A new role for l-ascorbic acid: michael donor to α,β-unsaturated carbonyl compounds

7rhakhm Vd. 39, No. 13. pp. 2137 to 2145. 1983 Pnatcd In GreoI Britain.

-/83

A NEW ROLE FOR L-ASCORBIC ACID:

$3.00 + .a3 F’qamon Prm Ltd.

MICHAEL DONOR TO o,B-UNSATURATED

CARBONYL COMPOUNDS1

GABOR FODOR", REGINA ARNOLD AND TIVADAR MOHACSI National Foundation for Cancer Research Laboratory at the Department of Chemistry, West Virginia University, Morgantown, WV

26506

ISABELLA KARLE AND JUDITH FLIPPEN-ANDERSON Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375

(Receivedin USA

14 January 1983)

Abstract - In a novel Michael-type reaction L-ascorbic acid (1) undergoes addition to acrolein to give the tricyclic hemiacetal lactone 3. The constitution and relative configuration of 3 was studied by a combination of NMR and IR spectroscopy. Ultimately, the structure of 3 was determined by X-ray crystallography. The absolute stereochemistry follows from the known chirality of C-4 and C-5 of L-ascorbic acid. A mechanism for the reaction, including its steric course, is proposed. Methyl vinyl ketone reacts with 1 in a similar fashion to give diketo lactone derivative 4. Upon the action of methanolic hydrogen chloride on 5 one anomer, the tricyclic methyl ketal lactone 5, forms. Structure 5 is closely related to 3. Synthetic and possible biological applications of the new reaction are-discussed. Recently one of us and his associates found

saturated lactone.

that L-ascorbic acid (1) undergoes enediol

the primary product (m.p. 114-116")

The elemental analysis of

acetal formation with 1,2-dicarbonyl

corresponds to the monohydrate of a 1:l adduct

compounds like methylglyoxal and their 2-4 vinylogues e.g., 4-keto'2-pentenal.

between acrolein and -1. However, the adduct can be dehydrated to product 2 (m.p. 150-152') via

In contrast, we now observed that acrolein

the ternary azeotrope with ethanol and

reacts with ascorbic acid in water to give a

benzene.

crystalline product that shows no olefinic

is the 1:l adduct.

protons and carbons, respectively, lnthe and "C

NMR spectrum.

'H

Both 2 and its monohydrate show identical

A single carbonyl

13C NMR spectra in DMSO-d. (Fig. 3).

band appears at 1780 cm-' in the IR and a signal at 175

Elemental analysis confirms that 2

A

noticeable feature is that most of the major

ppm in the r'C NMR spectrum.

signals appear as narrowly spaced "doublets"

These values suggest the presence of a

in the proton decoupled spectrum.

2137

This

2138

G. FODORet al.

points

to

isomers of

2 can

is

presence

are

of

in

with

that

MS0

solution.

(Fig.

from

4)

which

findings

hemiacetal

the

aldehyde

crystals

of

recrystallization

2 were

from

a structure

study

are

obtained

thus

crystallizes

feasible.

described

in

in

Details

the

of

c = 10.318(4)

the

monoclinic

A,

independent

A,

B -

using

structure

was

were

for

each

from

observed to

in

hydroxyl the

of

and

also

resultant

refinement

by

for

did

not

data

atoms

were after

the

C

well

coordinates

hydrogen

give

z1 IFoI-IFcl Z/F 1

on in

the

that

1,

I

using final

good

and O-9 on O-12. these

R factors,

Table and

1 lists

the

Beq values

for final for

our

analysis.

of

in FIp,.

1,

experimentally The molecule

b-membered

the

is

has

ring

and

consistent

cyclic

hemiacetal

the

configurations were

The absolute

C-4

the

rational

by

configu-

the

and C-5

to

at

established

by using

atoms

2’6.02’9]

know” of

ascorbic

new ehiral

name of

dodecane.

throughout

request

centers.

