Peptide bond formation using an enzyme mimicking approach

w

Tmahtdra Vol. 46. No. 20. pp. 7289.7300. 1990 F’nmcd m GKSI Briuin

PEPTIDE

BOND

FORMATION

Ccsare Gennari*, Dtpartimento

di Chimica

USING

AN ENZYME

Francesco Molinari.

Umberto

MIMICKING

Piarulli,

APPROACH’

Marcella

Organica e Industriale. Universid di Milano, via Venezian 21. 20133 Milano. Italy

4020/90 S3.Lw+.~ ~agmon Rers pk

Bartoletu

Centro CNR Sost. Org. Nat.,

(Received in UK 13 July 1990)

Abstmct: A man-made enzyme-model based on a concerted proton transfer step (btfunctional catalysis) which mimics the corresponding step in non-ribosomal pcptide synthesis was developed. Important features of the model are the following: (a) a bifunctional acid-base catalystfor thiolester aminolysis rate acceleration, (b) two thiol-containing arms mimicking the “swinging arms” of the enzyme, and (c) symmetry elements so that the process can be iterated with consequent formation of the polypeptide chain. Peptide bondfonnation was obtained by intramolecularly catalyzed thiolester ambwlysis to give 5 in 80% isolated yield (Scheme IV,V) and with at least a Id-fold rate acceleration in comparison with the corresponding non catalyzed process (4 - @(Scheme IV, Table I). The reaction is also 4-20 times farter than the analogous process 4 - 6 run in the presence of 0.1 M external catalyst (Et,N-Bu’COOH or 2-Pyridone). Important structural and reaction parameters are discussed. A second intramolecular aminolysis reaction gave tripeptide 8 in lower yield (35%) because of higher steric congestion in the transition state.

Dunng

the 1970’s the biosynthetic

pathway

occurring

enzyme”

(MW

ca.280.000)

primitive

than the usual ribosomal

are the following: enzyme.

polypeptide

are involved,

are cleaved

a “light

enzyme”

using ATP-activated

by enzyme-catalyzed

for example,

ca.lOO.000)

intramolecular

aminolysis

is used by Nature for the production

and thiol

of relatively

and more sequence

groups

with consequent

with consequent

does not

and a “heavy

steps of the biosynthetic

a-aminoacids

group, and (c) this process is iterated

about IO residues. Hem we report on a man-made enzyme-model

(MW

to peptides such

sequence appears to be much simpler

biosynthesis. 2b.3aThe fundamental

are synthesized

bond and of a free thiol chain. This pathway

Two enzymes.

and the biosynthetic

protein

(a) thiolesters

(b) thiolesters

of a peptide

or tRNA.

and leading

elucidated. 2’ Gramicidin-S biosynthesis,

as Tyrocidtne and Gramicidin-S was completely rely on the presence of ribosomes

in some mtcroorganisms

of the

formatton

formation

of the

small peptides. consisting

of

which mimics this sequence of reactions.

SH

SCO-Pro-Phe \ NH2

_A Previous from

attempts

the work

throlysis

to mimic

reported

of a terminal

while the intramolecular

-

’

SCOCH-iPr the thiolesrer

here. In particular p-niuobenzoate aminolysis

SCO-Val-Pro-Phe mediated

non-ribosomal

the catalytic

step in Koga’s

of an aminoacid-NHs’Br rate (S -

pepude

N acyl transfer)

7289

synthesi@

approach

group complexed

substantially

differ

is thiolester

formation

to a pendant

crown

is enhanced by standard buffering

by

ether,

with pivalic

7290

C. Gm~at

acid and uiethylamine In our work through

in benzene (vi&

thiolesters

infra).’

are easily synthesized

mixed anhydrides

ATP activation.

(e.g. DCC.QPP;

Our efforts

er al.

are directed

using standard chemistry

DPPA;

involving

carboxylic

acid activation

DEPC; etc.)6 in a way which is quite similar

toward catalysis

of the intramolecular

thiolester

to biosynthetic

aminolysis

step, which

is the key reaction for peptide bond formation.’

