The design and synthesis of redox core-alpha amino acid composites based on thiol-disulfide exchange mechanism and a comparative study of their zinc abstraction potential from [CCXX] boxes in proteins

Article abstract of DOI:10.1016/S0040-4020(02)00159-X

The design and synthesis of agents that can abstract zinc from their [CCXX] (C=cysteine; X=cysteine/histidine) boxes by thiol-disulfide exchange-having as control, the redox parities of the core sulfur ligands of the reagent and the enzyme, has been illustrated, and their efficiency demonstrated by monitoring the inhibition of the transcription of calf thymus DNA by E. coli RNA polymerase, which harbors two zinc atoms in their [CCXX] boxes of which one is exchangeable. Maximum inhibition possible with removal of the exchangeable zinc was seen with redox-sulfanilamide-glutamate composite. In sharp contrast, normal chelating agents (EDTA, phenanthroline) even in a thousand fold excess showed only marginal inhibition, thus supporting an exchange mechanism for the metal removal.

Full text of DOI:10.1016/S0040-4020(02)00159-X

TETRAHEDRON
Pergamon
Tetrahedron 58 $2002) 2861±2874
The design and synthesis of redox core±alpha amino acid
composites based on thiol±disul®de exchange mechanism and a
comparative study of their zinc abstraction potential from
[CCXX] boxes in proteins
Subramania Ranganathan,a,b,p K. M. Muraleedharan,a Parimal Bharadwaj,c Dipankar Chatterjid
and Isabella Karlee,p
aDiscovery Laboratory, Organic III, Indian Institute of Chemical Technology, Hyderabad 500 007, India
bJawaharlal Nehru Centre for Advanced Scienti®c Research, Bangalore 560 012, India
cDepartment of Chemistry, Indian Institute of Technology, Kanpur 208 016, India
dMolecular Biophysics Unit, Indian Institute Science, Bangalore 560 012, India
eLaboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375-5341, USA
Respectfullydedicated to Darshan Ranganathan who passed awayon her 60th birthday$4 June 2001)
Received 2 October 2001; revised 7 January2002; accepted 7 February2002
AbstractÐThe design and synthesis of agents that can abstract zinc from their [CCXX] $Cˆcysteine; Xˆcysteine/histidine) boxes by thiol±
disul®de exchange-having as control, the redox parities of the core sulfur ligands of the reagent and the enzyme, has been illustrated, and their
ef®ciencydemonstrated bymonitoring the inhibition of the transcription of calf thymus DNA by E. coli RNA polymerase, which harbors two
zinc atoms in their [CCXX] boxes of which one is exchangeable. Maximum inhibition possible with removal of the exchangeable zinc was
seen with redox±sulfanilamide±glutamate composite. In sharp contrast, normal chelating agents $EDTA, phenanthroline) even in a thousand
fold excess showed onlymarginal inhibition, thus supporting an exchange mechanism for the metal removal. q 2002 Elsevier Science Ltd.
All rights reserved.
1.Introduction
making the packaging of virions in the HIV virus defective
and dysfunctional.2
The genesis of the present work pertains to recent reports on
the fact that, in the maturation of HIV virus, a 55-residue
protein called nucleocapsid protein $NCp7) plays a very
important role in the proper packaging of HIV-RNA.
Viral RNA, which provides all the information for its func-
tion, can be packaged onlyif this protein is present. The
sequence of this protein is presented in Chart 1, A. The
protein contains two zinc ®nger modules, where the metal
is harbored in the [CCXX] boxes.1 Concerted efforts to
remove zinc from these ef®ciently, led to the identi®cation
of disul®des of the type B $Chart 1) as useful agents to
disrupt the NCp7 conformation bythe removal of the zinc
present, thus making the protein inoperative and thereby
The ef®ciencywith which zinc is removed from A by B, in
the context of work from our laboratory,3 provided an
opportunityto design agents that could abstract zinc present
in the [CCXX] boxes of enzymes, by the thiol±disul®de
exchange mechanism $Chart 2).4
Thus, B $Chart 1) could react with an appropriate [CCXX]
box harboring zinc in proteins, to form the octahedral
complexes, which could break up, effecting oxidation in
the [CCXX] box with concomitant removal of B as a zinc
complex. The rationalization of the overall process in this
manner brings to focus, the redox parityof the S±S system
present in A and B $Chart 2).
The redox core in B was redesigned to interact favorably
with the zinc ®nger modules, bylinking with diverse amino
acid residues. This was considered important, since, in the
removal of zinc from [CCXX] boxes, the amino acid side
chains could playa major role in stabilizing the transition
state, byinteraction with residues present in the zinc
Keywords: thiol±disul®de exchange; RNA polymerase; dithiobisbenza-
mides; benzisothiazolones.
Corresponding authors. Address: DiscoveryLaborator,y Organic III,
Indian Institute of Chemical Technology, Hyderabad 500 007, India.
Tel.: 191-40-7160123x2648; fax: 191-40-7160757;
e-mail: rangan@iict.ap.nic.in
p
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020$02)00159-X

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S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
Chart 1.
protein/enzyme. In the zinc ®nger±DNA recognition, the
importance of these interactions has been recognized.5
The ease with which zinc complexes can be prepared
from a [CCXX] box made it interesting to generate these,
in an exchange process, leading to minimalistic zinc ®nger
motifs byattachment of a histidine to the redox system in B.
This approach has yielded good results.
2.Results and discussion
2.1. Synthesis and crystal structures
The reaction of 2,20 dithiodibenzoyl chloride $2) with
freshlyprepared amino acid methly esters of Ala, Aib,
Val, Pro, Phe and Met, followed bycolumn chromatography
afforded the expected bisamides 3±8 $Chart 3).
Surprising results were obtained from amino acids that have
a proton source in the side chain. Theyafforded benzisothia-
zolones $9, 11, 13±15: Chart 4) arising from cleavage of the
S±S bond, with concomitant peptide NH participation.
Tyrosine and threonine, in addition, gave the normal
bisamides 10 and 12 $Chart 4).
In spite of sustained interest in redox systems of the type B
$Chart 1), no crystal structure of this class of compound is
available. Fortunately, the threonine linked bis amide 12
and the abnormal product 11 crystallized from CHCl3/
CH3CNˆ1:1. The resulting X-raystructures are presented
in Figs. 1 and 2.
The structure of 12 clearlyshowed the near orthogonal
disposition of the two ligands of the disul®de unit and, as
envisaged in Chart 2. Of additional interest is the general-
ized hydrogen bonding seen, involving the amide unit,
Chart 2.

