Peptide synthesis with α-(difluoromethyl)-substituted α-amino acids

Article abstract of DOI:10.1135/cccc20021533

Methodology for the incorporation of α-(difluoromethyl)-substituted α-amino acids into N- and C-terminal positions of dipeptides as well as into position 2 of tri- and tetrapeptides is described.

Full text of DOI:10.1135/cccc20021533

Peptide Synthesis
1533
PEPTIDE SYNTHESIS WITH α-(DIFLUOROMETHYL)-SUBSTITUTED
α-AMINO ACIDS
a
b1
c
c
Thomas MICHEL , Beate KOKSCH , Sergei N. OSIPOV , Alexander S. GOLUBEV ,
d
b2,
Joachim SIELER and Klaus BURGER
*
a
Jenapharm, Otto-Schott-Strasse 15, D-07745 Jena, Germany
Department of Organic Chemistry, University of Leipzig, Johannisallee 29, D-04103 Leipzig,
b
1
2
Germany; e-mail: koksch@organik.chemie.uni-leipzig.de, burger@organik.chemie.uni-leipzig.de
Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov str. 28, GSP-1,
V-334, 117813 Moscow, Russia
Department of Inorganic Chemistry, University of Leipzig, Linnéstrasse 3, D-04103 Leipzig,
Germany
c
d
Received Jun e 20, 2002
Accepted October 11, 2002
In memoriam of Professor Miloš Hudlický.
Meth odology for th e in corporation of α-(difluorom eth yl)-substituted α-am in o acids in to N-
an d C-term in al position s of dipeptides as well as in to position 2 of tri- an d tetrapeptides is
described.
Keyw ord s: Peptides; α,α-Disubstituted am in o acids; α-Fluoroalkyl α-am in o acids; Peptide
couplin g reagen ts; Fragm en t con den sation ; Fluorin ated com poun ds.
In corporation of α,α-disubstituted am in o acids in to key position s of pep-
tides is an efficien t strategy to retard proteolytic degradation an d to stabi-
lize secon dary structure elem en ts1. α-(Fluoroalkyl)-substituted α-am in o
acids are a special class of α,α-disubstituted am in o acids2. A fluoroalkyl
group in α-position of an am in o acid exerts con siderable polarization ef-
fects on n eigh borin g substituen ts. However, th is effect stron gly depen ds on
th e n um ber of fluorin e atom s presen t in th e α-(fluoroalkyl) substituen t as
can be seen on com parison of pKa (NH2) values for alan in e 9.8, 3-fluoro-
alan in e 9.8, 3,3-difluoroalan in e 8.4 an d 3,3,3-trifluoroalan in e 5.3 3. Th e re-
m arkable gap of ∆ = 3.1 between 3,3,3-trifluoro- an d 3,3-difluoroalan in e
sh ould result in m ajor differen ces with respect to reactivity an d m olecular
properties.
Recen tly it was disclosed th at h ydroph obic in teraction s are far m ore es-
sen tial in ligan d/receptor in teraction s th an assum ed, wh ile th e in fluen ce of
Collect. Czech. Chem. Commun. (Vol. 67) (2002)
doi:10.1135/cccc20021533

1534
Michel et al.:
h ydrogen bon din g was overestim ated4. Th e m ech an ism of h ydroph obic in -
teraction s, h owever, was n ever elucidated in detail5. Side ch ain ph en yl
groups play an essen tial role in ligan d/receptor in teraction s. Wh en th e aro-
m atic system in teracts with th e side ch ain of Val, Leu, Ile an d Ala, th e aro-
m atic π-system fun ction s as a h ydrogen bon d acceptor. Th ese in teraction s
are den oted as CH/π in teraction s, a con cept wh ich h as been recen tly estab-
lish ed by Nish io et al.6 In th e series CF3, CHF2, CH2F, CH3, on ly th e CHF2
an d CH2F groups can react as both h ydrogen bon d don or an d h ydrogen
bon d acceptor7, with th e CHF2 group bein g th e m ost poten t h ydrogen
bon d don or in th is series8. Furth erm ore, fluoroalkyl groups m ay act as
coordin ative sites in m etal com plexes.
Recen tly, we dem on strated th at th e in corporation of α-(trifluorom eth yl)
am in o acids (α-Tfm am in o acids) in peptides n ot on ly im proves proteolytic
stability an d in duces secon dary structure m otifs, but also im proves
lipoph ilicity9 en h an cin g in vivo absorption an d im provin g perm eability
th rough certain body barriers. 19F NMR spectroscopy is an efficien t tool for
con form ation al studies of fluorin e-con tain in g peptides an d for th e elucida-
tion of m etabolic processes10. Th e spectra can be recorded even in water
an d un der cell-like con dition s.
Th e m ost con ven ien t syn th esis of α-Tfm am in o acids is based on an
am idoalkylation of carbon n ucleoph iles with h igh ly electroph ilic acyl-
im in es of 3,3,3-trifluoropyruvate11. Th is route en ables th e syn th esis of
α-Tfm am in o acids with orth ogon al protective groups12. A sligh tly m odified
approach provides access to h om och iral α-Tfm am in o acids via alkylation
of in situ form ed h om och iral cyclic α-(trifluorom eth yl)-substituted acyl-
im in es with C-n ucleoph iles13 an d via alkylation of trifluorom eth yl-
substituted acylim in es with h om och iral carban ion s14
. An alogously,
α-(difluorom eth yl) α-am in o acids (α-Dfm am in o acids), th e virtually un -
kn own α-(ch lorodifluorom eth yl) an d α-(brom odifluorom eth yl) α-am in o ac-
ids were obtain ed via addition of C-n ucleoph iles to acylim in es of
correspon din g partially fluorin ated pyruvates15.
Th e low n ucleoph ilicity of th e am in o group an d th e bulkin ess of th e
trifluorom eth yl group of α-Tfm am in o acids requires m odification of stan -
dard protocols for peptide syn th esis2. On ly H-(α-Tfm )Gly-OMe an d
H-(α-Tfm )Ala-OMe 16 can be coupled by classical m eth odology obtain in g
reason able yields. However, for α-Tfm am in o acids with bulkier side ch ain s,
all classic activation m eth ods exam in ed so far h ave turn ed out to be un suc-
cessful or resulted in substan tial epim erization of th e adjacen t n on -
fluorin ated am in o acid17
.
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1535
To circum ven t th e above syn th etic problem s, we decided to in troduce
α-Dfm am in o acids for peptide m odification to im prove n ucleoph ilicity,
wh ile retain in g th e positive profile of th e fluoroalkyl group. On th e basis of
th e pKa (NH2) values, we expected th e n ucleoph ilicity to be of th e order of
α-m eth yl-substituted α-am in o acids, exh ibitin g steric strain sim ilar to
α-am in oisobutyric acid (Aib). Th erefore, we tested th e efficien cy of a series
of curren tly used couplin g m eth ods for in corporation of α-Dfm am in o ac-
ids in to th e N- an d C-term in al position of peptides.
RESULTS AND DISCUSSION
N-Terminal Incorporation
In corporation of α-Dfm am in o acids in to th e N-term in al position of pep-
tides can be successfully ach ieved eith er by activation of Z-protected α-Dfm
am in o acids with diisopropylcarbodiim ide (DIC) via oxazolon es (1→2→3)
or on activation with DIC/1-h ydroxy-7-azaben zotriazole (HOAt) via th e
step of an activated ester (Sch em e 1). On activation of Boc-protected α-Dfm
am in o acids with DIC, N-carboxyan h ydrides (NCAs) are form ed via retro
en e reaction from oxazolon es11c,18
.
O
R1
H-Yaa-OR2
12 h
XF2C
O
DIC,
15 min
N
OBzl
R1 CF2X
Z
2
Z-(α-CF2X)Xaa-Yaa-OR2
N
H
COOH
3
DIC,
HOAt
10 min
H-Yaa-OR2
30 min
1
R1 CF2X
COAt
Z
N
H
SCHEME 1
In corporation of α-Dfm am in o acids in to N-term in al position of peptides (X = H, Cl, Br)
Attem pts to con vert Z-protected α-Dfm am in o acids on treatm en t with
DIC to sym m etric an h ydrides19 failed, on ly th e correspon din g oxazolon es
were obtain ed. Th is type of in tram olecular cyclization is favored in th e
presen ce of an addition al α-substituen t (gem in al dim eth yl effect)20
.
Epim erization via oxazolon es is a m ajor problem in peptide syn th esis21.
Th is problem is irrelevan t with α,α-disubstituted α-am in o acids because of
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1536
Michel et al.:
th e absen ce of an α-proton . Wh ile dipeptide form ation via rin g open in g of
oxazolon es with am in o acid esters (H-Yaa-OR) 2→3 is a slow process an d
n eeds reaction tim es of 12–14 h at room tem perature, carboxylic group acti-
vation with DIC/HOAt results in quan titative form ation of th e HOAt ester
(19F NMR an alysis) with in about 10 m in , wh ich is th en reacted in situ with
am in o acid esters at room tem perature to give th e dipeptide ester in excel-
len t yields with in an oth er 30 m in . Un der th ese reaction con dition s,
oxazolon e form ation could n ot be observed. In Table I som e represen tative
exam ples are sum m arized.
Com pared with th e oxazolon e route, carboxylic group activation of
Z-protected α-Dfm am in o acids in th e presen ce of additives is a m ore effi-
cien t couplin g m eth od, ch aracterized by sh orter reaction tim es an d sligh tly
h igh er yields. Th is protocol is n ot lim ited to peptide syn th esis in solution .
C-Terminal Incorporation
As expected, in corporation of α-Dfm am in o acids in to th e C-term in al posi-
tion of peptides is m ore difficult th an in to th e N-term in al position . Th ere-
fore, we tested a series of curren tly used peptide couplin g tech n iques, like
th e acid fluoride an d acid ch loride m eth od, wh ich seem to be exception ally
well suited for in corporation of sterically h in dered am in o acids in to pep-
tides22. Furth erm ore, we tested reagen ts like brom otris(pyrrolidin -1-
TABLE I
Yields of dipeptides with α-Dfm am in o acids in N-term in al position
No.
