Experiment 8: Lobry de Bruyn Transformation

Lobry de Bruyn transformation is the process involved when glucose reserves has been utilized and the fructose and
mannose reserve has not been used yet, hence the body will device means to arrange the molecular form of
fructose and mannose to that of glucose to meet the demands needed by the body or vice-versa.

Objective:
To demonstrate through in vitro tests how such sugar transformation occur.

Observation (30 Observation

sec) (1min)

- Cherry-red color
- Cherry-red color

Yellow Ppt Cherry-red color

Orange-red color

Add 4mL of fresh Seliwanoff's reagent to each tube. Shake and immerse in a boiling water bath. After 30 seconds of
heating, examine the tubes. Record results. Observe again after 1 minute of heating.

Results:
1 (Sat. Ba (OH) + 5% glucose) —Straw colored solution with red ring — Reddish brown solution with red ring
2 (Sat. Ba (OH) +5% fructose) — Blood red colored solution — Dark Red Colored Solution
3 (5% glucose + water) — Dark Straw colored solution — Reddish brown colored solution
4 (5% fructose + water) — Wine red colored solution — Mahogany red colored solution
DISCUSSION:
Experiment 1 (Glucose + Ba(OH)2):
Initial (30 seconds):
Straw-colored solution with a red ring. The straw color signifies dissolved glucose. The red ring likely suggests
early formation of fructose due to the fast reaction with Ba(OH)2 (a strong base).
Final (1 minute):
Reddish-brown solution with a red ring. This indicates an ongoing Lobry de Bruyn transformation. Glucose is
isomerized to fructose (reddish-brown contribution) while some unreacted glucose and other aldoses (like
mannose) might still be present (red ring contribution).

Experiment 2 (Fructose + Ba(OH)2):

Initial (30 seconds):
Blood-red colored solution. Fructose, being a ketose, directly reacts with Seliwanoff's reagent present in Ba(OH)2,
resulting in the characteristic red color.
Final (1 minute):
Dark red colored solution. The color deepening could be due to increased fructose-Seliwanoff reaction or slight
caramelization from heating during the experiment.

Experiment 3 (Glucose + Water):

Initial (30 seconds):
Dark straw colored solution. This simply indicates dissolved glucose.
Final (1 minute):
Reddish-brown colored solution. This suggests slow, uncatalyzed isomerization of some glucose to fructose over
time. The extent of transformation would be significantly lower compared to Experiment 1 due to the absence of a
catalyst.

Experiment 4 (Fructose + Water):

Initial (30 seconds):
Wine-red colored solution. Similar to Experiment 2, the initial color arises from the direct reaction of fructose with
Seliwanoff's reagent.
Final (1 minute):
Mahogany red colored solution. Again, the deepening color could be due to further fructose-Seliwanoff reaction of
caramelization from heating.

Key Points and Further Analysis

● Ba(OH)2 significantly accelerates the Lobry de Bruyn transformation. Comparing Experiments 1 & 3
(glucose) and 2 & 4 (fructose) demonstrates the dramatic impact of the base catalyst on the reaction rate.
● Seliwanoff's reagent confirms the presence of ketoses. The red color in Experiments 1 and 2 directly confirms
the formation of fructose, while the absence of red in 3 and 4 indicates minimal fructose generation without the
base catalyst.
● Heating might contribute to color changes. Caramelization or condensation reactions from heating could
contribute to the final color deepening in all experiments, independent of the Lobry de Bruyn transformation.

The Lobry de Bruyn transformation is not primarily a mechanism for the body to convert fructose and mannose reserves
into glucose when glucose reserves are depleted. While such interconversion can occur, it's not the main function in
healthy individuals.
● The Lobry de Bruyn transformation is a chemical reaction that interconverts aldoses (sugars with an aldehyde
group) and ketoses (sugars with a ketone group) through an enediol intermediate.
● It occurs naturally in various biological systems, including human physiology, but not as a primary response to
depleted glucose reserves.
 ● In the human body, glucose is the primary fuel source for most cells. When dietary glucose is not readily
available,gluconeogenesis (synthesis of new glucose from non-carbohydrate sources) is the main mechanism to
maintain blood sugar levels.
● The conversion of fructose and mannose to glucose through the Lobry de Bruyn transformation is a minor
contribution compared to gluconeogenesis.

