New Reagents for the Development of Latent Fingerprints results when compared with the previous compounds. Since it is known that replacement of a phenyl ring by a thiophene ring in

The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: New Reagents for the Development of Latent

Fingerprints Author(s): Madeleine M. Joullie Ph.D. Document No.: 179287 Date Received: 01/17/2000 Award Number: 92-IJ-CX-K0154 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally-funded grant final report available electronically in addition to traditional paper copies.

Opinions or points of view expressed are those

of the author(s) and do not necessarily reflect the official position or policies of the U.S.

Department of Justice.

Grant award number: 92-IJ-CX-KO154

Project Title: New Reagents for the Development of Latent Fingerprints

Estimated completion date: January 1, 1995

Project contact person: Dr. Richard Rau

Contact person's telephone number: (202) 307-6394

F.

Grantee name and address: Professor Madeleine M. Joullie

Department of Chemistry, University of Pennsylvania, Philadelphia, PA 1 91 04- I ,

6323

Project description:

la. The presentation of physical evidence is the core of any case in our

criminal justice system. Irrefutable evidence which can establish the identity of

the perpetrator is the prosecution's goal. DNA fingerprinting is currently at the

forefront of many criminal cases; however, there is still debate, even among

forensic experts, regarding the handling of samples, population matches, and

reproducibility of results. In many instances, no DNA samples can be recovered

e \ -

from a crime scene, or the samples are old or damaged. DNA fingerprinting is

also a costly and time-consuming process, and is often unavailable to the

average law enforcement officer working on a full workload of cases. In contrast,

fingerprints are known to be unique to each individual and a definite number of

"matches" between a print obtained as evidence and one on record have been

established as acceptable. The basics of obtaining usable prints are standard

among forensic scientists and law enforcement officers. The computerized

collection of prints of known criminals which is on-line will drastically decrease

the time spent searching for a match, and will allow for searches against a

broader population. One of the most affordable and useful reagents for

visualization of latent fingerprints on porous surfaces such as paper, wood, and

; . l c 3 r ;-c 1y 0 F

This document is a research report submitted to the U.S. Department of Justice.This report has not been published by the Department. Opinions or points of viewexpressed are those of the author(s) and do not necessarily reflect the officialposition or policies of the U.S. Department of Justice.

walls is ninhydrin (2,2-dihydroxy-l,3-indanedione). Ninhydrin was first made in

191 0 by the English chemist Siegfried Ruhemann,l who also investigated its

reaction with amines and amino acids to form a colored compound known as

Ruhemann's Purple (RP).

- OH

0 0 0-

Ninhydrin

or 2,2-dihydroxy-l,34ndanedione

Ruhemann's Purple (RP)

The significance of this discovery to the development of latent fingerprints

went unnoticed until 1954 when Oden and von Hoffsten reported the use of

ninhydrin as a fingerprint development reagent.2 The reaction between

ninhydrin and the amino acids secreted from the ecrine glands form

Ruhemann's purple (RP).3 Although the relative amount of amino acids in a

fingerprint residue is low compared to the amount of salts and fatty acids, they

form a unique, lasting picture of an individual.

Ib. In 1991, Almog reported that ninhydrin and modified ninhydrins were

the best reagents for use on paper.4 Therefore, it was our goal to enhance the

properties of ninhydrin via structural modifications to create a superior print

development reagent which would be useful to professionals involved in

identification of latent fingerprints. We proposed to synthesize different

ninhydrin analogs to be tested for latent prints visualization.

Ha. A variety of fingerprint-developing reagents have been reported. For

fresh prints, fuming superglue fixes prints on non-porous surfaces such as


metals. Dusting with a fine powder also aids in visualization of fresh prints.

However, criminals rarely leave their mark cleanly and on surfaces which are

conveniently treated to afford clear prints. Evidence may also not be uncovered

until after the prints have aged significantly. In 1990, Pounds and co-workers

introduced the reagent 1,8-diazafIuorenone (DFO), which is commercially

available and used in the United K i n g d ~ m . ~ , ~ Unlike ninhydrin, which reacts

with the amino acids in a fingerprint residue to produce a violet-colored print,

DFO gives only a weakly colored red print. The main feature of this reagent is its

ability to give a fluorescent print with little background interference. This "glow"

can be seen when the fingerprint is illuminated with a light of certain

wavelength. Ninhydrin itself exhibits a weaker fluorescence when the originally

developed print is treated with a metal salt solution such as zinc chloride, but

has the advantage of providing an initial print that is highly colored.