The mechanism

and kinetics

bases of those results reported shown

in Scheme

determining

thiolesters

process, the reaction

energy

p-nitrophenyl). or by concerted proton-transfer to catalyze catalysts solutions,

have been studied

R’

is alkyl

mechanism

or a weakly

are high energy

substrates

in apolar

and thiolester

solvents

aminolysis

(a rate constant

changing

R’

from

an alkyl

group

can be tentatively

to a strongly

by the elimination-TS

at the TS level from the ammonium

assistance through

several alkyl

are bicarbonate,

thiolester

an eight-membered

ring (bifunctional

inter- and intramolecular

the monoanion

and 2-hydroxypyridines

of phosphate,

(e.g. 2-pyridone)

cation

substituted

electron-withdrawing

0 Rl R

S’

+ R2-NH,

kl +_ k.1

interirradiate 2 I

I

‘~y$-$++q

addition-TS

eliminallon-TS

(e.g.

anion.8i A concerted

see Scheme II) has been used

phosphonates.

fast

group

in protic solvents (water).‘”

to the sulfide

acids (e.g. pivalic

Scheme I

for a

rate is to decrease the elimination

catalysis,

~o,vents 3b.8b.lO.l I

favored

was reported

aminolyses. 4S*9 The most widely

or carboxylic

as

the rate

(see Scheme

is a thermodynamically

Other ways to catalyze the process are by general acid-base catalysis proton-transfer

described

group,

of 1.5~10~~ sec”M”

case in benzene). 5d One way to increase the reaction

by

in some detaiL8 On the

electron-withdrawing

and the reaction appears to be controlled

is very sluggish

simple intermolecular

aminolyses

in the literature 8hc.d*e the reaction

I. In most cases, when

step is thiol elimination

I). Although

activation

of ester and thiolester

methyl

used bifunctional

arsonate

in aqueous

acid, acetic acid) in aprotic

7291

Peptide bond formation

Scheme II 0

R’