Dipeptide
Meth od
Yield, %
3a
3b
3b
3c
4a
4b
4c
4d
5
Z-(α-CF2H)Ala-Gly-OtBu
Z-(α-CF2H)Ala-Ph e-OtBu
Z-(α-CF2H)Ala-Gly-OtBu
Z-(α-CF2H)Ala-Pro-OtBu
Z-(α-CF2Cl)Ala-Gly-OtBu
Z-(α-CF2Cl)Ala-Ph e-OtBu
Z-(α-CF2Cl)Ala-Pro-OtBu
Z-(α-CF2Cl)Ph e-Gly-OtBu
Z-(α-CF2Br)Ala-Ph e-OtBu
DIC
73
75
90
90
78
83
93
85
86
DIC
DIC/HOAt
DIC/HOAt
DIC
DIC
DIC/HOAt
DIC
DIC
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1537
yl)ph osph on ium h exafluoroph osph ate (PyBroP), [(7-azaben zotriazol-1-
yl)oxy]tris(pyrrolidin -1-yl)ph osph on ium h exafluoroph osph ate (PyAOP),
[(d im eth ylam in o)(1H-1,2,3-triazolo[4,5-b]p yrid in -1-yl)m eth ylid en e]d i-
m eth ylam m on ium h exafluoroph osph ate N-oxide (HATU) an d N,N,N′,N′-
tetram eth ylfluoroform am idin ium h exafluoroph osph ate (TFFH). Th e cou-
plin g reagen ts were tested syn th esizin g th e m odel peptide Fm oc-Ph e-
(α-Dfm )Ala-OMe (6a) (Sch em e 2). A stan dard protocol was used: H-(α-Dfm )-
Ala-OMe (0.38 g, 2.5 mmol), (ethyl)diisopropylamine (DIEA) (0.33 g, 2.5 mmol)
an d activated Fm oc-Ph e-OH (5.0 m m ol) were stirred in DMF at room tem -
perature for 12 h . Th e progress of th e reaction s was con trolled by 19F NMR
spectroscopy an d RP-HPLC. Th e results obtain ed are sum m arized in Table II.
Fmoc-Phe-OH
+
H(α-CF2H)Ala-OMe
Fmoc-Phe-(α-CF2H)Ala-OMe
6a
SCHEME 2
Syn th esis of m odel peptide 6a usin g differen t activation strategies
Con ven tion al carbodiim ide m eth odology with additives such as HOBt or
active esters like pen tafluoroph en yl esters gave un satisfactory results in th e
stan dard couplin g reaction . Moderate to respectable yields were obtain ed
TABLE II
Yields (%) of Fm oc-Ph e-(α-Dfm )Ala-OMe 6a usin g differen t couplin g m eth ods. Yields were
determ in ed by 19F NMR spectroscopy
Yield (%) at reaction tim e
(h )
Yield (%) at reaction tim e
(h )
Meth od
Meth od
TFFH
0.5
3
12
0.5
3
12
Fm oc-Ph e-F
Fm oc-Ph e-Cl
Fm oc-Ph e-OPfp
HBTU
55
65
85
>95
<5
>95
<5
5
15
55
60
75
70
t
MA; BuOCOCl
<5
15
40
55
55
80
TBTU
5
45
<5
25
HATUa
PyBroP
DIC/HOAt
PyAOP
75
90
45
45
>95
>95
60
BOP
<5
30
DIC/HOBt
PyBOP
<5
<10
35
>95
>95
<10
70
a
Secon d activation after 30 m in .
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1538
Michel et al.:
with the first-generation uronium salt and phosphonium salt coupling reagents,
e.g. [(ben zotriazol-1-yl)(dim eth ylam in o)m eth yliden e]dim eth ylam m on ium
h exafluoroph osph ate N-oxide (HBTU), [(ben zotriazol-1-yl)oxy]tris(dim eth yl-
am in o)ph osph on ium h exafluoroph osph ate (BOP), [(ben zotriazol-1-yl)oxy]
tris(pyrrolidin -1-yl)ph osph on ium h exafluoroph osph ate (PyBOP), O-(ben zo-
triazol-1-yl)-1,1,3,3-tetram ethyluronium tetrafluoroborate (TBTU). The m ixed
an h ydride activation with isobutyl ch loroform ate gave acceptable yields as
well as th e activation with brom otris(pyrrolidin -1-yl)ph osph on ium h exa-
fluoroph osph ate, were described to give good to excellen t yields even in
couplin g reaction s of two am in o acids with h igh steric requirem en ts22
.
Yields h igh er th an 95% were obtain ed for th e above m en tion ed m odel reac-
tion with in 3 h . On activation with DIC/HOAt th e dipeptide 6a was form ed
in n early quan titative yield. Usin g HATU th e reaction was com plete (19F
NMR an alysis) already after 1 h at room tem perature, wh en a secon d activa-
tion was perform ed after 30 m in .
Acid ch lorides h ave been already successfully applied to couplin g reac-
tion s of α,α-disubstituted am in o acids. However, th eir application is lim ited
to am in o acid derivatives con tain in g n o acid labile protectin g groups23. An -
oth er problem usin g th e acid ch loride m eth od arises from th e ten den cy of
am in o acids to be con verted to oxazolon es in th e presen ce of tertiary
am in es24. A racem ization test of th e m odel peptide obtain ed from
Fm oc-Ph e-Cl usin g th e m eth od of Bayer et al.25 was perform ed. After h ydro-
lysis of th e peptide with DCl/D2O, th e N-(trifluoroacetyl)-protected
isopropyl ph en ylalan in ate was syn th esized by stan dard protocols an d an a-
lyzed by ch iral GC-MS (colum n s precoated with Ch irasil-Val 26). Th e loss of
stereoch em ical in tegrity was sh own to be less th an 0.15% 27. A serious
drawback for th e acid ch loride strategy is th eir in com patibility with Boc-
an d -OtBu-protection . Th erefore, it can n ot be applied to th e h igh ly effi-
cien t Fm oc/-OtBu strategy.
In con trast, th e correspon din g acid fluorides do n ot suffer from th ese
lim itation s (Sch em e 3). Th e acid fluorides are gen erally obtain ed from th e
correspon din g acids on reaction with cyan uric fluoride an d pyridin e28 or
with (dieth ylam in o)sulfur trifluoride (DAST) in th e absen ce of a base29
Fm oc-protected am in o acid fluorides can be used in th e case of -OtBu, Boc-
.
Fmoc-Xaa-F
+
H-(α-CF2X)Ala-OMe
Fmoc-Xaa-(α-CF2X)Ala-OMe
6a-6c
SCHEME 3
C-Term in al in corporation of α-Dfm am in o acids via Fm oc-am in o acid fluorides
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1539
an d trityl-protected am in o acid derivatives30. An oth er advan tage of acid
fluorides com pared with th e correspon din g acid ch lorides is th eir h igh er
stability to m oisture an d th eir low cyclization ten den cy to give oxazolon es
in th e presen ce of tertiary am in es31.
Carpin o et al. in troduced TFFH 32, a reagen t for th e in situ preparation of
acid fluorides from N-protected am in o acids. However, it h as been dem on -
strated th at th e application of th e in situ tech n ique resulted in lower yields
of th e m odel peptide 6a.
Acyl tran sfer reaction s to H-(α-Dfm )Ala-OMe with am in o acids with h igh
steric requirem en ts such as Leu, Val an d Aib turn ed out to be problem atic.
Of th e five m eth ods tested (PyBroP, DIC/HOAt, 7-azaben zotriazol-1-yloxy-
tris(pyrrolidin -1-yl)ph osph on ium h exafluoroph osph ate (PyAOP), HATU,
Fm oc-Xaa-F), on ly PyBroP an d Fm oc-Xaa-F (Xaa = Ph e, Leu, Val) gave th e
dipeptides in good to excellen t yields (Table III). However, experim en ts to
syn th esize Fm oc-Aib-(α-Dfm )Ala-OMe usin g th e couplin g m eth odology
h ave been un successful so far.
Alth ough , givin g on ly about 65–70% yields, th e m ixed an h ydride
m eth od usin g isobutyl ch loroform ate as couplin g reagen t is attractive for
th e syn th esis of Boc- an d Z-protected dipeptides with (α-Dfm ) am in o acids
TABLE III
Syn th esis of Fm oc-, Z- an d Boc-protected dipeptides with (α-Dfm )Ala an d (α-Dfm )Ph e in
C-term in al position
Couplin g reagen t/Yield, %
No.
Product
PyBroP
Fm oc-Xaa-F
6a
6b
6c
Fm oc-Ph e-(α-Dfm )Ala-OMe
Fm oc-Leu-(α-Dfm )Ala-OMe
Fm oc-Val-(α-Dfm )Ala-OMe
>95a/75b
80a/58b
65a/47b
95a/81b
>95a/72b
80a/62b
Isobutyl ch loroform ate
6d
6e
6f
Z-Ph e-(α-Dfm )Ala-OMe
67b
65b
72b
67b
Z-(R)-Ph e-(α-Dfm )Ala-OMe
Boc-Tyr(OtBu)-(α-Dfm )Ala-OMe
Boc-Gly-(α-Dfm )Ph e-OMe
6g
a
b
Yield determ in ed by 19F NMR spectroscopy; isolated yield.
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1540
Michel et al.:
in C-term in al position because of its sim plicity an d in expen sive reagen ts
(Sch em e 4). Several solven t m ixtures THF/DMF, eth yl acetate/DMF an d
CH2Cl2/DMF (7 : 1–10 : 1) can be used. Th e presen ce of DMF results in
h igh er yields an d sh orter reaction tim es (12–16 h ) due to better solubility of
th e am in o acid derivatives.
R1
R1
R2 CF2H
H2N CO2Me
H
N
PG
CO2Me
PG
O
O
+
N
H
N
H
O R2 CF2H
6d-6g
O
O
SCHEME 4
C-Term in al in corporation of α-Dfm am in o acids via m ixed an h ydride m eth od
Th e resultin g dipeptides 6a–6g carry orth ogon al protective groups. Th ere-
fore, th ey are valuable buildin g blocks for peptide syn th esis useful in frag-
m en t con den sation .
Tripeptides h avin g (α-CF2H)-Xaa or (α-CF2Cl)-Xaa in position 2 are
readily obtain able from Z- an d Boc-protected dipeptides 3–5 (Sch em e 5).
R1
O
R2
H
N
Fmoc
R
N
H
N
H
F HC CH
O
O
2
3
Route A Route B
SCHEME 5
Two differen t strategies for th e syn th esis of tripeptides with α-Dfm am in o acids in position 2
Th e Z-protectin g group was cleaved by h ydrogen olysis in th e presen ce of
Pd/C. Th en , th e dipeptide ester was reacted at room tem perature un der in -
ert gas with th e correspon din g Fm oc-am in o acid fluorides in a solven t m ix-
ture of CH2Cl2/DMF (2 : 1) in th e presen ce of DIEA (Route A, Sch em e 6).
Fin ally, th e tert-butyl ester was deblocked with 50% TFA in CH2Cl2 to give
th e Fm oc-protected tripeptides.
In a secon d approach (Route B, Sch em e 7) we started from dipeptides 6.
After cleavage of m eth yl ester with LiOH in a CH3OH/H2O m ixture (3 : 1) at
5 °C, [2+1]- an d [2+2]-fragm en t con den sation s with am in o acid am ides an d
dipeptide am ides on treatm en t with DIC/HOAt provide ready access to tri-
an d tetrapeptides with α-Dfm am in o acids in position 2 (Table IV).