1.3,7-trioxa-

the

Chemical name:

This

numbering

text.

However,

upon

Abstracts

suggcstcd

the

(3S,3aR,SaS,6R)-hexahydro-

R and 3.94% refined “on-

which

Fig.

2 fiall

within

The angles

around

C-2,

indicate

that

form

in

and angles

values.

the

without ring

(the

has

C-2,

C-9,

34.1(5)O).

Both

conformation

the

C-2,

C-6

the

“flaps”;

by C-2,

O-3,

and C-2

is

C-2,

bond

C-4

and C-5

of

and C-9 The ring

O-1

packing

rings torsion is

intermolecular

the

the

in

also angle

influenced hydrogen

are

of

is

in a”

(the

the

bonds.

rings

by C-6,

the H-6,

two

Crystal

presence O-5

5-

C-6,

-31.2(5)‘). the

from

formed

five-membered

between

by

&r

about

atoms

formed

other

cc.*

is is

plane

one

plane

In the

5-

twisted

these

of

junction is

rings

that

out

the

angle

and are

such

chair

junction

5-membered

is

out

at

able

The h-

flattened

torsion

C-6

and C-9 was

strain.

ring

O-9

expected C-6

fusion

h-membered

are

system

a somewhat

envelope

C-8

ring

The ring

and

O-3,

fused

significant

conformation. membered

ring.

the

a

centers

was deduced

membered 3.24%

the

This

of

as a reference

C-7, and Rw -

so

WFO =

R+.

on O-5

to

stereodiagram

The relative

chiral

membered

parameters

map showed

calculated

of

[

X-ray

to

and COH angles

a disordered

Zw(jFo\-iFc/)’

the

the product

2-(3-oxopropyl)-3-oxo-I.-gulono-

illustrated

were

on just

refine

hydrogens

parameters

R =

five

elucidated addition

one

Bond distances

The hydrogens

difference the

factors

hydrogen

the

[3,4-blpyran-S-one.

deviations

positional

O-H distances

and indicated

where

from

3,5a,8-trihydroxy-211,5H-furo-[3’,2’:2,3]furo-

map calculated

refined.

A subsequent

for

using

unreasonable.

Structure

and a

and calculated

the

rings.

of

14.3.2.0

w

Hydrogen

their

oxygens

positions

thermal

has

from

structure

following

802

the

rings,

(2).

the

were calculated All

the

was used

minimized

standard

refinement.

cycles

then

were

estimated

a difference

and 0 atoms were

the

&oxo-(55,9S,12R)-trihydroxy-(2R,6E)-tricyclo-

refined

weights,

in

5-membered

We choose

thermal were

of

coordinates.

fused

acid

was

the

Gilardi7.

the

several

and

atoms

available

analysis

shown

chirality

The

methods

procedure

intensity)

according

located

are

by computer

ration

a graphite

The function

where

collected

symbolic

and

least-squares

least-squares

in

that

draw”

lac tone

non-centrosymmetric

C and 0 atom

0RFXLS3.

(derived

for

observed

for

atoms,

“on-hydrogen

formula

3,6-ketal

A,

beam. the

The coordinates

full-matrix

used

using for

group

diffracto-

with

incident

solved

Cw(IF,I-IF&)‘,

of

the

procedure5

crystals.

program6

coordinates

carbon

Anisotropic

the

factors

structural

with

and 2 = 2.

X-ray

CuKa radiation on

space

101.8(l)’

reflections

monochromator

the

between

two

-3

b = 7.526(S)

on a NICOLET P3F automatic

by

comparison

determined

following

The compound

a = 6.293(3)

addition

to

map coordinates

for

three

P21 with

refined

hydrogens.

parameters

be

by X-ray

became

Crystalloarauhv.

factors

difference

the bonded

The X-ray

paragraph.

meter

the

by

methanol-ether,

determination

crystallography

802

hydrogens

authors.