I R

~~~+x~

K

R* N’ H

H

A typical M pivalic

thiolester

N.--H.. I\

aminolysis

is usually carried out in benzene at room temperature

catalyzed

aminolyses

weak carboxylic

reaction.5d~8h (c) a-pyridone

of acid (>o.

I M) the rate acceleration decreases because the nucleophilic

IS almost as effective

is less reactive,5d*8h (e) the solution

acid and E13N shift the equilibrium

a 3.4 fold rate acceleration and amine concentrations II is evident

(dependent 20-fold

in the presence of 0.15

towards the inactive

on the amount offree

and acetic acid strongly

as acetic acid. 8h (d) with increasing

can be buffered

cases.’

: (a) EQN alone has little or

are the following

actds such as pivaltc

ammolysis

of pivalic

H

Rate constants are usually around 2-6x10 5 sec.’ for the intramolecular

features of the bifunctionally

and therefore

ex Y-

R’SH

activity, 8h (b) relatively

no catalytic

+

R2

acid and 0. I5 M Et,N.

lmpottant

+

y

amine is protonated

by added EI,N;

however,

uiethylammonium

the

IO a greater extent. increasing

pivalate

pivalic acid) is achieved

catalyze

concentrations

amounts

and therefore only

upon increasing

the acid

(from 0.005 M to 0. I M).sd

from these data that the rate acceleration

obtained with 0.1 M pivalic

acid and 0.1 M EI~N cannot

be pushed much further.

We have developed features:

a bifunctional

(a) a bifunctional

thiol-containing

arms which

acid-base mimic

catalyst catalyst

the “swinging

process can be iterated with consequent

Startmg

from

(Scheme III). with

commercially

Monoacylation

BOC-Ala

available

of Et,N

high dilution

thiol

thiolester

of a polypeptide

2.6-dihydroxybcnzorc (using DCC/4-PPh

IV).

neutralize

groups

and possessing

aminolysis

and (c) C2-symmetry

acid, dlthiols

(b) two

so that the

I and 2 were easily synthesized

or DEPCb acnvation,

W-55%)

and acylatton

3 and 4 (Scheme IV). Transacylation

the TFA.salt)

the following

rate acceleration,

chain.

ester groups of 3 (I: I TFA-CH,CI,.

(1.5 mol.equiv.)(to

(lx 10 ’ M)(Scheme

pendant

arms” of the enzyme.‘.2.3

YO-95%) gave compounds

removal of the BOC and the t-butyl additron

formation

with Z.Gly

(DPPA/D,MF,6

bearing

for promoting

O”C, 100%). evaporation

to a benzene solution

again

was effected

by

of solvent, and

of the intermediate

under

7292

c. GENNARI et 01.

Scheme III. Synthesis of dithiols 1 and 2. Jn=3[ HO(CH,),OH +C

d_.

BnO(CH2)20H 9

BnO(CH2)20(CH2),0H

d_

b120_

BnO(CH2)20W2)30THP 10 e/21 or 22 BnO(CH&O(CH&OTs 12

11 R

Y-(CH,),WH,),

HO(CH,),OH

&

,WH,),WH,),-Y

HO(CH,),OMP 19

COOBu’

HO(CH,),OH

A

+

d

HO(CH,),OTBDMS

O(CH,),-Y

I HO(CH,),O(CH,),OH

-

Y= OBn R=C02But

14 Y= OBn R=COfle

Y= OH R=C02But Y= SAC R=C02Bu’

16 Y-OH R=C02Me 16 Y-SAC R-C02Me

hL

1

Y-SH

2 Y=SH

R=C02Bu’

R=C02Me

COOMe

L

23

Y-G+,),0

13 15 17

TsO(CH,)~OTHP 20

COOH

R

f /

gt

TsO(CH,),OTBDMS 24

e

rn< 25 g i 27 29

Y= OTBDMS R= C02Bu’ 26 Y= OTBDMS R=CO,Me Y= OH R=C02Bu’ 28 Y= OH R=CO,Me Y= SAC R=C02Bu’ 30 Y= SAC R=C02Me

