Ile3 an d Tyr2-Ph e3 peptide bon ds, respectively. Our in -
ten tion was to evaluate th e im portan ce of in tram olecular h ydrogen bon d
between th e carbon yl oxygen of Tyr2 residue an d th e am ide proton of Asn 5
residue. Th is bon d was design ed to stabilize th e β-turn im portan t for th e in -
teraction of both th e h orm on es with receptors9,10. Th e Asn 5 residue h as
been proposed to be n ecessary for biological activity of oxytocin an d
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Methyleneoxy Isosters of Oxytocin and Vasopressin
375
vasopressin 11 due to its side-ch ain in teraction with th e receptor bin din g
site (even th ough som e fin din gs12,13 proposed a m ore exten sive exploration
of structure–fun ction relation s at th is residue because of th e poten cy foun d
in Asp5 con tain in g an alogues). Also th e Tyr2 residue was postulated to ex-
h ibit an essen tial fun ction in th e in teraction of both th e h orm on es with
correspon din g receptors an d th e side-ch ain of th is residue was design ed to
play an im portan t role in th e bioactive con form ation 14–16. Th erefore, th e
in troduction of th e CH2O surrogate between position s 2 an d 3 could in -
crease relative flexibility of th e cyclic h exapeptide parts in correspon din g
h orm on e an alogues an d th us in terfere with design ed con form ation s, ex-
cludin g both th e an alogues from a proper in teraction with correspon din g
receptor system . Th e in crease in th e flexibility of th e cyclic part could also
worsen con dition s for stackin g of Tyr2 an d Ph e3 arom atic rin gs in vaso-
pressin , design ed for its biologically active m odel10.
RESULTS AND DISCUSSION
Our syn th etic strategy was based on preparation of correspon din g pseudo-
dipeptides H-Tyrψ[CH2O]Ile-OH (3a) an d H-Tyrψ[CH2O]Ph e-OH (3b) an d
th eir furth er utilization as th e buildin g blocks in th e solid-ph ase syn th esis
of both th e h orm on e isosteric an alogues 2a an d 2b.
In th e syn th esis of th e pseudodipeptides 3a an d 3b we utilized th e
in tram olecular m odification of th e William son reaction , providin g
stereoch em ically defin ed com poun ds17. Th e startin g com poun d for both
th e C2-alkylation reaction s, (5S)-5-[4-(ben zyloxy)ben zyl]-4-(tert-butoxy-
carbon yl)m orph olin -3-on e (5), was prepared by th e reaction of O-ben zyl-
tyrosin ol with eth yl ch loroacetate affordin g th e (5S)-5-[4-(ben zyloxy)-
ben zyl]m orph olin -3-on e (4), followed by N-protection with di-tert-butyl
dicarbon ate.
Th e con den sation of 5 with butan on e provided a m ixture of stereo-
isom ers of 6 in good yield. Elim in ation of th e h ydroxy group led to a m ix-
ture of two regioisom ers (7 : 3) with th e double bon d in differen t position s.
Th e m ajor product was isolated as a m ixture of E- an d Z-stereoisom ers 7a
1
an d 7b in th e 1 : 1 ratio, accordin g to H NMR an d HPLC an alyses; th e m i-
n or product 8 was Z-isom er on ly. Th e separation of th e diastereom ers usin g
flash ch rom atograph y was n ot successful an d th erefore we decided to con -
tin ue with th is m ixture an d to separate th e diastereom ers later on . Th e cat-
alytic h ydrogen ation of th e 7a/7b m ixture provided a m ixture of th e
diastereom ers of th e N-protected 2,5-substituted m orph olin -3-on e 9 due to
th e presen ce of Ile an d aIle side-ch ain s. Hydrolysis of th e lactam rin g fol-
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

376
Mařík, Buděšínský, Slaninová, Hlaváček:
BnO
O
BnO
OH
1. NaH
2. ClCH2COOEt
3. ∆
N
H
O
NH2
4
Boc2O/DMAP
BnO
O
N
O
1. LHDMS
1. LHDMS
2. butanone
2. benzaldehyde
Boc
5
Ph
C
CH3
BnO
O
N
OH
BnO
O
C OH
H
CH2CH3
O
O
N
Boc
Boc
10
6
TEA/∆
MsCl/TEA
R1
Ph
CH3
O
N
H
BnO
O
BnO
O
R2
H
+
CH3
O
N
O
N
O
Boc
Boc
Boc
11
7a, R1 = CH3; R2 = CH2CH3
7b, R1 = CH2CH3; R2 = CH3
8
H2/Pd
H2/Pd
CH3
CH
Ph
HO
O
CH2
HO
O
CH2CH3
N
O
N
O
Boc
Boc
12
9
1. 6 M HCl
1. 6 M HCl
2. FmocOSu
2. FmocOSu
CH3
CH
CH2CH3
Ph
HO
O
CH2
HO
O
COOH
COOH
NH
NH
Fmoc
Fmoc
3b
3a
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Methyleneoxy Isosters of Oxytocin and Vasopressin
377
lowed by in troduction of th e Fm oc protectin g group provided th e desired
pseudodipeptide 3a wh ich was used for th e syn th esis of th e oxytocin an a-
logue 2a.
Th e quen ch in g of th e en olate gen erated from 5 with ben zaldeh yde af-
forded 10, followed by elim in ation of th e h ydroxy group providin g substi-
tuted m orph olin -3-on e 11, on ly as th e Z-isom er. Th is com poun d, after
catalytic h ydrogen ation , afforded stereoselectively (2S,5S)-2-ben zyl-4-
(tert-butoxycarbonyl)-5-(4-hydroxybenzyl)m orpholin-3-one (12) (d.e. > 99%).
Th e stereoselectivity of th is reaction is in duced by th e ben zyl substituen t at
th e C5 position wh ich adopts th e pseudoaxial con form ation as a con se-
quen ce of m in im izin g th e pseudoallylic A1,3 strain caused by N-Boc protect-
in g group18. Fin ally, acid h ydrolysis of th e m orph olin -3-on e rin g an d
in troduction of th e Fm oc protectin g group provided H-Tyrψ[CH2O]Ph e-OH
(3b) wh ich was used in th e syn th esis of [8-argin in e]vasopressin an alogue
2b.
Solid-ph ase syn th esis of th e m eth ylen eoxy an alogues 2a an d 2b of
oxytocin 1a an d [8-argin in e]vasopressin 1b was carried out on a Rin k am ide
resin 19 usin g Nα-Fm oc protected am in o acids in th ree-fold excess: Gly, Leu
or Arg(Mtr), Pro, Cys(Trt), Asn (Trt), Gln (Trt). DIC with HOBt as con den sa-
tion agen ts in DMA were used, in th e presen ce of DIEA. In each step, th e
Fm oc deprotection was perform ed with 20% piperidin e in DMF. In both
th e syn th eses, th e N-protected pseudodipeptides 3a or 3b, with th eir ph e-
n olic h ydroxy groups un protected, were coupled in two-fold excess with
th e growin g peptide ch ain s on th e resin usin g a m ixture of TOTU, HOBt,
an d DIEA. Th e peptides were detach ed from th e resin by treatm en t with a
TFA–TIS m ixture in DCM. Th e side-ch ain deprotection in oxytocin an a-
logue 2a was perform ed with a TFA–water–EDT–TIS m ixture an d in
vasopressin an alogue 2b with a TFA–th ioan isole–ph en ol–water–EDT–TIS
m ixture for a prolon ged tim e due to th e presen ce of th e Arg(Mtr) residue.