Single

X-Ray

atoms,

the

structure

group

acrolein.

this

hydrogen

hydroxyl

between

These

a cyclic

arises

related

mutarotatio”

an equilibrium

3 in

consistent

two closely

Indeed,

be observed

of

moiety

of

solutio”.

indicative

isomers

of

the

in

of is

3

a donor

A new role for L-ascorbic acid

2139

Table 1.

Fractional Coordinates and Equivalent Isotropic Thermal Parameters with e.s.d.'s in Parentheses. --.\*om x 2 v 8eq ~_____-0.3529(4) 0.2379(j) 0.1204(4) 0.2057(6) 0.4037(7) 0.6029(4) 0.3816(6) 0.24?6(4) 0.0714(7) -0.0665(5) 0.0834(5) -0.1240(4) 0.1752(7) O.U73(7) 0.4X5(6) 0.6042(4)

O(1) C(2) O(3) C(4) C(5) O(5) C(6) O(7) C(8) O(8) C(9) O(9) C(10) C(11) C(12) O(12)

0.5957(7) 0.759L 0.7605(7) 0.8843(9) 0.9697(8) O-8954(7) 0.9252(7) 1.0638(6) 0.990?(9) 1.0844(7) 0.7917(a) 0.7134(7) 0.7223(9) 0.5295(8) 0.5204(8) 0.6156(8)

2.5(l) 2.3(l) 2.9(l) 3.5(2) 2.9(l) 3.1(l) 2.8(l) 3.3(2) 3.0(l) 3.9(2) 2.6(l) 2.9(l) 3.1(l) 3.3(2) 3.2(l) 3.9(2)

----

--____

=

B eq

Fig.

0.1958(Z) 0.1806(3) 0.0505(2) -0.0302(4) 0.0552(4) 0.0337(3) 0.1955(4) 0.2375(3) 0.2762(4) 0.3054(3) 0.2762(4) 0.2319(3) 0.4137(4) 0.4059(4) 0.3256(4) 0.3970(3)

1.

; z r oii ai*.l. J 1.i

Stereodiar:ram of the structural formula and stercocnnfiguracion of 3 as rlr,~~r,:i:~i~d h':k-rav ~if~Fr;~ctior,.

d

10 1OQ.Z

Fig. C.

119.7 b01

aund lenp,ths (e.s.d. 0.003

X)

01-2-6 j-2-9

101.7 t12.e

and bond angles (e.s.d. 0.5*) in 3.

2140

G. FODORer al. Table 2.

Fractional Coordinates for H Atoms with e.s.d.'s in Parentheses.

Atom

x

Y

H(4) H(4) H(5) H(6) H(l0) H(lO) H(U) H(ll) H(l2) H(O5) H(O9) H(012)

0.248(9) 0.082(8) 0.410(8) 0.531(8) 0.302(9) 0.065(7) 0.120(8) 0.286(8) 0.479(8) .635 -.174 .682 .664

0.811(8) 0.979(8) 1.103(9) 0.907(9) 0.801(9) 0.736(9) 0.450(9) 0.478(8) 0.377(9) .945 .754 .616 .591

to the ring oxygen O-l with the He**0

-0.113(6) -0.063(6) 0.053(S) 0.261(S) 0.452(S) 0.471(S) 0.373(S) 0.506(6) 0.306(S) -.023 .159 .325 .478

DISCUSSION

distance at 2.10 A, the O***O distance at 2.84 A an

2

the O-H***0 angle at 166.7".

After the structure of the tricyclic O-9

lactone 2 was unequivocally determined tie

is a donor to O-5 with an H***O distance of

were able to confirm and complete the

1.99 A, and O***O distance of 2.72 A and an

interpretation of its spectra.

O-H.**0 angle of 151.4".

The hydrogen on

All major signals in the "C

NMR spectrum

O-12 appears to be disordered between two

have been assigned to the two anomers of 2.

positions.

