Structural modifications of the active site in teicoplanin and related glycopeptides. 2. Deglucoteicoplanin-derived tetrapeptide

Article abstract of DOI:10.1021/jo9506746

The deglucoteicoplanin-derived tetrapeptide (TDTP), a key synthon suitable for the synthesis of modified glycopeptide antibiotics differing in the structure of the active site, was prepared from the product (RH-TD) of reductive hydrolysis of the 2,3-peptide bond of deglucoteicoplanin (TD) upon selective oxidation of the newly formed hydroxymethyl group and following simultaneous removal of amino acids 1 and 3 by double Edman degradation. The oxidation of the alcohol function of residue 2 in RH-TD was accomplished (Jones reagent) after protection of the two free amino groups as tert-butyl BOC carbamates and of most of phenolic hydroxy groups as benzyl CBZ carbonates. Esterification of the C-terminal carboxy group of intermediate di-BOC-RH-TD allowed the formation at the end of the process of the tetrapeptide (TDTP-Me) protected at one carboxy group as methyl ester. Selective protection of the primary N⁴- and N²-amino groups of TDTP-Me as BOC and CBZ carbamates, respectively, followed by removal of the BOC function, afforded a more suitable intermediate (N²-CBZ-TDTP-Me) for the synthesis of new glycopeptides.

Full text of DOI:10.1021/jo9506746

J . Org. Chem. 1996, 61, 2151-2157
2151
Str u ctu r a l Mod ifica tion s of th e Active Site in Teicop la n in a n d
Rela ted Glycop ep tid es. 2. Deglu coteicop la n in -Der ived
Tetr a p ep tid e
Adriano Malabarba,* Romeo Ciabatti, Michele Maggini,† Pietro Ferrari, Luigi Colombo, and
Maurizio Denaro
Marion Merrell Dow Research Institute, Lepetit Center, Via R. Lepetit 34, 21040 Gerenzano (Varese), Italy
Received April 10, 1995 (Revised Manuscript Received October 8, 1995X)
The deglucoteicoplanin-derived tetrapeptide (TDTP ), a key synthon suitable for the synthesis of
modified glycopeptide antibiotics differing in the structure of the active site, was prepared from
the product (RH-TD) of reductive hydrolysis of the 2,3-peptide bond of deglucoteicoplanin (TD)
upon selective oxidation of the newly formed hydroxymethyl group and following simultaneous
removal of amino acids 1 and 3 by double Edman degradation. The oxidation of the alcohol function
of residue 2 in RH-TD was accomplished (J ones reagent) after protection of the two free amino
groups as tert-butyl BOC carbamates and of most of phenolic hydroxy groups as benzyl CBZ
carbonates. Esterification of the C-terminal carboxy group of intermediate d i-BOC-RH-TD allowed
the formation at the end of the process of the tetrapeptide (TDTP -Me) protected at one carboxy
group as methyl ester. Selective protection of the primary N4- and N2-amino groups of TDTP -Me
as BOC and CBZ carbamates, respectively, followed by removal of the BOC function, afforded a
more suitable intermediate (N2-CBZ-TDTP -Me) for the synthesis of new glycopeptides.
The mechanism of action of glycopeptide antibiotics1
in part depends on the structure of amino acids 1 and 3.
A current strategy to overcome the emerging resistance
to glycopeptides in pathogenic bacteria2 is based on the
replacement of these amino acids with new amino acids
or other moieties suitably selected to interact with the
modified target3 present in resistant organisms. The
recent discovery of a selective method for the reductive
hydrolysis of the amide bond between amino acids 2 and
3 in teicoplanin, its pseudoaglycons and aglycon (TD,
Figure 1), and related glycopeptides, with sodium boro-
hydride in EtOH/H2O 35/65 solution,4 provided the op-
portunity to remove amino acids 1 and 3 by Edman
degradation. The resulting tetrapeptide derivatives are
potential synthons for the synthesis of new families of
glycopeptides differing in the structure of their active
site.5
F igu r e 1. Structure of TD and RH-TD (with proton nomen-
clature).
In this paper, the preparation of the deglucoteicopla-
nin-derived tetrapeptide (TDTP , Figure 2) from the
product (RH-TD) of reductive hydrolysis of the 2,3-
peptide bond of TD is described. Suitable methods for
the selective protection and deprotection of the two
primary amino groups and of the C-terminal carboxy
group of the tetrapeptide chain are also reported.
Resu lts a n d Discu ssion
Two different synthetic pathways could be followed to
prepare TDTP from RH-TD.
(i) Procedure A (Scheme 1): the amino and phenolic
hydroxy groups of RH-TD are properly protected before
oxidation of the primary alcohol function of residue 2.
After deprotection, the resulting pentapeptide derivative
F igu r e 2. Structure of TDTP and TDTPA.
(TDP P ) is submitted to a double Edman degradation6
step for the simultaneous removal of amino acid frag-
ments 1 and 3.
(ii) Procedure B (Scheme 2): it consists of Edman
degradation of RH-TD as a preliminary step. The
resulting tetrapeptide alcohol (TDTP A, Figure 2) is then
oxidized after proper protection of the susceptible amino
and phenolic functions. Final deprotection steps give
TDTP .
† Present address: Department of Organic Chemistry, University
of Padova, Via Marzolo, 1-35100 Padova, Italy.
