Chiroptical properties of fluorescamine condensation compounds with dipeptides in situ

Vol. 74, No. 2, 1977

BIOCHEMICAL

CHIROPTICAL

AND BlOPHYSlCAl

RESEARCH COMMUNICATIONS

PROPERTIES OF FLUORESCAMINE CONDENSATION COMPOUNDS WITH DIPEPTIDES

--IN SITU

V. Toome, B. Wegrzynski and J. Chemical Research Department Hoffmann-La Nutley,

Received

November

14,

Dell

Roche Inc.

New Jersey

07110

1976

SUMMARY The free camine absorption

maxima which

of a dipeptide

reacts

in the

380 nm region.

A simple

of --in situ determination amino acids of the dipeptides

of their

chromophoric

efficiently

chromophores

allows

the NH*-terminal

properties

group

to form pyrrolinone-type

(FLURA @I

described of

amino

test

with

fluores-

with

long

tube

procedure

the absolute based

wavelength is

configuration

on the chiroptical

derivatives.

INTRODUCTION We have

recently

spirolfuran-2(3N), the

determination

amino

on the application

of the absolute

acids

--in situ. of a dipeptide

group

reported

l'-phthalan)-3,3'-dione configuration

Fluorescamine (3)

of fluorescamine,

I as a chromophoric also

of primary

reacts

to form pyrrolinone-type

(1)

efficiently

4-phenyl-

reagent

for

and secondary

with

chromophores

the amino II:

C\H(Rl)-CONH-CH(R2)-COOH H2N-CH(R+-CONH-CH(R2)-COOH >

6H5

d

II

I

This under

mild

reaction

can be obtained

Copyright A,, rid,,.

is

from

simple

and fast

Chromophore

conditions. the

reaction

0 1977 by Academic Press, Inc. nf ronm,fr,rt;nn ;n nnll ,,Y”.M “““““..“A

II

and can be performed is

mixtures

825

chiroptically without

active isolation

in

test

tubes

and CD spectra of the product.

(2)

BIOCHEMICAL

Vol. 74, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

EXPERIMENTAL A.

Reagents The dipeptides

Sciences

Group

(Fluram 9

and were from

from

from

Mallinckrodt

from

Inc.

Co.

to Clark

Biochemicals

purification.

Roche

Scientific

according Chemical

Vega-Fox

further

Hoffmann-La

the Fischer

M) was prepared

B.

purchased

used without

was obtained

and methanol 0.05

were

Fluorescamine

and spectral

The phosphate

and Lubs

and ICN Life

(4)

grade

buffer

using

dioxane

(pH 8.0,

AR-grade

chemicals

Works.

Method Two milliliters

added

of a 0.004M

to 2 ml of a 0.002M

solution

of a peptide

reaction

mixture

a 0.1 cm cell concentration), Model

buffer tide

CD spectra

phosphate

buffer

some peptides

in phosphate

buffer

is

stirred

If

for

length,

in dioxane is rapidly -2 and 0.5 x 10m6M)

mixer,

depending

tube.

The

transferred

to

on the dipeptide

on a JASCO Spectropolarimeter,

necessary,

the

1:l

reaction

mixtures

are

v/v.

procedure with

10

in a test

on a Vortex

pH 8/dioxane tube

between

pH 8.0

recorded

pH 8/methanol

2 ml of a 0.004M

mixture

are

the general

in a test

fluorescamine

buffer

-ca 15 set of different

450 and 240 nm.

pH 8 is mixed

added

for

of

may range

phosphate

a cell

and the

with For

stirred into

20 between

diluted

in 0.05M

is (or

solution

(concentration

is

modified:

4 ml of phosphate

2 ml of a 0.002M 1:1 v/v

solution

of fluorescamine

15 set

and the CD spectra

solution

and to this

of a dipep-

mixture is rapidly The reaction

in dioxane.

are measured

as described

above. In two cases the

reaction

tion

was also

carried

of fluorescamine

a dipeptide absorption if

are

in organic 4 hrs

difficult

reagent,

longer

than

and L-phenylalanyl-L-isoleucine) with

to obtain if

cm are

used.

below its

2 ml of a 0.004M

2 ml of a 0.002M

the CD spectra

especially 0.1

solvents:

was mixed

After

of the

cells

out

in dioxane

in methanol.

The spectra or

(L-lysyl-L-tryptophan

were

solu-

solution

of

recorded.