In a 1993 report by Cantu et. al., one reagent previously prepared by our

group, thieno[f]ninhydrin, was reported to provide a print which was initially as

highly colored as ninhydrin, but after treatment with a zinc chloride solution, it

gave a print which exhibited stronger fluorescence than DF0.7 When the print is

initially developed with a solution of the reagent containing acetic acid, a

fluorescent complex was observed without the use of a metal for secondary

development. Thus, we had succeeded in preparing a reagent which combined

the most desirable properties of reagents known at that time. However, since

the compound was difficult to make, and would be too expensive to produce on

a commercial scale, we had to design a reagent which would possess the

properties of a superior reagent but would be amenable to a cost-effective,

la rge-scale product ion.


1,8-DiazafIuorenone (DFO) Thieno[f]nin hydrin

Ilb. We next created a list of desirable properties of a superior reagent.

There must be a rapid rate of reaction with the amino acids in the fingerprint,

which would produce a colored print. The developed print must be fluorescent,

preferably without secondary treatment with a metal salt solution. The maximum

fluorescence emission should occur at a wavelength greater than 550

nanometers to avoid competition with optical brighteners in paper, and to match

the visual acuity of the human eye. The fluorescence should also exhibit a high

quantum efficiency. The reagent must be soluble in solvents that do not smear

the print, and fluorinated solvents which were previously used are now banned.

The compound must be stable on storage for long periods of time. The reagent

must be non toxic and safe to handle. It should be economical, and its synthesis

suited to large-scale preparations. Unfortunately, one cannot predict a priori

which reagent will possess superior properties. Therefore, we proposed to

synthesize a variety of reagents and have them evaluated by forensic scientists

at the United States Secret Service and other law enforcement agencies.

’

Ilc. Until recently, protocols available for the synthesis of ninhydrin

analogs were inefficient. Our group reported novel approaches to the synthesis

of benzo[f]ninhydrin, 5-methoxyninhydrin, 5-(methylthio)ninhydrin, and

thieno[f]ninhydrin. We then synthesized 5-phenylninhydrin in which the

conjugation of a phenyl group with the ninhydrin nucleus was expected to result

in an absorption at longer wavelengths. However, this reagent gave


disappointing results when compared with the previous compounds. Since it is

known that replacement of a phenyl ring by a thiophene ring in some

triarylmethane dyes such as Malachite Green produces a marked bathochromic

shift, we set out to prepare the 5-(2-thienyl)ninhydrin (2-THIN), which has

proven to be a superior reagent. However, based on our experience with

reagents which were designed to be superior and showed properties only

equal to ninhydrin itself, we are still unable to predict which reagents will

possess the properties required for a superior reagent.

0 0 Benzo[f]ninhydrin 5-Methoxyninhydrin

0 \ % / / \ O H O H M e w : H / 0

I I

0 5-Phenylninhydrin 5-( Met h y It h io)n in hydrir

Illa,b. The methodology developed for the synthesis of the aryl-

substituted ninhydrin analogs, most notably "2-THIN", is shown in Scheme 1.

A palladium-catalyzed cross-coupling reaction of 5-bromo-ninhydrin dimethyl

acetal and an aryl boronic acid, aryl tributylstannane, or an electron-sufficient

aryl group is the key to these syntheses. We investigated several routes to a

common coupling partner (5-bromoninhydrin dimethyl acetal), and are now

able to prepare this compound on a large scale that is commercially viable


, " . I*.W

I

(Scheme 2). Preparation of the 2-thienyl boronic acid and the 2,5-

bis(tributylstanny1)-thiophene are shown in Scheme 3. The "2-THIN" reagent

can also be prepared by the reaction of thiophene with the bromoacetal, in an

improved yield.* The synthesis of "2-THIN" was performed on a 100 gram scale

at Vinfer Ltd. in Belfast, Ireland last year. The company is interested in the

commercial production of this superior reagent. A list of compounds prepared

by the new methods we developed is shown in Table 1.

One of the synthetic steps responsible for a decreased yield is the last

step, the deprotection of the dimethyl acetal to afford the free ninhydrin. Much

time and effort has been spent on improving this reaction, but even with

common and exotic deprotection protocols known to organic chemists, side

reactions which give undesired products are still a problem.

We have designed and synthesized several superior reagents which

contain dual reactive sites (Schemes 4 and 5) The resulting polymeric ,

Ruhemann's purple formed would be expected to extend the conjugation of the

system, and increase the fluorescence of the developed print. Unfortunately,

many of these "Janus" ninhydrins had poor solubility in acceptable solvents for

fingerprint development, and thus showed poor performance as reagents.