X Abstract published in Advance ACS Abstracts, February 15, 1996.
(1) Parenti, F.; Cavalleri, B. Drugs Future 1990, 15, 57.
(2) J ohnson, A. P.; Uttley, A. H. C.; Woodford, N.; George, R. C. Clin.
Microbiol. Rev. 1990, 3, 280.
(3) Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep., 1992, 9, 199.
(4) Malabarba, A.; Ciabatti, R.; Kettenring, J .; Ferrari, P.; Ve´key,
K.; Bellasio, E.; Denaro, M. J . Org. Chem. Submitted for publication.
(5) Malabarba, A.; Ciabatti, R. J . Med. Chem. 1994, 37, 2988.
(6) Edman, P. Acta Chem. Scand. 1950, 4, 277.
0022-3263/96/1961-2151$12.00/0 © 1996 American Chemical Society

2152 J . Org. Chem., Vol. 61, No. 6, 1996
Sch em e 1. Syn th esis of TDTP (P r oced u r e A)
Malabarba et al.
Sch em e 2. Syn th esis of TDTP (P r oced u r e B)
As reported below, although these approaches were
both successful, procedure A was more suitable to prepare
TDTP when the phenolic hydroxy groups were protected
as benzyl carbonates.
P r ot ect ion of t h e Oxid a t ion -Sen sit ive F u n c-
tion a l Gr ou p s in RH-TD a n d TDTP A. In RH-TD and
TDTP A there are several functions, besides the hy-
droxymethyl moiety of residue 2, which are potentially

Deglucoteicoplanin-Derived Tetrapeptide
J . Org. Chem., Vol. 61, No. 6, 1996 2153
susceptible to oxidation: the two primary amino groups,
the benzylic OH of residue 6, and all the phenolic hydroxy
groups. Preliminary tests carried out on N1-BOC-TD
methyl ester,7 suitably permethylated8 at the phenolic
OH’s, showed that the benzylic OH of residue 6 was
resistant to oxidation under both basic and acidic condi-
tions and that only five of the six phenolic OH’s needed
protection.9
nation under hydrogenation conditions (1 atm, 5% Pd/
C) used to remove the CBZ groups from Zn -d i-BOC-
TDTP .
It follows that procedure A is more suitable than B, in
terms of overall yields and number of reaction steps, for
preparing TDTP when using the CBZ as phenol-protect-
ing group. Although the CBZ moiety could be also used
for the protection of the amino groups, the BOC group
was preferred in this case as it was more easily removed
from the N-carbamate derivatives (d i-BOC-TDP P or d i-
BOC-TDTP ) than the CBZ group.13,14
Oxid a tion of th e Hyd r oxym eth yl F u n ction to
Ca r boxy Gr ou p . Several conditions were tried before
selecting the most suitable method for the oxidation of
the hydroxymethyl group of residue 2 of Zn -d i-BOC
derivatives of RH-TD and TDTP A. Due to the protec-
tion of the phenolic OH’s as benzyl carbonates, basic
conditions had to be avoided. Different oxidation re-
agents were investigated under the more appropriate
acidic conditions. When CrO3 in glacial or aqueous AcOH
was used, a large excess of oxidizer was necessary to
complete the reaction. The best results were obtained
by treatment of the above protected tetra- or pentapep-
tide-alcohol derivatives with the J ones reagent (H2-
CrO4-H2SO4/H2O)15 at room temperature in THF solu-
tion. Under these conditions, the oxidation was fast and
occurred without deprotection/decomposition of amino
groups or phenolic OH’s.
E d m a n Degr a d a t ion of R H-TD a n d TDP P . The
diphenyl ether 1,3-diamino acid was removed from RH-
TD or TDP P by a double Edman procedure. Accord-
ingly, reaction of RH-TD or TDP P with 2.5 equiv of
phenyl isothiocyanate in pyridine/water 1/1 afforded the
corresponding mixed N1,N3-di-isothiourea derivative which,
upon treatment with dry TFA at room temperature, gave
tetrapeptide TDTP A or TDTP in high yields.16
Selective P r otection of On e Ca r boxy a n d th e Tw o
P r im a r y Am in o Gr ou p s in TDTP . TDTP is a poten-
tial intermediate for the synthesis of new families of
glycopeptides by introduction of appropriate amino acids
in positions 1 and 3. This process would require protec-
tion of one amino group of TDTP prior to coupling of the
other primary amine with the carboxy function of a
N-protected amino acid. The protection of the carboxy
group of residue 7 of TDTP is also necessary to prevent
intermolecular side reactions in the final macrocycliza-
tion step to give a new hexapeptide or heptapeptide
compound.