S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
2863
Chart 3.
which is augmented bythe hdyrogen bonding with the
threonine hydroxyl group. These interactions are likely to
hold the disul®de group exposed, thus making its approach
to the enzyme metal center facile $Chart 2).
spectrum. The isolation of the benzisothiazolones is signi®-
cant in the sense that, although the formation of such
compounds has been speculated in the fragmentation of
bisamides $Chart 1, C), theyhave never been isolated
pure and adequatelycharacterized.
The X-raystructure of the benzisothiazolone 11 $Fig. 2)
showed no surprises. This class of compounds is stable
and therefore these molecules are potentiallybetter drug
candidates, compared to the dimeric bisamides.
Hitherto, the bisamide!benzisothiazolone change was
rationalized on the basis of a nucleophilic attack bythe
amide nitrogen on the dithio bond, resulting in S±S
cleavage to benzisothiazolone on the one hand and the
corresponding ±SH compound on the other. This
mechanism would envisage loss of 50% of the starting
compound. The fact that, in the case of threonine, not
onlythe benzisothiazolone $ 11) was secured in 59%, but
also, the bisamide $12) was isolated in 31% yield, would
require a revision of this mechanism. We propose that, in
the cases where there is a proton source available in the side
chain, the protonated intermediate undergoes rupture to give
the benzisothiazolone on the one hand and the ±SH species
on the other. The process operates in a cyclic form by the
transformation of the latter byoxidation, to the bisamide.
This pathwaywould account for as much as 100% yields of
benzisothiazolones $Chart 5). The facile oxidation of the
intermediate SH compounds to the bisamides has been
experimentallyveri®ed.
The benzisothiazolone 9 from tyrosine gave crystals from
CHCl3/CH3CNˆ1:1. The X-raystructure presented in Fig. 3
shows an orthogonal disposition of the tyrosine aromatic
residue. Here also the S±N bond involved in the thiol±
disul®de exchange is exposed.
The bisamides and benzisothiazolones can be spectro-
scopicallydistinguished. In their FAB mass spectra, the
bisamides displayprotonated mass peaks and those
complexed with sodium ions. Theyinvariablyshow base
peaks corresponding to the clean rupture of the S±S
bonds. The fact that these were actuallygenerated in the
mass spectrometer was evidenced bythe fact that, they
appeared uncomplexed with the proton, as is always the
case with parent ions. In sharp contrast, the benzisothia-
zolones gave peaks corresponding to the mass with a proton.
The consequences of two alterationsÐthe introduction of
spacer between the redox core and the amino acid, and the
tethering of the terminiÐthat could have a bearing on the
proclivityfor exchange, were examined.
A characteristic feature that distinguishes the bisamides
from the benzisothiazolones is the appearance of the Ca
proton at, respectively, ,4.5 and ,5.5 ppm in the NMR

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S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
Chart 4.
The sulfanilamide±redox composite $16), prepared from 2
and sulfanilamide, on coupling with Boc-Glu$gOMe)OH
afforded the redox±spacer±aminoacid composite 17
$Chart 6). This coupling is noteworthysince amidation
involving a charge depleted SO2NH2 unit is rare.
the system by tethering, fragmentation occurs via formation
of a dehydro Ala on the one hand and an S±S cleavage on
the other, and this byprocesses, similar to that for benz-
isothiazolone formation $Chart 5), is transformed in to 18
and 19 $Chart 8).
The reaction of 2 with freshlyprepared cystine-di-OMe
afforded three compounds, namely, the desired tethered
system 20 and fragmentation products 18 and 19 $Chart
7). Compound 19 crystallized from EtOAc/hexaneˆ1:1.
The crystal structure of 19 $Fig. 4) consists of, in the unit
cell, a heterodimer arising from strong hydrogen bonding of
the nearly cis oriented amides to form an 8-ring non-
covalent assembly. Such dimer formation was also seen in
the mass spectrum of 19, which showed a signi®cant peak at
475 $2M1H)1. Compounds of type 19 are potent angio-
tensin II inhibitors in their own right.6 Compounds 18 and
19 arise from fragmentation of the composite 20.
Methanolic solutions of pure 20, admixed with triethyl-
amine, rapidlygenerated the fragmentation products. It is
suggested that, arising from steric constraints imposed on
The reaction of 2 with o-phenylenediamine afforded
the benzisothiazolone 21, the symmetrical monocyclic
212 adduct 22 and none of the 1:1 tethered system
$Chart 9).
Compound 21 crystallized from EtOAc/hexaneˆ1:1. The
crystal structure is presented in Fig. 5. Unlike the other
examples $9 and 11), the molecule 21 is nearlyplanar
with proximal alignment of the amino group to the sulfur
atom, rather than to the carbonyl of the benzisothiazolone.
The mass spectrum of 22 exhibited a peak at 756 $M1H)1.
This compound is interesting in that, within the con®nes of a
Ar±S±S±Ar periphery, the cavity is highly programmed for
the uptake of ions, possessing as it does, four rigidlyaligned
peptide groups.