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1541
Leu
(α-CF H)Ala
Pro
2
t
H
O Bu
Z
Z
H
OH
DIC, HOAt
t
O Bu
H , Pd/C
2
t
Fmoc
F
O Bu
DIEA
t
Fmoc
Fmoc
O Bu
TFA
OH
SCHEME 6
Route A, syn th esis of 7a/ 1
Wh en α-(fluoroalkyl) α-am in o acids are in troduced in peptide syn th esis
as racem ic m ixtures or peptide fragm en ts as diastereom er m ixtures, pairs of
diastereom ers are obtain ed wh ich can be readily separated by flash ch rom a-
tograph y an d m edium pressure liquid ch rom atograph y (MPLC). For exam -
ple, separation of th e diastereom er m ixtures of dipeptides 3c, 4c, 6d an d 6e
an d of tripeptides 7a–7c an d 8 h as been successfully perform ed18b. Th e
h om och iral pairs of tripeptides 7a/ 1, 7a/ 2–7b/ 1, 7b/ 2–7c/ 1, 7c/ 2 an d 8/ 1,
8/ 2 obtain ed by flash ch rom atograph y or MPLC of th e diastereom er m ix-
tures are iden tical to th ose obtain ed via h om och iral reagen ts (e.g.
3a/ 1→7a/ 1 an d 3a/ 2→7a/ 2).
(R)-Phe
(α-CF H)Ala
(R)-Ala
2
Z
OMe
H , Pd/C
2
OMe
OMe
OH
Z
Z
Z
Z
H
OH
DIC/HOAt
LiOH, CH OH/H O
3
2
NH
NH
H
2
DIC/HOAt
TFA
2
SCHEME 7
Route B, syn th esis of 7c/ 1
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1542
Michel et al.:
TABLE IV
Syn th esis of tri- an d tetrapeptides with α-CF2H an d α-CF2Cl am in o acids in position 2
Yield
%
No. Product
Educt
7a Fm oc-Leu-(α-Dfm )Ala-Pro-OtBu
7b Z-Ph e-(α-Dfm )Ala-Ala-NH2
Z-(α-Dfm )Ala-Pro-OtBu
Z-Ph e-(α-Dfm )Ala-OMe
Z-(R)-Ph e-(α-Dfm )Ala-OMe
Z-(α-CF2Cl)Ala-Pro-OtBu
75
85
7c Z-(R)-Ph e-(α-Dfm )Ala-(R)-Ala-NH2
8
9
Fm oc-Leu-(α-CF2Cl)Ala-Pro-OtBu
Boc-Tyr(OtBu)-(α-Dfm )Ala-Ph e-Ph e-NH2 Boc-Tyr(OtBu)-(α-Dfm )Ala-Ph e-OMe 78
Th e absolute con figuration of th e (fluoroalkyl)-substituted am in o acids
was determined for 7a/ 1 Fmoc-Leu-(R)-(α-Dfm )Ala-Pro-OtBu, for 7c/ 1 Z-(R)-Phe-
(R)-(α-Dfm )Ala-(R)-Ala-NH2 and for 8/ 1 Fm oc-Leu-(S)-(α-CF2Cl)Ala-Pro-OtBu.
Th e con figuration of th e α-fluoroalkyl α-am in o acid can be iden tified di-
rectly from th e X-ray structure33, sin ce th ere are two in depen den t ch iral ref-
eren ce cen ters presen t in th e m olecule (Table V). In th e case of 7a/ 1 th e
ch iral referen ce cen ters are (S)-Leu an d (S)-Pro. Th e SRS-con figuration was
determ in ed for 7a/ 1. Sin ce 7a/ 1 was syn th esized from 3a/ 1, th e latter h as
RS-con figuration . Con sequen tly, 3c/ 2 is SS an d 7a/ 2 is SSS-con figured. A
sim ilar argum en tation was applied to elucidation of th e stereoch em istry of
dipeptides 4c/ 1, 4c/ 2 an d 6e/ 1 an d 6e/ 2.
Molecular structure of peptide 8/ 1 is sh own in Fig. 1. Th e sam e atom
n um berin g was ch osen for 7a/ 1 an d 8/ 1. A com parison of th e torsion an -
gles sh ows th at th e α,α-disubstituted am in o acid h as th e sam e con form a-
tion in both peptides (Table VI).
Com poun ds 7a/ 1 an d 8/ 1 were foun d to be isom orph ic (Table V). Th e
structure of both peptides differs on ly in th e type of h alogen substituen ts.
Despite th e fact th at 8/ 1 bears an addition al ch lorin e atom , th e differen ce
of th e cell volum es was sh own to be on ly 28.8 Å3. Accordin g to th e volum e
in crem en t system of Im m irizi an d Perin i33, th e differen ce in volum es be-
tween a h ydrogen atom (6.9 Å3) an d a ch lorin e atom (26.7 Å3) is 19.8 Å3.
Th erefore, th e differen ce between th e cell volum es sh ould be 80 Å3 calculat-
in g with four form ula un its with in th e space group Cc. In term olecular h y-
drogen bon ds between th e atom s N(1)–H(1N)–O(1) an d N(2)–H(2N)–O(2)
were foun d to be a special feature of th ese strctures. Figure 2 sh ows th e
in term olecular h ydrogen bon ds alon g th e m on oclin ic axis b, correspon din g
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1543
TABLE V
Crystal data for 7a/ 1, 7c/ 1, an d 8/ 1
Param eters
8/ 1
7a/ 1
7c/ 1
Form ula
C
34H42ClF2N3O6
C34H43F2N3O6
627.7
C24H38F2N4O5·CH3OH
M
662.2
223
522.55
213
Tem perature, K
223
Crystal system
m on oclin ic
C2
m on oclin ic
C2
m on oclin ic
P21
Space group
Un it cell dim en sion , Å
a
39.5751(6)
5.9819(10)
14.6786(3)
102.51(1)
3392.4(1)
4
39.236(5)
5.9509(8)
14.764(2)
102.65(1)
3363.6(7)
4
12.52760(10)
8.2386(2)
14.3409(3)
112.510(1)
1367.4
b
c
β, °
V, Å3
Z
2
D
calc, g cm –3
1.296
1.240
1.269
µ (MoKα), m m –1
2θ, °
0.171
0.092
0.100
54.4
50.0
54.0
Num ber of reflection s
collected
9006
7740
12118
Num ber of un ique
reflection s
5012 (Rin t = 0.0317)
6069
4493
Num ber of observed
reflection s
5596
2526
3960
Data used/param eters
refin ed
6069/415
4493/414
5012/463
R1, wR2(obs)
0.0456, 0.1146
1.116
0.0923, 0.1434
1.099
0.0375, 0.0788
1.079
Goddn ess-of-fit on F2
Largest differen ce peak
an d h ole, e Å3
0.386 an d –0.255
0.224 an d –0.284 0.206 an d –0.207
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1544
Michel et al.:
data are given in Table VI. Obviously, stron g in term olecular h ydrogen
bon ds dom in ate th e m olecular structure an d cause th e isom orph ism of
both peptides. Rem arkably, in α-(CF2Cl)-substituted peptide 8/ 1, we could
TABLE VI
Torsion an gles of 7a/ 1 an d 8/ 1
Fm oc-Leu-Xaa-Pro-OtBu
Xaa = (R)-(α-CF2H)Ala
Fm oc-Leu-Xaa-Pro-OtBu
Xaa = (S)-(α-CF2Cl)Ala
Am in o acid
An gle
Ph e
Xaa
Pro
Φ+1
Ψ+1
Φ+2
Ψ+2
Φ+3
Ψ+3
–93.3
–28.3
41.8
–98.3
–26.9
49.3
58.3
48.6–78.0
–75.3
–8.7
–8.3
C30
C24
C31
C29
C23
C25
C32
C33
C26
Cl1
C28
C22
C27
C21
C34
C7
F1
F2
O1
C14
O3
C2
C6
C15
O4
N2
O6
N1
C1
N3
C3
C5
C13
O2
C16
C11
C4
C17
C18
C12
C8
O5
C10
C19
C9
C20
FIG. 1
Molecular structure of Fm oc-Leu-(S)-(α-CF2Cl)Ala-Pro-OtBu (8/ 1)
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1545
fin d
a
weak in tram olecular h ydrogen bon d between th e atom s
C(7)–H(7B)–F(1) as well as an in term olecular h ydrogen bon d between
C(15)–H(15A)–F(2) (Table VII).
TABLE VII
Stron g h ydrogen bon ds for 7a/ 1 an d 8/ 1 (Å an d °)
D–H···A
d(D–H)
d(H···A)
d(D···A)
<(DHA)
Peptide 7a/ 1
N(1)–H(1N)···O(1)a
N(2)–H(2N)···O(2)a
N(2)–H(2N)···O(2)
Peptide 8/ 1
0.90
0.90
0.90
2.42
2.12
2.45
3.134(8)
2.870(7)
2.823(8)
136.2
140.5
104.9
N(1)–H(1N)···O(1)a
N(2)–H(2N)···O(2)a
N(2)–H(2N)···O(2)
C(7)–H(7B)–F(1)b
C(15)–H(15A)–F(2)b
0.77
0.75
0.77
0.94
0.97
2.52
2.17
2.47
2.44
2.52
3.126(3)
2.917(3)
2.827(3)
2.80
136.8
171.0
109.8
101.9
141.9
3.34
Sym m etry tran sform ation s used to gen erate equivalen t atom s: a) x, y – 1, z; b) x, 1 + y, z.
O1a
O2a
O2
O1
N1a
N1
N2
N2a
FIG. 2
Stron g in term olecular h ydrogen bon ds of 8/ 1; for h ydrogen bon d param eters, see Table VI
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1546
Michel et al.:
Th e crystal structure of 7c/ 1 is sh own in Fig. 3. Th is peptide structure is
ch aracterized by five stron g in term olecular h ydrogen bon ds between th e
carbon yl an d am in o groups (Fig. 4). Data are given in Table VIII.