Closure of th e disulfide bon ds in both th e peptide an alogues was carried
out by oxidation with K3Fe(CN)6 at pH 8. After desaltin g an d purification ,
both th e an alogues were an alyzed an d ch aracterized by an alytical HPLC,
AAA, FAB MS, an d 1H NMR an d 13C NMR spectroscopies. In th e oxytocin
an alogue 2a, h owever, we obtain ed a n on -separable m ixture of two
stereoisom ers th at differ in con figuration at β-carbon of th e side ch ain in
position 3 of th e peptide an d correspon d to oxytocin an d [3-alloisoleucin e]-
oxytocin isosteric an alogues.
Th e uteroton ic, pressoric an d an tidiuretic activities of th e n ewly syn th e-
sized m eth ylen eoxy an alogues 2a an d 2b were tested an d com pared with
th e activities of th e paren t n euroh ypoph yseal h orm on es 1a an d 1b (Table I).
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

378
Mařík, Buděšínský, Slaninová, Hlaváček:
Both th e an alogues were en tirely in active in all th e tests used in bioassay.
Th ese results are in accordan ce with th ose obtain ed from th e experim en ts
on an alogues with m eth ylen eam in o surrogate in th e sam e position 20
(Table I). As we indicated above, in the case of oxytocin analogue 2a, a m ix-
ture of two n on -separable stereoisom ers differin g in con figuration at β-car-
bon of th e side ch ain in position 3 an d correspon din g to oxytocin an d
[3-alloisoleucin e]oxytocin an alogues was, in fact, bioassayed. Even th ough
th e uteroton ic activity of th e latter is by about on e order of m agn itude
lower th an th at of oxytocin 21, it is still sign ifican t (m oreover, on ly 30% of
th e allo-form was presen t in th e m ixture). Th e absen ce of th e biological po-
ten cy in an alogue 2a can be th erefore explain ed sim ilarly to an alogue 2b
by a sign ifican t con form ation al ch an ge caused by in troduction of th e flexi-
ble m eth ylen eoxy peptide bon d surrogate, un able to form h ydrogen bon d
between th e Asn 5 am ide proton an d Tyr2 carbon yl oxygen . In th e proposed
con form ation al m odel of oxytocin , based on NMR studies, th is H-bon d is
assum ed to stabilize a β-turn , in volvin g th e Tyr-Ile-Gln -Asn sequen ce. Th is
β-turn structure h as been suggested to be on e of buildin g elem en ts in over-
all con form ation n ecessary for recogn ition by correspon din g receptors an d
tran sduction of biological activities, as well9–12,22–25. By in troducin g th e
m eth ylen eoxy surrogate, form ation of th is h ydrogen bon d was avoided an d
th e flexible twen ty-m em bered disulfide rin g could n ot adopt a proper con -
form ation for th e in teraction with th e receptor.
TABLE I
Biological activities of oxytocin (1a), [8-argin in e]vasopressin (1b) an d th eir an alogues
Peptide
UTa
BPb
ADc
Ref.
OTd (1a)
450e
27
0
5
5
38
21
[alle3]OT
~0.18
0.046
[Tyr2ψ[CH2O]Ile3]OT (2a)
[Tyr2ψ[CH2NH]Ile3]OT
[Arg8]VPf (1b)
0
0
0
0
0
20
38
17
0
412
465
0
[Tyr2ψ[CH2O]Ph e3,Arg8]VP (2b)
[Tyr2ψ[CH2NH]Ph e3,Arg8]VP
0
0
0
0
20
a
b
c
d
e
Uteroton ic activity; pressor activity; an tidiuretic activity; OT, oxytocin ; biological ac-
tivity values are given in IU m g–1
;
VP, vasopressin .
f
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Methyleneoxy Isosters of Oxytocin and Vasopressin
379
Th e active con form ation of th e twen ty-m em bered rin g of [8-argin in e]-
vasopressin (1b) is close to th e active con form ation of oxytocin 26–28. Con se-
quen tly, in th e case of vasopressin an alogue 2b, th e β-turn in volvin g th e
Tyr-Ph e-Gln -Asn sequen ce also can n ot be form ed due to th e presen ce of th e
m eth ylen eoxy isosteric bon d an d th e resultin g con form ation of th e rin g is
disfavored for in teraction with receptor.
Th e NMR m easurem en t of both th e an alogues 2a an d 2b was perform ed
in DMSO sin ce th is solven t was described to preven t a con form ation al aver-
agin g in th e peptide backbon e of th e oxytocin h exapeptide rin g in water.
Also β-turn region s of con form ers of [1-(3-m ercaptopropan oic acid)]-
oxytocin in th e crystal structure were described to correspon d to th e β-turn
region s proposed in th e m odel for oxytocin an d its 1-deam in o an alogue in
DMSO (ref.29).
Th e NMR an alysis (Tables II an d III) con firm ed our previous presum ption
th at th e flexibility of th e twen ty-m em bered rin gs in th e an alogues 2a an d
2b h as in creased by in troducin g th e m eth ylen eoxy peptide bon d surrogate.
Tem perature coefficien ts of am ide proton ch em ical sh ifts of m eth ylen eoxy
an alogs an d paren t peptides are com pared in Table IV. Th e observed h igh er
n egative values of th e tem perature coefficien t of th e Asn 5 am ide proton in
th e m eth ylen eoxy an alogues, wh en com pared with th ose of th e paren t h or-
m on es 1a, 1b, in dicate th at th e am ide proton of Asn 5 in th e m eth ylen eoxy
an alogues is n ot in volved in th e h ydrogen bon d th at leads to th e in creased
flexibility of th e twen ty-m em bered rin g. Moreover, th e NOE con tact be-
tween αCH of Cys1 an d Cys6 observed in [8-argin in e]vasopressin 1b disap-
peared in th e case of th e m eth ylen eoxy an alog 2b. In th e sam e m an n er, th e
NOE con tact between αCH of Tyr2 an d Asn 5 of oxytocin 1a was n o lon ger
observed in th e correspon din g m eth ylen eoxy an alogue 2a. Th us, th e essen -
tiality of th e h ydrogen bon d in th e twen ty-m em bered rin g stabilizin g bio-
logically active con form ation of oxytocin an d vasopressin was con firm ed.