S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
2865
Figure 1. $a) Crystal structure of 12. $b) Extended hydrogen-bonded assembly of 12 in the crystal lattice. The distances in the hydrogen bonds $dashed lines)
Ê
Ê
Ê
Ê
are O2±O1ˆ2.895 A, N1±O1ˆ2.949 A. O2s±O1sˆ3.102 A, N1s±O1sˆ2.908 A.
2.2. Preliminary assessment of the design
comparison purposes, the enzyme activity without the
reagent was taken as 100% and the outcome from other
experiments are made based on this standard. To enable a
direct comparison, the results from normal chelating
agents like EDTA and o-phenanthroline at much higher
concentrations $,£104) are also presented in Fig. 6,
which also incorporates results from use of a 10-fold excess
of selected reagents. At higher concentrations $,£104), all
the reagents totallyinhibited transcription.
Evidence for the operation of the two main features of the
design, namely, zinc abstraction by thiol±disul®de
exchange and the importance of the amino acid ligand
present in the composite were sought bymonitoring the
course of transcription of calf thymus DNA by E. coli
RNA polymerase7 after treatment with either the dithio-
bisbenzamides $3±8, 10, 12, 16, 17 and 20) or benziso-
thiazolones $11, 14, and 15).8 The latter class has not been
envisaged for use in metal abstraction.
The effect of zinc removal from yeast RNA polymerase II
on transcription pro®le has been studied.9 Of the two zinc
atoms present in the enzyme, one could be removed rather
The results are presented in a graphical format in Fig. 6. For
Figure 2. Crystal structure of 11.

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S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
Figure 3. Crystal structure of 9.
easily, leading to a mono zinc depleted modi®ed enzyme
which still showed ,35% of original activity. The removal
of both zinc atoms requires rather drastic conditions and the
resulting totallyzinc depleted enzyme does not show any
transcription activity. Recent studies with E. coli RNA
polymerase10 has led to remarkablysimilar conclusions.
The 1407 residue b0 fragment harbor in their [CCXX]
boxes, the two zinc atoms. Whilst the zinc is bound tightly
in the N-terminal location, and could not be exchanged
readily, that present at the C-terminal can be exchanged
and reconstituted with onlypartial loss of activity. Present
studies with E. coli RNA polymerase has clearly shown that,
with excess of the reagent, both zinc ions are removed,
resulting in the total inhibition of transcription and with
two equivalents of the reagent, onlyone zinc is removed,
resulting in the retention of a third of the activityof the
native enzyme.
With an equivalent of the reagent for each zinc ion, the
inhibition varied from 1% for Pro-bisamide $6) to 62% for
the core±sulfanilamide±Glu composite 17. The 62%
inhibition by 17 represents the removal of one zinc from
the enzyme. This threshold was reached by the Ala-
bisamide $3) and the Met-bisamide $8) at a 10-fold increased
concentration.
A striking observation from Fig. 6 is that the reagents
studied here are far superior in the removal of metal,
compared to the traditional chelators like EDTA and
o-phenanthroline. Even at enhanced concentrations
[EDTA: £12,500; o-phenanthroline: £6250], theywere
not able to effect signi®cant removal of the metal, which
provides strong support for the thiol±disul®de exchange
mechanism envisaged here $Chart 2) and rules out a simple
chelation path. This understanding will be helpful in the
Chart 5.

S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
2867
Chart 6.
further development of proper reagents for the abstraction of
zinc.
subunit of E. coli RNA polymerase, that harbor the
exchangeable zinc in a [CCCH] box10 $Chart 10), as many
as 12 are hydrophobic $others: Gly, 3; polar, 4; acidic, 3;
basic, 4 and CCCH box).
Fig. 6 neatlydivides the reagents studied in this work into
two categories. Those which are on the left side of
o-phenanthroline and EDTA $at vastlyenhanced concentra-
tions) and the others to the right of it. The former constitute
a class having decreased capacityfor inhibition of
transcription process by E. coli RNA polymerase and the
latter, a vastlyenhanced inhibitorypro®le. It cannot be a
coincidence that a better pro®le is shown byhydrophobic
amino acid derivatives $Phe, Val, Ala, Aib, Tyr). This
preference pro®le stronglysupports the notion that amino
acids attached to the redox core can playa role in the
stabilization of the transition state involved in the exchange
$Chart 2).
The importance of amino acid attachment in the design is
again highlighted with the pairs 16 and 17. Compound 16
exhibited the maximum potential in the removal of zinc
from NCp7. However, with RNA polymerase, the
activityis modest. This pro®le is greatlyenhanced from
31 to 62% inhibition bysimple attachment of the glutamate
residue.
The near complete lack of activityof the proline bisamide 6
clearlybrings out the important role of the peptide NH in the
exchange process. Contraryto expectations, the cystine-
di-OMe tethered composite 20 exhibited poor activity
$13%), compared to the structurallyclosest open
compound from Ala $3) $47%). Minimum energyconforma-
tions of the cyclic compound 20 and the open 3 derived
using simulated annealing molecular dynamics $MD)
The fact that hydrophobic amino acids are able to promote
such stabilization indicates the importance of such inter-
actions in the vicinityof the [CCXX] box in E. coli RNA
polymerase. Of the 30 residue C-terminal segment of the b0

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S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
Chart 7.
Chart 8.
suggest that approach to the redox active aromatic disul®de
core is greatlyhindered in the case of the cyclic compound
20.11
A novel ®nding of the present work is the clear establish-
ment of the abilityof benzisothiazolones to abstract zinc,
most likelyinvolving the S±N bond in the exchange $Chart
11). Interestingly, such an exchange with the imidazole
containing isothiazolone 15 would result in complex,
reminiscent of a minimalistic zinc ®nger motif3 $Chart
12). The observed exceptional activityof 15 could arise
from this combination of properties. In view of the known
biological pro®le of isothiazolones, this propertysuggests
further avenues for exploration.
On the basis of observations presented here, it is suggested
that the removal of zinc present in [CCXX] boxes of
proteins/enzymes takes place by a thiol±disul®de exchange
mechanism $Chart 2). Thiol±disul®de exchange is an
important process in biological systems,4 the rami®cations
of which are being increasinglyunderstood and exploited.
The most attractive feature of this process is that the metal
transfer takes place on a common template, leading to clean
reactions, avoiding leakage of open ligands.
Figure 4. Crystal structure of 19 as hydrogen bonded dimer. The two
Ê
N±OvC distances $dashed lines) are 2.895 and 2.910 A.

S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
2869
Chart 9.
Figure 5. Crystal structure of 21.