The two free amino groups were protected as tert-butyl
carbamates by reaction of RH-TD (Scheme 1) or TDTP A
(Scheme 2) with di-tert-butyl dicarbonate [(BOC)2O] at
room temperature in dioxane/water 1/1 solution, in the
presence of NaHCO3. The BOC protective group was
resistant to oxidation, and in the final deprotection step,
it was removed from d i-BOC-TDP P or d i-BOC-TDTP
by mild acidic treatment (TFA), giving TDP P or TDTP
with highest yields. The benzyloxycarbonyl (CBZ) group
was found to be the most appropriate for the selective
protection of the phenolic OH’s of d i-BOC-R H -TD
(Scheme 1). It was resistant to oxidation with the J ones
reagent10 and easily removed from resulting per-protected
compound (Zn -d i-BOC-TDP P ) under hydrogenolysis con-
ditions (1 atm, 5% Pd/C) which were well tolerated by
the rest of the molecule. The per-CBZ-protected deriva-
tive (Zn -d i-BOC-RH-TD) was prepared by reaction of d i-
BOC-RH-TD with a large excess of benzyl chloroformate
(CBZ-Cl) at room temperature in dioxane/water 1/1
solution, in the presence of Cs2CO3.11 The CBZ group
was also used to protect the phenolic OH’s in d i-BOC-
TDTP A (Scheme 2), but in this case12 a preliminary
protection of the primary alcohol of residue 2 was
necessary to prevent coupling of the hydroxymethyl
function with the CBZ group. This was accomplished by
forming an oxazolidine ring upon reaction of the N-BOC-
ethanolamine moiety of residue 2 with 2,2-dimethoxypro-
pane in anhydrous Me2CO at room temperature in the
presence p-toluenesulfonic acid (Ts-OH) as the condensa-
tion catalyst. Protection of the phenolic-OH’s with CBZ-
Cl under anhydrous conditions (DMSO) in the presence
of Cs2CO3 yielded the per-CBZ phenyl carbonate deriva-
tive Zn -d i-BOC-TDTP A-oxa zolid in e. The hydroxym-
ethyl group was then regenerated by mild acidic treat-
ment (1 N HCl, 25 °C, 90 min) to give Zn -d i-BOC-
TDTP A which was amenable to oxidation (Jones reagent)
to afford Zn -d i-BOC-TDTP . In contrast to the pen-
tapeptide (d i-BOC-TDP P ), tetrapeptide d i-BOC-TDTP
once formed was in part (∼20%) susceptible to dechlori-
A TDTP derivative (N2-CBZ-N4-BOC-TDTP -Me) se-
lectively protected at the C-terminus of the tetrapeptide
chain as methyl ester and at the amino groups of residues
2 and 4 as CBZ and BOC carbamates, respectively, was
obtained according to the procedure of Scheme 3. Es-
terification of the carboxy group of d i-BOC-RH-TD with
MeI in DMF in the presence of KHCO3 provided a methyl
(7) Malabarba, A.; Trani, A.; Ferrari, P.; Pallanza, R.; Cavalleri, B.
J . Antibiot. 1987, 40, 1572.
(8) The phenolic-OH’s of N1-BOC-TD methyl ester were protected
as methyl ethers using a large excess of MeI in DMF, in the presence
of K2CO3.
(9) The number of phenolic-OCH3’s present in permethylated N1-
BOC-TD methyl ester was determined by NMR. The position of the
unprotected phenolic group resistant to oxidation was not determined.
(10) (a) Bowden, K.; Heibron, I. M.; J ones, E. R. H.; Weedon, B. C.
L. J . Chem. Soc. 1946, 39. (b) Bladon, P.; Fabian, J . M.; Henbest, H.
B.; Koch, H. B.; Wood, G. W. J . Chem. Soc. 1951, 2402. (c) Bowers, A.;
Halsall, T. G.; J ones, E. R. H.; Lemin, A. J . J . Chem. Soc. 1953, 2548.
(11) Suitable protection of the phenolic groups was also obtained in
anhydrous conditions, in DMSO/THF 4/1 solution, using a large excess
of K2CO3 and/or TEA as the acid-acceptor agents, but with longer
reaction times. The use of K2CO3 in aqueous media was unsuitable
since the CBZ moiety on ring 4 was susceptible, once formed, to
hydrolysis under the resulting basic reaction conditions. Another
advantage of the Cs2CO3 method is that the reaction mixture dioxane/
water is the same used in the preparation of di-BOC carbamates, thus
allowing a one-pot synthesis of Zn -d i-BOC-RH-TD from RH-TD.
(12) In d i-BOC-RH-TD the protection of the hydroxymethyl group
of residue 2 was unnecessary.
(13) Under the conditions used (H2, 1 atm., 10% Pd/C) in the
hydrogenolysis of the N′,N′′-di-CBZ derivatives of TDP P and TDTP
the chlorine atoms on rings 2 and 6 were also affected, to a greater
extent in TDTP than TDP P , leading to dechlorinated byproducts.
(14) The number of phenolic-OH’s which could be protected as BOC
carbonates was insufficient, under all tried conditions, for the oxidation
step.
(15) The J ones reagent, “standard” solution, was prepared according
to the procedure described in Fieser & Fieser: Reagents for Organic
Synthesis; Vol. 1: J ohn Wiley & Sons, Inc.: New York, 1967; Vol. 1, p
142.
(16) Although the 1,3-diphenyl ether dihydantoine moiety was not
isolated, its formation can be inferred according to the well-known
mechanism of Edman degradation.

2154 J . Org. Chem., Vol. 61, No. 6, 1996
Malabarba et al.