It is eviden t th at in creasin g th e peptides m etabolic stability to en zym atic
degradation by in troduction of th e m eth ylen eoxy isosteric bon d was n ot ef-
fective in th e isosteric an alogues 2a an d 2b of oxytocin an d [8-argin in e]-
vasopressin . It is, in con trast, to flexible lin ear oostatic peptides30 wh ere th e
in troduction of th is surrogate in creased th eir stability an d caused th e accel-
eration an d en h an cem en t of th eir biological activity6. However, th e sam e
structure ch an ge, m ade in th e backbon e of peptides with relatively ordered
con form ation like th at in th e cyclic part of n euroh ypoph yseal h orm on es,
apparen tly h am pers a proper con form ation of peptides an d th eir recogn i-
tion by th e correspon din g receptor system . Th is con sequen tly leads to loss
of biological activity of th e peptides. From th e syn th etic poin t of view, our
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

380
Mařík, Buděšínský, Slaninová, Hlaváček:
TABLE II
Proton NMR data of peptides 1a, 1b an d 2a, 2b in DMSO-d6
2a
Resi-
due
Param eter
1a
1b
2b
m ajor
m in or
Cys-1 NH2
8.27
4.00
8.21
4.05
7.51
8.22
4.07
αH
3.51
βH2(Jαβ)
3.37 (5.7);
3.08 (7.1)
3.40 (a);
3.11 (a)
3.02 (5.6);
2.66 (8.0)
3.43 (4.3);
3.10 (7.5)
Tyr-2
NH
8.48
8.47
8.50
8.60
8.50
αH(JNα)
βH2(Jαβ)
4.62 (8.5)
3.96 (7.8)
2.88 (7.3)
3.95 (8.0)
2.71 (7.0)
4.35 (a)
3.81 (8.1)
3.17 (3.8);
2.76 (10.8)
2.85 (5.2);
2.62 (9.6)
2.65 (8.1);
2.53 (6.8)
o-H
7.12
6.68
7.03
6.69
9.12
6.91
6.62
6.65
6.58
m-H
OH
9.20
~9.20
–
9.09
ψ[CH2O]
NH
–
3.52; 3.41
–
3.57; 3.43
–
3.44; 3.28
–
Ile-3
8.02
8.39
(Ph e) αH (JNα)
βH (αβ)
3.88 (a)
1.79 (5.9)
3.53 (a)
3.60 (a)
4.27 (7.0)
3.91 (a)
1.74
3.18 (4.7);
2.92 (10.1)
3.03 (4.2);
2.90 (8.7)
γH2
β-CH3
1.45; 1.12
1.23
0.88
0.88
–
–
–
–
0.90
0.86
–
–
–
γ-CH3
–
o-H
7.22
7.30
7.21
8.08
4.02 (6.5)
1.87
2.12
7.34
6.84
7.26
m-H
–
–
7.33
p-H
–
–
7.24
Gln -4 NH
αH (JNα)
βH2(Jαβ)
γH2
8.24
3.95 (5.4)
1.83–1.90
2.16
7.26
6.74
8.04
8.00
8.19
4.24 (7.7)
4.28 (~8.0)
4.20 (7.5)
1.92 (4.8, 9.0)
2.17
1.91 (5.0, 9.0)
2.18
CONH2
7.29
7.31
6.79
6.81
6.82
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Methyleneoxy Isosters of Oxytocin and Vasopressin
381
TABLE II
(Continued)
2a
Resi-
due
Param eter
1a
1b
2b
m ajor
8.08
m in or
8.12
Asn -5 NH
αH (JNα)
7.76
4.49(7.4)
2.63 (6.7)
2.59 (6.7)
7.33
7.97
4.48 (7.0)
2.56 (6.5)
2.52 (6.5)
7.23
8.10
4.52 (7.2)
4.53 (7.8)
2.62 (6.4)
2.52 (5.5)
7.50
βH2 (Jαβ)
2.60 (6.6) 2.57 (6.6)
2.53 (5.6)
CONH2
7.46
7.43
6.86
7.01
6.72
7.02
Cys-6 NH
8.28
8.02
8.07
8.29
7.91
αH (JNα)
βH2 (Jαβ)
4.72 (77.6)
4.79 (8.6)
4.75 (7.8)
4.76 (8.2)
3.18 (6.5);
3.03 (6.7)
3.10 (3.0); 2.93 (9.5)
3.12 (5.8);
2.95 (7.6)
3.07 (3.2);
2.90 (9.9)
Pro-7
αH
4.32
4.31
4.33
4.32
βH2 (Jαβ)
2.03 (3.0);
1.83 (9.0)
2.06 (4.0); 1.86 (8.6)
2.02 (3.6);
1.87 (8.4)
2.07 (4.1);
1.86 (8.5)
γH2
1.80–1.90
3.59; 3.44
7.96
1.91
1.87
3.64; 3.56
8.20
1.90; 1.86
3.63; 3.56
8.09
δH2
3.58
Leu-8
(Arg)
NH
7.95
αH2 (JNα)
βH2 (Jαβ)
γH
4.18 (7.6)
1.51
4.18 (7.6)
4.19 (7.6)
4.18 (7.5)
1.51 (5.4, 9.4)
1.76 (5.0; 8.6) 1.75 (6.1, 8.7)
1.64
1.62
1.58; 1.52
–
1.52
–
δ-CH3
δ-CH2
δ-NH
0.89; 0.83
–
0.89; 0.84
–
3.07
3.10
7.50
7.87
–
–
7.51
Gly-9
NH
7.81
7.81
8.07
αH2 (JNα)
3.65 (6.0);
3.54 (5.6)
3.65 (6.2) 3.55 (5.6)
3.66 (5.9);
3.60 (5.6)
3.67 (5.9);
3.61 (5.7)
CONH2
7.07
6.98
7.05
6.98
7.13
6.98
7.14
7.00
a
Th e J-value could n ot be determ in ed.
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

382
Mařík, Buděšínský, Slaninová, Hlaváček:
TABLE III
13C ch em ical sh ifts of peptides 1a, 1b an d 2a, 2b in DMSO-d6
2a
Residue Param eter
1a
1b
2b
m ajor
m in or
Cys-1
Tyr-2
CO
166.85
51.95
40.35
171.98
54.95
36.73
127.71
130.15
115.29
156.10
–
166.30 166.39
52.18
168.03
54.43
44.05
170.96
54.84
36.73
127.73
129.92
115.03
155.86
–
166.08
52.03
41.43
–
Cα
Cβ
41.35
CO
–
Cα
51.55
51.28
36.18
128.21
129.96
115.15
155.72
69.55
172.15
82.73
39.60
–
Cβ
35.93
i-C
128.49
130.13
115.37
155.95
o-C
m-C
p-C
ψ[CH2O]
CO
69.99
70.53
Ile-3
171.54
59.56
35.81
25.06
15.60
11.16
–
172.05
171.83
55.46
36.62
–
(Ph e)
Cα
84.96
37.45
83.57
37.76
Cβ
Cγ
24.64
β-CH3
γ-CH3
i-C
15.11
11.38
14.68
11.85
–
–
–
–
–
–
–
–
138.04
129.16
128.21
126.33
170.82
53.21
27.25
31.52
174.18
137.59
129.36
128.31
126.51
170.76
52.87
27.19
31.66
174.26
o-C
–
m-C
p-C
–
–
Gln -4
CO
171.25
54.42
26.46
31.61
174.48
171.28
52.67
Cα
Cβ
27.36
Cγ
31.93
CONH2
174.35 174.38
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Methyleneoxy Isosters of Oxytocin and Vasopressin
383
TABLE III
(Continued)
2a
Residue Param eter
1a
1b
2b
m ajor
m in or
Asn -5
CO
Cα
170.84
50.38
36.37
171.89
167.91
51.64
41.20
172.03
60.29
29.05
24.59
46.94
172.34
51.53
40.10
24.36
23.18
21.66
–
171.23
173.48
50.09
35.92
171.21
170.82
50.96
40.94
171.79
59.96
28.96
24.48
46.84
171.77
52.36
28.47
24.92
40.00
172.27
49.51
35.50
171.01
168.28
50.00
38.92
171.74
59.86
29.12
24.46
46.87
171.49
52.52
28.48
25.06
40.49
49.70
36.58
171.79
168.51
50.10
40.20
171.30
60.06
29.19
24.55
46.93
172.26
51.55
40.00
24.39
23.19
21.69
–
Cβ
CONH2
CO
Cα
Cys-6
Pro-7
Cβ
CO
Cα
Cβ
Cγ
Cδ
Leu-8
(Arg)
CO
Cα
Cβ
Cγ
Cδ
(N>C=N)
CONH2
Cα
157.34
171.67
42.05
156.86
171.10
41.97
Gly-9
171.10
42.17
171.01
42.13
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

384
Mařík, Buděšínský, Slaninová, Hlaváček:
work sh ows com patibility of m eth ylen eoxy pseudodipeptide buildin g
blocks with m eth ods com m on ly used in th e solid-ph ase peptide syn th esis.