TABLE VIII
Hydrogen bon ds for 7c/ 1
D–H···A
d(D–H)
d(H···A)
d(D···A)
<(DHA)
N(1)–H(1NA)···O(71)a
N(4)–H(4N)···O(5)b
N(1)–H(2NB)···O(1)c
0.77(4)
0.80(3)
0.87(3)
2.20(4)
2.09(3)
2.15(3)
2.936(3)
2.875(3)
2.885(4)
159(4)
168(3)
143(3)
O(99)–H(99O)···O(3)d
N(2)–H(2N)···O(71)
N(3)–H(3N)···O(99)
0.76(5)
0.89(3)
0.82(3)
2.14(5)
2.14(4)
2.10(3)
2.806(3)
3.012(3)
2.918(3)
146(4)
165(3)
172(4)
Sym m etry tran sform ation s used to gen erate equivalen t atom s: a) –x + 1, y – 1/2, –z; b) –x + 2,
y + 1/2, –z; c) –x + 1, y + 1/2, –z; d) x, y + 1, z
C74
C63
C67
C73
C75
O72
C61
C64
C66
C6
C62
C65
C76
N4
C77
C72
C7
C71
C5
O71
O5
N3
C42
C4
C21
F1
N2
C3
C41
C2
O4
C1
F2
N1
O1
FIG. 3
Molecular structure of Z-(R)-Ph e-(R)-(α-Dfm )Ala-(R)-Ala-NH2 (7c/ 1)
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1547
FIG. 4
Stron g in term olecular h ydrogen bon ds of 7c/ 1, for h ydrogen bon d param eters, see Table VIII
100
2
1
90
3
80
4
70
60
6
50
5
40
30
20
10
0
0
2
4
6
8
10
Time, h
12
FIG. 5
Com parison of th e efficien cy of curren tly used peptide couplin g reagen ts. 1 HATU, 2 PyAOP,
3 DIC/HOAt, 4 PyBOP, 5 HBTU, 6 DIC/HOBt
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1548
Conclusion
Michel et al.:
(α-Dfm )Ala as well as (α-Tfm )Ala represen t ch iral Aib surrogates. Th erefore,
both of th ese am in o acids th em selves an d th eir peptides are of curren t in -
terest as buildin g blocks for th e syn th esis of m odified peptides an d
peptaiboles34. We tested som e curren tly used peptide couplin g reagen ts for
th e N- an d C-term in al in corporation of α-(CF2H), α-(CF2Cl) an d α-(CF2Br)
am in o acids in to peptides (Fig. 5). We foun d th at N-term in al in corporation
can be perform ed with out an y problem s usin g th e reagen t com bin ation
DIC/HOAt, wh ile for C-term in al in corporation th e reagen t com bin ation s
DIC/HOAt, HATU, PyBroP an d PyAOP are superior to DIC/HOBt, HBTU an d
PyBOP. Am in o acid fluorides an d ch lorides gave excellen t yields in
C-term in al peptide couplin g reaction s of α-Dfm am in o acids. Th e applica-
tion of α-(fluoroalkyl) am in o acids in th e solid ph ase syn th esis is reported
elsewh ere.
EXPERIMENTAL
Gen eral
1H NMR spectra were recorded at 200 an d 300 MHz with Me4Si as in tern al stan dard.
13C NMR spectroscopy was perform ed at 50, 75 an d 100 MHz. 19F NMR spectra were ob-
tain ed at 188 an d 282 MHz with trifluoroacetic acid as extern al stan dard, down field sh ifts
bein g design ated as positive. Ch em ical sh ifts (δ) are given in ppm , couplin g con stan ts (J)
in Hz. Mass spectra were obtain ed usin g EI ion ization at 70 eV. Optical rotation s were
m easured with a Sch m idt an d Haen sch Polartron ic-D polarim eter; [α]D values are given in
10–1 deg cm 2 g–1. Meltin g poin ts were determ in ed on a Boetius apparatus. CCD diffracto-
m eter (AXS Bruker) was used for X-ray m easurem en ts (MoKα radiation , 0.71069 A at room
tem perature).
All reaction s were routin ely m on itored by 19F NMR spectroscopy, TLC or HPLC. An alyti-
cal TLC was perform ed usin g Merck precoated silica gel 60F-254 plates (0.25 m m ). For flash
ch rom atograph y, silica gel 60 (30–60 µm ) was used with h exan es/eth yl acetate as solven t
system s.
Organ ic solven ts were dried an d distilled prior to use.
Gen eral Procedure for In corporation of α-(Difluorom eth yl)-Substituted Am in o Acids
in to N-Term in al Position of Peptides
Method A: Activation with DIC. To a solution of a Z-protected α-(difluorom eth yl) am in o
acid (2 m m ol) in a 2 : 1 m ixture of CH2Cl2/DMF (7 m l) DIC (0.27 g, 2.1 m m ol) was added
an d stirred at room tem perature for 15 m in , th en th e correspon din g am in o acid ester (3 m m ol)
an d DIEA (0.35 g, 2.7 m m ol) in a 2 : 1 m ixture of CH2Cl2/DMF (7 m l) were added. After stir-
rin g for 12 h , th e reaction m ixture was acidified with 10% citric acid to pH 3–4. Th e solu-
tion was partition ed between H2O (20 m l) an d CH2Cl2 (30 m l). Th e organ ic layer was
wash ed with 10% citric acid (20 m l), brin e (20 m l), 1 M NaHCO3 (20 m l), brin e (20 m l) an d
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1549
dried over an h ydrous MgSO4. Evaporation of th e solven t afforded a m ixture of two dia-
stereom ers. Th e crude product was purified by recrystallization from CHCl3/h exan es or by
flash ch rom atograph y (SiO2, EtOAc/h exan es).
Method B: Activation with DIC/HOAt. A solution of a Z-protected α-(difluorom eth yl)
am in o acid (2 m m ol) in a 2 : 1 m ixture of CH2Cl2/DMF (7 m l), was stirred with DIC (0.27 g,
2.1 m m ol) an d HOAt (0.29 g, 2.1 m m ol) at room tem perature for 5 m in . A solution of th e
correspon din g am in o acid ester (3 m m ol) an d DIEA (0.35 g, 2.7 m m ol) in a 2 : 1 m ixture of
CH2Cl2/DMF (7 m l) was added to th e vigorously stirred reaction m ixture in on e portion . Af-
ter 15–20 m in stirrin g at room tem perature, th e products were isolated; for work-up see
above. Th e spectral an d an alytical data are iden tical to peptides obtain ed by m eth od A.
tert-Butyl N-{N-[(Ben zyloxy)carbon yl]-(RS)-2-(difluorom eth yl)alan yl}glycin ate (3a)
Method A: Yield 0.57 g (73%). MS (FAB, m/z), calculated for C18H24F2N2O5: 386.39; foun d:
409.2 [M + Na]+. M.p. 114–116 °C. RF 0.22 (AcOEt/h exan es 1 : 3). 1H NMR (200 MHz,
2
3
2
CDCl3): 1.47 (s, 9 H); 1.70 (s, 3 H); 3.90 (dd, JHH = 18.5, JHH = 4.9, 1 H); 3.98 (dd, JHH
=
3
2
18.5, JHH = 4.9, 1 H); 5.10 (s, 2 H); 5.97 (s, br, 1 H); 6.39 (t, JHF = 56.2, 1 H); 6.71 (s, br,
1 H); 7.36 (s, 5 H). 13C NMR (50 MHz, CDCl3): 18.6; 28.4; 42.9; 61.6 (t, JCF = 23.0); 67.6;
2
83.2; 114.7 (t, JCF = 249.0); 128.6; 128.8; 129.1; 136.3; 155.5; 168.8; 169.0. 19F NMR
1
2
(188 MHz, CDCl3): –52.20 (ddABX
2JHF = 56.0, 1 F).
,
2JFF = 280.0, JHF = 56.0, 1 F); –50.91 (ddABX
,
2JFF = 280.0,
tert-Butyl ambo-N-{N-[(Ben zyloxy)carbon yl]-2-(difluorom eth yl)alan yl}ph en ylalan in ate (3b)
Method B: Yield 0.86 g (90%). MS (MALDI-TOF), calculated for C25H30F2N2O5: 476.52;
foun d: 499.18 [M + Na]+; 515.14 [M + K]+. M.p. 114–120°C. RF 0.46 (AcOEt/h exan es 1 : 3).
3
1H NMR (200 MHz, CDCl3): 1.41/1.42 (s, 9 H); 1.58/1.62 (s, 3 H); 3.12 (d, JHH = 6.0, 2 H);
2
4.74 (m , 1 H); 5.08 (s, 2 H); 6.02 (s, br, 1 H); 6.36/6.37 (t, JHF = 50.4, 1 H); 6.76 (m , 1 H);
2
7.10–7.35 (m , 10 H). 13C NMR (50 MHz, CDCl3): 18.5; 28.3; 38.1/38.2; 54.5; 61.5 (t, JCF
=
1
22.0); 67.5; 83.2; 114.6 (t, JCF
= 250.0); 127.6; 128.5/128.6; 128.8; 128.9; 129.1;
130.0/130.1; 136.2/136.4; 155.4; 168.3/168.4; 170.3/170.4. 19F NMR (188 MHz, CDCl3):
2
–50.58/–50.92 (ddABX
,
2JFF = 288.0, JHF = 50.0, 1 F); –51.71/–51.03 (ddABX
,
2JFF = 288.0,
2JHF = 50.0, 1 F).
tert-Butyl ambo-N-{N-[(Ben zyloxy)carbon yl]-2-(difluorom eth yl)alan yl}prolin ate (3c)
Method B: Yield 0.77 g (90%). MS (MALDI-TOF), calculated for C21H28F2N2O5: 426.46;
foun d: 449.4 [M + Na]+; 465.5 [M + K]+. Separation of th e diastereom ers: MPLC; eluen t:
AcOEt/h exan es 2 : 9.
Diastereom er 1 (3c/ 1): M.p. 140 °C. [α ]2D5 –94.0 (c 1, CH2Cl2). RF 0.40 (AcOEt/h exan es 1 : 2).
1H NMR (300 MHz, CDCl3): 1.44 (s, 9 H); 1.55 (s, 3 H); 1.72 (m , 2 H); 1.85 (m , 1 H); 1.96
2
(m , 1 H); 3.43 (m , 1 H); 3.63 (m , 1 H); 4.42 (m , 1 H); 5.12 (m , 3 H); 6.36 (t, JHF = 56.2,
2
1 H); 7.35 (m , 5 H). 13C NMR (50 MHz, CDCl3): 16.6; 25.8; 28.1; 28.3; 47.5; 61.1 (t, JCF
=
1
19.0); 61.6; 67.7; 81.6; 114.6 (t, JCF = 249); 128.9; 129.0; 129.1; 136.4; 155.2; 167.7; 171.7.
2
19F NMR (282 MHz, CDCl3): –52.44 (ddABX
,
2JFF = 280.0, JHF = 56.0, 1 F); –53.50 (ddABX
,
2
2JFF = 280.0, JHF = 56.0, 1 F).
Diastereom er 2 (3c/2): M.p. 132–133°C. [α ]2D5 –24 (c 1, CH2Cl2). RF 0.32 (AcOEt/hexanes 1 : 2).
1H NMR (300 MHz, CDCl3): 1.44 (s, 9 H); 1.79 (s, 3 H); 1.81 (m , 2 H); 1.95 (m , 2 H); 3.57
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1550
Michel et al.:
2
(m , 1 H); 3.68 (m , 1 H); 4.38 (m , 1 H); 5.11 (s, 2 H); 5.78 (s, 1 H); 6.23 (t, JHF = 56.2, 1 H);
2
7.35 (m , 5 H). 13C NMR (50 MHz, CDCl3): 17.4; 26.1; 28.2; 28.3; 48.0; 62.5 (t, JCF = 19);
1
62.6; 67.5; 81.6; 115.4 (t, JCF = 249); 128.7; 129.0; 129.1; 136.5; 155.1; 167.5; 171.6.