Sch em e 3. Syn th esis of N2-CBZ-N4-BOC-TDTP -Me, N4-BOC-TDTP -Me a n d N2-CBZ-TDTP -Me
ester derivative (d i-BOC-RH-TD-Me) which, after pro-
tection of the phenolic functions as benzyl carbonates,
oxidation of the residue 2-CH2OH, and deprotection of
the phenolic and amino groups, gave the pentapeptide
monomethyl ester TDP P -Me. Edman degradation of
TDP P -Me afforded the tetrapeptide monomethyl ester
TDTP -Me. The amino group of residue 4, more nucleo-
philic17 than that of residue 2, was selectively protected
as BOC carbamate upon treatment of TDTP -Me with
an equimolecular amount of (BOC)2O at -5 °C in a water/
dioxane 1/1 mixture in the presence of NaHCO3. Under
these conditions, the mono-N4-BOC derivative (N4-BOC-
TDTP -Me) was obtained with high (>95%) yields, while
the disubstituted N2,N4-d i-BOC-TDTP -Me was the
main product (>50%) when the reaction was carried out
at room temperature. Final triprotected derivative N2-
CBZ-N4-BOC-TDTP -Me was prepared by reaction of N4-
BOC-TDTP -Me with CBZ-Cl (1 equiv, room tempera-
ture) in DMF in the presence of KHCO3. The selective
removal of the BOC protective group from N2-CBZ-N4-
BOC-TDTP -Me (TFA, 0 °C) afforded a further dipro-
tected compound (N2-CBZ-TDTP -Me) particularly suit-
able for the selective introduction of new amino acids in
position 3.18
FAB-MS spectrometry. Among protected intermediates,
only the d i-BOC derivatives of TDP P and TDTP were
1
purified and their structures confirmed by H NMR and
FAB-MS.19
1
The most characteristic H NMR signals of the core
molecules and their protecting groups, when present, are
given in Table 1 in comparison with the proton assign-
ments for TD hydrochloride. With respect to the TD
spectrum, strong changes are found, as expected, in the
right-hand part of the structure of TDTP , in particular
in the chemical shift values of protons x2, x4, 4b, and to
a lesser extent, 4f and x5. The remaining left-hand part
of the molecule of TDTP appears to be substantially
unchanged. The differences in their 1H NMR spectra
between TDTP and TDTP A are mainly shown by a
strong chemical shift change of proton x2 and by a minor
variation in the chemical shift of proton 4b. Significant
changes are also present in the spectrum of TDP P with
respect to that of TD, in the region from x1 to x4 and 4b.
The attributions were based on the phase sensitive
double quantum filter (Bruker COSYPHDQ micropro-
gram) and on the well-established spectra-structure
correlations in the teicoplanin field.20 The FAB MS
Str u ctu r e Elu cid a tion . The structures of TDTP and
TDTP -Me, their synthetic precursors TDTP A, TDP P ,
and TDP P -Me, and the BOC and CBZ derivatives of
TDTP -Me were determined by NMR spectroscopy and
(19) The structure of d i-BOC-RH-TD, which was obtained suf-
ficiently pure for the next reaction steps but was not further purified
for analysis, was confirmed by FAB MS only. The per-protected Zn
compounds and the oxazolidine derivatives of TDTP A, as well as the
diisothiourea intermediates of the Edman degradation procedures,
were only characterized on the basis of their HPLC retention time (tR).
Their chromatographic behavior is in accordance with the proposed
structures.
(20) (a) Barna, J . C. J .; Williams, D. H.; Stone, D. J . M.; Leung,
T.-W. C.; Doddrell, D. M. J . Am. Chem. Soc. 1984, 106, 4895. (b) Hunt,
A. H.; Molloy, R. M.; Occlowitz, J . L.; Marconi, G. C.; Debono, M. J .
Am. Chem. Soc. 1984, 106, 4891. (c) Malabarba, A.; Ferrari, P.; Gallo,
G. G.; Kettenring, J .; Cavalleri, B. J . Antibiot. 1986, 39, 1430.
(17) In TDTP -Me, as well as TDTP , the residue 4-NH2 appeared
to be generally more susceptible to acylation than the residue 2-NH2
upon treatment with different acylating agents. In some cases, this
allowed the selective coupling of the residue 4-NH2 of TDTP -Me or
TDTP with activated N-protected amino acid esters without protection
of the residue 2-NH2 (data not reported).
(18) Malabarba, A.; Ciabatti, R. Int. Patent WO94-26780, Nov 24,
1994 (to Gruppo Lepetit S.p.a.).