Lobry de Bruyn–Alberda van Ekenstein rearrangement involves the reciprocal interconversion of carbohydrates into their
isomers in an alkaline solution through the enediolic intermediate. It is a glycochemical reaction catalyzed by hydroxide,
irrespective of the counterions, and depends only on the base concentration and temperature. The reaction mechanism
takes place via the transformation reactions. This reaction is used in the preparation of carbohydrate isomers on the basis
of available carbohydrate molecules.

RATIONALE & PRINCIPLE OF LOBRY DE BRUYN TEST:

● Rationale: This test detects the presence of aldoses (sugars with an aldehyde functional group) based on their
ability to isomerize (rearrange their structure) into ketoses (sugars with a ketone functional group) under alkaline
conditions.
● Principle: The aldose reacts with alkaline solution (sodium carbonate or bicarbonate) to form an enediol
intermediate. This intermediate can then rearrange to form a ketose isomer. The presence of the ketose is then
confirmed by further tests specific to ketoses (e.g., Seliwanoff's test).

RATIONALE & PRINCIPLE BEHIND SELIWANOFF'S TEST REAGENT

● Rationale: This test specifically detects ketoses based on their ability to form furfural derivatives upon heating with
concentrated hydrochloric acid. These derivatives react with resorcinol to produce a cherry-red colored complex.
● Principle: The ketose is dehydrated by concentrated hydrochloric acid to form hydroxymethylfurfural. This furfural
derivative then condenses with resorcinol in the presence of excess acid to form the colored complex. This
reaction is specific to ketoses and does not occur with aldoses.

We used Seliwanoff’s reagent (resorcinol + HCl) to each tube to indicate any molecular rearranged or transformed.
Selwanoff’s test is more sensitive to ketosis for ketones are easily dehydrated by HCl at boiling temperature, the ketosis
form 5- hydroxymethyl furfural and later will react with resorcinol giving a cherry red color. Aldoses react the same way but
do so at a very slow rate.

RATIONALE & PRINCIPLE BEHIND BA(OH)2

● Barium hydroxide (Ba(OH)2) is not typically used as a specific test for sugars. However, it can be used as a
general base to raise the pH of a solution, which can influence the behavior of different molecules. In the context
of sugar testing, a basic environment can accelerate the isomerization of aldoses to ketoses in the Lobry de Bruyn
test.
RATIONALE & PRINCIPLE BEHIND GLUCOSE
● Glucose is not a test itself, but rather a specific type of sugar. It is an aldose and therefore does not directly react
with Seliwanoff's reagent to produce a cherry-red color. However, glucose can undergo isomerization to fructose
(a ketose) in the Lobry de Bruyn test, which would then react with Seliwanoff's reagent.

RATIONALE & PRINCIPLE BEHIND FRUCTOSE

● Fructose is a ketose. In the presence of Seliwanoff’s reagent, its reaction should yield a cherry-red complex
indicating the presence of ketose. This indicates that test tube 1 (Ba(OH)2 + glucose + seliwanoff’s reagent)
containing glucose has undergone tautomerization and converted into D-fructose, and with its reaction with the
HCl from the seliwanoff’s reagent, would result into 5-hydroxymethylfurfural then condenses with resorcinol,
yielding a cherry red colored complex.
● It is a ketose and therefore reacts directly with Seliwanoff's reagent to produce a cherry-red color. This property is
used in various tests to specifically identify fructose.

RATIONALE & PRINCIPLE BEHIND WATER

● Water plays a crucial role in facilitating chemical reactions, including those involved in sugar testing. The solvent
properties of water allow for dissolution of reagents and sugars, enabling their interaction and subsequent
reactions.

Reagents:
● Glucose: An aldose sugar commonly found in nature.
● Barium hydroxide (Ba(OH)2): A strong base that provides OH- ions for the reaction.
● Water (H2O): The solvent in which the reaction occurs.