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S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
Figure 6. E. Coli RNA polymerase inhibition with dithiobisbenzamides [2,20-dtbis$AA)OMe] and benzisothiazolones [Isot$AA)OMe] $AAˆamino acid).
3.Experimental
3.1. Materials and methods
All amino acids used were of l-con®guration. Melting
points were recorded on a Fisher±Johns melting point
apparatus and are uncorrected. Optical rotations were
measured with an automatic JASCO polarimeter; concen-
trations are given in grams/100 mL. Infra red spectra were
recorded on a Perkin±Elmer/1600-FT spectrometer either as
neat liquids or as KBr pellets and prominent peaks are
expressed in cm21. 1H and 13C NMR spectra were recorded
on Bruker WM-300 NMR spectrometer. The chemical shifts
are expressed in d $ppm) with TMS at 0.000 as the internal
reference. FAB MS were obtained on a JEOL SX-120/DA-
6000 instrument using m-nitrobenzylalcohol as the matrix.
C, H, N analysis was carried out in Vario EL elemental
Chart 10.
Chart 11.

S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
2871
with distilled water $1£10 mL) $in the case of Trp and His,
the acid wash was not done). The organic layer was dried
$MgSO4), evaporated in vacuo and the residue chromato-
graphed on a silica gel using hexane±EtOAc as eluent.
When present, the benzisothiazolones eluted earlier than
2,20-dithiobisbenzamides. Characteristic spectral features
of the products $3±15) obtained on using various amino
acids $AA) are presented below.
3.3.1. Compound 3. Dithiobisbenzamide: AAˆAla; white
29
powder; yield: 25%; [a]D ˆ210.00 $c 1, CHCl3); mp 104±
1
1068C; IR $KBr): 3306, 3012, 1749, 1651, 1545; H NMR
$CDCl3) d 1.56 $d, Jˆ7 Hz, 6H, CH3), 3.8 $s, 6H,
COOCH3), 4.7±4.8 $m, 2H, CaH), 6.78 $d, Jˆ3 Hz, 2H,
NH), 7.2±7.77 $m, 8H, ArH); FAB MS $m/z) $%): 477
$10) $MH)1, 499 $5) $M1Na)1, 238 $100) $M/2); Anal.
Found: C, 55.01; H, 5.38; N, 5.80. Calcd for
C22H24N2O6S2: C, 55.46; H, 5.04; N, 5.88.
3.3.2. Compound 4. Dithiobisbenzamide: AAˆAib; white
powder; yield: $24%); mp 178±1808C; IR $KBr): 3245,
2989, 1748, 1634, 1539; 1H NMR $CDCl3) d 1.69 $s,
12H, CH3), 3.79 $s, 6H, COOCH3), 6.76 $brs, 2H, NH),
7.2±7.76 $m, 8H, ArH); FAB MS $m/z) $%): 527 $7)
$M1Na)1, 252 $100) $M/2); Anal. Found: C, 57.55; H,
5.34; N, 4.80. Calcd for C24H28N2O6S2: C, 57.14; H, 5.55;
N, 5.55.
Chart 12.
analyzer. Reactions were monitored wherever possible by
TLC $silica gel/G $Merck) and the eluent $invariablyethyl
acetate/hexane) ratio adjusted to give Rf values in the range
of 0.4±0.6. Column chromatographywas done on silica gel
$100±200 mesh) columns, which were generallymade from
a slurryin hexane or a mixture of hexane and ethyl acetate.
Products were eluted with a mixture of ethyl acetate/hexane.
3.3.3. Compound 5. Dithiobisbenzamide: AAˆVal; white
29
powder; yield: 63%; mp 148±1508C; [a]D ˆ23.00 $c 1,
CHCl3); IR $KBr): 3293, 2963, 1744, 1634, 1529; 1H NMR
$CDCl3) d 0.98 $d, Jˆ7 Hz, 6H), 1.0 $d, Jˆ7 Hz, 6H), 2.3
$m, 2H, CbH), 3.79 $s, 6H, COOCH3) 4.81 $m, 2H, CaH),
6.63 $d, Jˆ3 Hz, 2H, NH), 7.2 $dd, Jˆ8, 0.5 Hz, 2H), 7.3
$dd, Jˆ8, 0.52 Hz, 2H), 7.6 $d, Jˆ8 Hz, 2H), 7.75 $d,
Jˆ8 Hz, 2H) $aromatic); FAB MS $m/z) $%): 533 $44)
$MH)1, 555 $10) $M1Na)1, 266 $100) $M/2); Anal.
Found: C, 58.58; H, 6.01; N, 5.03. Calcd for
C26H32N2O6S2: C, 58.64; H, 6.01; N, 5.26.
3.2. Synthesis
3.2.1. 2,20-Dithiodibenzoyl chloride 52). A mixture of 2,20-
dithiodibenzoic acid $1) $2 g, 6.53 mmol) and SOCl2
$24 mL) was re¯uxed for 24 h, evaporated, the residue
dissolved in CH2Cl2 $5 mL), evaporated in vacuo, triturated
with dryhexane, ®ltered under protection from moisture,
washed with dryhexane and dried over P O5 to afford
2
1.5 g $67%) of 2 $waxysolid).
Mp: 1488C $Lit.2a mp 150±1528C); IR $KBr): 1721, 1600,
3.3.4. Compound 6. Dithiobisbenzamide: AAˆPro; pale
1631, 1415; H NMR $CDCl3) d 1.88±2.32 $m, 8H, Cb
1
yellow solid; yield: 43%; IR $neat): 2961, 1752, 1652,
1560; H NMR $CDCl3±DMSO-d6) d 7.4 $dd, Jˆ13.8,
1
0.5 Hz, 2H), 7.6 $dd, Jˆ13.8, 0.5 Hz, 2H), 7.7 $dd, Jˆ10,
and CgH2), 3.3±3.44 $m, 4H, CdH2), 3.79 $s, 6H,
COOCH3), 4.69 $m, 2H, CaH), 7.18±7.33 and 7.71±7.74
$m,m, 8H, ArH); FAB MS $m/z) $%): 529 $59) $MH)1,
551 $13) $M1Na)1, 264 $100) $M/2); Anal. Found: C,
59.51; H, 5.54; N, 4.92. Calcd for C26H28N2O6S2: C,
59.09; H, 5.30; N, 5.30.
0.5 Hz, 2H), 8.08 $dd, Jˆ10, 0.5 Hz, 2H) $aromatic).
3.3. The reaction of 2,20-dithio dibenzoylchloride with a
aminoacid methyl esters: isolation of 2,20-dithiodi-
benzoyl bis-aminoacid methyl esters 53±8, 10 and 12)
and N-5benzisothiazolone) amino acid methyl esters 59,
11 and 13±15) 5general procedure)
3.3.5. Compound 7. Dithiobisbenzamide: AAˆPhe; yellow
29
A solution of 2,20-dithiodibenzoyl chloride $2) $1 g,
2.915 mmol) in dryCH 2Cl2 $25 mL) and 0.9 mL
$6.413 mmol) of triethylamine were simultaneously added
dropwise to an ice cooled and stirred solution of the free
amine of the amino acid methyl ester $generated in situ by
the addition of triethylamine $6.413 mmol) to an ice cooled
and stirred suspension of the amino acid methyl ester hydro-
powder; yield: $36%); mp 174±1768C; [a]D ˆ1112.00 $c
1
1, CHCl3); IR $KBr): 3285, 3036, 1742, 1640, 1533; H
NMR $CDCl3) d 3.3 $dq, Jˆ14, 3 Hz, 4H, CbH2), 3.78 $s,
6H, COOCH3), 5.1 $m, 2H, CaHs), 6.57 $d, 2H, NHs), 7.15±
7.74 $m, 16H, Phe ArHs1dithio ArHs), 7.75 $d, 2H,
dithioArHs); FAB MS $m/z) $%): 629 $30) $MH)1, 651 $7)
$M1Na)1, 314 $100) $M/2); Anal. Found: C, 65.03; H, 5.05;
N, 4.20. Calcd for C34H32N2O6S2: C, 64.96; H, 5.09; N, 4.45.
chloride $6.413 mmol) in dryCH Cl2 $60 mL). $In the case
2
of histidine methyl ester dihydrochloride, 12.8 mmol of
triethylamine was used.) The reaction was stirred for 48 h
at room temperature, washed successivelywith cold satu-
rated NaHCO3 $3£10 mL), 1 M H2SO4 $3£10 mL) and then
3.3.6. Compound 8. Dithiobisbenzamide: AAˆMet;
29
yellow powder; yield: $75%); mp 206±2088C; [a]D
18.00 $c 1, CHCl3); IR $KBr): 3265, 3070, 1755, 1640,
ˆ