EXPERIMENTAL
Protected am in o acids were purch ased from Bach em (Buben dorf, Switzerlan d) or were pre-
pared in our laboratory followin g gen eral protocols31. In th e Nα-Fm oc-am in o acid prepara-
tion s, th e prescribed pH of th e reaction m ixtures was m ain tain ed usin g pH m eter with an
autom atic titrator (Radiom eter, Copen h agen , Den m ark). O-Ben zyltyrosin ol, {(2S)-2-am in o-
3-[4-(ben zyloxy)ph en yl]propan -1-ol}, was purch ased from Advan ced Ch em Tech (Louisville,
U.S.A.) an d th e Rin k am ide resin from Calbioch em –Novabioch em AG (Läufelfin gen , Switzer-
lan d). Meltin g poin ts were m easured on a Kofler block an d were n ot corrected. Colum n
ch rom atograph y was carried out with Silica gel 60 (Merck, Darm stadt, Germ an y). Organ ic
solution s im m iscible with water were dried over an h ydrous Na2SO4. Solven ts were evapo-
rated in vacuo on a rotary evaporator (bath tem perature 30 °C); DMF was evaporated at
30 °C an d 150 Pa. An average degree of substitution on th e resin was determ in ed from a
resin weigh t in crease after loadin g th e Fm oc-Gly-OH, spectroscopically by th e m easurem en t
of th e diben zofulven e–piperidin e com plex absorption after th e Fm oc-group cleavage with
5% piperidin e in DCM–DMF 1 : 1 for 10 m in an d 20% piperidin e in DMF for 15 m in , an d
by th e quan titative AAA of glycin e loaded on th e resin . Optical rotation s were m easured on
a Perkin –Elm er 141 MCA polarim eter at 22 °C an d [α]D values are given in 10–1 deg cm 2 g–1
.
For m easurem en t of IR spectra (ν, cm –1), a Bruker IFS 88 in strum en t (Karlsruh e, Germ an y)
was used. Th e sam ples for am in o acid an alysis were h ydrolyzed with 6 M HCl con tain in g 3%
of ph en ol at 110 °C for 20 h . AAA were perform ed on
a Bioch rom 20 in strum en t
(Ph arm acia, Sweden ). Molecular weigh ts of th e peptides were determ in ed usin g m ass spec-
TABLE IV
Tem perature coefficien ts of backbon e am ide proton s ch em ical sh ifts (in ppb/°C) of oxytocin
(1a), [8-argin in e]vasopressin (1b) an d th eir isosteric an alogues 2a, 2b
2a
Residue
1a
Residue
1b
2b
m ajor
–3.3
m in or
–3.6
Tyr-2
Ile-3
–1.2
–5.7
–3.1
–1.5
–6.2
–4.4
–4.0
Tyr-2
Ph e-3
Gln -4
Asn -5
Cys-6
Arg-8
Gly-9
–5.3
–7.1
–1.5
+0.5
–8.2
–4.4
–4.4
–4.3
–
–
Gln -4
Asn -5
Cys-6
Leu-8
Gly-9
–4.0
–2.6
–2.7
–3.6
–3.7
–1.0
–2.8
–5.4
–3.6
–3.7
–1.6
–3.2
–7.4
–3.7
–3.7
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Methyleneoxy Isosters of Oxytocin and Vasopressin
385
troscopy with FAB tech n ique (Microm ass, Man ch ester, En glan d). For HPLC, a Spectra Ph ys-
ics in strum en t with an SP 8800 pum p, an SP 4290 in tegrator an d a Th erm o Separation
Products Spectra 100 UV detector were used. Th e com poun ds were purified by preparative
HPLC on a 25 × 1 cm colum n , 10 µm Vydac RP-18 (Th e Separation s Group, Hesperia, CA,
U.S.A.), flow rate 3 m l m in –1, detection at 220 n m usin g 0–100% gradien t of ACN in 0.05%
aqueous TFA, 60 m in . Th e an alytical HPLC was carried out on a 25 × 0.4 cm colum n : 5 µm
LiCh rosph er WP-300 RP-18 (Merck, Darm stadt, Germ an y), flow rate 1 m l m in –1, detection at
200 n m , usin g th e 0–90% gradien t of ACN in 0.05 aqueous TFA, 60 m in , un less oth erwise
stated. 1H NMR spectra were m easured on a Varian UNITY-200 (at 200 MHz) an d/or
UNITY-500 (at 500 MHz) spectrom eter. 13C NMR spectra were m easured on a Varian
UNITY-500 spectrom eter (at 125.7 MHz) at room tem perature un less oth erwise stated.
(5S)-5-[4-(Ben zyloxy)ben zyl]m orph olin -3-on e (4)
A m ixture of O-ben zyltyrosin ol (5.0 g, 20 m m ol) in THF (300 m l) an d NaH (1.0 g, 25 m m ol;
60% dispersion in m in eral oil) was stirred at room tem perature un der n itrogen for 30 m in ,
an d th en eth yl ch loroacetate (2.44 g, 20 m m ol) was added dropwise in 5 m in . After 30 m in
of stirrin g, th e reaction m ixture was refluxed for 3 h , cooled an d poured in to 1 M HCl
(300 m l). Th e product was extracted with EtOAc (3 × 100 m l), th e com bin ed extracts were
wash ed with water (3 × 100 m l) an d brin e (3 × 100 m l), dried an d th e solven t was evapo-
rated. Th e crude product was crystallized from a PE–EtOAc m ixture affordin g a yield 2.2 g
(51%) of wh ite solid, m .p. 120–123 °C; [α]D –5.21 (c 0.3, MeOH). 1H NMR (CDCl3, 200 MHz):
2.61 dd, 1 H, J = 13.7, 9.1 an d 2.85 ddd, 1 H, J = 13.7, 5.2, 1.4 (C(5)-CH2); 3.55 dd, 1 H, J =
11.6, 7.0 an d 3.94 ddd, J = 11.6, 3.7, 1.4 (C(6)-CH2); 3.72 m , 1 H, J = 9.1, 7.0, 5.2, 3.7
(C(5)H); 4.14 d, 1 H, J = 16.0 an d 4.22 d, 1 H, J = 16.0 (C(2)H2); 5.06 s, 2 H (CH2 (Bn ));
5.78 bs, 1 H (NH); 6.94 m , 2 H an d 7.10 m , 2 H (C6H4); 7.30–7.50 m , 5 H (C6H5). For
C
18H19NO3 (297.3) foun d FAB MS, m/z: 298.1 (M+ + 1).