2
19F NMR (282 MHz, CDCl3): –49.65 (ddABX
,
2JFF = 280.0, JHF = 56.0, 1 F); –52.00 (ddABX
,
2
2JFF = 280.0, JHF = 56.0, 1 F).
tert-Butyl ambo-N-{N-[(Ben zyloxy)carbon yl]-2-(ch lorodifluorom eth yl)alan yl}glycin ate (4a)
Method A: Yield 0.66 g (78%). MS (FAB; m/z), calculated for C18H23ClF2N2O5: 420.84;
foun d: 443.4 [M + Na]+. M.p. 83–85 °C. RF 0.27 (AcOEt/h exan es 1 : 3). 1H NMR (200 MHz,
2
3
2
CDCl3): 1.47 (s, 9 H); 1.95 (s, 3 H); 3.90 (dd, JHH = 18.4, JHH3 = 4.8, 1 H); 4.01 (dd, JHH
=
3
18.4, JHH = 4.8, 1 H); 5.10 (s, 2 H); 6.00 (s, br, 1 H); 6.66 (t, JHH = 4.8, 1 H); 7.36 (s, 5 H).
13C NMR (50 MHz, CDCl3): 18.2; 28.4; 43.2; 67.1 (t, JCF = 25.0); 67.7; 83.3; 128.7; 128.8;
2
129.1; 129.7 (ddABX
CDCl3): 16.32 (dAB
,
1JCF = 301.0, 303.0); 136.3; 154.7; 166.5; 168.7. 19F NMR (188 MHz,
,
2JFF = 163.0, 1 F); 17.30 (dAB 2JFF = 163.0, 1 F).
,
tert-Butyl ambo-N-{N-[(Ben zyloxy)carbon yl]-2-(ch lorodifluorom eth yl)alan yl}-
ph en ylalan in ate (4b)
Method A: Yield 0.85 g (83%). MS (FAB, m/z), calculated for C25H29ClF2N2O5: 510.96;
foun d: 511.6 [M + H]+. M.p. 89–93 °C. RF 0.10 (AcOEt/h exan es 1 : 10). 1H NMR (200 MHz,
CDCl3): 1.39/1.41 (s, 9 H); 1.84/1.90 (s, 3 H); 3.05 (m , 2 H); 4.72 (m , 1 H); 5.08 (s, 2 H);
2
6.05/6.09 (s, 1 H); 6.62/6.69 (d, JHH = 7.2/7.2); 7.25 (m , 10 H). 13C NMR (50 MHz, CDCl3):
2
18.1/18.3; 28.3; 38.1; 54.9; 67.0/67.2 (t, JCF = 25.0); 67.6; 83.2/83.3; 127.6; 128.7;
2
128.8/128.9; 129.0; 129.1; 129.8 (t, JCF
=
304); 136.3; 136.4; 154.7/154.8; 166.0;
2JFF = 165.0, 1 F), 17.54/17.61
170.3/170.4. 19F NMR (188 MHz, CDCl3): 16.36/16.54 (dAB
(dAB = 165.0, 1 F).
,
tert-Butyl ambo-N-{N-[(Ben zyloxy)carbon yl]-2-(ch lorodifluorom eth yl)alan yl}prolin ate (4c)
Method B: Yield 0.87 g (93%). MS (MALDI-TOF), calculated for C21H27ClF2N2O5: 460.90;
foun d: 483.0 [M + Na]+; 498.9 [M + K]+. Separation of th e diastereom ers: MPLC; eluen t:
AcOEt/h exan es 2 : 9.
Diastereom er 1 (4c/ 1): M.p. 148 °C. [α ]2D5 –54 (c 1, CH2Cl2). RF 0.21 (AcOEt/h exan es 1 : 3).
1H NMR (300 MHz, CDCl3): 1.43 (s, 9 H); 1.60–2.00 (m , 4 H); 1.93 (s, 3 H); 3.38 (m , 1 H);
3.74 (m , 1 H); 4.36 (m , 1 H); 5.13 (s, 2 H); 5.66 (s, 1 H); 7.37 (m , 5 H). 13C NMR (APT;
2
75 MHz, CDCl3): 19.4; 25.8; 27.7; 27.8; 48.3; 61.6; 67.4; 67.7 (t, JCF = 23.0); 81.2; 128.5;
1
128.6; 129.8 (t, JCF = 301.0); 135.8; 154.0; 163.8; 171.1. 19F NMR (282 MHz, CDCl3): 17.07
2
2
(d, JFF = 164.0, 1 F); 18.50 (d, JFF = 164.0, 1 F).
Diastereom er 2 (4c/ 2): M.p. 162–164 °C. [α ]2D5 –74 (c 1, CH2Cl2). RF 0.16 (AcOEt/h exan es
1 : 3). 1H NMR (300 MHz, CDCl3): 1.44 (s, 9 H); 1.55–2.00 (m , 4 H); 1.86 (s, 3 H); 3.52 (m ,
1 H); 3.61 (m , 1 H); 4.38 (m , 1 H); 5.13 (s, 2 H); 5.44 (s, br, 1 H); 7.36 (m , 5 H). 13C NMR
2
(75 MHz, CDCl3): 18.8; 25.6; 27.8; 27.9; 47.8; 61.9; 66.1 (t, JCF = 23.0); 67.4; 81.2; 128.4;
1
128.5; 128.6; 130.5 (t, JCF = 304.0); 135.9; 154.1; 164.2; 171.1. 19F NMR (282 MHz, CDCl3):
18.19 (s, br, 2 F).
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1551
tert-Butyl N-{N-[(Ben zyloxy)carbon yl]-(RS)-2-(ch lorodifluorom eth yl)ph en ylalan yl}-
glycin ate (4d )
Method A: Yield 0.85 g (85%). MS (MALDI-TOF), calculated for C24H27ClF2N2O5: (496.94);
foun d: 519.06 [M + Na]+; 535.04 [M + K]+. M.p 118–119 °C. RF 0.19 (AcOEt/h exan es 1 : 6).
2
3
1H NMR (200 MHz, CDCl3): 1.51 (s, 9 H); 3.26 (d, JHH = 13.8, 1 H); 3.94 (d, JHH = 4.4,
2
2
2
2 H); 4.36 (d, JHH = 13.8, 1 H); 5.04 (d, JHH = 12.4, 1 H); 5.24 (d, JHH = 12.4, 1 H); 6.37
(s, br, 1 H); 6.76 (m , 1 H); 7.20 (m , 5 H); 7.37 (s, 5 H). 13C NMR (75 MHz, CDCl3): 28.1;
2
33.7; 43.0; 67.0; 70.4 (dd, JCF = 24.0, 26.0); 83.3; 127.5; 128.3; 128.4; 128.6; 129.5 (ddABX
,
1JCF = 301.0, 308.0); 130.0; 133.1; 136.2; 154.2; 164.5 (d, JCF = 4); 168.0. 19F NMR
(188 MHz, CDCl3): 16.76 (dAB
2JFF = 169.0, 1 F); 20.60 (dAB 2JFF = 169.0, 1 F).
3
,
,
tert-Butyl ambo-N-{N-[(Ben zyloxy)carbon yl]-2-(brom odifluorom eth yl)alan yl}-
ph en ylalan in ate (5)
Method A: Yield 0.96 g (86%). MS (FAB, m/z), calculated for C25H29BrF2N2O5: 555.41;
foun d: 555.5 [M + H]+. M.p. 98–99 °C. RF 0.49 (AcOEt/h exan es 1 : 3). 1H NMR (300 MHz,
CDCl3): 1.39/1.42 (s, 9 H); 1.84/1.91 (s, 3 H); 3.11 (m , 2 H); 4.74 (m , 1 H); 5.10 (s, 2 H;
3
5.96/6.03 (s, br, 1 H); 6.56/6.63 (d, JHH = 6.9/7.2, 1 H); 7.10–7.40 (m , 10 H). 13C NMR
2
(50 MHz, CDCl3): 17.9/18.1; 27.9; 37.8; 54.5; 67.2; 67.6/67.7 (t, JCF = 22.0); 82.9/83.0;
1
123.7/123.8 (t, JCF = 315.0); 127.2; 128.2; 128.3/128.4; 128.5/128.6; 129.5; 129.6; 135.7;
135.8/135.9; 155.0; 165.3; 169.7/169.8. 19F NMR (282 MHz, CDCl3): 22.67/22.93 (dAB 2JFF
, =
165.0, 1 F); 24.26/24.28 (dAB,
2JFF = 165.0, 1 F).
Gen eral Procedure for In corporation of α-(Fluoroalkyl)-Substituted Am in o Acids
in to C-Term in al Position of Peptides (6, 7)
Method A: Fm oc-am in o acid fluoride m eth od. A solution of Fm oc-protected am in o acid
fluoride (2.5 m m ol) in a 2 : 1 m ixture of CH2Cl2/DMF (7 m l) was added to th e correspon d-
in g C-protected α-(fluoroalkyl)-substituted am in o acid or dipeptide (1 m m ol) an d DIEA
(0.33 g, 2.5 m m ol) in a 2 : 1 m ixture of CH2Cl2/DMF (7 m l) an d stirred at room tem perature
for 16 h . Th e reaction m ixture was acidified with 10% citric acid to pH 3–4 an d extracted
with CH2Cl2 (3 × 10 m l). Th e com bin ed organ ic layer was wash ed with 10% citric acid
(10 m l), brin e (10 m l), 5% NaHCO3 (10 m l) an d brin e (10 m l), th en dried over an h ydrous
MgSO4 an d evaporated un der reduced pressure. Th e crude products were purified by
recrystallization from CHCl3/h exan es or by flash ch rom atograph y (SiO2, EtOAc/h exan es).
Method B: Mixed an h ydride m eth od.
A solution of th e N-protected am in o acid
(2.05 m m ol) in a 10 : 1 m ixture of AcOEt/DMF (7 m l) was cooled to –30 °C an d n eutralized
with N-m eth ylm orph olin e (NMM; 0.2 g, 2 m m ol). Isobutyl ch loroform ate (0.3 g, 2.20 m m ol)
was added at –15 °C. After 10 m in at –15 °C, a solution of a precooled α-(fluoroalkyl)-substi-
tuted am in o acid or peptide ester (1 m m ol) in a 10 : 1 m ixture of AcOEt/DMF (5 m l) was
added. Th e reaction m ixture was stirred at –15 °C for 2 h an d th en warm ed up to room tem -
perature After 12–16 h stirrin g at room tem perature peptides were isolated. Work-up proce-
dure, see m eth od A.
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1552
Michel et al.:
Meth yl ambo-N-{N-[(Fluoren -9-ylm eth oxy)carbon yl]ph en ylalan yl}-2-(difluorom eth yl)-
alan in ate (6a)
Method A: Yield 0.42 g (81%); 1 : 1 m ixture of two diastereom ers. MS (FAB, m/z), calcu-
lated for C29H28F2N2O5: 522.55; foun d: 523.5 [M + H]+; 545.5 [M + Na]+. M.p. 140–143 °C.