The minor peaks can be attributed to a small

In one of its disordered

positions it does not form any hydrogen

amount of bicyclic lactone free aldehyde

bonds.

which is also formed in solution (Fig. 3).

In the other position it is a donor

to a hydrogen bond to O-9 with an He**0

The fact that 2 and its monohydrate have

distance of 1.84 A. an O***O distance of

identical NMR spectra in DMSO-d. indicates

2.74 A and an O-H***0 angle of 153.0".

The

only other intermolecular approach less than

that the water molecule is loosely bound to 2, since the DMSO-water interactions are

a van der Waals' separation is between C-2

stronger than the intermolecular association

and O-5 at 3.14 A.

of water and compound 2.

a. b. C.

d. e. f. g. h. i.

Fig. 3.

Proton decoupled "C

NMR spectrum of 3 in DMSO-de.

29.3, 29.9 32.0, 32.3 73.6 74.8 85.2, 85.3 87.2, 87.5 99.6, 100.0 106.0, 106.8 174.9. 175.1

A new role for L-ascorbic acid NXR spectrum

The “C

of

the monohydrate

Da0 shows the same pattern There are only

signals.

(2 2 ppm) downfield lactone

carbonyl

the conclusion Involved

as

the hydrate

in hydrogen

geometry

membered rings appears

the two fused

as a broadened

to

proton

reached

rapidly

Michael

and thereby

can be followed

escapes 4).

of

were taken at 365 nm, because

opposite

is larger

is

solvent

Time zero

refers

is added to -3.

is possible dissolved,

before

Since rotation

be determined

acid

completely of pure 1 at

experimentally.

The initial

reading

(t = 4.5)

but changes

rapidly

towards

positive

of

lead

Concerning

to the

ascorbic

that acid

1

acrolein

Hence the addition

The ultimate

involving

from the

from the side ring

C-3 and O-6 of ascorbic

to the thermod~namfcally of

Furthermore,

in the crystal

the two five-membered

hydroxyl

prefers

lattice,

is noteworthy

that

favored rings. the

an equatorial

away from the ketofuranose the Michael

-2b

Scheme

acts

the steric

one can infer

&s-junction

It

acid

a

nucleophilic

the product

C-2 of

leads

position

values.

ascorbic

to C-5 and C-6.

hemiacetal

is negative

J_.

stereoselective.

closure

no measurement

the sample is

the optical

t = 0 cannot

to the time the

to the keto

is essentially

of -_ 2b should

the reaction

configuration

the rotation lengths.

This

wherein

carbon

approaches

wave-

the conjugated

leading

A concerted

product

measurements

at shorter

to make C-2 the

of O-6 upon C-3 and O-3 upon the

course

The

,221.

reaction

tricyclic

the change of

(Fig.

(vie

In our case

around C-Z is

increased

aldehyde

the equilibrium

Las an

two potentially

whjch can attack

s

reaction

of _3.

O-3 and C-2. density

as the donor.

be observed

with

bond of acrolein

attack

In DMSO, however,

rotation

sites:

aldehyde

with a hydroxyl

We assume that

detection.

reactive

nucleophile

resonance.

in methanol.

nucleophile

double

a possible

can be regarded

ambident

sufficiently

five-

at 6 5.6.

of 2 cannot

in the

the anomerfc

the formation

the X-electron

the C-S proton

singlet

overlaps

The mutarotation is

of

for Due to

at 6 4.5,

- maybe

- which forms

of establishing

mechanism for

mono-

except

The C-l.? hemiacetal

This peak partly proton

singlet

be due to a

species

Scheme 1 outlines to

The C-6 proton

peak.

coupling

is negligible.

aldehydc

ascorbate-3-anion

the water

as a sharp

the rigid

is

bonding

of 2 and its

could

equilibrium.

molecule.