(5S)-5-[4-(Ben zyloxy)ben zyl]-4-(tert-butoxycarbon yl)m orph olin -3-on e (5)
To a solution of 4 (2.0 g, 6.7 m m ol) in DCM (30 m l), di-tert-butyl dicarbon ate (4.5 g,
20 m m ol) an d DMAP (2.5 g, 20 m m ol) were added at 0 °C an d th e reaction m ixture was
stirred at room tem perature for 3 h , wash ed with 20% citric acid (3 × 50 m l) an d brin e (3 ×
50 m l), dried an d evaporated to dryn ess. Th e crude product was purified on a Silica gel 60
colum n usin g a PE–EtOAc (65 : 35) m ixture givin g a yield of 1.6 g (67%) of 5, m .p. 81–83 °C;
[α]D –2.2 (c 0.46, MeOH). 1H NMR (CDCl3, 200 MHz): 1.59 s, 9 H (Boc); 2.98 m , 2 H
(C(5)-CH2); 3.57 dd, 1 H, J = 12.5, 2.5 an d 3.80 ddd, 1 H, J = 12.5, 1.5, 1.0 (C(6)H2); 4.15 m ,
1 H (C(5)H); 4.16 d, 1 H, J = 17.1 an d 4.35 d, J = 17.1 (C(2)H2); 5.05 s, 2 H (CH2 (Bn ));
6.93 m , 2 H an d 7.18 m , 2 H (C6H4); 7.30–7.50 m , 5 H (C6H5). For C23H27NO5 (397.4) foun d
FAB MS, m/z: 398.2 (M+ + 1).
(2RS,5S)-5-[4-(Ben zyloxy)ben zyl)-4-(tert-butoxycarbon yl)-2-(RS)-2-h ydroxybutan -2-yl)-
m orph olin -3-on e (6)
To a solution of LHDMS (2.5 m l of 1 M solution in THF, 2.5 m m ol) in THF (5 m l), protected
m orph olin -3-on e 5 (1.0 g, 2.5 m m ol) in THF (5 m l) was added dropwise at –78 °C un der
n itrogen . Th e resultin g m ixture was stirred for 30 m in , butan on e (0.23 m l, 2.5 m m ol) was
added at th is tem perature an d stirrin g con tin ued for an oth er 2 h . Th e reaction m ixture was
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

386
Mařík, Buděšínský, Slaninová, Hlaváček:
poured in to 20% citric acid (50 m l) an d th e aqueous ph ase was extracted with EtOAc (3 ×
50 m l). Th e collected organ ic extracts were wash ed with brin e, dried an d EtOAc was evapo-
rated. Th e crude product was purified on a Silica gel 60 colum n usin g a PE–EtOAc (7 : 3)
m ixture to obtain 0.8 g (68%) of colorless oily 6 as a m ixture of diastereom ers (ca 3 : 2)
wh ich was used for n ext reaction with out purification . 1H NMR (CDCl3, 200 MHz), m ajor
isom er: 0.97 t, 3 H, J = 7.3 an d 1.70 m , 2 H (CH3CH2); 1.31 s, 3 H (CH3); 1.58 s, 9 H (Boc);
2.96 m , 2 H (C(5)-CH2); 3.58 m , 1 H an d 3.84 m , 1 H (C(6)H2); 4.13 m , 1 H (C(5)H); 4.47 s,
1 H (C(2)H); 5.05 s, 2 H (CH2 (Bn )); 6.94 m , 2 H an d 7.15 m , 2 H (C6H4); 7.30–7.50 m , 5 H
(C6H5); m in or isom er: 0.94 t, 3 H, J = 7.3 an d 1.70 m , 2 H (CH3CH2); 1.21 s, 3 H (CH3);
1.55 s, 9 H (Boc); 2.80 dd, 1 H, J = 13.0, 9.6 an d 3.05 dd, 1 H, J = 13.0, 4.0 (C(5)-CH2);
3.60 m , 1 H an d 3.87 m , 1 H (C(6)H2); 4.26 m , 1 H (C(5)H); 4.29 s, 1 H (C(2)H); 5.05 s, 2 H
(CH2 (Bn )); 6.94 m , 2 H an d 7.15 m , 2 H (C6H4); 7.30–7.50 m , 5 H (C6H5). For C26H35NO6
(469.6) foun d FAB MS, m/z: 470.2 (M+ + 1). IR (CHCl3): ν(OH) 3 467.
(2E)- an d (Z)-(5S)-5-[4-Ben zyloxy)ben zyl]-2-(butan -2-yliden )-4-(tert-butoxycarbon yl)-
m orph olin -3-on e (7a an d 7b)
To a solution of th e 6 (0.8 g, 1.7 m m ol) an d TEA (1.2 m l, 5.1 m m ol) in DCM (20 m l),
m eth an esulfon yl ch loride (0.6 m l, 5.1 m m ol) was added dropwise at 0 °C. Th e reaction m ix-
ture was th en allowed to warm up to room tem perature an d stirred for 2 h after wh ich it
was wash ed with 20% citric acid (2 × 50 m l) an d brin e (2 × 50 m l). Th e organ ic ph ase was
dried an d evaporated to give a wh ite solid. Un der th ese con dition s a spon tan eous elim in a-
tion of correspon din g m esylate proceeded. Th e purification on a Silica gel 60 colum n usin g
a PE–EtOAc (9 : 1) afforded a m ixture of E- an d Z-isom ers (ca 1 : 1) with a yield 0.28 g (36%)
of 7a an d 7b; [α]D –52.1 (c 0.36, MeOH). 1H NMR (CDCl3, 500 MHz): 1.04 t, 3 H, J = 7.4 an d
2.25 m , 2 H (Et (Z)); 1.10 t, 3 H, J =7.2, 2.38 dq, 1 H, J = 12.4, 7.2 an d 2.75 dq, 1 H, J = 12.4,
7.2 (Et (E)); 1.84 s, 3 H (Me (E)); 2.15 s, 3 H (Me (Z)); 1.56 s, 9 H (t-But); 2.90 m , 1 H, J =
13.0, 10.6, 2.2 an d 2.96 m , 1 H, J = 13.0, 4.6, 1.6, 1.0 (C(5)-CH2); 3.78 m , 1 H, J = 11.8, 8.0,
2.2, 1.0 an d 3.90 m , 1 H, J = 11.8, 4.2, 1.6 (C(6)H2); 4.25 m , 1 H (C(5)H); 5.05 s, 2 H (CH2
(Bn )); 6.93 m , 2 H an d 7.16 m , 2 H (C6H4); 7.43 m , 2 H, 7.39 m , 2 H an d 7.33 m , 1 H
(C6H5). 13C NMR (CDCl3, 125.7 MHz, APT): 11.76 an d 12.84 (CH3 (Et)); 17.82 an d 18.71
(CH3); 27.18 an d 27.08 (CH2 (Et)); 28.08 (3 × CH3 (Boc)); 36.10 (C(5)-CH2); 56.83 an d 56.81
(C(5)); 64.33 an d 64.28 (CH2 (Bn )); 70.05 (C(6)); 83.27 an d 83.22 (C(Boc)); 94.76 (C(2)=C<);
138.67 (C(2)); 115.07 (2 × CH); 130.46 (2 × CH); 129.65 an d 157.74 (C6H4); 136.69 (C);
128.58 (2 × CH); 127.42 (2 × CH) an d 127.96 (CH) (C6H5); 151.20 (C=O (Boc)); 159.35
(C(3)). For C27H33NO5 (451.6) foun d FAB MS, m/z: 452.2 (M+ + 1). IR (CHCl3): ν(C=C) 1 617.