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S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
1
1546; H NMR $CDCl3) d 2.10 $s, 6H, ±SCH3), 2.1±2.33
$m, 4H, CbH2), 2.63 $m, 4H, CgH2), 3.81 $s, 6H, COOCH3),
4.94 $m, 2H, CaH), 6.93 $d, Jˆ3 Hz, NH), 7.25 $dd, Jˆ8,
0.5 Hz, 2H), 7.4 $dd, Jˆ10, 0.5 Hz, 2H) 7.6 $d, Jˆ8 Hz,
2H), 7.8 $d, Jˆ8 Hz, 2H) $ArH); FAB MS $m/z) $%): 597
$48) $MH)1, 619 $11%) $M1Na)1, 298 $100) $M/2); Anal.
Found: C, 51.90; H, 5.30; N, 4.28. Calcd for C26H32N2O6S2:
C, 52.34; H, 5.37; N, 4.70.
$%): 537 $12) $MH)1, 559 $12) $M1Na)1, 268 $100) $M/2);
Anal. Found: C, 53.61; H, 5.20; N, 4.98. Calcd for
C24H28N2O8S2: C, 53.73; H, 5.22; N, 5.22.
3.3.11. Compound 13. Benzisothiazolone: AAˆSer; white
powder; yield: $41%); mp 153±1558C; Anal. Found: C,
51.74; H, 5.41; N, 5.56; Calcd For C11H11NO4S; C, 52.17;
29
H, 4.34, N, 5.33; [a]D ˆ14.00 $c 0.5, CHCl3); IR $KBr):
1
3466 $br), 3241, 2922, 1765, 1627, 1451, 1331; H NMR
$CDCl3) d 3.2 $t, Jˆ5 Hz, 1H, OH), 3.9 $s, 3H, COOCH3),
4.15, 4.4 $m,m, 2H, CbH2), 5.4 $t, Jˆ4 Hz, 1H, CaH), 7.4
$dd, Jˆ8, 4 Hz, 1H), 7.6 $m, 2H), 8.1 $d, Jˆ8 Hz, 1H)
$aromatic); FAB MS $m/z) $%): 254 $100) $MH)1, 276 $7)
$M1Na)1; Anal. Found: C, 51.56; H, 4.25; N, 5.24. Calcd
for C11H11NO4S: C, 52.17; H, 4.34; N, 5.53.
3.3.7. Compound 9. Benzisothiazolone: AAˆTyr; pale
29
yellow needles; yield: $29%); mp 188±1908C; [a]D
263.00 $c 1, CHCl3); IR $KBr): 3421 $br), 3161, 1752,
ˆ
1
1636, 1599, 1518, 1449; H NMR $CDCl3) d 3.15 $dd,
Jˆ10, 4 Hz, 1H), 3.5 $dd, Jˆ10, 2 Hz, 1H, CbH2) 3.76 $s,
3H, COOCH3), 5.75 $m, 1H, CaH), 6.7 $d, Jˆ8 Hz, 2H), 7.0
$d, Jˆ8 Hz, 2H) $, Tyr), 7.29, 7.54, 7.94 $m,m,m, 4H, ArH);
13C NMR $CDCl3) d 36.78 $Cb), 52.79 $Ca), 57.03
$±OCH3), 115.66±155.66 $C aromatic), 166.24 $±COO±),
170.25 $Ar-CO); FAB MS $m/z) $%): 330 $100) $MH)1, 352
$7) $M1Na)1 659 $6) $2M1H)1; Anal. Found: C, 61.89; H,
4.64; N, 3.98. Calcd for C17H15NO4S: C, 62.00; H, 4.56; N,
4.26.
3.3.12. Compound 14. Benzisothiazolone: AAˆTrp;
29
yellow solid; yield: $25%); [a]D ˆ275.00 $c 1, CHCl3);
IR $neat): 3420 $br), 3357 $br), 2930, 1748, 1663, 1552,
1
1453, 1308, 1240; H NMR $CDCl3) d 3.5 $m, 2H, CbH2),
3.74 $s, 3H, COOCH3), 5.78 $m, 1H, CaH), 7.2, 7.35, 7.6,
8.1 $m,m,m,m, 4H, ArH); 13C NMR $CDCl3) d 27.62 $Cb),
52.8 $Ca), 55.97 $±OCH3), 110.12±141.19 $C aromatic),
165.92 $±COO), 170.69 $Ar-CO); FAB MS $m/z) $%):
353 $66) $MH)1, 375 $11) $M1Na)1, 705 $4) $2M1H)1;
Anal. Found: C64.31; H4.34; N, 7.80. Calcd for
C19H16N2O3S: C, 64.77; H, 4.55; N, 7.95.
3.3.8. Compound 10. Dithiobisbenzamide: AAˆTyr; white
solid; yield: $19%); Anal. Found: C, 61.52; H, 5.01; N, 4.71;
mp 229±2318C; Calcd For C34H32N2O8S2: C, 61.81; H,
29
4.84; N, 4.24; [a]D ˆ128.00 $c 0.5, CHCl3); IR $KBr):
1
3366 $br), 3279, 3022, 1735, 1640, 1512, 1438; H NMR
$CDCl3±DMSO-d6) d 3.14 $m, 4H, CbH2), 3.74 $s, 6H,
COOCH3), 4.8 $m, 2H, CaH), 6.7 $d, Jˆ8 Hz, 4H), 7.1 $d,
Jˆ8 Hz, 4H) $Tyr H), 7.2, 7.4, 7.6, 7.8 $m,m,m,m, 8H,
ArH), 8.01 $br, 2H, NH), 8.89 $s, 2H, OH); 13C NMR
$CDCl3) d 35.56 $Cb), 51.38 $Ca), 53.55 $±OCH3),