240 nm because

concentration

of the high

is higher

than

0.004M

RESULTS AND DISCUSSION For

the reasons

in phosphate the peptide trations phore arising

pH 8-8.5

concentration

fluorescamine dard

buffer

previously

is (<10-4M)

conditions

require

a 20-40

stable at least The UV absorption reaction

analytical are

for l-2 spectra of

fold

(5), with

in the range for

the reactions

the

and reacted

sufficient

is

from

is

discussed

fluorescamine

of O.Ol-O.OOlM, but purposes,

excess

complete

the dipeptides

of

within

the

fluorescamine

with

826

a twofold excess of lower dipeptide concen-

reagent

dipeptides,

When

in dioxane.

one minute

hrs. of the pyrrolinone-type

are dissolved

Under

(6).

stan-

and the chromo-

chromophores (3,6)

II,

show maxima

Vol. 74, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Cotton Effects

in CD Spectra of Reaction Products of Dipeptides Fluorescamine

--in Situ

-1st Dipeptide -

-2nd

ml (e)x10 -3

Solventa

Cotton Effect nm (0)x10 -3

rml (elx10 -3

D-Alanyl-D-alanine D-Alanyl-L-alanine L-Alanyl-D-alanine L-Alanyl-D-phenylalanine D-Alanyl-D-phenylalaniEe L-Arginyl-L-isolcucine L-Arginyl-L-isoleucine a-D-Glutamyl-D-glutamic Glycyl-L-leucine Glycyl-D-leucine Glycyl-L-phenylalanine Glycyl-D-phenylalanina L-Leucyl-Galanine GLeucyl-L-proline L-Lysyl-L-trj'ptophan L-Lysyl-L-tryptophan LPhenylalanyl-L-isoleucine L-Phenylalanyl-L-isoleucine L-Valyl-L-valine L-Valyl-L-valine D-Valyl-D-valine D-Valyl-D-valine D-Valyl-L-valine D-Valyl-L-valine

(a)

U = phosphate buffer pH 8/dioxane 1:l v/v,; B/M = phosphate buffer pH 8/dioxane/methanol 62.5:25:12.5 v/v.; M = dioxane/methanol 1:l v/v., reaction time 4 hrs.

(b)

Under standard t 4.80

at 280-285 these

electronic amino

derivatives W

transitions

afford

maxima.

the UV spectra

386 403 380 380 390 378 385 378 387 385 387

acid

Cotton effect

+ 8.75 t11.20 -11.02 -14.82 + 7.20 - 5.80 - 6.16 t 8.22 - 4.65 + 5.03 - 6.55 + 7.28 - 8.15 -20.58 -10.21 - 7.85 -15.65 -30.19 - 7.19 - 6.67 + 6.45 + 6.39 + 8.27 + 7.35

An additional

Cotton

show minima),

presumably

effects

effect

-36.00 -26.81 t26.85 t18.78 -27.60 t66.15 t34.42 -28.11 + 8.47 - 8.58 + 5.15 - 5.06 +28.41 i37.19 429.62 t36.00 + 1.05 +14.05 t45.03 t33.19 -43.65 -32.80 -43.15 -31.63

As expected, of the a-C atom of the

and consequently Cotton

268 276 276 275 276 264 264 264 265 270 278 279 269 270 272 264 285 281 270 268 270 268 270 272

nm (E = 6000-7000).

the chirality

of a dipeptide with

324 325 326 323 333 303 315 322 327 321 336 336 318 318 325 316 324 315 315 317 316 315 319 319

is observed at 336 ran, (G) x10w3 =

and at 380-390 recognize

CD spectra

+15.58 + 6.25 - 6.39 - 3.65 + 5.15 -29.40 -16.05 + 8.88 - 8.06 + 7.14 - 2.27 + 3.10 - 5.11 - 7.61 t 9.20 - 5.63 + 4.51 -12.20 t 4.11 - 5.15 - 3.90 t 4.78 - 4.70 t 5.80

390

an additonal

nm (s = 16000-18000)

NH2-terminal the

conditions

384 382 381 380 376 381 382 385 383 385 388 386

-3rd

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

acid

B B B B E B B/M B B B B B B B B M B M B BFI B B/M B BFi

with

the chromophoric

located

is observed

in the at 315-325

due to a coupled

region

of

nm (here

oscillator

mechanism

(7). A number spectra sign, Figures

were

of dipeptides

recorded

and intensity 1 and 2 the

L-proline,

of the Cotton spectra

L-valyl-L-valine The

were

reacted

with

in the above-mentioned

pyrrolinone-type

effects

fluorescamine

spectral are

D-valine

chromophores

have

827

The position, in Table I. In

summarized

of D-alanyl-Galanine, and D-valyl-

L-alanylare three

and the CD

range.

D-alanine,

L-leucyl-

shown. characteristic

Cotton

Vol. 74, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

40

1

30

24 20 16 28 12

/

IOe6-

nh z -

4+2-

0 -246-

eIO12-

*.;

26[1”“““‘1”““‘1”“‘1 200 250

Fig.