Deglucoteicoplanin-Derived Tetrapeptide
J . Org. Chem., Vol. 61, No. 6, 1996 2155
Ta ble 1. Assign m en ts of Sign ifica n t 1H NMR Sign a ls in DMSO-d 6 in Com p a r ison w ith TD (TMS,
In ter n a l Refer en ce, δ in p p m )
di-BOC-
TDPP
di-BOC-
TDTP
N4-BOC- N2-CBZ- N2-CBZ-N4-BOC-
proton
TD
TDPP
TDPP-Me TDTPA
TDTP TDTP-Me TDTP-Me TDTP-Me
TDTP-Me
Phe-CH2 2.87, 3.35 2.97, 2.84 3.12, 3.00 3.12, 3.04
2.90
3.34
4.20
4.46
4.62
5.12
5.16
5.55
6.28
6.43
6.95
8.05
8.53
8.53
9.09
2.99, 2.80 3.07
3.06
4.03
4.20
4.53
4.61
5.16
5.12
5.55
6.09
6.42
6.87
8.45
8.45
8.67
9.16
3.70
3.07
4.10
4.20
4.52
4.52
5.12
5.42
5.50
6.12
6.48
6.52
3.06
4.20
4.20
4.54
4.60
5.15
5.17
5.56
5.98
6.42
6.85
3.05, 2.82
4.19
4.19
4.54
4.54
5.12
5.46
6.09
6.44
x2
x6
x7
x5
z6
x4
4f
7f
7d
4b
w2
w4
w7
w5
4.92
4.10
4.42
4.33
5.10
5.60
5.08
6.24
6.39
5.50
8.10
7.53
8.40
8.38
3.88
4.20
4.39
4.57
5.10
5.22
5.51
4.18
4.23
4.44
4.62
5.11
5.69
5.48
4.17
4.23
4.51
4.62
5.11
5.72
5.46
4.07
4.19
4.46
4.53
5.10
5.42
5.46
6.26
6.42
6.50
4.20
4.22
4.45
4.62
5.12
5.18
5.58
6.28
6.41
6.87
8.35
8.45
8.45
9.12
6.53
6.27
6.27
3.70
COOCH3
CH2(OH)
t-Bu-CH3
CBZ-CH2
x1
3.71
1.27
3.71
5.13
3.69
2.90
1.37, 1.33
1.33, 1.26
1.24
5.11
5.47
4.91
Ta ble 2. An a lytica l Da ta
formula MW (av)
spectra of the above compounds were in accordance with
the proposed structures, as shown in Table 2.
FAB MS
[MH]+
compd
di-BOC-RH-TD
di-BOC-TDPP
TDPP
TDPP-Me
TDTPA
C68H65Cl2N7O22 1403.2244 1402.4 ( 0.1
C68H63Cl2N7O23 1417.2078 1416.3 ( 0.1
C58H47Cl2N7O19 1216.9714 1216.2 ( 0.1
C59H49Cl2N7O19 1230.9984 1230.3 ( 0.1
C42H37Cl2N5O13 890.7036 890.2 ( 0.1
C52H51Cl2N5O18 1104.9235 1104.3 ( 0.1
Exp er im en ta l Section
The 1H NMR experiments were recorded at 500 MHz in
DMSO-d6 solution, added with or without TFA. The positive
ion low-resolution (RP ) 2000; 10% valley definition) FAB MS
spectra were obtained using 8 kV accelerating voltage. The
samples were dissolved in a DMSO/thioglycerol 1/1 mixture.
Reaction products were purified by reversed-phase column
chromatography on silanized silica gel (0.063-0.2 mm), ac-
cording to the following procedure: 1 g of crude compound was
dissolved in 20-30 mL of a MeCN/H2O (1/1) mixture, the
solution was adjusted at pH 5.5 with solid HCO2NH4, and then
H2O was added dropwise under stirring until precipitation
started. After a few drops of MeCN were added, the resulting
cloudy solution was loaded on a column of 50 g of silanized
silica gel in the same solvent mixture. Elution was carried
out according to a linear gradient from 5-10% to 40-70% of
MeCN in H2O, in 10-15 h, at a flow rate of 200-300 mL/h,
while collecting 20 mL fractions; those containing pure com-
pound were pooled, and enough 1-BuOH was added to obtain,
after evaporation of most solvent at 40 °C under reduced
pressure, a concentrated dry butanol solution (or suspension).
Upon addition of Et2O the precipitated solid was collected,
washed with Et2O, and dried in vacuo at room temperature
overnight. Reactions, column eluates, and final products were
checked by HPLC performed on a column (125 × 4 mm)
prepacked with LiChrospher RP-8 (5 µm). Chromatograms
were recorded at 254 nm. Elutions were carried out by mixing
eluent a, MeCN, with eluent b, 0.2% aqueous HCO2NH4,
according to linear gradients programmed as follows:
di-BOC-TDTP
TDTP
C42H35Cl2N5O14 904.687
904.2 ( 0.1
TDTP-Me
C43H37Cl2N5O14 918.7141 918.2 ( 0.1
C48H45Cl2N5O16 1018.8324 1018.2 ( 0.1
C51H43Cl2N5O16 1052.8498 1052.2 ( 0.1
N4-BOC-TDTP-Me
N2-CBZ-TDTP-Me
N2-CBZ-N4-BOC-TDTP-Me C56H51Cl2N5O18 1152.9681 1152.3 ( 0.1
of RH-TD (HPLC, method A, tR 11.6 min) in 400 mL of a
dioxane/water 1/1 mixture was added 20 mL of a 1 M aqueous
solution of NaHCO3 at room temperature followed by a solution
of 9 g (∼40 mmol) of (BOC)2O in 100 mL of the same above
dioxane/water 1/1 mixture. After the mixture was stirred at
room temperature for 5 h, 250 mL of H2O was added, and the
resulting solution was adjusted at pH 4 with 1 N HCl and then
extracted with EtOAc (2 × 200 mL). The organic layer was
washed with H2O (2 × 100 mL), dried over Na2SO4, and
concentrated at room temperature under reduced pressure to
a small volume (∼50 mL). On adding Et2O (450 mL), the
precipitated solid was collected and dried in vacuo at room
temperature overnight to give 27 g (95% yield) of the title
compound: HPLC, method B, tR 11.3 min.