RF 0.20 (AcOEt/h exan es 1 : 3). Racem ization test: 0.13% (±0.01%) (R)-Ph e. 1H NMR (300 MHz,
CDCl3): 1.48/1.51 (s, 3 H); 3.02/3.11 (m , 2 H); 3.78/3.79 (s, 3 H); 4.20 (m , 1 H); 4.42 (m , 1 H);
2
4.45 (m , 2 H); 5.28 (s, br, 1 H); 6.18/6.20 (t, JFH = 56.0, 1 H); 6.30/6.42 (s, 1 H); 7.15–7.83
2
(m , 13 H). 13C NMR (75 MHz, CDCl3): 16.3; 38.2; 47.2; 53.2; 55.9/56.0; 61.0/61.2 (t, JCF
=
1
23/23); 67.2; 112.8/113.2 (t, JCF = 249/249); 120.0; 125.0; 127.1; 127.8; 128.9; 129.4; 136.0;
141.4; 143.6/143.7; 155.9; 169.2; 171.0. 19F NMR (282 MHz, CDCl3): –52.72/–52.42 (ddABX
,
2
2
2JFC = 282.0, JFH = 56.0, 1 F); –50.57/–50.51 (ddABX 2JFC = 282.0, JFH = 56.0, 1 F).
,
Meth yl ambo-N-{N-[(Fluoren -9-ylm eth oxy)carbon yl]leucyl}-2-(difluorom eth yl)-
alan in ate (6b)
Method A: Yield 0.35 g (72%); PyBroP: 0.28 g (58%); 1 : 1 m ixture of two diastereom ers.
MS (MALDI-TOF), calculated for C26H30F2N2O5: 488.53; foun d: 511.2 [M + Na]+; 527.1 [M +
K]+. Oil. RF 0.43 (AcOEt/h exan es 1 : 3). 1H NMR (300 MHz, CDCl3): 0.94 (m , 6 H); 1.57 (s,
3
3 H); 1.60 (m , 3 H); 3.76/3.78 (s, 3 H); 4.22 (t, JHH = 6.6, 1 H); 4.24 (m , 1 H); 4.44 (m , 2 H);
3
2
5.13 (d, JHH = 7.2, 1 H); 6.25/6.26 (t, JFH = 56.2, 1 H); 6.96 (s, 1 H); 7.27–7.80 (m , 8 H).
13C NMR (75 MHz, CDCl3): 16.2/16.4 (s/t, JCF = 4); 22.0, 22.8; 24.6; 40.8/40.9); 47.2; 53.1;
53.2; 60.9 (t, JCF = 22.0); 67.2; 112.7/112.8 (t, JCF = 247.0, 247.0); 120.0; 124.7/125.0;
127.1; 127.6/127.8; 141.3; 143.6/143.7; 156.4; 169.5/169.6; 172.4. 19F NMR (282 MHz,
CDCl3): –53.00/–52.79 (ddABX
282.0, JFH = 56.0, 1 F).
3
2
1
2
,
2JFC = 282.0, JFH = 56.0, 1 F); –50.54/–50.52 (ddABX
,
2JFC
=
2
Meth yl ambo-N-{N-[(Fluoren -9-ylm eth oxy)carbon yl]valyl}-2-(difluorom eth yl)-
alan in ate (6c)
Method A: Yield: 0.29 g (62%); PyBroP: 0.23 g (47%); 1 : 1 m ixture of two diastereom ers.
MS (MALDI-TOF), calculated for C25H28F2N2O5: 474.50; foun d: 497.0 [M + Na]+; 513.0 [M +
K]+. Oil. RF 0.34 (AcOEt/h exan es 1 : 3). 1H NMR (300 MHz, CDCl3): 0.96 (m , 6 H); 1.59 (s,
3
3 H); 2.15 (m , 1 H); 3.78/3.80 (s, 3 H); 4.04 (m , 1 H); 4.23 (t, JHH = 6.6, 1 H); 4.44 (m , 2 H);
2
5.32/5.35 (s, br, 1 H); 6.17/6.28 (t, JFH = 56.0, 1 H); 6.65 (s, 1 H); 7.29–7.80 (m , 8 H).
13C NMR (75 MHz, CDCl3): 16.4/16.5; 17.7; 19.0; 30.9/31.0; 47.2; 53.1; 60.1; 60.7/61.0 (t,
1
2JCF = 22.0, 22.0); 67.2; 112.7 (t, JCF = 247); 120.0; 125.0; 127.1; 127.8; 141.3; 143.7/ 143.8;
156.4; 169.4/169.5; 171.3. 19F NMR (282 MHz, CDCl3): –52.87/–52.48 (ddABX
,
2JFC = 282.0,
2
2JFH = 56.0, 1 F); –50.63/–50.53 (ddABX
,
2JFC = 282.0, JFH = 56.0, 1 F).
Meth yl ambo-N-{N-[(Ben zyloxy)carbon yl]ph en ylalan yl}-2-(difluorom eth yl)-
alan in ate (6d )
Method B: Yield 0.29 g (67%); 1 : 1 m ixture of diastereom ers. MS (FAB, m/z), calculated for
C22H24F2N2O5: 434.44; foun d: 435.4 [M + H]+. Separation of th e diastereom ers by flash ch ro-
m atograph y, eluen t: AcOEt/h exan es 1 : 3.
Diastereom er 1 (6d/ 1): M.p. 79–80 °C. [α ]D +6 (c 1, CH2Cl2). RF 0.16 (AcOEt/h exan es 1 : 3).
2
1H NMR (300 MHz, CDCl3): 1.45 (s, 3 H); 3.04 (d, JHH = 7.2, 2 H); 3.76 (s, 3 H); 4.47 (m ,
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1553
2
2
1 H); 5.08 (s, 2 H); 5.43 (d, JHH = 8.2, 1 H, NH); 6.17 (t, JHH = 55.6); 6.60 (s, 1 H, NH);
7.15–7.40 (m , 10 H). 13C NMR (75 MHz, CDCl3): 16.1 (t, JCF = 3.0); 38.2; 53.1; 55.9; 60.9 (t,
3
2JCF = 23); 67.3; 112.8 (1JCF = 247.0); 127.2; 128.0; 128.3; 128.6; 128.8; 129.4; 136.1; 156.1;
169.4; 171.0. 19F NMR (282 MHz, CDCl3): –53.01 (ddABX
,
2JFC = 282.0, JFH = 56.0, 1 F);
2
2
–50.10 (ddABX
,
2JFC = 282.0, JFH = 56.0, 1 F).
Diastereom er 2 (6d/ 2): M.p. 84–86 °C. [α ]D –11 (c 1, CH2Cl2). RF 0.12 (AcOEt/h exan es 1 : 3).
3
1H NMR (300 MHz, CDCl3): 1.48 (s, 3 H); 3.05 (d, JHH2 = 7.2); 3.76 (s, 3 H); 4.48 (m , 1 H);
3
5.09 (s, 2 H); 5.42 (d, JHH = 7.2, 1 H, NH); 6.19 (t, JFH = 56.0, 1 H); 6.72 (s, 1 H, NH);
3
7.15–7.35 (m , 10 H). 13C NMR (100 MHz, CDCl3): 15.7 (t, JCF = 3.0); 37.7; 52.7; 55.5; 60.5
2
1
(t, JCF = 23); 66.8; 112.4 (t, JCF = 247.0); 126.8; 127.6; 127.9; 128.2; 128.4; 129.0; 135.6;
155.7; 169.0 (d, JCF = 4.0); 172.4. 19F NMR (282 MHz, CDCl3): –52.60 (ddABX
2JFH = 56.0, 1 F); –50.41 (ddABX
,
2JFC = 282.0,
3
2
,
2JCF = 282.0, JFH = 56.0, 1 F).
Meth yl ambo-N-{N-[(Ben zyloxy)carbon yl]-(R)-ph en ylalan yl}-2-(difluorom eth yl)-
alan in ate (6e)
Method B: Yield 0.29 g (67%); 1 : 1 m ixture of diastereom ers. MS (FAB, m/z), calculated for
C22H24F2N2O5: 434.44; foun d: 435.4 [M + H]+. Separation of th e diastereom ers by flash ch ro-
m atograph y, eluen t: AcOEt/h exan es 1 : 3.
Diastereom er 1 (6e/ 1): M.p. 79–81 °C. [α ]D –4 (c 1, CH2Cl2). RF 0.16 (AcOEt/h exan es 1 : 3).
2
3
1H NMR (300 MHz, CDCl3): 1.47 (s, 3 H); 3.02 (dd, JHH = 14.0, JHH = 7.3, 1 H); 3.11 (dd,
2JHH = 14.0, JHH = 6.4, 1 H); 3.78 (s, 3 H); 4.43 (m , 1 H); 5.08 (d, JHH = 12.0, 1 H); 5.12 (d,
3
2
2JHH = 12.0, 1 H); 5.29 (m , 1 H, NH); 6.16 (t, JFH = 56.5, 1 H); 6.39 (s, br, 1 H, NH);
2
7.20–7.40 (m , 10 H). 13C NMR (75 MHz, CDCl3): 16.2 (t, JCF = 3.0); 38.2; 53.1; 55.9; 60.9 (t,
3
2JCF = 23.0); 67.3; 112.8 (t, JCF = 247); 127.2; 128.0; 128.3; 128.6; 129.4; 136.1; 156.1; 169.3
1
(d, JCF = 3.0); 171.0. 19F NMR (282 MHz, CDCl3): –52.83 (ddABX
,
2JFF = 282.0, JFH = 56.0,
3
2
2
1 F); –50.59 (ddABX
,
2JFF = 282.0, JFH = 56.0, 1 F).
Diastereom er 2 (6e/ 2): M.p. 85–86 °C. [α]D +12 (c 1, CH2Cl2). RF 0.12 (AcOEt/h exan es 1 : 3).
1H NMR (300 MHz, CDCl3): 1.49 (s, 3 H); 3.08 (m , 2 H); 3.78 (s, 3 H); 4.43 (m , 1 H); 5.08 (d,
2
2
2JHH = 12.3, 1 H); 5.13 (d, JHH = 12.3, 1 H); 5.29 (m , 1 H, NH); 6.19 (t, JFH = 56.0, 1 H);
6.44 (s, br, 1 H, NH); 7.18–7.40 (m , 10 H). 13C NMR (75 MHz, CDCl3): 16.1; 38.3; 53.1; 56.0;
2
1
61.0 (t, JCF = 23.0); 67.2; 112.8 (t, JCF = 249.0); 127.2; 128.0; 128.3; 128.6; 128.7; 129.4;
136.1; 156.1; 169.3 (d, JCF = 3); 171.1. 19F NMR (282 MHz, CDCl3): –52.49 (ddABX 2JFF
, =
3
282.0, JFH = 56.0, 1 F); –50.52 (ddABX,
2JFF = 282.0, JFH = 56.0, 1 F).