1.

spectra

CD

(----),

of the --in situ L-alanyl-D-alanine

fluorescamine

effects acid

between

the

in 0.05

400 and 260 nm.

of the dipeptide

effect

(at sign

403-375

of the

experimental

Cotton

amino

acids

of D-configuration

acids

of L-configuration.

the

sign

of the

Cotton

on the configuration tide. minor

Evidently influence

interesting glycine (Table

the

that

of D-alanyl-L-alanine

buffer

(--- -)

pH 8/dioxane,

l:l,

the

336-308

285-264

nm) are

nm) is positive. derived

first

amino Cotton

negative, Within from

of the

amplitude

pyrrolinone-type amino

acid

chromophore

intensities

of the Cotton

828

the

NH2-terminal NH2-terminal results,

depends

only dipep-

amino acid has only Furthermore, it is

effects.

characteristics

I).

whereas

of the corresponding

of the COOH-terminal of the Cotton

are

transmitted

effects

are

with

v/v.

when the NH2-terminal

of the chromophores

the NH2-terminal

the

mixtures

cases,

one (at (at

the configurational although

450

are mirror images of those derived from As can be seen from the experimental

configuration

on the

moiety,

second

effects of

400

and L-leucyl-L-proline

M phosphate

effect

the CD curves

amino

( -)

350

has an L-configuration,

nm) and the

third

nm

reaction

In most

derivative

error,

300

through reduced

a the

BIOCHEMICAL

Vol. 74, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

5or

50 """""""""""'II 200 250

Fig.

CD spectra

2.

of

300

nm

the --in situ reaction (-- -) with

pH 8.0/dioxane

camine

buffer/dioxane/methanol

in phosphate

(see

entries

effect

in the

with

17,

and D-valyl-L

encountered 19,

regarding

21 and 23 in Table

(8).

in 0.05 of the

valine

I).

sign

is positive

for

the derivative

and negative

for

those

in Fig. chromophoric

2.

The same sequence

fluorescamine

with

of the

first

cases

Cotton the Cottor

of a dipeptide an NH2-terminal

of first

derivatives

fluorcsv/v.

In these

acid

mixtures

with

62.5:25:12.5

the

(--)

M phosphate

reaction

(-.-)

nm area

of these first Cotton effects is sensitive 2) and the exception to the general (Fig.

differences moiety

L-amino

as demonstrated

solvent

15,

403-375

an NH2-terminal

The sign

were

of L-valyl-L-valine

The spectra

(----)

effect

acid,

v/v.

450

fluorescamine

of L-valyl-L-valine

A few exceptions

was observed

1:l

400

mixtures

and D-valyl-L-valine buffer

350

Cotton

of o-amino

with D-amino

effects acids

(1).

to the polarity of the rule may be caused by the

in hydration or by differences of the conformation But in any case, the sign of the -__ second and __third

of the dipeptide Cotton effect can

Vol. 74, No. 2, 1977

be safely terminal

used amino

BIOCHEMICAL

for

the determination

acid

of a dipeptide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the using

absolute

configuration

the following

Configuration

Sign

of the NH~

empirical

rule:

of the -2nd and 3rd

Cotton

Effects

L D

@ 0

As in the case advantage

of this

conditions, with

of primary

fluorescamine

as little

(1) method

as 0.1-1.0

fluorescamine

and secondary

and useful

is

ug/ml

its

(2) amj.no acids,

simplicity.

of dipeptides

CD spectra

00

of the

Under has been

reaction

the main standard

routinely

mixtures

reacted

were

obtained.

ACKNOWLEDGEMENTS We thank

Drs.

A. Felix

and M. Weigele

for

helpful

(1976)

discussions

and

suggestions. REFERENCES 1.

Toome,

V.,

Commun., 2.

Toome,

V.,

Commun., 3. 4. 5.

Weigele,

M.,

DeBernardo,

J. Amer.

Chem. Sot., W. M.,

DeBernardo, 163,

8.

and Reymond,

G.

B.,

and Dell,

(1976)

S. L.,

34,

Biochem.

Riophys.

Res.

H. A.

Weigele,

Stein,

S.,

J.

Tengi,

J.

Biochem.

P.,

Biophys.

and Leimgruber,

Res. W. (1972)

5927-5928.

and Lubs,

S., P.,

M.,

(1916) Toome,

J. Biol. V.,

and Udenfriend,

Chem.,

Manhart, S. (1974)

K.,

2,

479-484.

Leimgruber,

Arch.

W.,

Biochem.

Biophys.,

391-399.

Toome,

V.,

Letters, 7.

Wegrzynski, 598-602

Clark,

B.,

206-211.

7&

Bohlen, 6.

Wegrzynski, 2,

Schellman, Temperature exceptions

DeBernardo, 1,

S.,

Manhart,

K.,

and Weigele,

M. (1974)

Analytical

437-443. J. A.

(1968)

and pH studies to the general

Accounts are

Chem. Res., planned

rule.

830

for

l-,

144-151.

the clarification

of the observed