Reactions:
● Base-catalyzed enolization: The OH- ions from Ba(OH)2 abstract a proton from the C-2 position of glucose,
forming an enolate intermediate. This intermediate has a double bond between C-2 and C-3 and a negative
charge on the oxygen atom at C-2.
● Intramolecular rearrangement: The double bond of the enolate intermediate shifts, resulting in a flip of the
hydroxyl group at C-1. This creates a new enolate intermediate with the double bond between C-1 and C-2.
● Protonation: The newly formed enolate intermediate is protonated by water, forming a mixture of glucose and its
C-2 epimer (mannose). This is a reversible step.
● Further enolization and rearrangement: Both glucose and mannose can undergo the same enolization and
rearrangement steps described above. However, due to the different configuration at C-2, the resulting enolate
intermediates will have different stabilities.
● Selective protonation: The enolate intermediate derived from mannose is less stable and therefore protonates
faster than the one from glucose. This leads to the preferential formation of fructose, a ketose isomer of glucose.
● Equilibrium: The Lobry de Bruyn transformation is an equilibrium reaction. This means that glucose, fructose, and
mannose can interconvert continuously, with fructose being the most stable product under these conditions.

Role of Reagents:

● Ba(OH)2: Provides OH- ions that catalyze the enolization and rearrangement steps. It does not participate directly
in the reaction but influences the reaction rate and equilibrium position.
● Water: Acts as the solvent for the reaction and also serves as the source of protons for the final step.

Overall Outcome:
The Lobry de Bruyn transformation starting with glucose in a Ba(OH)2 solution ultimately leads to a mixture of
aldoses and ketoses at equilibrium. However, fructose, the ketose isomer of glucose, is the most stable product
under these conditions, resulting in its predominant formation.

Additional Notes:
 ● The reaction rate is dependent on the temperature and concentration of the Ba(OH)2 solution.
● The Lobry de Bruyn transformation is not specific to glucose and can occur with other aldoses and ketoses.
● This explanation provides a simplified overview of the reaction. The actual mechanism involves multiple steps and
intermediates.

PRINCIPLE
(OH)2 acts as a base. At slight basic solutions, sugars undergo molecular change known as TAUTOMELobry de Bruyn
transformation is the process involved where the glucose reserves of the body are molecularly rearranged to form
glucose. This transformation can happen vice versa.

Seliwanoff’s Reagent:
We used Seliwanoff’s reagent (resorcinol + HCl) to each tube to indicate any molecular rearranged or transformed.
Selwanoff’s test is more sensitive to ketosis for ketones are easily dehydrated by HCl at boiling temperature, the ketosis
form 5- hydroxymethyl furfural and later will react with resorcinol giving a cherry red color. Aldoses react the same way but
do so at a very slow rate.

● Seliwanoff's reagent detects ketoses more readily than aldoses. In your experiment, the color change indicates
the presence of fructose (a ketose) formed from other sugars like glucose (an aldose) through the Lobry de Bruyn
transformation.
● While understanding the Lobry de Bruyn transformation is valuable, it's important to distinguish its in vitro
demonstration from its specific role in human physiology.The body prioritizes other mechanisms like
gluconeogenesis for maintaining glucose homeostasis.

The Seliwanoff reagent was not actually used directly in the Lobry de Bruyn transformation itself. The Lobry de Bruyn
transformation only involves glucose, Ba(OH)2, and water, leading to an equilibrium mixture of various sugars including
fructose.

However, the Seliwanoff reagent can be used as a confirmatory test to identify the presence of fructose, which is one of
the products formed in the Lobry de Bruyn transformation. This is because it is specific to ketoses like fructose and gives
a distinct cherry-red color when they react.

Therefore, it is indistinguishable whether the presence of fructose in the final mixture is from the starting glucose
undergoing Lobry de Bruyn transformation or if it was present initially. The Seliwanoff test would simply detect the
presence of fructose, irrespective of its origin.