The ban on the very useful chlorofluorocarbons such as

trichlorotrifluoroethane has greatly disrupted police work. 9,10 Therefore, a

search for acceptable substitute solvents has been initiated but so far it has not

proven fruitful. We have found that some derivatives of ninhydrin, resulting from

its reaction with higher molecular weight alcohols (hemiacetals), undergo the

same color-forming reactions and are more soluble in organic solvents such as

ethyl acetate, methylene chloride, or toluene than ninhydrin (Table 11). This

approach affords a solution to the ban on chlorofluorocarbons.


, I

IVa. To summarize, several synthetic approaches were developed and

evaluated to provide a total of 21 new substituted ninhydrins. These reagents

were evaluated for fingerprint visualization by forensic experts at the US Secret

Service and other law enforcement agencies both in this country, England,

Switzerland, Australia, and Israel. The new methodology developed afforded

two complimentary reagents, which displayed similar fingerprint developing

properties (2-THIN and 3-THIN). 2-THIN was produced on a large scale by

Vinfer Ltd. in North Ireland to investigate its potential for commercialization.8

I ,

Finally, because of the importance of formulations in the development of

new fingerprint visualization reagents and as a potential solution to the

problems caused by the ban on chlorofluorocarbons, we developed structural

modifications of ninhydrin-based reagents to expand their solubilities in suitable

organic solvents (methylene chloride, toluene, hexanes etc.).

IVb. Our research has resulted in improved reagents for latent fingerprint

development. Additionally, it has provided a suitable alternative to the technical

problems created by the ban on chlorofluorocarbons. Our results are useful to

all law enforcement agencies both in this country and abroad.

Bibliography

(1)

(2)

(3)

Ruhemann, S. Trans. Chem. SOC. 1910, 97, 2025.

Oden, S.; von Hofsten, B. Nature 1954, 773, 449.

Joullie, M. M.; Thompson, T. R.; Nemeroff, N. H. Tefrahedron

Report Number 300, Tetrahedron 1991, 47, 8791 -8830.

Almog, J.; Hirshfeld, A.; Frank, A.; Sterling, J.; Leonov, D. J.

Forensic Sci. 1991, 36, 104.

Grigg, R.; Mongkolaussavaratana, T.; Pounds, C. A.; Sivagnanam,

S. Tetrahedron Lett. 1990, 37, 7215-7218.

(4)

(5)


(6) Pounds, C. A.; Grigg, R.; Mongkolaussavaratana, T. Journal of

Forensic Sciences 1990, 35, 169-1 75.

Cantu, A.; Leben, D. A.; Joullie, M. M.; Hark, R. 'R. J. Forensic /dent. (7)

1993, 43, 44-66.

(8) Hark, R. R.; Hauze, D. B.; Petrovskaia, 0.; Joullie, M. M.;g Jaouhari,

R.; McComiskey, P. Tetrahedron Lett. 1994, 35, 771 9-7722.

Hamer, M. New Scientist 1994, 5.

Zurer, P. Chem. Eng. News 1995, 9.

(9)

(1 0)


Scheme 1

HBr HOAC, H20 - heat, 86%

0

BrGk / OMe Me

0

0

Pd(PPh3)s, Na2C0, - PhCH3, EtOH, A,

96%

+

S

0 S

Scheme 3

1. Mg, THF, reflux

2. B(OMe),,THF

4. recrystallization (H20)

-78 "C to RT 3.10% H2S04

(59%)

1. n-BuLi (2 eq) hexane

7

TMEDA (2 eq) 2. Bu3SnCI (2 eq) 3. distillation (in vacuo)

(78%)

bu35n O S n B u ,


Scheme 2

/ OMe Me

0 \

Me

Br2

Fe, 12 Me (70 %yield) ,

1. HN03 (aq.) 7

2. KMn04, NaOH(aq.) 3. distillation

(56 - 58 % yield)

1. ethyl acetoacetate EtSN, Ac20

7

2. HCI (aq.) (46 - 75 % yield)

1. EtSN, EtOH, -20 "C 7

2. tosyl azide (68 - 75 % yield)

tert- b u t y I h y poc h lor i t e MeOH

7

(59-74 % yield)