In th e ch rom atograph y purification described above, an isom er (2RS,5S)-5-[4-(ben zyloxy)-
ben zyl]-2-[(Z)-(but-2-en -2-yl)-4-tert-(butoxycarbon yl)m orph olin -3-on e (8) was separated
1
(0.1 g). H NMR (CDCl3, 200 MHz): 1.56 d, 3 H, J = 6.7 (CH3); 1.58 s, 9 H (Boc); 1.69 bs, 3 H
(CH3); 2.91 dd, 1 H, J = 13.4, 10.4 an d 3.05 dd, 1 H, J = 13.4, 4.3 (C(5)-CH2); 3.56 dd, 1 H,
J = 12.2, 2.5 an d 3.79 dd, 1 H, J = 12.2, 2.7 (C(6)H2); 4.14 m , 1 H (C(5)H); 4.58 bs, 1 H
(C(2)H); 5.05 s, 2 H (CH2 (Bn )); 5.52 bq, 1 H, J = 6.7 (=CH); 6.92 m , 2 H an d 7.18 m , 2 H
(C6H4); 7.30–7.46 m , 5 H (C6H5).
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Methyleneoxy Isosters of Oxytocin and Vasopressin
387
(2S,5S)-2-(RS)-(Butan -2-yl)-4-(tert-butoxycarbon yl)-5-(4-h ydroxyben zyl)-
m orph olin -3-on e (9)
To a solution of th e 7a an d 7b (0.2 g, 0.44 m m ol) in MeOH (200 m l), Pd-black (10 m g) was
added un der argon an d h ydrogen ation was carried out at room tem perature for 4 h . Th e cat-
alyst was rem oved by filtration an d th e solven t was evaporated to give 0.16 g (99%) of col-
orless oil, wh ich was a m ixture of stereoisom ers due to th e presen ce of Ile an d aIle
side-ch ain s in th e 7 : 3 ratio. 1H NMR (CDCl3, 500 MHz; on ly sign als of th e m ajor isom er
wh ich could be fully assign ed are given ): 0.96 t, 3 H, J = 7.2 an d 1.45 m , 2 H (CH3CH2);
1.09 d, 3 H, J = 6.8 an d 2.26 m , 1 H (CH3CH); 1.58 s, 9 H (Boc); 2.98 m , 2 H (C(5)-CH2);
3.54 dd, 1 H, J = 12.4, 2.5 an d 3.80 dd, 1 H, J = 12.4, 1.0 (C(6)H2); 4.03 d, 1 H, J = 2.4
(C(2)H); 4.10 m , 1 H (C(5)H); 4.96 bs, 1 H (OH); 6.80 m , 2 H an d 7.14 m , 2 H (C6H4).
13C NMR (CDCl3, 125.7 MHz; on ly sign als of th e m ajor isom er wh ich could be fully as-
sign ed are given ): 12.29 an d 26.13 (CH3CH2); 15.88 an d 37.75 (CH3CH<); 28.05 (3 × CH3
(Boc)); 37.35 (C(5)-CH2); 57.36 (C(5)); 63.98 (C(6)); 83.68 (>C< (Boc)); 83.99 (C(2)); 115.52
(2 × CH); 130.72 (2 × CH); 129.57 (C) an d 154.51 (C) (C6H4); 151.49 (C=O (Boc)); 170.15
(C(3)). For C20H29NO5 (363.2) foun d FAB MS, m/z: 364 (M+ + 1).
Fm oc-Tyrψ[CH2O]Ile-OH (3a)
A m ixture of substituted m orph olin -3-on e 9 (0.16 g, 0.44 m m ol) an d 6 M HCl (10 m l)
was refluxed for 6 h an d th en h eated in a sealed tube at 100 °C for 4 h to com plete th e
h ydrolysis. After coolin g, th e aqueous ph ase was wash ed with Et2O (2 × 5 m l) an d evapo-
rated to dryn ess to give 72 m g (59%) of th e epim er m ixture of h ydroch lorides of
H-Tyrψ[CH2O]Ile-OH an d H-Tyrψ[CH2O]aIle-OH. 1H NMR (D2O, 200 MHz): 0.89 t, J = 7.6
an d 0.92 t, J = 7.6 (γ-CH3 (Ile + aIle)); 0.91 d, J = 7.6 an d 0.97 d, J = 7.0 (β-CH3 (Ile + aIle));
1.30 m , 1 H an d 1.48 m , 1 H (γ-H2 (Ile + aIle)); 1.86 m , 1 H (β-H, (Ile + aIle)); 2.97 m , 2 H
(β-H2 (Tyr)); 3.53 dd, 1 H, J = 11.6, 7.0 an d 3.70 m , 1 H (ψ-CH2-O); 3.70 m , 1 H (α-H (Tyr));
3.82 d, J = 5.2 an d 3.94 d, J = 3.7 (α-H (Ile + aIle)); 6.89 m , 2 H an d 7.20 m , 2 H (C6H4
(Tyr)). FAB MS, m/z: 282 (M+ + 1).
To a suspen sion of th e pseudodipeptide h ydroch lorides (72 m g, 0.26 m m ol) in water
(4 m l), a Fm oc-OSu (95 m g, 0.28 m m ol) in dioxan e (4 m l) was added at 0 °C, th e solution
was adjusted to pH 8.5 with 10% Na2CO3 an d m ain tain ed at room tem perature for 3 h .
Dioxan e was evaporated, th e aqueous ph ase was wash ed with Et2O (3 × 15 m l), acidified
with 1 M HCl to pH 3 an d th e separated product was extracted with EtOAc (3 × 15 m l). Th e
collected organ ic extracts were wash ed with brin e (3 × 15 m l), dried an d EtOAc was evapo-
rated to yield 0.11g (84%) of th e colorless oily N-protected epim er m ixture of pseudo-
dipeptides 3a; [α]D +14.3 (c 0.1, MeOH). For C30H33NO6 (503.6) foun d FAB MS, m/z: 504.2
(M+ + 1).
(2RS,5S)-5-[4-(Ben zyloxy)ben zyl]-4-(tert-butoxycarbon yl)-2-(RS)-[h ydroxy(ph en yl)m eth yl]-
m orph olin -3-on e (10)
Th e com poun d was prepared from 5 (1 g, 2.5 m m ol) in THF (5 m l) an d LHMDS (2.5 m l of
1 M solution in THF, 2.5 m m ol) in THF (5 m l) accordin g to th e procedure described for 6 by
quen ch in g th e en olate with ben zaldeh yde (0.27 m l, 2.5 m m ol). Th e crude product was puri-
fied on a Silica 60 colum n usin g a PE–EtOAc (75 : 25) to yield 0.98 g (78%) of pure 10.