of

appears

process

rotation

levorotatory

the free

This allows

in DMSO-d6 are identical

the size

transient

C-5 and the

the C-5 hydroxyl

The ‘H NMRspectra hydrate

for

resonance. that

The negative

“doublet”

t.wo significant

shifts:

donor

a

with

in

2141

ring. addition

G. FODOR et al.

2142 step

takes

place

at

No basic

catalyst

was used

as

provides

sufficient

act

as

shifted

of

somewhat

in aqueous 10

with

solution

at

subsequent to

form

lactone

in a recent ascorbic line

acid

dimer

pcntacyclic

the

tendency

M_etil

with

;lroceeded

with

ascorbic

Clycosidation

a

affords

ketounderscore

there

Once

the

scope

of

this

compound

corresponds and

to the

the

have

carbonyl cm-‘).

(Fig.

5)

been

shows at

lactone

214.7

is In

sharp

singlet

the

177.9

6 2.1 to

singlet

at

can

d 4.4

are

ketone

Two hydroxyl

1.

carbons

singlet

to

a methoxyl

peak

appears

correct

the

at

in

the

tricyclic

perfect

the

lactone

‘H YX??

6 3.7

group; at

6) but

ppm and

The

another

6 1.4.

elemental

are

(Fig.

(112.8

a sharp

the TR

The analysts

agreement methyl

with

ketal

two (1700 in

to Tbl.s

signal

CH,OH 2aHCI

’ the is

at

s

108.3

(DMSO-d.)

belongs

to

the

function;

be ascribed proton

cm-‘,

D20

A hemiketal

-4.

a keto

_4

carbonyl

ppm.

by the

adjacent

proton.

There

structure

protons

CllHlsOI

the

carbonyl

instead.

the

lacks

in

ppm indicates

4

lactone.

In

hemiketal

IR spectrum

‘H NMR spectrum at

for

structure

ascorbic

ppm in addition

indicated

ppm.

cyclic

one

at

with

of

which

ketone.

NMR spectrum

only

carbonyl

consistent

the “C

isolated

and

a keto

present

shows

spectra

which cm-’

NMR spectrum

for

Two ketal

methyl

1700

of

2-(3-oxobutylj-3-oxo-

its

formed. in

The

resonance

carbon

or

bands

1760

adduct

the

L-gulonolactone, could

1:l

be

CloHI,O,

a,@-unsaturated

either

principle,

could for

“C

173.8

corresponding sharp

analyzed

at

ppm) are

spectrum

as Michael

at

no signal

resonance

114.8

Yethyl

serve

In the

is

methanol)

product

absorption

carbonyl.

we

(2% HCl in

a crystalline

carbonyl spectrum.

on C-3.

acid. to

4

acceptor.

acid

CH,=CH-C-CH,

L-

formation

the

was chosen

perfectly

fl

+

1

and C-3

was clarified,

new reaction

A crystalline

or

a

contains

as acceptor.

vinyl

that

pH _ 4,

pii 7.4.

The crystal-

group

investigate

ketone

at

in

Jackson

of

examples

hemiketal

acrolein

to

solution at

of

place

role two

acid,

also These

ketone

acid

takes

was described

synthesis 11

and a carbonyl

vinyl

reaction

the

towards

O-6

formation

The reaction

buffer

_1 is

A similar

dehydro-L-ascorbic

rings.

between

O-6

intermediate

structure, 12

furanose

exclusive

of

benzylation

examples

between

2-O-phosphate.

of

ascorbic

for

and C-3

3.

the

Earlier

was observed.

on

aqueous

O-6

was

C-2

In both

paper

by the 5.

upon

The formation

pH 4.

cyclization

bicyclic

between

product

completely

The preference

the

however,

acid

on the

a hemiketal

D1O.

3-(hydroxymethyl)indol

had reported acid.

disappear

with

in a phosphate

plays

are,

reactions.