hc

Y-SH

1

R=CO+u’

2

Y= SH R=CO,Me

e 121 H0(CH2),0(CH2),0TBDMS

-f-w

m~33

TsO(CH,),O(CH,),OTBDMS 32 Y= OTBDMS R= C02Bu’

g c”,

;I

31 R O(CH,),O(CH,),-Y

h<

l

-

zyc RR==y&‘;t

Y= SH R&O&’

Reagents and average yields: a) NaH. BnBr. THF. Bu,NI cat (50%) b) Bu,NHSO,. 50% NaOH In H20. loluene (84%) c) MeOH. PPTS (95%) d) TsCI. C,H,N. 4-DMAP cat (65%) e) Cs,CO,. DMF (60.91%) I) H2, Pd-C. MeOH (99%) g) AcSH. DEAD. PPh,. THF (74.90%) h) MeONa, MeOH (65%) i) DHP. CH,CI,. PTSA cat. (50%) I) NaH. THF. TBDMSCI (70%) j) Me,NCH(OBu’),. benzene (65%) k) MeOH. H,SO, (65%) m) TBAF. THF (95%)

1293

Peptide bond formation

R

Scheme IV

0(CH&-X-(CH2)2SH

HSKH,),-X-KH,)R n-2.3

1 A=C029Ut

x - CH2.0

2 RdqMe

4 R=C02Mo

I

1. TFA-CH$$ , O’C 2. benzene, E13N

H

0

OICH&-X-W&

NWH,),-X43+3@ 5 R=CO$t 6 R=CO$Je I

H

0

R

CWX,),-X-(CHd,S

S(CH&-X-KHz)@

7 R=C02H 1. TFA-W&I,

, 0%

R

0(CH2),-X-(CH2)2SH

“j NHZ

Scheme V

-5 (n=3, X=0)

Peptide

bond

eight-membered

formation

is proposed

to occur

with

ring via the properly oriented carboxylic

catalysis

rapid

acid (Scheme V).”

gave dipeptide 5 (80% isolated yield) with rate constants of 8xlO%ec sec.’ (X=CH,.

by

tlR= 3 h and 50 min), and with ur least u Id-fold

’ (X=0,

proton

Experiments

transfer

through

an

with n=3. R=COtH

tlR= 2 h and 25 min) and 5xlO.s

rule acceleration

in comparison with the

C.GENNm er al.

corresponding

noncatalyzed

processes [4 -

6. n=3, X=0

6 months (k < 5x10 8 WC-‘)]. This acceleration (e.g.l.5xld).

I3 but shows

the feasibility

by rate constants

Rxn

Product

Important more polar (DBU)

aminolyses

n

X

3 3 3 3

0 CH2 0 CR2

8x10-5 5x10-5 <5x1 o-8 <5x1 o-8

3 3

0 0

;.;;;;I;

3 3 2

CH2 CH2 0

2:9x10-6 7x10-5

than Et,N,

going

from

n=3. X=CH,

than the

and are approximately

20

to n=3, X=0.

External

(a) bifunctional

(b) weaker

(Pyridine.

(more than 2) or incomplete

(e) CPK

and (f) no significant

rate difference

R=CO,H,

k=7xlO

models

(Scheme

IV,

catalysts

is inhibited

2,6-Lutidine)

neutralization

tend to slow down

of the TFA-salt

and 23-membered

for the best orientation

by

or stronger

(less

there is a 1.6-3.2 fold rate acceleration

suggest that the 2l-

V), allow

was observed

[M]

ButCOOH LO.11 Et3N LO.11 IO.11 2-Pyridone ButCOOH [O-l] Et3N IO.11 [O.l] 2-Pyridone __

the following:

DMSO),

catalyst -__ ---

in the arms makes a difference:

catalysis,

on

macrocyclic

for the bifunctional

between the n=2 and n=3 series (e.g. n=2. X=0,

5 sec.‘).

A new BOC-protected Unfortunately

the

steric hindrance

and formation

aminoacid second

(e.g. BOC-Ala) intramolecular

at the a-carbon

of the desired

Ala-Ala-S-(Spacer)-S-Ala-Gly-Z, working

’ (X=CH,),

(c) more than 2 or less than I mol. equiv. of Et,N

to n=2,3

VI).‘4

include DMF.

states, corresponding

unfavorable

@in)

575

transition

X=0).

set

3300 1860 3900 165

formation

than I), (d) the presence of oxygen

in other cases faster

I M pivalic acid and 0.1 M EtsN which are