1H NMR (CDCl3, 200 MHz): 1.55 s, 9 H (Boc); 2.08 dd, 1 H, J = 13.1, 4.0 an d 2.54 ddd, 1 H,
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J = 13.1, 4.0, 1.5 (C(5)-CH2); 3.53 ddd, 1 H, J = 12.5, 2.5, 1.5 an d 3.72 dd, 1 H, J = 12.5, 1.0
(C(6)H2); 3.62 d, 1 H, J = 4.3 (OH); 3.99 m , 1 H, J = 11.0, 4.0, 2.5, 1.0 (C(5)H); 4.42 d, 1 H,
J = 4.6 (C(2)H); 5.02 s, 2 H (CH2 (Bn )); 5.27 dd, 1 H, J = 4.6, 4.3 (C(2)-CH); 6.87 m , 2 H an d
6.98 m , 2 H (C6H4); 7.25–7.52 m , 10 H (2 × C6H5). For C30H33NO6 (503.6) foun d FAB MS,
m/z: 504.2 (M+ + 1). IR (CHCl3): ν(OH) 3 606, 3 458.
(Z)-(5S)-2-Ben zyliden e-5-[4-(ben zyloxy)ben zyl]-4-(tert-butoxycarbon yl)-
m orph olin -3-on e (11)
A reaction of 10 (0.98 g, 1.95 m m ol) with m eth an esulfon yl ch loride (0.3 m l, 3.9 m m ol) in
th e presen ce of TEA (0.54 m l, 3.9 m m ol), accordin g to th e procedure described in th e 7a, 7b
preparation , afforded th e correspon din g m eth an esulfon yl derivative wh ich , on h eatin g with
TEA (0.47 m l, 3.4 m m ol) to 100 °C for 30 m in , un derwen t elim in ation to give 0.78 g (83%)
of 11 as a wh ite solid, m .p. 103–106 °C; [α]D –52.1 (c 0.4, MeOH). 1H NMR (CDCl3,
200 MHz): 1.58 s, 9 H (Boc); 2.94 dd, 1 H, J = 12.5, 10.2 an d 3.06 dd, 1 H, J = 12.5, 5.5
(C(5)-CH2); 4.05 dd, 1 H, J = 11.6, 1.8 an d 4.16 dd, 1 H, J = 11.6, 1.5 (C(6)H2); 4.41 m , 1 H
(C(5)H); 5.05 s, 2 H (CH2 (Bn )); 6.95 s, 1 H (C(2)=CH); 6.95 m , 2 H an d 7.20 m , 2 H (C6H4);
7.30–7.50 m , 8 H an d 7.77 m , 2 H (2 × C6H5). For C30H31NO5 (485.6) foun d FAB MS, m/z:
486.2 (M+ + 1). IR (CHCl3): ν(C=C) 1 617.
(2S,5S)-2-Ben zyl-4-(tert-butoxycarbon yl)-5-(4-h ydroxyben zyl)-
m orph olin -3-on e (12)
Th e m orph olin -3-on e 11 (0.78 g, 1.62 m m ol) was h ydrogen ated in MeOH (200 m l) usin g
con dition s described in th e 9 preparation to give 0.57 g (89%) of colorless oily 12. [α]D
–55.3 (c 0.2, MeOH). 1H NMR (CDCl3, 500 MHz): 1.56 s, 9 H (Boc); 2.20 dd, 1 H, J = 13.4,
10.7 an d 2.56 ddd, 1 H, J = 13.4, 4.2, 1.5 (C(5)-CH2); 3.24 dd, 1 H, J = 14.0, 5.6 an d 3.27 dd,
1 H, J = 14.0, 4.5 (C(2)-CH2); 3.54 ddd, 1 H, J = 12.5, 2.5, 1.5 an d 3.69 dd, 1 H, J = 12.5, 1.2
(C(6)-CH2); 4.00 m , 1 H, J = 10.7, 4.2, 2.5, 1.2 (C(5)H); 4.40 dd, 1 H, J = 4.5, 5.6 (C(2)H);
4.78 bs, 1 H (OH); 6.74 m , 2 H an d 6.96 m , 2 H (C6H4); 7.23 m , 1 H an d 7.32 m , 4 H (C6H5).
For C23H27NO5 (397.5) foun d FAB MS, m/z: 398.2 (M+ + 1). IR (CHCl3): ν(OH) 3 597.
Fm oc-Tyrψ[CH2O]Ph e-OH (3b)
Com poun d 12 (0.57 g, 1.44 m m ol) was h ydrolyzed with 6 M HCl (30 m l) un der con dition s
used in preparation of th e 3a to give 0.25 g (52%) of pseudodipeptide h ydroch loride, m .p.
162–163 °C; [α]D –7.03 (c 0.2, MeOH). 1H NMR (D2O, 200 MHz): 2.78 d, 2 H, J = 7.6 (β-H2
(Tyr)); 3.00 dd, 1 H, J = 14.0, 8.7 an d 3.20 dd, 1 H, J = 14.0, 4.3 (β-H2 (Ph e)); 3.34 dd, 1 H,
J = 10.7, 4.6 an d 3.66 dd, 1 H, J = 10.7, 3.0 (ψ-CH2-O); 3.54 m , 1 H (α-H (Tyr)); 4.20 dd, 1 H,
J = 8.7, 4.3 (α-H (Ph e)); 6.82 m , 4 H (C6H4); 7.30–7.50 m , 5 H (C6H5). FAB MS, m/z: 316
(M+ + 1). Th is com poun d (0.32 g, 1 m m ol) in water (15 m l) was reacted with Fm oc-OSu
(0.34 g, 1.0 m m ol) in dioxan e (15 m l) usin g th e sam e procedure as described in preparation
of 3a to yield 0.44 g (86%) of th e colorless oily N-protected pseudodipeptide 3b; [α]D –41.3
(c 0.2, MeOH). For C33H31NO6 (537.6) foun d FAB MS, m/z: 538.2 (M+ + 1).