Br*: /

0

Brw 0

0

+ - N=N ,

0


I

Scheme 4 > COpH

CHO

LiAIH4, THF - A, 2 days

20%

CHO CO2H CH20H C02H I 1

malonic pyridine acid > piperidine, 90°C

90%

\

C02H ? 0

0

1

1. H2, Pd/C,MeOH - 2. LiOH, MeOH 1

THF, H20 90% Q

1 c02h

Se02, HOAc

7

100 "C 15%

HO

OH


. . I

Scheme 5

1 . 0 9 , MeOH, -30°C - 2. HOAc, Nal

80%

OHC 8 H02C

C02H HOPC

0

Se02, HOAc - 100 "C 15%

'CHO malonic acid

pyridine - piperidine, 90°C

55%

10% Pd/C, H2 - Na2C03, H20

90%

PPA, 80°C - 80%

~

0

0


Table 1 1

New ninhydrin analogs

Y

X

0

X = Y =

2 CH3S

3 Ph

4 CH3O

H

H

H

5 HO H

H H 10 11 -0CH20-

12

OH

1 Br*: I t "

I

0

" 0

7 <:"

8

HO

13

0


.. 'Table I

New ninhydrin analogs (continued)

X =

6,144 6,18-22

0

8

X =

6 m S

2-THIN

14

15

16

18

19

(3 S '

Me

Et 20

21

22

Me

h\


xwH / 0 OH

X

17 H

18 H

19 H

20 H

21 H

22 H

Table II Hemiacetals

- x*R / OH

ROH

A 0

17 - 23

R

Me

Et

n-Pr

i-Pr

n-Bu

i-Am

23 2-(5ethyl-)thienyl- Et


I

Grant award number: 92-IJ-CX-KO154

Project Title: New Reagents for the Development df Latent Fingerprints

Estimated completion date: January 1, 1995

Project contact person: Dr. Richard Rau

Contact person's telephone number: (202) 307-6394 \

Grantee name and address: Professor Madeleine M. JoulliC

Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19 104-6323 I

E $ E L ~ T w E 5 L - " ~ " f

Project description:

The presentation of physical evidence is the core of any case in our criminal justice

system. Irrefutable evidence which can establish the identity of the perpetrator i's the

prosecution's goal. DNA fingerprinting is currently at the forefront of many criminal cases;

however, there is still debate, even among forensic experts, regarding the handling of

samples, population matches, and reproducibility of results. In many instances, no DNA

samples can be recovered from a crime scene, or the samples are old or damaged. DNA

fingerprinting is also a costly and time-consuming process, and is often unavailable to the

average law enforcement officer working on a full workload of cases. In contrast,

fingerprints are known to be unique to each individual and a definite number of "matches"

between a print obtained as evidence and one on record have been established as

, I ,I

: acceptable. The basics of obtaining usable prints are standard among forensic scientists and

law enforcement officers. The computerized collection of prints of known criminals which

is on-line will drastically decrease the time spent searching for a match, and will allow for

searches against a broader population. One of the most affordable and useful reagents for

visualization of latent fingerprints on porous surfaces such as paper, wood, and walls is

ninhydrin, a compound first made in 1910 by the English chemist Siegfried Ruhemann,l

who also investigated its reaction with amines and amino acids to form a colored compound

known as Ruhemann's Purple (RP).


,

, I

I 1

The significance of this discovery to the development of latent fingerprints went

unnoticed until 1954 when OdCn and von Hoffsten reported the use of ninhydrin as a

fingerprint development reagent.2 The reaction between ninhydrin and the amino acids

secreted from the ecrine glands form Ruhemann's purple (RP).3 Although the relative

amount of amino acids in a fingerprint residue is low compared to the amount of salts and \

fatty acids, they form a unique, lasting picture of an individual. 1 .

In 1991, Almog reported that ninhydrin and modified ninhydrins were the best

reagents for use on paper.4 Therefore, it was OUT goal to enhance the properties of

ninhydrin via structural modifications to create a superior print development reagent which I

would be useful to professionals involved in identification of latent fingerprints. We

proposed to synthesize different ninhydrin analogs to be tested for latent prints

visualization. '

4

In 1990, Pounds and co-workers introduced the reagent 1,s-diazafluorenone

(DFO), which is commercially available and used in the united K i n g d ~ m . ~ , ~ Unlike

ninhydrin, which reacts with the amino acids in a fingerprint residue to produce a violet-

colored print, DFO gives only a weakly colored red print. The main feature of this reagent

is its ability to give a fluorescent print with little background interference. This "glow" can

be seen when the fingerprint is illuminated with a light of certain wavelength. Ninhydrin

itself exhibits a weaker fluorescence when the originally developed print is treated with a

metal salt solution such as zinc chloride, but has the advantage of providing an initial print

that is highly colored.