C-2

in

and 6.8

confirmed

product

acid

L-ascorbic

L-ascorbic

cyclization

_1

and

example

There

analogous

6’ :
of

are

tricyclic

any

ascorbic

ascot-b&en: at

exchange

to

equilibria

stable

donor.

alkylated’

6 5.6

reaction

dissociation

find

where

a Michael

of

more

not

literature

of

various

the

We could

acid. water

dissociation

As the

towards

formation

the

Since

ascorbate-3-anions

“starters”. the

ascorbic

necessary.

a solirent,

progresses,

of

pH -. 4 of

is

to

the methyl the

the

resonances

Structure We are

2 is basing

C-4

on analogies

at

the

methyl

closely our

with ketal

related

to

configurational -3. carbon

compound assignments

The configuration was deduced

at from

2.

2143

A new role for L-ascorbic acid

180150. 120SO-

min

Fig. 4.

Mutarotation

of _? in DMSO (c,

1.5).

3.

b. C.

d.

I

e. f. 8. h.

5.

Proton

decoupled

“C

NMR spectrum

of

5 in

0

50

100

150

200 Fig.

I

I

I

wm

17.8 30.2 37.1 74.1 76.4 78.1 87.5 108.3

D.0

(D:p-dioxane

reference).

a.

a

b. c. d. e. f. 8. h. 1’:

23.3 31.2 35.1 53.9 73.2 75.5 77.6 86.6 114.8 112.8

k.

173.8

r* i 200 ppm

I

Fig.

6.

Proton

decoupled

100 ‘*C

NHR spectrum

I

I

I

150

50 of

2 In D,O

fD:p-dioxane

0 reference).

2144

G. FODORet

al.

Dteiding models and especially space filling

might be possible by co-administration of

Catali" models of the two possible anomets

cyclophosphamide and L-ascorbic acid.

of -5.

The methoxyl group in g-position is

much preferred stetically.

In the tetta-

hydropyran ring assuming a twist-boat

The

spectrum

of

the

shows

that

nated

by some

Similar methyl mixture

-5 is

the

material major

unteacted

attempts

glycoside of

crude

“C

to

resulted

NMR

potential structure could be transformed into

contami-

other functionalities.

-4. convert in

-3 into

a

intermediate for syntheses.

a syrupy

products which shows three

3.6) of the 'H NMR spectrum.

EXPERIMENTAL

After partial

chromatographic separation spectral data of the individuai fractions suggest that two anomeric C-12 methyl glycosides were formed together with a dimethyl acetal-methyl ketal derived from the aldehyde form of 2.

The

separation and purification of these products is in progress. Conclusions.

This is the first report on L-

ascorbic acid as a novel powerful Michael catbanion donor towards two reactive a.@ unsaturated carbonyl compounds.

Further

studies are already under way to evaluate the scope of

this reaction.

We are

interested in using biologically significant a,B-unsaturated aldehydes like 4-hydroxypentenal as acceptors.

Assuming

that

the

addition is reversible in UiUO a"

adduct with 4-hydtoxypentenal could have 13 cancetostatic activity. Unsaturated nittiles and esters. and also q&nones,

shall

be investigated as potential Michael acceptors. The fact that the reaction with actolein takes place under almost physiological conditions suggests that ascorbic acid might be useful as a detoxifying agent in vivo.

Acrolein is a metabolite of the

widely used anticancer agent cyclophosphamide.

Melting points were determined on an Electrothermal melting point apparatus and ate uncorrected. IR spectra were recorded on a Beckman IR 10 spectrophotometet. A PetkinElmer Model 141 M Polarimetet was used for the optical rotation measurements. 13c NMR spectra were taken on a Varian CFT-20 specttometer at 20 MHz. The ‘H NMR spectra were recorded on a Vatian EM-360 (60 MHz) spectto-

meter and on a Vatian Cm-20 spectrometer at go MHZ. Elemental analysis were performed by Galbtaith Laboratories, Inc., Knoxville, Tennessee. L-(+)-ascorbic acid, actolein (97%) and methyl vinyl ketone (technical grade) were purchased from Aldrich and used without further purification. A standard solution of Tillmans’ reagent (TR) i.e., 2,6dichlotoindophenol sodium salt,17 was used for the titration of _1.