2
2
Meth yl ambo-N-{N-[(Ben zyloxy)carbon yl]-O-tert-butyltyrosin yl}-2-(difluorom eth yl)-
alan in ate (6f)
Method B: Yield 0.34 g (72%); m ixture of diastereom ers. MS (Maldi-TOF), calculated for
C23H34N2F2O6: 472.53; foun d: 495.4 [M + Na]+; 511.4 [M + K]+. Oil. RF 0.25 (AcOEt/h exan es
1 : 3). 1H NMR (300 MHz, CDCl3): 1.32 (s, 9 H); 1.42 (s, 9 H); 1.48/1.50 (s, 3 H); 2.99 (m , 2 H);
2
3.78 (s, 3 H); 4.30 (m , 1 H); 5.03 (m , 1 H, NH); 6.18/6.20 (t, JFH = 56.0, 1 H); 6.55/6.62 (s, 1 H,
NH); 6.93 (d, JHH = 8.3, 2 H); 7.10 (dd, JHH = 1.9, 8.3, 2 H). 13C NMR (APT; 75 MHz, CDCl3):
2
16.0; 28.2; 28.8; 37.3; 52.9; 55.4/55.5; 60.8 (t, JCF = 24.0); 78.3; 80.3/80.4; 112.7/112.8 (t,
1JCF = 247.0); 124.3; 129.8; 131.1/131.2; 154.4; 155.6; 169.4/169.5; 171.6. 19F NMR
2
(282 MHz, CDCl3): –47.51/–47.26 (ddABX
,
2JFC = 282.0, JFH = 56.0, 1 F); –45.08/–45.00
2
(ddABX,
2JFC = 282.0, JFH = 56.0, 1 F).
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1554
Michel et al.:
Meth yl N-{N-[(tert-Butyloxy)carbon yl]glycyl}-(RS)-2-(difluorom eth yl)-
ph en ylalan in ate (6g)
Method B: Yield 0.26 g (67%). MS (FAB, m/z), calculated for C18H24N2F2O5: 386.39; foun d:
387.4 [M + H]+. Oil. RF 0.16 (AcOEt/h exan es 1 : 3). 1H NMR (200 MHz, CDCl3): 1.42 (s, 9 H);
2
2
3.31 (d, JHH = 13.5, 1 H); 3.76 (m , 2 H); 3.81 (d, JHH = 12.8, 1 H); 3.86 (s, 3 H); 5.05 (s, br,
2
1 H, NH); 6.51 (t, JHH = 55.5); 6.93 (s, 1 H, NH); 7.03–7.07 (m , 2 H); 7.27 (m , 3 H).
13C NMR (75 MHz, CDCl3): 28.2; 34.8 (d, JCF = 3.0), 44.7; 53.1; 66.3 (t, JCF = 22); 80.4;
3
2
3
112.8 (ddABX
,
1JCF = 250.0); 127.5; 128.6; 129.8; 133.6; 155.9; 168.3 (d, JCF = 3.0); 170.0.
19F NMR (188 MHz, CDCl3): –50.50 (ddABX
,
2JFF = 281.0, JHF = 56.0, 1 F); –49.37 (ddABX
,
2
2JFF = 281.0, JHF = 56.0, 1 F).
2
tert-Butyl ambo-N-(N-{N-[(Fluoren -9-ylm eth oxy)carbon yl]leucyl}-(R)-2-(difluorom eth yl)-
alan yl)prolin ate (7a/ 1)
To a solution of (R)-H-(α-CF2H)Ala-Pro-OtBu (0.29 g, 1 m m ol), prepared from 3c/ 1, in a 2 : 1
m ixture of CH2Cl2/DMF (5 m l) Fm oc-Leu-F (0.90 g, 2.5 m m ol) an d DIEA (0.32 g, 2.5 m m ol)
were added at room tem perature. Th e m ixture was stirred for 16 h . Th en th e pH was ad-
justed to 3–4 on addition of citric acid (10% aqueous solution ) an d th e m ixture was ex-
tracted with CH2Cl2 (3 ×). Th e com bin ed organ ic layer was wash ed with citric acid, with
saturated NaCl solution , with H2O, an d fin ally dried with an h ydrous NaSO4. After evapora-
tion of th e solven t in vacuo, 7a/ 1 was purified by flash ch rom atograph y. MS (MALDI-TOF),
calculated for C34H43F2N3O6: 627.72; foun d: 650.6 [M + Na]+; 666.6 [M + K]+. Yield 0.49 g
(78%). M.p. 201–202 °C. [α ]2D5 –77 (c 1, CH2Cl2). RF 0.53 (AcOEt/h exan es 1 : 1). 1H NMR
(300 MHz, CDCl3): 0.95 (m , 6 H, Leu H); 1.44 (s, 9 H); 1.45–2.00 (m , 7 H); 1.60 (s, 3 H);
3
3.54 (m ); 4.19 (m , 2 H); 4.42 (m , 3 H); 5.12 (d, JHH = 7.4, NH); 6.34 (s, 1 H, NH); 6.40 (t,
2
3
1 H, JHF = 56.2); 7.30–7.80 (8 H). 13C NMR (50 MHz): 15.8 (t, JCF = 3.0); 22.0; 22.8; 24.6;
2
1
25.6; 27.9; 28.0; 40.9; 47.1; 47.2; 53.4; 60.6 (t, JCF = 22.0); 61.2; 67.4; 81.2; 113.7 (t, JCF
249.0); 120.1; 125.0; 127.2; 127.8; 141.3; 143.7; 156.4; 166.6; 171.3; 172.1. 19F NMR
(282 MHz, CDCl3): –52.80 (ddABX
2JHF = 56.0, 1 F).
=
2
,
2JFF = 282.0, JHF = 56.0, 1 F); –51.68 (ddABX
,
2JFF = 282.0,
tert-Butyl ambo-N-(N-{N-[(Fluoren -9-ylm eth oxy)carbon yl]leucyl}-(S)-2-(difluorom eth yl)-
alan yl)prolin ate (7a/ 2)
Syn th esis from (S)-H-(α-CF2H)Ala-Pro-OtBu (0.29 g, 1 m m ol), prepared from 3c/ 2 an d
Fm oc-Leu-F (0.90 g, 2.5 m m ol). Yield 0.48 g (77%). M.p. 178–180 °C. [α ]2D5 –44 (c 1, CH2Cl2).
RF 0.46 (AcOEt/h exan es 1 : 1). 1H NMR (300 MHz, CDCl3): 0.94 (m , 6 H); 1.45 (s, 9 H);
1.40–2.15 (m , 7 H); 1.73 (s, 3 H); 3.66 (m , 2 H); 4.21 (m , 2 H); 4.40 (m , 3 H); 5.35 (m , 2 H,
2
NH); 6.31 (t, JHF = 56.0, 1 H); 7.26–7.77 (8 H). 13C NMR (75 MHz, CDCl3): 16.6; 21.8, 22.9;
2
24.7; 25.7; 29.9; 27.9; 41.2; 47.1; 47.7; 53.8; 62.2 (t, JCF = 22.0); 62.2; 67.3; 81.5; 114.4 (t,
1JCF = 248.0); 120.0; 125.0; 127.1; 127.8; 141.3; 143.7; 156.3; 166.7; 171.2; 172.4. 19F NMR
2
(282 MHz, CDCl3): –50.89 (ddABX
2JHF = 56.0, 1 F).
,
2JFF = 282.0, JHF = 56.0, 1 F); –48.95 (ddABX
,
2JFF = 282.0,
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1555
ambo-N-(N-{N-[(Ben zyloxy)carbon yl]ph en ylalan yl}-(S)-2-(difluorom eth yl)alan yl)-
alan in am ide (7b/ 1)
A solution of th e N-protected dipeptide ester (2 m m ol) in m eth an ol (3 m l) after addition of
a solution of LiOH/H2O (0.42 g, 10 m m ol) in a 2 : 1 H2O/CH3OH m ixture (12 m l) was
stirred at 5 °C for 15 h . Th e reaction m ixture was con cen trated in vacuo, adjusted to pH 3–4
by addition of dilute citric acid, an d extracted with eth er (3 × 25 m l). After dryin g with an -
h ydrous MgSO4, th e organ ic solven t was evaporated in vacuo. Th e crude com poun d was di-
rectly used for th e n ext reaction step. To a solution of th e crude N-protected dipeptide in
a 2 : 1 solven t m ixture of CH2Cl2/DMF (7 m l), DIC (0.27 g, 2.1 m m ol) an d HOAt (0.29 g,
2.1 m m ol) were added at room tem perature. After 10 m in H-Ala-NH2 (0.18 g, 2 m m ol) was
added. Th e reaction m ixture was stirred un til th e startin g m aterial was con sum ed (19F NMR
an alysis). Th en th e reaction m ixture was adjusted to pH 3–4 by addition of dilute citric acid
an d extracted with CH2Cl2 (3 × 35 m l). Th e com bin ed organ ic layer was wash ed successively
with dilute citric acid, con cen trated NaCl solution , NaHCO3 solution (5%) an d fin ally with
water. After dryin g with an h ydrous MgSO4, th e organ ic solven t was evaporated in vacuo, th e
peptides were purified by recrystallization from CHCl3/h exan es or by flash ch rom atograph y.
7b / 1 was syn th esized from Z-Ph e-(S)-(α-CF2H)Ala-OMe (6d / 1) (0.87 g, 2 m m ol) an d
H-Ala-NH2 (0.18 g, 2 m m ol). MS (MALDI-TOF), calculated for C24H28N4F2O5: 490.50; foun d:
513.0 [M + Na]+; 529.0 [M + K]+. Yield 0.64 g (65%). M.p. 184 °C. [α]D +8 (c 1, CH3OH). RF
3
0.10 (CHCl3/CH3OH 20 : 1). 1H NMR (300 MHz, CDCl3): 1.38 (d, JHH = 7.3, 3 H); 1.43 (s,
3
3
3 H); 2.95 (dd, JHH = 8.3, 14.0, 1 H); 3.12 (dd, JHH = 6.8, 14.0, 1 H); 4.32 (m , 2 H); 5.01 (d,
2JHH = 12.1, 1 H); 5.10 (d, JHH = 12.1, 1 H); 5.66 (m , 2 H, 2 NH); 6.22 (t, JFH = 56.0, 1 H);
2
2
2
6.76 (s, 1 H, NH); 7.06 (s, 1 H, NH); 7.19 (d, JHH = 6.1, 1 H, NH); 7.26–7.33 (m , 10 H).
13C NMR (75 MHz, CDCl3): 17.2 (t, JCF = 3.0); 21.1; 37.3; 49.5; 57.4; 61.4 (t, JCF = 21.0);
3
2
1
67.5; 113.8 (t, JCF = 249); 127.5; 128.0; 128.5; 128.7; 129.0; 129.1; 135.5; 135.8; 156.9;
168.9; 172.6; 174.9. 19F NMR (282 MHz, CDCl3): –51.42 (d, JFH = 56.0, 1 F); –51.37 (d,
2
2JFH = 56.0, 1 F).
ambo-N-(N-{N-[(Ben zyloxy)carbon yl]ph en ylalan yl}-(R)-2-(difluorom eth yl)alan yl)-
alan in am ide (7b/ 2)
7b / 2 was syn th esized from Z-Ph e-(R)-(α-CF2H)-Ala-OMe (6d / 2) (0.87 g, 2 m m ol) an d
H-Ala-NH2 (0.18 g, 2 m m ol). Yield 0.70 g (72%). M.p. 152–153 °C. [α]D –8.3 (c 1, CH3OH).