145 230 ---

CH,CN,

because of carboxylate

times

(Table I).

tl/p

2x10-5

amines are less effective

the reaction,

k (set-‘)

(e.g. CH,CI,.

to those reported

are also 4-8

in benzene (IO 3M).

features of these transformations solvents

and 6.2~10’~

by 0.1 M 2-Pyridone

no reaction product delected after

small, compared

The reactions

run in the presence of 0.

of 2x10 5 sec’*(X=O)

times faster than the teactions catalyzed

Table 1. intramolecular

is still relatively

of the process.

analogous processes 4 + 6 (n=3. R=COzMe) characterized

or CH,. R=COzMe,

on a new macrocyclic

derived

should give higher rate accelerations

aminolysis

(Scheme

of the acyl group (CHMe

tripeptide

model

can then be added (DPPA/DMF,85%)

from system

8 (n=3,

X=0)

intermolecular with

reduced

IV)

appears

to

proceeds

in low yield

aminolysis,

(35%);

are also isolated.

conformational

freedom

R,=H(g09 0

dipeptide

by

Scheme 5 and

We are currently

of the two arms which

q

R3~HN$$~$&Rz

be complicated

vs. CH, of the first aminolysis,

and reduce the amount of side products formed.

Scheme VI

to give 7 (n=3,

yield)

R’= Me (3;9’0 yield)

1296

C. Cimm

er al.

mixture was then diluted with ethyl acetate and washed with I N HCI. The organic layer was dried and the solvent evaporated; the crude product was purified by flash chromatography (n-hcxant-ethyl acetate 7:3) to give tosyke 12 in 85% yield. ‘H NMR (CDCI,) 6: 1.87-2.08 (2H, m), 2.41 ( 3H. s), 3.41-3.59 (6H. m), 4.17(2H, t, J=6.67Hz), 4.54 (2H, s). 7.34 (ZH, d, J=8.35), 7.34 (SH. bs). 7.78 (2H. d, J=8.35). Tosvlate 20. Purified by flash chromatography (n-hexane-ethyl acetate 7:3). Yield: 70%. ‘H NMR (CDCl ) S: 1.41-1.70 (6H. m), 1.86-1.94 (2H, m), 2.41 (3H, s). 3.32-3.50 (2H. m). 3.70-3.82 (2H, m). 4.26 (24. t, J=585Hz). 4.40-4.49 (IH. m), 7.36 (2H. d. J=8.35Hz). 7.80 (2H. d, J=8.35Hz). Tosvlatc 24. Putifted by flash chromatography (n-hexane-ethyl acetate 9: I). Yield: 85%. ‘H NMR (CDCl ) S: 0.04 (6H, s), 0.89 (9H, s). 1.25-1.51 (6H, m), 1.62-1.71 (2H, m). 2.41 (3H. s), 3.57 (2H. t. J=6.50Hz). 4.02 (2k. t, J=6.5OHz). 7.36 (2H. d. J=8.35Hz), 7.78 (2H. d, J=8.35Hz). Tosvlate 32. Purified by flash chromatography (n-hexane-ethyl acetate 85: 15). Yield: 79%. ‘H NMR (CDCI,) 6: 0.05 (6H. s), 0.90 (9H, s), 2.41 (3H. s), 3.50-3.80 (6H, m), 4.30 (2H, t, J=6.30Hz), 7.36 (2H. d. J=8.35Hz). 7.80 (2H. d. J=8.35Hz). General Drocedure for the synthesis of Diphenolic ethers 13. 14.25.26,33. Dibenzvlderivate 13. A solution of diphenol 21 (0.220 g. 1.046 mmol)% dry DMF (8 ml) was treated with cesium carbonate (Cs_COs) (1.022 g. 3.138 mmol); after stirring for 30 min. a solution of tosylate 20 (0.800 g, 2.197 mmol) in dry DMF (2 ml) was added. The reaction mixture was stirred for 10 hr at room temperature, then diluted with ethyl acetate (50 ml) and washed with a saturated aq. NH&I solution. The organic extracts were dried, evaporated and the crude product was purified by flash chromatography (n-hexane-ethyl acetate 65:35) to give 13 in 78% yield. (Modified procedure from ref. 17). ‘H NMR (CDCI,) 6: 1.58 (9H. s), 2.02-2.17 (4H, m), 3.61-3.70 (12H. m). 