Monohydrate

of 3. To a stirred solution of 20 g (0.11 mol) ascorbic acid (1) in 100 ml H1O under a Nz atmosphere 5.8 g (0.10 mol) actolein was added dropwise. Throughout and after the addition the temperature was not allowed to rise above 25”. A white ptecipitate usually formed within two hours. If not, crystallization was induced by seeding. The suspension was stirred at room temperature until titration (with TR solution) of alfquots taken from the supetnatant liquid indicated a quantitative consumption of 1. After being kept in the refrigerator overnight the solid was filtered, washed and dried. 13 g monohydrate of 3, m.p. 111-113’ was obtained. Recrystallization from 95% ethanol gave transparent prisms, m.p. 114-116”. The supetnatant liquid was freeze-dried and the residual solid recrystallized from 95% ethanol to afford a” additional crop (5.1 g) of the monohydrate. Total yield: 18.1 g, 72%. Anal. Calcd. for C.H,.zO,*HzO: Found : C. 43.24; H. 5.60; 0, 51.16. c, 43.37; H, 5.71; 0, 51.03.

Protection against some toxic side-effects

1,3,7-Trioxa-&oxo-

which have been linked to acrolein was

G+tricyclo-

achieved by co-administration of sodium 214 or N-acetylmetcaptoethane sulfonate 15 cysteine. In both cases a free sulfhydtyl group is believed to react with the double bond of actolein to form a non-toxic addition compound. non-toxic substance

We shall try to

explore the use of 3 as a new chital

distinct peaks in the methoxy region (6 3.4-

Michael

derivative with a chital tertiary hydtoxyl. The side chain or the lactone ring of such a

clearly

product

a Robinson

annellation leading to a cyclohexenone

conformation the B-methoxy group is placed in equatorial position.

In addition, compound 3 in the diketo form appears to be set up for

Since 2 proved to be a 16 , a similar protection

(5% 9% lZE)-trikydroxy-

[4.3.2.0

2J 6. 02, ‘I-dodecane

(25 (2).

10 g (0.04 mol) monohydrate of 2 was dissolved in 140 ml hot absolute ethanol, 50 ml dry benzene was added and the mixture was After removal of the ternary and distilled. binary azeottope the residual dry ethanol solution was concentrated to about 60 ml. Compound 2 crystallized as white needles, IR (~gt!, 7g, 75%. m.p. 150-152’. Yield: 3580-3100 (broad, vOH). 2960 and 1780 cm-‘.