114.67±155.35 $C aromatic), 166.70 $±COO±), 171.36
$Ar-CO); FAB MS $m/z) $%): 661 $49) $MH)1, 683 $10)
$M1Na)1, 330 $100) $M/2); Anal. Found: C, 61.78; H,
5.21; N, 4.18. Calcd for C34H32N2O8S2: C, 61.82; H, 4.85;
N, 4.24.
3.3.13. Compound 15. Benzisothiazolone: AAˆHis;
yellow solid; yield: $56%; mp 240±2428C; IR $KBr): 3446
$br), 3025, 1650, 1455, 1340; 1H NMR $CDCl3±DMSO-d6)
d 3.43 $m, 2H, CbH2), 3.75 $s, 3H, COOCH3), 5.62 $m, 1H,
CaH), 6.73 $br, Im 4H), 7.38 $br, 1H, Im 2H), 7.8 $br, Im
NH); FAB MS $m/z) $%): 304 $100%) $MH)1; Anal. Found:
C, 55.55; H, 4.19; N, 14.21. Calcd for C14H13N3O3S: C,
55.45; H, 4.29; N, 13.86.
3.4. Preparation of 2,20-dithiobis-[40-5sulfamoyl)-
benzanilide] 516)
3.3.9. Compound 11. Benzisothiazolone: AAˆThr; white
1
CHCl3); IR $KBr): 3496 $br), 2992, 1732, 1670, 1450; H
29
solid; yield: $59%); mp 144±1468C; [a]D ˆ280.00 $c 0.5,
To 2.58 g $14.99 mmol) of sulfanilamide in 50 mL of
pyridine at 0±58C was added in drops, 2 g $5.83 mmol) of
2,20-dithiodibenzoyl chloride $2) in 20 mL of dryCH Cl2.
NMR $CDCl3) d 1.25 $d, Jˆ7 Hz, 3H, CH3), 3.1 $m, 1H,
OH), 3.77 $s, 3H, COOCH3), 4.72 $m, 1H, CbH), 5.41 $m,
1H, CaH), 7.4 $dd, Jˆ8, 0.5 Hz, 1H), 7.6 $d, Jˆ8 Hz, 1H),
7.7 $d, Jˆ8 Hz, 1H), 8.1 $d, Jˆ8 Hz, 1H) $ArH); 13C NMR
$CDCl3) d 19.21 $Cg), 52.86 $Cb), 60.95 $Ca), 67.38
$±OCH3), 120.1±142.1 $C aromatic), 166.8 $±COO),
169.42 $Ar-CO); FAB MS $m/z) $%): 268 $100) $MH)1,
290 $21) $M1Na)1 535 $3) $2M1H)1, 557 $2)
$2M1Na)1; Anal. Found: C, 54.08; H, 4.86; N, 5.01.
Calcd for C12H13NO4S: C, 53.93; H, 4.86; N, 5.24.
2
The mixture was stirred at 0±258C for 18 h, ®ltered, the
solid washed with 1N HCl $3£10 mL), water $3£10 mL),
dried, suspended in a mixture of DMF $20 mL) and EtOH
$25 mL), ®ltered and precipitated from the ®ltrate with 9 mL
of 5% NaHCO3. The product was collected by®ltration,
washed with water and then with EtOH and dried to afford
1.2 g of 16 $microcrystalline white powder).
Yield: 34%; mp 3008C $lit.2a; mp 311±3128C; IR $KBr):
3373, 3265, 1661, 1600, 1526, 1317, 1175; 1H NMR
$CDCl3±DMSO-d6) d 7.1 $s, 4H, SO2NH2), 7.31±7.46 $m,
4H, ArH), 7.76±7.95 $m, 12H, ArH), 10.7 $s, 2H, NH);
Anal. Found: C, 50.37; H, 3.57; N, 8.75. Calcd for
C26H22N4O6S4: C, 50.81; H, 3.58; N, 9.12.
3.3.10. Compound 12. Dithiobisbenzamide: AAˆThr;
29
white solid; yield: $31%); mp 182±1848C; [a]D ˆ240.00
$c 0.5, CHCl3); IR $KBr): 3527 $br), 3331, 2980, 1751,
1633, 1541, 1439, 1259; 1H NMR $CDCl3) d 1.31 $d,
Jˆ6 Hz, CH3), 2.86 $br, 2H, ±OH), 3.79 $s, 6H,
COOCH3), 4.4 $br, 2H, NH), 4.7 $d, Jˆ4 Hz, 2H, CbH),
4.8 $d, Jˆ4 Hz, 2H, CaH), 7.25, 7.4, 7.75, 7.85
$m,m,m,m, 8H, ArH); 13C NMR $CDCl3) d 19.81 $Cg),
51.78 $Cb), 57.99 $Ca), 66.99 $±OCH3), 125.29±137.15 $C
aromatic), 167.5 $±COO), 170.71 $Ar-CO); FAB MS $m/z)
3.4.1. 2,20-Dithiobis [405sulfamido{Boc-Glu5gOMe)})
benzanilide] 517). A solution of Boc-Glu$gOMe)OH
$0.836 g, 3.2 mmol) $prepared in two steps from Glu] in
dryDMF $30 mL) was admixed with DCC $0.725 g,
3.52 mmol), left stirred for 5 min, admixed with