4.09 (4H. t, J=6.67Hz). 4.58 (4H. s), 6.53 (2H. d. J=7.75Hz). 7.18 (lH, t. J=7.75Hz), 7.37 (IOH, bs). Dibenzvlderivate 14. Purified by flash chromatography (n-hexane-ethyl acetate 6:4). Yield: 72%. ‘H NMR (CDCI,) 6: 2.02-2.19 (4H, m). 3.60-3.70 (12H. m). 3.88 (3H. s). 4.08-4.16 (4H. m). 6.56 (ZH, d. J=7.95Hz). 7.22 (1H.T. J=7.95Hz), 7.37 (10H. bs). Protected diol 25. Purified by flash chromatography (n-hexane-ethyl acetate 9:l). Yield: 84%. ‘H NMR (CDCI,) 6: 0.10 (12H. s). 0.94 (18H. s), 1.35-1.58 (12H. m). 1.56 (9H. s). 1.68-1.83 (4H. m), 3.60 (4H. t. J=6.50Hz), 3.98 (4H. t, J=6.50Hz). 6.49 (2H. d, J=7.75Hz), 7.18 (IH. t, J=7.75Hz). Protected diol 26. Purified by flash chromatography (n-hexane-ethyl acetate 9:l). Yield: 60%. ‘H NMR (CDCI,) 6: 0.10 (12H. s). 0.94 (18H. s). 1.32-1.60 (12H, m), 1.56 (9H, s), 1.68-1.83 (4H. m), 3.60 (4H. I. J=6.50Hz). 3.98 (4H. t. 6.50Hz). 6.49 (2H, d, J=7.75Hz). 7.18 (I H, t, J=7.75Hz). Protected diol 3_3. Purified by flash chromatography (n-hexane-ethyl acetate 8:2). Yield: 91%. ‘H NMR (CDCI ) 6: 0.09 (12H. s), 0.90 (18H. s). 1.52 (9H, s). 3.48-3.89 (12H. m). 4.08 (4H. t. J=6.09Hz), 6.51 (2H. d. J=79Okz), 7.19 (IH, t. J=7.90Hz). General urocedure for the synthesis of diols 15 and 16. Diol 15. A suspension of palladium on activated charcoal (10% Pd) (95 mg. 0.088 mmol) in MeOH (1 ml) was treated with a solution of 13 (264 mg, 0.444 mmol) in McOH (3 ml). The reaction mixture was stirred under hydrogen atmosphere for I hr. the mixture was filtered on a celite pad and the solvent evaporated. The crude product was purified by flash chromatography (methylene chloride-methanol 95:5) to give 15 in 99% yield. ‘H NMR (CD&./D 0) 6: 1.58 (9H. s), 2.01-2.19 (4H. m). 3.52 (4H. t. J=550Hz), 3.53-3.71 (8H. m), 4.05-4.17 (4H. m). 6.53 (2H.d. 3=7.75Hz). 7.20 (IH. t, J=7.75Hz). Diol 16. Purified by flash chromatography (ethyl acetate-methanol 98:2). Yield: 99%. ‘H N MR (CDC13/D,O) S: 2.02-2.18 (4H. m). 3.54-3.72 (12H. m), 3.91(3H. s), 4.1 I (4H. t. J=5.95Hz). 6.56 (2H. d. J=7.95Hz), 7.22 (IH. t, J=7.95Hz). General oroccdure for the synthesis of dithiolesters 17. l&29.30,35. Dithiolester 17. To a solution of PPh, (1.785 g, 2.72 mmol) in dry THF (8 ml) diethyl azodicarboxylate (DEAD) (I.067 ml. 6.81 mmol) was slowly added at 0°C. After stirring for 20 min. ‘he reaction mixture was treated with a solution of diol 15 ( I. 128 g. 2.72 mmol) and thioacetic acid (A&H) (0.484 ml, 6.8 I mmol) in dry THF (5 ml). The resulting solution was stirred for 1 hr at 0°C and at room temperature for 2 hr. The solvent was evaporated and the crude product was purified by flash chromatography (n-hexane-ethyl acetate 7:3) 10 give 17 in 84% yield. (Modified procedure from ref. 18). ‘H NMR (CDCI,) 6: 1.58 (9H. s), 2.0?(4H, t. J=6.50Hz), 2.32 (6H. s), 3.08 (4H. t, J=6.80Hz). 3.57 ( 4H. t. J=650Hz), 3.61 (4H, t. J=6.8OHz). 4.09 (4H. t, J=650Hz). 6.52 (2H. d. J=7.75Hz). 