In a 1993 report by Cantu et. al., one reagent previously prepared by our group,

thieno[flninhydrin, was reported to provide a print which was initially as highly colored as

ninhydrin, but after treatment with a zinc chloride solution, it gave a print which exhibited

stronger fluorescence than DF0.7 When the print is initially developed with a solution of

the reagent containing acetic acid, a fluorescent complex was observed without the use of a

metal for secondary development. Thus, we had succeeded in preparing a reagent which : This document is a research report submitted to the U.S. Department of Justice.This report has not been published by the Department. Opinions or points of viewexpressed are those of the author(s) and do not necessarily reflect the officialposition or policies of the U.S. Department of Justice.

combined the most desirable properties of reagents known at that time. However, since the

compound was difficult to make, and would be too expensive to produce on a commercial

scale, we had to design a reagent which would possess the properties of a superior reagent

but would be amenable to a cost-effective, large-scale production.

Until recently, protocols available for the synthesis of ninhydrin analogs were

inefficient. Our group reported novel approaches to the synthesis of benzo[flninhydrin, 5-

methoxyninhydrin, 5-(methylthio)ninhydrin, and thieno[flninhydrin. We then synthesized

5-(2-thienyl)ninhydrin (2-THIN), which has proven to be a superior reagent.

The ban on the very useful chlorofluorocarbons such as trichlorotrifluoroethane has

greatly disrupted police work.','' Therefore, a search for acceptable substitute solvents

has been initiated but so far it has not proven fruitful. We have found that some derivatives

of ninhydrin, resulting from its reaction with higher molecular weight alcohols

(hemiacetals), undergo the same color-forming reactions and are more soluble in organic

solvents such as ethyl acetate, methylene chloride, or toluene than ninhydrin. This

approach affords a solution to the ban on chlorofluorocarbons.

To summarize, several synthetic approaches were developed and evaluated to

provide a total of 21 new substituted ninhydrins. These reagents were evaluated for

fingerprint visualization by forensic experts at the US Secret Service and other law

enforcement agencies both in this country, England, Switzerland, Australia, and Israel. The

novel methodology developed afforded two complimentary reagents, which displayed

similar fingerprint developing properties (2-THIN and 3-THIN). 2-THIN was produced on

a large scale by Vinfer Ltd. in North Ireland to investigate its potential for

commercialization. 8

Finally, because of the importance of formulations in the development of new

fingerprint visualization reagents and as a potential solution to the problems caused by the

ban on chlorofluorocarbons, we developed structural modifications of ninhydrin-based


.- =

I

I

4 1

reagents to expand their solubilities in suitable organic solvents (methylene chloride,

toluene, hexanes etc.).

Our research has resulted in improved reagents for latent fingerprint development.

Additionally, it has provided a suitable alternative to the technical problems created by the

ban on chlorofluorocarbons. Our results are useful to all law enforcement agencies both in

this country and abroad.

6

I I , a

Bibliography

(1)

(2)

Ruhemann, S . Trans. Chem. SOC. 1910,97, 2025.

Odkn, S.; von Hofsten, B. Nature 1954,173, 449.

(3)

Report Number 300, Tetrahedron 1991,47, 8791-8830.

JoulliC, M. M.; Thompson, T. R.; Nemeroff, N. H. Tetrahedron I

'

, I t f

(4) Almog, J.; Hirshfeld, A.; Frank, A.; Sterling, J.; Leonov, D. J.

Forensic Sci. 1991,36, 104.

(5 ) Grigg, R.; Mongkolaussavaratana, T.; Pounds, C. A.; Sivagnanam,

S. Tetrahedron Lett. 1990, 31 , 72 15-72 18.

(6) Pounds, C. A.; Grigg, R.; Mongkolaussavaratana, T. Journal of

Forensic Sciences 1990, 35, 169- 175.

(7) Cantu, A.; Leben, D. A.; JoulliC, M. M.; Hark, R. R. J. Forensic Ident.

1993,43, 44-66.

(8) Hark, R. R.; Hauze, D. B.; Petrovskaia, 0.; JoulliC, M. M.; Jaouhari,

R.; McComiskey, P. Tetrahedron Lett. 1994,35, 77 19-7722.

(9) Hamer, M. New Scientist 1994, 5.

(10) Zurer, P. Chem. Eng. News 1995, 9.

I


Documents

New Reagents for the Development of Latent Fingerprints results when compared with the previous compounds. Since it is known that replacement of a phenyl ring by a thiophene ring in