S. Ranganathan et al. / Tetrahedron 58 62002) 2861±2874
2873
2,20-dithiobis[40-$sulfamoyl)benzanilide] $16) $0.984 g,
1.6 mmol) in dryDMF $10 mL) and the reaction left stirring
for 48 h. Solvents were evaporated in vacuo, the residue
dissolved in CHCl3 $150 mL), ®ltered, the ®ltrate washed
48.70; H, 4.08; N, 4.89. Calcd for C22H22N2O6S4: C, 49.07;
H, 4.08; N, 5.2.
3.6. Reaction of 2,20-dithiodibenzoyl chloride with
o-phenylene diamine: isolation of benzisothiazolone 521)
and the 212 adduct 522)
successivelywith cold saturated NaHCO $2£15 mL), 1 M
3
H2SO4 $2£15 mL), distilled water $1£10 mL), dried
$MgSO4) and chromatographed on silica gel using hexane/
EtOAc $1:2) as eluent to afford 0.5 g of 17 and 0.7 g of
unreacted 16 $yellow solid).
To a stirred and ice-cooled mixture of o-phenylenediamine
$0.157 g, 1.45 mmol) and triethylamine $0.45 mL,
3.2 mmol) in dryCH 2Cl2 $40 mL) was added 2,20-dithiodi-
benzoyl chloride $0.5 g, 1.457 mmol) in CH2Cl2 $20 mL)
dropwise. The reaction mixture was left stirred at room
temperature for 2 days, washed successively with cold satu-
rated NaHCO3 $2£15 mL), distilled water $1£15 mL), dried
$MgSO4), evaporated in vacuo and chromatographed on
silica gel. Elution with hexane/EtOAc $1:1) afforded 21
and 22.
Yield: 98% $based on the starting material recovered); mp
29
113±1158C; [a]D ˆ210.0 $c 0.5, CHCl3); IR $KBr): 3333,
1
2982, 1701, 1593, 1533, 1330, 1162; H NMR $CDCl3) d
1.41 $s, 18H, BocCH3), 1.67±2.10 $m, 4H, Glu CbH2),
2.32±2.35 $m, 4H, Glu CgH2), 3.63 $s, 6H, COOCH3),
4.34 $m, 2H, CaH), 5.13 $s, ±SO2±NH±CO±), 5.29 $d,
Jˆ2 Hz, 2H, BocNH), 7.5±7.85 $m, 16H, ArH), 8.3 $br,
2H, NH); FAB MS $m/z): 1123 $M1Na)1; Anal. Found:
C, 52.67; H4.57; N, 7.75. Calcd for C48H56N6O16S4: C,
52.36; H, 5.09; N, 7.64.
3.6.1. Compound 21. Yellow solid; yield: 66%; IR $neat)
3434, 3336, 1681, 1635, 1504, 1458, 1334; 1H NMR
$CDCl3) d 2.83 $br, 2H, NH2), 6.8 $m, 2H), 7.2 $m, 2H),
$OPDA, H), 7.4 $dd, Jˆ10, 5 Hz, 1H), 7.6 $dd, Jˆ8, 4 Hz,
1H), 7.7 $dd, Jˆ8, 4 Hz), 8.1 $d, Jˆ8 Hz, 1H) $ArH); FAB
MS $m/z) $%): 243 $100) $MH)1, 485 $5) $2M1H)1, 507 $1)
$2M1Na)1; Anal. Found: C, 64.12; H, 3.87; N, 11.75.
Calcd for C13H10N2OS: C, 64.46; H, 4.13; N11.57.
3.5. The reaction of 2,20-dithiodibenzoyl chloride with
cystine-di-OMe: isolation of 18, 19 and 20
A solution of 2,20-dithiodibenzoyl chloride $2) $1 g,
2.915 mmol) in dryCH 2Cl2 $30 mL) and triethylamine
$0.9 mL, 6.4 mmol) were added dropwise, simultaneously,
to an ice cooled and stirred solution of cystine-di-OMe
$generated in situ bythe dropwise addition of triethylamine
$0.9 mL, 6.4 mmol) to an ice cooled and stirred suspension
of cystine-di-OMe´2HCl $1.1 g, 3 mmol) in dry CH2Cl2
$70 mL)). The reaction mixture was left stirred for 4 days
at room temperature, washed successivelywith cold satu-
rated NaHCO3 $2£20 mL), 1 M H2SO4 $2£20 mL), distilled
water $1£20 mL), dried $MgSO4), evaporated under
reduced pressure and chromatographed on silica gel. Elution
with hexane/EtOAc $2:1) gave 18, 19 and 20.
3.6.2. Compound 22. Pale yellow powder; yield: 5%; mp
2158C $dec); IR $KBr): 3472, 1662, 1605, 1526, 1451, 1315;
1H NMR $CDCl3±DMSO-d6) d 7.24±8.19 $m, 24H, ArH),
9.93±10.12 $brm, 4H, NH); FAB MS $m/z) $%): 757 $22)
$MH)1.
3.7. X-Ray diffraction analysis
The crystallographic parameters for the crystals are: $9)
Ê
Ê
C17H15NO4S, sp. gr. P32, aˆbˆ9.450$2) A, cˆ15.614$3) A,
Zˆ3, Mo rad., R1ˆ0.101; $11) C12H13NO4S, sp. gr. P212121,