Here's why Seliwanoff is not used directly in Lobry de Bruyn:

Purpose: The Lobry de Bruyn transformation aims to observe the interconversion of aldoses and ketoses, not just identify
them. Seliwanoff is specific to ketoses and wouldn't provide information about the overall interconversion process.
Interference: The strong acid present in the Seliwanoff reagent could potentially interfere with the delicate equilibrium
established in the Lobry de Bruyn transformation, potentially altering the final product distribution.

Interconversion of Sugars (Tautomerization) Glucose,. Fructose and mannose are interconvertible in weak alkaline
solutions such as Ba(OH)2 or Ca(OH)2. It is because weak alkaline solutions of sugars undergo molecular change known
as “tautomerization” where H atoms migrate from one carbon to another to form “enediol” compounds. All sugars give the
same enediol w/c tautomerizes to all 3 sugars. This interconversion of related sugars by the action of dilute alkali is
referred to as “Lobry de Bruyn” - “Van Ekenstein” reaction.

1. WHAT IS THE PURPOSE OF THE ADDITION OF BA(OH)2?

Ba(OH)2 acts as a base. At slight basic solutions or weak alkaline (light basic) solutions, sugars undergo molecular
change known as TAUTOMERIZATION (epimerization)
In the experiment you described, Ba(OH)2 acts as a weak base that catalyzes the Lobry de Bruyn transformation.
Under these in vitro conditions, it promotes the interconversion of sugars present in the solution.

2. WHERE DOES LOBRY DE BRUYN TRANSFORMATION OCCUR?

While the experiment demonstrates the reaction, the main site of this transformation in humans is not the intestines. It
primarily occurs in the liver, which plays a crucial role in carbohydrate metabolism.

It often occurs in the intestines. It occurs when a person is on a carbohydrate restricted diet or the person has ingested a
lot of fruit sugar in which the body transforms it from fructose into glucose.

3. WHAT ARE THE MECHANISMS

INVOLVED AND THE CONDITIONS THAT COULD BRING SUCH TRANSFORMATION?
The Lobry de Bruyn transformation involves two main mechanisms:

● Aldose-ketose interconversion: This interconverts between sugars with different functional groups
(e.g.,glucose to fructose).

Aldose-ketose interconversion:

This interconverts between sugars with different functional groups. An example is the conversion of glucose (an aldose)
to fructose (a ketose):

● Step 1: The C1 hydroxyl group of the aldose undergoes enolization,losing a proton to form a double bond
between C1 and C2.
● Step 2: The proton is added back to either C2 or C3, forming an enediol intermediate.
● Step 3: Depending on where the proton is added, a different ketose or epimer is formed. In the case of glucose
to fructose, the proton adds to C3, generating fructose.

● Epimerization: This changes the configuration of one hydroxyl group around a specific carbon atom, creating a
different sugar isomer (e.g., glucose to mannose).

Epimerization:

This involves changing the configuration of one hydroxyl group around a specific carbon atom, creating a different sugar
isomer. For example, converting glucose to mannose changes the hydroxyl group configuration at C2:

● Step 1: Similar to aldose-ketose interconversion, enolization occurs at the C2 hydroxyl group of glucose.
● Step 2: Protonation can happen at either C2 (retaining D-glucose) or C1 (forming D-mannose).

Conditions:

● The reaction proceeds readily in weakly alkaline solutions (pH 8-10), such as those created by Ba(OH)2 used in
your experiment.
● Enzymes can also catalyze the transformation under physiological conditions in living organisms.

Additional Information:

Lobry De Bruyn transformation is the process involved where the glucose reserves of the body have been exhausted and
fructose and mannosereserves in the body are molecularly rearranged to form glucose, This transformation can happen
vice-versa.

Seliwanoff’s reagent (resorcinol+HCL) wa sused to each tube to indicate any molecular rearrangement between
glucose+fructose. The amountof color change can qualitatively indicate the amount ofsigar being rearranged or
transformed. Seliwanoff’s test is more sensitive to ketones for ketones are easily dehydrated by HCL at boiling
temperature. The ketosus form 5-hydroxymethylfurfural and the latter will react with resorcinol giving a cherry red color.
Aldose reacts the same way but does so at a very slow rate.