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Methyleneoxy Isosters of Oxytocin and Vasopressin
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[Tyr2ψ[CH2O]Ile3]oxytocin (2a) an d [Tyr2ψ[CH2O]Ph e3]vasopressin (2b)
Th e solid ph ase syn th esis of oxytocin an d vasopressin an alogues 2a an d 2b was carried out
on a Rin k am ide resin 19 (1 g) usin g, with regard to an average substitution of th e Gly resi-
due (0.52 m m ol g–1) on th e resin , a th ree-fold excess (1.56 m m ol) of Nα-Fm oc protected
am in o acids: Gly, Leu, Arg(Mtr), Pro, Cys(Trt), Asn (Trt) an d Gln (Trt) assem bled stepwise to
th e resin . Th e couplin g of th e am in o acids was m ediated with 3 equivalen ts of DIC, HOBt
an d DIEA in DMA (20 m l) un til th e Kaiser test32 was n egative. In each step, th e Fm oc
deprotection was perform ed with 20% piperidin e in DMF (2 × 20 m in ) an d wash in g was car-
ried out with DMF (3 × 20 m l) an d DMA (3 × 20 m l). In th e last step, th e protected
pseudodipeptides 3a or 3b in two-fold excess were coupled to th e growin g peptide ch ain s
usin g a 2.5-fold m olar excess of a TOTU, HOBt, an d DIEA m ixture. Fin ally, th e peptide resin
was wash ed th ree tim es with 20 m l of DMA, isopropyl alcoh ol, DMF, Et2O an d was dried in
a desiccator. Th e peptides were detach ed from th e resin by treatm en t with a TFA (10%)–TIS
(1%) m ixture in DCM (30 m l) for 10 m in an d with 5% TFA in DCM at room tem perature for
20 m in . After evaporation to dryn ess, th e side-ch ain deprotection of th e oxytocin an alogue
2a was perform ed by treatm en t of th e residue with
a TFA (94%)–water (2.5%)–EDT
(2.5%)–TIS (1%) m ixture for 1.5 h , an d th at of th e vasopressin an alogue 2b by treatm en t
with TFA (81.5%)–th ioan isole (5%)–ph en ol (5%)–water (5%)–EDT (2.5%)–TIS (1%) m ixture
at room tem perature for 6 h . Th e reaction m ixtures were con cen trated to 10% of th eir origi-
n al volum es, th e crude peptides were precipitated by addition of Et2O an d separated by fil-
tration . A closure of disulfide bon ds in both th e peptide an alogues was carried out by
oxidation of th eir solution s in 1 M AcOH with 6 · 10–3 M K3Fe(CN)6, at pH 8 adjusted with
NH4OH, to a perm an en t yellow color. Th e oxidation was followed by desaltin g on an
Am berlite CG-50 colum n an d lyoph ilization , yieldin g th e crude oxytocin (40 m g) an d
vasopressin (43 m g) an alogues, wh ich were purified by preparative HPLC. Oxytocin an a-
logue 2a (25 m g, 0.025 m m ol): HPLC reten tion tim e 23.2 m in ; m . p. 170–175 °C; AAA: Asp
0.9, Glu 1.0, Gly 1.0, Leu 1.0, Pro + Cys 1.4, Cys 0.5. 1H an d 13C NMR data are given in
Tables II an d III. For C43H67N11O12S2 (994.2) foun d FAB MS, m/z: 994 (M+ + 1). Vasopressin
an alogue 2b (24 m g, 0.022 m m ol): HPLC reten tion tim e 24.7 m in ; m .p. 153–157 °C; AAA:
Asp 1.0, Glu 1.0, Gly 1.0, Arg 1.0, Pro + Cys 1.5, Cys 0.5. 1H an d 13C NMR data are given in
Tables II an d III. For C46H66N14O12S2 (1 071.2) foun d FAB MS, m/z: 1 071 (M+ + 1).
Biological Activity Assessm en t
Peptides were tested for uteroton ic activity in vitro in th e previously described rat uteroton ic
test for oxytocin accordin g to Holton 33 an d Mun sick34, eith er in th e absen ce of Mg2+ or in
th e presen ce of 1 m M Mg2+ in th e bath in g solution on uteri from 3–5 differen t rats (Wistar,
Kon árovice, Czech Republic). Fem ale rats were estrogen ized 24–48 h before th e experim en t.
Cum ulative dose respon se curves were con structed usin g data from experim en ts in wh ich
doses were added successively to th e organ bath in doublin g con cen tration s an d in 1 m in
in tervals with out th e fluid bein g ch an ged un til th e m axim al respon se was obtain ed. Th e ac-
tivity was determ in ed by com parin g th e th resh old doses of th e stan dard an d th e an alogue
(IU m g–1 or EC50). In th e case of th e an tagon istic activity, th e dose of th e an alogue was ap-
plied to th e organ bath 1 m in prior to th e stan dard dose of oxytocin . Th e an tagon istic activ-
ity was expressed as EC50 or pA2, i.e. th e con cen tration of th e an alogue, wh ich reduced th e
effect of th e double dose of agon ist, in our case oxytocin , to th e effect of dose x, an d th e
n egative decadic logarith m of th e EC50, respectively. Th e peptides were tested for pressor po-
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Mařík, Buděšínský, Slaninová, Hlaváček:
ten cy in th e pressor test accordin g to Dekan ski35. Th e test was perform ed usin g 4-am in o-
ph en yl ph en yl eth er-treated m ale rats un der ureth an e an aesth esia (doses applied i.v.). Tests
to estim ate an tidiuretic or diuretic properties were con ducted on con scious h ydrated m ale
rats in th e m odified Burn test36,37 in a stan dard way. Th e an im als fasted for 16 h were
weigh ed an d th en given a water load (4% of body weigh t). Im m ediately after th e water load,
th e tested substan ces were applied s.c. Th e rats were th en placed in to in dividual m etabolic
cages, an d th eir urin e was collected over a 5 h period. Th e tim e was determ in ed in wh ich
th e rats excreted h alf of th e water load. Syn th etic oxytocin an d argin in e vasopressin were
used as stan dards in th e relevan t tests.
NMR Spectra Measurem en t
NMR spectra of oxytocin 1a, vasopressin 1b an d th eir an alogues 2a an d 2b were m easured
on a Varian UNITY-500 spectrom eter (1H at 500 MHz, 13C at 125.7 MHz) in DMSO. Stan -
dard algorith m based on com bin ation of 2D-COSY, 2D-TOCSY an d 2D-ROESY spectra was
used for com plete structure assign m en t of proton s. Tem perature coefficien ts of am ide NH
proton s were determ in ed from 1D-proton spectra m easured in th e in terval 20–40 °C. Carbon
sign als were assign ed usin g th e correlation via J(C,H) couplin gs observed in 2D-HMQC an d
2D-HMBC spectra. NMR param eters are sum m arized in Tables II–IV.
Abbreviation s Used
AAA, am in o acid an alysis; ACN, aceton itrile; APT, attach ed proton test; Boc, tert-butoxy-
carbon yl; Bn , ben zyl; COSY, correlation spectroscopy; DCM, dich lorom eth an e; DIC, N,N-di-
isopropylcarbodiim ide; DIEA, N,N-diisopropyleth ylam in e; DMA, N,N-dim eth ylacetam ide;
DMAP, 4-(dim eth ylam in o)pyridin e; DMF, N,N-dim eth ylform am ide; DMSO, dim eth yl
sulfoxide; EDT, eth an e-1,2-dith iol; Et2O, dieth yl eth er; EtOAc, eth yl acetate; FAB MS, fast
atom bom bardm en t m ass spectrom etry; Fm oc, [(fluoren -1yl)m eth oxy]carbon yl; HMBC,
h eteron uclear m ultiple bon d correlation ; HMQC, h eteron uclear m ultiple quan tum coh er-
en ce; HOBt, 1-h ydroxyben zotriazole; LHMDS, lith ium bis(trim eth ylsilyl)am ide; MsCl,
m eth an esulfon yl ch loride; Mtr, 2,3,6-trim eth yl-4-m eth oxyben zen esulfon yl; OSu, succin im id
ester; PE, ligh t petroleum ; ROESY, rotatin g fram e NOESY spectroscopy; TIS, triisopropyl-
silan e; TOCSY, total correlation spectroscopy; TOTU, O-cyan oeth oxycarbon ylm eth yliden e
am in o-1,1,3,3,-tetram eth yl uron ium tetrafluoroborate; TFA, trifluoroacetic acid; TEA,
trieth ylam in e; THF, tetrah ydrofuran ; Trt, triph en ylm eth yl. Th e n om en clature an d sym bols
of am in o acids follow publish ed recom m en dation s of th e IUPAC/IUB Join t Com m ission on
Bioch em ical Nom en clature (Eur. J. Biochem. 1984, 138, 9–37).
This research was carried out under the research project Z4 055 905 and was supported by grants
of the Grant Agency of the Czech Republic (grants No. 203/98/1330 and No. 203/00/0955) and by
the Ministry of Education, Youth and Sports of the Czech Republic (LB98233).
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