'H NMR (DMSO-d., 80 MHz) 6 1.7-2.4 !m, 4H),

2145

A new role for L-ascorbic acid 3.8-4.3 (m, 3H), 4.45 (s, lH), 5.58 (br s, 2H, partially disappears upon addition of D*O), 6.4-6.8 (-2H. DnO exchangeable peaks). I'C NMR (DMSO-d.): see Fig. 3. [al;' = +34.4" + 0.3" (c, l-in methanol). Mutarotation (c, 1.5 in DMSO): [a]::. = -36 o + + 184' (4.5 min -+ 3 hrs); see Fig. 4. Anal. Calcd. for C. 46.55 H, 5.17 0, 48.28. CeH1207: Found: C, 46.34 H, 5.26 0, 48.05. 1.6 g (7 mmol) of 2 in Acetalization of 3. 25 ml 2% methanol?k hydrogen chloride was stirred at room temperature for 8 hours. The solution was neutralized with powdered silver carbonate, filtered and dried (NalSO,). The methanol was rerrPved in vacua. The residue, a colorless syrup, was subjected to column chromatography on neutral alumina with benzene-ethanol (3:l) as eluent. TLC on alumina sheets (using the same eluent) indicated that two materials with Rf 0.25 and Rf 0.13 had been separated. 'H NMR spectra of the two isolated syrupy products showed three methoxy methyls (6 3.4, 3.5, 3.6) for one material (Rf 0.25), while the other product (Rf 0.13) had one methoxy peak at 6 3.5. So far the purification and characterization has not been completed. 3,6-Hemiketal of 2-(3-oxobutyZl-3-0x0-G gulonolactone 12). It was found advantageous to introduce methyl vinyl ketone in he vapor phase following a method by DeBoer. 15 20 g (0.11 mol) ascorbic acid (1) was dissolved in 100 ml H=O. Dry nitrogen gas (flow rate ‘-40 ml/min) was bubbled through 8.8 g (0.12 mol) methyl vinyl ketone and then introduced into the ascorbic acid solution through a fritted disc. The temperature was kept below 25'. After about 10 hours all the ketone - except for a small residue of non-volatile materialhad been transferred. Stirring of the homogeneous reaction mixture at 0" was continued until titration of aliquots indicated a constant amount (26%) of unreacted 1. Hence the conversion of 1 was 74%. Some inreacted methyl vinyl ketone had to be removed in UQCUO before the reaction mixture was freeze-dried. The crude product was extracted with hot ethyl acetate (ratio: 10 ml per 1 g). A white crystalline solid separated from the extract. After cooling and filtration 13.7 g of product 3 were obtained. The yield was 66% based on 1 consumed. Recrystallization from ethyi acetate yielded transparent crystals, m.p. 134-136". Direct recrystallization of the crude product from ethyl acetate gave pure 4 in a somewhat lower yield. IR (KBr) 7380, 3260, 3010, 2950, 2900, 1760 and 1700 cm-'. 'H NMR (DMSO-d., 80 MHz) 6 1.87 (m, 2H), 2.07 (s, 3H), 2.5 (m. 2H), 3.8-4.2 (m. 3H), 4.43 (s. 1H). 5.62 (br s, 2H), 6.79 (s, 1H). The signals at 5.62 and 6.79 disappear completely upon addition of D?O. "C NMR (D,O): See Fig. 5. [ali = +41" + 0.3" (c, 1 in methanol). Anal. Calcd. forC1aH1z.0,: C, 48.78; H. 5.69; 0, 45.53. Found: C. 48.91; H. 5.77; 0. 45.32. 1,3, ?-Trioza-&oxo- (5% SSI-dihydroxy-lZR(?)metbxy-12E(?I-methyZ(24 6l+tricycZo[4.3.2.0 2a6.02Jg]-dodecane 15). 1.5 g (6.1 mmol) of 4 was dissolved in ?5 ml anhydrous methanol containing 2% hydrogen chloride and stirred for 7 hours at room temperature. The yellow solution was neutralized with

powdered silver carbonate, cooled, filtered and dried (NazSOb). Removal of the solvent in vacua gave a syrup which partly crystallized. The crude product was recrystallized twice from ethyl acetate to'afford 0.5 g (31%) of pure 5, colorless crystals, m.p. 154-156'. IR (KBr): 3440. 3330, 2950 and 1730 cm-'. 'H NMR (DMSO-da, 60 MHz) 6 1.40 (s. 3H), 1.6-2.3 (m, 4H), 3.68 (s. 3H), 3.84.3 (m, 3H), 4.43 (s. lH), 5.3 and 5.82 (DIO see "C NMR (D,O) exchangeable protons). Fig. 6. [old = +53.7' + 0.3" (c, 1 in methanol). Anal. Calcd. for C1,H160,: C, 50.77; H. 6.15; 0, 43.08. Found: C, 50.69; H, 6.24; 0, 43.17. The work done at West Acknowledgements. Virginia University was supported entirely by a grant from the National Foundation for Cancer Research (NFCR). Thanks are due to Professor Albert Szent-Gysrgyi for calling our attention to the reaction of ascorbic acid with acrolein. REFERENCES 1.

Part of this work will be included in the Ph.D. dissertation of R.A.

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