Tautomerization (Interconversion of Sugars)

Glucose, fructose and mannose are interconvertible in weak alkaline solutions as Ba(OH)O2 or Ca(OH)2. It is because
weak alkaline solutions of sugars undergo molecular change known as tautomerization where H atoms migrate from one
carbon to another to form “enediol” compounds. All sugars give the sane enediol which tautomerizes to all 3 sugars. This
interconversion of related sugars by the action of dilute alkali is referred to as “LOBRY DE BRUYN” reaction.

Role in Human Physiology:

● The Lobry de Bruyn transformation contributes to maintaining carbohydrate homeostasis by interconverting

available sugars.However, it is not the primary mechanism for replenishing depleted glucose reserves.
● Gluconeogenesis, the synthesis of glucose from non-carbohydrate precursors, plays a more significant role in
this process.
● The transformation likely occurs in the liver during various metabolic processes involving dietary intake,hormonal
regulation, and energy demands.

Beyond the Basics:

● The Lobry de Bruyn transformation can be coupled with other reactions, such as phosphate esterification, to
generate more complex sugar derivatives.
● It plays a role in the breakdown of dietary sugars in the intestine and the biosynthesis of various carbohydrates in
different tissues.
● Understanding this reaction is crucial for studying carbohydrate metabolism, its connection to various diseases,
and the development of therapeutic strategies.

Conditions triggering this transformation in vivo are not primarily related to depleted glucose reserves but rather complex
metabolic processes involving hormonal regulation, dietary intake, and energy demands.

2 Mechanisms of Lobry de Bruyn:

1. Aldose ketose Transformation
2. Epimerization
In the context of organic chemistry, epimerization refers to the process of converting an epimer into its diastereomeric
counterpart.

Here's a breakdown of the key terms:

● Epimer: Two molecules are considered epimers if they differ in the configuration of only one stereogenic center.
Stereogenic centers are atoms within a molecule that have four different substituents attached to them. The
configuration at a stereogenic center can be R or S, and epimers will have opposite configurations at their single
point of difference.
● Diastereomers: These are stereoisomers that are not mirror images of each other. They have different spatial
arrangements of their atoms but do not have a superimposable mirror image. So, while epimers are a specific
type of diastereomer, not all diastereomers are epimers.

Therefore, epimerization involves changing the configuration at a single stereogenic center, leading to a diastereomeric
molecule. Epimerization can happen spontaneously but often requires catalysts or specific conditions depending on the
molecule.

**Heating serves primarily to accelerate the reaction. The transformation, although possible at room temperature,
proceeds much faster with elevated temperatures. This is a general principle in chemistry, as thermal energy increases
the average kinetic energy of molecules, increasing the likelihood of successful collisions and reactions.

Seliwanoff's Test:
Heating is crucial for the reaction between Seliwanoff's reagent and ketoses (like fructose). This reaction involves several
steps, including dehydration of the ketose and condensation with resorcinol, which are facilitated by elevated
temperatures.
Regarding the timing (30 seconds vs. 1 minute):

**Comparing the results at different time points helps gauge the reaction progress. In the Lobry de Bruyn transformation, a
color change from straw to reddish-brown signifies the formation of fructose and other products. Observing the color
intensity or shade difference between 30 seconds and 1 minute can indicate the reaction rate and extent of conversion.
**In Seliwanoff's test, the intensity of the red color also reflects the concentration of ketoses present. By comparing color
intensity after 30 seconds and 1 minute, you can assess the rate of reaction between the ketose and the reagent.

Limitations to Consider:

The provided observations don't explicitly mention using Seliwanoff's reagent in the Lobry de Bruyn setup. If it was indeed
used, its presence might interfere with the transformation due to its acidic nature.
Interpreting color changes qualitatively has limitations. Quantitative measurements of starting and final sugar
concentrations would provide a more accurate picture of the transformation extent.