Ê
Ê
Ê
aˆ9.705$2) A, bˆ10.181$2) A. cˆ12.433$3) A, Zˆ4, Mo
3.5.1. Compound 18. Yellow unstable solid; yield: 3%; IR
$neat): 3245, 2948, 1742, 1640, 1539, 1445, 1236; 1H NMR
$CDCl3) d 3.4 $d, Jˆ5 Hz, 1H), 3.8 $d, Jˆ5 Hz, 1H) $CbH2),
3.86 $s, 3H, COOCH3), 4.2 $m, 2H, CaH), 7.36±7.85 $m,
4H, ArH); FAB MS $m/z) $%): 302 $34) $MH)1, 324 $5)
$M1Na)1, 268 [M2SH], 236 [M2$S±S±H)].
rad., R1ˆ0.059; $12) C24H28N2O8S2, sp. gr. P21, aˆ
Ê
Ê
Ê
5.041$4) A, bˆ20.271$4) A, cˆ12.769$4) A, bˆ91.77$4)8,
Zˆ2, Cu rad., R1ˆ0.097, extremely®ne needle crystal with
0.06£0.03 mm2 cross-section; $19) C22H22N2O6S2 $dimer),
Ê
Ê
sp. gr. P21/n, aˆ7.747$1) A, bˆ19.889$2) A, cˆ
Ê
14.482$1) A, bˆ94.65$1)8, Zˆ4 $dimer), Cu rad., R1ˆ
Ê
0.051; $21) C13H10N2OS, sp. gr. Pbca, aˆ19.109$5) A,
3.5.2. Compound 19. Pale yellow thick prisms; yield: 17%;
mp 150±1528C; IR $KBr): 3191, 2962, 1755, 1661, 1458,
1384; 1H NMR $CDCl3) d 3.0 $dd, Jˆ10, 5 Hz, 1H), 3.6 $dd,
Jˆ10, 3 Hz,1H) $CbH2), 3.78 $s, 3H, COOCH3), 4.08 $m,
1H, CaH), 6.8 $br, 1H, NH), 7.4 $m, 2H), 7.6 $m, 1H), 7.7
$m, 1H) $ArH); FAB MS $m/z) $%): 238 $100) $MH)1, 260
$4) $M1Na)1, 475 $15) $2M1H)1 $hydrogen bonded
dimer), 497 $1) $2M1Na)1; Anal. Found: C, 56.06; H,
4.70; N, 5.66. Calcd for C11H11NO3S: C, 55.69; H, 4.64;
N, 5.9.
12
Ê
Ê
bˆ15.817$7) A, cˆ7.642$7) A, Zˆ8, Mo rad., R1ˆ0.056.
Acknowledgements
Financial assistance from Indian National Science
Academy, New Delhi and Department of Biotechnology,
New Delhi is gratefullyacknowledged, as well as the Of®ce
of Naval Research, USA and NIH grant 30902.
3.5.3. Compound 20. Yellow powder; yield: 31%; mp 212±
References
1
2148C; IR $KBr): 3279, 3063, 1742, 1640, 1539, 1337; H
NMR $CDCl3) d 3.55 $d, 4H, CbH2), 3.9 $s, 6H, COOCH3),
4.94 $m, 2H, CaH), 6.1 $d, Jˆ10 Hz, 2H, NH), 6.8 $dd,
Jˆ10, 0.5 Hz, 2H), 7.0 $dd, Jˆ10 Hz, 2H), 7.6 $dd, Jˆ10,
5 Hz, 2H), 8.1 $dd, Jˆ10, 0.2 Hz, 2H) $ArH); FAB MS $m/z)
$%): 539 $25) $MH)1, 561 $6) $M1Na)1; Anal. Found: C,
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ase $5 mL from 4 mM stock) in 5 mL of 1£transcription buffer
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3
0.2 mM CTP, 0.2 mM GTP, 0.05 mM UTP, 50 mM H UTP,
Calf thymus DNA, 1 mg/mL. The solution was made up using
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2
10 min each), distilled water $2£200 mL, 5 min each), and
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11. The conformations of cyclic, 20 and the open analog 3 have
been studied using simulated annealing molecular dynamics.
Initial pro®les were developed based on the diphenyl-
disulphide conformations found in the Cambridge Structural
Database. In the lowest energyconformation thus derived the
disul®de linkage was inaccessible because of it being buried.
In the open analogue 3, similar studies suggested an open
access to the disul®de bridge involved in the exchange.
12. Crystallographic data for the structures reported, have been
deposited as supplementarypublication nos. CCDC 175203
$12), CCDC 175204 $19 dimer), CCDC 17611 $9), CCDC
17612 $11), CCDC 17613 $21) at the Cambridge crystallo-
graphic Data Center. Copies of the data can be obtained free
of charge on application to the Director, CCDC, 12 Union
Road, and Cambridge CB2 1EZ, UK $fax: 144-1223-336-
033; e-mail: deposit@cdc.cam.ac.uk).
6. $a) Crescenza, A.; Botta, M.; Corelli, F.; Santini, A.; Ta®, A.
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