Di-BOC-TDP P . A solution of 22.4 g (∼16 mmol) of the
above compound and of 16 g (∼50 mmol) of Cs2CO3 in 1 L of
a dioxane/water 1/1 mixture was stirred at room temperature
for 1 h, and then a solution of 23 mL (∼160 mmol) of CBZ-Cl
in 100 mL of dry THF was added dropwise over 30 min. After
being stirred at room temperature overnight, the reaction
mixture was poured into a stirred mixture of EtOAc/H2O 1/1
(1.5 L). The aqueous phase was adjusted at pH 3 with 1 N
HCl, the organic layer was separated and dried over Na2SO4,
and then the solvent was evaporated at room temperature
under reduced pressure to give an oily residue which was
slurried with Et2O. The resulting solid material was collected
to yield 27 g of per-CBZ derivative Zn -d i-BOC-RH-TD as a
crude mixture of two main compounds (HPLC, method C, tR
29.8, 32.3 min).21 This product was dissolved in 400 mL of
THF, and 90 mL of J ones reagent, “standard” solution,15 was
added dropwise under vigorous stirring, in 1.5 h, while the
temperature was maintained at 20-25 °C. After 30 min, the
time (min)
0
10
23
20
30
35
40
45
method A:
method B:
method C:
% b in a
% b in a
% b in a
5
26
47
64
35
60
75
75
75
85
35
75
85
5
20
40
20 33
40 52
Pure deprotected pentapeptide (TDP P and TDP P -Me) and
tetrapeptide (TDTP A, TDTP , and TDTP -Me) derivatives
were analyzed for C, H, N, and Cl on samples previously dried
at 140 °C under N2 atmosphere. The analytical results
obtained for the above elements were within (0.4% of the
theoretical values. The solvent content (<7%, mainly water)
and inorganic residue (<0.2%) were determined by thermo-
gravimetry (TG), at 140 °C, and after the samples were heated
at 900 °C in O2 atmosphere, respectively.
(21) It was assessed that all per-CBZ derivatives of d i-BOC-RH-
TD with tR > 20 min (HPLC, method C) were suitable for the next
oxidation step, while more hydrophilic homologous compounds were
degraded upon treatment with the J ones reagent.
P r ep a r a tion of TDTP F ollow in g P r oced u r e A (Sch em e
1). Di-BOC-RH-TD. To a stirred solution of 24 g (∼20 mmol)

2156 J . Org. Chem., Vol. 61, No. 6, 1996
Malabarba et al.
resulting dark suspension was poured into 2.5 L of a stirred
mixture EtOAc/H2O 1/1. The organic layer was separated,
washed several times with a 1 N solution of Na2S2O5 (to
complete decomposion of peroxides), and then dried over Na2-
SO4. Evaporation of solvents at room temperature under
reduced pressure yielded 25 g of Zn -d i-BOC-TDP P as a crude
mixture of two main compounds (HPLC, method C, tR 21.5,
24.7 min). A solution of this product in 1 L of a MeOH/DMF/
AcOH 5/2/2 mixture was hydrogenated (1 atm, 25 °C) in the
presence of 12.5 g of 5% Pd/C. About 1.5 L of H2 was absorbed
within 2 h; afterwards, the catalyst was filtered off and MeOH
was evaporated at room temperature under reduced pressure.
The resulting solution was diluted with 2.5 L of H2O and
extracted with 2.5 L of 1-BuOH. The organic layer was
separated, washed with H2O (2 × 1 L), and then concentrated
at 45 °C under reduced pressure to a final volume of ∼100
mL. Upon addition of Et2O (500 mL), the precipitated solid
was collected (∼15 g) and purified by reversed-phase column
chromatography, yielding 5.6 g (∼25%) of the title compound:
HPLC, method B, tR 6.0 min.
TDP P . To remove the BOC protective groups, the above
compound (5.5 g) was dissolved in 50 mL of dry TFA and the
resulting solution was stirred at room temperature for 1.5 h.
Then, the solvent was evaporated at room temperature under
reduced pressure and the oily residue was slurried with EtOAc
to obtain a solid product which was collected, washed several
times with Et2O, and dried in vacuo at room temperature (over
KOH) to give the title compound (5.5 g, ∼100%), as the ditri-
fluoroacetate: HPLC, method A, tR 9.0 min.
separated, washed with H2O (2 × 250 mL), dried over Na2-
SO4, and then concentrated at 30 °C under reduced pressure
to a small volume (∼25 mL). Upon addition of Et2O (250 mL),
the precipitated solid was collected and dried in vacuo at room
temperature overnight to yield 8.7 g (100%) of title compound
pure enough for the next step: HPLC, method B, tR 12.2 min.