3
RF 0.08 (CHCl3/CH3OH 20 : 1). 1H NMR (300 MHz, CDCl3): 1.36 (d, JHH = 7.14, 3 H); 1.58 (s,
3
3
3 H); 2.95 (dd, JHH = 8.7, 14.2, 1 H); 3.12 (dd, JHH = 5.7, 14.2, 1 H); 4.30 (m , 1 H); 4.41 (m ,
3
3
1 H); 5.00 (d, JHH = 12.1, 1 H); 5.13 (d, JHH = 12.1, 1 H); 5.53 (s, 1 H, NH); 5.58 (s, 1 H,
NH); 5.94 (t, JFH = 55.1, 1 H); 6.89 (s, 2 H, NH); 7.18–7.35 (m , 10 H).19F NMR (282 MHz,
2
2
2
CDCl3): –53.20 (ddABX
1 F).
, ,
2JFF = 274.0, JFH = 55.0, 1 F); –49.96 (ddABX 2JFF = 274.0, JFH = 55.0,
N-(N-{N-[(Ben zyloxy)carbon yl]-(R)-ph en ylalan yl}-(R)-2-(difluorom eth yl)alan yl)-
(R)-alan in am ide (7c/ 1)
7c/ 1 was syn th esized from Z-(R)-Ph e-(R)-(α-CF2H)Ala-OMe (6e/ 1) (0.87 g, 2 m m ol) an d
(R)-H-Ala-NH2 (0.18 g, 2 m m ol). MS (MALDI-TOF), calculated for C24H28N4F2O5: 490.50;
foun d: 513.0 [M + Na]+; 529 [M + K]+. Yield 0.86 g (88%). M.p. 166–168 °C (decom p.). [α]D
–7.4 (c 1, CH3OH). RF 0.12 (CHCl3/CH3OH 20 : 1). 1H NMR (300 MHz, CDCl3): 1.35 (d,
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

1556
Michel et al.:
3
3JHH = 7.4, 3 H); 1.43 (s, 3 H); 2.95 (m , 1 H); 3.12 (dd, JHH = 7.6, 14.0, 1 H); 4.33 (m , 2 H);
2
2
4.99 (d, JHH = 12.3, 1 H); 5.07 (d, JHH = 12.3, 1 H); 5.88 (s, 1 H, NH); 6.18 (s, 1 H, NH);
2
6.22 (t, JFH = 56.0, 1 H); 6.80 (s, 1 H, NH); 6.93 (s, 1 H, NH); 7.15–7.34 (m , 11 H, 10 HAr
,
NH). 19F NMR (282 MHz, CHCl3): –51.82 (ddABX
,
2JFF = 278.0, JFH = 56.0, 1 F); –50.97
2
2
(ddABX
,
2JFF = 278.0, JFH = 56.0, 1 F).
N-(N-{N-[(Ben zyloxy)carbon yl]-(R)-ph en ylalan yl}-(S)-2-(difluorom eth yl)alan yl)-
(R)-alan in am ide (7c/ 2)
7c/ 2 was syn th esized from Z-(R)-Ph e-(S)-(α-CF2H)Ala-OMe (6e/ 2) (0.87 g, 2 m m ol) an d
(R)-H-Ala-NH2 (0.18 g, 2 m m ol). Yield 0.80 g (82%). Oil. [α]D +9 (c 1, CH3OH). RF 0.07
3
(CHCl3/CH3OH 1 : 20). 1H NMR (300 MHz, CDCl3): 1.39 (d, JHH = 7.1, 1 H); 1.63 (s, 3 H);
3
3
2.95 (dd, JHH = 8.6, 15.0, 1 H); 3.12 (dd, JHH = 5.2, 13.0, 1 H); 4.31 (m , 1 H); 4.43 (m , 1 H);
2
3
5.01 (d, JHH = 12.1, 1 H); 5.14 (dd, JHH = 12.1, 1 H); 5.45 (s, 1 H, NH); 5.56 (s, 1 H, NH);
6.94 (t, JFH = 56.0, 1 H); 6.82 (s, 1 H, NH); 6.94 (s, 1 H, NH); 7.19 (d, JHH = 6.2, 1 H, NH);
7.26–7.34 (m , 10 H). 19F NMR (282 MHz, CDCl3): –53.29 (ddABX
2
3
2
,
2JFF = 273.0, JFH = 56.0,
2
1 F); –49.90 (ddABX
,
2JFF = 273.0, JFH = 56.0, 1 F).
tert-Butyl ambo-N-(N-{N-[(Fluoren -9-ylm eth oxy)carbon yl]leucyl}-(S)-2-(ch lorodifluorom eth yl)-
alan yl)prolin ate (8/ 1)
8/ 1 was syn th esized from Fm oc-Leu-OH (0.71 g, 2 m m ol) an d (S)-H-(α-CF2Cl)Ala-Pro-OtBu
(4c/ 1) (0.65 g, 2 m m ol). MS (MALDI-TOF), calculated for C34H42N3ClF2O6: 662.17; foun d:
684.4 [M + Na]+; 700.4 [M + K]+. Yield 0.83 g (63%). M.p. 158–159 °C. [α ]2D5 –54 (c 0.5,
MeOH). RF 0.33 (AcOEt/h exan es 1 : 2). 1H NMR (300 MHz, CDCl3): 0.91 (m , 6 H); 1.43 (s,
9 H); 1.25–2.00 (m , 7 H); 1.94 (s, 3 H); 3.53 (m , 1 H); 3.69 (m , 1 H); 4.21 (m , 2 H); 4.42 (m ,
3
3 H); 5.19 (d, JHH = 6.3, 1 H, NH); 7.11 (s, 1 H, NH); 7.25–7.80 (m , 8 H). 19F NMR
(282 MHz, CDCl3): 17.41 (2JFF = 165.0, 1 F); 19.00 (2JFF = 165.0, 1 F).
tert-Butyl ambo-N-(N-{N-[(Fluoren -9-ylm eth oxy)carbon yl]leucyl}-(R)-2-(ch lorodifluorom eth yl)-
alan yl)prolin ate (8/ 2)
8/ 2 was syn th esized from Fm oc-Leu (0.71 g, 2 m m ol) an d (R)-(α-CF2Cl)Ala-Pro-OtBu (4c/ 2)
(0.65 g, 2 m m ol). Yield 0.76 g (57%). M.p. 178 °C (decom p.). [α ]2D5 –143 (c 1, MeOH). RF 0.33
(AcOEt/h exan es 1 : 2). 1H NMR (300 MHz, CDCl3): 0.95 (m , 6 H); 1.44 (s, 9 H); 1.40–2.00
3
(m , 7 H); 1.85 (s, 3 H); 3.54 (m , 2 H); 4.21 (m , 2 H); 4.42 (m , 3 H); 5.14 (d, JHH = 7.4, 1 H,
3
NH); 6.72 (s, 1 H, NH); 7.25–7.80 (m , 8 H). 13C NMR (APT; 75 MHz, CDCl3): 18.4 (t, JCF
=
2
3.0, CH3); 22.0; 22.8; 24.6; 25.8; 27.8; 28.0; 40.5; 47.1; 47.7; 53.9; 61.8; 65.8 (t, JCF = 22.0);
1
67.3; 81.2; 122.1 (t, JCF = 308.0); 120.1; 125.0; 127.1; 127.9; 141.4; 143.7; 156.5; 163.7;
171.1. 19F NMR (282 MHz, CDCl3): 18.55 (2JFF = 162.0, 1 F); 18.98 (2JFF = 162.0, 1 F).
ambo-N-[N-(N-{N-[(tert-Butoxy)carbon yl]-O-tert-butyltyrosin yl}-2-(difluorom eth yl)alan yl)-
ph en ylalan yl]ph en ylalan in am ide (9)
Syn th esis from ambo-N-Boc-OtBu-Tyr-(α-CF2H)Ala-OMe (0.95 g,
2 m m ol) (6f) an d
H-Ph e-Ph e-NH2 (0.62 g, 2 m m ol). MS (MALDI-TOF), calculated for C40H51N5F2O7: 751.87;
foun d: 774.3 [M + Na]+; 790.3 [M + K]+. Yield 1.17 g (78%); 1 : 1 m ixture of two diastereo-
m ers. Oil. RF 0.22 (CHCl3/CH3OH 20 : 1). 1H NMR (300 MHz, CDCl3): 1.13/1.16 (s, 9 H);
Collect. Czech. Chem. Commun. (Vol. 67) (2002)

Peptide Synthesis
1557
1.32/1.35 (s, 9 H); 1.54/1.58 (s, 3 H); 2.65–3.63 (m , 6 H); 4.03 (m , 1 H); 4.78 (m , 1 H); 4.91
2
(m , 1 H); 6.18 (t, JFH = 56.0, 1 H); 6.57 (s, 1 H, NH); 6.62 (s, 1 H, NH); 6.84 (s, 1 H, NH);
6.95–7.40 (m , 16 H, HAr, NH); 7.44 (s, 1 H, NH). 19F NMR (282 MHz, CDCl3): –52.55/-52.91
2
2
(ddABX, ,
2JFF = 282.0, JFH = 56.0, 1 F); –51.21 (ddABX 2JFF = 282.0, JFH = 56.0, 1 F).
Data Collection an d Structural Refin em en t for 7a/ 1, 7c/ 1 an d 8/ 1
Crystallograph ic data are given in Table I. Th e data (λ MoKα = 0.71073 Å) were collected
with a CCD diffractom eter (AXS Bruker). All observed reflection s are used for determ in ation
of th e un it cell param eters. Em pirical absorption correction was carried out with SADABS 35
Structures were solved by direct m eth ods. Th e structures were refin ed an isotropically for all
n on -h ydrogen atom s usin g program SHELX97 36. Hydrogen atom s were refin ed in calculated
position s.
.
CCDC 187347 (for com poun d 7a/ 1), CCDC 187349 (for com poun d 7c/ 1) an d CCDC
187348 (for com poun d 8/ 1) con tain th e supplem en tary crystallograph ic data for th is paper.
Th ese data can be obtain ed free of ch arge via www.ccdc.cam .ac.uk/con ts/retrievin g.h tm l
(or from th e Cam bridge Crystallograph ic Data Cen tre, 12, Un ion Road, Cam bridge, CB2
1EZ, UK; fax: +44 1223 336033; or deposit@ccdc.cam .ac.uk).
We thank the Deutsche Forschungsgemeinschaft, the Stiftung Volkswagenwerk and the Fonds der
Chemischen Industrie for financial support and Aventis AG, Frankfurt/Main for generous supply of
chemicals. Special thanks are due to Ms J. Ortwein for NMR experiments.
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Peptide Synthesis
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Collect. Czech. Chem. Commun. (Vol. 67) (2002)