7.20 (IH. t, J=7.75Hz). Dithiolester 18. Purified by flash chromatography (n-hexane-ethyl acetate 65:35). Yield: 87%. ‘H NMR (CDCl,) 6: 2.03 (4H. t. J=6.50Hz), 2.37 (6H. s), 3.09 (4H. I. J=6.80Hz). 3.54.3.68 (XH, m), 3.88 (3H. s). 4.04 (4H. t. J=6.50 Hz), 6.58 (2H, d, J=7.95Hz). 7.22 (1H. I. J=7.95Hz). Dithiolester 29. Purified by flash chromatography (n-hexane-dietyl ether 7:3). Yield: 90%. ‘H N MR (CDCI,) 6: 1.47-1.78 (16H. m), 1.51 (9H. s). 2.32 (6H. s), 2.90 (4H,t. J=h.SOHz). 3.98 (4H. t, J=6.50Hz), 6.49 (2H. d.

C. GENNARler al.

Dial 27. Compound 25 (0.607 g, 0.95 mmol) was dissolved in a 1.0 M solution of tetrabutylammonium fluoride(TBAF) in THF (3.8 ml). The reaction mixture was stirred for 1 hr at room temperature and then diluted with ethyl acetate (40 ml) and washed with water (IO ml); the organic extracts were dried and evaporated. The crude product was purified by flash chromatography (n-hexane-ethyl acetate 3:7) to give 27 in 99% yield. ‘H NMR (CDCl@,O) 6: 1.39-1.94 (16H. m), I.51 (9H, s), 3.67 (4H. t, J=6.50Hz), 3.99 (4H. t, J=6.5OHz), 6.52 (2H. d. J=7.75Hz), 7.20 (IH, I. J=7.75Hz). Dial 28. Purified by flash chromatography (n-hexanecthyl acetate 15:85). Yield: 99%. tH NMR (CDCI@@) 6: 1.37-1.77 (16H, m). 3.57 (4H. t. J=6.50Hz). 3.90 (3H. s). 3.99 (4H. t, J=6.50Hz). 6.50 (2H. d, J=8.04Hz$7.22 (IH. I. J=8.04Hz). Dial 34. Purified by flash chromatography (ethyl acetate-MeOH 97:3). Yield: 76%. tH NMR (CDCI I.52 (9H. s), 3.51-3.72 (8H. m), 3.82 (4H.t. J=5.40Hz). 4.19 (4H. t, J=5.40Hz), 6.57 (2H. d, J=7.9OHz). s/”.2$$ t, J=79OHz). General Procedure for the synthesis of Dithiolester 3 and 4. Dithiolester 3 (n=3. X=01. Dithiol 1 (n=3, X=0) (436 mg, 0.966 mmol) was added to a stirred solution of Z-GIy (202 mg. 0.966 mrnol) and DCC (199 mg. 0.966 mmol) in dry methylene chloride (I5 ml) at rOOm temperature. After 5 min 4-Pyrrolidinopyridine (4-PP) (2 I .5 mg, 0. I45 mmol) was added and the solution stirred for 4 hr. The solvent was then filtered and evaporated: the crude product was purified by flash chromatography (n-hexane-ethyl acetate I:]) to give the mono-Z-Gly derivative in 50% yield. mono-Z-Gly derivative (n=3, X=0. R=COOtBu) : ‘H NMR (CDCI,) 6: 1.59 (9H. s). 1.95-2.04 (4H. m), 2.64 (2H. dt, J=6.77.6.50Hz). 3.12 (2H. t. 6.5OHz), 3.51-3.68 (8H. m), 4.01-4.18 (6H, m), 5.18 (2H, s), 6.53 (2H, dd, J=7.15Hz). 7.21 (IH. T.J=7. I5Hz). 7.48 (5H. bs). A solution of this compound (438 mg. 0.688 mmol) in dry DMF ( 7 ml) was treated with diphenylphosphoryl azide (DPPA) (253 ~1. I. 170 mmol). Ala-Boc (195 mg, 1.030 mmol) and triethylamine (144 )I]. I.030 mm~I) at O’C. After stirring for 2 hr the solution was diluted with ethyl acetate (50 ml) and washed with brine (25 ml); the organic extracts were dried and evaporated. The crude product was purified by flash chromatography (n-hexane-ethyl acetate 55:45) to give dithiolester 3 (n=3, X=0) in 92% yield. (Modified procedure from ref. 21). ‘H NMR (CDCI,) 6: 1.36 (3H. d. J=6.80Hz), 1.46 (9H. s), 1.57 (9H, s), 1.93-2.04 (4H, m). 