Di-BOC-TDTP . To a stirred suspension of 6 g (∼5.5 mmol)
of the above compound in 500 mL of dry Me2CO were added
120 mL of 2,2-dimethoxypropane and 0.27 g (∼1.4 mmol) of
p-toluenesulfonic acid. After the mixture was stirred at room
temperature for 1.5 h, a solution of 0.24 g of NaHCO3 in 4 mL
of H2O was added and solvents were evaporated at 35 °C under
reduced pressure. The solid residue (d i-BOC-TDTP A-ox-
a zolid in e, 6.4 g: HPLC, method b, tR 17.9 min) was collected
and dissolved in 300 mL of a dioxane/water 1/1 mixture, and
5.5 g (∼17 mmol) of Cs2CO3 was added. The resulting solution
was stirred at room temperature for 1 h, and then a solution
of 7.5 mL (∼50 mmol) of CBZ-Cl in 50 mL of dry THF was
added dropwise in 45 min while the mixture cooled to 5-10
°C. After being stirred at room temperature overnight, the
reaction mixture was poured into a stirred mixture EtOAc/
H2
O 1/1 (500 mL). The aqueous phase was adjusted at pH
4.3 with glacial AcOH, the organic layer was separated and
dried over Na2SO4, and then the solvent was evaporated at
room temperature under reduced pressure. The oily residue
was suspended in 250 mL of MeCN, and 25 mL of 1 N HCl
was added at room temperature. After being stirred at room
temperature for 1.5 h, the reaction mixture was poured into
500 mL of H2
O and the resulting suspension was extracted
with EtOAc (500 mL). The organic layer was washed with 1
N NaHCO3 until the pH of the aqueous washings was neutral,
and then it was washed with H2O (2 × 250 mL), dried over
Na2SO4, and concentrated at 30 °C under reduced pressure to
a small volume (∼30 mL). Upon addition of Et2O (300 mL),
the precipitated solid was collected (Zn -d i-BOC-TDTP A, 5.7
g: HPLC, method C, tR 27.1 min) and oxidized with the J ones
reagent as described above (procedure A), obtaining 5.3 g of
crude (85% HPLC titer) per-CBZ derivative Zn -d i-BOC-TDTP
(HPLC, method C, tR 18.7 min). Removal of the CBZ protective
groups under the previously described conditions (1 atm, 25
°C, 2.5 g of 5% Pd/C) yielded a 80/15/5 mixture (3.5 g) of crude
(∼85%, by HPLC) d i-BOC-TDTP and corresponding mono-
and didechlorinated derivatives, respectively. Purification by
reversed-phase chromatography gave 1.1 g (∼17%) of pure title
compound: HPLC, method b, tR 7.9 min).
TDTP . A solution of the above compound (1.1 g) in 25 mL
of dry TFA was stirred at room temperature for 1.5 h, and
then the solvent was evaporated at 30 °C under reduced
pressure. The oily residue was slurried with EtOAc (50 mL)
and then with Et2O (100 mL), obtaining a solid product which
was collected, washed with Et2O, and dried in vacuo at room
temperature overnight, yielding 1.1 g (∼100%) of pure title
compound as the bistrifluoroacetate: HPLC, method B, tR 6.8
min.
P r ep a r a tion of N2-CBZ-TDTP -Me (Sch em e 3). TDP P -
Me. To a stirred solution of 30 g (∼21 mmol) of d i-BOC-RH-
TD in 200 mL of DMF was added 2.5 g of KHCO3 followed by
a solution of 22 mL (∼23 mmol) of MeI in 30 mL of DMF. After
being stirred at room temperature overnight, the reaction
mixture was poured into 1 L of H2O. The resulting cloudy
solution was adjusted at pH 3 with 1 N HCl and extracted
with 1 L of a EtOAc/1-BuOH 1/1 mixture. The organic layer
was separated, washed several times with H2O to neutral pH,
and then concentrated at 40 °C under reduced pressure to a
small (∼50 mL) volume. Upon addition of Et2O (300 mL), the
precipitated solid was collected and dried in vacuo at room
temperature overnight, yielding 30 g (∼100%) of d i-BOC-RH-
TD-Me pure enough for the next step. HPLC, method B, tR
16.1 min. The above compound was protected at the phenolic
hydroxy groups following the same procedure described previ-
ously for Zn -d i-BOC-RH-TD, obtaining 36 g of Zn -d i-BOC-
RH-TD-Me as crude mixture of two main compounds (HPLC,
method C, tR 30.3, 32.5 min), which was submitted to oxidation
with the J ones reagent as described above, yielding 34.3 g of
crude Zn -d i-BOC-TDP P -Me (HPLC, method C, tR 22.3, 25.8
min). Then the CBZ groups were removed from this product
TDTP (by d ou ble Ed m a n d egr a d a tion of TDP P ). To a
stirred solution of the above product (5.5 g, ∼4 mmol) in 75
mL of a pyridine/water 1/1 mixture was added 1.1 mL (∼9
mmol) of phenyl isothiocyanate at room temperature. After 3
h, the reaction mixture was poured into 200 mL of H2O, and
the resulting cloudy solution was adjusted at pH 3 with 1 N
HCl and then was extracted with EtOAc (2 × 200 mL). The
organic layer was discarded, and the aqueous phase was
extracted again with 1-BuOH (200 mL). The butanol layer
was separated, washed with H2O (2 × 200 mL), and then
concentrated at 40 °C under reduced pressure to a small
volume (∼20 mL). Upon addition of Et2O (100 mL), the
precipitated solid was collected (5.8 g, crude diisothiourea:
HPLC, method A, tR 11.3 min) and redissolved in 100 mL of
dry TFA. The resulting solution was stirred at room temper-
ature for 1.5 h, and then the solvent was evaporated at 30 °C
under reduced pressure. The oily residue was purified by
reversed-phase chromatography under the usual conditions,
yielding 0.95 g (∼23%) of the title compound: HPLC, method
A, tR 6.8 min.
P r ep a r a tion of TDTP F ollow in g P r oced u r e B (Sch em e
2). TDTP A (by d ou ble Ed m a n d egr a d a tion of RH-TD).
To a stirred solution of 13.5 g (∼11 mmol) of RH-TD in 270
mL of a pyridine/water 1/1 mixture was added 2.8 mL (∼23
mmol) of phenyl isothiocyanate at room temperature. After 7
h, the reaction mixture was poured into 500 mL of H2O, and
the resulting cloudy solution was adjusted at pH 3 with 1 N
HCl and then extracted with EtOAc (2 × 500 mL). The organic
layer was discarded, and the aqueous phase was extracted
again with 1-BuOH (500 mL). The butanolic layer was
separated, washed with H2O (2 × 300 mL), and then concen-
trated at 40 °C under reduced pressure to a small volume (∼50
mL). Upon addition of Et2O (300 mL), the precipitated solid
was collected (14.3 g, crude diisothiourea: HPLC, method A,
tR 13.5 min) and redissolved in 200 mL of dry TFA. The
resulting solution was stirred at room temperature for 1.5 h,
and then the solvent was evaporated at 30 °C under reduced
pressure. The oily residue was purified by reversed-phase
chromatography under the usual conditions, yielding 7.5 g
(∼75%) of the title compound: HPLC, method A, tR 8.3 min.
Di-BOC-TDTP A. To a stirred solution of 7.2 g (∼8 mmol)
of the above compound and 11 g of NaHCO3 in 250 mL of a
dioxane/water 1/1 mixture was added a solution of 4 g (∼18.5
mmol) of (BOC)2O in 30 mL of dioxane dropwise at 0-5 °C.
Stirring was continued at room temperature for 5 h, and then
the reaction mixture was adjusted at pH 4 with 1 N HCl and
extracted with 300 mL of EtOAc. The organic layer was

Deglucoteicoplanin-Derived Tetrapeptide
J . Org. Chem., Vol. 61, No. 6, 1996 2157
by hydrogenolysis (1 atm, room temperature, in the presence
of 15 g of 5% Pd/C added in two portions), obtaining 24.5 g of
crude d i-BOC-TDP P -Me (HPLC, method B, tR 8.3 min). This
compound was dissolved in 300 mL of TFA, and the resulting
solution was stirred at room temperature for 30 min. Evapo-
ration of the solvent under reduced pressure at 30 °C yielded
an oily residue which was slurried with EtOAc to give a solid
(∼21 g) which was purified by reversed-phase column chro-
matography, obtaining 9.7 g (∼38%, overall yield) of the title
compound (HPLC, method A, tR 11.3 min).
N2-CBZ-N4BOC-TDTP -Me. To a stirred solution of 1.55
g (∼1.5 mmol) of N4-BOC-TDTP -Me in 50 mL of a dioxane/
water 1/1 mixture was added 1.4 g of NaHCO3 followed by a
solution of 0.23 mL of CBZ-Cl in 5 mL of dioxane (added
dropwise at room temperature in 5 min). After 10 min, the
reaction mixture was poured into 200 mL of a stirred H2O/
EtOAc 1/1 mixture. After the aqueous phase was adjusted at
pH 3 with 1 N HCl, the organic layer was separated, dried
over Na2SO4, and concentrated at 30 °C under reduced
pressure to a small (∼20 mL) volume. Upon addition of Et2O
(100 mL), the precipitated solid was collected, obtaining 1.75
g (∼100%) of pure title compound (HPLC, method B, tR 12.7
min).
N2-CBZ-TDTP -Me. A solution of 0.6 g of the above
compound in 10 mL of TFA was stirred at room temperature
for 15 min. Afterwards, the solvent was evaporated at 30 °C
under reduced pressure. The oily residue was slurried with
Et2O, and the solid which separated was collected, washed
with Et2O, and dried in vacuo at room temperature overnight
to yield 0.65 g (∼100%) of pure title compound (HPLC, method
B, tR 8.0 min), as the trifluoroacetate.
TDTP -Me. Edman degradation of 6.5 g of TDP P -Me (6.5
g) was carried out as described previously for TDTP , yielding
1.35 g (∼25%) of title compound (HPLC, method A, tR 10.3
min), as the bistrifluoroacetate.
N4-BOC-TDTP -Me. A solution of 2.8 g (∼3 mmol) of
TDTP -Me (2TFA) and of 0.75 g of NaHCO3 in 100 mL of a
dioxane/water 1/1 mixture was stirred at -5 °C while a
solution of 0.62 g of (∼3 mmol) of (BOC)2O was added dropwise,
in 1 h, in 20 mL of dioxane. Stirring was continued at -5 °C
for an additional 7 h, and then the reaction mixture was
poured into 300 mL of H2O. The resulting cloudy solution was
adjusted at pH 3 with 1 N HCl and extracted with 150 mL of
EtOAc. The organic layer was separated, dried over Na2SO4,
and evaporated at 30 °C under reduced pressure, obtaining
0.9 g of d i-BOC-TDTP -Me (HPLC, method A, tR 21.5 min)
which was treated with 10 mL of TFA to regenerate starting
TDTP -Me. The aqueous phase was extracted with 100 mL
of 1-BuOH, obtaining, after evaporation of butanol at 45 °C
under reduced pressure, 1.6 g (∼50%) of pure title compound
(HPLC, method A, tR 15.0 min).
Su p p or tin g In for m a tion Ava ila ble: Combustion analy-
sis data (1 page). This material is contained in libraries on
microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
J O9506746