3.03-3.14 (4H. m), 3.45-3.52 (8H. m), 4.01-4.11 (6H, m). 4.29-4.41 (IH. m), 5.18 (2H, s), 6.52 (2H. dd, J=7.I5Hz). 7.21 (IH. I, J=7.15Hz), 7.48 (SH.bs). In alI the reaction sequences leading to the formation of dithiolesters 3 and 4. mono-Z-Gly derivatives were isolated and purified (details below), and then transformed as described above. mono-Z-Gly derivative (n=3. X=0. R=COOMe). Purified by (lash chromatography (n-hexane-ethyl acetate 55:45). Yield: 50%. ‘H NMR (CDCI,) S: 1.57 (IH. t. J=7.83Hz), 1.89-2.05 (4H. m), 2.58 (2H. dt. J=7.83.6.50Hz), 3.09 (2H, t, J=6.5OHz). 3.52-3.60 (8H, m), 3.90 (3H. s), 4.01-4.09 (6H. m), 5.14 (2H. s). 5.41-5.52 (IH, m). 6.55 (2H. dd. J=7.95Hz), 7.23 (IH. 1,J=7.95Hz). 7.37 (5H. bs). Dithiolester 4 (n=3. X=0). Purified by flash chromatography (n-hexane-ethyl acetate 53:47). Yield: 90%. ‘H NMR (CDCI,) 6: 1.44 (9H, s). 1.93-2.04 (4H. m), 3.01-3.12 (4H, m), 3.47-3.53 (8H. m). 3.90 (3H. s), 3.99-4.14 (6H m), 4.35-4.46 (IH, m). 5.18 (2H, s). 5.51-5.65 (IH, m), 6.51 (2H. dd. J=7.95Hz), 7.21 (IH, t, J=7.95Hz). 7.37 (5H, bs). mono-Z-Glv derivative (n=3. X=CH,. R=COOtBu). Purified by flash chromatography (n-hexaneethyl acetate 7:3). Yield: 49%. ‘H NMR (CD&) 6: 1.38-1.81 (l7H. m), 1.59 (9H. s), 2.54 (2H. dt, J=7.22.6.50Hz). 2.92 (2H. 1; J=6.5OHz), 3.97 (4H. I. J=6.50Hz); 4.12 (2H. d, J=6.67Hz), 5.18 (2H. s). 5.79-5.92 (IH, m). 6.53 (2H. d. J=7.15Hz), 7. I8 (1 H.I. J-7.15Hz). 7.37 (5H. bs). Dithiolester 3 (n=3. X=CHd. Purified by flash chromatography (n-hexaneethyl acetate 65:35). Yield: 91%. ‘H NMR (CDCI ) 6: 1.38-1.81 (16H. m). 1.46 (9H. s), 1.59 (9H, s), 2.88 (2H, I, J=7.52Hz). 2.91 (2H. t, J=7.22Hz). 3.97 14H, I. J=6.50Hz), 4.12 (2H.d. J=6.62Hz). 4.29-4.46 (IH. m). 4.93-5.04 (IH. m), 5.18 (2H, s). 5.29-5.40 (I H, m). 6.53(2H.d, J=7.18Hz). 7. I8 (I H, I, J=7. I8Hz). 7.37 (5H, bs). mono-Z-Glv derivative (n=3. X=CH,. R=COOMe). Purified by flash chromatography (n-hexane-ethyl acetate 65:35). Yield: 50%. ‘H NMR (CDCI,) S: 1.22-1.86 (19H. m). 2.56 (2H. dt, 5=6.67.6.50Hz), 2.97 (2H. t. J=6.50Hz), 3.90 (3H, s), 3.97 (4H. I, J=6.50), 4.13 (2H. d, J=6.6OHz), 5.63 (2H. s), 5.28-5.42 (IH. m). 6.57 (2H. d, J=7.77Hz). 7.22 (I H. t, J=7.77Hz), 7.37 (5H. bs). Dithiolester 4 (n=3. X=CH,). Purified by flash chromatography (n-hexane-ethyl acetate 6:4). Yield: 92%. ‘H NMR (CDCI,) 6: 1.22-1.86 (IgH, m), 1.41 (9H, s). 2.81-2.97 (4H. m), 3.90 (3H. s), 3.97 (4H. I. J=6.50). 4.13 (2H. d, J=6.6OHz). 4.30-4.52 (IH. m). 4.91-5.09 (IH, m), 5.18 (ZH, s), 5.24-5.532 (IH. m), 6.51 (2H. d. J=7.77Hz), 7.22 (I H, t, J=7.77Hz), 7.37 (5H. bs). mono-Z-Glv derivative (n=2, X=0. R=COOtBu). Purified by flash chromatography (n-hexane-ethyl acetate I:]). Yield: 51%. ‘H NMR (CDCI,) 6: 1.59 (9H. s). 2.68 (2H. dt, J=6.50,6.78Hz). 3.12 (2H. t, J=6.50), 3.66 (2H, 1. J=6.50Hz), 3.78 (2H. t, 6.lOHz). 4.08-4.21 (6H. m). 5.18 (2H. s). 5.39-5.51 (IH, m), 6.56 (2H. d, J=7.98Hz),

C.GENNARI er al.

7300

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D.J.Kerman.

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